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WORLD HEAL1H ORGANZATION
INTERNATONAL AGENCY FOR RESEARCH ON CANCER
IARC MONOGRAHS
ON THE
EVALUATION OF CARCINOGENIC
RISKS TO HUMAS
Pharmaceutical Drugs
VOLUME 50
This publication represents the views and expert opinions

of an IAC Working Group on the
Evaluation of Carcinogenic Risks to Rumans,
which met in Lyon,
17-24 October 1989
1990
lARe MONOGRAHS
ln 1969, the International Agency for Research on Cancer (IAC) initiated a

programme on the evaluation of the carcinogenic risk of chemicals to humans
involving the production of critically evaluated monographs on individual
chemicals. ln 1980 and 1986, the programme was expanded to include the evalua-
tion of the carcinogenic risks associated with exposures to complex mixtures and
other agents.
The objective of the programme is to elaborate and publish in the form of

monographs critical reviews of data on carcinogenicity for agents to which humans
are known to be exposed, and on specific exposure situations; to evaluate these data
in terms of human risk with the help of international working groups of experts in
che mi cal carcinogenesis and related fields; and to indicate where additional
research efforts are needed.
This project is supported by PHS Grant No. 6 U01 CA33193-06 awarded by the
US National Cancer Institute, Department of Health and Human Services.
Additional support has been provided since 1986 by the Commission of the
European Communities.
C9lnternational Agency for Research on Cancer 1990
ISBN 92 832 1250 9

ISSN 0250-9555
AlI rights reserved. Application for rights of reproduction or translation, in part

or in toto, should be made to the International Agency for Research on Cancer.
Distributed for the International Agency for Research on Cancer
by the Secretariat of the World Health Organization
PRINTD lN 1RE UK
eONTNTS
Nom TO TH READER ............................................. 5
LIST OF PARTICIPANS .............................................. 7
PREAMLE
Background ...................................................... Il
Objective and Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " Il
Selection of Topics for Monographs ................................. 12
Data for Monographs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 13
The Working Group . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 13
Working Procedures ............................................... 14
Exposure Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 14
Biological Data Relevant to the Evaluation of Carcinogenicity to
Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " 16
Evidence for Carcinogenicity in Exerimental AnimaIs ................ 17
Other Relevant Data in Experimental Systems and in Humans ......... 19
Evidence for Carcinogenicity in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21
Summary of Data Reported ........................................ 24
Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 25
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 29
GENERA REMAKS ............................................... 33

TH MONOGRAPHS
Antineoplastic an.d immunosuppressive agents
Azacitidine ................................................... 47
Chlorozotocin .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " 65
Ciclosporin ........... -. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 77
Prednimustine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 115
Thiotepa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 123
'fichlormethine (Trimustine hydrochloride) . . . . . . . . . . . . . . . . . . . . .. 143
CONTNTS
Antimicrobial agents
153
Ampicillin . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 . 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0
Chloramphenicol 0 0 0 0 . 0 0 0 0 0 0 0 0 0 . 0 0 0 0 . 0 0 0 . 0 . 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 . . 0 0 169
Nitrofural (Nitrofurazoile) 0 0 0 . . 0 . 0 0 0 0 0 0 0 0 . . . 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 . 0 0 0 195
Nitrofurantoin 0 0 0 0 0 0 . 0 0 0 0 . 0 . 0 0 . 0 0 0 0 0 0 0 0 . 0 . 0 0 0 0 0 0 0 0 0 0 0 0 _ 0 . . . . 0 0 211
Other drugs
coimeti°do
ine .. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0 . 0 0 . 0 0 0 . . 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 0 . . . 0 235
Dantron (Chrysazn; 1,8-Dihydroxyanthraquinone) 000000 0 0 0 0 0 .000 265
Furosemide (Frusemide) 0 0 0 0 0 .. 0 0 0 0 0 0 0 0 0 .. .. . 0 0 0 .. 0 .. 0 0 0 0 .. 0 0 0 277
293
Hydrochlorothiazde 0 0 0 0 0 0 . 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 . 0 0 0 0 0 0
Paracetamol (Acetaminophen) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 307
SUMMAY OF FINAL EVALUATIONS 000000000.0000..0.000000000000 333

APPENDIX 10 SUMMAY TABLE OF GENETIC AN RELATED
EFFECTS 0 0 . . . . . 0 0 0 0 0 . 0 0 0 . . 0 . . . 0 0 . 0 0 . 0 0 0 0 0 0 . 0 0 0 . . 0 0 0 0 0 . 0 0 0 0 0 0 . 0 . 0 0 0 335
APPENDIX 2. ACTIVI PROFILES FOR GENETIC AN RELATED

EFFECTS 0 0 0 0 . . . . 0 . . 0 0 0 . . . . . . 0 0 0 0 . 0 0 . 0 0 0 . 0 . . . 0 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 . . 0 0 0 337
SUPPLEMENTARY CORRIGENDA TO VOLUMES 1-49. 0 0 . 0 . . . 0 0 . . 0 0 385
CUMULATIVE INDEX TO THE MONOGRAHS SERIES 0 0 0 0 0 . 0 . 0 0 0 0 387

NOTE TO THE READER
The term 'carcinogenic risk' in the lARe Monographs series is taken to mean the
probabilty that exposure to an agent wil lead to cancer in humans.
Inclusion of an agent in the Monographs does not imply that it is a carcinogen,
only that the published data have been examined. Equally, the fact that an agent has
not yet been evaluated in a monograph does not me
an that it is not carcinogenic.
The evaluations of carcinogenic risk are made by international working groups
of independent scientists and are qualitative in nature. No recommendation is
given for regulation or legislation.
Anyone who is aware of published data that may alter the evaluation of the
carcinogenic risk of an agent to humans is encouraged to make this information
available to the Unit of Carcinogen Identification and Evaluation, International
Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon Cedex 08,
France, in order that the agent may be considered for re-evaluation by a future
Working Group.
Although every effort is made to prepare the monographs as accurately as
possible, mistakes mayoccur. Readers are requested to communicate any errors to
the Unit of Carcinogen Identification and Evaluation, so that corrections can be
reported in future volumes.
-5-
lAe WORKING GROUP ON THE EVALUATION
OF eARelNOGENie RISKS TO HUMAS:
PHARAeEUTICAL DRUGS
Lyon, 17-24 October 1989
LIST OF PARTICIPANTS
Members
I.N. Chernozemsky, National Oncological Centre, Medical Academy, Darvenitza,
Sofia 1156, Bulgaria
L. Fiore-Donati, Istituto di Anatomia e Istologia Patologica, Verona University,
Policlinico Borgo Roma, 37100 Verona, Italy
G.D. Friedman, Division of Research, Kaiser Permanente Medical Care Program,
Northern California Region, 3451 Piedmont Avenue, Oakland, CA 94611, USA
B. Holmberg, Department of Toxicology, National Institute of Occupational
Health, 171 84 SoIn a, Sweden
L.J. Kinlen, Cancer Research Campaign Epidemiology Unit, University of
Edinburgh, 15 George Square, Edinburgh EH8 9JZ, UK
M. Marselos, Department of Pharmacology, Medical School, University of
loannina, loannina 45110, Greece
M. Mattila, Department of Clinical Pharmacology, University of Helsinki,
Department of Clinical Pharmacology, University of Helsinki, Paasikivenkatu
4, 00250 Helsinki, Finland
G. Obe, Universitat GSH Essen, Fachbereich 9, Department of Genetics, PO Box
103 764, 4300 Essen 1, Federal Republic of Germany
J.H. Olsen, Danish Cancer Registry, Rosenvaengets Hovedvej 35, Box 839, 2100
Copenhagen ø, Denmark
N. ~ Popova, Laboratory of Carcinogenic Substances, Ali- Union Cancer Research
Centre, Kashirskoye Shosse 24, 115478 Moscow, USSR
-7-
8 IARC MONOGRAHS VOLUME 50
J.-l Seiler1, Swiss Federal Research Station for Fruit-Growing, Viticultui1e and
Horticulture, 8820 Wadenswil, Switzerland
S. Shapiro, Boston University Medical School, Slone Epidemiology Unit, 1371
Beacon Street, Brookline, MA 02146, USA
S.M. Sieber, Division of Cancer Etiology, National Cancer Institute, Building 31,
Room 1 lA03, Bethesda, MD 20892, USA
M. Sors a, Institute of Occupation al Health, Topeliuksenkatu 41 a A, 00250 Helsinki,
Finland (Vice-ehairperson)
R. Stahlmann, Institut fur Toxikologie und Embryonal Pharmakologie der Freien
Universitat Berlin, Garystrasse 1-9, 100 Berlin 33, Federal Republic of Germany
B. Stewart, Children's Leukaemia and Cancer Research Unit, The Prince of Wales
Children's Hospital, High Street, Randwick, NSW 2031, Australia
F.M. Sullvan, Department of Pharmacology, Guy's Hospital Medical Sch(Xl,
London SEI 9Rl: UK
J. Weissinger, Office of Drug Evaluation (HFD-502), Center for Drug Evaluation
and Research, Food and Drug Administration, 560 Fishers Lane, Rockville,
MD 20857, USA
G .M. Willams, American Health Foundation, Dana Road, Valhalla, NY 10595,
USA (ehainnan)
K. Yokoro, Hiroshima University, Institute of Nuclear Medicine, 1-2-3 Kasumi,
Minami-ku, Hiroshima 734, Japan
Representatives and observers2

Representative of the Interntional Federation of Pharmaceutical Manufacturers'
Associations
E. Longstaff, Safety Medicines Department, ICI Pharmaceuticals, Mereside,
Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
Representative of the US Pharmaceutical Manufacturers' Association
M.J. Tidd, Norwich Eaton Pharmaceuticals Inc., PO Box 191, Norwich, NY
13815-0191, lJS)\
lPresent address: Interkantonale Kontrol1stelle fur Heilmittel (IKS), Erlachstras 8, 300 Bern,
Switzerland
2Unable to attend, M.-Th. van der Venne, Commission of the European Communities, Health and
Safety Directorate, Bâtiment Jean Monnet (C4/83), BP 1907, 292 Luembourg, Grand Duchy of
Luembourg
PARCIPANS 9
Secretariat
A. Aitio, Laboratory of Biochemistry, Institute of Occupational Health, Arinatie
3, 00370 Helsinki, Finland
H. Bartsch, Unit of Environmental Carcinogenesis and Host Factors
X. Bosch, Unit of Field Intervention Studies
E. Cardis, Unit of Biostatistical Research and Informatics
J. Cheney, Editorial, Translation and Publication Servces
M. Coleman, Unit of Descriptive Epidemiology
M. Friesen, Unit of Environmental Carcinogenesis and Host Factors
E. Heseltine, Montignac, France
J. Jongen, Unit of Mechanisms of Carcinogenesis
J. Kaldor, Unit of Biostatistics Research and Informatics
v: Krutovskikh, Unit of Mechanisms of Carcinogenesis
K. LAbbé, Unit of Analytical Epidemiology
D. McGregor, Unit of Carcinogen Identification and Evaluation
D. Mietton, Unit of Carcinogen Identification and Evaluation
R. Montesano, Unit of Mechanisms of Carcinogenesis
S. Narod, Unit of Mechanisms of Carcinogenesis
G. Nordberg, Unit of Carcinogen Identification and Evaluation
C. Partensky, Unit of Carcinogen Identification and Evaluation
I. Peterschmitt, Unit of Carcinogen Identification and Evaluation, Geneva,
Switzerland
D. Shuker, Unit of Environmental Carcinogenesis and Host Factors
L. Shuker, Unit of Carcinogen Identification and Evaluation
L. Tomatis, Director
H. Vainio, Unit of Carcinogen Identification and Evaluation
J. Wilboum, Unit of Carcinogen Identification and Evaluation
Secretari assistance
J. Cazaux
M. Lézère
S. Reynaud
lAe MONOGRAHS PROGRAME ON THE
EVALUATION OF eARelNOGENie RISKS TO HUMAS1
PREAMBLE
1. BACKGROUND
ln 1969, the International Agency for Research on Cancer (!AC) initiated a
programme to evaluate the carcinogenic risk of chemicals to humans and to
produce monographs on individual chemicals. The Monographs programme has
since been expanded to include consideration of exposures to complex mixures of
an
chemicals (which occur, for example, in some occupations and as a result of hum
habits) and of exposures to other agents, such as radiation and viruses. With
Supplement 6(1), the title of the series was modified from lARe Monographs on the
Evaluation of the earcinogenic Risk of ehemicals to Humans to lARe Monographs
on the Evaluation of earcinogenic Risks to Humans, in order to reflect the widened
scope of the programme.
The criteria established in 1971 to evaluate carcinogenic risk to humans were
adopted by the working groups whose deliberations resulted in the first 16 volumes
of the lARe Monographs series. Those criteria were subsequently re-evaluated by
working groups which met in 1977(2), 1978(3), 1979(4), 1982(5) and 1983(6). The
present preamble was prepared by two working groups which met in September
1986 and January 1987, prior to the preparation of Supplement 7(7) to the
Monographs and was modified by a working group which met in November 1988(8).
2. OBJECTIV AND SCOPE
The objective of the programme is to prepare, with the help of international
working groupsof experts, and to publish in the form of monographs, critical
IThis project is supported by PHS Grant No. 5 UO 1 CA33193-07 awarded by the US National Cancer
Institute, Deparment of Health and Human Servce, and with a subcntract to Tracor Technology
Resurce,Ine. Since 1986, this progrmme has also been supported by the Commission of the Euro-
pean Communities.
-11-
12 lARe MONOGRAHS VOLUME 50
reviews and evaluations of evidence on the carcinogenicity of a wide range of human

exposures. The Monographs may also indicate where additional research efforts are
needed.
The MonographS represent the first step in carcinogenic risk assessment, which
involves examination of aIl relevant information in order to assess the strength of
the available evidence that certain exposures could alter the incidence of cancer in
humans. The second step is quantitative risk estimation, which is not usually
attempted in the Monographs. Detailed, quantitative evaluations of epidemio-
logical data may be made in the Monographs, but without extrapolation beyond the
range of the data available. Quantitative extrapolation from experimental data to
the human situation is not undertaken.
These monographs may assist national and international authorities in making
risk assessments and in formulating decisions concerning any necessary preventive
measures. The evaluations of IARC working groups are scientific, qualitative
judgements about the degree of evidence for carcinogenicity provided by the
available data on an agent. These evaluations represent only one part of the body
of
information on which regulatory measures may be based. Other components of
regulatory decisions may vary from one situation to another and from country to
country, responding to different socioeconomic and national priorities. Therefore,
no recommendation is given with regard to regulation or legilation, which are the
responsibility of individual govemments and/or other international organizations.
The lARe Monographs are recognized as an authoritative source of
information on the carcinogenicity of chemicals and complex exposures. A users'
survey, made in 1988, indicated that the Monographs are consulted by various
agencies in 57 countries. Each volume is generally printed in 40 copies for
distribution to governments, regulatory bodies and interested scientists. The
Monographs are also available via the Distribution and Sales Service of the World
Health Organization.
3. SELECTION OF TOPICS FOR MONOGRAPHS

cern
Topics are selected on the basis of two main criteria: (a) that they con
agents and complex exposures for which there is evidence of human exposure, and
(b) that there is some evidence or suspicion of carcinogenicity. The term agent is
used to include individual chemical compounds, groups of che mi cal compounds,
physical agents (such as radiation) and biological factors (such as viruses) and
mixtures of agents such as occur in occupation al exposures and as a result of
personal and cultural habits (like smoking and dietary practices). Chemical
analogues and compounds with biological or physical characteristics similar to
those of suspected carcinogens may also be considered, even in the absence of data
on carcinogenicity.
PREAMBLE 13
The scientific literature is surveyed for published data relevant to an

assessment of carcinogenicity; the IAC surveys of chemicals being tested for
carcinogenicity(9) and directories of on-going research in cancer epidemiology(10)
often indicate those exposures that may be scheduled for future meetings. An
ad-hoc working group convened by IAC in 1984 gave recommendations as to
which che cals and exposures to complex mixures should be evaluated in the
mi
lARe Monographs series(11,12).

As significant new data on subjects on which monographs have already been
prepared become available, re-evaluations are made at subsequent meetings, and
revised monographs are published.
4. DATA FOR MONOGRAPHS

The Monographs do not necessarily cite all the literature concerning the subject
of an evaluation. Only those data considered by the Working Group to be relevant
to making the evaluation are included.
WIth regard to biological and epidemiological data, only reports that have
been published or accepted for publication in the openly available scientific
literature are reviewed by the working groups. ln certain instances, government
agency reports that have undergone peer review and are widely available are
considered. Exceptions may be made on an ad-hoc basis to inc1ude unpublished
reports that are in their final form and publicly available, if their inclusion is
considered pertinent to making a final evaluation (see pp. 25 et seq.). ln the sections
on chemical and physical properties and on production, use, occurrence and
analysis, unpublished sources of information may be used.
5. THE WORKING GROUP

Reviews and evaluations are formulated by a working group of experts. The
tasks of this group are five-fold: (i) to ascertain that aIl appropriate data have been
collected; (ii) to select the data relevant for the evaluation on the basis of scientific
merit; (ii) to prepare accu rate summaries of the data to enable the reader to follow
the reasoning of the Working Group; (iv) to evaluate the results of experimental and
epidemiological studies; and (v) to make an overall evaluation of the carcinogenicity
of the exposure to humans.
Working Group participants who contributed to the considerations and
evaluations within a particular volume are listed, with their addresses, at the
beginning of each publication. Each participant who is a member of a working
group serves as an individual scientist and not as a representative of any
organization, government or industry. ln addition, representatives from national
and international agencies and industrial associations are invited as observers.
6. WORKING PROCEDURES
Approximately one year in advance of a meeting of a working group, the topics
of the monographs are announced and participants are selected by IAC staff in
consultation with other experts. Subsequently, relevant biological and
epidemiological data are collected by IARC from recognized sources of
information on carcinogenesis, including data storage and retrieval systems such as
CHEMICAL ABSTRCTS, MEDLlNE and TOXLlNE-including EMIC and
ETIC for data on genetic and related effects and teratogenicity, respectively.
The major collection of data and the preparation of first drafts of the sections
on chemical and physical properties, on production and use, on occurrence, and on
analysis are carried out under a separate contract funded by the US National
Cancer Institute. Efforts are made to supplement this information with data froID
other national and international sources. Representatives from industrial
associations may assist in the preparation of sections on production and use.
Production and trade data are obtained from governmental and trade
publications and, in some cases, by direct contact with industries. Separate
production data on sorne agents may not be available because their publication
could disclose confidential information. Information on uses is usually obtained
from published sources but is often complemented by direct contact with
manufacturers.
Six months before the meeting, reference material is sent to experts, or is used
by IAC staff, to prepare sections for the first drafts of monographs. The complete
first drafts are compiled by IARC staff and sent, prior to the meeting, to aIl
participants of the Working Group for review.
The Working Group meets in Lyon for seven to eight days to discuss and
finalize the texts of the monographs and to formulate the evaluations. After the
meeting, the master copy of each monograph is verified by consulting the original
literature, edited and prepared for publication. The aim is to publish monographs
within -nine months of the Working Group meeting.
7. EXPOSURE DATA
Sections that indicate the extent of past and present human exposure, the
sources of exposure, the persons most likely to be exposed and the factors that
contribute to exposure to the agent, mixure or exposure circumstance are included
at the beginning of each monograph.
Most monographs on individual chemicals or complex mixures include
sections on chemIcal and physical data, and production, use, occurrence and
analysis. ln other monographs, for example on physical agents, biological factors,
occupation al exposures and cultural habits, other sections may be included, such
PREAMBLE 15
as: historical perspectives, description of an industry or habit, exposures in the work

place or chemistry of the complex mixture.
The Chemical Abstracts Services Registry Number, the latest Chemical
Abstracts Primary Name and the IUPAC Systematic Name are recorded. Other
synonyms and trade names are given, but the list is not necessarily comprehensive.
Some of the trade names may be those of mixtures in which the agent being
evaluated is only one of the ingredients.
Information on chemical and physical properties and, in particular, data
relevant to identification, occurrence and. biological activity are included. A
separate description of technical products gives relevant specifications and
includes available information on composition and impurities.
The dates of first synthesis and of first commercial production of an agent or
mixture are provided; for agents which do not occur naturally, this information may
allowa reasonable estimate to be made of the date before which no human exposure
to the agent could have occurred. The dates of first reported occurrence of an
exposure are also provided. ln addition, methods of synthesis used in past and
present commercial production and different methods of production which may
give rise to different impurities are described.
Data on production, foreign trade and uses are obtained for representative
regions, which usually include Europe, Japan and the USA. It should not, however,
be inferred that those areas or nations are necessarily the sole or major sources or
users of the agent being evaluated.
Some identified uses may not be current or major applications, and the
coverage is not necessarily comprehensive. ln the case of drugs, mention of their
therapeutic uses does not necessarily represent current practice nor does it imply
judgement as to their clinical efficacy.
Information on the occurrence of an agent or mixure in the environment is
obtained from data derived from the monitoring and surveilance of levels in
occupation al environments, air, water, soil, foods and animal and human tissues.
When available, data on the generation, persistence and bioaccumulation are also
included. ln the case of mixtures, industries, occupations or processes, information
is given about all agents present. For processes, industries and occupations, a
historical description is also given, noting variations in chemical composition,
physical properties or levels of occupational exposure with time.
Statements concerning regulations and guidelines (e.g., pesticide registrations,
maxmal levels permitted in foods, occupation
al exposure limits) are included for
some countries as indications of potential exposures, but they may not reflect the
most recent situation, since such limits are continuously reviewed and modified.
The absence of information on regulatory status for a country should not be taken to
imply that that country does not have regulations with regard to the exposure.
The purpose of the section on analysis is to give the reader an overview of
current methods cited in the literature, with emphasis on those widely used for
regulatory purposes. No critical evaluation or recommendation of any of the
methods is meant or implied. Methods for monitoring human exposure are also
given, when available. The IAC publishes a series of volumes, Environmental
earcinogens: Methods of Analysis and Expsure Measurement(13), that describe
validated methods for analysing a wide variety of agents and mixures.
8. BIOLOGICAL DATA RELEVANT TO THE EVALUATION OF CARCINO-
GENICITY TO HUMANS
The term 'carcinogen' is used in these monographs to denote an agent or
mixture that is capable of increasing the incidence of malignant neoplasms; the
induction ofbenign neoplasms may in some circumstances (see p. 18) contribute to
the judgement that the exposure is carcinogenic. The terms 'neoplasm' and
'tumour' are used interchangeably.
Some epidemiological and experimental studies indicate that different agents
may actat different stages in the carcinogenic process, probably by fundamentally
different mechanisms. ln the present state of knowledge, the aim of the Monographs
is to evaluate evidence of carcinogenicity at any stage in the carcinogenicprocess
independently of the underlying mechanism involved. There is as yet insufficient
information to implement classification according to mechanisms of action(6).
Definitive evidence of carcinogenicity in humans can be provided only by
epidemiological studies. Evidence relevant to human carcinogenicity may also be
provided by experimental studies of carcinogenicity in animaIs and by other
biological data, particularly those relating to humans.
The available studies are summarized by the Working Group, with particular
regard to the qualitative aspects discussed below. ln general, numerical findings
are indicated as they appear in the original report; units are converted when
necessary for easier comparison. The Working Group may conduct additional
analyses of the published data and use them in their assessment of the evidence and
may include them in their summary of a study; the results of such supplementary
analyses are given in square brackets. Any comments aIe also made in square
brackets; however, these are kept to a minimum, being restricted to those instances
in which it is felt that an important aspect of a study, directly impinging on its
interpretation, should be brought to the attention of the reader.
For experimental studies with mixures, consideration is given to the
possibility of changes in the physicochemical properties of the test substance
during collection, storage, extraction, concentration and delivery. Either chemical
PREAMBLE 17
or toxicological interactions of the components of mixures may result in nonlinear

dose- response relationships.
An assessment is made as to the relevance to human exposure of samples
tested in exprimental systems, which may involve consideration of: (i) physical and
chemical characteristics, (ii) constituent substances that indicate the presence of a
c1ass of substances, (ii) tests for genetic and related effects, including genetic
activity profiles, (iv) DNA adduct profiles, (v) oncogene expression and mutation;
suppressor gene inactivation.
9. EVDENCE FOR CARCINOGENICITY lN EXPERIMENTAL ANlMALS
For several agents (e.g., 4-aminobiphenyl, bise chloromethyl)ether,
diethylstilboestrol, melphalan, 8-methoxysoralen (methoxsalen) plus ultra -violet
radiation, mustard gas and vinyl chloride), evidence of carcinogenicity in
experimental animaIs preceded evidence obtained from epidemiological studies or
case reports. Information compiled from the first 41 volumes of the lARe
Monographs(14) shows that, of the 44 agents and mixures for which there is
suffcíent or limited evidence of carcinogenicity to hum ans (see p. 26), all 37that have
been tested adequately experimentally produce cancer in at least one animal
species. Although this association cannot establish that all agents and mixures
that cause cancer in experimental animaIs also cause cancer in humans,
nevertheless, in the absence of adequate data on humans, it is biologically plausible
and prudent to regard agents and mixures for which there is suffcíent evidence (see
p. 27) of carcinogenicíty in exerimental animaIs as if they presented a carcinogenic rik
to humans.
The monographs are not intended to summarize all published studies. Those
that are inadequate (e.g., too short a duration, too few animaIs, poor survival; see
below) or are judged irrelevant to the evaluation are generally omitted. They may be
mentioned briefly, particularly when the information is considered to be a useful
supplement to that of other reports or when they provide the only data available.
Their inclusion does not, however, imply acceptance of the adequacy of the
experimental design or of the analysis and interpretation of theIr results.
Guidelines for adequate long-term carcinogenicity experiments have been outlIned
(e.g., 15).
The nature and extent of impurities or contaminants present in the agent or
mixure being evaluated are given when avaIlable. Mention is made of aIl routes of
exposure that have been adequately studied and of all species in which relevant
experiments have ben performed. Animal strain, sex, numbers per group, age at
start of treatment and survival are reported.
EXperiments in which the agent or mixure was administered in conjunction
with known carcinogens or factors that modify carcinogenic effects are also
reported. Experiments on the carcinogenicity of known metabolites and derivatives

may be included.
(a) Qualitative aspects

An assessment of carcinogenicity involves several considerations of qualitative
importance, including (i) the experimental conditions under which the test was
performed, including route and schedule of exposure, species, strain, sex, age,
duration of follow-up; (ii) the consistency of the results, for example, across species
and target organes); (ii) the spectrum ofneoplastic response, from benign tumours
to malignant neoplasms; and (iv) the possible role of modifying factors.
Considerations of importance to the Working Group in the interpretation and
evaluation of a particular study include: (i) how clearly the agent was defined and, in
the case of mixures, how adequately the sample characterization was reported; (ii)
whether the dose was adequately monitored, particularly in inhalation experiments;
(iii) whether the doses used were appropriate and whether the survival of treated
animaIs was simIlar to that of con troIs; (iv) whether there were adequate numbers of
animaIs per group; (v) whether animaIs of both sexes were used; (vi) whether
animaIs were allocated randomly to groups; (vii) whether the duration of
observation was adequate; and (viii) whether the data were adequately reported. If
available, recent data on the incidence of specific tumours in historical controls, as
well as in concurrent controls, should be taken into account in the evaluation of
tumour response.
When benign tumours occur together with and originate from the same cell
type in an organ or tissue as malignant tumours in a particular study and appear to
represent a stage in the progression to malignancy, It may be valid to combine them
in assessing tumour incidence. The occurrence of lesions presumed to be
preneoplastic may in certain instances aid in assessing the biological plausibilty of
any neoplastic response observed.
Of the many agents and mixures that have been studied extensively, few
induced only benign neoplasms. Benign tumours in experimental animaIs fre-
quently represent a stage in the evolution of a malignant neoplasm, but they may be
'endpoints' that do not readily undergo transition to malignancy. However, if an
agent or mixture is found to induce only benign neoplasms, it should be suspected of
being a carcinogen and it requires further investigation.
(h) Quantitative aspects
The probabilty that tumours wil occur may depend on the species and strain,
the dose of the carcinogen and the route and period of exposure. Evidence of an
increased incidence of neoplasms with increased level of exposure strengthens the
inference of a causal association between the exposure and the development of
neoplasms.
PREAMBLE 19
The form of the dose-response relationship can vary widely, depending on the
particular agent under study and the target organ. Since many chemicals require
metabolic activation before being converted into their reactive intermediates, both
metabolic and pharmacokinetic aspects are important in determining the
dose-response pattern. Saturation of steps such as absorption, activation,
inactivation and elimination of the carcinogen may produce nonlinearity in the
dose-response relationship, as cou Id saturation of processes such as DNA
repaire 16,17).
(c) Statistical analysis of long-term exeriments in animaIs

Factors considered by the Working Group inc1ude the adequacy of the
information given for each treatment group: (i) the number of animaIs studied and
the number examined histologically, (ii) the number of animaIs with a given tumour
type and (iii) length of survvaL. The statistical methods used should be clearly
stated and should be the generally accepted techniques refIned for this
purpose(17,18). When there is no difference in survival between control and
treatment groups, the Working Group usually compares the proportions of animaIs
developing each tumour type in each of the groups. Otherwise, consideration is
given as to whether or not appropriate adjustments have been made for differences
in survivaL. These ad justments can include: comparisons of the proportions of
tumour-bearing animaIs among the 'effective number' of animaIs alive at the time
the first tumour is discovered, in the case where most differences in survival occur
before tumours appear; life-table methods, when tumours are visible or when they
may be considered 'fatal' because mortality rapidly follows tumour development;
and the Mantel- Haenszel test or logis tic regression, when occult tumours do not
affect the animaIs' risk of dying but are 'incidental' findings at autopsy.
ln practice, classifying tumours as fatal or incidental may be difficult. Several
survival-adjusted methods have been developed that do not require this
distinction(17), although they have not been fully evaluated.
10. OTHER RELEVANT DATA lN EXPERIMENTAL SYSTEMS AND
HUMANS
(a) Structure-activity considerations

This section describes structure-activity correlations that are relevant to an
evaluation of the carcinogenicity of an agent.
(b) Absorption, distribution, excretwn and metabolism

ConCise information is given on absorption, distribution (including placental
transfer) and excretion. Kinetic factors that may affect the dose-reponse
relationship, such as saturation of uptake, protein binding, metabolic activation,
detoxification and DNA repair processes, are mentioned. Studies that indicate the
metabolic fate of the agent in experimental animaIs and hum

ans are summarized
briefly, and comparisons of data from animaIs and humans are made when
possible. Comparative information on the relationship between exposure and the
dose that reaches the target site may be of particular importance for extrapolation
between species.
(c) Toxicity
Data are given on acute and chronic toxic effects (other th
an cancer), such as
organ toxicity, immunotoxicity, endocrine effects and preneoplastic lesions. Effects
on reproduction, teratogenicity, feto- and embryotoxicity are also summarized
briefly.
(d) Genetic and related effects
Tests of genetic and related effects may indicate possible carcinogenic activity.
They can also be used in detecting active metabolites of known carcinogens in
human or animal body fluids, in detecting active components in complex mixtures
and in the elucidation of possible mechanisms of carcinogenesis.
The adequacy of the reporting of sample characterization is considered and,
where necessary, commented upon. The available data are interpreted critically by
phylogenetic group according to the endpoints detected, which may include DNA
damage, gene mutation, sister chromatid exchange, micronuclei, chromosomal
aberrations, aneuploidy and cell transformation. The concentrations (doses)
employed are given and mention is made ofwhether an exogenous metabolic system
was required. When appropriate, these data may be represented by bar graphs
(activity profiles), with corresponding summary tables and listings of test systems,
data and references. Detailed information on the preparation of these profiles is
given in an appendix to those volumes in which they are used.
Positive results in tests using prokaryotes, lower eukaryotes, plants, insects and
cultured mammalian cells suggest that genetic and related effects (and therefore
possibly carcinogenic effects) could occur in mammals. Results froID such tests
may aIso give information about the types of genetic effect produced and about the
involvement of metabolic activation. Some endpoints described are clearly genetic
in nature (e~g., gene mutations and chromosomal aberrations), others are to a
greater or lesser degree associated with genetic effects (e.g., unscheduled DNA
synthesis). ln-vitro tests for tumour-promoting activity and for cell transformation
may detect changes that are not necessarily the result of genetic alterations but that
may have specific relevance to the process of carcinogenesis. A critical appraisal of
these tests has been published(15).
Genetic or other activity detected ifi the systems mentioned above Is not always
manifest in whole mammals. Positive indications of genetic effects in experimental
mammals and in humans are regarded as being of greater relevance than those in
PREAMBLE 21
other organisms. The demonstration that an agent or mixture can induce gene and
chromosomal mutations in whole mammals indicates that it may have the potential
for carcinogenic activity, although this activity may not be detectably expressed in
any or all species tested. Relative potency in tests for mutagenicity and related
effects is not a reliable indicator of carcinogenic potency. Negative results in tests
for mutagenicity in selected tissues from animaIs treated in vivo provide less weight,
partly because they do not exclude the possibilty of an effect in tissues other than
those examined. Moreover, negative results in short-term tests with genetic
endpoints cannot be considered to provide evidence to mIe out carcinogenicity of
agents or mixures that act through other mechanisms. Factors may arise in many
tests that could give misleading results; the se have been discussed in detail
elsewhere(15).
The adequacy of epidemiological studies of reproductive outcomes and
genetic and related effects in humans Is evaluated by the same criteria as are
applied to epidemiological studies of cancer.
11. EVIDENCE FOR CARCINOGENICITY lN HUMANS
(a) rypes of studies considered
Three types of. epidemiological studies of cancer contribute data to the
assessment of carcinogenicity in humans-cohort studies, case-control studies and
correlation studies. Rarely, results from randomized trials may be available. Case
reports of cancer in hum ans are also reviewed.
Cohort and case-control studies relate individual exposures under study to the
occurrence of cancer in individuals and provide an estimate of relative risk (ratio of
incidence in those exposed to incidence in those not exposed) as the main measure
of association.
ln correlation studies, the units of investigation are usually whole populations
(e.g., in particular geographical areas or at particular times), and cancer frequency
is related to a summary measure of the exposure of the population to the agent,
mixure or exposure circumstance under study. Because individual exposure is not
documented, however, a causal relationship is less easy to infer from correlation
studies than from cohort and case-control studies.
Case reports generally arise from a suspicion, based on clinical experience,
that the concurrence of two events ~ that is, a particular exposure and occurrence of
a cancer - has happened rather more frequently than would be expected by chance.
Case reports usually lack complete ascertainment of cases in any population,
definition or enumeration ofthe population at risk and.estimation of the expected
number of cases in the absence of exposure.
The uncertainties surounding interpretation of case reports and correlation
studies make them inadequate, except in rare instances, to forff the sole basis for
inferring a causal relationship. When taken together with case-control and cohort
studies, however, relevant case reports or correlation studies mayadd materially to
the judgement that a causal relationship is present.
Epidemiological studies of benign neoplasms and presumed preneoplastic
lesions are also reviewed by working groups. They may, in some instances,
strengthen inferences drawn from studies of cancer itself.
(h) Quality of studies considered

It is necessary to take into account the possible roles of bias, confounding and
chance in the interpretation of epidemiological studies. By 'bias' is meant the
operation of factors in study design or execution that lead erroneously to a stronger
or weaker association than in fact exists between disease and an agent, mixure or
exposure circumstance. By 'confounding' is meant a situation in which the
relationship with disease is made to appear stronger or to appear weaker than it
truly is as a result of an association between the apparent causal factor and another
factor that is associated with either an increase or decrease in the incidence of the
disease. ln evaluating the extent to which these factors have been minimized in an
individual study, working groups consider a number of aspects of design and
analysis as described in the report of the study. Most of these considerations apply
equally to case-control, cohort and correlation studies. Lack of clarity of any of
these aspects in the reporting of a study can decrease its credibility and its
consequent weighting in the final evaluation of the exposure.
Firstly, the study population, disease (or diseases) and exposure should have
been well defined by the authors. Cases in the study population should have been
identified in a way that was independent of the exposure of interest, and exposure
should have been assessed in a way that was not related to disease status.
Secondly, the authors should have taken account in the study design and
analysis of other variables that can influence the risk of disease and may have been
related to the exposure of interest. Potential confounding by such variables should
have been dealt with either in the design of the study, such as by matching, or in the
analysis, by statistical adjustment. ln cohort studies, comparisons with local rates
of disease may be more appropriate than those with national rates. InternaI
comparisons of disease frequency among individuals at different levels of exposure
should also have been made in the study.
Thirdly, the authors should have reported the basic data on which the
conclusions are founded, even if sophisticated statistical analyses were employed.
At the very least, they should have given the numbers of exposed and unexposed
cases and con troIs in a case-control study and the numbers of cases observed and
expected in a cohort study. Further tabulations by time since exposure began and
other temporal factors are also important. ln a cohort study, data on aIl cancer sites
PREAMBLE 23
and aIl causes of death should have been given, to avoid the possibility of reporting
bias. ln a case-control study, the effects of investigated factors other than the
exposure of interest should have been reported.
Finally, the statistical methods used to obtain estimates of relative risk,
absolute cancer rates, confidence intervals and significance tests, and to adjust for
confounding should have been clearly stated by the authors. The methods used
should preferably have been the generally accepted techniques that have been
refined since the mid- 1970s. These methods have been reviewed for case-control
studies(19) and for cohort studies(20).
(c) Quantitative considerations
Detailed analyses of both relative and absolute risks in relation to age atfirst
exposure and to temporal variables, such as time since first exposure, duration of
exposure and time since exposure ceased, are reviewed and summarized when
avaIlable. The analysis of temporal relationships can provide a useful guide in
formulating models of carcinogenesis. ln particular, such analyses may suggest
whether a carcinogen acts early or late in the process of carcinogenesis( 6), although
such speculative inferences cannot be used to draw firm conclusions concerning the
mechanism of action and hence the shape (linear or otherwise) of the dose-response
relationship below the range of observation.
(d) eriteria for causa/ity
After the quality of individual epidemiological studies has been summarized
and assessed, a judgement is made concerning the strength of evidence that the
agent, mixure or exposure circumstance in question is carcinogenic for humans. ln
making their judgement, the Working Group considers several criteria for causality.
A strong association (Le., a large relative risk) is more likely to indicate causality
than a weak association, although it is recognized that relative risks of small
magnitude do not imply lack of causality and may be important if the disease is
common. Associations that are replicated in several studies of the same design or
using different epidemiological approaches or under different circumstances of
exposure are more likely to represent a causal relationship th an isolated
observations from single studies. If there are inconsistent results among investi-
gations, possible reasons are sought (such as differences in amount of exposure),
and results of studies judged to be of high quality are given more weight than those
from studies judged to be methodologically less sound. When suspicion of
carcinogenicity arises largely from a single study, these data are not combined with
those from later studies in any subsequent reassessment of the strength of the
evidence.
If the risk of the disease in question increases with the amount of exposure, this
is considered to be a strong indication of causality, although absence of a graded
24 lARC MONOGRAHS VOLUME 50
response is not necessarily evidence against a causal relationship. Demonstration

of a decline in risk after cessation of or reduction in exposure in individuals or in
whole populations also supports a causal interpretation of the findings.
Although a carcinogen may act upon more than one target, the specificity of an
association (i.e., an increased occurrence of cancer at one anatomical site or of one
morphological type) adds plausibility to a causal relationship, particularly when
excess cancer occurrence is limited to one morphological type within the same
organ.
Although rarely available, results from randomized trials showing different
rates among exposed and unexposed individuals provide particularly strong
evidence for causality.
When several epidemiological studies show little or no indication of an
association between an exposure and cancer, the judgement may be made that, in
the aggregate, they show evidence of lack of carcinogenicity. . Such a judgement
requires first of all that the studies giving rise to it meet, to a sufficient degree, the
standards of design and analysis described above. Specifically, the possibilty that
bias, confounding or misclassification of exposure or outcome could explain the
observed results should be considered and excluded with reasonable certainty. ln
addition, all studies that are judged to be methodologically sound should be
consistent with a relative risk of unityfor any observed level of exposure and, when
considered together, should provide a pooled estimate of relative risk which is at or
near unity and has a narrow confidence interval, due to sufficient population size.
Moreover, no individual study nor the pooled results of aIl the studies should show
any consistent tendency for relative risk of cancer to increase with increasing level of
exposure. It is important to note that evidence oflack of carcinogenicity obtained iil
this way from several epi demiological studies can apply only to the type( s) of cancer
studied and to dose levels and intervals between first exposure and observation of
disease that are the same. as or less than those observed in all the studies.
Experience with human cancer indicates that, in some cases, the period from first
exposure to the development of clinical cancer is seldom less than 20 years; latent
periods substantially shorter than 30 years cannot provide evidence for lack of
carcinogenici ty.
12. SUMMARY OF DATA REPORTED

ln this section, the relevant experimental and epidemiological data are
summarized. Only reports, other than in abstract form, that meet the criteria
outlined on p. 13 are considered for evaluating carcinogenicity. Inadequate studies
are generally not summarized: such studies are usually identified by a
square-bracketed comment in the text.
PREAMBLE 25
(a) Exsures
Human exposure is summarized on the basis of elements such as production,
use, occurrence in the environment and determinations in human tissues and body
fluids. Quantitative data are given when available.
(h) Exerimental carcinogenicity data

Data relevant to the evaluation of carcinogenicity in animaIs are summarized.
For each animal species and route of administration, it is stated whether an
increased incidence of neoplasms was observed, and the tumour sites are indicated.
If the agent or mixture produced tumours after prenatal exposure or in single-dose
experiments, this is also indicated. Dose-response and other quantitative data may
be given when available. Negative findings are also summarized.
(c) Human carcinogenicity data

Results of epidemiological studies that are considered to be pertinent to an
assessment of human carcinogenicity are summarized. When relevant, case reports
and correlation studies are also considered.
(d) Other relevant data

Structure-activity correlations are mentioned when relevant.
Toxicological information and data on kinetics and metabolIsm in
experimental animaIs are given when considered relevant. The results of tests for
genetic and related effects are summarized for whole mammals, cultured
mammalian cells and nonmammalian systems.
Data on other biological effects in humans of particular relevance are
summarized. These may include kinetic and metabolic considerations and
evidence of DNA binding, persistence of DNA lesions or genetic damage in
exposed humans.
When available, comparisons of such data for humans and for animaIs, and
particularly animaIs that have developed cancer, are described.
13. EVALUATION
Evaluations of the strength of the evidence for carcinogenicity arising from
human and experimental animal data are made, using standard terms.
It is recognized that the criteria for these evaluations, described below, cannot
encompass all of the factors that may be relevant to an evaluation of
carcinogenicity. ln considering aU of the relevant data, the Working Group may
assign the agent, mixure or exposure circumstance to a higher or lower category
than a strict interpretation of these criteria would indicate.
(a) Degrees of evidence for carcinogenicity in humans and in exerimental

animaIs and supporting evidence
It should be noted that these categories refer only to the strength of the
evidence that an exposure is carcinogenic and not to the extent of its carcinogenic
activity (potency) nor to the mechanism involved. A classification may change as
new information becomes available.
An evaluation of degree of evidence, whether for a single substance or a
mixure, is limited to the materials tested, and these are chemically and physically
defined. When the materials evaluated are considered by the Working Group to be
sufficiently closely related, they may be grouped for the purpose of a single
evaluation of degree of evidence.
(i) Human carcinogenicity data

The applicabilty of an evaluation of the carcinogenicity of a mixure, process,
occupation or industry on the basis of evidence from epidemiological studies
depends on the variability over time and place of the mixures, processes,
occupations and industries. The Working Group seeks to identify the specific
exposure, process or activity which is considered most likely to be responsible for
any excess risk. The evaluation is focused as narrowly as the available data on
exposure and other aspects permit.
The evidence relevant to carcinogenicity from studies in humans is classified
into one of the following categories:
Suffcient evidence of carcinogenicity The Working Group considers that a

causal relationship has been established between exposure to the agent, mixure or
exposure circumstance and human cancer. That is, a positive relationship has been
observed between the exposure and cancer in studies in which chance, bias and
confounding could be ruled out with reasonable confidence.
Liriited evidence of carcinogenicity A positive association has been observed
between exposure to the agent, mixture or exposure circumstance and cancer for
which a causal interpretation is considered by the Working Group to be credible,
but chance, bias or confounding could not be ruled out with reasonable confidence.
Inadequate evidence of carcinogenicity The avaIlable studies are of insufficient
quality, consistency or statistical power to permit a conclusion regarding the
presence or absence of a causal association.
Evidence suggesting lack of carcinogenicity There are several adequate studies
covering the. full range of levels of exposure that human beings are known to
encounter, which are mutually consistent in not showing a positive association
between exposure to the agent, mixture or exposure circumstance and any studied
cancer at any observed level of exposure. A conclusion of 'evidence suggesting lack
of carcinogenicity' is inevitably limited to the cancer sites, conditions and levels of
PREAMBLE 27
exposure and length of observation covered by the available studies. ln addition,

the possibilty of a very -small risk at the levels of exposure studied can never be
excluded.
ln sorne instances, the above categories may be used to classify the degree of
evidence for carcinogenicity for specific organs or tissues.
(ii) Experimental carcinogenicity data
The evidence relevant to carcinogenicity in experimental animaIs is classified
into one of the following categories:
Suffcient evidence of carcinogenicity: The Working Group considers that a
causal relationship has been established betweeri the agent or mixure and an
increased incidence of malignant neoplasms or of an appropriate combination of
benign and malignant neoplasms (as described on p. 18) in (a) two or more species
of animaIs or (b) in two or more independent studies in one species carried out at
different times or in different laboratories or under different protocols.
Exceptionally, a single study in one species might be considered to provide
sufficient evidence of carcinogenicity when malignant neoplasms occur to an
unusual degree with regard to incidence, site, type of tumour or age at onset.
ln the absence of adequate data on humans, it is biologically plausible and
prudent to regard agents and mixtures for which there is suffcient evidence of
carcinogenicity in experimental animaIs as if they presented a carcinogenic risk to
humans.
Limited evidence of carcinogenicity The data suggest a carcinogenic effect but
are limited for making a definitive evaluation because, e.g., (a) the evidence of
carcinogenicity is restricted to a single experiment; or (b) there are unresolved
questions regarding the adequacy of the design, conduct or interpretation of the
study; or (c) the agent or mixture increases the incidence only of benign neoplasms
or lesions of uncertain neoplastic potential, or of certain neoplasms which may
occur spontaneously in high incidences in certain strains.
Inadequate evidence of carcinogenicity The studies cannot be interpreted as
showing either the presence or absence of a carcinogenic effect because of major
qualitative or quantitative limitations.
Evidence suggesting lack of carcinogenicity Adequate studies involving at least
two species are avaIlable which show that, within the limits of the tests used, the
agent or mixture is not carcinogenic. A conclusion of evidence suggesting lack of
carcinogenicity is inevitably lImited to the species, tumour sites and levels of
exposure studied.
(iii) Supporting evidence of carcinogenicity
Other evidence judged to be relevant to an evaluation of carcinogenicity and of
sufficient importance to affect the overall evaluation is then described. This may
include data on tumour pathology, genetic and related effects, structure-activity

relationships, metabolism and pharmacokinetics, physicohemical. parameters,
chemical composition and possible mechanisms of action. For complex exposures,
including ocupational and industrial expsures, the potential contribution of
carcinogens known to be present as well as the relevance of materials tested are
considered by the Working Group in its overall evaluation of human
carcinogenicity. The Working Group also determines to what extent the materials
tested in experimental systems are relevant to those to which humans are exposed.
The available experimental evidence may help to specify more precisely the causal
factor( s).
(h) Overall evaluation

Finally, the body of evidence is considered as a whole, in order to reach an
overall evaluation of the carcinogenicity to hum
ans of an agent, mixure or
circumstance of exposure.
An evaluation may be made for a group of chemical compounds that have been
evaluated by the Working Group. ln addition, when supPOrting data indicate that
other, related compounds for which there is no direct evidence of capacity to induce
cancer in animaIs or in humans may also be carcinogenic, a statement describing
the rationale for this conclusion is added to the evaluation narrative; an additional
evaluationmay be made for this broader group of compounds if the strength of the
evidence warrants it.
The agent, mixure or exposure circumstance is described according to the
wording of one of the following categories, and the designated group is given. The
categorization of an agent, mixure or exposure circumstance is a matter of
scientific judgement, reflecting the strength of the evidence derived from studies in
humans and in experimental animaIs and from other relevant data.
Group 1 - The agent (mixre) is carcinogenic to humons.
The exsure circumstance entails exsures that are carcinogenic to humans.
This categoiy is used onlywhen there is suffcient evidence of carcinogenicity in
humans.
Group 2
This category includes agents, mixures and exposure circumstances forwhich,
at one extreme, thedegree of evidence of carcinogenicity in hum
ans is almost
sufficient, as well as those for which, at the other extreme, there are no human data
but for which there is exprimental evidence of carcinogenicity. Agents, mixures
and exposure circumstances are assigned to either 2A (probably carcinogenic) or
2B (possibly carcinogenic) on the basis of epidemiological, experimental and other
relevant data.
PREAMBLE 29
Group lA-The agent (mixure) is probably carcinogenic to humans.

The exsure circumstance entails exsures that are probably carcinogenic to
humons.
This category is used when there is limited evidence of carcinogenicity in
humans and suffcient evidence of carcinogenicity in experimental animaIs.
Exceptionally, an agent, mixure or exposure circumstance may be classified into
this category solely on the basis of limited evidence of carcinogenicity in humans or
of suffcient evidence of carcinogenicity in experimental animaIs strengthened by
supporting evidence from other relevant data.
Group 2B-The agent (mixre) is possibly carcinogenic to humans.
The exsure circumstance entails exsures that are possibly carcinogenic to humans.
This category is generally used for agents, mixures and exposure
circumstances for which there is limited evidence of carcinogenicity in humans in the
absence of suffcient evidence of carcinogenicity in experimental animaIs. It may
also be used when there is inadequate evidence of carcinogenicity in humans orwhen
hum an data are nonexistent but there is suffcient evidence of carcinogenicity in
experimental animaIs. ln sorne instances, an agent, mixure or exposure circum-
stance for which there is inadequate evidence of or no data on carcinogenicity in
hum ans but limited evidence of carcinogenicity in experimental animaIs together
with supporting evidence from other relevant data may be placed in this groupe
Group 3- The agent (mixure, expsure circumstance) is not classifiable as to its
carcinogenicity to humans.
Agents, mixtures and exposure circumstances are placed in this category when
they do not fall into any other groupe
Group 4 - The agent (mixure, exsure circumstance) is probably not carcinogenic to
humons.
This category is used for agents, mixures and exposure circumstances for
which there is evidence suggesting lack of carcinogenicity in hum
ans together with
evidence suggesting lack of ciircinogenicity in experimental animaIs. ln some
instances, agents, mixures or exposure circumstances for which there is inadequate
evidence of or no data on carcinogenicity in hum ans but evidence suggesting lack of
carcinogenicity in experimental animaIs, consistently and strongly supported by a
broad range of other relevant data, may be classified in this groupe
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PREAMLE 31
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VoL. 8. Some Metals: As, Be, Cd, Cr, Ni, Pb, Se, Zn (lARC Scientific Publications No. 71).
Edited by I.K. O'Neil, P. Schuller & L. Fishbein (1986)
VoL. 9. Passive Smoking (lARC Scientific Publications No. 81). Edited by LK. O'Neil,
K.D. Brunnemann, B. Dodet & D. Hoffmann (1987)
VoL. 10. Benzene an Alkylated Benzenes (lARC Scientific Publications No. 85). Edited
by L. Fishbein & I.K. O'Neil (1988)
14. Wilboum, J., Haroun, L., Heseltine, E., Kaldor, J., Partensky, C. & Vainio, H. (1986)
Response of experimental animaIs to human carcinogens: an analysis based upon
the IARC Monographs Programme. Carcinogenesis, 7, 1853-1863
15. Montesano, R., Bartsch, H., Vainio, H., Wilboum, J. & Yamasaki, H., eds (1986)
Long-term and Short-term Assays for Carcinogenesis - A Critical Appraisal (IARC
Scientific Publications No. 83), Lyon, IARC
16. Hoel, D.G., Kaplan, N.L. & Anderson, M.W. (1983) Implication of nonlinear kietics
on risk estimation in carcinogenesis. Science, 219, 1032-1037
17. Gart, J.J., Krewski, D., Lee, EN., Throne, R.E. & Wahrendod, J. (1986) Statistical
Methods in Cancer Research, Vol.3, The Design and Analysis of Long-term Animal
Expriments (IARC Scientific Publications No. 79), Lyon, IARC
18. Peto, R., Pike, M.C., Day, N.E., Gray, R.G., Lee, EN., Parish, S., Peto, J., Richards, S.
& Wahrendod, J. (1980) Guidelines for simple, sensitive significance tests for
carcinogenic effects in long-term animal experients. In: lAC Monographs on the
Evaluation of the Carcinogenic Risk of Chemicals to Humans, Supplement 2,
Long-term and Short-term Screening Assays for Carcinogens: A Critical Appraisal,
Lyon, pp. 311-426
19. Breslow, N.E. & Day, N.E. (1980) Statistical Methods in Cancer Research, VoL. 1, The
Case-control Studies (IARC Scientific Publications No. 32), Lyon, IARC
Analysis of
20. Breslow, N.E. & Day, N.E. (1987) Statistical Methods in Cancer Research, VoL. 2, The
Design and Analysis o/Cohort Studies (IARC Scientific Publications No. 82), Lyon,
IARC
GENERA REMAS
ON THE SUBSTANeES eONSIDERED
This fiftieth volume of the lARe Monographs comprises monographs on five

antineoplastic agents, four antimicrobial agents, two diuretics, ciclosporin (an
immunosuppressant), cimetidine (used in the treatment of gastric and duodenal
ulcers), paracetamol (a popular analgesic and antipyretic drug) and dantron (a
laxative). Many pharmaceutical drugs were evaluated in previous lARe Mono-
graphs (see Table 1), including sorne of those covered in this volume. Azacitidine
and trichlormethine-both antineoplastic agents-and nItrofural-an anti-
bacterial drug-were re-evaluated because new data on carcinogenicity in experi-
mental animaIs had been pub Ii shed since the earlier evaluations; thiotepa and
chloramphenicol were re-evaluated largely because of new data on carcino-
genicity in humans.
Table 1. Pharmaceutical agents evaluated in the lARC Monographs
Agent Vol. no. Year Evaluationa
Ruman Animal Overall

Anaethetics
Anaesthetics (unspecified mixres) Suppl. 7 1987 1 3
Chlorofonn Suppl. 7 1987 1 S 2B
Cyc1opropane Suppl. 7 1987 1 ND 3
Diethyl ether Suppl. 7 1987 1 ND 3
Divinyl ether Suppl. 7 1987 1 ND 3
Enfluràne Suppl. 7 1987 1 1 3
Fluroxene Suppl. 7 1987 1 ND 3
Halothane Suppl. 7 1987 1 1 3
lsoflurane Suppl. 7 1987 1 l 3
Methoxyurane Suppl. 7 1987 1 1 3
Nitrous oxide Suppl. 7 1987 1 1 3
Trichloroethylene Suppl. 7 1987 1 L 3
Angeies and anti-injlammaory agents
Aurothiogluco 13 1977 ND L 3
Oxhenbutazone 13 1977 ND ND 3
-33-
Table 1 (contd)
Ruman Animal Ove raIl
Analgesies and anti-injlammatory agents lcontd)

Paracetamol (Acetaminophen) 50 199 1 L 3
Phenacetin Suppi. 7 1987 L S 2A
Analgesie mixres containing phenacetin Suppi. 7 1987 S L 1
Phenazopyrdine hydrochlonde Suppl. 7 1987 1 S 2B
Phenylbutazne Suppl. 7 1987 1 ND 3
Antibacteri dmgs
Ampicilln 50 199 L
1 3
Chloramphenicol 50 199 L 1 2A
Chrysoidine Suppl. 7 1987 L
1 3
Dapsone Suppl. 7 1987 1 L 3
Dihydroxyethylfuratnzine 24 1980 ND 1 3
Ethionamide 13 1977 ND L 3
Isoniazid (lsonicotinic acid hydrazide) Suppl. 7 1987 L
1 3
Nitrofural (Nitrofurazone) 50 199 L
1 3
Nitrofurantoin 50 199 1 L 3
1-( (5~Nitrofurfryìidene )amino )-2-imidazoli- 7 1974 ND S 2B
dinone (Nifuradene)
N-( 4-5-Nitro-2-furyl)-2-thiazolyl)acetamide 7 1974 ND S 2B
(Furothiazle)
Panfuran S (a formulation with dihydroxy- 24 1980 ND S 2B
methylfuratnzine and several other
compounds)
Penicillc acid 10 1976 ND L 3
Rifampicin 24 1980 ND L 3
Sulfafrazle (Sulphisoxazole) Suppl. 7 1987 1 1 3
Sulfamethoxazle Suppi. 7 1987 l L 3
Antineoplastic drugs
Actinomycin D (Dactinomycin) Suppi. 7 1987 1 L 3
Adnamycin (Doxorubicin) Suppl. 7 1987 1 S 2A
Azacitidine (5-Azacyidine) 50 199 ND S 2A
Azasnne 10 1976 ND S 2B
N ,N-Bis(2-chloroethyl)-2-naphthylamine Suppl. 7 1987 S L 1
(Chlomaphazne)
Bischloroethyl nitrosurea (BNU) Suppl. 7 1987 L S 2A
Bleomycins Suppl. 7 1987 1 L 2B
1,4-Butanediol dimethanesulfonate (Myleran) Suppl. 7 1987 S L 1
Chlorambucil Suppl. 7 1987 S S 1
GENERA REMARKS ON THE SUBSTANCES CONSIDERED 35
Table i (contd)

Human Animal Overall
Antineoplastic dmn (contd)

1-(2-hloroethyl)-3-cclohexyl-1-nitrosurea Suppl. 7 1987 1 S 2A
(CCNU) (Lmustine)
1-(2-Chloroethyl)-3-( 4-methylcyclohexyl)-l- Suppl. 7 1987 S L 1
nitrosurea (Methyl-CCNU)
Chlorotrianisene (see Hormones)
Chlorozotocin 50 199 ND S 2A
Cisplatin Suppl. 7 1987 1 S 2A
Cyclophosphamide Suppl. 7 1987 S S 1
Dacarbazne Suppl. 7 1987 1 S 2B
Daunomycin (Daunorubicin) 10 1976 ND S 2B
Diethylstilbostrol (see Hormones)
Ethynodiol diacetate (see Hormones)
5- Fluorouracil Suppl. 7 1987 1 1 3
17a-Hydroxyrogesterone caproate
(see Hormones)
Isphosphamide 26 1981 ND L 3
Mannomustine 9 1975 ND L 3
Medphalan 9 1975 ND 1 3
Megestrol acetate (see Hormones)
Melphalan Suppl. 7 1987 S S 1
6- Mercaptopurine Suppl. 7 1987 1 1 3
Merphalan 9 1975 ND S 2B
Methotrexate Suppl.7 1987 1 1 3
Mitomycin C 10 1976 ND S 2B
MOppb and other combined chemotherapy Suppl. 7 1987 S 1 1
including alkylating agents
Nitrogen mustard Suppl. 7 1987 L S 2A
Nitrogen mustard N-oxide 9 1975 ND S 2B
Norethisterone (se Hormones)
Prednimustine 50 199 ND 1 3
Prednisne (see Hormones)
Procbazne hydrochloride Suppl. 7 1987 1 S 2A
Streptozotocin (Streptozocin) 17 1978 ND S 2B
Treoulphan Suppl. 7 1987 S ND 1
Trichlormethine (frimustine hydrochloride) 50 199 ND S 2B
Triethylene glycol diglycidyl ether (Ethoglucid) 11 1976 ND L 3
Tris( azridinyl)-pa-benzouinone Suppl. 7 1987 1 L 3
(friazquone)
Table i (contd)

Hum an Animal Overall
Antineoplastic drugr (contd)

Tris(l-azridinyl)phosphine sulphide 50 199 S S .1
(liotepa)
2,4,6- Tri 1-azridinyl)-s-triazne 9 1975 ND L 3
Uracil mustard (Uramustine) Suppl. 7 1987 1 S 2B
Vinblastine sulphate Suppl. 7 1987 1 1 3
Vincristine sulphate Suppl. 7 1987 1 1 3
Antifungal, antiprotozo and antipaitic agents
Chloroquine 13 19TI ND 1 3
DDT (Clofenotane) Suppl. 7 1987 1 S 2B
Furazlidone (also antibacterial 31 1983 ND 1 3
Griseofulvin Suppl. 7 1987 ND S 2B
'Y-Hexachlorocc1ohexane (Lindane) Suppl. 7 1987 1 L 2B
Hycanthone mesylate 13 19TI ND 1 3
Metronidazole (also antibacterial Suppl. 7 1987 1 S 2B
Niridazle 13 1977 ND S 2B
Pyrmethamine 13 19TI ND L 3
Trichlorfon (Metrifonate) 30 1983 ND 1 3
Antiseptic agents
Acridine orange 16 1978 ND 1 3
Acriflavinium chIo ride 13 19TI ND 1 3
Benzoyl peroxide (see Derratological agents)
Chryidine Suppl. 7 1987 1 L 3
Eugenol (used in dentistry) 36 1985 ND L 3
Hexachlorophene 20 1979 ND 1 3
Hydrogen peroxide 36 1985 ND L 3
Phenol 47 1989 1 1 3
Proflavine salts 24 1980 ND 1 3
ß-Propiolactone 4 1974 ND S 2B
Sealet Red 8 1975 ND 1 3
Tannic acid and tannins 10 1976 ND L 3
Dennatological agents
pa-Aminobenzoic acid 16 1978 ND 1 3

Arnic salts (Fowler's solution) (also Suppl. 7 1987 S L ie
antineoplastic)
Benzoyl peroxide (also antiseptic) 36 1985 1 1 3
Canthardin 10 1976 ND L 3
Coal-tar Suppl. 7 1987 S S 1
GENERA REMARKS ON TIE SUBSTANCES CONSIDERED 37
Table i (contd)
Agent Vol. no. Year EvaluationQ
Dennatologial agents (contd)

Diacetylaminoaztoluene 8 1975 ND 1 3
Dithranol (Anthralin) 13 1977 ND 1 3
Hydroquinone 15 1977 ND 1 3
8-Hydroxyquinoline (also antiseptic) 13 1977 ND 1 3
5-Methoxyralen Suppl. 7 1987 1 S 2A
8-Methoxyralen (Methoxsalen) + UV Suppl. 7 1987 S S 1
Resrcinol 15 19TI ND 1 3
Safole (Oil of Sasafas) 10 1976 ND S 2B
Selenium and selenium compounds 9 1975 1 1 3
Tannic acid and tannins (see Antiseptic agents)
4,5' ,8- Trimethylpsralen Suppl. 7 1987 1 1 3
Dm8f for treating anaemia
Iron-dextran complex Suppl. 7 1987 1 S 2B
Iron-dextrin complex 2 1973 ND L 3
Iron-sorbitol-citric acid complex 2 1973 ND 1 3
Saccharated iron oxide 2 1973 ND L 3
Dm8f for treating cardiovascular disorders
Clofibrate (hyprcholesterolaemia) Suppl. 7 1987 1 L 3
Furosmide (Frumide) (hyprtension) 50 199 1 1 3
Hydralazne (hyprtension) Suppl. 7 1987 1 L 3
Hydrochlorothiazde (hyprtension) 50 199 1 1 3
Phenoxybenzamine hydrochloride (hypr- 24 1980 ND S 2B
tension)
Reserpine (hyprtension) Suppl. 7 1987 1 L 3
Spironolactone (hyprtension) Suppl. 7 1987 1 L 3
Dm8f for treating centra nelVOUS disorders
Diazpam (anxety) Suppl. 7 1987 1 1 3
Oxazpam (anxety) 13 1977 ND L 3
Phenelzine sulphate (depresion) Suppl. 7 1987 1 L 3
Phenobarbital (epilepsy) Suppl. 7 1987 1 S 2B
Phenytoin (epilepsy) Suppl. 7 1987 L L 2B
Dm8f for treing thyroid disorders
Methylthiouracil 7 1974 ND S 2B
Propylthiouiacil Suppl. 7 1987 1 S 2B
Thiouracil 7 1974 ND L 3
Thiourea 7 1974 ND S 2B
38 IARC MONOGRAHS VOLUM 50
Table 1 (contd)

Human Animal Ove raIl
Honnones
Androgenie (an abolie) steroids
Oxetholone Suppl. 7 1987 L ND 2A
Testosterone Suppl. 7 1987 L S 2A
Oestrogens, progestins and combinations Suppl. 7 1987
Oestrogens
Nonsteroidal oetrogens S 1c
Diethylstilbostrol S S 1
Dienoetrol L
Hexoetrol S
Chlorotrianisene 1
Steroidal oestrogens S 1C
Oestrogen replacement therapy S ic
Conjugated oetrogens L
OestradioI-17ß and esters S
Oestriol L
Oestrone S
Ethinyloestradiol S
Mestranol S
Progestins 1 2Bc
Medroxyrogesterone acetate 1 S 2B
Chlonnadinone acetate L
Dimethisterone 1
Ethynodiol diacetate L
17a-Hydroxyrogesterone caproate 1
Lynoetrenol 1
Megestrol acetate L
Norethisterone S
Norethynodrel L
Norgestrel 1
Progesterone S
Oestrogen-progestin combinations
Sequential oral contraceptives S ie
,
Dimethisterone and oestrogens 1
Combined oral contraceptives S id
Chlonnadinone acetate and oetrogens L
Ethynodiol diacetate and oetrogens L
Lynoetrenol and oetrogens 1
GENERA REMARKS ON THE SUBSTANCES CONSIDERED 39
Table i (contd)
Honnones (contd)
Megestrol acetate and oestrogens L
Norethisterone and oestrogens L
Norethynodrel and oestrogens S
Norgestrel and oestrogens 1
Progesterone and oestrogens L
Oestrogen-progestin replacement therapy 1 3
Anti-ostrogens
Clomiphene citrate Suppl. 7 1987 1 1 3
Other hormones
Prednisone Suppl. 7 1987 1 1 3
Immunosuppreants
Azathioprine Suppl. 7 1987 S L 1
Ciclosporin (Cyclosporin A) 50 199 S L 1
Traditional remedies of herbal origin

Dantron (Chryazn, 1,8-Dihydroxyanthra- 50 199 ND S 2B
quinone) (Æoe sp., Cassia senna, Rhamnus
purshianus or Cascar sagrda, and Rheum
offcinale)
Hydroxynkirkine (Crotalaia sp.) 10 1976 ND 1 3
Isatidine (Senecio sp.) 10 1976 ND L 3
Jacobine (Senecio sp.) 10 1976 ND 1 3
Monocrotoline (Crotalaia sp.) 10 1976 ND S 2B
Petasitenine (Petasites japonicus) 31 1983 ND L 3
Retrorsine (Senecio sp.) 10 1976 ND L 3
Riddelline (Crotalar sp.) 10 1976 ND 1 3
Seneciphyllne (Senecio sp. and Crotalar sp.) 10 1976 ND ND 3
Senkirkine (Senecio kirkii) 31 1983 ND L 3
Symphytine (Symphytum offcinae) 31 1983 ND 1 3
Misce/laneous dm¡n and exrimental agents
2-Amino-5-(5-nitro-2-furyl~1,3,4- 7 1974 ND S 2B
thiadiazole (gastritis)
Angelicin + UVA (skin disorders) 40 1986 ND L 3
Cimetidine (pptic ulcer) 50 199 1 1 3
4,4' -Dimethylangelicin + UV A 40 1986 ND ND 3
(skin disorders)
4,5'-Dimethylangelicin + UVA 40 1986 ND L 3
(skin disrders)
Table 1 (contd)

Miscellaneous drugs and exrimental agents (contd)
Disulfiram (alcoholism) 12 1976 ND 1 3
Investigational oral contraceptives Suppl. 7 1987 S L Id
Laiocine (emetic) 10 1976 ND S 2B
5-Methylangelicin + UVA (skin disorders) 40 1986 ND L 3
N-Methyl-N-nitrosurea (antineoplastic) 17 1978 ND S 2A
7-Methylpyrdo(3,4- )psoralen (skin disorders) 40 1986 ND 1 3
Nafenopin (hyprcholesterolaemia) 24 1980 ND S 2B
Pyrdo(3,4- )psoralen (skin disorders) 40 1986 ND 1 3
Sodium diethyldithiocbamate (nickel 12 1976 ND 1 3
poisoning)
4,4',6- Trimethylangelicin + UV A (skin 40 1986 ND ND 3
disorders )
i-terinar drugs
Acriflavinium chloride (see Antiseptic agents)
pa-Aminobenzoic acid (see Dennatological
agents)
5-Amino-2-nitrothiazole 31 1983 ND L 3
Aranilc acid Suppl. 7 1987 S L ie
Diacetylaminoazotoluene (see Dennatological
agents)
Furazolidone (see Antifungal, antiprotozoan and
antiparasitic agents)
Hexachloroethane 20 1979 ND L 3
Hexachlorophene (see Antisptic agents)
lron-dextran complex (see Drugs for treating
anaemia)
Lead arnate Suppl. 7 1987 S L ie
5-(orpholinomethyl)-3-( (5-nitrofurfryl- 7 1974 ND S 2B
idene )amino )-2-oxazolidinone
(Furaltadone)
Nithiazde 31 1983 ND L 3
5-Nitro-2-furaldehyde semicabazne 7 1974 ND 1 3
N-( 4-5-Nitro-2-furyl)-2-thiazolyl) acetamide
(see Antibacterial drugs)
GENERA RE MARKS ON ïHE SUBSTANCES CONSIDERED 41
Table 1 (contd)

Ruman Animal Ove raIl
Veterina drngr (contd)

Nitrovin 31 1983 ND 1 3
Selenium (see Dermatological agents)
Urethane 7 1974 ND S 2B
ll, inadequate; 5, sufficient; L, Iimited. For definitions of the sybols used, see Preamble, pp. 26-29.
6combined therapy with nitrogen mustard, vincristine, procrbazine and prednisone
9òis evaluation applies to the group of chemicals as a whole and not necssrily to aH individual chemicals
within the group.
tlere is also conclusive evidence that thes agents have a protective effect against cancers of the ovary and
endometrium.
eAccording to the overall evaluation of arsnic compounds
Derivatives of chloramphenicol without the N02 moiety have been developed;

of these, thiamphenicol has been used extensively, but florfenicol is not used in man.
Thiamphenicol and florfenicol were not considered in this volume, however,
because there appear to be no published data with regard to their carcinogenicity.
Similarly, ranitidine and famotidine are used therapeutically like cimetidine; but
monographs on ranitidine and famotidine and their nitrosated derivatives were also
not prepared due to a lack of relevant published studies.
ln clinical use and in formulations, salts, esters and complexes of drugs are
often designated by the name of the parent compound; this is the case with ampi-
cilin and chloramphenicoL. ln the case of nitrofurantoin, products of different
crystal size have been synthesized. While the Working Group attempted to
distinguish these alternative forms, in some instances insufficient information was
avaIlable to do so.
The primary source of human exposure to drugs is from their use in therapy.
Other types of exposure may also occur, however: persons employed in the
manufacture of drugs may be exposed, as well as nursing and other staff responsible
for the preparation and administration of compouiids and staff responsible for the
care of treated patients. Veterinary use of drugs may result in their entry into the
human food chain.
For the drugs considered here, as for many others, studies of human
carcinogenicity present difficult problems. The symptoms of an undiagnosed
cancer may prompt the use of drug, which is subsequently suspected as its cause.
Alternatively, the condition for which the drug therapy is prescribed may itself be a
risk factor for cancer. An additional problem is that patients commonly receive
more than. one drug, and determination of the carcinogenicity of any single drug
may not be feasible. Repeated reference is made in this volume to hypothesis-
generating studies. These refer to sets of data containing information on many
drugs and many outcomes, in which multiple comparisons are made. Statistically
significant associations (p ~ 0.05) are noted, but in terms of probabilty theory
many such associations may be due to chance. For this reason, the p values given in
the text must be interpreted with caution, and independent examination of
associations identified in hypothesis-generating studies is particularly desirable.
This situation is substantially different from that in which a prior hypothesis exists
before the data are analysed.
An increasing number of agents, including pharmaceutical drugs, have been
shown to inhibit cancer development in animal models. Such properties may le
ad
to new possibilties for cancer treatment and prevention. The Working Group
noted that in long-term experiments with paracetamol, nitrofurantoin and nitro-
fural, reductions in tumour incidence were seen at some sites in some animal
species, although such reductions may have other interpretations thanan inhibition
of tumour induction.
Exposure can generally be much more accurately measured for drugs than for
other agents suspected or identified as human carcinogens, and therapeutic doses
used in hum ans are often close to those tested in experimental animaIs. However, as
is the policy in the lARe Monographs, no attempt was made to quantify cancer risk
at specifie dose levels. Asstated in the Preamble, the Monographs represent the first
stage in carcinogenic risk assessment. Subsequent stages, not attempted in the
Monographs, may involve quantitative determinations. By extrapolation of
avaIlable epidemiological data, and possibly experimental data, estimations of risk
may be attempted for specific populations in respect of particular carcinogens.
Such information may be a factor in regulatory or legislative processes, but no
recommendation concerning these processes Is given in the Monographs. However,
the Working Group responsible for the present monographs observed that
inference of carcinogenic hazard was likely to be a major factor in decision-making .
regarding the usage of many of the drugs considered.
Many (if not most) regulatory decisions concerning putative carcinogens
necessitate consideration not only of perceived hazard but also of the benefit
derived from particular chemicals. It is crucial, therefore, that decisioiis on the
availabilty of drugs include assessment not only of potential carcinogenicity but
also the health benefit derived from their usage.
THE MONOGRAHS
ANINEOPLASTIC AND
IMMUNOSUPPRESSlVE AGENTS
AZACITIDINE
This substance was considered bya previous Working Group, in October 1980,
under the title 5-azacytidine (IAC, 1981). Since that time, new data have become
available, and these have been incorporated into the monograph and taken into
consideration in the present evaluation.
1. ehemical and Physical Data

1.1 Synonyms
ehem. Abstr. Services Reg. No.: 320-67-2
ehem. Abstr. Name: 1,3,5-Triazin-2(1H-one,4-amino-1-ß-ribofuranosyl
Synonym: Antibiotic U 18496; 5-azacytidine; ladakamycin; NSC 102816;
lJ- 18496; WR- 183027
1.2 Structural and molecular formulae and molecular weight
NH2
Á
N "' N
IL L
'N~O
HOH2C
C8H 12N40S MoL. wt: 244.2
1.3 Chemical and physical properties of the pure substance

From Winkley and Robins (1970), unless otherwse specified
(a) Description: White crystalline powder
-47-
(b) Me/ting-point: 235-237°C (decmposes)

(c) Optical rotation: ( 0: J if . + 26.6 ° C (c = 1.00; in water)
(d) Solubility Soluble in warm water (40 mg/ml), cold water (14 mg/ml), 0.1 N
hydrochloric acid (28 mg/ml) and 0.1 N sodium hydroxide (43 mg/ml);
soluble in 35% ethanol (14.2-15.0 mg/l), acetone (1 mg/ml), chloroform
(1 mg/ml), hexane (1 mg/ml) and dimethyl sulfoxide (52.7 mg/ml) (von
Hoff et al., 1975)
(e) Spectrosocopy data: Ultraviolet, infrared and nuclear magnetIc resonance
spectra have been reported (Beisler, 1978).
(j Stability Very unstable in aqueous media, rapid degradation to complex
products occurring within hours of
dissolution in intravenous solutions at
room temperature (Reynolds, 1989)
1.4 Technical products and impurities
Trade name: Mylosar

Azacitidine is available as a lyophilized powder in vials containing 100 mg of
the compound with 100 mg mannitol for reconstitution as injections of 5 mg/ml (von
Hoff et al., 1975).
2. Production, Occurrence, Use and Analysis
2.1 Production and occurrence
(a) Production
Azacitidine, a pyrimidine analogue of cytidine with a nitrogen substituted for a
5-carbon, can be isolated from a culture of the bacterium Streptoverticillium
ladakanus, but has also been prepared by synthetIc methods. One reported method
involved treatment of the trimethylsilyl derivative of 4-amino- 1,3,5-triazn-2-one
with 2,3,5-tri-O-acetyl-D-ribofuranosyl bromide, followed by deacetylation to give
azacitidine.(Winkley & Robins, 1970).
Azacitidine is synthesized in the Federal Republic of Germany (Chemical
Information Servces, 1989-90).
(b) Occurrence
Azacitidine is produced by the bacterium Streptoverticillium ladakanus
(Winkley & Robins, 1970).
2.2 Use
Azacitidine is a cytostatic agent. It has ben used mainly in the treatment of
acute leukaemia, either as intravenous or intramuscular injections or as
AZAcmDINE 49
intravenous infusions at a daIly level of 40-750 mg/m2 (Weiss et al., 1972; Skoda,
1975; von Hoff et aL., 1975, 1976; von Hoff & Slavik, 1977; Wade, 1977; Glover et aL.,
1987; Reynolds, 1989). It is used alone, or in combination with vincristine,
vinblastine, prednisone, cytarabine or amsacrine, at a daily dose of 50- 150 mg/m2
azacitidine. It has also been tested for use in the treatment of a variety of solid
tumours (Glover et al., 1987).
2.3 AnaJysis
Azacitidine can be quantified in blood by microbiological assay (Pittillo &

Woolley, 1969) and in plasma by high-p-erformance liquid chromatography with
ultraviolet detection (Rustum & Hoffman, 1987).
3. Biological Data Relevant to the Evaluation of

earcinogenic Risk to Humans
3.1 Carcinogenicity studies in animaIs
(a) lntraperitoneal injection

Mouse: ln a screening assay based on the accelerated induction of leukaemia
in a strain highly susceptible to development of this neoplasm, 40 AK female mice,
two months of age, were given six intraperitoneal injections of azacitidine at 1.5
mg/kg bw (purity unspecified) over 20 days, and, because of toxicity, six injections of
azacitidine at 0.8 mg/kg bw over the following 30 days. Alhtreated mice had died of
leukaemia by 60 days. A control group of 40 females survived free of disease for the
observation time of 120 days (Vesely & Cihák, 1973).
ln a screening assay based on the accelerated induction of lung tumours in a
strain highly susceptible to developmentof this neoplasm, three groups of ten male
and ten female NHe mice, six to eight weeks of age, received intraperitoneal
injections of azacitidine (purity unspecified), in a vehicle composed of saline,
polysorbate-SO, carboxyethyl cellulose and benzyl alcohol, three times a week for
eight weeks (total doses, 33, 62 and 90 mglg bw (which was the maxmum tolerated
dose)). Control groups received 24 intraperitoneal injections of 0.1 ml vehicle or
were untreated. AlI animaIs were killed 24 weeks after the first injection. The
numbers of mice with lung tumours, calculated on the basis of survivors of each sex,
were 6/11 (54%), 5/15 (33%) and 8/19 (42%) in the groups receiving the high, mid
and low doses, respectively. The results for untreated and vehicle-treated groups
were expressed only as per cent tumour incidence; thus, 22% (males) and 17%
(females) of untreated controls and 26% (males) and 23% (females) of
vehicle-treated controls developed lung tumours. The number of lung tumours per
mouse (counted grossly) in animaIs of each sex treated with the highest dose was
0.73 :f 0.22 (SE), which was significantly higher (p ~ 0.05) than that in untreated
(males, 0.22 :f 0.03; females, 0.17 :f 0.02) or vehicle-treated (males, 0.25 f: 0.05;
females, 0.23 :f 0.04) control mice. With lower doses, the increase in the number of
lung tumours per mouse was not statistically significant (Stoner et al., 1973).
Groups of 35 male and 35 female B63F1 mice, 38 days of age, received
intraperitoneal injections of azcitidine at 2.2 or 4.4 mg/g bw (~99% pure) in
buffered saline three times a week for 52 weeks. Groups of 15 male and 15 female
mice were untreated or recived the vehicle only. Survving mice were killed at 81 or
82 weeks. AlI high-dose females died before week 62, with no significant increase in
the incidence of any tumour; of the low-dose females, 17/35 survved untIl
termination of the experiment. Among males, 7/35 of the high-dose group and 13/35
of the low-dose group survved to the end of the study. The overall numbers of
survivors in untreated and vehicle-treated groups were 25/30 and 20/30,
respectively. ln female mice of the low-dose group, lymphocytic and granulocytic
neoplasms of the haematopoietic system were observed in 17/29 animaIs examined
histologically, at a highly significant incidence (p ~ 0.(01) compared with the
vehicle-control group (0/14); 10 of the treated animaIs had granulocytic tumours
(nine sarcomas, one leukaemia). A malignant lymphocytic lymphoma was obseived
in 1/15 untreated controls. No increase in the incidence of tumours was observed in
male mice (National Cancer Institute, 1978).
Groups of 50 male and 50 female BALB/c/Cb/Se mice, eight weeks of age, were
given intraperitoneal injections of azacitidine at 2.0 mg/kg bw in saline (99% pure)
once a week for 50 weeks. Control groups received injections of saline. Mer 25
weeks, survival was reduced in exposed animaIs of each sexe The incidence of
lymphoreticular neoplasms was increased, occurring in 1250 (p ~ 0.01) males and
36/50 (p ~ 0.001) females, compared to 3/50 and 6/50 in control males and females,
respectively. The incidence of lung adenomas was increased in treated males (27/50
versus 12/50 fp ~ 0.01 D but not in females. Mammary gland adenocarcinomas and
adenoacanthomas were found in 7/50 treated females and in none of the controls.
The incidence of skin - tumours was increased in treated animaIs of each sex,
occurring in 3/50 treated males compared to 0/50 controls fp ~ 0.05) and in 7/50
treated females compared to 1/50 controls fp ~ 0.01, log rank test) (Cavaliere et al.,
1987). (The Working Group noted that adenocanthomas are not described as
mammary tumours in reference sources; see Turusov (1973, 1976).)
Rat: Two groups of 12 or 8 male Fischer rats, weighing 160-180 g, were given
intraperitoneal injections of azacitidine at 2.5 or 10 mglg bw (purity unspecified) in
saline twice a week for nine months. A control group of 12 male rats was maintained
without treatment. AlI rats were killed at 18 months. Interstitial-ceU testicular
AZACffDlNE 51
tumours were found in 1/8 high-dose animaIs and 9/12 low-dose animals còmpared
to 0/12 controis. ln the high-dose group, two squamous-cll carcinomas of the skin
and one skin appendage tumour at the site of injection were found, compared to
none in controls (Carr et al., 1984). (The Working Group noted the small number of
animaIs tested, and the absence in controls of testicular tumours, which occurred
commonly in a secnd, shorter study by the same investigators (sec below).)
Groups of 10, 10 or 100 young aduit male Fischer rats, weighing 100-160 g,
recived intraperitoneal injections of azacitidine at 0.025, 0.25 or 2.5 mg/kg bw in
saline (purity unspeified) three times a week for one year. A control group of 50
rats was injected with saline. At one year, when the study was terminated, 87/100 of
animaIs at the high dose and 10/10 in each of the lower-dose groups were still alive.
The highest dose increased the incidence of testicular interstitiai-cell tumours to
56/87, compared to 10/49 in controls (p ~ 0.(01). No other tumour was observed in
controis. ln the highest dose group, other tumours noted were four lymphomas,
four renal tumours, one lung tumour, three skin tumours, two mesotheliomas and
two sarcomas (Carr et al., 1988). (The Working Group noted the short duration of
the experiment and the small numbers of animaIs in some groups.)
(h) Transplacental administration

Mouse: Groups of 32-37 pregnant NMI mice received intraperitoneal
injections of azacitidine at 1 or 2 mglg bw in saline (purity unspecified) on day 12,
14 or 16 of gestation. A group of 53 control dams was injected with saline. The
number of stilbirths was increased at the high dose; survivai of offspring was
decreased in aIl expsed groups. ln exposed progeny, increased percentages of
tumour-bearing animaIs and increased incidences of Ieukaemias and lymphomas,
lung tumours and liver tumours were seen in some groups (see Table 1). Some
increases in the incidence of soft-tissue sarcomas were also seen (Schmahl et al.,
1985).
(c) Administration in comhination with other compounds

Rat: ln the experiment by Carr et al. (1984), described above, groups of 6-10
male Fischer rats were given N-nitrosodiethylamine at 50 mglg bw 18 h after
partial hepatectomy, alone or with azacitidine at 2.5 or 10 mglg bw by
intraperitoneal injection. Liver tumours were found in 2/10 and 8/10 animaIs given
the low and the high dose of azacitidine, respectively, but not in the group given the
nitroso compound alone.
Table 1. Incidences of tumours in the progeny of NMRI mice given azciti-

dineby intraperitoneal injectiontl
Treatment Se No. of Leukaemias Lung tumours rier tumours
animaIs and Iymphomas
mgl bw day of No. % No. % No. %
gestation
1 12 Males 165 81 49.1 30 18.2 15 9.1

Females 15 80 50.6 33 20.9 6 3.8
2 12 Males 113 28 24.8 22 19.5 11 9.7
Females 110 26 23.6 22 20.0 9 8.2
1 14 Males 178 42 23.6 29 16.3 12 6.7
Fern ales 171 26 15.2 31 18.1 20 11.7
2 14 Males 97 9 9.3 46 47.4 11 11.3
Females 101 14 13.9 43 42.6 7 6.9
1 16 Males 153 97 63.4 81 529 14 9.2
Fern ales 160 98 61.3 99 61.9 8 5.0
2 16 Males 158 67 42.4 78 49.3 18 11.4
Fern ales 151 57 37.7 82 54.3 5 3.3
Con troIs Males 293 84 28.7 57 19.5 14 4.8
Fern ales 279 82 29.4 53 19.0 11 3.9
aprorn Schrnahl et al. (1985)
3.2 Other relevant data

(a) Exerimental sytems
(i) Absorption, distriution, cxretion and metabolism
Blood levels of azacitidine, determined by biological activity, in mice peaked
within 0.5 h after intraperitonealor oral administration. Maxmal concentrations of
azcitidine in blood after administration at 50 mglg bw were about 2 J.g/ml after
oral administration and 43 l.g/ml after intraperitoneal injection (Neil et al., 1975).
ln a study using a microbiölogical assay, maxmal concentrations were found
in blood 15 min after intraperitoneal injection of 9.5 and 4.75 mglg bw (LDio and
0.5 LDio) to mice. Elimination was rapid, and no azcitidine was detected in blood
1 h after injection of the high dose or 30 min after injection of the low dose. No drug
was detected in liver, lung, brai n, spleen or kidneys (Pittillo & Woolley, 1969).
ln a further study, 14c activity in bloo diminished rapidly in mice after
intraperitoneal administration of labelled azacitidine (Raska et al., 1965). The
half-time for azacitidine and its radioactive metabolites was calculated by von Hoff
and Slavic (1977) to be 3.8 h; radioactivity was retained in lymphatic organs.
AZAcmDINE 53
As reported in an abstract, 50% of a dose (amount and route unspecified)

administered to mice was excreted in the urine within 8 h; of the excreted
radioactive material, 4% was associated with unchanged azacitidine. Six additional
radioactive metabolites were found (Coles et al., 1975). ln beagle dogs, azacitidine,
5-azacytosine, urea and guanidine were observed after intravenous administration
of azacitidine at 0.5 mg/kg bw; 33% of the administered dose was excreted in urine
by 4 h (Coles et al., 1974). ln rabbits, most of the radioactivity (25-40%) was
excreted in the urine after intravenous administration of labelled azacitidine at 15
mg/kg bw; only small amounts were excreted via the bile (Chan et al., 1977).
Azacitidine is phosphorylated and inhibits uridine kinase and orotidylic acid
hydroxylase (von Hoff et al., 1975, 1976). It is readily deaminated in biological
systems to 5-azauridine, which is degraded further (Cihák, 1974; Neil et al., 1975;
Glover & Leyland-Jones, 1987).
(ii) Toxic effects
As reported in an abstract, the intraperitoneal LDso for azacitidine in mice
was 116 mg/kg bw and the oral LDso, 572 mg; fIve daily doses increased the toxicity
considerably (Palm & Kensler, 1971).
After phosphorylation, azacitidine is incorporated into DNA and RNA in
L1210 leukaemia cells in vitro (Li et al., 1970); it inhibits DNA synthesis in the liver of
partially hepatectomized rats. Intraperitoneal injection of azacitidine at 10
lLmol/lOO g bw inhibited thymidine kinase and thymidylate kinase in rat liver
(Cihák & Vesely, 1972).
Azacitidine is cytotoxic to Friend eryhroleukaemia cells (Hickey et al., 1986),
L1210 leukaemia cells (Li et al., 1970) and normal rat hepatocytes (Carr et aL., 1988)
in vitro; after a dose of 1 X 10-4 M, 32% survival of rat hepatocytes was observed
within 24 h.
(ii) Hypmethylation and effects on gene exression
After incorporationinto DNA, azacitidine inhibits DNA methyl transferase
noncompetitively, blocking cytosine methylation in newly replicated DNA. Since
hypomethylation patterns in DNA are related to gene expression, this may be the
mechanism by which azacytidine induces a range ofbiological effects (Glover et al.,
1987). A number of in-vitro and in-vivo studies have shown that azacitidine
treatment affects both differentiation (Constantinides et aL., 1978; Taylor & Jones,
1979; Tsao et al., 1984; Csordas & Schauenstein, 1986; Liu et al., 1986; Sémat et al.,
1986; Rothrock et al., 1988) and gene expression (Tennant et al., 1982; Harrison et al.,
1983; Rothrock et al., 1983; Sugiyama et al., 1983; deI Senno et al., 1984; Waalkes &
Poirier, 1985; Castelaz et al., 1986; Hickey et al., 1986; Hoshino et al., 1987;
Ishikawa et al., 1987; Price-Haughey et al., 1987; Carr et al., 1988; Stephanopoulos et
al., 1988; Wagner et al., 1988).
(iv) Effects on reproduction and prenatal toxicity

Intraperitoneal administration of azcitidine at 1.5-2.5 mg/kg bw to mice for
various periods during pregnancy induced very high or total resorption of
conceptuses when treatment was given in the preimplantation period up to day 6;
after this time, the incidence of resorptions was only slightly greater than the control
level (Svata et al., 196; Seifertová et al., 1968). Other workers have shown that single
intraperitoneal doses of 1-2 mg/kg to mice during the period of embryogenesis can
cause a high resorption rate and malformations in the majority of surviving fetuses,
including major central nervous system defects, facial clefts and limb defects
(Schmahl et al., 1984; Takeuchi& Takeuchi, 1985).
Intraperitoneal injection of azcitidine at 1-4 mglg to mice at latet stages of
pregnancy, especially on day 15, can result in morphological changes in the brain
(Langman & Shimada, 1971), and behavioural changes can be detected in offspring
when tested as adults (Rodier et al., 1973; Langman et al., 1975; Rodier, 1979).
The primary mechanism by which azacitidine causes malformations in rats is
thought to be induction of cell death, but inhibition of some but not all of the effects
of azacitidine by administration of caffeine indicates that more th an one
mechanism may be involved (Kurishita & Ihara, 1987a,b).
(v) Genetic and related effects
ln Escherichia coli, azcitidine caused DNA damage (Bhagwat & Roberts,
1987) and prophage induction (Barbe et aL., 1986). It was mutagenic to E. coli (Fucik
et al., 1965; LaI et al., 1988) and induced base-pair but not frameshift mutations in
Salmonella tyhimurium (Marquardt & Marquardt, 1977; Podger, 1983; CalI et al.,
1986; Levin & Ames, 1986; Schmuck et al., 1986).
Azacitidine induced mitotic recombinations, mitotic gene conversions and
reverse mutations but not mitotic chromosome loss in Saccharomyces cerevisiae
(Zimmermann & Scheel, 1984). It induced mitotic recmbinations, deletions and
gene mutations in the wing spot assay in Drosophila melanogaster (Katz, 1985) and
chromosomal aberrations in root meristem cells of Vicia laba (Fucik et al., 1970).
Azcitidine inhibited DNA synthesis in Chinese hamster CHO cells (Tobey,
1972) and induced DNA strand breaks in HeLa cells (Snyder & Lachmann, 1989).
It induced mutations at the hprt locus in Chinese hamster V79 cells iD one study (at
5 l1M; Marquardt & Marquardt, 1977) but not in another (at 40 l1M; Landolph &
Jones, 1982). It did not iDduce mutation at the hprt locus in Syrian hamster BHK
cells (Bouck et al., 1984), primary rat tracheal epithelial cells (Walker & Nettesheim,
1986) or mouse lymphoma 15178Y cells (at 4 l1M; McGregor et al., 1989).
Azacitidine induced mutations at the hprt and tk loci in human fibroblasts (CalI et
al., 1986) and at thetk locus of mouse lymphoma 15178Y cells (Amacher & Turner,
1987; McGregor et al., 1989). It did not induce ouabain-resistant mutations in
AZAcrnDINE 55
mouse C3H 10T1f, Chinese hamster V79 (Landolph & Jones, 1982), Syrian hamster
BHK (Bouck et al., 1984) or primary rat tracheal epithelial cells (Walker &
Nettesheim, 1986).
Azcitidine induced sister chromatid exchange in a cloned hamster cell line
(Banerjee & Benedict, 1979), inCHO cells (Hori, 1983) and in human peripheral
lymphoces in vitro (only one concentration, 8 J.M, was tested) (Lavia et al., 1985).
ln another study, azcitidine did not induce sister chromatid exchange in hum
an
lymphoces (up to 9 J.M; loannidou et al., 1989). It induced chromos
omal
aberrations in Chinese hamster Don cells (Karon & Benedict, 1972) and in hum
an
peripheral lymphocytes in vitro (only one concentration, 8 J.M, was tested) (Lavia et
al., 1985) but not in hum an lymphoblasts (10 J.M; Call et al., 1986).
Azcitidine induced transformation in mouse C3H110rh (Benedict et al.,
1977), Syrian hamster BHK (Bouck et al., 1984), mouse BALB/313 (Yasutake et al.,
1987) and primary rat tracheal epithelial cells (Walker & Nettesheim, 1986).
Azacitidine did not induce dominant lethal mutation in male mice after
administration at 5 and 10 mglg bw intraperitoneally (Epstein et al., 1972).
(h) Humans
The toxicity, cytostatic activity and mechanism of action of azacitidine have
been reviewed (Cihák, 1974; von Hoff & Slavik, 1977; Glover & Leyland-Jones,
1987).
(i) Pharmcokinetics
Afer an intravenous injection of radiolabelled azacitidine, the ~-phase
half-time of radioactivity was 16-33 min (Israeli et al., 1976), and the ß-phase
half-time was 3.4-6.2 h (Troetel et al., 1972; Israeli et al., 1976). Afer 30 min, less th
an
2% of the plasma radioactivity cohromatographed with azcitidine; at least two
different metabolites or decmposition products were detected by thin-Iayer
chromatography (Israeli et al., 1976), and 73-98% of the injected radioactivity was
detected in the urine within three days (Israeli et al., 1976). Similar results were
obtained by Troetel et al. (1972).
Less than 1% of radiolabelled azacitidine was bound to human serum albumin
in vitro (Israeli et al., 1976).
(ii) Adverse effects

The major toxic. effects of the clinical use of azcitidine have been
gastrointestinal, haematological and hepatic (von
Hoff et al., 1976; von Hoff &
Slavik, 1977; Reynolds, 1989). Leukopenia is generally the dose-limiting toxicity; in
a compilation of several studies with a total of 821 patients, the incidence of
leukopenia (total leukoce count, less than 1500/mm3) was 34% and was
dose-related. Thromboopenia has ben reported less frequently (von Hoff et al.,
1976; von Hoff & Slavik, 1977). Fatal hepatic damage was reported in four patients
with previous hepatic dysfunction, who had ben treated with azcitidine (Bellet et
al., 1973).
(ii) Effects on reproduction and prenatal toxicity
No data were avaIlable to the Working Group.
(iv) Genetic and related effects
No adequate study was avaIlable to the Working Group.
3.3 Case reports and epidemiological studies of carcinogenicity to humans

4. Summary of Data Reported and Evaluation
4.1 Exposure data
Azacitidine is a cytostatic agent that has been used since the 1970s for the
treatment of acute leukaemia.
4.2 Experimental carcinogenicity data

Azacitidine was tested for carcinogenicity by intraperitoneal injection in four
studies in mice and in two studies in rats and by transplacental exposure in one
study in mice. ln one study in mice, it accclerated the development of leukaemias; in
the two long-term studies and in the transplacental study, it increased the incidence
of lymphoid neoplasms. ln one of the long-term studies, the incidence of lung
adenomas was increased in male mice and that of skin tumours in mice of each sexe
ln the transplacental study in mice, it also increasrd the incidences oflung and liver
tumours. It accelerated the induction of lung tumours in mice. ln rats, it increased
the incidence of testicular tumours.
Intraperitoneal administration of azacitidine to rats enhanced the
development of liver tumours induced by N-nitrosodiethylamine.
4.3 H uman carcinogenicity data

During the early stages of gestation, azcitidine induces embryomortality in

mice; during the organogenesis period, multiple, gross structural malformations
AZAClllNE 57
can be induced; and during later stages of gestation, mainly central nervous system
defects have been induced in mice.
Azcitidine is readily deamInated to azuridine and further degraded. It is
incorporated into DNA and alters gene expression. ln humans, it causes
leukopenia.
Azcitidine causes hypmethylation of DNA both in vivo and in vitro.
ln one study, azacitidine did not induce dominant lethal mutations in mice.
Contradictory results have ben reported with respet to the induction of
chromosomal aberrations and sister chromatid exchange in human cells. ln single
studies, azcitidine induced gene mutations and DNA strand breaks in human
cells. It induced chromosomal aberrations in Chinese hamster cells, sis
ter
chromatid exchange in cloned Chinese hamster cells, gene mutations in Chinese
hamster and mouse lymphoma cells and transformation in various cell lines. It
induced mitotic recmbination and mutations in Drosophila. Azacitidine Induced
chromosomal aberrations in Vicia loba. ln Saccharomyces cerevisiae, it induced
gene mutations and mitotic recmbination but not chromosomal loss. It induced
mutations and DNA damage in Salmonella tyhimurium and Escherichia coli. (See
Appendix 1.)
4.5 Evaluation1
There is suffcient evidence for the carcinogenicity of azacitidine in

experimental animaIs.
ans on the carcinogenicity of

No data were available from studies in hum
azacitidine.
ln making the overall evaluation, the Working Group also took note of the
following information. Azcitidine is active in a broad spectrum of assays for
genetic and related effects, including those involving mammalian cells.
Furthermore, azcitidine, a pyrimidine analogue, is incorporated into DNA,
causing hypmethylation.
Overall evaluation
Azacitidine is probably carcinogenic to humons (Group lA).
1Por desription of the italicize terms, se Preamble, pp. 2629.

58 IAC MONOGRAHS VOLUM 50
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AZAcmDINE 61
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62 lAC MONOGRAHS VOLUME 50
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eHLOROZOTOelN
1. ChemicaI and Physical Data
1.1 Synonyms
ehem. Abstr. Services Reg. No.: 54749-905

ehem. Abstr. Name: D-Glucose, 2-( H(2-chloroethyl)nitrosoaminoJcarbonyll-
amino )-2-deoxy-
Synnym: D-Glucopyranose, 2-( ~ ((2-chloroethyl)nitrosoamino )carbonyl l_
amino )-2-deoxy-l-(2-chloroethyl)- 1 -nitroso-3-(D-glucos- 2-yl)urea; 2-(3-( chlo-
roethyl)-3-nitrosoureido)-2-deoxy-D-glucopyranose; DCNU; NSC- 178248
~CH2~H H
H
OH H
HO OH/NO
H HNCONCH2CH2C1
C9H 16CIN 307 MoL. wt: 313.69

From Windholz (1983), unless otherwise specified
(a) Description: Ivory crystals
(b) Melting-point: 147-148°C (decomposes); 14O-141°C (decomposes)

(c) Solubility Soluble in water; decmposition in aqueous solution has been
studied (Montgomery et al., 1975).
(d) Spectroscopy data: Infra-red and nuclear magnetic resonance spectra
have been reported (Johnston et al., 1975).
-65-
(e) Stability Stable ( .: 5% decomposition by ultraviolet spectroscopy) in

solution at room temperature (22-25 0 C) for 3 h and at 2-80 C for 24 h;
powder is stable for 24 months under refrigeration
(f Partition coeffcient: Pc = 3 (octanol:water) (Johnston et al., 1975)
Trade name: Dome

Chlorozotocin is avaIlable as a lyophilized powder in vials containing 50 mg of
the compound with 48 mg citric acid and sodium hydroxide to adjust the pH
(National Cancer Institute, 1988).

Chlorozotocin is synthesized by nitrosation of the urea derivative prepared
from D-glucosamine and 2-chloroethylisocyanate (Johns ton et al., 1975). It is
reported to be produced in the USA.
Chlorozotocin is not known to occur naturally.
2.2 Use
Chlorozotocin is a cytostatic agent. It can be used in the treatment of cancers
of the stomach, large bowel, pancreas and lung, melanoma and multiple myeloma. It
has been given intravenously, at doses of 100-225 mg/m2 (Samson et al., 1982; Smith
et al., 1982; Bukowski et al., 1983; Haas et al., 1983; Forman et al., 1984;
Gastrointestinal Tumor Study Group, 1985). No indication for its use was given by
Reynolds (1989).
2.3 Analysis
A colorimetric method for the analysis of chlorozotocin in plasma has been

reported (Hoth et al., 1978; Kovach et al., 1979).

(a) lntraperitoneal administration

Rat: Groups of 20 male and 20 female Sprague-Dawley rats, 100 days old, were
given intraperitoneal injections of chlorozotocin (synthesized according to
CHLOROZOTOCIN 67
standard methods) at 0.4 or 2.0 mg/kg bw once a week for up to 80 days. Control
groups of 20 rats of each sex received injections of Cremophor EL: ethanol:saline in
a ratio of 1: 1:2 volume parts. The median survival times in days were as follows:
control males, 724; low-dose males, 463; high-dose males, 307; control females, 750;
low-dose females, 694; high-dose females, 346. Sarcomas and mesotheliomas of the
peritoneal cavity occurred in 13/20 fp cC 0.001) and 14/20 fp cC 0.001, Fisher's exact
test) high- and low-dose males, respectively, compared to 0/20 controls, and in 16/20
fp cC 0.001) and 10/20 fp = 0.002, Fisher's exact test) high- and low-dose females,
respectively, compared to 1/20 controls (Habs et al., 1979).
(b) lntravenous administration

Rat: Groups of 30 male Wistar rats (age unspecified) were given intravenous
injections of chlorozotocin at 9.5, 19 or 38 mg/m2every sIxweeks for 10 applications.
A group of 120 con troIs received Cremophor EL:ethanol:water in a ratio of
1.5:1.5:20 volume parts. The median survival times in days were as follows: high
dose, 474; median dose, 590; low dose, 583; controls, 621. AnimaIs were observed for
lire. Malignant tumours of the nervous system, lung and forestomach were found in
4,5 and 4% of treated animaIs compared to 1, 0 and 1% of controls, respectively
(Eisenbrand & Habs, 1980; Eisenbrand et al., 1981; Zeller et al., 1982). (The
Working Group noted the poor survival and limited reporting.)

The toxicity of chlorozotocin has been reviewed (Schein et al., 1976; Macdonald
et al., 1980; Wang et al., 1981; Eisenbrand, 1984; Eisenbrand et al., 1986; Johnston &
Montgomery, 1986).
(i) Absorption, distribution, exretion and metabolism
(ii) Toxic effects
The LDso of chlorozotocin within 60 days in Sprague-Dawley rats was 27.2
mglg bw after intraperitoneal injection and 22.5 mg/kg bw after intravenous
injection (Fiebig et al., 1980).
ln one study of acute toxicity, the LDso after intravenous injection in BDFi
mice was 24.9 mglg bw for males and 30.3 mg/kg bw for females. ln animaIs of
each sex, tubular necrosis of the kidney and cast formation were observed, as weIl as
splenic lymphoid atrophy. ln the same study, a dose of 6 mg/kg bw was lethal to
beagle dogs after five days, and 3 mglg bwafter 19 days. Renal dysfunction with
tubular necrosis also occurred in these animaIs. Elevated serum levels of alanine
aminotransferase and alkaline phosphatase were observed in dogs injected
repeatedly with chlorozotocin. Nephrotoxicity was also seen in rhesus monkeys

given 40 mg/kg intravenously (Gralla et al., 1979).
ln Fischer rats, a lethal subcutaneous injection of chlorozotocin at 40 mglg
bw caused renal necrosis in the cortex and, subsequently, necrosis of papillary
collecting ducts. At sublethal doses, hyprtrophy and karyomegaly were observed
in collecting duct cells (Kramer et al., 1986). ln Fischer rats given a subcutaneous
injection of chlorozotocin at 25 or 40 mglg bw, no necrosis was observed in
papilary collecting ducts, although karyomegaly was observed (Dees & Kramer,
1986).
Central nervous system vascular necrosis was observed in beagle dogs treated
with chlorozotocin at 1.5-2.0 mg!g bwonce a week for two weeks or with a single
intraventricular dose of 10 mg/kg bw (Levin et al., 1985).
Chlorozotocin affects cell cycle progression in Chinese hamster CHO cells.
Non-cycling G 1-arrested cells were the most sensitive; traverse from G 1 to S was not
affected, and chlorozotocin doubled the time for completion of DNA synthesis.
Small quantities of polyploid cells were produced (Tobey et al., 1975). Chloro-
zotocin at a concentration of 20 J-M induced differentiation and inhibited cell
growth of mouse neuroblastoma N-18 cells (Yoda et al., 1982). DNA synthesis in
L1210 leukaemia cells was almost completely inhibited (96%) within 24 h of an
intraperitoneal administration to BD2F i mice (Anderson et al., 1975). ln vitro,
DNA synthesis in L1210 leukaemia cells was inhibited by 68% (Fox et al., 1977).
Single intraperitoneal injections of chlorozotocin at 15 mg/kg bw (maxmal
nonlethal dose) to BDFi mice slightly decreased peripheral white blood cell counts
(Schein et al., 1976). Similar observations were made in CD2F i mice (Fox et al.,
1977; Macdonald et al., 1980). Intraperitoneal injections of chlorozotocin at 20
mg/kg bw to mice reduced peripheral lymphoce counts by 50% in three days.
Spleen weights were decreased by about 40%, and the response to mitogens was
markedly reduced (Fisher et aL., 1980).
ln another study in mice, chlorozotocin was shown to have immunomodulating
activity. The IgM plaque-forming cell response was suppressed when the drug was
injected four days before immunization; furthermore, hypersensitivity to oxazolone
treatment was increased by about 30% when animaIs were injected intraperi-
toneally with chlorozotocin four days before sensitization. Treatment with chloro-
zotocin in vivo inhibited the proliferative response of spleen cells to mitogens and
stimulated the chemIluminescence of peritoneal macrophages (Florentin et al.,
1983).
Chlorozotocin exerts its toxic and other adverse effects through the formation
of mono- and bifunctional alkylating agents. It also carbamoylates proteins via an
isocyanate intermediate formed upon decmposition (Eisenbrand, 1984).
CHLOROZOTOCIN 69
Alkylation of nuclear chromatin in HeLa cells has been observed, and there was
preferential alkylation ofDNA associated with the nuc1eosome core partic1e (Tew et
al., 1978).
(iii) Effects on reproduction and prenatal toxicity

Chlorozotocin induced base-pair substitutions but not frameshift mutations
in Salmonella tyhimurium in the presence and absence of an exogenous metabolic
system (Zimmer & Bhuyan, 1976; Franza et al., 1980; Suling et al., 1983). It induced
mitotic gene conversion in Saccharomyces cerevisiae (Siebert & Eisenbrand, 1977)
and sex-linked recessive mutations in Drosophila melanogaster (Kortselius, 1978).
Chlorozotocin alkylated DNA in mouse leukaemia L1210 cells (Panasci et al.,
1979; Ahlgren et al., 1982). It induced DNA strand breaks in L1210 cells (Ewig &
Kohn, 1977; Alexander et al., 1986) and in V79 Chinese hamster cells (Erickson et al.,
1978), and interstrand cross-links in DNA of mouse leukaemia L1210 cells (Ewig &
Kohn, 1977) and of human embryo cells (Erickson et al., 1980). It induced mutation
at the hprt locus in V79 Chinese hamster cells (Bradley et al., 1980) and sister
chromatid exchange in mouse leukaemia L1210 cells (Siddiqui et al., 1988) and in 9L
rat brain tumour cells (Tofilon et aL., 1983).
Chlorozotocin at a single intraperitoneal dose of 100 J.mol/kg induced DNA
strand breaks and interstrand cross-links in bone-marrow cells of Wistar rats
treated in vivo (Bedford & Eisenbrand, 1984).
(h) Humans
(i) Pharmacokinetics
After an intravenous dose of chlorozotocin at 120 mg/m2, the disappearance
curve of the N-nitroso group froID the circulation exhibited three successive
exponential phases, with half-times of 3-4.5 min, 6-12 min and 18-30 min.
Twenty-four hours after administration of either ethyl- or glucose-Iabelled
chlorozotocin, 82-84% ofthe blood-borne radioactivitywas bound to protein; after
seven days, 2% of the peak radioactivity value was detected in the blood. By 48 h,
50% of the radioactivity from (ethyl-14C)chlorozotocin and 58% of that froID
(glucose-14c)chlorozotocin was excreted in the urine; only 5-8% was excreted as the
intact drug (Hoth et al., 1978).
Thrombocytopenia, leukopenia, elevated aminotransferase activity, nausea
and vOIDiting were seen in patients after intravenous administration of
chlorozotocin, generally at doses of 120 mg/m2 or higher (Hoth et al., 1978;
Bukowski et al., 1983; Haas et al., 1983; Forman et al., 1984; Schutt et al., 1984;
Gastrointestinal Tumor Study Group, 1985).

4. Summary or Data Reported and Evaluation
4.1 Exposure data
Chlorozotocin has been used as a cytostatic drug for the treatment of cancers
at a variety of sites.

Chlorozotocin was tested for carcinogenicity in single experiments in rats by
intraperitoneal and intravenous injection. Intraperitoneal administration induced
a high incidence of sarcomas and mesotheliomas in the peritoneal cavity in rats of
each sexe The study by intravenous administration was inadequate for evaluation.
4.3 "uman carcinogenicity data

No datawere avaIlable to the Working Group.
Chlorozotocin alkylates DNA and protein and causes DNA interstrand

cross-links. ln humans, it induces leukopenia and thrombocytopenia; in animaIs, it
suppresses the bone marrow and affects immune response.
It is hepatotoxic in both hum ans and experimental animaIs.
Chlorozotocin induced DNA damage in bone-marrow cells of rats in vivo. It
induced DNA damage in human, mouse and Chinese hamster cells in vitro, sister
chromatid exchange in mouse and rat cells and gene mutation inChinese hamster
cells. It induced sex-linked recessive lethal mutations in Drosophila and gene
CHLOROZOTOCIN 71
conversion in Saccharomyces cerevisiae. Chlorozotocin induced mutations in

Salmonella tyhimurium. (Se Appendix 1.)
4.5 Evaluation 1
There is suffcient evidence for the carcinogenicity of chlorozotocin in experi-

mental animaIs.
No data were available from studies in humans on the carcinogenicity of
chlorozotocin.
following information. Chlorozotocin is an alkylating agent and is structurally
related to other chloroethyl nitrosoureas, one of which, 1-(2-chloroethyl)-3-
( 4-methylcyclohexyl)- 1 -nitrosourea (methyl-CCNU), is carcinogenic to humans
(Group 1) and two ofwhich, bischloroethyl nitrosourea (BCNU) and 1-(2-chloro-
ethyl)-3-cyclohexyl-l-nitrosourea (CCNU), are probably carcinogenic to hum
ans
(Group 2A) (IAC, 1987). Chlorozotocin has given consistently positive results in a
broad spectrum of assays for genetic and related effects, inc1uding those involving
mammalian cells.
Overall evaluation
Chlorozotocin is probably carcinogenic to humans (Group 2A.
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Caner Res., 42,265-268
Alexander, J.A., Bowdon, B.J. & Wheeler, G.P. (1986) DNAdamage in cultured L1210 cells
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Andersn, 1:, McMenamin, M.G. & Schein, P.S. (1975) Chlorozotocin,
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2719-2725
lFor desription of the italicized terms, se Preamble, pp. 2629.

Bukowski, R.M., McCracken, J.D., Balcerzk, S.R & Fabian, C.J. (1983) Phase II study of
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Chlorowtoc, an anti-tumour agent lackig bone marrow toxicity at therapeutic
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CHLOROZOTOCIN 73
Fox, P.A., Panasci, L.C. & Schein, P.S. (1977) Biological and biochemical properties of
1-(2-chloroethyl)-3-(ß-D-glucopyrnosyl)-I-nitrosourea (NSC D 254157), a nitroso-
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Franza, B.R., Jr, Oeschger, N.S., Oeschger, M.P. & Schein, P.S. (1980) Mutagenic activity of
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Gastrointestinal Thmor Study Group (1985) Phase II trials of maytansine, low-dose
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Gralla, E.J., Fleischman, R.W. & Luthra, Y.K. (1979)
Toxicology studies in mice, beagle dogs
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Haas, C.D., Stephens, R.L., Bukowski, R.M., Studkey, WJ., McCracken, J.D., Gagliano,
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CHLOROZOTOCIN 75
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eieLOSPORIN
1.1 Synonyms
ehem. Abstr. Services Reg. No.: 59865-13-3 (cyclosporin A); 79217-60-0 (cyclo-
sporine)
ehem. Abstr. Name: tR-(R *,R *-(E)H-L-Cyclic(L-alanyl-D-alanyl-N-methyl-
L-leucyl-N-methyl- leucyl-N-methyl- L-valyl-3-hydroxy-N,4-dimethyl- L-2-ami-
no-6-octenoyl- L-~-aminobutyl-N-methylglycyl-N-methyl- L-leucyl- L-valyl-
N-methyl-L-Ieucyl)
Synnym: Cyclosporin A; cyclosporine; dyclosporin; OL-27-40; cycloH(E)-
(2S,3R,4R)- 3-hydroxy-4-methyl-2-( methylamino )-6-octenoyl)- L- 2-aminobuty-
ryl-N-methylglycyl-N-methyl- L-leucyl- L-valyl-N-methyl- L-Ieucyl- L-alanyl - D-
alanyl-N-methyl- L-Ieucyl-N-methyl-L-Ieucyl-N-methyl- L-valyl); cyclot (4-(E)-
but -2-enyl-N,4-dimethyl- L-threonyl)- L-homoalanyl(N-methyl-glycyl) (N-me-
thyl- L-Ieucyl)- L-valyl(N-methyl- L-Ieucyl)- L-alanyl- D-alanyl-(N-methyl- L-
leucyIXN-methyl- L-IeucYIXN-methyl- L-valyl))
CH3, /H
cI;
Il
/ c~ (J
H CH2
CH3" / CH3 HO C, 1
CH , / 'Y CH3 CH3

1 CH3" / CH3 CH 1
CH2 CH3 CH CH3 1 CH2 CH3
CH"
1 "1
CH;
1"11 1 1 1 1 : 1 1
CH3-N-CH-CO-N - CH-C-N - CH-CO-N-CH-C-N-CH2
1 H. 0.H1
OC AAl0 AAU 0 AAl H AA 0 AA3 CO
CH-CH2-CH AA9 N-CH3
CH3-N 1 AAS AA7 1 AA6 1/ AAS 1 AA4
OC-CH - N-CO-CH-N-C-CH - N-C-CH-N-CO-CH
1 1 1 1/ 1 1 1 1
CH3 H CH3 0 CH2 CH3 CH CH2
, -1 CH('CH31
, -- - - -CH3
-- --CH3
, /CH,
CH3/CH,
CH3
C:62l1iii~ ii()i2 MoL. wt: 1202.64
-77-
78 IAC MONOGRAHS VOLUM 50

From Ruegger et al. (1976), Windholz (1983) and Hassan and Al Yahya (1987)
(a) Description: White prismatic crystals from acetone; neutral, hydro-
phobie, cyclic non-polar oligopeptide composed of Il amino acid resi-
dues. The X-ray crystallographic structure is known.
(b) Melting-point: 148-151°C (natural); 149-150°C (synthetic)
(c) Optical rotation: ((I)ît = _244° (c = 0.6inehloroform); ((I)~= -189°
(c = 0.5 in methanol)
(d) Solubility Neutral; ri
ch in hydrophobic amino acids; insoluble in water
and n-hexane; very soluble in all other organic solvents
(e) Spectroscopy data: Ultraviolet, infrared, nuclear magnetie resonanee and
mass spectra have ben reported.
if Stability Stable in solution at temperatures below 30°C; sensitive to light,
eold and oxidization (Reynolds, 1989)
Trade names: Sandimmun; Sandimmune

Ciclosporin is available in bottles containing 100 mg/ml in an olive oil-based
solution and 12.5% ethanol for oral administration, and in ampoules containing 50
mg/ml with 33% ethanol and 650 mg polyoxethylated castor oil for intravenous
injection (Barnhart, 1989).
The isolation of cyclosporins A and C from the fungus Tolypcladium inflatum

Gams has been described (Ruegger et al., 1976), and the biosynthesis of ciclosporin
has been reported (Kobel & Traber, 1982; Kobel et al., 1983; Bilich & Zoher, 1987).
It is also produced synthetically from N-methyl-e-9-amino acid with subsequent
additions of appropriate peptides, followed by cyclization (Hassan & Al- Yahya,
1987).
Ciclosporin is manufactured commercially in Switzerland (Reynolds, 1989).
Cyclosporins (mostly A and C) are produced by the fungi Tolypcladium
inflatum Gams and T. cylindrosporum and by other fungi isolated from soiL.
2.2 Use
Ciclosporin is an immunosuppressive agent. It is used extensively in the
prevention and treatment of graft-versus-host reactions in bone-marrow
CICLOSPORIN 79
transplantation, and for the prevention of rejection of kidney, heart and liver
transplants. It has also been tested for the therapy of a large variety of other
diseases in which immunological factors may have a pathogenetic role, including
Graves' disease, uveitis, Crohn's disease, ulcerative colitis, chronic active hepatitis,
primary bilary cirrhosis, diabetes melltus, myasthenia gravis, sarcoidosis,
dermatomyositis, systemic lupus eryhematosus and psoriasis (CaIne et al., 1978,
1979; Powles et al., 1980; Merion et al., 1984; Kahan et al., 1985; Reynolds, 1989).
The usual oral dose of ciclosporin is 18 mg/kg daily, beginning 12 h before
transplantation and continuing for one to two weeks. Dosage may subsequently be
reduced to 5- 10 mg/kg or less. Ciclosporin may also be given intravenously, usually
at one-third of the oral dose. This drug is often given for several months to
transplant recipients (Reynolds, 1989).
2.3 Analysis
Ciclosporin has been measured in pharmaceutical preparations by

high-performance liquid chromatography (HPLC; US Pharmacopeial Convention,
Inc., 1989).
Ciclosporin and its metabolites have also been measured in biological fluids
using HPLC (Awni & Maloney, 1988; Christians et al., 1988a,b; Birckel et al., 1988),
and ciclosporin has been monitored in whole blood by radioimmunoassay
(Donatsch et al., 1981; Vine & Bowers, 1987). Vine and Bowers (1987) provided a
critical summary of HPLC methods used to measure ciclosporin in biological
fluids, and Hassan and Al- Yahya (1987) reviewed the methods for analysing
ciclosporin. Radioimmunoassay kits for the analysis of ciclosporin in plasma are
available, and their performance has been compared to that of HPLC analyses
(Vernilet et al., 1989; Wolf et al., 1989).

(a) Oral administration

Mouse: Groups of 50 male and 50 female OF1 mice, weighing 26-39 and 19-
28 g, respectively, were fed ciclosporin at 1, 4 or 16 mg/kg of diet for 78 weeks, at
which time all survivors were killed. An untreated group of 50 males and 50 females
served as controls. AIl mice were necropsied, and all macroscopic lesions were
examined histologically. Mortality was higher In high-dose females (60%) than in
controls (40-50%) and in other treated groups (42-52%). No increase in the

incidence of tumours was observed in treated mice (Ryffel et al., 1983).
ln a screening assay based on the accelerated induction of leukaemia in a
strain highly susceptible to development of this neoplasm, 30 male AKR mi ce, six
weeks of age, were fed cic1osporin at 150 mg/kg of diet. The first thymic lymphoma
in treated mice was noted at week 17; these tumours ocurred in 13/18 animais killed
between 20 and 29 weeks (p = 0.00) and in 9/9 killed between 30 and 34 weeks £p =
et only, the first
0.005, Fisher's exact test). ln 22 mice that received the basal di
thymic lymphoma was noted at week 23, and the incidences of these tumours in
animaIs kiled between 20 and 29 weeks and 30 and 34 weeks were 2/12 and 3/9,
respectively (Hattori et al., 1986).
Rat: Groups of 50 male and 50 female OFA rats, weighing 242-326 and 169-244
g, respectively, were fed ciclosporin at 0.5, 2 or 8 mg/kg bw of diet for 95 weeks
(males) and 105 weeks (females), at which time the experiment was terminated. An
untreated group of 50 males and 50 females served as controls. All animaIs were
necropsied, and aIl macroscopic lesions were examined histologically. Mortality
rates were 68% in controls, 74% in low- and mid-dose groups, and 86% in the
high-dose groupe No increase in tumour incidence was observed in treated rats
(Ryffel et al., 1983). (The Working Group noted the high incidence of tumours in the
controls, which may have reduced the sensitivity of the assay.)
(b) Administration with other treatments
Mouse: A group of 39 male Swiss Webster mice and 13 male C57Bl/6J mice, six
to seven weeks of age, were given a single whole-body 'Y-irradiation of 350 rad and
ten days later were fed cic1osporin (purity unspecified) at 150 mg/kg of diet for 35
weeks, at which time all survivors were killed and autopsied. A group of 26 male
Swiss Webster and 14 male C57Bl/6J mi ce received the same irradiation and were
maintained on basal diet. Two groups of 18 male Swiss Webster and 12 male
C57BI/6J mice received no irradiation and were maintained on control diet or were
given ciclosporin at 150 mg/kg of diet. No tumour was observed in either of the
strains of mice irradiated and maintained on basal diet alone or in either strain that
received no radiation and were fed diets containing ciclosporin. Of the Swiss
Webster mice that were irradiated and fed diets containing ciclosporin, 18/39 ( 46%)
(p ~ 0.001, Fisher's exact test) developed lymphoid tumours, primarIly in the spleen
and mesenteric lymph nodes, within an average latent period of 24 weeks. The
tumours were interpreted as B-immunoblastic lymphomas with plasmacytoid
features. Four of the 39 (10%) mIce developed classical thymic lymphomas within
an average latent period of 23.7 weeks. Of the C57Bl/6 mice irradiated and fed diets
containing ciclosporin, 7/13 (54%) (p ~ 0.002, Fisher's exact test) developed thymic
CICLOSPORIN 81
lymphomas within an average latent period of 27.4 weeks. No spleen or lymph node
lymphoma developed in this strain (Hattori et al., 1988).
Two groups of 13 male Swiss Webster mice, six to seven weeks old, received a
single intraperitoneal injection of 1 glg bw urethane. One week later, ciclosporin
(purity unspecified) was administered at 150 mglg of diet. Two groups of 15 or 14
mice not receiving injections of urethane were fed the basal di et or ciclosporin at
150 mg/kg of di et. AIl animaIs were killed 22 weeks after the beginning of treatment.
No significant difference in the number of lung adenomas was found between the
groups receiving urethane and ciclosporin and those receiving urethane alone
(Shinozuka et al., 1988). (The Working Group noted the small number of animaIs
used and the short duration of the study.)
Groups of 28-41 male Swiss Webster mice, six to seven weeks of age,received a
single intraperitoneal injection of N-methyl-N-nitrosourea (MNU) at 0, 12.5 or 25
mg/kg bw (vehicle unspecified) and one week later were fed either basal diet or
ciclosporin (purity unspecified) at 150 mg/kg of diet for 35 weeks. Mice treated with
MNU and ciclosporin had four- and eight-fold higher incidences of thymic
lymphomas, respectively, than mice treated with either dose of MNU alone ( .c 2%)
(figures not given). Thymic lymphomas did not develop in mice treated with
cic1osporin alone or maintained on basal diet (Shinozuka et aL., 1988). (The Working
Group noted the incomplete reporting of the study.)
Rat: Groups of 10- 12 male Sprague- Dawley rats, weighing 100- 120 g, received a
0 or 25 mg/kg bw MNU in 10% ethanol and citrate
single intraperitoneal injection of
buffer; one week later, they were fed basal diet or ciclosporin (purity unspecified) at
110 mg/kg of diet for 34 weeks, at which time the experiment was terminated.
Autopsies were carried out on aU rats killed during the course or at the end of the
experiment, and tissues from the thymus, mesenteric lymph nodes, intestinal
lymphoid plaques, spleen, lung, kidney and liver were examined histologically. Of
the rats receiving MNU and ciclosporin, 6/10 developed intestinal adeno-
carcinomas in the region of intestinal lymphoid plaques: two in the lower portion of
the Ileum and four in the ascending and transverse colon; two of the latter had two
tumours each in the colon. The first tumour appeared in week 23 of the study. Of
the rats receiving MN alone, 1/12 developed an intestinal adenocarcinoma in
week 33 of the study (p .c 0.05). No intestinal tumour was observed in rats receiving
ciclosporin or basal diet alone, but in rats treated with ciclosporin alone, atypical
epithelial proliferations of the intestinal mucosa associated with hyperplasia of
gut-associated lymphoid structures was observed (Perera et al., 1986). (The
Working Group noted the small number of animaIs used.)
Rat: Young male Wistar rats, weighing 62-80 g, were divided into six groups:
group 1 (five animaIs) recived daily subcutaneous injections of ciclosporin (purity
unspecified) at 10 mg/kg bw in olive oil during week 1; group 2 (15 animaIs) recived
daily subcutaneous injections of ciclosporin at 10 mglg bw in olive oil during week

1, followed by administration of N-methyl-N' -nitro-N-nitrosoguanidine (MNNG).
at 83 J.g/ml in the drinking-water ad libitum from week 3 to 28; group 3 (15 animaIs)
received MNNG in the drinking-water from week 3 to 28; groups 4 and 5 (15 animaIs
per group) received MNNG in the drinking-water in weeks 3-28 and daily
subcutaneous injections of cic1osporin at 10 mglg bw during week 15 or during
week 30; group 6 (ten animaIs) served as untreated controls. AIl survving animaIs
were sacrificed in week 39. No rat in group 1 or 6 died during the experiment, and no
tumour was found in any animal in these groups. ln group 2, 7/9 survving rats had a
total of 14 tumours (one intestinal carcinosarcoma, 13 adenocarcinomas of the
stomach and small intestine; me an number of tumours per rat, 1.56). ln group 3,
8/12 survivors had a total of 12 tumours (mostly adenocarcinomas of the stomach,
small intestine or both; mean number of tumours per rat, 1.00). ln group 4, 10/13
survivors had a total of 19 tumours (18 adenocarcinomas of the stomach, small
intestine or both, and one large-cell lymphoma involving coliac lymph nodes, liver
and spleen; mean number of tumours per rat, 1.46). ln group 5, 10/12 survivors had
a total of 20 tumours (one carcinosarcoma, 19 adenocarcinomas of the stomach,
small intestine or both; mean number of tumours per rat, 1.67). No statistical
difference in the incidence of tumours was observed among groups 2-5 (Johnson et
al., 1984).
Monkey. A group of 55 macaques (age and sex unspecified) that had received
cardiac or heart-Iung allografts and had survived the first two post-operative weeks
received daily intramuscular injections of ciclosporin (purity unspecified) at 25
mg/kg bw in miglyol 812 (an oil base) for 14 days, after which they were treated
either every other day or daily with intramuscular injections of 17 mg/kg bw
ciclosporin continuously. Eight subgroups were formed: group 1 (16 animaIs)
received no treatment other than ciclosporin; group 2 (nine animaIs) was treated
concurrently with 2 mg/kg bw azathioprine; group 3 (six animaIs) had previously
received daily injections of 10 mg/kg bw rabbit antithymocyte globulin on
post-operative days 0-7; group 4 (13 animaIs) received concurrent weekly treatment
with 14 mg/kg bw antithymocyte globulin, azathioprine and methylprednisolone;
group 5 (11 animaIs) had received total lymphoid radiation at a dose of 100 rads per
day (total dose, 600-1800 rads) prior to operation; group 6 (ten animaIs) receIved
injections of azathioprine plus methylprednisolone; group 7 (23 animaIs) receIved
azathioprine, methylprednisolone and antithymocyte globulin; and group 8 (nine
animaIs) received azathioprine, antithymoce globulin and total lymphoid
irradiation. No lymphoma was observed among animaIs receiving treatment other
than with ciclosporin (groups 6-8). Of the animaIs treated with ciclosporin alone or
in combination with other immunosuppressive agents, B-cell lymphomas
developed in 12/55 monkeys fp ~ 0.001, Fisher's exact test): 2/16 treated with
CICLOSPORIN 83
ciclosporin alone (group 1), 4/9 with cic1osporin plus azathioprine (group 2), 1/6
with ciclosporin plus antithymocyte globulin (group 3), 2/13 with ciclosporin,
. antithymocyte globulin, azthioprine and methylprednisolone (group 4), and 3/11
with ciclosporin and total lymphoid radiation (group 5). Viral particles were noted
within the endoplasmic reticulum of plasmacytoid cells in 6/8 tumours froID
animaIs treated with ciclosporin alone or in combination with other immuno-
suppressive agents. The authors noted that the incidence ofspontaneous haema-
topoietic neoplasms in nonhuman primates is generally considered to be low,
although outbreaks of lymphomas have been reported among macaques (Bieber et
al., 1982).

The experimental toxicology of cic1osporin has been widely reviewed (e.g.,
Feutren & Bach, 1987; Aszalos, 1988; Grace, 1988; de Groen, 1988; Humes &
Jackson, 1988; Kahan et al., 1988a,b; Mihatsch et al.~ 1988a,b).
(i) Absorption, distribution, excretion and metabolism
The toxicokinetics and toxicodynamics of ciclosporin have been reviewed
(Wood et al., 1983; Maurer, 1985; Wood & Lemaire, 1985; Grevel, 1986a,b; Lemaire
et al., 1986).
Orally administered ciclosporin (in olive oil) was rapidly absorbed in dogs and
rats. About 50% of a single dose reached the circulation (plasma levels determined
by radioimmunoassay) in both species; there was no tendency for accumulation in
beagle dogs after repeated daily administration for a year (Ryffel et al., 1983).
A single oral administration of 82 mg/kg bw to WAGlRij rats resulted in levels
of 80 J-g/ gin liver, kidney and brain 3 and 7 h after administration. Slowelimination
occurred thereafter: even after five days, significant amounts (10 J-glg) were
detected. A short time after oral administration, 3.5 J-glml of ciclosporin were
detected in blood, and the levels remained almost the same for about two days; 2%
of the administered dose was eliminated unchanged in bile and 2% in urine (Nooter
et aL., 1984a). About 2% of an oral dose of ciclosporin was absorbed into the
intestinal lymphatic system in rats (Ueda et al., 1983).
Pharmacokinetic studies were also performed after intravenous administra-
tion of 20,40 or 80 mg/kg bw to WAG/Rij rats (Nooter et al., 1984b). Elimination of
ciclosporin at the lowest dose was best described by a two-compartment model (tYí:
6 min and 16.5 h); at the higher dose levels, a three-compartment model best
described the observed data. Urine and bile excretion was 10 and 20% of the total
administered dose. The bioavailabilty of ciclosporin in Wistar rats increased with
increasing oral dose. Daily oral administration of 4 mg/kg bw was necessary to
84 lAC MONOGRAHS VOLUM 50
young rats, while 7.5 mg/g bw per

maintain plasma levels at about 130 nglml in very
day were needed in one-month-old animals (Levy-Marchal et al., 1988).

Absorption of orally administered tritium-labelled ciclosporin by Sprague-
Dawley and Wistar rats was slow and was not affected by the vehicle. The degree of
absorption was about 30%. Labelled ciclosporin was widely distributed throughout
the radiolabel was 46 h after dosing
the body. The terminal elimination half-time of
with 10 mglg bw daily in olive oil for 21 days; elimination from kidney and liver
had a half-time of 70-100 h. Accumulation of the parent compound was evident
after repeated treatments, with high levels in kidney, liver, bloo and lymph nodes
and particularly in skin and adipose tissue (Wagner et al., 1987).
ln male CD-COBS rats treated intravenously, bloo concentrations during
elimination were best described by a three-cmpartment model, with half-times of
0.11 h, 1.8 h and 23.8 h. The apparent distribution volume ranged from 4.88 to 6.84
l/kg. Elimination was almost entirely by hepatic metabolism (Sangall et al., 1988).
Total body clearance was lower in obese rats than in lean Zucker rats (Brunner et al.,
1988).
A non-linear pharmacokinetic behaviour was seen in New Zealand white
rabbits injected intravenously. The volume of distribution at steady state increased
with increasing dose (Awni & Sawchuk, 1985). The mean half-time after intra-
venous administration of 15 mglg bw to male New Zealand rabbits was 229.7 min
(D'Souza et al., 1988).
ln rabbits, the concentrations of ciclosporin in blood were about 100 nglml
from day 43 to 120 after repeated subcutaneous injections; the calculated
absorption half-time was 33 days following injection with 20 mg/g twice a week
during days 7-29 of the experiment (Shah et al., 1988). ln BALB/c mice injected
subcutaneously with 12.5, 50 or 20 mglg bw, ciclosporin was detected (by
radioimmunoassay) in every organ investigated (Boland et al., 1984). The organs in
mice that are susceptible to toxicity (e.g., brain, kidney, liver) retained ciclosporin
after intraperitoneal injection (Belitsky et al., 1986).
Following oral, intraperitoneal, subcutaneous or intravenous administration
of radiolabelled ciclosporin to C57BI mice, a high initial concentration of radiolabel
was observed in liver, pancreas, salivary glands, spleen and fat tissuebywhole-body
autoradiography. Relatively high levels were retained in liver, bone marrow, thymus
and lymph nodes. ln kidney, the radiolabel was confined to the outer zone and outer
medulla. No radioactivity was seen in the central nervous system or in fetuses
(Backman et al., 1987, 1988).
When ciclosporin was mixed with human or rat blood in vitro, 50% was found
in eryhrocytes, 15% iD leukocytes and 3040% in plasma. At concentrations of
CICLOSPORIN 85
25- 100 nglml in human plasma, 65-80% of tritiated ciclosporin was associated with
lipoproteins (Lemaire & Tilement, 1982; Niederberger et al., 1983).
Ciclosporin is extensively metabolized by cytochrome P450-mediated
oxidation, hydroxylation and N-demethylation (Maurer et al., 1984; Maurer, 1985;
Burke & Whiting, 1986; Maurer & Lemaire, 1986; Bertault-Peres et aL., 1987;
Wagner et al., 1987). Figure 1 shows some characteristics of the metabolites that
have been isolated. The numbers in the following text refer to the amino acids and
metabolites identified in the figure.
Fig. 1. Structures and molecular weights or metabolites or ciclosporin that have
been isolateda
R1 H ,/ct
Il
/CZa
H CH2
1
CH~ / CH3 HO CH,
CH CH~ / CH3 'l'Y CH3 CH3
CH
1
ß 1
CH2 CH3 CH CH3 CH2 CH3

1 1
CH3-N-CH-CQ-N
1 1
CH - C- N
~ 1 1
CH-CO-N-CH-C-N-CH2
CH3 OC AA10
1
AA11 0
Il
AA 1 1 AA Il AA3 1
H 0 CO
1 . 1 1
R -C-CH2-CH AA9 N-R2

1 1
CH3 N-CH3 H
1 AAS AA7 1 AA6 ~ AA5 7 AA4 1
QC-CH - N-CQ-CH-N-C-CH - N-C-CH-N-CQ-CH
1 1 Il 1 1 1 1
/,
1
CH3 H CH3 Q CH2 CH3 CH CH2
CH3 CH3
1_________ l
/,
1 1
C C,
CH3 1 CH3 CH; 1 CH3
R4 R3
Fig. i (contd)
Metabolite A Ai A2 R3 A4 Other Molecular
no. modification weight
Ciclosporin H CH3 CH3 H H 1202.64

1 OH CH3 CH3 H H 1218.64
8 OH CH20H CH3 H H 1234.64
9 OH CH3 H H OH 1220.62
10 OH CH3 CH3 OH H 1234.64
13 Hydroxylated and N-demethylated derivative of ciclosporin 1204.62
16 OH CH3 CH3 H OH 1234.64
17 H CH20H CH3 H H 1218.64
18 H CH20H CH3 H H 0 1218.64
/ \
CH CH-CH2 of AA 1
ß e ~
22 H CH3 H H H 1188.62
25 H CH20H H H H 1202.64
26 OH CH20H CH3 H H 0 1204.62
/ \
CH CH-CH2 of AA1
ß e ~
203-218 H COOH CH3 H H 1234.64
tlrom Maurer & Lemaire (1986)
AlI ciclosporin metabolites from dog urine and from rat bile and faeces
retained the intact cyclic oligopeptide structure of ciclosporin. Conjugations with
sulfuric or glucuronic acid were not detected (Maurer et al., 1984). Using perfused
rab bit liver, 27 metabolites were characterized, including three dihydrodiol
metabolites probably derived from epoxide intermediates (Wallemacq et al., 1989a).
An ~,ß-unsaturated carboxylic acid metabolite of amino acid 9 (AA9) was
isolated in rabbit urine after intravenous administration of ciclosporin (Hartman et
al., 1985). ln a study on ciclosporin metabolism in rats, parent ciclosporin
predominated over metabolites in blood. Metabolite i was found to be the major
one in this species. Intraperitoneal injections of phenobarbital and methyl predni-
solone to Wistar rats receiving daily subcutaneous treatments with ciclosporin
decreased ciclosporin levels in blood (Pell et al., 1988). ln rats injected intra-
venously, covalently bound ciclosporin was detected in protein fractions ofliver and
kidney homogenates, and phenobarbital treatment enhanced adduct formation.
CICLOSPORIN 87
Covalent binding to protein was found in vitro after incubation of labelled

ciclosporiß with a rat liver microsomal fraction in the presence of NADPH.
Binding also ocurred in isolated hepatocytes. SKF-525A inhibited the covalent
binding, and glutathione depletion increased cic1osporin binding to protein
(N agelkerke et aL., 1987).
No association of radioactivity was observed with cellular proteins or with
DNA in liver homogenates from mice administered the drug parenterally
(Backman et al., 1987, 1988).
(ii) Toxic effects
ce, rats and

The LDs() for ciclosporin after a single oral administration to mi
rabbits were 2.3, 1.5 and :; 1.0 g!g bw, respectively. The corresponding figures
after a single intravenous administration were 107, 25 and :; 10 mg/kg bw. Toxic
signs were hyperventilation, drowsiness and muscular spasms. After oral adminis-
tration, weight loss and diarrhoea were noted (Ryffel et al., 1983, 1986).
Daily subcutaneous injections of ciclosporin into BALB/c mice at a dose of20
mg!g bw per day resulted in a median survival time of about 13 days. Nephro-
toxicity, hypocellularity of the thymus, lymph nodes and spleen and fatty changes in
the liver were observed; no abnormality of femoral bone marrow was found (Boland
et al., 1984).
Histological findings in OFA rats fed a diet containing ciclosporin for 13 weeks
included leukocosis, lymphopenia, hypochromic anaemia, monocytosis and
eosinopenia without myelotoxic effects. Lymphoid tissues were atrophied. Doses
of 45 mg!g bw per day and more produced nephrotoxicity and hepatotoxicity. A
chronic nonspecific gingivitis with atrophy of periodontal tissue was observed in
treated rats. Nephrotoxicity and hepatotoxicity were also observed among rats
administered ciclosporin orally for 104 weeks (Ryffel et al., 1983).
OF1 mice were given ciclosporin in the diet at 1.4 and 16 mg!g per day for 78
an other mice and
weeks. Females given the high dose had higher mortality rates th
had haematological changes without myelotoxic signs (Ryffel et al., 1983).

NZW and RB rabbits treated subcutaneously with ciclosporin at 15 mg/kg bw
daily had weight loss and reduced food and water intake. High mortality was
observed within 60 days of treatment, and animaIs had distended stomachs and
intestines (Gratwohl et al., 1986).
Afer intravenous treatment at 45 mg!g bw day for four weeks, cyomolgus
monkeys showed bloo chemistry changes, marked neurological side-effects, and
degenerative changes in kidney and lIver. Rhesus monkeys tolerated high oral doses
of ciclosporin (2030 mglg bw) for 13 weeks, with small functional and
histopathological changes (Ryfel et al., 1983).
The renal effects of ciclosporin in experimental systems have been studied

extensively and reviewed (Sullvan et al., 1985; Ryffel & Mihatsch, 1986; Humes &
Jackson, 1988).
The severity of histological changes in the kidneys of rats receiving

subcutaneous injections daily for up to 30 days were directly correlated with tissue
levels of ciclosporin (Kumar et al., 1988).
Ciclosporin induced marked renal vasoconstriction in rats (Kaskel et al., 1988;
Monaco et al., 1988; Stanley Nahman et al., 1988) and sheep (Friedman et al., 1988).
Various defects in renal function accompanied the vasoconstriction, inc1uding
decreased glomerular filtration rate (Whiting et al., 1982; Sabbatini et al., 1988;
Tejani et al., 1988); decreased sodium reabsorption (Whiting & Simpson, 1988),
impairment of the diluting capacity of the thick ascending lImb of the loop of RenIe
(Gnutzmann et al., 1986) and release of cellular enzymes into the urine (Whiting et
al., 1986).
Sprague-Dawley rats given ciclosporin at 50 or 100 mg/kg bw per 48 h over 21

days by gastric intubation had elevated serum urea and creatinine levels, and
urinary N-acetyl-ß-D-glucosaminidase activity was increased (Thomson et aL.,
1981; Whiting et al., 1982). The renal and hepatic functional disturbances were
reversible (Thomson et al., 1981). There was cytoplasmic vacuolization of the
proximal tubule, swollen cells and cell necrosis - the latter at the higher dose.
Vacuolization was due to dilatation of smooth and rough endoplasmic reticulum.
The number oflysosomes was increased, and myeloid bodies were present (Whiting
et al., 1982).
Rats given ciclosporin at 20 or 40 mg/kg bw in the diet showed augmentation of

autoplagic vacuoles, lipid drops and loss ofmicrovili in the proximal nephron as
well as prenecrotic damage of proximal tubular S2 and S3 cclls (Pfaller et al., 1986).
Similar observations were made by Verani (1986), Jackson et al. (1987), Dieperinket
al. (1988), Gilum et al. (1988), Jackson et al. (1988) and Starklint et al. (1988a,b),
although strain differences have been reported (Duncan et al., 1986).
When ciclosporin was given by gavage at 30 mg/kg bw per day to
Sprague- Dawley rats for four weeks, serum testosterone levels were decreased by
50%; this change was reversible (Sikka et al., 1988).
Rats injected intraperitoneally with ciclosporin at 5, 10 or 15 mg/kg bw for one
or three weeks had significantly raised levels of serum bile acids. Both bile
salt-dependent and independent-flow were decreased (Stone et al., 1988).
Ciclosporin markedly decreased pancreatIc insulin content and insulin release
in rats administered the drug by intramuscular injection for two weeks (Hahn et al.,
1986). Electron microscopy demonstrated cytoplasmic degranulation, nuclear
CICLOSPORIN 89
inclusions and cistemal dilatation of endoplasmic reticulum and of the Golgi

apparatus in pancreatic ß cells (Hamaguchi et al., 1988).
When Sprague-Dawley rats were fed ciclosporin at 150 mg/kg of diet, their
thymuses and lymph nodes were smaller after eight weeks. Proliferative changes
were observed in gut-associated lymphoid tissue, with mitotically active
lymphocytes that displayed local tissue invasion and destruction (Demetris et al.,
1984).
Orál administration of immunosuppressive doses of ciclosporin reduced the
trabecular bone volume of Sprague-Dawley rats. Osteolast number and bone
resorption were significantly increased at low (7.5 mglg bw per day) and high (15
mglg bw per day) doses of ciclosporin (Movsowitz et al., 1988).
Thromboxane syothesis in rats and its excretion in urine were increased by
ciclosporin treatment (Perico et al., 1986a,b; Coffman et al., 1987; Benigni et al.,
1988; Rogers et al., 1988). Prostaglandin production was stimulated by ciclosporin
(Coffman et al., 1987), and administration of prostaglandin Ei (Ryffel et al., 1986) or
its analogues (Paller, 1988a,b) reduced the nephrotoxicity of ciclosporin. A throm-
boxane synthetase inhibitor (CGS 12970) also prevented nephrotoxicity in rats
(Smeesters et al., 1988a,b).
Ciclosporin affected protein synthesis in vivo and in vitro (Backman et al., 1988;
Buss et al., 1988), altered hepatic glycogen metabolism (Betschart et al., 1988) and
inhibited P450-dependent metabolism in vivo (Augustine & Zemaitis, 1986;
Moohhala & Renton, 1986).
It induced dose-dependent malonaldehyde production in rat renal
microsomes (Inselmann et al., 1988). It bound with high affinity to cyclophilin, a
low-molecular-weight cytosolic protein that occurs ubiquitously in eukaryotic cells
and is thought to be a regulator of T- and B-cell activation (Harding &
Handschumacher, 1988; Quesniaux et al., 1988).
Ciclosporin inhibited T-Iymphocyte proliferation (Borel et al., 1977) but did
not affect protein kinase C. It inhibited the augmentation of ornithine
decarboxylase levels in mouse skin induced by phorbol ester (EIder et al., 1988) and
interfered with intracellular calcium metabolism (for reviews, see Aszalos, 1988;
Bijsterbosch et al., 1988).
ln routine studies to evaluate the safety of ciclosporin, oral administration at
1.5,5 or 15 mg/kg bw to male and female rats daily from before mating (males, 12
weeks; females, two weeks) until weaning had no adverse effect on reproduction. ln
rats administered ciclosporin at 10-30 mglg bw ~orally from day 6 to 15 of
gestation, there was no embryotoxic effect at doses up to 17 mg/kg bw. At 30 mglg
bw, which was clearly toxic to the mother, high rates of embryolethality (90%)
ocurred, average fetal weights were lower than those of controls and skeletal
retardations were seen frequently, but there was no increase in the frequency of
minor or major anomalies. At higher doses, embryolethalty was 100%. ln a
similarly designed study in rabbits, using doses of 10-30 mglg bw, no adverse
effect was observed up to 30 mglg. At 100 mglg and above, maternaI toxicity was
seen, with an increased frequency of resorptions; however, no major or minor
anomalywas found. ln a peri-/postnatal study in rats at three dose levels (5,15, and
45 mglg bw), a distinct increase in pre-/perinatal and early postnatal mortality of
offspring was observed at the highest dose level (Ryfel et al., 1983).
Two further studies confirm the toxic effects of ciclosporin on rat fetuses after
daily exposure during late gestational stages at a maternally toxic dose (25 mg/g).
Fetal kidneys that cou Id be examined showed evidence of ciclosporin-induced
proximal tubular-cll damage (Brown et al., 1985; Mason et al., 1985).
When ciclosporin was administered subcutaneously for 14 days at daily doses
of 10, 20 and 40 mglg bw to sexually mature male rats, dose-dependent changes in
body and reproductive organ weights were noted. Histological examination of the
testis showed degenerative changes, and sperm counts and motilty were decreased
in all three treated groups. Rats treated with the two highest doses were infertile
(Seethalakshmi et al., 1987). This effect was reversible after withdrawal of the drug
(Seethalakshmi et al., 1988).
Ciclosporin did not induce mutation in Salmonella tyhimurium in either the
presence or absence of an exogenous metabolic system (Matter et al., 1982).
It did not induce mutations at the hprt locus of Chinese hamster V79 cells in the
presence or absence of an exogenous metabolic system (Zwanenburg et al., 1988). It
induced sister chromatid exchange in human peripheral lymphocytes in vitro
(Yuzawa et al., 1986, 1987).
At doses up to 100-300 mglg, ciclosporin did not induce chromosomal
aberrations or micronuclei in bone-marrow cells of CD- 1 mice or Chinese hamsters
in vivo, or unscheduled DNA synthesis (dose unspeified) or dominant lethal
mutations in CD-1 mice (Matter et al., 1982).
(h) Humons
(i) Phanncokinetics
The kinetics of ciclosporin has been reviewed (Bowers et al., 1986; Grevel,
1986a,b; Lemaire et al., 1986; Vine & Bowers, 1987; Grevel, 1988; McMillan, 1989).
ln studies on the kinetics of ciclosporin, radioimmunoassay and liquid
chromatography have generally been used. If not indicated otherwise, the data
CICLOSPORIN 91
given below are from studies in which high-performance liquid chromatography

analysis was used, which is the most specific for ciclosporin.
Absorption of orally administered ciclosporin is variable and low: the oral
bioavailability was 35 :i II % in heart transplant patients (Venkataramanan et aL.,
1986), 36 :i 17% in adult uraemic patients (Grevel et al., 1989) and 27 :l 20% in
41 renal transplant recipients; it was ~ 10% in 17% ofthese subjects (Ptachcinski et
al., 1985). Peak bloo ciclosporin concentrations were reached between 1 and 8 h
after oral dosing (Beveridge et al., 1981; Ptachcinski et al., 1985; Venkataramanan et
al., 1986).
Ciclosporin is rapidly and widely distributed; distribution half-times after

intravenous administration have been reported to be 0.1 :i 0.03 h (Follath et aL.,
1983) and 0.3-0.5 h (Yee et al., 1984). The steady-state apparent volume of
distribution is large, and means of 2.7-5.1 l/kg have been calculated (Follath et aL.,
1983; Yee et al., 1984; Ptachcinski et al., 1985; Venkataramanan et al., 1986; Clardyet
al., 1988). Concentrations of ciclosporin in rejected kidney were higher than
preoperative values in the blood of three patients (Kahn et aL., 1986; Rosano et al.,
1986). High concentrations of ciclosporin and its metabolites are found in, e.g., fat,
gall-bladder, liver, gastrointestinal tract and pancreas (Atkinson et al., 1983a;
Kahan et al., 1983a; Ried et al., 1983).
After the distribution phase, two further first-order disappearance phases
may be discerned, with half-times of approximately 1 and 16 h, respectively (Follath
et al., 1983). Even in a case of acute overdose of cic1osporin (500 mg), saturation of
clearance was not observed (Schroeder et al., 1986). Clearance of ciclosporin from
the blood is rapid: in bone-marrow transplant recipients with normal liver and
kidney function, clearance of 12.8 :i 1.6 ml/min per kg was reported; in those with
elevated serum bilirubin but normal renal function, it was 9.8 :i 2.1 ml/min per kg.
ln another study, however, no relationship was noted between the disappearance of
ciclosporin from the blood and the degree of impairment of hepatic function in
patients with primary bilary cirrhosis (Robson et al., 1984). ln renal and heart
transplant recipients, average clearance values of 6.5 and 5.7 ml/min per kg were
reported (Ptachcinski et al., 1985; Venkataramanan et al., 1986), while in patients
with renal failure clearance was 369 ml/kg per h (6.15 ml/min per kg) (Follath et al.,
1983). ln healthy subjects, a value of 51 ml/h per kg (8.5 ml/min per kg) was reported
(Grevel et al., 1986); in this study, however, the radioimmunological assay method
was used, which provides an underestimate of clearance (Grevel et al., 1989).
After administration of tritiated ciclosporin to two patients, 6% of the dose
was recvered in the urine (Maurer et al., 1984; Maurer, 1985; Lemaire et al., 1986).
ln healthy volunteers, approximately 0.1-0.2% of a dose was excreted in the urine as
unchanged ciclosporin (Beveridge et al., 1981; Maurer & Lemaire, 1986).
More ciclosporin and ciclosporin metabolites were detected in the bile than in
urine after intravenous and oral administrations (Kahan et al., 1983b;
Venkataramanan et al., 1985). Unchanged ciclosporin is a minor component in the
bile (mean, 0.29% of an oral dose) (Venkataramanan et al., 1985).
The concentration of cic1osporin in blood cells is approximately double that in
the plasma (Follath et al., 1983). The majority of ciclosporin and/or its metabolites
in serum is bound to different lipoprotein fractions (Mraz et al., 1983; Gurecki et al.,
1985). After treatment of pregnant women with ciclosporin, it was detected in cord
blood at concentrations somewhat lower than those in maternaI blood (Lewis et al.,
1983; Venkataramanan et al., 1988; Rose et al., 1989). Cic1osporin has also been
detected in breast milk (Lewis et al., 1983).
The first study of the metabolism of ciclosporin in humans was performed by
Maurer et al. (1984), who isolated and identified nine ether-extractable metabolites
from the urine of two patients who had received a single oral dose of 300 mg
3H-ciclosporin. AlI identified metabolites retained the intact cyclic peptide
structure; the sites on the molecule that are changed by metabolism are indicated in
Figure 1. The primary metabolites were products of hydroxylation; the secondary
metabolites identified were products of oxidation or demethylation of oxidized
primary metabolites or of a cyclization reaction. Similar oxidized cic1osporin
metabolites have been identified in the blood and bile of patients treated with
ciclosporin (Hartman et al., 1985; Rosano et al., 1986; Lensmayer et al., 1987a,b;
Wallemacq et al., 1989a,b; Wang et al., 1989). Twenty-seven ciclosporin metabolites
were identified in human bile; these included a vicinal dihydrodiol and a
demethylated vicinal dihydrodiol, suggesting that an epoxide is the intermediate
(Wallemacq et al., 1989a).
ln addition to metabolites generated by oxidation, demethylation and
cyclization reactions, three further metabolites have been isolated in which the
double bond in amino acid 1 (AAI in Fig. 1) is probably saturated (Wang et al.,
1989). This metabolite and metabolites 1,8, 17 and 203-218 (Fig.l) were reported to
be the major metabolites of ciclosporin in human bile (Hartman et al., 1985;
Maurer, 1985; Wang et al., 1989; Maurer & Lemaire, 1986). A sulfate conjugate of
ciclosporin was also identified in human bile and plasma (Henricsson et al., 1989).
Metabolite 17 was the main metabolite in human blood, and metabolites l, 8 and 21
were the other major ones (Maurer, 1985; Maurer & Lemaire, 1986; Rosano et al.,
1986). Metabolite 17 was the main metabolite detected in kidney (Rosano et al.,
1986).
A cytochrome P450 isolated from human liver catalysed the formation of
mono- and dihydroxylated and demethylated metabolites from ciclosporin
(Combalbert et al., 1989). This cytochrome is encoded by the gene P450IIIA, as is
CICLOSPORIN 93
nifedipine oxidase; it is induced by treatment with rifampicin (Kronbach et al., 1988;

Combalbert et al., 1989).
(ii) lmmunosuppressive action

The pharmacological effects of cic1osporin on the human immune system have
been reviewed (Thomson, 1983; Shevach, 1985; Drugge & Handschumacher, 1988;
Kerman, 1988; Kahan, 1989; Lorber, 1989). The ratio of T-helper cells to
T-suppressor cells was decreased in renal transplant recipients during treatment
with ciclosporin and prednisolone (Kerman et al., 1987). Production of ~-inter-
feron, )'-inteneron and interleukin-2 by isolated leukocytes was decreased in renal
and heart transplant patients reciving ciclosporin and prednisolone, as compared
to healthy volunteers (Dupont et al., 1985).
Many studies have ben published on the immunosuppressive effects of
ciclosporin since the detection (Borel et al., 1977) of its biological and c1inical
significance in the early 1970s (for review, see Feutren & Bach, 1987). Its
immunosuppressive effects have been demonstrated experimentally to lead to
tolerance of tissue grafts (Morris et al., 1980; Pennock et al., 1981; Bain et al., 1988;
Chisholm & Bevan, 1988; Finsen et al., 1988; Kimura et al., 1988; Lear et al., 1988;
White & Lim, 1988; for reviews, see Lorber, 1986; Tutschka, 1986; Hopt et al., 1988;
Kahan et al., 1988a,b) and to affect a variety of experimental autoimmune diseases,
such as uveitis (Nordmann et al., 1986; Dinning et al., 1987; Mahlberg et al., 1987;
Caspi et al, 1988a,b; Kaswan et al., 1988), myasthenia gravis (for review sec Feutren
& Bach, 1987; for a tabular summary, see Gunn et al., 1988), mercuric
chloride-induced glomerulonephritis (Aten et al., 1988), allergic encephalomyelitis
(Polman et al., 1988) and serum sickness nephritis (Shigematsu & Koyama, 1988).
Ciclosporin is preferentially active on proliferating T cells (White et al., 1979)
and selectively inhibits T-helper cell function (Caspi et al., 1988a,b) while sparing
T-suppressor cell activities (Kupiec-Weglinski et aL., 1984; Bucy, 1988). It inhibits
the production of interleukin-2 (Larsson, 1980; Bunjes et al., 1981; Caspi et al.,
1988b; Tracey et al., 1988) from T-helper cells and of interleukin-1 from splenic
adherent cells (Bunjes et al., 1981). Ciclosporin metabolites also suppressed
concanavalin A-stimulated human peripheral bloo mononuclear cell proliferation
(Cheung et al., 1988).
Ciclosporin was bound to a low-affinity site (KD = 3-6 x 10-7 M) on human
splenic T-Iymphoces in vitro, while B-Iymphoces showed both a high-affinity
(KD = 2 x 10-9 M) and a low-affnity binding site (LeGrue et al., 1983).
Ciclosporin depressed the synthesis of )'-inteneron by human thymocytes and
T-Iymphocytes in vitro (Reem et al., 1983; McKenna et al., 1989), as weIl as the
synthesis of lymphotoxin and tumour necrosis factor by lymphoces activated in
mixed-lymphoce culture or by concanavalin A (McKenna et al., 1989; Szturm et
al., 1989). Ciclosporin reduced T-cell growth factor (interleukin-2) gene trans-
cription in a c10ned human leukaemic T-cellline (Krönke et al., 1984) and binding of
radiolabelled human recombinant interleukin-2 to high-affinity receptors in human
T-Iymphocytes (Povlsen et al., 1989). CiclosPOrin also inhibited the release of
)'-interferon from alloactivated human peripheral blood mononuclear cells (Bishop
& Hall, 1988).
han et al.,
The adverse effects of ciclosporin therapy have been reviewed (Ka
1985; Bennett & Norman, 1986; Myers, 1986; Keown et al., 1987; Mihatsch et al.,
1988a,b; Racusen & Solez, 1988; Schachter, 1988; Weidle & Vlasses, 1988;
Dieperink, 1989; Mihatsch et al., 1989; Reynolds, 1989; Steinmuller, 1989).
The first report on the use of ciclosporin in the treatment of renal allograft
rejection (CaIne et al., 1978) documented nephrotoxicity, hepatotoxicity and
hirsutism as side-effects of the therapy. Nephrotoxicity has since been amply
documented as the most prevalent and serious complication of ciclosporin therapy,
in recipients of kidney transplants (CaIne et al., 1979; Klintmalm et al., 1981a,b;
Merion et al., 1984) and in other transplant recipients (Powles et al., 1980; Klintmalm
et al., 1981b; Shulman et al., 1981; Atkinson et al., 1983b; Hows et al., 1983; Myers et
al., 1984). Morphological changes related to ciclosporin administration include
diffuse interstitial fibrosis (associated with oligo- or anuria), tubular toxicity,
peritubular capilary congestion and a combination of the last two. These two
changes have been associatcd with acute renal damage; acutely impaired renal
function was not, however, necessarily accompanied by microscopic changes.
Arteriolopathy and interstitial fibrosis with tubular atropy, or a combination of the
two, have been attributed to chronic ciclosporin toxicity (Mihatsch et al., 1988a,b,
1989). Mechanisms of the renal toxicity of ciclosporin have been reviewed (Bennett
et aL., 1988; Dieperink et al., 1988; Grace, 1988; Neild, 1988; Benigni et al., 1989).
Mild functional disturbances of the liver have been reported in 20-40% of
treated patients (Klintmalm et al., 1981a; Kahan et al., 1985).
Other side-effects reported include gastrointestinal disturbances, hirsutism,
acne, gingival hyperplasia, neurotoxicity, altered blood coagulability, hypertension,
electrolyte changes and gout. Anaphylactoid reactions have occurred following
intravenous administration of preparations containing ciclosporin (Kahan et al.,
1985; Bennett & Norman, 1986; Wei dIe & Vlasses, 1988; Lin et al., 1989; Reynolds,
1989).

ln two of three published reports of babies born to mothers treated throughout
pregnancy with ciclosporin (Lewis et al., 1983; Klintmalm et al., 1984; Endler et al.,
CICLOSPORIN 95
1987), growth was retarded. However, whether this effect was due to the drug or to
the general condition of the mother is uncertain.
A group of 25 kidney transplant patients recived daily oral treatment with
ciclosporin at 12- 14 mglg bw (reduced to 4 mg/g)combined with variable doses of
prednisolone for over one year (Fukuda et al., 1987). ln an extension of this study
(Fukuda et al., 1988), the number of patients was increased to 40. More patients
receiving ciclosporin had chromosomal aberrations in their peripheral lymphocytes
(68% and 48% in the two studies, respectively) than did 50 healthy individuals (0%)
or 50 haemodialysis patients (2%). (The Working Group n.oted the poor reporting
of the studies and that cells were cultured for 72 h.)
Unscheduled DNA synthesis was reported to be elevated in the lymphocytes of
kidney transplant patients treated with ciclosporin (dose and length of treatment
unspecified) in comparison with those from healthy individuals (Petitjean et al.,
1986). (The WorkingGroup noted the incomplete reporting of the study.)
3.3 Case reports and epidemiological studies of carCÎnogenicity to humans
(a) ease reports

Numerous case reports have been published of neoplasms occurring in organ
transplant recipients who received only ciclosporin, without azathioprine or
cytotoxic agents. The majority of these neoplasms were lymphomas, commonlyof
the gastrointestinal tract (Thiru et al., 1981; Beveridge et al., 1984; Bencini et al.,
1985; Bloom et al., 1985; Castro et al., 1985; Thompson et aL., 1985; Walker et al.,
1989), but Kaposi's sarcoma and skin cancers have also been reported (Thompson
et al., 1985; Gorg et al., 1986; Arico et al., 1987; Cockburn, 1987; Bencini et aL., 1988;
Civati et al., 1988). Malignancies at other sites have also been seen (Maung et al.,
1985; Thompson et al., 1985). Regression of lymphomas when the drug was dis-
continued has sometimes been reported (Bencini et al., 1988).
ln the most recent report from a registry of organ transplant recipients who
developed tumours (Penn & Brunson, 1988), 412 tumours had been recorded in
ciclosporin-treated patients. Of these, the most frequently reported were
lymphoma (29%), skin cancer (22%) and Kaposi's sarcoma (11%). (The Working
Group noted that the size of the underlying population was unknown; but, given the
low incidence of Kaposi's sarcoma in the general population, the number of cases in
this registry is strikingly large.)
Cockburn and Krupp (1989) described the occurrence of 186 neoplasms in

organ transplant recipients treated with ciclosporin and reported to the drug
manufacturer. The most frequent malignancies were lymphomas and leukaemias
(55 cases) and Kaposi's sarcoma (26 cases). The lymphomas were found predo-
minantly in the gastrointestinal tract.
(h) eohort studies

Anderson et al. (1978) reported that among 143 cardiac transplant recipients
treated with ciclosporin and other immunosuppressive agents, six developed
lymphomas.
CaIne et al. (1979) followed up 34 organ transplant recipients treated with
cic1osporin, six of whom had also received a cyclophosphamide derivative; three
lymphomas developed-two in patients treated with ciclosporin only and one in a
patient treated with both drugs.
Starzl et al. (1984) reported lymphoproliferative lesions (15 lymphomas, two
other lesions) that ocurred during follow-up in eight of 315 renal transplant, four of
129 heart transplant, three of 48liver transplant and two of six heart-Iung transplant
patients treated, in general, with cic1osporin alone. ln seven renal transplant
patients with these lesions who were operated on for bowel perforation,
ter-
discontinuation of ciclosporin treatment resulted in tumour regression, as de
mined by a second laparotomy.

Bencini et al. (1986) followed 67 renal transplant recIpients treated with
ciclosporin for 1-17 months (mean, 3.2 months); one developed a squamous
epithelioma and one, skin nodules thought to be a lymphoIla.
Sheil et al. (1987) reported three-year results of a trial of ciclosporin in renal
transplant patients. One malignant melanoma and one adenocarcInoma of the
remaining kidney were observed among 140 renal transplant patients receiving
long-term treatment with ciclosporin alone, while no tumour was reported among a
further 140 patients who received treatment wIth ciclosporin alone for three months
followed by treatment with azathioprine.
Smith et al. (1989) reported that lymphomas developed in two of 712 organ
transplant patients who received azathioprine, none of 160 treated with ciclosporin
and seven of 132 who received both.
Cockburn and Krupp (1989) followed up 40 organ transplant recipients
treated with ciclosporin and compared observed with expected numbers based on
population rates. Increased risks were noted for lymphoma (relative risk, 27.5; Il
cases observed), skin cancers (6.8; Il) and urinary-tract cancers (5.9; Il). (The
Working Group noted that it was not clear that the only immunosuppressive
treatment received was ciclosporin.)
Table 1 summarizes the studies in which lymphomas were reported in
transplant patients who had received cIclosporin but not azathioprine or cytotoxic
drugs. The Working Group estimated upper IImits for the expected values (not
CICLOSPORIN 97
Table 1. Non-Hodgk.n's Iymphomas in organ transplant patients treated

with ciclosporin (without azathioprine or cytotoxic drugs)
No. of patients Maxmal follow-up Non-Hodgkn's lymphomas Reference
(yeaTS)
Expteda Obsrved
28 1.S 0.02 2 CaIne et al. (1979)

498 4 1.0 11 Starzl et al. (1984)
67 1.S O.OS 0 Bencini et al.
(1986)
120 Sb 0.3 0 Sheil et al. (1987)
160 S 0.4 0 Smith et al. (1989) ,
873 (total) 1.8 13
aAs estimated by the Working Group

hMean, as given in paper
provided in the original papers), on the basis of assumptions adverse to a causal

relationship, as follows:
(i) When the total period of follow-up was not given, the time of observation
of every patient was equivalent to the maxmal observation time of the
relevant study.
(ii) The incidence rate for any age group below 70 years was the highest
published in the Connecticut Tumor Registry (higher than in any UK or
Australian registry), I.e., 50/100 00 per year (Muir et al., 1987).
(iii) AIl patients followed up received only ciclosporin. ln fact, it is known
that sorne had received other agents, but only patients with lymphomas
who had not received other agents were included in the count of observed
cases.
Even with the above assumptions, the occurrence of lymphomas was
remarkably high.
(The Working Group noted that in many studies no information on dose,
survival or follow-up time was given for any group, and it was difficult to compare
rates. As is clear from estimates of expected numbers made by the Working Group,
however, the incidence of lymphoma in the cohort studies is remarkably high. ln
addition, Kaposi's sarcoma has figured prominently in case reports. It is also
noteworthy that lymphomas regressed following discontinuation of ciclosporin in
two studies. A higher incidence of lymphomas was noted when ciclosporin was
used in combination with other imunosuppressive agents, as was a frequent

practice soon after its introduction (Anderson et al., 1978; CaIne et al., 1979; Kinlen,
1982; Beveridge et aL., 1984). This is consistent with other evidence that the intensity
of immunosuppression has an important influence on lymphoma incidence.)
4.1 Exposure data
Ciclosporin has been used as an immunosuppressive agent since the

mid-1980s.

Ciclosporin was tested for carcinogenicity by oral administration in two
studies in mice and in one study in rats. ln one study in mice, it accelerated the
development of leukaemias; tumours were not induced in a chronic bioassay. ln
rats, negative results were obtained in a study with limited sensitivity.
Ciclosporin enhanced the development of lymphomas induced in two strains
of male mice by single whole-body irradiation or N-methyl-N-nitrosourea. ln
grafted macaques, ciclosporin increased the incidence of lymphomas, a neoplasm
that occurs extremely infrequently in this species of monkeys. When given in
combination with various other immunosuppressive regimens, ciclosporin induced
a substantial increase in the incidence of lymphomas when compared to
immunosuppressive regimens excluding ciclosporin. This drug also enhanced the
incidence of intestinal adenocarcinomas induced in male rats by N-methyl-
N-nitrosourea.
ln case reports, both lymphomas and Kaposi's sarcoma have been associated
frequently with exposure to ciclosporin. Four cohort studies recorded a high
incidence of lymphoma in organ transplant rccipients; in two of these, ciclosporin
was given without azathioprine or cytotoxic drugs. ln several cases, there has been
well-documented regression of lymphoma following withdrawal of the drug.

Ciclosporin induced dose-dependent changes in reproductive organ weights in
male rats and caused sterilty at high doses. Fetal mortality was observed in rats
and rabbits when the drug was administered during the second half of gestation at
maternally toxic doses. No other sign of embryo- or fetotoxicity was noted.
CICLOSPORIN 99
Ciclosporin is rapidly absorbed and widely distributed in humans and in

experimental animaIs. It is extensively metabolized by the cytochrome P450 system.
Adverse effects include nephro- and hepatotoxicity. The compound is
immunosuppressive, resulting in tolerance to tissue grafts; its main effect is on the
early proliferation of T -cells.
ln a single study, ciclosporin was reported to increase the incidence of
chromosomal aberrations in the lymphocytes of kidney transplant patients.
omal
Cic1osporin did not induce dominant lethal mutations in mice, chromos
aberrations in the bone marrow of Chinese hamsters or micronuc1ei in the bone

marrow of Chinese hamsters or mice in vivo. It induced sister chromatid exchange
in hum an peripheral lymphocytes in vitro but did not induce gene mutations in
Chinese hamster cells. Ciclosporin did not induce mutations in Salmonella
tyhimurium. (Se Appendix 1.)
4.5 Evaluation
1
There is suffcient evidence for the carcinogenicity of ciclosporin in humans.

There is limited evidence for the carcinogenicity of ciclosporin in experimental
animaIs.
Overall evaluation
Ciclosporin is carcinogenic to humans (Group 1).
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CICLOSPORIN 103
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Seethalakshmi, L., Diamond, D.A., Malhotra, R.K., Maznitis, S.G., Kumar, S. & Menon,
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3), 1005-1010
Shah, A.K., Sawchuk, R.J., Gratwohl, A., Baldomero, H. & Speck, B. (1988) Subcutaneous
absorption of cyclosporie in rabbits. Tranplan. Proc., 20 (Suppl. 2), 710-714
Sheil, A.G.R., Flavel, S., Disney, AP.S., Mathew, 1:H. & Hall, B.M. (1987) Cancer
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Tranplan. Proc., 19, 2214-2216
Shevach, KM. (1985) The effects of cyclospri A on the immune system. An. Re
Immnol., 3, 397-423
Shigematsu, H. & Koyama, A. (1988) Suppressive effect of cyclospri A on the induction of
chronic serum sickness nephritis in the rat. Acta pathol. jpn., 38, 11-19
Shinozuka, H., Ratton, A., Gil, TJ., III & Kunz, H.W (1988) Experiental models of
malignancies after cyclosprie therapy. Tranplan. Proc., 20,893-899
Shulman, H., Strier, G., Deeg, H.J., Kennedy, M., Storb, R. & Thomas, E.D. (1981)
Nephrotoxicity of cyclospri A after allogeneic marrow transplantation. Glomerular
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Sika, S.C., Koyle, M.A., Swerdloff, R.S. & Rajfer, J. (1988) Reversibility of cyclosprie-
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Smeesters, C., Chaland, P., Giroux, L., Moutquin, J.M., Etienne, P., Douglas, E, Corman, J.,
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Stanley Nahman, N., Cosio, EG., Mahan, J.D., Henry, M.L. & Ferguson, R.M. (1988)
Cyclosporie nephrotoxicity in spntaneously hyprtensive rats. Tranplanation, 45,
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Starklint, H., Dieperik, H., Kemp, E. & Leyssac, P.P. (1988) Ultrastructural study of
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Thir, S., CaIne, R.Y. & Nagington, J. (1981) Lymphoma in renal allograft patients treated
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Thomson, A.W., Whiting, :PH., Blair, J.T, Davidson, R.J.L. & Simpson, J.G. (1981)
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cyclospori A
and their reversaI following drug withdrawaL. Tranplanation, 32, 271-277
CICLOSPORIN 113
Tracey, D.E., Hardee, M.M., Richard, K.A & Paslay, J.W. (1988) Pharmacological inhibition
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Tranplantation, 46 (Suppl.), 118S-121S
114 lARC MONOGRAHS VOLUME 50
White, D.J.G., Plumb, AM., Pawelec G. & Brons, G. (1979) Cyclospri A: an

immunosuppressive agent preferentially active against proliferating T cells.
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Measurement of cyclosprie concentrations in whole bloo: HPLC and
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Woo, A.J., Maurer, G., Niederberger, W. & Beveridge, 1: (1983) Cyclosporie:
phannacokietics, metabolism, and drug interactions. Tranplant. Proc., 15 (Suppl. 1),
240-2412
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intravenous cyclosprie in bone marrow transplant recipients. Tranplanation, 38,
511-513
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cyclosprie. Tranplanation, 42, 61-63
Yuzawa, K., Fukao, K., Iwasaki, Y. & Hamaguchi, H. (1987) Mutagenicity of cyclosprie
against human cells. Tranplan. Proc., 19, 1218-1220
Zwanenburg, 1:S.B., Suter, W. & Matter, B.E. (1988) Absence of genotoxic potential for
cyclosporie in experiental systems. Tranplan. Proc., 20 (Suppl. 2), 435-437
PREDNIMUSTINE
1.1 Synonyms

ehem. Ahstr. Name: Pregna- 1,4-diene-3,20-dione, 21 -( 4-t 4-(bis(2-chloro-
ethyl)amino )phenyl l-l-oxybutoxy)- 1 1, 17 -dihydroxy-(l 1ß)-
Synonym: Prednisolone, 21 -( 4-Wara(bis(2-chloroethyl)-~ß-amino )phenyl ì-
butyrate); 11ß,17,21 -trihydroxypregna- 1,4-diene-3,20-dione-21 -(1 -wara-(bis-
(2-chloroethyl)amino)phenyObutyrate); Leo 1031; NSC 134087
1.2 Structural and molecular rormulae and molecular weight
ïWCO - (CH,), -0 N(CH,æ,cI),

c=o
CH3
C3sI4sCliN06 MoL. wt: 64.66
1.3 Chemical and physical properties or the pure substance

From WIndholz (1983), unless otherwise specified
(a) Description: Crystals from methanol-water
(b) Me/ting-point: 163- 164 0 C
(c) Optica/ rotation: (o:)fi = +92.90 (c = 1.06 in chloroform)
-115-
(d) Solubility Practically insoluble in water; soluble in ethanol, acetone,

chloroform and methanol (Reynolds, 1989)
Trade names: Mostarine; Sterecyt; Stéréoyt
Prednimustine can be produced by the esterification of chlorambucil with

prednisolone (Fex et aL., 1970). It is synthesized in Sweden.
Prednimustine is not known to occur naturally.
2.2 Use
Prednimustine is a cytostatic agent. It has been used in the treatment of

various malignancies, including chronic lymphatic leukaemia and non-Hodgkin's
lymphomas, at dailyoral doses of 140-20 mg for three to five days or continuously
at 20-30 mg per day (Reynolds, 1989; Szanto et al., 1989). It has also been tested for
use in the treatment of breast cancer (Lober et al., 1983; Rankin et al., 1987).
2.3 Analysis
Prednimustine has been quantified in plasma by high-performance liquid

chromatography (Newell et al., 1979). It has also been quantified after hydrolysis to
chlorambucil, by gas chromatography-mass spectrometry (J akhammer et al., 1977)
and high-performance liquid chromatography (Workman et al., 1987).


Rat: Four groups of 30 female Sprague- Dawley rats, 100 days of age, received
prednimustine (purity unspecified) at 12 mg/kg bw in a vehicle consisting of
PREDNIMUSTIE 117
carboxymethylcellulose, 1Ween 80 and glucose in water by gavage once, twice, 4.5 or

nine times per month for 18 months. The last group had significantly reduced
ne times
survivaL. A group of 12 female rats recived the vehicle alone by gavage ni
per mon th for 18 months. A significant increase (p .c 0.01; Peto test: Peto et al.,
1980) in the incidence of squamous-cell carcinomas of the external auditory canal
was observed (controls, 0/30; once per month, 0/30; twice per month, 1/30; 4.5 times
per month, 2/30; nine times per month, 2/30). No increase in the incidence of other
tumours was observed (Berger et al., 1985, 1986).
(b) earcinogenicity of metablites
Chlorambucil has ben evaluated in the lARe Monographs (!AC, 1975, 1981,
1987).

(a) Exrimental sytems
Following a subcutaneous injection of radiolabelled prednimustine at 20
mg/kg bw to female Wistar rats, radioactivity appeared gradually in blood plasma
over 48 h. The levels of chlorambucil and phenylacetic mustard in plasma were
below 5 i.M. Radioactivity levels in all tissues studied were lower than those in
plasma; in the small intestine, activity peaked at 2-4 h after administration. No or
little radioactivity was detected in bone marrow (Newell et al., 1981).
When radiolabelled prednimustine was injected intravenously to baboons, low
urinary and biliary excretion was observed. The radioactivity in blood and kidney
decreased with time, but it was stable in the liver over the observation period of 6 h.
ln muscle, prostate, lung, spleen and seminal vesicles, however, radioactivity levels
rose after 4 and 6 h (Kirdani et al., 1978).
Prednimustine is hydrolysed completely in vitro by rat plasma esterases to
chlorambucil and prednisolone (Wilkinson et al., 1978). A cholesterol ester of
chlorambucil, originating from prednimustine by acyltransferase-catalysed trans-
esterification, was detected when prednimustine was incubated with human, rat or
dog plasma in vitro. The same ester was identified in plasma of dogs after
intravenous injection in vivo (Gunnarsson et al., 1984).
(ii) Toxic effects
ln an acute lethality study, survival of Wistar rats given prednimustine at 128
mglg bw subcutaneously was 70% after 21 days. The drug was less toxic than
chlorambucil and less toxic than chlorambucil and prednisolone given in combi-
nation (Harrap et al., 1977).
ln subacute toxicity experiments, the mortality caused by daily oral
administrations of prednimustine for four weeks to Sprague-Dawley rats was low
compared to that induced by chlorambucil and prednisolone given together.

Mortality in prednimustine-treated animaIs was about 10% at dose levels of 32 and
64 mglg bw. Dose-related lymphopenia was observed, and spleen and adrenal
weights were reduced (Fredholm et al., 1978).
No symptom of toxicity was observed during a life-time carcinogenicity study
with prednimustine given to Sprague-Dawley rats at 12 mglg bwone to nine times
per month for 18 months (Berger et al., 1986).
Prednimustine caused a d~se-dependent dccrease in survival in Chinese
hamster V79-4 cells; it was at least three times as toxic as chlorambucil throughout
the dose range (Hartley-Asp et al., 1986). Dose-dependent cell death was also
observed in the hormone-sensitive S49 mouse lymphoma cell line after incubation
for 24 h with prednimustine at 10- M up to 5 X 10-7 M prednimustine (Harrap et
al., 1977).

No data were available to the Working Group.
(h) Humans
When prednimustine was given orally at doses up to 20 mg, no unchanged
drug could be detected in blood (Newell et al., 1979; Ehrsson et al., 1983; Gaver et al.,
1983; Newell et al., 1983; Oppitz et al., 1989) or In urine (Kirdani et al., 1978). When
prednimustine was given orally at 20 mg, no chlorambucIl or phenylacetic mustard
was detected in the circulation (Newell et al., 1979, 1983); however, when a higher
dose (100 or 20 mg) was given, chlorambucil was detccted in blood (Ehrsson et al.,
1983; Oppitz et al., 1989). Mter a dose of 20 mg, phenylacetic mustard was also
identified in the circulation (Oppitz et al., 1989), and, after an oral dose of 100 mg,
free prednisolone was detected (Sayed et al., 1981). The concentrations of chloram-
bucil and its metabolites and of prednisolone detected in the circulation after an
oral dose of prednimustine were lower than those after equimolar doses of chloram-
bucil and prednisolone given separately (Sayed et al., 1981; Ehrsson et al., 1983;
Oppitz et al., 1989). Mter a single oral dose of prednimustine, the peak values of
chlorambucil and phenylacetic acid mus tard in the serum were reached later and
the disappearance half-time was longer than after administration of chlorambucil
and prednisolone separately (Ehrsson et al., 1983; Oppitz et al., 1989). TInee to six
hours after a single oral dose of 40 mg/m2 radiolabelled prednimustine, 50% of the
plasma radioactivity could be extracted into organic solvents; the extractable
proportion decreased with time and was -c 10% after 12-18 h. The terminal
PREDNIMUSTINE 119
half-time of ten days for prednimustine-derived radioactivity in plasma could be

attributed to these covalently bound metabolites (Gaver et aL., 1983).
When a trace amount of double-Iabelled prednimustine cl4c in the bischloro-
ethyl group, 3H at positions 6 and 7 of the steroid par) was administered intra-
venously, 14c and 3H in the urine cohromatographed partially during the first hour
after the injection but were fully separated thereafter, indicating that intact
prednimustine is excreted in the urine only immediately after injection (Kirdani
et al., 1978).

Leukopenia and thromboytopenia seem to be dose-dependent and may limit
the dose that can be used. Nausea and vomiting are frequent (Könyves et al., 1975;
Lober et al., 1983; Rankin et al., 1987; Szanto et al., 1989).
(iii) Effects on reproductin and prenatal toxicity
No adequate studies were available to the Working Group.

4.1 Exposure data
Prednimustine has been used as a cytostatic drug, mainly for the treatment of
malignancies of lymphatic tissue.
Prednimustine given by oral administration to rats induced a low but
significant increase in the incidence of squamous-cell carcinomas of the external
auditory canaL.
4.3 Human carcinogenicity data

ln humans, prednimustine causes leukopenia and thrombocytopenia; in

experimental animaIs, it causes lymphopenia. It is hydrolysed to chlorambucil and
prednisolone in vivo. (See Appendix 1.)
4.5 Evaluation!
There is inaequate evidence for the carcinogenicity of prednimustine in

prednimustine.
Ove rail evaluation
Prednimustine is not classif/e as to ifs carcinogenicity to humans (Group 3).
5. References
Berger, M.R., Habs, M. & Schmähl, D. (1985) Compative carciogenic activity of

prednimustine, chlorambuci, prednislone and chlorambuci plus prednisne in
Sprague-Dawley rats. Arch. Geschwulstforsch., 55, 429-442
Berger, M.R., Habs, M. & Schmähl, D. (1986) Long-term toxicology effects of
prednimustine in comparison with chlorambucil, prednislone, and chlorambucil plus
prednisolone in Sprague-Dawley rats. Sernn. Oncol., 13 (Suppl. 1),8-13
Ehrsson, H., Wallin, L, Nilsson, S.-O. & Johansson, B. (1983) Pharmacokietics of
chlorambucil in man after administration of the free drug and its prednislone ester
(Pednimustine, Leo 1031). Eur. 1. clin. Ph(Jol., 24, 251-253
Fex, H.J., Hogberg, K.B. & Könyves, I. (1970) Corticosteroid p-bis(2-chloroethyl)-
aminophenylcarbxylates as antitumor agents. Chem. Abstr., 73, 391
Fredholm, B., Gunnarsson, K., Jensen, G. & Müntzing, J. (1978) Mammaiy tumour
inhibition and subacute toxicity in rats of prednimustine and of its molecular
components chlorambucil and prednisolone. Acta phol. toxicol., 42, 159-163
Gaver, R.C., Deeb, G., Pittman, K.A., Issel, B.F., Mittelman, A. & Smyth, R.D. (1983)
Disposition of orally administered 14C-prednimustine. Caner Chemother. Phaol.,
Il, 139-143
Gunnarssn, EO., Johansson, S.-Å. & Svenssn, L. (1984) Cholesterol ester formation by
transesteriication of chlorambucil: a novel pathway in drug Metabolisme Xenobiotica,
14, 569-574
Harrp, K.R., Riches, EG., Gilby, E.D., Sellwoo, S.M., Wilkison, R. & Könyves, 1. (1977)
Studies on the toxicity and antitumour activity of prednimustine, a prednislone ester
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Hartley-As, B., Gunnarsson, :PO. & Liljekvst, J. (1986) Cytotoxicity and metabolism of
prednimustine, chlorambucil and prednislone in a Chinese hamster cellline. Cancer
Chemother. Phaol., 16, 85-90

PREDNIMUSTINE 121
IARC (1975) lAC Monograph on the Evaluation of Carcinogenic Risk of Chemicals to Man,
Vol. 9, Some Azridines, N-, S-, an O-Mustards an Selenium, Lyon, pp. 125-134
IARC (1981) lAC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to
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Overall Evaluations of Carcinogenicity: An Updating o/IARC Monographs Volumes 1 to
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Jakhammer, L, Olsson, A. & Svenson, L. (1977) Mass fragmentographic determination of
prednimustine and chlorambucil in plasma. Acta phar. suee., 14, 485-496
Kidani, R.Y., Murphy, G."P & Sandberg, AA (1978) Sorne metabolic aspects of a nitrogen
mustard of prednisolone. Oncology, 35, 47-53
Könyves, 1., Nordenskjöld, B., Plym Forshell, G., de Schryer, A. & Westerberg-Larsson, H.
(1975) Preliminaiy clinical and absorption studies with prednimustine in patients with
mammaiy carcinoma. Eur. 1 Caner, Il, 841-84
Loeber, J., Mouridsen, H.L, Christiansen, LE., Dombemowsky,"P, Mattsson, W. & Roerth,
M. (1983) A phase III trial comparig prednimustine (LEO 1031) to chlorambucil plus
prednisolone in advanced breast cancer. Cancer, 52, 1570-1576
Newell, D.R., Hart, L.1. & Harrap, K.R. (1979) Estimation of chlorambucil, phenyl acetic
mustard and prednimustine in human plasma by high-pedormance liquid
chromatography.l Chromatogr., 164, 114-119
Newell, D.R., Shepherd, C.R. & Harrp, K.R. (1981) The pharmacokietics of
prednimustine and chlorambucil in the rat. Caner Chemother. Pharacol., 6, 85-91
Newell, D.R., Calvert, A.H., Harrap, K.R. & McElvain, LJ. (1983) Studies on the
pharmacokietics of chlorambucil and prednimustine in man. Br.
1 clin. Pharacol., 15,
253- 258
Oppitz, M.M., Musch, E., Malek, M., Rueb, H.P., von Unruh, G.E. & Los, u. (1989) Studies
on the pharmacokietics of chlorambucil and prednimustine in patients using a new
high-pedormance liquid chromatographic assay. Cancer Chemother. Phaol., 23,
208-212
Peto, R., Pike, M.C., Day, N.E., Gray, R.G., Lee, -lN., Parih, S., Peto, J., Richards, S. &
Wahrendod, J. (1980) Guidelines for simple sensitive significance tests for carcino-
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Assays for Carcinogens: A Cntical Appraisal (lAC Monographs on the Evaluation of the
Carcinogenic Risk o/Chemical to Human, Suppl. 2), Lyon, IARC, pp. 311-426
Ranki, E.M., Harvey, C., Knoght, R.K. & Rubens, R.D. (1987) Phase II tril of
prednimustine as first-line chemotherapy in patients with advanced breast cancer.
Caner Treat. Rep., 71, 1107-1108
Reynolds, J.E.F., ed. (1989) Marindale. The Exra Phacopoia, 29th ed., London, The
Sayed, A., Van Hove, W. & Vermeulen, A (1981) Prednisolone plasma levels after oral
administration of prednimustineCB. Oncology, 38, 351-355
Szanto, 1., Fleischmann, 1: & Eckhardt, S. (1989) Prednimustine treatment in malignant
lymphomas. Oncology, 46, 205-207
Wilkisson, R., Gunnarsson, :PO., Plym-Forshell, G., Renshaw, J. & Harrap, K.R. (1978)
The hydrolysis of prednimustine by enzyes from normal and tumour tissues. ln:
Davi, W. & Harrap, K.R., eds, Advaies in Tumour Prevention, Detection an
ChaacteriZlion, Amsterdam, Excepta Medica, pp. 26-273
Windholz, M., ed. (1983) The Merck Index 10th ed., Rahway, NJ, Merck
& Co., pp.1l09-1110
Workman, :P, Oppitz, M., Donaldson, J. & Lee, EY.E (1987) High-pedormance liquid
chromatography of chlorambucil analogues. 1 Chromaogr., 422, 315-321
THIOTEPA
This substance was considered by previous working groups, in April 1975 and
March 1987, under the title tris(l -azridinyl)phosphine sulphide (!AC, 1975, 1987).
Since that time, new data have beme available, and these have been incorporated
into the monograph and taken into consideration in the present evaluation.
1.1 Synonyms

ehem. Abstr. Name: Azridine, 1,1' 1" -phosphinothioylidynetris
Synnym: NSC-6396; phosphoric tri(ethyleneamide); TESPA; thiophospha-
mide; thiotriethylenephosphoramide; triazridinylphosphine sulfide; N,N' N"-
tri- 1,2-ethanediylphosphorothioic triamide; N,N' Nil -tri- 1,2-ethanediylthio-
phosphoramide; trie ethyleneimino )thiophosphoramide; meta-triethylenethio-
phosphoramide; N,N' N" -triethylenethiophosphoramide; meta-tris( aziridin-1-
yl)phosphine sulfide; triethylenethiophosphorotriamide; tris-(l-azridinyl)-
phosphine sulfide; tris(l-azridinyl)phosphine sulphide; tris-( ethyleneimino)-
thiophosphate; TSPA; WR-45312
H2C - CH2
" /
N
H2~ 1 /CH2
1 N- P-
H2C/ IL
N,I
CH2
s
C6l 12N3PS MoL. wt: 189.23
-123-

From Windholz (1983) and Barnhart (1989), unless otherwse indicated
(a) Description: White, crystallne solid; fine white crystallne flakes from
pentane or ether
(b) Melting-pint: 51.5°C; 52-57°C (Reynolds, 1989)
(c) Solubility 1:8 in water; 19 g/100 ml water at 25 ° C; soluble in ethanol,
diethyl ether, benzene and chloroform
(d) Stability At temperatures above 2-8°C, thiotepa polymerizes and
becomes inactive. The bulk drug is stable (up to two years) at 2-8 ° C, is
unstable in acid and is sensitive to light. Aqueous solutions of 10 mg/ml
are stable for five days at 2-8 ° C. Thiotepa is stable in alkaline solution.
Trade names: Ledertepa, Onco Thiotepa, Tespamin; Thio-TEPA; Tifosyl

Thiotepa is available in vials containing 15 mg thiotepa, 80 mg sodium chloride
and 50 mg sodium bicarbonate; when reconstituted, the pH is 7.6 (Barnhart, 1989).
Thiotepa has been prepared by the addition of trichlorophosphine sulfide to

azridine and triethylamine (Kuh & Seeger, 1954) and by the addition of azridine to
phosphorus oxychloride (Bestian, 1950). Thiotepa is synthesized in J apan.
Thiotepa is not known to occur naturally.
2.2 Use
Thiotepa is a cytostatic agent. It has been used in the treatment of lymphomas

and a variety of solid tumours, such as those of breast and ovary; it has also been
used in cases of urinary bladder malignancies, meningeal carcinomatosis and
various soft-tissue tumours (Wright et al., 1958; Hollster & Coleman, 1980; Hagen
et al., 1987; Reynolds, 1989). Thiotepa is administered intramuscularly, intra-
venously and intrathecally; other parenteral routes (e.g., intratumoral injections)
have also been used. It has been used as instillations in cases of urinary bladder
carcinoma (Hollster & Coleman, 1980). Thiotepa has been used recently at high
doses in combination chemotherapy with cyclophosphamide in patients with
lHIOTEPA 125
refractory malignancies treated with autologous bone transplantation (Henner et

al., 1987; Lazarus et al., 1987; Willams et al., 1987; Ackland et al., 1988; Eder et al.,
1988; Willams et al., 1989).
The initial dosage of thiotepa has generally been 5-40 mg (3-23 mg/m2i at one-
to four-weekly intervals (Wright et al., 1958; Cohen et al., 1986; Hagen et al., 1987);
doses up to 75 mg/m2 have been used in children (Heideman et al., 1989). The
dosage is generally adjusted on the basis of changes in leukocyte counts. High-dose
therapy has involved daily doses in excess of 1100 mg/m2 (Lazarus et aL., 1987).
2.3 Analysis
Thiotepa has been determined in pharmaceutical preparations by

colorimetric titration (US Pharmacopeial Convention, Inc., 1989) and in biological
fluids by chromatography (Egorin et al., 1985; Hagen et al., 1985; McDermott et al.,
1985) and high-performance liquid chromatography (Sano et al., 1988).

The carcinogenicity of antineoplastic drugs, including thiotepa, in animaIs has

been reviewed (Berger, 1986).
(a) 1 ntraperitoneal administration

Mouse: ln a screening assay based on the accelerated induction of lung
tumours in a strain highly susceptible to development of this neoplasm, three
groups of ten male and ten female strain A/e mice, six to eight weeks of age,
received intraperitoneal injections of thiotepa (purity, 95-99%) in 0.1 ml of purified
tricaprylin three times perweek for fourweeks (total doses, 19,47 and 94 mg/kg bw).
A group of 80 males and 80 females recived 24 injections of 0.1 ml of tricaprylin
alone. AlI mice were killed 24 weeks after the first injection. The incidences of lung
tumours in treated mice were 16/20, 10/20 and 11/20 in the groups receiving the high,
mid and low doses, respectively, compared to 28% and 20% in male and female
controls. The numbers of Iung adenomas per mouse were significantly higher in the
high-dose (1.50;p .c 0.001) and mid-dose (0.74;p .c 0.05) groups in comparison to
male (0.24) and female (0.20) controls (Stoner et al., 1973).
Groups of 35 male and 35 female B6C3F1 mice, six weeks of age, received
intraperitoneal injections of thiotepa (purity, 98.0:f 1.0%) at 1.15 or 2.3 mg/kg hw
three times a week for up to 52 weeks and were observed for an additional 34 weeks.
Two groups of 15 males and 15 females were untreated or recived injections of
phosphate-buffered saline vehicle only and served as matched controls. Pooled
troIs were also used, by adding 15 animals of each sex taken froID a
vehicle con
bioassay on another chemicaI. By 43 weeks, al high-dose females had died, and, by

56 weeks, all high-dose males had died. At weeks 8687, 15/35 low-dose males, 17/35
low-dose females, 7/15 vehic1e-control males and 1215 vehicle-cntrol females were
stil alive, at which time the study was terminated. Beause of early deaths,
statistical analyses were based only on time-adjusted incidences of tumours,
eliminating those mice that had died before week 52. The incidences of malignant
lymphoma and lymphocytic leukaemia combined were significantly greater in
high-dose animaIs (32/32 females, 26/28 males; p c: 0.001, Cochrane-Aritage test,
Fisher's exact test) in comparison with vehicle and poled controls (0/14 and 0/29
females; 1/8 and 1/18 males) (National Cancer Institute, 1978). (The Working
Group noted the poor survival among the high-dose animaIs and that the study
design involved controls pooled from different studies.)
Rat: Groups of 35-39 male and 31-35 female Sprague-Dawley rats, aged 35,42
or 58 days, received intraperitoneal injections of thiotepa (purity, 98.0 :f 1.0%) at
0.7, 1.4 or 2.8 mg/kg bw three times a week for up to 52 weeks and were observed for
addi tional periods of time. Two groups of ten males and ten females were untreated
or received injections of buffered saline alone at 2.5 mllkg bw and served as
controls. A lower-dose group was started 69 weeks after the beginning of the
original study, together with two addition al control groups. Pooled vehicle controls
were also used, by adding ten rats of each sex from bioassays on other chemicals.
AlI high-dose males had died by week 19 and all high-dose females by week 21.
Treatment of mid-dose groups was terminated at week 34, and animaIs were
observed until weeks 78-81, at which time all of them had died. AIl other groups
were observed until weeks 82~87. Because of early deaths, statistical analyses were
based only on time-adjusted incidences of tumours, eliminating those rats that had
died before week 52. Malignant lymphomas, lymphocytic leukaemia and granu-
locytic leukaemia were observed in 6/34 low-dose (poled controls, 0/29; p = 0.020)
and 6/16 mid-dose (pooled controls, 0/30; p c: 0.001) males. Uterine adeno-
carcinomas were found in 7/21 mid-dose females (pooled controls, 0/28; p = 0.(01)
and 2/29 low-dose females but not in corresponding lower-dose controls. The
incidence of adenocarcinomas of the mamm.ary gland was significantly increased in
mid-dose females (8/24; pooled controls, 1/28; p = 0.00), but this tumour was also
observed in one lower-dose poled control and in 3/10 lower-dose untreated
controls. The incidences of neuroepitheliomas or nasal carcinomas (three in
low-dose males, two in low-dose females, two in mid-dose females) were not
statistically significantly increased, although they did not occur among
THIOTEPA 127
corresponding controls or among the 388 pooled vehicle controls (National Cancer
Institute, 1978). (The Working Group noted the high mortality among high- and
mid-dose groups, which necessitated the later inclusion of the lower dose-treated
group, and that the study design included controls pooled from different studies.)
(b) 1 ntravenous administration
Rat: A group of 48 male BR46 rats, 100 days of age, received weekly
intravenous injections of thiotepa (purity and vehicle unspecified) at 1 mg/kg hw for
52 weeks. A group of 89 untreated males served as controls. Of the treated animaIs,
30 were stil alive when the first tumour appeared, compared to 65 controls.
Malignant tumours developed in 9/30 treated animaIs (two sarcomas of the
abdominal cavity, one lymphosarcoma, one 'myelosis', one seminoma, one
fibrosarcoma and one haemangioendothelioma of
the salivary gland, one mammary
sarcoma, one phaeohromocytoma) and in 4/65 controls (three mammary
sarcomas, one phaeochromocytoma) (p -c 0.01). Benign tumours occurred in 5/30
treated and 3/65 control animaIs (Schmahl & Osswald, 1970; Schmahl, 1975). (The
Working Group noted the short latency of tumour induction.)
(a) Exerimental systems
One hour after intraperitoneal injection of thiotepa at 9.3 mg/kg bw into
Sprague-Dawley rats, radioactivity was found in plasma (5.4%), peritoneal fluid
(26%), urine (1.9%), kidney (0.7%), liver (3.8%), lung (0.6%) and muscle (25.9%)
(Litterst et al., 1982). ln another study, 5 min after intravenous or intraarterial
injection of labelled thiotepa in Sprague-Dawley rats, slightly higher levels of
radioactivity were found in plasma, heart, kidneys and lungs, compared to other
organs; 94-98% of radioactivity administered intravenously was excreted in urine
within 8.5 h. Most of the urinary radioactivity was associated with unchanged
thiotepa; tris(l-azridinyl)phosphine oxide (tepa) was responsible for about 30% of
the radioactivity (Boone et al., 1962).
ln female mongrel dogs, 75-85% of an intravenous dose of labelled thiotepa
was recvered in the urine; only 0.2-0.3% unchanged thiotepa was found (Mellett
et al., 1962). Following intravenous (at 3 mglg bw) or oral (at 6 mglg qw)
administration of thiotepa to dogs, about 13% of the dose was excreted as tepa. The
plasma level of tepa was about 1.2 Ilglml2 h after intravenous injection of thiotepa.
The authors concluded that 50% of the administered thiotepa was absorbed
(Mellett & Woos (196).
A biexponential decline in thiotepa concentration in plasma was seen during
the first hours after intravenous injection of thiotepa at 5 mglg bw in
Swiss-Webster mice. The half-time was 0.21 min for the first phase and 9.62 min for
the second (Egorin et al., 1984).
Mter an intravenous dose of thiotepa to rhesus monkeys, equilbrium with
plasma levels in lumbar and ventricular cerebrospinal fluid was obtained rapidly.
After intravenous administration, the total body clearance of thiotepa was about 35
ml/min (Strong et al., 1986).
The major urinary metabolite in rats, rabbits and dogs following a single
intravenous injection of 32p-thiotepa was tepa, which is also an alkylating agent.
Most of the radioactivity in mouse urine, however, was rccovered as inorganic
phosphate. ln mice and rats, a small proportion of radioactivity was detected in
most tissues nine days after an intravenous injection of thiotepa; higher levels were
detected in blood of rats (Craig et al., 1959).
Mter addition of thiotepa to sera from patients and healthy individuals, about
10% was bound to protein (Hagen & Nilsen, 1987).
(ii) Toxic effects
The LDso of thiotepa in rats was about 9.5 mg/kg bw by intravenous injection
and about 8.8 mg after intraarterial injection (Boeme et al., 1962). The LDso in mice
was 400 mg/kg bw 24 h after an intraperitoneal injection. The acute lethality after
1 h and 24 h was markedly increased by intraperitoneal injection of 60 mg/kg bw
pentobarbital shortly after the thiotepa injection (Munson et aL., 1974). Pre-
treatment of mice with 40 mg/kg bw SKF525A also enhanced the acute lethality of
thiotepa (Mellett & Woods, 196).
Thiotepa caused a dose-dependent inhibition of the growth of P388 murine
leukaemia cells in culture (Miler et al., 1988).
When rats were given thiotepa at 4 mg/kg bw by intraperitoneal injection on

gestation day 12, teratogenic effects occurred in the offspring (Murphy et al., 1958).
(The Working Group noted that the details given in the paper were insufficient to .
assess the significance of the effect.j
ln an extensive study of the effects of thiotepa in pregnant mice, Tanimura
(1968) demonstrated both dose-related and time-related effects. Prenatal mortality
was most pronounced following intraperitoneal injection of 5- 10 mg/kg bw on days
7.5 and 8.5 of gestation, and fetal growth was suppressed after injection on days
10.5-12.5 of gestation. The lowest single teratogenic dose was shown to be 1.0 mglg
bw; the dose that caused 100% incidence of malformed fetuses was 10.0 mg/kg. The
malformations observed were exencephaly, spina bifida, cleft palate, kinky tail and
digit alterations.
lHIOTEPA 129

Thiotepa was mutagenic to Salmonella tyhimurium TA1535 (Benedict et al.,
1977a) and TA 100 (Pak et al., 1979) but gave contradictory results in TA98 (Bruce &
Heddle, 1979; Pak et al., 1979) in the absence of an exogenous metabolic system.
Rats perfused with thiotepa produced urine that was mutagenic to S. tyhimurium
(Pak et al., 1979). ln the host-mediated assay in mice, thiotepa was mutagenIc to S.
tyhimurium TA1535 (Arni et al., 1977) and G46 (Devi & Reddy, 1980).
Thiotepa induced forward mutations to 8-azaguanine resistance inAspergillus
nidulans (Bignami et al., 1982) and chromosomal aberrations (Kihlman, 1975;
Sturelid & Kihlman, 1975; Popa et al., 1976) and sister chromatid exchange
(Kihlman, 1975) in root meristem cells of Vicia laba. It induced sex-linked recessive
lethal mutations in Drosophüa melanogaster (Lüers & Röhrborn, 1965; Fahmy &
Fahmy, 1970) and dominant lethaI mutations in Aedes aegyti (Rodriguez &
Rodriguez, 1985).
Thiotepa induced unscheduled DNA synthesis in unstimulated human
peripheral lymphocytes (Titenko, 1983). It induced mutations at the hprt locus in
Chinese hamster V79 cells (Paschin & Kozachenko, 1982), and, in a host-mediated
assay with mice and mouse lymphoma L5178Y cells, it induced resistance to
thymidine and methotrexate (Le, 1973).
Thiotepa induced sister chromatid exchange in mouse cells (Andersen, 1983),
a cloned hamster cell line (Banerjee & Benedict, 1979), Chinese hamster celIs
(Chebotarev & Selezneva, 1979; Chebotarev et al., 1980; Selezneva et al., 1982) and
peripheral lymphocytes of rhesus monkeys (Kuzin et al., 1987) and hum
ans
(Littlefield et al., 1979; Mourelatos, 1979; Chebotarev & Listopad, 1980; Listopad &
Chebotarev, 1982; Shcheglova & Chebotarev, 1983a). It induced chromosomal
aberrations in a cloned hamster cell line (Benedict et al., 1977b), in Chinese hamster
CHO cells (Maier & Schmid, 1976; Sturelid, 1976), in peripheral lymphocytes of
rabbits (Bochkov et al., 1982) and in human peripheral lymphocytes in vitro
(Hampcl et al., 196; Bohkov & Kuleshov, 1972; Bohkov et al., 1972; Chebotarev,
1974; Kirichenko, 1974; Kirichenko & Chebotarev, 1976; Yakovenko & Nazarenko,
1977; Bochkov et al., 1979; Wolff & Artynyan, 1979; Yakovenko & Kagramanyan,
1982; Shcheglova & Chebotarev, 1983a). Thiotepa induced morphological
transformation of C3H/10rh cells (Benedict et al., 1977b).
Thiotepa induced DNA cross-links in chick embryos (McCann et al., 1971). It
induced sister chromatid exchange (Shcheglova & Chebotarev, 1983b) and
chromosomal aberrations (Malashenko & Surkova, 1974a,b, 1975; Sram, 1976;
Lenard et al., 1979; Malashenko & Surkova, 1979; Shcheglova & Chebotarev,
1983b) in bone marrow of mice treated in vivo. It induced mIcronuclei in the bone
marrow of rats (Setnikar et al., 1976) and mice (Maier & Schmid, 1976; loan et al.,
1977; Bruce & Heddle, 1979; Lenard et al., 1979) and chromosomaI aberrations in
peripheral lymphocytes of rabbits (Bohkov et al., 1982) and rhesus monkeys (Kuzn
et al., 1987) in vivo. Treatment of pregnant mice with thiotepa led to chromosomal
aberrations in embryonic liver cells (Korogodina et al., 1979; Korogodina &
S'yakste, 1981).
Thiotepa induced dominant lethal mutations (Machemer & Hess, 1971;
Epstein et al., 1972; Setnikar et al., 1976; Sram, 1976; Semenov & Malashenko, 1981)
and chromosomal aberrations in spermatogonia (Malashenko & Beskova, 1988)
and spermatocytes (one dose) (Devi & Reddy, 1980; Meistrich et al., 1982) in mice in
vivo. Treatment of male mice with thiotepa led to chromosomal aberrations in
preimplantation embryos (one dose) (Malashenko et al., 1978a; Semenov &
Malashenko, 1979). Thiotepa also induced sperm abnormalities (Bruce & Heddle,
1979) and heritable translocations (one dose) (Malashenko & Surkova, 1974b;
Semenov & Malashenko, 1977; Malashenko et al., 1978b; Malashenko & Goetz
1981) in mice in vivo. Thiotepa produced liver protein variants in Fi fetuses derived
from treated male mice (one dose) (Paschin & Ambrossieva, 1984).
(h) Humans
(i) Pharmcokinetics
Because of acid instability, absorption of thiotepa after oral administration is
erratic and incomplete (Mellet et al., 1962). After an intravenous bolus injection of
thiotepa at 12 mg/m2, a biexponential disappearance from the plasma was
observed; the second-phase half-time was 73.7 min (Egorin et al., 1985).
Disappearance half-times of 1.3-2.1 h were reported in further studies (McDermott
et al., 1985; Cohen et al., 1986; Hagen et al., 1987; Henner et al., 1987; Hagen et al.,
1988; Heideman et al., 1989) after intravenous or intramuscular administration. At
dose levels in excess of 25 mg/m2 (Heideman et al., 1989), 180 mg/m2 (Henner et al.,
1987) and 4.8 mg/kg (Ackland et al., 1988), the plasma clearance of thiotepa was
reported to decline with increasing dose. However, in one study with high doses
(45- 1215 mg/m2), no dose-dependence of kinetics was reported (Lazarus et aL.,
1987). The volume of distribution of thiotepa has been rePOrted to be approximately
501 (Cohen et al., 1986; Henner et al., 1987; Hagen et al., 1988; Heidemann et al.,
1989).
After an intravenous injection of thiotepa in paediatric patients, the
cerebrospinal fluid:plasma ratio of thiotepa was 0.92 (Heideman et al., 1989). After
intraventricular administration of thiotepa, the ratio of thiotepa concentrations in
cerebral ventricular fluid:plasma was almost 100 (Strong et al., 1986); in another,
similar study, it was approximately 20 (Grochow et al., 1982). The urinary excretion
of unchanged thiotepa is complete usually within 8 h of the injection, and less than
1.5% of the dose is excreted in the urine unchanged (Egorin et al., 1985; Hagen et aL.,
1985; Cohen et al., 1986; Hagen et al., 1987). Five minutes after an intravenous
lHIOTEPA 131
injection of thiotepa, tepa was observed in the blood; after 12 min, the
concentration of tepa in the bloo was higher than that of thiotepa. The proportion
of thiotepa in urine was 1.5%, and that of tepa was 4.2%; other alkylating
metabolites represented another 23.5% of the dose administered (Cohen et al.,
1986).

The toxic effect of thiotepa that limits the dose that can be given is
myelosuppression, characterized by granulocopenia and thrombocytopenia;
disturbances in hepatic and renal function, neurotoxicity, nausea and vomiting
were uncommon at dose levels of approximately 75 mglm2 or less (Wright et al.,
1958; Heideman et al., 1989). ln high-dose therapy with autologous bone-marrow
transplantantion, central nervous system disturbances, hepatic damage, infections,
nausea, vomi ng, diarrhoea, mucositis, skin rashes, haemorrhagic cystitis and
ti
cardiomyopathy may be severe (Lazrus et al., 1987; Willams et al., 1987, 1989).
Severe myelosuppression has also been described after intravesicular instilations
of thiotepa (Bruce & Edgcomb, 1967; Watkins et al., 1967; Hollster & Coleman,
1980).

Use of thiotepa in the third tri
mes ter of pregnancy had no adverse effect on the
progeny (Nicholson, 1968; Sweet & Kinzie, 1976). ln a report of the effects of
treatment of women with stage-II and stage-III Hodgkin's disease with
radiotherapy and chemotherapy with TVP (thiotepa, vinblastine, vincristine,
procarbazne and prednisone), menstrual function ceased in two of four women
aged 35-44 years but continued in all 30 women under 35 years of age. Ten of the
women had a total of 12 babies, all with normal development (Lacher & Toner,
1986).
As reported in an abstract, transient azoospermia occurred in a man treated
with thiotepa; the effect was reversed when the dose interval was increased from
monthly to three-monthly dosing (Bayar et al. 1978).
Five patients who received a total dose of thiotepa at 40-100 mg had 9.5 :l
1.07% aberrant cells in peripheral lymphocytes 24 h after the last treatment,
compared with 1.4 :l 0.1% in a control group (Selezneva & Korman, 1973).

Many case reports have been made of cancer occurring following treatment
with thiotepa (!AC, 1975; Nakanuma et al., 1976; Anon., 1977; Hollster &
Coleman, 1980; Sheibani et al., 1980; Easton & Poon, 1983; Silberberg & Zarrabi,
1987). AlI report the ocurrence of nonlymphocic leukaemia, and usually thiotepa
was the only chemotherapeutic agent administer~d.
No increased risk of secnd malignancies was found among 470 patients with
colorectal cancer randomized to low-dose (four doses of 0.2 mglg bw) adjuvant
therapy with thiotepa, followed for 3102 person-years (30 secnd noncolorectal
malignancies observed, 31.4 expted; Boice et al., 1980). No increased risk of
second malignancies was found among 90 patients with breast cancer randomized
to adjuvant therapy with thiotepa for one year (at 0.8 mglg bw in divided doses
followed by 0.2 mglg bw weekly maintenance); afr an average follow-up of
approximately five years, five nonskin, nonbreast cancers had ocurred in 5819
person-years among 90 treated subjects compared with six in 4746 person-years
among the 77 nonexposed patients (Kadinal & Donegan, 1980). (Te Working
Group considered these two studies to be too small to provide useful information.)
Kaldor et al. (199) compared 114 cases of leukaemia that developed in
patients previously diagnosed with ovarian cancer, with 342 controls with ovarian
cancer who had survived as long as the cases and who were matched by age and year
of diagnosis of ovarian cancer. Chemotherapy (without radiotherapy) was
associated with a relative risk of 12 (95% confidence interval, 4.4-32) compared to
treatment by surgery only. For nine cases and Il controls, the only chemotherapy
was thiotepa; 21 cases and 187 controls had had no chemotherapy. The matched
relative risks were 8.3 and 9.7 in a lower- and a higher-dose group, and these were
significantly different from 1.0 (p .: 0.01). ln the samestudy, four other alkylating
agents known to be carcinogenic (melphalan, chlorambucil, cyclophosphamide and
treosulphan; sec IAC, 1987) were independently associated with significantly
increased risks for leukaemia.
4.1 Exposure data
Thiotepa is a cytostatic agent that has been used in the treatment of malignant
lymphomas and solid tumours, in a wide range of doses.

Thiotepa was tested for carcinogenicity by intraperitoneal administration in
mice and rats and by intravenous administration in male rats. ln mi ce, it induced an
increased incidence of lung tumours and lymphoproliferative malignancies in mice
of each sexe ln rats, intraperitoneal administration induced an increased incidence
of lymphoproliferative malignancies in males and of uterine adenocarcinomas and
THIOTEPA 133
mammary carcinomas in females. Intravenous administration to male rats induced

tumours at a variety of sites.
Several cases of leukaemia following treatment with thiotepa alone have been
reported. One case-control study has shown a strong association between risk for
leukaemia and treatment with thiotepa.
ln one study, there was no evidence that thiotepa therapy adversely affected
subsequent fertility in women. Thiotepa is embryotoxic to mice and rats, and
embryo- and fetolethality and gross structural abnormalities were induced during
organogenesis after single intraperitoneal injections.
Thiotepa is converted to alkylating metabolites in vivo. It suppresses the bone
marrow in humans.
ln one study, increased frequencies of chromosomal aberrations were
observed in peripheral lymphocytes of patients receiving thiotepa.
Thiotepa induced chromosomal aberrations in germ cells, sperm
abnormalities and dominant lethal mutation in mice in vivo. It induced micronuc1ei
in the bone marrow of .rats and mice, chromos omal aberrations in bone-marrow
cells and liver cells of mice and in peripheral lymphocytes of rabbits and rhesus
monkeys and sister chromatid exchange in bone-marrow cells of mice in vivo.
Thiotepa induced DNA damage in chick embryos. It induced chromosomal
aberrations in cloned hamster cells, in Chinese hamster cells and in human cells,
sister chromatid exchange in human, mouse, Chinese hamster and rabbit cells, gene
mutations in Chinese hamster cells and unscheduled DNA synthesis in human
peripheral lymphocytes in vitro. It induced cell transformation in mouse cells.
Thiotepa induced sex-linked recessive lethal mutations in Drosophila and sister
chromatid exchange and chromosomal aberrations in Vicia faba. It induced gene
mutations in Aspergillus nidulans and Salmonella tyhimurium. (See Appendix 1.)
4.5 Evaluationl
There is suffcient evidence for the carcinogenicity of thiotepa in humans.

There is suffcient evidence for the carcinogenicity of thiotepa in experimental
animaIs.

Overall evaluation
Thiotepa is carcinogenic to humans (Group 1).
s. References
Ackland, S.E, Choi, K.E., Ratain, M.J., Egori, M.J., Wiliams, S.E, Sinkule, J.A. & Bitran,
J.D. (1988) Human plasma pharmacokietics of thiotepa followig administration of
high-dose thiotepa and cyclophosphamide.l clin. Onco/.,6, 1192-1196
Andersen, O. (1983) Effects of col combustion products and metal compounds on sister
chromatid exchange (SCE) in a macrophagelike cell line. Environ. Bealth Perspect., 47,
239-253
Anon. (1977) Case records of the Massachusetts General HospitaL. Case 28-1977. New
Engl.l Med., 297, 102-106
Ami, E, Mantel, 1:, Deparade, E. & Müller, D. (1977) Intrasanguine host-mediated assay
with Salmonella typhimurium. Mutat. Res., 45, 291-307
Banerjee, A. & Benedict, WE (1979) Production of sister chromatid exchanges by various
cancer chemotherapeutic agents. Cancer Res., 39, 797-799
Bamhart, E. (1989) Physician's Desk Reference, 43rd ed., Oradell, NJ, Medical Economies, p.
1152
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THIOTEPA 135
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THIOTEPA 137
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lHIOTEPA 139
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TRIeHLORMETHINE (TRIMUSTINE HYROeHLORIDE)
This substance was considered by a previous Working Group, in April 1975,

under the title trichlorotriethylamine hydrochloride (IARC, 1975). Since that time,
new data have beome available, and these have been incorporated into the
monograph and taken into consideration in the present evaluation.
1.1 Synonyms

ehem. Abstr. Name: Ethanamine-2-Chloro-N,N-bis(2-chloroethyl) hydro-
chloride
Synonym: HN31; HN3 hydrochloride NSC-30211; R-47; SK-100; tri(ß-
chloroethyl)amine hydrochloride; trichlorotriethylamine hydrochloride;
2,2' ,2" -trichlorotriethylamine hydrochloride; trimustine1; tris(2-chloroethyI)-
amine hydrochloride; tris(ß-chloroethyl)amine hydrochloride; tris(2-chloro-
ethyl)amine monohydrochloride; tris(ß-chloroethyl)amine monohydrochlo-
ride; tris-N-Iost; TS- 160
C6H 12CI3N.HCI
"
/CH2-CH2CI
CICH2- CH2- N . HCI
CH2-CH2CI
MoL. wt: 241.0
lThis name is also us for the free bas, trichlorotriethylamine.
-143-
1.3 Chemical and physical properties or the pure substance

From Reynolds (1982) and WIndholz (1983)
(a) Description: Crystals
(b) Melting-point: 130-131°C
(c) Solubility Very soluble in water; soluble in ethanol
(d) Stability Aqueous solutions deteriorate rapidly.
Trade names: Lekamin; Sinalost; Trilekamin; Trimitan

Trichlormethine can be prepared by treating triethanolamine with thionyl
chloride (Ward, 1935). No current manufacturer is known.
Trichlormethine is not known to occur naturally.
2.2 Use and therapy
Trichlormethine is a cytostatic agent. It was first used in the treatment of
Hodgkin's disease and leukaemias in 1946 (Gooman et al., 1946) and subsequently
for other neoplastic diseases (Bratzel et al., 1963; Bundesverband der phar-
mazeutischen Industrie, 1969). It was recognized in the 1977 and 1982 editions of
Martindale. The Exra Pharmcopoeia, but not in the 1989 edition (Wade, 1977;
Reynolds, 1982, 1989).
2.3 Analysis
A colorimetric method in which 4-(4' -nitrobenzyl)pyridine is used as the

analytical reagent has been used to analyse for various alkylating agents.
Trichlormethine may also be determined by thin-Iayer chromatography (Epstein et
aL., 1955; Petering & Van Giessen, 1963; Sawicki & Sawicki, 1969).

The Working Group was aware of a short letter (Griffin et aL., 1950) in which
experiments were described with mice and rats injected subcutaneously with
trichlormethine.
TRICHLORMElHINE 145
Subcutaneous administration
Mouse: A group of 20 mice (age, strain and sex unspeified) received weekly
subcutaneous injections of trichlormethine (purity unspeified) at 1 mglg bw in
aqueous solution for ten weeks, afer which time only four mice were alive and
treatment was terminated. At surval times of 548-567 days, one of the four mice
had a lung adenoma, one had a Iung carcinoma and one had a lung carcinoma and a
spindle-cll sarcoma at the site of injection. ln a control group of 40 untreated mIce
killed between 14 and 18 months of age, six animaIs had lung adenomas, two had
hepatomas and three had enlarged lymph nodes (Boyland & Horning, 1949). (The
Working Group noted the very small number of survving animaIs.)
Rat: Groups of ten male and ten female random-bred SPF Wistar rats, two
months of age, recived daily subcutaeous injections of trichlormethine (purity
unspeified) at 0.1 or 0.25 mglg bw or weekly subcutaneous injections of 1 mglg
bw in water for six months and were observed for one year after termination of
treatment. Total doses were approximately 16.5, 40-42 and 24 mglg bw in the three
treated groups, respectively. A control group of ten male and ten female rats
received injections of 0.3 ml water only for six months. Survival was decreased in
males reciving daily injections of trichlormethine. The incidences of sarcomas
(mostly spindle-cell type) at the injection site were: males - low daily, 7/10 ip -c
0.0015); high daily, 8/10 fp = 0.00); weekly, 5/10 fp = 0.016); females - low daily,
7/10 fp ~ 0.(015); high daily, 7/9 fp = 0.007); weekly, 4/10 ip = 0.04). ln the group
receiving 0.25 mglg bw daily, three males and one female had a mucus-secreting
intestinal adenocarcinoma. Tumours were not seen in controls (Sýkora et al., 1981).
(a) Exrimental sytems

(i) Absorption, distriution, exretion and metabolism
(ii) Toxic effects
The LDs() for mice, rats, rabbits and dogs after dermal application of
trichlormethine were 7, 4.9, 19 and 1 mglg bw, respectively. After subcutaneous
injections in saline, the LDso for mice was 2.0 mglg bw. The LDsos after
intravenous injections were 0.7 mglg for rats and 2.5 mg/kg bw for rabbits
(Anslow et al., 1947).
Trichlormethine caused vomiting, anorexia and blood-containing faeces in
dogs a few hours after a single intravenous injection of 1 mg/kg bw. Coma preceded
death caused by anoxia as a consequence of peripheral circulatory failure (Houck
et al., 1947).
Decreased peripheral lymphocyte counts were observed in rabbits injected

intravenously (Friederici, 1955) and in mice in jected subcutaneously (Boyland &
Horning, 1949) with trichlormethine.
This compound caused cross-links in membrane proteins and haemoglobin In
human eryhrocytes in vitro (Wildenauer & Weger, 1979; Ankel et al., 1986); it
alkylated nucleic acids in vitro (Szinicz et al., 1981).
Trichlormethine inhibited DNA synthesis and induced mutations at the hprt
locus of Chinese hamster V79 cens (Slamenova et al., 1983). It induced
chromosomal aberrations in transplanted Walker rat carcinoma cens (Boyland et
al., 1948) and transplanted Ehrlich and Krebs tumour cells (Koller, 1969) following
intraperitoneal injection into animaIs carrying these cens. (The Working Group
noted that these early papers on transplanted tumour cells did not permit detailed
evaluation.) A single intraperitoneal treatment with trichlormethine at 5 mg/kg
induced dominant lethal mutations in mice (Sýkora & Gandalovicova, 1978).
(h) Humans
(i) Pharmcokinetics
Lymphopenia, granulocytopenia, thrombocytopenia, anaemia, nausea and
vomiting and thrombophlebitis in the vein receiving the infusion were reported
after use of trichlormethine (Goodman et al., 1946).

4.1 Exposure data
'fichlormethine is a cyostatic agent that has been used since 1946 for the
treatment of leukaemia and lymphoma.
TRICHLORMETHINE 147

Trichlormethine was tested for carcinogenicity by subcutaneous injection in
mice and rats. The study in mice was inadequate for evaluation. ln rats,
trichlormethine induced a high incidence of sarcomas (mostly spindle-cell type) in
animaIs of each sex at the site of subcutaneous injection, as well as a few intestinal
adenocarcinomas; neither tumour type was seen in controls.
ln single studies, trichlormethine induced dominant lethal mutations in mice
and gene mutations in Chinese hamster cells. (Sec Appendix 1.)
4.5 Evaluation 1
There is suffcient evidence for the carcinogenicity of trichlormethine in

trichlormethine.
Overall evaluation
Trichlormethine is lXssibly carcinogenic to humans (Group 2B).
5. References
Ankel, E.G., Ring, B.J., Lai, C.-S. & Holcenberg, J.S. (1986) The lack of effects of alkylating
agents on mammalian cell membranes. /n. 1 TIssue React., 8, 347-354
Anslow, WP., Kamovsky, D.A, Jager, B.\' & Smith, H.W (1947) The toxicity and
pharmacological action of the nitrogen mustards and certin related compounds. 1
Phaol. ex. Ther., 91, 224-235
Boyland, E. & Homing, E.S. (1949) The induction of tumours with nitrogen mustards. Br. L
~ane~ 3, 118-123
Boyland, E., Clegg, J.W, Koller, P.C., Rhoden, E. & Warwck, O.H. (1948) The effects of
chloroethylamines on tumours, with specl reference to bronchogenic carcinomas. Br.
1 ~aner, 2, 17-29
lFor desription of the italicize terms, se Preamble, pp. 2629.

Bratzel, R.P., Ross, R.B., Gooridge, LH., Huntress, ~L, Flather, M.L & Johnson, D.E.
(1963) Survey of nitrogen mustards. Caner Chemothe Rep., 26, 1-322
Bundesverband der pharmazeutishen Industrie (1969) Rote Lie (Red Lit), Frankfurt
Epstein, J., Rosenthal, R.W. & Ess, R.J. (1955) Use of -y-(4-nitrobnzl)pyrdine as analytical
reagent for ethyleneimines and alkylating agents. An. Chem.,27, 1435-1439
Friederici, L. (1955) Der Einuss von Sulfonamiden, Stickstoff-Lot, TEM und
Aminopteri auf das Blut und die blutbildenden Organe des Kaninchens. (Te
inuence of sulfonamides, nitrogen mustard, triethanomelamine and aminopterie on
the bloo and haematopoietic tissues of raits (Ger.).) Folia hamaol., 73,49-74
Gooman, L.S., Wintrobe, M.M., Dameshek, W., Gooman, MJ., Gilman, M.A &
McLennan, M.L (1946) Nitrogen mustard therapy. Use of methylbis(beta-
chloroethyl)amine hydrochloride and trita-chloroethyl)amine hydrochloride for
Hodgki's disease, lymphosarcoma, leukemia and cert alled and misllaneous
disrders.l Am. med. ksoc., 132, 126132
Grifin, A.C., Brandt, E.L. & Thtum, E.L. (1950) Nitrogen mustards and cancer-inducing
agents. L Am. med. ksoc., 144, 571
Houck, C.R., Crawford, B., Bannon, J.H. & Smith, H. ~ (1947) Studies on the mechanism of
death in dogs after systemic intoxication by the intravenous injection of
methylbis(ß-chloroethyl)amine or tris(ß-chloroethyl)amine. L Phaol. ex. Ther.,
90, 277-292
IARC (1975) lAC Monograph on the Evaluation of the Carcinogenic Rik of Chemical to
Man, Vol 9, Some Azridines, N-, S- an O-Musards an Selenium, Lyon, pp. 229-23
Koller, P.C. (1969) Mutagenic alkylating agents as growth inhibitors and careinogens. Mutai.
Re., 8, 199-20
Peterig, H.G. & Van Giessen, G.L. (1963) Colorietrie method for determination of uraci
mustard and related alkylating agents. L phar. Sei., 52, 1159-1162
Reynolds, J .E.F., 00. (1982) Marindale. Th Exra Phaopoia, 28th ed., London, The
Reynölds, J .E.F., 00. (1989) Marindale. Th Exra Phaopoia, 29th ed., London, The
Sawicki E. & Sawicki, C.R. (1969) Analysis of alkylating agents: application to air pollution.
An. NY. Acad. Sei., 163, 895-921
Slamenova, D., Dusinska, M., Budayova, E. & Gabelova, A (1983) The genetic effects of the
cyostatie drug TSl60 on Chinese hamster fibroblasts in vitro. Mutat. Re., 116,431-44
Sýkora, 1. & Gandalovicova, D. (1978) Dominant-Iethal asy of selected cyostatics.
Neoplas, 25, 523-533
Sykora, 1., Yortel, ~, Marhan, O. & Dyterová, A (1981) Carciogenicity of
trichloromethine hydrochloride (fS-I60 Spofa) and morphological damage after its
intraaniotic injection. Neoplas, 28, 565-574
Szinicz, L., Albrecht, GJ. & Weger, N. (1981) Effec ofvarious compounds on the reaction
of tri(2-chloroethyl)amine with nbonucleie acid in vitro and on its toxicity in mice.
ArTnttel.-forsch. (Drg Re.), 31, 1713-1717
TRICHLORMETHINE 149
Wade, A., ed. (1977) Marindale. Th Exra Phaopoia, 27th ed., London, The
Ward, K. (1935) The chloriated ethylamines-a new tye of vesicant. 1 Am. chem. Soc., 57,
914-916
Wildenauer, D. & Weger, N. (1979) Reactions of the triunctional nitrogen mustard
tris(2-chloroethyl)amine (HN3) with human eryhroce membranes in vitro. Biochem.
Pharacol.,28,2761-2769
Windholz, M., ed. (1983) The Merck Index 10th ed., Rahway, NJ, Merck & Co., p. 1379
Witten, B., Magaha, E.P. & Wiliams, W.A. (196) Chemical wadare. ln: Kik, R.E. &
Othmer, D.F., eds, Enclopedia of Chemical Teclmlogy, 2nd ed., VoL. 4, New York,
John Wiley & Sons, pp. 871-875
ANIMICROBIA AGENTS
AMPielLLIN
1. Chemical and Physical Data
1.1 Synonyms
ehem. Abstr. Services Reg. No.: 69-53-4; 7177-48-2

(tri
hydrate); 69-52-3 (sodium
salt)
ehem. Abstr. Name: 4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic acid,
6-((aIninophenylacetyl)amino)-3,3-dimethyl-7-oxo-, t25-(20!, 5~, 6ß(S*))l-
Synnym: Anhydrous: (2S,5R,6R )--( (R )-2-Amino-2-phenylacetamido )-3,3-
dimethyl-7-oxo-4-thia-1-azabicyclo(3.2.0)heptane-2-carboxylic acid; ampicil-
linum; ampicilinum anhydricum; anhydrous ampicilln; (6R)-6-(0!-D-phe-
nylglycylamino)penicilanic acid. Trihydrate: aminobenzylpenicilin (3H20);
~-aminobenzyl-penicilin (3HiO); ampicillnum trihydricum; (sodium salt)
ampicillnnatrium; ampicilinum natricum
H H
CH3
~ ~ tCONH CH3
NH2 N ..
o ..
COGH
Cl~9N304S MoL. wt: 349.40; 403.46 (3HiO);

371.4 (sodium salt)

From Ivashkiv (1973), unless otherwise specified
(a) Description: White, crystallne powder; practicaIly odourless; also occurs
as trihydrate; pH of 10 glml aqueous solution, 3.5-6.0
-153-
(b) Me/ting-point: Ampicilin monohydrate melts with decomPOsition at

202°C; sodium ampicilin melts with decomposition at 205°C; sesqui-
hydrate and anhydrous ampicilin decompose at 199-202°C. The melting
range for ampicilin trihydrate with decomposition has been reported as
214.5-215°C and 202-20 cC.
(c) Optica/ rotation: Ampici//in monohydrate, (alfl +281° (c = 1 in HiO);
ampicilln sesquihydrate, (a) ~ + 283.1 ° (c in HiO); sodium ampicillin
(a)~ +20° (c = 0.2 in HiO); anhydrous ampicilin (a)~ +287.90
(c = 1 in HiO)
(d) So/ubi/ity The solubilties of anhydrous ampicilln, ampicilin trihydrate
and sodium ampicilin in various solvents are given in detail by Ivashkiv
(1973).
(e) Spectroscopy data: Ultraviolet, infrared, nuclear magnetic resonance and
mass spectra have been reported.
if Stabi/ity Ampicilin powders are stable when stored in a closed system at
43% and 81% relative humidity at room temperature for six weeks.
Ampicilin is also stable at 35 ° C in such closed systems for nine weeks.
Stability decreases significantly in the presence of sugars (Reynolds,
1989).
(g) Dissociation constant: pKa = 2.5, 7.3 (23 ° C)
Trade names: A-Cilin; Adobacilln; Aletmicina; Alpen; Alpen-N; Amblosin;

Amcil; Amcil-S; Am fi pen; Ampen; Amperil; Ampibel; Ampi-Biopharma;
Ampibiotic; Ampibronc Capsules; Ampicil; Ampicilat; Ampicillne; Ampiciman;
Ampicin; Ampicur; Ampifen; Ampi-Framan; Ampigal; Ampikel; Ampilag;
Ampilan; Ampiland; Ampilar; Ampilean; Ampilsa; Ampilwç; Ampinebiot;
Ampinova; Ampinoxi; Ampi-Oral; Ampiorus; Ampipenix; Ampi-Rol; Ampisint;
Ampi-Tablinen; Ampitex; Ampivax Ampi-Vial; Ampixlion; Ampi-Zoja;
Amplibios; Amplicid; Amplimedix; Amplipen; Amplipenyl; Ampliscocil;
Amplital; Amplizer; Anhypen; Anidropen; Antibiopen; Anticyl; Apo-Ampi;
Argoci na; Austrapen; Bemicina; Benusel Oral; Binotal; Bio-ampi; Biocellna;
ll
Bionacilin; Biosan; Bonapicilln; Bristin; Britapen; Britcin; CileraI; Cimexillin;

Citicil; Cuxacilin; Cymbi; D-Amp; Deripen; Diancina; Doktacilin; Domicillin;
DuraApicilln; Espectrosira; Espimin-Cilna; Eurocilin; Famicilin; Farmampil;
Fidesbiotic; Fortapen; Fuerpen; Germicilina; Geycilina; Globipen Balsamico;
Gobemcina; Gramcilina; Grampenil; Guicitrina; Helvecilln; Hostes; Iwacillin;
Lampocilina; Lifeampil; Marisilan; Maxcilna; Medicilln-D; Morepen; Napicil;
NC Ciln; Negmapen; Novoexpectro; Nuvapen; Omnipen; Omnipen-N; Overcilina;
tes; Pen-A; Pen Ampil; Penampil; Pen NN; Penberin; Pen-Bristol;
Panbiotic; Panes
AMPICILUN 155
Penbristol; Penbritin; Penbritine; Penbrock; Pénicline; Penimaster; Penimic;

Penimul; Peninovel; Penisint B.G.; Penisintex; Penorsin; Penrite; Pensyn; Pentrex;
Pentrexil; Pentrexyl; Pentrexyl-K; Petercilin; Pharcilln; Platocilina; Plumericin;
Poenbiotico; Polycilin; Prestacilna; Principen; Principen/N; Quimetam;
Racenacilln; Radiocilina; Resan; Rivocilln; Rosampline; Roscilin; Saicil;
Semicilin; Sernabiotic; Servciline; Sesquicilina; Sintopenyl; SK-Ampicilin;
SK-Ampicilin-N; Spectracil; Sumipanto; Supen; Suractin; Synpenin; Synthecilln;
Tauglicolcinna; Togram; Tokiocilin; Tolimal; Totaciclina; Totacilin; Totacilln N;
Totalciclina; Totapen; Trafarbiot; Trifacilna; Ukapen; Ultrabion; Urebion
Ampicilina; Valmingina; Viacilna-A; Vidopen
The following names have been used for multi-ingredient preparations
hydrate: Ampicin- PRB;
containing ampicilin, ampicilin salts and ampicilin tri
Ampiclox; Ampicyn; Flu-Amp; Magnapen; Nuvapen Reard; Orbecilna; Penbritin

KS; Pentrex-F; Polycilin-PRB; Principen with Probenecid; Pro-Biosan; Unasyn
tains 90-1050 lLg/mg ampicilin (calculated as
USP anhydrous ampicilln con
the anhydrous base), and the tri

tains 845-988 lLg/mg. Ampicilln is
hydrate con
available in 125-, 20-, 250- and 5OO-mg tablets that contain 90-120% labelled active
ingredient, in 125-, 250- and 5OO-mg capsules containing 90-120% labelled active
ingredient, and as oral suspensions of 100, 125 and 250 mg/5 ml containing 90-120%
of the labelled active ingredient and probenecid. The sodium salt of ampicilln is
available for injection in vials of 0.125, 0.25, 0.5, 1, 2 and 10 g.
Impurities of ampicilln that occur during preparation of the product are
D-( - )-a-phenylglycine and 6-aminopenicilanic acid. It has been reported that
sodium ampicilln in aqueous solution undergoes a reaction to form oligomeric
products (Van der Bijl et al., 1988).
Ampicilin is produced by the acylation of 6-aminopenicilanic acid with

D-( - )-a-phenylglycine by either microbiological or chemical synthesis (Ivashkiv,
1973). It was first marketed in 1961 in the UK. It is synthesized in Austria, Brazil,
Hungary, India, Italy, Japan, the Republic of Korea, Mexico, the NetherIands,
Romania, Spain, Sweden, Turkey, the USA and Yugoslavia (Chemical Information
Servces, 1989-90).
ln Sweden, ampicilln sales in 1988 were 0.05 defined daily doses per 100
inhabitants (Apoteksbolaget, 1988, 1989). ln 1988, over six milion new pres-
criptions of ampicilin were issued in the USA (La Piana Simonsen, 1989).
Ampicilin is not known to ocur naturally.
2.2 Use
Ampicilin is bactericidal and has a simIlar mode of action to that of
benzylpenicilin, although it has a broader spetrum of activity, covering several
additional gram-positive and gram-negative organisms. Ampicilin may have a
synergistic action with aminoglycosides and with the ß-Iactamase inhibitors
clavulanic acid and sulbactam (Foulds, 1986; Barnhar 1989).
The clinical indications for ampicilin cover a varety of infections, including
those of the respiratory and urinary tracts, gonorrhoea, meningitis, septicaemia and
enteric infections.
Exressed in various formulations as ampicilln equivalents, the usual oral
dosing is 0.25-1 g every 6 h. The disposition of ampicilln is altered in pregnancy,
and therefore higher doses may be required for severe infections in pregnancy
(Assael et al., 1979). Children may be given half the adult dose. The usual doses of
ampicilin given by injection are 500 mg every 4 or 6 h intramuscularly (painful), by
slow (5 min) intravenous injection or by intravenous infusion. Intrapleural,
intraperitoneal and intrathecal injections of ampicilin are used occasionally
(Reynolds, 1989).
2.3 Analysis
Ampicilln can be analysed in pharmaceutical preparations by micro-

biological, iodometric, colorimetric, high-performance liquid chromatographic
(US Foo and Drug Administration, 1988) and fluorometric assays (Barbhaiya &
Turner, 1976) and by gas chromatography-mass spetrometry (Wu et al., 1977).
Ampicilin can be analysed in biological fluids by high-performance liquid
chromatography (Miyazaki et al., 1983; Haginaka & Wakai, 1987; Abuirjeie &
Abdel-Hamid,1988).
3. Biological Data Relevant to the Evaluation or

Oral administration
Mouse: Groups of 50 male and 50 female B6C3F1 mice, seven to eight weeks of
age, were administered ampicilin tri hydrate (purity, 97%) by gavage at 0, 1500 or
AMPICILUN 157
300 mglg bw in corn oil on five days per week for 103 weeks. The animaIs were
maintained for a further one to two weeks, after which time they were killed. Weight
gain was similar in all groups, and no significant difference in survival was observed
in mice of either sex: at the end of the study period, 32/50, 21/50 and 20/50 males in
the control, low-dose and high-dose groups, respectively, and 34/50, 27/50 and 28/50
females in the control, low-dose and high-dose groups, respectively, were stil alive.
ln female mice, a slight increase in the incidence of benign lung tumours was
observed (control, 1/50; low-dose, 0/50; high-dose, 4/50; p = 0.049, incidental
tumour test). No increase in the incidence of any other neoplasm was recorded
(National Toxicology Program, 1987; Dunnick et al., 1989).
Rat: Groups of 50 male and 50 female Fischer 344/N rats, seven to eight weeks
old, were administered ampicilin tri hydrate (purity, 97%) by gavage at 0, 750 or
1500 mglg bw in corn oil on five days per week for 103 weeks. AnimaIs were
observed for a further one to two weeks, after which time they were killed. Mean
body weights of treated males and females were similar to those of controls. At the
end of the study, 31/50, 27/50 and 26/50 control, low-dose and high-dose males,
respectively, and 32/50, 31/50 and 31/50 control, low-dose and high-dose females,
respectively, were stil alive. An increase in the incidence of mononuclear-cell
leukaemia was observed in treated males: control, 5/50; low-dose, 14/50 (p = 0.019,
fe-table test; p = 0.024, life-table test
life-table test); high-dose, 13/50 (p = 0.029, Ii
for trend). A dose-related increase in the incidence of combined benign and

malignant phaeochromocytomas of the adrenal medulla was also observed in
males: control, 13/50; low-dose, 16/50; high-dose, 23/49 (p = 0.007, incidental
tumour test; p = 0.007, trend test for incidental tumours). The incidences of
mammary gland fibroadenomas in females were: control, 16/50; low-dose, 25/50 (p
= 0.019, incidental tumour test); high-dose, 19/50. No increase in the incidence of
tumours at other sites was observed (National Toxicology Program, 1987; Dunnick
et al., 1989). (The Working Group noted the high frequency of spontaneous tumours
and that the increase in the incidence of mammary gland fibroadenomas was not
dose-related.)

Following intraperitoneal injection to rats, ampicilln was distributed
throughout the major organ systems; the serum half-life was estimated to be 27 min
(Fabre, 1977). Assay of serum collected after a single subcutaneous dose of sodium
ampicilin at 10 mg/kg bw to guinea-pigs yielded ampicilin levels of approximately
10 J.g/ml at 5 min, which fell rapidly to less than 0.2 J.glml at 60 min (Young et al.,
1987).
(ii) Toxic effects

The intraperitoneal LDSO for ampicilin was 330 mglg bw for one-day-old
rats and 4500 mglg bw for83-day-old rats (Goldenthal, 1971). The oral LDSO in
rats was 10 glg bw and that in mice, 15.2 glg bw (Khosid et al., 1975). Deaths
ocurred in 63, 45 and 100% of rabbits that recived oral doses of ampicilin at 5, 15
and 50 mglg bw, respectively, for three consecutive days (Milhaud et al., 1976).
Ampicilln administered as a single oral or subcutaneous dose of up to 500
mglg bw had no noticeable toxic effect in mice or rats. Intravenous administration
of 20 mglg bw to mice caused muscle tremors, slowed respiration and mild
convulsions. No biochemical, haematological or histological abnormality was seen
in rats administered ampicilin at 100 or 500 mglg hw for 12 weeks (Brown &
Acred, 1961). Administration of25 mg/l in the drinking-water to four-week-old rats
for up to eight weeks resulted in an increase in body weight gain; no toxic effect was
noted (King, 1975).
Nabata et al. (1988) reported that intravenous exposures of rats to ampicilin at
120 mg/kg bw per day for 28 days were well tolerated. Intravenous administration
of sulbactam:ampicilin (1:2) at 90180 mg/kg bw for 28 days caused caecal
enlargement; deposition of glycogen-like droplets in the liver ocurred at the higher
dose levels.
The toxicity of ampicilin trihydrate has been studied in Fischer 344/N rats and
B6C3F1 mice (National Toxicology Program, 1987). ln 14-day studies of rats and
mice administered ampicilin at 20-240 mg/kg bw by gavage, dose-related clInical
signs included diarrhoea and excessive salivation in the high-dose rats immediately
after dosing. Diarrhoea of minimal severity was observed in high-dose mice given
240 mglg. No dose-related gross pathology or histopathology was observed in
either species.
ln 13-week studies, doses of 180-30 mg/kg bw were administered by gavage

on five days per week to rats and mice. AlI rats given 30 mglg bw and one of ten
male mice at either 20 mglg or 30 mglg had diarrhoea. No compound-related
pathology or histopathology was observed grossly in either species.
ln the two-year studies (see section 3.1), ampicilin at doses of 750 or 1500
mg/kg bw (rats) and 1500 or 30 mglg bw (mice) was administered by gavage on
five days per week for 103 weeks. Clinical signs observed in treated rats included
diarrhoea, excessive urination and chromodacryorrhoea; those in treated mice
included increased salivation and decreased activity. The incidence of C-cell
hyperplasia of the thyroid gland was increased in low-dose male and high-dose
female rats. High-dose male rats showed increased incidences of hyperkeratosis
AMPICILU 159
and acanthosis of the forestomach. ln male and female mi

ce, an increased
incidence of forestomach lesions, including ulcers, inflammation, hyprkeratosis,
acanthosis and evidence of fugal infection, was observed in expsed animaIs.
(iii) Effects on reproductin and prenatal toxicity

The absence of exprimenta details precluded assessment of the only study of
prenatal toxicity (Korzova et al., 1981).

Ampicilin induced lysogenic phage in Staphylococcus aureus (Manthey et al.,
1975). It did not induce a SOS response in Eschrichia coli PQ37 (Venier et al., 1989),
and no differential toxicity was observed in E. coli in the absence (Green & 1Weats,
1981) or presence of an exogenous metabolIc system (1eats et al., 1981; De Flora et
al., 1984). ln Salmonella tyhimurium plate incorpration tests, ampicilin was not
mutagenic in the presence or absence of an exogenous metabolic system (De Flora
et al., 1984; Mortelmans et al., 1986; National Toxicology Program, 1987).
Treatment of Vicia faba seeds with a 0.5% solution of ampicilin led to
chromosomal aberrations in root-tip meristem cells (Pras
ad, 1977).
Ampicilin did not induce mutation at the tk locus in L5178Y mouse lymphoma
cells in the presence or absence of an exogenous metabolIc system at concentrations
up to 500 iiglml (National Toxicology Program, 1987). No increase in the frequency
of sister chromatid exchange was observed in Chinese hamster CHO cells with
concentrations of ampicilin up to 1500 iiglml in the presence or absence of an
exogenous metabolic system (National Toxicology Program, 1987). Ampicilin did
not induce sister chromatid exchange in human lymphocytes in vitro (Jaju et al.,
1984). No chromosomal aberration was observed in Chinese hamster CHO cells
treated with ampicilin at 0-1500 iiglml in the presence or absence of an exogenous
metabolic system (National Toxicology Program, 1987). Ampicilin did not induce
chromosomal aberrations in human fibroblasts after 50 h of treatment with a
concentration of 40 iiglml (Byarugaba et al., 1975), but a dose of 28 l1g/ml induced
chromosomal aberrations in human peripheral lymphocytes in vitro (Jajuet al.,
1984). (Te Working Group noted the low concentration used in this test, as
compared to those of other reports.) It was reported in an abstract that ampicilln
did not induce chromosomal aberrations in human lymphocytes in vitro at
concentrations up to 10 mg/ml (Stemp et al., 1988).
It was reported in an abstract that ampicilln at single- or double-dose oral
regimens of 5 mglg did not induce micronuclei in rats treated in vivo (Stemp et al.,
1988).
(h) Humans
(i) Pharmcokinetics
The pharmacokinetics of ampicilin have ben reviewed (Barza & Weinstein,
1976).
Ampicilin is relatively stable in the acid contents of the stomach; anhydrous or
trihydrated ampicilin is absorbed incompletely from the gut after oral adminis-
tration. Peak concentrations in plasma (2-6 mgl after an oral dose of 500 mg) occur
within 1-2 h. Ester prodrugs (pivampicilin, bacampicilin) and the condensation
prodrug (hetacilin) of ampicilin are absorbed more readily th an ampicilin (Jusko
& Lewis, 1973; Lo et aL., 1974; Magni et al., 1978; Pennington & Crooks, 1983).
Ampicilin at 500 mg given by intramuscular injection as the sodium salt produced
plasma peaks of 7-14 mg/l within about 1 h (Doluisio et al., 1971).
Ampicilin is distributed widely, and therapeutic concentrations can be
achieved in soft tissues, including ascitic, pleural and joint fluids (Lewis & Jusko,
1975). Bacampicilln produces higher tissue concentrations than ampicilln
(Bronsveld et al., 1978). Only 20% of ampicilin is bound to plasma proteins (Barza
& Weinstein, 1976). It crosses the placenta (Hirsch et al., 1974; Kraybill et al., 1980),
and detectable concentrations of ampicilin occur in the milk of nursing mothers
(Chow & Jewesson, 1985).
Ampicilln is excreted via renal glomerular and tubular routes in the urine; its
plasma half-time is usually 1-2 h (Sjövall, 1985) but is longer in elderly people (Triggs
et aL., 1980). ln patients with renal failure, the half-time was as long as 20 h (Hori et
al., 1983).
Healthy subjects metabolize about 20% of a given dose (250-500 mg) of
ampicilin. Within 12 h, 7% of the total dose is excreted as metabolites in urine
(Cole et al., 1973; Haginaka & Wakai, 1987). Ampicilln is metabolized to
5R,6R-peniciloic acid and 5S,6R-peniciloic acid (Bi rd et al., 1983) and to
piperazine-2,5-dione after oral intake (Haginaka & Wakai, 1987). Other,
unidentified metabolites have been reported (Masada et al., 1979).

Skin rashes (Almeyda & Levantine, 1972) are the most commoIi side-effects of
ampicilln treatment and are either urticarial or maculopapular. The allergic
nature of the maculopapular rash is uncertain (Bierman et al., 1972; Campbell &
Soyka, 1977; Sokoloff, 1977; van Ketel, 1984). Non-allergie fever due to ampieilln
occurs rarely (Mackowiak & LeMaistre, 1987). The overall incidence of skin
reactions among a group of patients who received the drug between 1975 and 1982
was 59/1775 (3.3%) (Bigby et al., 1986), although higher incidences have been
reported. Unusually high incidences of skin rashes ocur during treatment with
AMPICILLIN 161
ampicilin of glandular fever and lymphatic leukaemia (Cameron & Richmond,

1971; Lambert et aL., 1972).
Ampicilin commonly affects the gastrointestinal tract, at least in children
(25-35%) (Feder, 1982). It has been reported to be one of the drugs most frequently
associated with pseudomembranous colitis (Gorbach, 1987). Seizures have been
reported after use of ampicilln in cases of underlying cerebral dysfunction (Serdaru
et al., 1982) or concomitant renal insufficiency resulting in high serum concen-
trations of ampicilln (Hodgman et al., 1984).
ln a study of 28 00 women belonging to a prepaid health plan in Seattle, W A

(USA), all drug prescriptions and aIl pregnancy outcomes were monitored between
July 1977 and December 1979. Among the liveborn babies of 6837women, 80 (1.2%)
had major congenital malformations. Four infants born to 309 women for whom
ampicilin had been prescribed in the first trimester had major malformations
(types not specified), giving a prevalence of 13 per 100, which was not significantly
different from the overall prevalence in the total population studied (12 per 1(0)
(Jick et al., 1981).
ln a second study of the same population covering January 1980 to June 1982,
6509 women had pregnancies ending in livebirths, and 105 (1.5%) of these had
major congenital malformations. Three infants born to 409 women for whom
ampicilin had been prescribed in the first trimester had major malformations
(types not specified), giving a prevalence of seven per 100, compared with an
overall prevalence in the entire group of 15 per 100 (Aselton et al., 1985).
ln a hospital study of Australian women, 7371 mothers had singleton
pregnancies in 1978-81; 106 of them had used amoxycilln or ampicilin (not
recrded separately) at sorne time during pregnancy: 211 had been treated in the
first trimes ter only and 73 in the first trimes ter and later. It was stated that there
was no evidence of any association between use of these drugs and the incidence or
type of congenital malformations, which were observed in 12 of the 28 (4.2%)
exposed babies, compared with the nonexposed (297/6311, 4.7%). There was no
association with use of these drugs and intrauterine growth retardation or perinatal
death, but there was a significant (p oi 0.01) difference in the rate of prematurity in
the users (8.9%) compared with nonusers (6.5%), which was not due to age or
differences in use of alcohoL. There was also a significant (p .. 0.001) increase in
the prevalence of low-birth-weight ( oi 2.5 kg) babies among users (9.6%) compared
with nonusers (6.6%), which was still significant (p .. 0.05) when controlled for
length of gestation (Colley et al. 1983). (The Working Group noted that the effects
might have been due to underlying infection in the mothers.)

No adequate study was available to the Working Group.

One case each of lymphoproliferative disease and Kaposi's sarcoma has ben
reported in association with use of ampicilin (Gordon & Luk, 1982; Brenner et al.,
1984).
Ampicilin was included in a hypothesis-generating cohort study designed to
screen a large number (215) of drugs for possible carcinogenicity, which covered
more than 140 () subscribers enrolled in July 1969 to August 1973 in a prepaid
medical care programme in northern Califomia (USA). Computer recrds of
persons to whom at least one drug prescription was dispensed were linked to cancer
records from hospitals and the local cancer registry. Observed numbers of cancers
were compared with expected numbers, standardized for age and sex, derived from
the entire cohort. Three publications have summarized the screening findings for
follow-up periods of up to seven years (Friedman & Ury, 1980), nine years
(Friedman & Ury, 1983) and 15 years (Selby et al., 1989). (The Working Group chose
to omit mention of associations based on fewer th an three cases.) Among 6706
persons who received ampicilin, an association was noted with subsequent skin
cancer (four cases observed, 0.9 expcted;p ~ 0.05) in the seven-year report. ln the
15-year report, an association was noted with lung cancer (48 cases observed, 27.3
expected; p ~ 0.(02). The latter association, although apparently not explained by
cigarette smoking in an analysis of smoking habits carried out specifically for
people taking ampicilin, was also seen for several other antibiotics. (Te Working
Group noted, as did the authors, that, since sorne 12 () comparisons were made in
this hypothesis-generating study, the associations should be verified independently.
Data on duration of use were not provided.)
4.1 Exposure data
Ampicilin is a broad-spetrum antibiotic and has ben used extensively to

treat bacterial infections since 1961. .
Ampicilin was tested for carcinogenicity by oral administration in mice and
rats. It increased the incidences of mononuclear-cll leukaemia and of
AMPICILLIN 163
phaeochromocomasof the adrenal medulla in male rats. A slight increase in the

incidence of benign lung tumours was observed in female mice.
4.3 H uman carcinogenicity data

ln a hypothesis-generating cohort study, use of ampicilin was associated with
the occurrence of lung and skin cancers, but these findings could have been due to
chance.

Use of ampicilin during the first tri mes ter of pregnancy has not been
associated with an increase in the incidence of major congenital malformations.
Ampicilln increased the frequency of chromosomal aberrations in human
lymphocytes but n(\t in human fibroblasts in vitro. It did not induce chromosomal
aberrations in Chinese hamster cells, mutations in mouse lymphoma cells or sister
chromatid exchange in human lymphocytes or in Chinese hamster cells. Ampicilln
induced chromosomal aberrations in Vicia faba. It was not mutagenic to Salmonella
tyhimurium and did not induce differential toxicity in Escherichia coli strains. (See
Appendix 1.)
4.5 Evaluation 1
There is inadequate evidence for the carcinogenicity of ampicilln in humans.

There is limiled evidence for the carcinogenicity of ampicilln in experimental
animaIs.
Overall evaluation
Ampicilin is not classijble as to its carcinogenicity ID humans (Group 3).
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Almeyda, J. & Levantine, A. (1972) Drug reactions. xix. Adverse cutaneous reaetions to
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AMPICILN 165
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Fabre, J. (1977) Pharmacoetique tisulae de la doxycycline comparée à celle d'autres
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5-8
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AMPICILLIN 167
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eHLORAPHENieOL
This substance was considered by previous working groups, in October 1975
and March 1987 (IAC, 1976, 1987a,b). Since that time, new data have become
available, and these have been incorporated into the monograph and taken into
consideration in the present evaluation.

1.1 Synonyms
ehem. Ahstr. Services Reg. No.: 56-75-7

ehem. Ahstr. Name: Acetamide, 2,2-dichloro-N-(2-hydroxy-1-(hydroxyme-
thyl)-2-( 4-nitrophenyl)ethyl)-(R-(R *,R *))-
Synnym: 2,2- Dichloro-N-( ( ~R,ßR )-ß-hydroxy-~-hydroxymethyl-4-nitrophe-
nethyl)acetamide; D-( - )-threo-2-dichloroacetamido-1-para-nitrophenyl-1,3-
propanediol; D-threo-N-dichloroacetyl- 1 -para-nitrophenyl- 2-amino-l,3-pro-
panediol; D-threo-( -)- 2,2-dichloro-N-(ß-hydroxY-~-(hydroxymethyl)-pra-ni-
trophenethyl) acetami de; D-threo-N-(1, l' -dihydroxy-1-para-nitrophenyliso-
propyl)dichloroacetamide; D-( - )-threo-para-nitrophenyl- 1 -dichloroacetami-
do- 2-propanediol-( 1,3)
N02
HOCH
1
HCNHCOCHCI2
1
CH20H
CiiHiiCliNiOs MoL. wt: 323.14
-169-

Data from Szulczewski and Eng (1975) and Al-Badr and EI-Obeid (1986),
unless otherwse specified
(a) Description: White to greyish-white or yellowish-white fine crystallne
powder or fine crystals, needles or elongated plates. Of the four possible
stereoisomers, only the CYR,ßR (or D-threo) form is active (Anon., 1979).
(b) Melting-point: 149-153°C (sublimes in high vacuum)
(c) Optical rotation: (a)Õ7 = + 18.60 (4.86% in ethanol)
(d) Solubility 1:40 in water at 25°C; aqueous solutions are neutral; 1:6 in
propylene glycol at 25 0 C; very soluble in methanol, ethanol, butanol, ethyl
acetate, acetone; fairly soluble in diethyl ether (Windholz, 1983)
(e) Spectroscopy data: Ultraviolet, infrared, nuclear magnetic resonance and
if Stability Stable in the solid state as a bulk drug and when present in solid
dosage forms. Reasonable precautions taken to prevent excessive
exposure to light or moi sture are adequate to prevent significant
decomposition over an extended periode ln solution, chloramphenicol
undergoes a number of degradative changes related to pH, temperature,
photolysis and microbiological effects.
(g) Reactivity: The nitro group is readily reduced to the amine.
Trade names: Ak-Chlor; AIcon Opulets Chloramphenicol; Amphicol;

Antibiopto; Aquamycetin; Arcomicetina; Biomicin; Bioticaps; Cafenolo;
Cébénicol; Chemicetina; Chemyzin; Chlomin; Chloramex; Chloramol; Chloratets;
Chlorcol; Chlorofair; Chloromycetin; Chloroptic; Chlorsig; Cloramffen;
Cloramplast; Clorbiotina; Clorfenicol Wolner; Clorofenicina; Cloromicetin;
Cloromisol; Cloromoin; Cloroptic; Cutispray No. 4; Doctamicina; Econochlor;
Espectro Medical; Farmicetina; Fenicol; Globenicol; Hortfenicol; 1 -Chlor; Iprobiot;
Isopto Fenicol; Kamaver; Kemicetina; Kemicetine; Kloramfenikol Minims;
Labamicol; Lennacol; Leukomycin; Levomicetina; Lomecitina; Micoclorina;
Micodry; Minims Chloramphenicol; Mycetin; Mychel; Nevimycin; Normofenicol;
Novochlorocap; Ocu-Chlor; Of talent; Oleomycetin; Opclor; Ophtaphénicol;
Ophthochlor; Paidomicetina; Pantofenicol; Pantovernil; Paraxin; Paraxin Succinat
A; Pentamycetin; Plastodermo; Quemicetina; Ranphenicol; Rivomycine; Septicol;
Sificetina; Sintomicetina; Sno Phenicol; Solnicol Ercé; Solu- Paraxin; Sopamycetin;
Spersanicol; Succicaf; Synthomycetine; Thilocanfol; Tifomycine; Tramina;
Troymycetin; Vernacetin
CHLORAPHENICOL 171
Many fixed combinations also contain chloramphenicoL.

Chloramphenicol is often formulated as the cinnamate, palmitate (1.7 g
equivalent to 1.0 g chloramphenicol) or sodium succinate salt (US Pharmacopeial
Convention, 1975; Reynolds~ 1989). Preparations are available as capsules (50, 100
and 250 mg; USP grade contains 90120% of the labelled amount of active
ingredient), ear drops (solution in propylene glycol), eye drops (0.5% solution or
sterile, dry mixure of chloramphenicol and suitable buffers containing 90- 130% of
the labelled amount of chloramphenicol; US Pharmacopeial Convention, Inc., 1975)
and eye ointment (1% chloramphenicol; USP grade contains 90- 130% of the
labelled amount of active ingredient); and as the palmitate in a suspension for oral
administration (USP 5 ml, 30 mg/ml,. containing 90- 120% of the labelled amount of
active ingredient) and the succinate in vials of 1 g for injection (USP grade
containing 90- 115% of the labelled amount of active ingredient).
Chloramphenicol is an antibiotic produced by Streptomyces venezuelae

(Ehrlich et al., 1947). The crystallne antibiotic substance was isolated by Bartz in
1948 (Goodman & Gilman, 1970), and, in 1949, its structural determination
(Rebstock et al., 1949) and chemical synthesis (Controulis et al., 1949) were reported.
Chloramphenical can be synthesized by condensation of para-nitrobenzoyl
chloride with ethyl malonate to give para-nitroacetophenone, followed by
bromination in acetic acid to form para-nitro-O!-bromoacetophenone, and reaction
of this with hexamethylene tetramine, followed by hydrolysis to give
para-nitro-~-aminoacetophenone; subsequent acetylation of the amine group and
condensation with formaldehyde give a hydroxymethyl group alpha to the amine
groupe Treatment with aluminium isopropylate reduces the keto group to a
secondary alcohol, and, after deacetylation, condensation of the amine group with
methyl dichloroacetate gives chloramphenicol (Anon., 1969). Chemical syntheses
of chloramphenicol usually include a resolution step to separate stereoisomers.
ln J apan, production by a fermentation process has also been described. The
process resulted from the discovery and isolation of a new strain of microbe and
does not require separation of stereoisomers (Anon., 1972).
Chloramphenicol is synthesizcd in Brazil, China, Czechoslovakia, the Federal
Republic of Germany, Hungary, Italy, India, Israel, J apan, Mexico, Romania, South
Africa, Spain and the USSR and has also been produced in France; Switzerland, the
UK and the USA. Commercial production of chloramphenicol in the USA was fIrst
reported in 1948 (US Tarff Commission, 1949; Chemical Information Servces,

1989-90).
ln Sweden, 584 780 packages of chloramphenicol were sold in 1988
(Apoteksbolaget, 1988, 1989). ln Finland, sales of chloramphenicol in 1987 were
0.01 defined dàily doses per 100 inhabitants (Finnish Committee on Drug
Information and Statistics, 1988).
Chloramphenicol can be isolated from Streptomyces venezuelae in soiL.
2.2 Use
Chloramphenicol is an antimicrobial agent recmmended for serious
infections in which the location of the infection, susceptibilty of the pathogen or
poor response to other therapy indicate restricted antimicrobial options. It has
been used since the 1950s for a wide range of microbial infections, including tyhoid
fever and other forms of salmonellosis, and central nervous system, anaerobic and
ocular infections (Bartlett, 1982; Sande & Mandell, 1985).
The usual dosage of chloramphenicol is 50 mglg daily in divided doses up to
two to four weeks (Bartlett, 1982; Sande & Mandell, 1985). ln certain indications,
e.g. cys tic fibrosis, treatment has been continued for years (Harley et al., 1970).
An allowed daily intake (ADI) could not be set for chloramphenicol because of
the dose-independence of chloramphenicol-induced aplastic anaemia (FAO/-
WHO, 1969; FAO/WHO Exrt Committee on Foo Additives, 1988).
Chloramphenicol is believed to have been widely used as a veterinaiy
antibiotic, despite legal controls in many countries, and there have been a few
reports of residual amounts in various animal products (Allen, 1985). ln countries
in which its veterinary use is permitted, food regulations require withdrawal periods
so as to avoid residues in the final product (FAO/WO, 1969; FAO/WO Expert
Committee on Food Additives, 1988).
2.3 Analysis
Methods for the analysis of chloramphenicol have ben reviewed (Wenk et al.,
1984; Al-Badr & El-Obeid, 1986). The compound has been determined in serum by
high-performance liquid chromatography (Ryan et al., 1984; So et al., 1987;
Meatherall & Ford, 1988) and enzyme immunoassay (Schwart et al., 1988).
Chloramphenicol has been analysed in pharmaceutical preparations using
microbiological turbidimetric and spectrophotometric assays (US Foo and Drug
Administration, 1988; US Pharmacopeial Convention, Inc., 1989).
Analytical methods for chloramphenicol residues in meat, milk and eggs have
been reviewed (Allen, 1985). The methods include high-performance liquid
chromatography (Schmidt et al., 1985) and radioimmunoassay (Arnold et al., 1984;
Arnold & Somogyi, 1985; Hock & Liemann, 1985).
CHLORAPHENICOL 173


Mouse: ln a study reported in an abstract, groups of 50 male and 50 female
BALB/c mice, six weeks of age, were administered chloramphenicol (purity
unspecified) at 0, 500 or 20 mgl in drinking-water for 104 weeks, at which time all
survivors were killed. The incidences of lymphomas in mice of each sex (combined)
were 3% in controls, 6% in low-dose animaIs and 12% in high-dose animaIs (p .c
0.05). The incidences of other types of tumour were simIlar in treated and control
animaIs (Sanguineti et al., 1983). (The Working Group noted the incomplete
reporting of the study.)
As reported in the same abstract, groups of 50 male and 50 female C57BI/6N
mice, six weeks of age, were administered chloramphenicol (purity unspecified) at 0,
500 or 20 mg/l in drinking-water for 104 weeks, at which time aIl survivors were
killed. The incidences of lymphomas in mice of each sex (combined) were 8% in
controls, 22% in low-dose animaIs (p .c 0.05) and 23% in high-dose animaIs (p .c
0.01). The incidences of malignant liver-ceIl tumours in mi ce of each sex (combined)
were: control, 0; low-dose, 2/90; and high-dose, 11/91 (p .c 0.01) (Sanguineti et al.,
1983). (The Working Group noted the incomplete reporting of the study.)
(h) lntraperitoneal administration
Mouse: Two groups of 45 male BALB/c x AF 1 mice, six to eight weeks of age,
received four intraperitoneal injections of 0.25 ml acetone in distiled water. After a
2O-week rest period, one group received daily intraperitoneal injections of
chloramphenicol (purity unspecified) at 0.25 ml (2.5 mg) in 0.9% saline solution on
five days per week for five weeks. The mice were killed on day 350. Con
troIs
received injections of saline solution only. No increase in the incidence of tumours
was observed (Robin et al., 1981). (The Working Group noted the short duration of
treatment and observation.)
(c) Administration with knwn carcinogens

Mouse: Two groups of 45 male BALB/c x AF 1 mice, six to eight weeks of age,
received intraperitoneal injections every two weeks of four doses of 0.5 mg
busulphan (l,4-butanediol dimethanesulfonate) in 0.25 ml acetone. After a 2O-week
rest period (on day 183 of the experiment), one group rcceived chloramphenicol
(purity unspccified) at 2.5 mg on five days per week for five weeks. On day 350 of the
experiment, all surviving mice were killed. The incidence of lymphomas was 13/37
in the combined treatment group compared with 4/35 in a group treated with
busulphan alone (p = 0.02, Fisher's exact test) (Robin et al., 1981). (The Working
Group noted the short duration of the experiment.)
(a) Experimental sytems

ln dogs, chloramphenicol was readily absorbed after oral administration of 50
mg/kg bw, giving plasma levels of 16.5 Jlglml 2 h after dosing (Watson, 1972, 1977a).
Similar findings were made in rabbits (Cid et aL., 1983).
Five minutes after intravenous administration of 14(-chloramphenicol to
newborn pigs at 0.52 mglg bw, most tissues had higher levels of 14( label than the
blood; however, levels of chloramphenicol in bone marrow did not reach those
noted in serum (Appelgren et al., 1982).
Chloramphenicol and its metabolites were excreted in the urine of rats after
oral dosing; up to 70% of an oral dose may be excreted in this way (Glazko et al.,
1949). About 0.4% of an intramuscuIar dose of 40 mg/kg to rats was detected in the
bile within 4 h (Kunii et al., 1983). ln newborn pigs, most of an intravenous dose of
chloramphenicol was excreted in the urine (Appelgren et al., 1982). Following
intravenous administration to goats, 69% of the dose was excreted in the urine
within 12 h (Javed et al., 1984).
Chloramphenicol was detected in the milk of goats and caUle after parenteral
administration (Roy et al., 1986); however, after oral administration (dose
unspecified) to cattle, no chloramphenicol was detected in milk (De Corte-Baeten
& Debackere, 1976).
ln addition to free chloramphenicol and the glucuronide, the oxamic acid,
alcohol, base, acetylarylamine and arylamine metabolites have been found in the
urine of rats given intramuscular doses of 3H-chloramphenicol (the 1R,2R-isomer).
On the basis of recovered radioactivity, the major metabolItes were assumed to be
chloramphenicol base (",26%) and the acetylarylamine derivative (l-20%) (Bories
et al., 1983).
ln dogs, chloramphenicol base and chloramphenicol glucuronide conjugate
were reported to be the major metabolites (Glazko et al., 1950). Chloramphenicol,
the glucuronide conjugate and the oxamic acid, acetylarylamine, arylamine and
base derivatives were found in the urine of goats given intramuscular injections of
chloramphenicol (Bories et al., 1983).
The glucuronide is the main metabolic product in isolated rat hepatocytes
exposed to chloramphenicol (Silciano et al., 1978). A study using perfused rat liver
CHLORAPHENICOL 175
and rat liver microsomes indicated that the arylamine derivative may undergo
N-oxidation to form nitrosochloramphenicol (Ascherl et al., 1985).
(ii) Toxic effects
The intravenous and intraperitoneal LDs() for single doses of

chloramphenicol in albino mice were 20 and 1320 mglg bw, respectively. The
intravenous LDso in rats was 170 mglg bw. Lethal amounts of chloramphenicol
given orally or parenterally produced respiratory failure (Gruhzit et al., 1949). ln
rats treated with chloramphenicol at 50 and 100 mg/kg bw, the lipid content of the
liver increased and the activities of aspartate and alanine aminotransferases in
serum were elevated (MandaI et al., 1982).
After three groups of ten three-month-old Swiss mice were given daily
intraperitoneal injections of chloramphenicol at 20, 40 or 100 mg/kg bw for three
months, splenomegaly, hepatomegaly, lymph adenopathy and hypertrophy of the
thymus occurred in a dose-dependent fashion (German & Lo, 1962).
Chloramphenicol caused decreased entry into S-phase in dividing
bone-marrow cells of mice treated in vivo (Ben es et al., 1980). The drug had a
deleterious effect on bone-marrow recovery in mice after X-irradiation (Benes et al.,
1980; Vacha. et al., 1981) and after busulfan treatment in one study (Morley et al.,
1976) but not another (Pazdernik & Corbett, 1980). Bone-marrow damage has been
described in cats and dogs after 14-21 days' treatmentwith chloramphenicol (Penny
et al., 1967; Watson, 1977b; Watson & Middleton, 1978; Watson, 1980). Effects
included vacuolation of the myeloid and eryhroid precursors and bone-marrow
hypoplasia in cats, and suppression of eryhropoiesis and a reduced rate of
granulocyte formation but not bone-marrow vacuolation in dogs.
Chloramphenicol caused dose-related inhibition of eryhroid and granulocytic
colony forming units obtained from LA 1 mice (Yunis, 1977).
Chloramphenicol and nitrosochloramphenicol inhibited DNA synthesis in rat
bone-marrow cells in vitro. This effect was reversible with chloramphenicol but not
with the nitroso comPOund. Similarly, the nitroso compound but not
chloramphenicol bound irreversibly to bone-marrow cells (Gross et al., 1982). ln
another study in vitro, chloramphenicol and nitrosochloramphenicol had no effect
on mouse haematopoietic precursor cells (Pazdemik & Corbett, 1979).
Several studies have demonstrated an effect of chloramphenicol on
mitochondrial protein synthesis. ln vitro, chloramphenicol inhibited mitochondrial
protein synthesis in rat liver and rab
bit bone marrow (Summ et aL., 1976;
Abou-Khali et al., 1980). Nitrosochloramphenicol inhibited rat mitochondrial
DNA polymerase in vitro, whereas the arylamine derivative and chloramphenicol
itself did not (Lim et al., 1984).

High oral doses of chloramphenicol of 500-20 mg/g to rats and mice and of
500 and 100 mg/g to rab bits produced high incidences of embryonic and fetal
deaths and fetal growth retardation in all three speies. Teratogenic effects-
predominantly umbilcal hernia-were observed only in rats. The pregnant ani-
maIs showed no toxic sign, except that those given the highest dose gained
significantly less weight than controls (Fritz & Hess, 1971).
Groups of eight pregnant albino mice were given chloramphenicol orally at 25,
50, 100, or 20 mg/kg bw in 10 ml distiled water over the third stage of pregnancy for
seven days. AnimaIs were allowed to give birth, and the young were tested for
conditioned avoidance response, electroshock seizure threshold and performance
in open-field tests. Dose-related effects were seen in all three elements of the test:
progeny of chloramphenicol-treated dams had reduced learning abilty, higher
brain seizure threshold and poorer performance in the open-field test (Al-Hachim
& Al-Baker, 1974).
Chloramphenicol was also investigated for its effects on avoidance learning in
rats. Four groups of 15 pregnant Wistar rats each were treated as follows:
chloramphenicol was given subcutaneously at 50 mg/kg bw on days 7-21 of
gestation; chloramphenicol was given subcutaneously at 50 and 100 mg/kg bw to
pups for the first three days after birth; and the fourth group served as 'controls. No
adverse effect on pregnancy or postnatal weight gain was seen, but when the animaIs
were 60 days old, they had significant impairment of avoidance learning (Bertolini
& Poggioli, 1981).
The genetic toxicology of chloramphenicol has been reviewed (Rosenkranz,
1988).
Chloramphenicol did not induce lysogenic phage in Staphylococcus aureus
(Manthey et al., 1975). It did not induce differential toxicity in Escherichia coli
(Slater et al., 1971; Shimizu & Rosenberg, 1973; Longnecker et al., 1974; Venturini &
Monti-Bragadin, 1978; Mitchell et al., 1980; Leifer et al., 1981), Salmonella
tyhimurium (Nader et al., 1981; Pall & Hunter, 1985), Proteus mirabilis (Adler et al.,
1976) or Baci//us subtilis (Kada et al., 1972; Suter & Jaeger, 1982), although a
contradictory positive result was obtained in the rec assay with E. coli (Suter &
Jaeger,1982). Chloramphenicol gave negative results in the SOS chromotest in E.
coli (Mamber et al., 1986). It induced breaks in DNA of E. coli Bir and S.
tyhimurium TA1976 (Jackson et al., 1977). It did not induce mutations in E. coli
(Hemmerly & Demerec, 1955) and was not mutagenic in plate incorporation assays
with S. tyhimurium in the presence or absence of an exogenous metabolic system
(Brem et al., 1974; McCann et al., 1975; Mortelmans et al., 1986). ln a liquid
CHLORAPHENICOL 177
pre-incubation assay, chloramphenicol did not induce reversions in E. coli; it did,

however, induce forward mutations to aztidine-2-carboxylic acid resistance in the
same bacterial strain. ln the same assay system, chloramphenicol was weakly
mutagenic to S. tyhimurium TA98 in the presence or absence of an exogenous
metabolic system (Mitchell et al., 1980).
Chloramphenicol induced petite mutations in haploid strains of
Saccharomyces cerevisiae (Weislogel & Butow, 1970; Willamson et al., 1971) but not
in diploid strains (Carnevali et al., 1971).
Treatment of Arabidopsis seeds with chloramphenicol did not induce lethal
mutations (Müller, 1965). Chloramphenicol induced chromosome breakage in
root-tip meristem cells of germinating barley (Yoshida et al., 1972) and Vìcia faba
seeds (Prasad, 1977). It did not induce micronuclei in pollen tetrads of Tradescantia
paludosa (Ma et al., 1984).
Chloramphenicol did not induce sex-linked recessive lethal mutations in
Drosophila melanogaster treated either by injection (Clark, 1%3) or by feeding
(Nasrat et al., 1977).
It inhibited DNA synthesis in human lymphoblastoid cell lines (Yunis et al.,
1973), in rat bone-marrow cells (Gross et al., 1982) and in mouse Ehrlich ascites cells
(Freeman et al., 1977). DNA strand breaks were induced in human lymphocytes by
chloramphenicol at 2.0 mM (Yunis et al., 1987) but not at 0.8 mM in a human
lymphoblastoid cell line, in human lymphocytes or in human bone-marrow cells
(Isildar et al., 1988). Chloramphenicol did not induce unscheduled DNA synthesis
in Syrian hamster embryo cells in the presence or absence of an exogenous
metabolIc system (Suzuki, 1987).
The drug induced mutations at the tk locus of L5178Y mouse lymphoma cells
in the presence and absence of an exogenous metabolic system (Mitchell et al., 1988;
Myhr & Caspary, 1988). It induced sister chromatid exchange in Syrian hamster
embryo cells (Suzuki, 1987) but not in human leukocytes (Pant et al., 1976). When
human white bloo cells were treated with low concentrations (10-40 llglml) of
chloramphenicol, a concentration-dependent increase in the number of cells with
chromosomal aberrations was observed (Mitus & Coleman, 1970). Chloram-
phenicol did not induce chromosomal aberrations in human lymphocytes (Jensen,
1972; Sasaki & Tonamura, 1973; Goh, 1979) or in human fibroblasts (Byarugaba et
al., 1975).
No morphological transformation was observed in Syrian hamster embryo
cells after treatment with chloramphenicol at 100-100 llg/ml (Suzuki, 1987).
Chloramphenicol did not reproducibly enhance the transformation of Syrian
hamster embryo cells by simian adenovirus SA 7 (Hatch et al., 1986).
Subcutaneous injections to C57B1/10 mice of chloramphenicol at 320 mglg bw

three times daily for three days led to inhibition of thymidine incorpnìtion in
bone-marrow cells (Benes et al., 1980). Intramuscular injections of chloram-
phenicol (three times 100 mglg bw) to Wistar rats did not induce chromosomal
aberrations in bone-marrow cells (Jensen, 1972). At 50 mglg bw, the drug induced
chromosomal aberrations in bone-marrow cells of mice (site of injection and
number of animaIs tested unspeified) (Manna & Bardhan, 1972, 1977). Intra-
muscular injection of chloramphenicol at 50 mglg to Swiss albino mice (number of
animaIs unspeified) induced chromosomal aberrations in mitotic and meiotic
germ line cells (Roy & Manna, 1981).
Chloramphenicol did not induce dominant lethal mutations in mice when
given twce at up to 1500 mglg intraperitoneally (Epstein & Shafner, 196; Ehling,
1971; Epstein et al., 1972) but did when given at 500 mglg bw (Sram, 1972).
(h) Humans
(i) Pharmcokinetics
Chloramphenicol is readily absorbed from the gastrointestinal tract after oral
administration of a crystallne powder of the active drug itself or a palmitate ester;
the latter is hydrolysed in the smaIl intèstine to active chloramphenicol before
absorption (Kauffman et al., 1981). Esters of chloramphenicol-for example, the
succinate-are converted to chloramphenicol in vivo (Salem et al., 1981). Peak levels
of 10-20 J-g/ml appear 2-3 h after administration of chloramphenicol orally at 15
mg/kg bw (see Bartlett, 1982).
Chloramphenicol is also weIl absorbed by infants and neonates after oral
administration. Serum (peak) concentrations of 20-24 J-glml were noted after oral
doses of 40 mg/kg bw to neonates. Infants given 26 mglg bw were found to have
peak concentrations of 14 J-g/ml (Mulhall et al., 1983).
ans, regardless of its route
Chloramphenicol is distributed extensively in hum
of administration. The compound has been found in heart, lung, kidney, liver,
spleen, pleural fluid, seminal fluid, ascitic fluid and saliva (Gray, 1955; Ambrose,
1984). It penetrates the blood-brain barrier, and its concentrations in cerebrospinal
fluid can reach about 60% of that in plasma (Friedman et al., 1979). The
concentrations in brain tissue equal or even exced those in plasma (Kramer et al.,
1969). Chloramphenicol easily crosses the placenta, and it is also excreted in breast
milk (Havelka et al., 1968).
Chloramphenicol has a half-time ranging from 1.6 to 4.6 h; using different
techniques and in different adult patients, apparent volumes of distribution ranging
from 0.2 to 3.1 l/kg have been measured (see Ambrose, 1984). The half-time is
considerably longer in neonates (Rajchgot et al., 1983): in one- to eight-day-old
CHLORAPHENICOL 179
infants the half-life ranged from 10 to over 48 h, and in 11-day- to eight-week-old

infants the range was 5- 16 h (Glazer et al., 1980).
Six hours after an intravenous dose of 500 mg chloramphenicol succinate, the
blood level was 4.5 iig/ml (2.8-6.9 iig/ml) in patients with chloramphenicol-induced
bone-marrow depression, while in the control group the mean level was 1.2 Mg/ml
(0-2.3 iig/ml). Such findings suggest that patients susceptible to the effects of
chloramphenicol on bone marrow may clear the drug from the blood more slowly
th an those who are not susceptible (Suhrland & Weisberger, 1969).
Chloramphenicol is excreted primarily in the urine (90%); up to 15% is
excreted as the parent compound and the remainder as metabolites, inc1uding
conjugated derivatives (Yunis, 1973; Burke et al., 1980; Ambrose, 1984). Glomerular
excretion is thought to be the major mechanism of excretion (Glazko et aL., 1949).
Approximately 48% of the chloramphenicol excreted in urine within 8 h of an
oral dosing was the glucuronide conjugate; only 6% was excreted as the parent
compound and 4% as the base derivative (Nagakawa et al., 1975; Baselt, 1982;
Bories et al., 1983). The alcohol derivative has been detected in the urine of neonates
(Dil et al., 1960).
Human liver microsomes have been shown -to reduce the nitro group of
chloramphenicol (Salem et al., 1981).
Chloramphenicol arylamide is formed by intestinal bacterial reduction of the
NOi group to NHi, which is acetylated and excreted in urine (Meissner & Smith,
1979). Oxamic acid (formed by oxidative dechlorination of the side chain) was
identified as a major metabolite in one human volunteer (Corpet & Bories, 1987).

The most important adverse effects of chloramphenicol involve the
haematopoietic system (as reviewed by the FAO/WHO Expert Committee on Food
Additives, 1988). Potentially fatal toxicity may develop in neonates exposed to
excessive doses of chloramphenicol (Sande & Mandell, 1985). This so-called 'grey
baby syndrome' may also occur in older children and in adults receiving doses
resulting in. serum concentrations of 40-20 iig/ml (see Bartlett, 1982). Other
adverse effects include hypersensitivity reactions, gastrointestinal complaints and
neurological complications after long-term treatment. Chloramphenicol canalso
precipitate haemolytic anaemia in subjects with glucose-6-phosphate
dehydrogenase deficiency (Robertson et aL., 1968).
Dose-dependent, reversible bone-marrow suppression affects primarily the
eryhroid series and occurs regularly when plasma concentrations of
chloramphenicol are 25 iiglml or higher (Scott et al., 1965; Yunis & Adamson, 1977).
Another haematological side-effect is rare, unpredictable, non-dose-related
aplastic anaemia, which often appears after the drug has been discontinued (Best,
1967).
The metabolite (or metabolites) responsible for the induction of aplastic
anaemia in human beings is unknown, but nitrosochloramphenicol has been
implicated (Nagai & Kanamuru, 1978; Yunis, 1988): it is known to be toxic to human
bone-marrow cells in vitro and, moreover, is more toxic than chloramphenicol itself
(Yunis et al., 1980a,b). Metabolites of chloramphenicol, such as dehydrochloram-
phenicol, produced by intestinal bacteria, are more than 2O-fold more cytotoxic
than the parent drug (Yunis, 1988).
There have been many case reports of the ocurrence of aplastic anaemia
following administration of chloramphenicol by various routes (Rosenthal &
Blackman, 1965; Nagao & Mauer, 1969; Carpenter, 1975; Yunis, 1978; Abrams et al.,
1980; Silver & Zuckerman, 1980; Flach, 1982; Fraunfelder et al., 1982; Plaut & Best,
1982; Issaragrisil & Pianki jagum, 1985; Korting & Kifle, 1985; Elberg & Hansen,
1986; von Muhlendahl, 1987). ln many of these cases, large doses had been taken
repeatedly over periods of many years before the onset of symptoms of aplastic
anaemia. Case-control studies have also suggested an association between
chloramphenicol use and aplastic anaemia (for review, see FAO/WHO Expert
Committee on Food Additives, 1988). A widely discussed causal association
between topical application of chloramphenicol eye-drops and aplastic anaemia
(Wade, 1972; Carptenter, 1975; Fraunfelder et al., 1982) has not been established.
ln the Collaborative Perinatal Project, in which drug intake and pregnancy
outcome were studied in a series of 50 282 women in 1959-65, 98 women had been
exposed to choramphenicol during the first trimester of pregnancy. There were
eight malformed children in the exposed group, giving a nonsignificant standard-
ized relative risk (RR) of 1.17. A total of 348 women had had exposure at any time
during pregnancy with no evidence of an increase in the incidence of congenital
malformations (Heinonen et al., 1977).
No adverse effect was reported in the children of 22 patients treated at various
stages of pregnancy with chloramphenicol (Cunningham et al., 1973).
No adequate study was available to the Working Group.
3.3 Case reports and epidemiological studies
Numerous case reports have been published of leukaemia ocurring following
chloramphenicol-induced aplastic anaemia (Edwards, 1969; Seaman, 1969; Goh,
1971; Cohen & Huang, 1973; Meyer & Boxer, 1973; HelIriegel & Gross, 1974; Modan
et al., 1975; IAC, 1976; Ellms et al., 1979; Witschel, 1986; IAC, 1987a); three case
CHLORAPHENICOL 181
reports have been published ofleukaemia following chloramphenicol therapy in the

absence of interceding aplastic anaemia (Humphries, 1968; Popa & Iordacheanu,
1975; Aboul-Enein et al., 1977).
Shu et ai. (1987) reported a case-cntrol study of 30 childhood leukaemia
cases (un der 15 years) notified to a population-based cancer registry in Shanghai,
China, during 1974-86, and 618 age- and sex-matched population controls.
Information was obtained from parents or guardians for lifetime use of selected
drugs, including prescribed chloramphenicol and syntomycin (a racemic mixure of
D- and L-chloramphenicol). The risk for aU tys ofleukaemia combined showed a
marked increase with accumulated use of chloramphenicol, yielding RRs of 1.7
(95% confidence interval, 1.2-2.5),2.8 (1.5-5.1) and 9.7 (3.9-24.1) for one to five days',
six to ten days' and more than ten days' treatment, respectively. The association was
present in a subgroup in which first use had ocurred more than five years prior to
diagnosis and in one in which last use had ben more than two years before
diagnosis. Significant trends in risk with dose were observed both for acute
lymphocytic leukaemia (56% of cases) and for acute nonlymphocytic leukaemia
(30%). An association with leukaemia was also seen for use of syntomycin (RR, 1.9;
1.1-3.2). (The Working Group noted that interviewwas undertaken up to ten years
after diagnosis, which adds to the possibility of differential recall between the
parents of cases and controls. Little information was available with regard to use of
other antibiotics, making it difficult to evaluate the possibilty of bias.)
4.1 Exposure data -

Chloramphenicol has been used widely as an antibiotic since the 1950s.
Veterinary use of chloramphenicol has resulted in the occurrence of residues in
animal-derived food.

No adequate study was available to evaluate the carcinogenicity of chloram-
phenicol to exprimental animaIs.
Intraperitoneal administration of chloramphenicol to mice enhanced the inci-
dence of lymphomas induced by 1,4-butanediol dimethanesulfonate.

Many case report have described an unusual succssion of leukaemia
following chloramphenicol-induced aplastic anaemia and bone-marrow
depression. Additional evidence for the association between use of chloram-

phenicol and leukaemia has come from a single large case-cntrol study in China,
which demonstrated a relationship with duration of expsure.
Use of chloramphenicol during the first trimester of pregnancy has not been
associated with an increase in the incidence of congenital malformations.
Chloramphenicol caused embryo- and fetolethality in mi ce, rats and rabbits.
ln humans, chloramphenicol causes aplastic anaemIa. ln both humans and
animaIs administered chloramphenicol, reversible suppression of the bone marrow
is frequent whenever the drug reaches relatively high plasma concentrations.
Chloramphenicol induced chromosomal aberrations in bone-marrow cells of
mice but not of rats treated in vivo. It induced chromosomal aberrations in meiotic
cells of male mice. Contradictory results were obtained in dominant lethal tests in
mice. ln human cells, chloramphenicol did not induce sister chromatid exchange or
chromosomal aberrations but gave contradictory results for DNA damage. It
induced sister chromatid exchange in Syrian hamster cells. Chloramphenicol
induced gene mutations in mouse lymphoma cells but did not induce DNA damage
in hamster cells. Chloramphenicol did not induce sex-linked recssive lethal
mutations in Drosophila. It induced chromosomal aberrations in plants. ln haploid
yeast, chloramphenicol induced petite mutations. ln most studies, chloram-
phenicol was not mutagenic to and. did not cause DNA damage in Salmonella
tyhimurium or Escherichia coli and did not induce DNA damage in Proteus
mirabilis or Bacillus subtilise (See Appendix 1.)
4.5 Evaluation!
There is limited evidence for the carcinogenicity of chloramphenicol in

humans.
There is inaequate evidence for the carcinogenicity of chloramphenicol in
following information. Chloramphenicol induces aplastic anaemia, and this
condition is related to the occurrence of leukaemia.
Overall evaluation
Chloramphenicol is probably carcinogenic to humans (Group lA).
IFor desription of the italicizeterms, se Preamble, pp. 2629.

CHLORAPHENICOL 183
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CHLORAPHENICOL 193
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NITROFURA (NITOFURAONE)
This substance was considered bya previous Working Group, in June 1974,
under the title 5-nitro-2-furaldehyde semicarbazne (!AC, 1974). Since that time,
new data have beme available, and these have ben incorporated into the mono-
graph and taken into consideration in the present evaluation.
1.1 Synonyms

ehem. Abstr. Name: Hydraz
necarboxami de, 2-i(5-nitro-2-furanyl)methyl-
ene)-
Synnym: 2-Furancarboxaldehyde; 5-nitrofuraldehyde semicarbazide; nitro-
furaldehyde semicarbazone; 5-nitro-2-furaldehyde semicarbazone; 5-nitro-
furan-2-aldehyde semicarbazone; 5-ni tro- 2- furancarboxaldehyde semicarba-
zone; 5-nitro-2-furfuraldehyde semicarbazone; 5-nitrofurfural semicarbazone;
5-ni tro- 2-furfural semicarbazone; (5-nitro-2-furfurylideneamino )urea; 1-(5-
nitro- 2- furfurylidene )semicarbazde
1.2 Structural and molecular formula and molecular weight
OiNLY CH = NNHCONH,
CSllN404 MoL. wt: 198.14

From Windholz (1983) and Reynolds (1989)
(a) Description: Pale-yellow needles
(h) Me/ting-point: 23-240°C (decomposition)
-195-
(c) Solubility Very slightly soluble (1:420) in water at pH 6.0-6.5, soluble in

alkaline solutions; slightly soluble in ethanol (1:590), propylene glycol
(1:350), acetone (1:415), dimethylformamide (1:15) and polyethylene
glycol (1:86); almost insoluble in chloroform (1:2700) and benzene
(1:43500)
(d) Spectroscopy data: Infrared and ultraviolet spectra have been reported.
(e) Stability Stable in solid state at less than 40° C when protected from light;
darkens with prolonged exposure; discolours on contact with alkali
Trade names: Acutol; Aldomycin; Alfucin; Amifur; Babrocid; Beafurazna;

Becafurazne; Biofuracina; Biofurea; Chemofuran; Chixn; Cocafurin; Coxistat;
Dermofural; Dyazne; Eldezol F -6; Fedacin; Flavazne; Fracine; Furaciln;
Furacilinum; Furacilln; Furacin; Furacin-E; Furacine; Furacinetten; Furacin-HC;
Furacoccid; Furacort; Furacycline; Furalcyn; Furaldon; Furalone; Furametral;
Furan-ofteno; Furaplast; Furaseptyl; Furaskin; Furazline; Furazin; Furazna;
Furazol W; Furazne; Furesol; Furfurin; Furosem; Fuvacilln; Germex; Hemofuran;
Ibiofural; Mammex; Mastofuran; Monafuracin; Nefco; NF-7; NFS; Nfz mix;
Nifucin; Nifurid; Nifuzon; Nitrazone; Nitreofural; Nitrofurastan; Nitrofurazan;
NSC-21oo; Nitrozone; Otofural; Otofuran; Rivafurazn; Rivopon-S; Sanfuran;
Spray-Dermis; Spray-foraI; Vabrocid; Veterinary nitrofurazne; Yatrocin
Nitrofural has been reported to contain 3% 5-nitro-2-furaldehyde azne as an
impurity (Morris et al., 1969). It is available in the USA as creams, ointments,
powders, solutions, sprays, suppositories and surgi cal dressings (Barnhart, 1989).
The action of nitrofural as a topical antibacterial agent was first reported in the
USA in 1944 (Dodd & Stilman, 1944), and the product was available for general use
in 1945 (Miura & Reckendorf, 1967). Commercial production in the USA was first
reported in 1955 (US Tariff Commission, 1956).
Nitrofural can be prepared by the reaction of 5-nitrofurfural with an aqueous
solution of a mixure of semicarbazde hydrochloride (sec IAC, 1987) and sodium
acetate (Stilman & Scott, 1947). It can also be synthesizcd from the reaction of
acetone semicarbazone or other semicarbazones with 5-nitrofurfuraldoxime (Gever
& O'Keefe, 196). It is synthesizcd in China, Hungary, India, Mexico and Spain
(Chemical Information Services, 1989-90).
NIOFU 197
Nitrofural is not known to ocur naturally.
2.2 Use
Nitrofural is a broad-spetrum bactericidal (Chamberlain, 1976). It also has

antiprotozoal and antiparasitic activities (Reynolds, 1982).
Nitrofural is used locally for the treatment of wounds, burns, ulcers and skin
infections; it has also ben applied localy to the ear, eye and bladder. Nitrofural is
used as a coidiostatic and antibacterial agent in farm animaIs, administered in
water or feed (Anon., 1979; Reynolds, 1989).
Oral administration has ben restricted to the treatment of late-stage African
tryanosomiasis that is refractory to melarsoproI. The dosage for adults is 500 mg
three or four times daily for five to seven days. ln addition, it has been given orally in
doses of 100 mg four times daily for five to six days in the treatment of acute
bacilary dysentery (Reynolds, 1982).
2.3 Analysis
Nitrofural has ben analysed in pharmaceutical preparations by

spetrophotometry (US Pharmacopieal Convention, Inc., 1980) and polarography
(Mishra & Gode, 1985). The separation and identification of nitrofural in
medicated feeds have been reviewed by Fishbein (1972). High-performance liquid
chromatography methods for analysing nitrofural in medicated feeds have also
been reported (Cieri, 1979; Thorpe, 1980).


Mouse: Groups of 50 male and 50 female B6C3F 1 mice, seven to eight weeks of
age, were administered nitrofural (99% pure) at 0, 150 or 310 mg/kg of diet for 103
weeks; aIl survving animaIs were killed at 112 weeks. The average amount of
nitrofural consumed per day was 14-16 mgJg bw for low-dose male and female
mice and 29-33 mgJg bw for high-dose animaIs. Survival of high-dose males was
troIs after week 88. At the end of the experiment, survival was:
lower than that of con
39/50,31/50 and 27/50 control, low-dose and high-dose males, and 39/50,40/50 and
35/50 control, low-dose and high-dose females. Ovarian atrophy was found in 7/47
controls, 44/50 low-dose and 38/50 high-dose mice. Granulosa-cell tumours of the
ovary developed in 4/50 low-dose and 9/50 high-dose females (p = 0.03, incidental
tumour test for trend) compared with 1/47 controls. The incidence of benign mixed
tumours of the ovary was 17/50 low-dose and 20/50 high-dose animaIs (p ~ 0.001,
incidental tumour test for trend); no such tumour ocurred among controls. No
significant difference in the incidence of other types of tumour was observed among
treated or control mice (National Toxicology Program, 1988; Kari et al., 1989).
Rat: A group of 30 female weanling Sprague-Dawley rats were administered
nitrofural (pharmaceutical grade) at 100 mg/kg of diet for 46 weeks (daily intake,
8-13 mg/rat), after which they were maintained on control diet for 20 weeks. A
control group of 30 rats received control diet for 66 weeks. Of the treated females
that lived 22 weeks or more, 2229 developed mammary fibroadenomas, compared
with 2/29 con troIs (Ertürk et al., 1970). (The Working Group noted that data on
survival were not given.)
Groups of 50 male and 50 female Fischer 344/N rats, six to seven weeks of age,
were administered nitrofural (99% pure) at 0, 310 or 620 mg/kg of diet for 103 weeks.
The average amount of nitrofural consumed per day was 11-12 mg/kg hw for
low-dose male and female rats and 24-26 mg/kg bw for high-dose animaIs. AlI
surviving animaIs were kiled at 111 weeks. Survival in high-dose males was lower
than that in controls after week 92. At the end of the experiment, survival was: 33/50,
30/50 and 20/50 controls, low-dose and high-dose males, and 28/50,37/50 and 31/50
controls, low-dose and high-dose females, respectively. Adenomas of the sebaceous
glands of the skin were observed in high-dose males only (4/50 high-dose versus 0/50
control; p = 0.067, incidental tumour test). Mammary fibroadenomas occurred in
8/49 control, 36/50 low-dose (p ~ 0.001, incidental tumour test) and 36/50 high-dose
females (p ~ 0.001, incidental tumour test; p ~ 0.001, incidental tumour test for
trend); adenocarcinomas were also observed in one control and two high-dose
females. Mononuclear-cell leukaemias occurred in 21/50 control males, 23/50
low-dose males and 6/50 high-dose males (p = 0.04, life-table test); 15/49 control
females, 2/25 low-dose females (p ~ 0.001) and 2/50 high-dose females (p ~ 0.001
life-table test). Testicular interstitial-cell tumours ocurred in 45/50 controls, 30/50
low-dose males (p ~ 0.001, incidental tumour test) and 28/50 high-dose males (p ~
0.001, incidental tumour test; p ~ 0.001, incidental tumour test for trend) (National
Toxicology Program, 1988; Kari et al., 1989).
(h) Transplacental administration

Mouse: A group of 20 pregnant ICR/Jcl mice, 10-12 weeks of age, reccived
three subcutaneous injections of nitrofural (purity unspeified) at 75 Jlg/g hw
suspended in 1% gelatin solution on days 13, 15 and 17 of gestation. Offspring were
foster-nursed by untreated dams and were killed 32 weeks afer birth. Treatment
NITOFURA 199
with nitrofural resulted in a marked reduction in the number of live births. At 32

weeks, 67/145 treated animaIs and 548/84 controls were stil alive. The incidence of
lung tumours was not significantly increased in nitrofural-treated mice as
compared with gelatin-treated controls. AlI tumours reported were papillary
adenomas of the lung (Nomura et al., 1984). (The Working Group noted the short
duration and limited reporting of the experiment; interlitter variation was not
recorded. )
A group of newborn ICR/Jcl mi ce, exposed transplacentally as described
above, recived a subcutaneous injection of nitrofural (purity unspecified) at 75
iig/g bw suspended in 1% gelatin solution within 12 h of birth; three further
injections were given on days 7, 14 and 21 after birth. A further group of mice
recived treatment with gelatin only, and another recived no treatment. At 32
weeks, 61/176 treated animaIs and 548/84 controls were stil alive. The number of
tumour-bearing micc was 1261 (19.7%; p c: 0.001, X2 test with Yates' correction
against gelatin controls) compared to 5/203 (2.5% ) untreated controls. AlI tumours
reported were papilary adenomas of the lung (Nomura et al., 1984). (The Working
Group noted the short duration and lImited reporting of the experiment; interlitter
variation was not recorded.)

Within 24 h after a single oral administration of 100 mg/kg bw 14C-nitrofural to
rats, about two-thirds of the radioactivity appeared in the urine, 26% in the faeces
and approximately 1 % in expired carbon dioxide; complete recovery of the
administered dose was observed after 96 h, less than 15% of the label being
recovered as unchanged parent comPOund (Tatsumi et al., 1971). Major metabolites
of nitrofural detected and identified in the urine of dosed rats included
hydroxylaminofuraldehyde semicarbazne, aminofuraldehyde semicarbazone and
4-cyano-2-oxobutyraldehyde semicarbazone (Paul et al., 196). The reduced
nitrofural metabolite, 4-cano-2-oxobutyraldehyde semicarbazne, was detected in
the urine of germ-free rats treated with the drug (Yeung et al., 1983). Binding of 14C
label to liver protein, DNA, ribosomal RNA and kidney protein was demonstrated
in rats after oral administration of 14c-nitrofural (Tatsumi et al., 1977).
Nitrofural is reduced by mouse liver homogenate and by several mammalian
cell lines, most efficiently under gas mixures containing 5% 02 or less (Paul et al.,
196; Olive & McCalla, 1975)
(ii) Toxic effects

Oral LDso values of 590 mglg bw in rats and 46 mg/g bw in mice have been
reported (Miyaji, 1971). Mice and rats recivIng 30 mglg hw or more orally
showed hyperirritabilty, tremors and seizures and died from respiratory arrest
(Krantz & Evans, 1945). ln mice and rats, subcutaneous injection of
large doses (3
g/kg bw) produced marked changes in the structure of the liver and kidney, but only
slight hepatic changes were seen after lethal oral doses (45 mg/kg bw for four to six
days) (Dodd, 1946).
Toxicity was studied by feeding diets containing nitrofural (99% pure) to
groups of F344/N rats and B6C3F 1 mice for 14 days, 13 weeks or two years. ln the
14-day studies, in which the doses ranged from 630 to 10 () mglg of diet,
nitrofural was more toxic to mice than to rats. ln the 13-week studies, doses for rats
ranged from 150 to 2500 mg/g of di et and for mice from 70 to 120 mg/g of diet. At
the higher doses, convulsive seizures and gonadal hypplasia were observed in both
species. Evidence oftoxicity in rats also included degenerative arthropathy. ln the
two-year studies (see section 3.1), nitrofural caused testicular degeneration
(atrophy of germinal epithelium and aspermatogenesis) in rats and degeneration of
vertebral and knee articular cartilage in rats of each sexe ln mice of each sex,
nitrofural administration induced stimulus-sensitive convulsive seizures, primarily
during the first year of study (National Toxicology Program, 1988; Kari et al., 1989).
The gonadotoxicity of nitrofural in male mice has ben recgnized for more
th an three decades. Nissim (1957) showed that administration to mice in the di et at
a dose equivalent to 375 mglg bw caused testicular atrophy. SimIlar degeneration
was observed in rat testis following daily doses of 100 mglg bw by gastric
intubation for seven days (Miyaji et al., 196).
ln male Sprague-Dawley rats given nitrofural in the diet at a dosage equivalent
to 64 mglg bw per day for 28 days, the mean weight of the testes was only 28% that
of the controls. Ali stages of spermatogenesis were affected, but Sertoli cells and
Leydig cells were not damaged (Hagenas et al., 1978).
Afer a single subcutaneous injection to ICR/Jcl mice of nitrofural at 300
mglg bw on day 10 of gestation, increased embryo- and fetomortality and
decreased fetal weight were observed compared with controls. A significant (p -c
0.(01) increase in the incidence of malformations was observed, predominantly
affecting the limbs, digits and taiL. Afer administration of nitrofural at 100 mglg
bw subcutaneously on days 9-11, the only significant effect observed was a
reduction in fetal weight (Nomura et al., 1984).
Pregnant CD1 mice were given nitrofural in the diet at doses equivalent to
6.3-82 mglg bw from days 6-15 of gestation. No teratogenic effect was observed,
NITOFURA 201
but there was increased fetal death and reduced fetal weight at the highest dose
(National Toxicology Program, 1988).

The genetic toxicology of nitrofurans has ben reviewed (Klemencic & Wang,
1978; McCalla, 1983).
Nitrofural inhibited DNA synthesis (Lu & McCalla, 1978) and caused
prophage induction in Escherichia coli (McCalla & Voutsinos, 1974). It induced
DNA strand breaks in E. coli (McCalla et al., 1971; Tu & McCalla, 1975; Wentzell &
McCalla, 1980) and in Salmonella tyhimurium strain TA1975 (McCalla et al., 1975).
Nitrofural induced differential toxicity in E. coli (Yahagi et al., 1974;
Haveland-Smith et al., 1979; Lu et al., 1979) and Bacillus subtilis (Tanooka, 1977) but
not S. tyhimurium (Yahagi et al., 1974).
Nitrofural induced mutations in E. coli (Zampieri & Greenberg, 196;
McCalla & Voutsinos, 1974; Yahagi et al., 1974; McCalIa et al., 1975; Tanooka, 1977;
Haveland-Smith et al., 1979; Lu et al., 1979; Ebringer & Bencova, 1980; Clarke &
Shankel, 1989) in the absence of an exogenous metabolic system, but not in strains
lacking nitroreductase activity (McCalla & Voutsinos, 1974; McCalla et al., 1975).
ln the presence of a microsomal preparation from Drosophila melanogaster,
nitrofural induced mutations in E. coli (Baars et al., 1980). It was not mutagenic to
S. tyhimurium strains TA1535, TA1536, TA1537 or TA1538 (Yahagi et al., 1974;
McCalla et al., 1975) but induced mutations in TA1535 in a fluctuation test (Green et
aL., 1977) and in plate incorporation tests, only in the presence of an exogenous
metabolic system (Zeiger et al., 1987; National Toxicology Program, 1988). Nitro-
fural induced mutations in S. tyhimurium TA100 and in TA98 in the presence and
absence of an exogenous metabolic system (Yahagi et aL., 1976; Goodman et aL.,
1977; Green et al., 1977; Chin et al., 1978; Rosin & Stich, 1978; Bruce & Heddle, 1979;
Haveland-Smith et al., 1979; Imamura et al., 1983; Obaseiki-Ebor & Akerele, 1986;
Ni et al., 1987; Zeiger et al., 1987; National Toxicology Program, 1988).
Nitrofural was mutagenic to Neurospora crassa (Ong, 1977) but not to
Aspergillus nidulans (Bignami et al., 1982).
Feeding of Drosophila melanogaster for three days with nitrofural at 5 mM did
not induce sex-linked recessive lethal mutations (Kramers, 1982).
Nitrofural inhibited DNA synthesis in mouse L-929 cells (Olive, 1979a,b). It
induced DNA strand breaks in human KB, Syrian hamster BHK-21 and mouse
L-929 cells (Olive & McCalla, 1975; Olive, 1978). No unscheduled DNA synthesis
was induced by nitrofural in either rat or mouse primary hepatocytes (Mori et al.,
1987) or in human fibroblasts (Tonomura & Sasaki, 1973).
Nitrofural induced mutations to 6-thioguanine resistance in Chinese hamster
lung (V79) cells (Olive, 1981) but not in Chinese hamster ovary (CHO) cells
(Anderson & Philips, 1985),either in the presence or absence of an exogenous

metabolic system. It induced mutations at the tk locus of mouse L5178Y lymphoma
cells (National Toxicology Program, 1988). Nitrofural induced sister chromatid
exchange in CHO cells in the presence and absence of an exogenous metabolic
system (National Toxicology Program, 1988). It induced chromosomal aberrations
in Chinese hamster lung cells in the presence and absence of an exogenous
metabolIc system (Matsuoka et al., 1979; Ishidate, 1988). ln CHO cells, however,
nitrofural induced chromosomal aberrations in the absence, but not in the
presence, of an exogenous metaboIic system (National Toxicology Program, 1988).
Nitrofural did not induce chromosomal aberrations in human lymphocytes in vitro
(Tonomura & Sasaki, 1973).
It was not active in micronucleus tests either in rats treated twce with 7.5-30
mglg bw intraperitoneally at 30 h and 6 h before they were killed (Gooman et aL.,
1977) or in mice treated with 150 mglg bw intraperitoneally on five consecutive
days (Bruce & Heddle, 1979). Nitrofural did not induce chromosomal aberrations
in bone-marrow cells of rats either after a single intraperitoneal injection of 60
mg/kg bw (Goodman et al., 1977) or after single oral doses of 4040 mglg bw or five
daily oral doses of 15-150 mglg bw (Anderson & Phillps, 1985).
Nitrofural did not induce sperm abnormalities in mice treated intraperi-
toneally with 15-150 mglg hw on five consecutive days (Bruce & Heddle, 1979).
(h) Humans
(i) Pharmcokinetics
Nitrofural is not significantly absorbed from skin or mucous membranes after
local administration (Marion-Landais et aL., 1975; Harvey, 1985).
Sensitization and generalized allergic skin reactions are known adverse effects
of topically administered nitrofuraL. ln a literature review of studies published in
1945-65, 176 (1.2%) cases of skin reactions were reported among 15 162 treated
patients (Glascok et aL., 1969; Reynolds, 1989).
Nausea, vomiting, joint pains, headaches and polyneuritis are typical toxic
effects after oral administration (Reynolds, 1989). Polyneuropathy is common
among trypanosomiasis patients treated with nitrofural (Cancado et al., 196;
Roberton & Knight, 196; Spencer et al., 1975).
Nitrofural has been reported to cause haemolytic anaemia in individuals with
glucose-6-phosphate dehydrogenase deficiency (see Prankerd, 1962).
NIOFU 203
expsed to nitrofuraI administered topically during the first trimester of pregnancy.

Fifteen malformed children were born in the expd group, giving a standardized
relative risk of 0.99 (Heinonen et al., 1977.
3.3 Case reports and epidemiologica studies of carcinogenicity to humans
ln a hypthesis-generating cohort study designed to screen a large number of
drugs for possible carcinogenicity (described in detail in the monograph on
ampicilin), 317 persons to whom at least one prescription for nitrofural had been
dispensed during 1969-73 were followed up for up to 15 years (Sclby et al., 1989). No
statistically significant assoiation was noted with cancer at any site or at aIl sites
combined. (Te Working Group note that the number of users was small and
therefore the power of the study to detect carcinogenic effects was probably low.
Data on duration of use were not provided.)

4.1 Exposure data
Nitrofural is an antibacterial agent used since 1945 mainly for the local
treatment of skin infections. It has ben used orally in the treatment of refractory
African trypanosomiasis.

Nitrofural was tested by oral administration in one study in mice and in two
studies in rats, and by transplacental administration to mice. Oral administration
to mice increased the incidence of granulosa-cell and benign mIxed tumours of the
ovary. ln rats, an increased incidence of mammaiy fibroadenomas was observed in
females in both studies. Two studies of transplacental administration of nitrofural
to mice were inadequate for evaluation.
ln a hypothesis-generating cohort study, use of nitrofural was not associated
with an increase in cancer incidence, but the power of the study was low.
One study did not provide evidence that topical use of nitrofural during
pregnancy is associated with birth defects. Nitrofural is gonadotoxic in male and
female mice and in male rats and is teratogenic in mice.
ln humans, nitrofural is poorly absorbed from skin and mucous membranes

after local administration. The drug binds to lIver protein and DNA as weIl as to
kidney protein in rats treated in vivo.
Nitrofural did not induce chromosomal aberrations in rats, micronuclei in
mice or rats or sperm abnormalities in mice. It induced sister chromatid exchange
in Chinese hamster cells in vitro; contradictory results were obtained on the
induction of chromosomal aberrations in mammalian cells. Nitrofural induced
DNA strand breaks in human, hamster and mouse cells but did not induce
unscheduled DNA synthesis in human, rat or mouse cells. Both positive and
negative results were obtained in gene mutation assays in rodent cells. Nitrofural
did not induce sex-linked recssive lethal mutations in Drosophila. It was
mutagenic to Neurospora but not to Aspergilus and induced differential toxicity in
Escherichia coli and Bacillus subtilis and mutations in E. coli and Salmonella
tyhimurium. (Sec Appendix 1.)
4.5 Evaluationl
There is inadequate evidence for the carcinogenicity of nitrofural in humans.

There is limited evidence for the carcinogenicity of nitrofural in experimental
animaIs.
Ove rail evaluation
Nitrofural is not classifble as to its carcinogenicity to humans (Group 3).
5. References
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cells in vitro andin vivo. Food. chem. Toxicol., 23, 1091-1098
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Baars, A.J., Blijleven, W.G.H., Mohn, G.R., Natarajan, A.L & Breimer, D.D. (1980)
Preliminaiy studies on the abilty of Drosophla microsomal preparations to activate
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Barnhart, E. (1989) Physician' Dek Reference, 43rd ed., Oradell, NJ, Medical Economies, p.
320
Bignami, M., Carere, A, Conti, G., Conti, L., Crebell, R. & Fabrii, M. (1982) Evaluation of
2 different genetic markers for the detection of frameshift and misense mutagens inA.
nidulan. Mutat. Res., 97, 293-302
IFor desription of the italicize tenns, se Preamble, pp. 2629.

NITOFURA 205
Bruce, W.R. & Heddle, J.A. (1979) The mutagenic activity of 61 agents as determined by the
micronucleus, Salmonella, and sperm abnormality assays. Cano 1. genet. Cytol., 21,
319-334
Cancado, J.R., Marr, U.D. & Brener, Z. (196) Clinical tril of 5-nitro-2-fur-
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Med. trop. Sao Paulo, 6, 12-16 rlTop. Dis. Bull., 61, 608)
Chamberlain, R.E. (1976) Chemotherapeutic properties of prominent nitrofurans. 1
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Chin, J.B., Sheinin, D.M.K. & Rauth, A.M. (1978) Screening for the mutagenicity of
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Cie ri, U.R. (1979) High pressure liquid chromatographic detection and estimation of
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5-nitro-2-furaldehyde semicarbazne. L Phaol. ex. Thr., 85, 324-331
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NITOFU 207
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NIOFU 20
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Mutagene, 9 (Suppl. 9), 1-110
NITROFURATOIN
1.1 Synonyms
ehem. Abstr. SeTVices Reg. No.: 67-20-9; 17140-81-7 (monohydrate); 54-87-5

(sodium salt)
Chem. Abstr. Name: 2,4-Imidazolidinedione, I-f((5-nitro-2-furanyl)methyl-
ene)amino l-
Synonyms: 1-((5- Nitrofurfurylidene )amino )imidazolidine-2,4-dione; 1-((5-
nitrofurfurylidene )amino )hydantoin
o
02N"L CH = N, )~
1 LN 1NHL
o
CSH6N40S MoL. wt: 238.16

From Cadwallader and Jun (1976) and Windholz (1983)
(a) Description: Pale orange-yellow needles from dilute acetic acid
(b) Melting-point: 270-272°C (decomposes)
(c) Solubility Solubilities of nitrofurantoin in many aqueous media and
organic solvents have been reported.
(d) Spectroscopy data: Ultraviolet, infrared and nuc1ear magnetic resonance
spectra have been reported.
(e) Stability: Tablets and suspension stable for fIve years at room temperature
in regular glass containers; crystals and solutions discoloured by al
kali
and by exposure to light
-211-
if Dissociation constant: pKa = 7.2
Trade names: Berkfurin; Chemiofuran; Chemiofurin; Cistofuran; Cyantin;

Cystit; Dantafur; Fua-Med; Furadantin; Furadantina; Furadantine; Furadöine;
Furadonine; Furandoninium; Furalan; Furantoin; Furantoina; Furatin; Furedan;
Furil; Furobactina; Furophen; Gurachel; Ituran; Ivadantin; Microdoin; Micturol
Simple; Nephronex; Nierofu; Nifuran; Nitrex; Nitrofor-50; Nitrofor-100;
Nitrofurantonum; Nitrofurin; Novofuran; N-Toin; Orafuran; Parfuran; Phenurin;
Sarodant; Trantoin; Trocurine; Urantoin; Urefuran; Uretoin; Uriston; Urizept;
Urodil; Urodin; Urolisa; Urolong; Urosagen; UroTablinen; Uro-Tablinen; Urotoin;
Uvamine; Welfurin; Zofurin
Macrocrystalline products: Furadantin MC; Macrodantin; Uvamin retard
Nitrofurantoin is available as tablets (50 mg and 100 mg) and as a suspension
(Barnhart, 1989; Reynolds, 1989). Impurities in the tablets include calcium
pyrophosphate, magnesium stearate, starch and sucrose; and those in the
suspension include carboxymethyl cellulose, sodium citric acid, glycerine,
magnesium aluminium silicate, methylparaben, propylparaben, saccharin (see
IARC, 1987a), sodium citrate and sorbitoL. Nitrofurantoin is available from at least
one manufacturer in macrocrystallne form (Cunha, 1988).
Nitrofurantoin can be prepared from 1 -aminohydantoin sulfate or hydro-

chloride and 5-nitro-2-furaldehyde diacetate in isopropyl alcohol (sec IAC, 1987b)
media (Cadwallader & Jun, 1976). It is synthesized in China, India, Italy, the
Netherlands and Spain (Chemical Information Services, 1989-90). ln Sweden, sales
of nitrofurantoin in 1988 were 0;09 defined daily doses per 100 inhabitants
(Apoteksbolaget, 1988, 1989).
Nitrofurantoin is not known to occur naturally.
2.2 Use
Nitrofurantoin is used extensively in the treatment and prophylaxs of

uncomplicated lower urinary-tract infections. The usual oral dose for adults is
50-100 mg four times daily, with meals and at bedtime. Treatment is usually
continued for 14 days. The daily dose for children is 5-7 mg/kg given in four divided
NITOFURAOIN 213
oral doses. The dosage is reduced if continued beyond 14 days or if used for
prophylaxs (Reynolds, 1989). ln long-term treatment, a dose as lowas 1 mglg may
be used (Lohr et al., 1977). A single dose of 50- 100 mg at bedtime may be sufficient
to prevent recurrences (Stamey et al., 1977).
2.3 Analysis
Analytical methods have been described for nitrofurantoin in pharmaceutical

formulations . using thin-Iayer chromatography (Cadwallader & Jun, 1976),
high-performance liquid chromatography (US Pharmacopeial Convention, Inc.,
1989), polarography (Surmann & Aswakun, 1985; Morales et al., 1987) and
electrochemical methods (Fogg & Ghawji, 1988). Methods for analysing the
compound in plasma and urine include high-performance liquid chromatography
(Vree et al., 1979), polarography (Morales et al., 1987) and electrochemical analysis
(Mason & Sandmann, 1976).


Mouse: Groups of 52-53 male and 54 female (C57BV6N x DBA/2N)F 1 (BDF 1)
mice, nine weeks of age, were administered nitrofurantoin (purity and crystallne
form unspecified) at 0, 750 or 300 mglg of di et for 104 weeks, when the experiment
was terminated. At that time, survival in males and females combined was 50.5%,
42.5% and 46.2% in control, low-dose and high-dose groups, respectively.
Administration of the high dose significantly lowered body weights in mice of each
sex in comparison with controls. ln males, a reduced incidence of hepatic
adenomas was observed: 6/53 controls, 1/52 low-dose and 0/52 high-dose mice (p =
0.014, Fisher's exact test). No increase in the incidence of tumours at any site was
observed (Ito et al., 1983).
Groups of 50 male and 50 female Swiss (Crl:CDR_1(ICR)BR) mice, about 50
days of age, recived nitrofurantoin (pharmaceutical grade macrocrystals) at 0, 50,
100 or 20 mglg of di et for 22 months. Increased mortality was observed in males
treated with the high-dose. ln males, the incidences of malignant lymphomas at aIl
sites were: controls, 2/50; low-dose, 6/50; mid-dose, 4/49; and high-dose, 10/50 fp =
0.014, Fisher's exact test; p = 0.012 Cochran-Armitage trend test) (Butler et al.,
199).
Groups of 50 male and 50 female B6C3F 1 mice, eight to nine weeks of age, were
fed nitrofurantoin (pharmaceutical grade) at 0, 130 or 2500 mg/kg of diet for 103
weeks. Survival at termination of the experiment was reduced in control females:
controls, 19/50; low-dose, 37/50; and high-dose, 37/50. Mean boy weights of male
and female high-dose mice were 12% lower than those of controls. ln females,
ovarian atrophy was seen in 0/50 control, 48/50 low-dose and 49/50 high-dose
animaIs. Controls had ovarian abscesses (18/50) and suppurative inflammation of
the uterus (11/50). There was no significant increase in the incidence of any
individual type of tumour; however, when tubular adenomas and benign mixed
tumours of the ovary are combined, the incidence is significant: 0/50 controls, 0/50
low-dose and 9/50 high-dose (p = 0.01, incidental tumour test) (National Toxicology
Program, 1989). (The Working Group noted the por survival and the high
incidence of ovarian abscesses in the controls.)
ln a study of ovarian atrophy, three groups of female B6C3F 1 mice, five to six
weeks of age, were given nitrofurantoin (pharmaceutical grade) at 0, 350 or 500
mg/kg bw daily in the diet for 64 weeks. Intermittent sacrifices were made at 4, 8, 13,
17 and 47 weeks; the numbers of mice stil alive at 65 weeks were 20 controls, 19
low-dose and 18 high-dose animaIs. Treated animaIs gained significantly less
weight than the controls. There was no increase in the incidence of neoplasms of the
reproductive system (the only tissues reported). By week 43, there was evidence of
ovarian atrophy in treated females; by the end of the study, the incidences were:
control, 0/20; low-dose, 18/19; and high-dose, 18/18 (Stitzel et al., 1989). (The
Working Group noted the short duration of the study and the small number of
animaIs used.)
Rat: A group ofweanling female Sprague-Dawley rats (36 animaIs alive at ten
weeks), weighing 40-72 g, was administered nitrofurantoin ('pure'; identity and
purity checked by infrared and ultraviolet absorption spectrophotometry,
melting-point and paper chromatography) at 1870 mg/kg of diet for 16 weeks, after
which time the dose was reduced to 100 mg/kg of diet in weeks 16-75 due to
impaired growth and premature mortality. The experiment was terminated at week
80. A group of untreated rats served as con troIs (30 alive at ten weeks). No increase
in tumour incidence was observed (Cohen et al., 1973). (The Working Group noted
the short duration of the experiment and the small number of effective animaIs.)
Two groups of 11-12 weanling, germ-free female Sprague-Dawley rats,
weighing 85- 100 g, were fed nitrofurantoin (extracted from pharmaceutical grade,
macrocrystallne nitrofurantoin) at 0 or 1880 mg/kg of diet' for 104 weeks. The
growth rate in treated rats was slightly retarded as compared with that in controls.
The median survival time was 96 weeks for controls and 90 weeks for treated
animaIs. The incidences of mammary fibroadenomas were 2/11 controls and 9/12
treated rats (p .( 0.01, Fisher's exact test). No increase in the incidence oftumours
NITOFURAOIN 215
at other sites was observed (Wang et al., 1984). (The Working Group noted the small
number of animaIs used.) .
Groups of 50 male and 50 female Fischer 34 rats, six to seven weeks of age,
were given nitrofurantoin (pharmaceutical grade) at 0, 60 or 13 mg/g bw
(females) and 0, 130 or 2500 mg/kg (males) of diet for 103 weeks. Mean body
weights were simIlar in control and treated animaIs. Survival at termination of the
experiment was: males-control, 24/50; low-dose, 27/50; and high-dose, 26/50;
females-control,25/50; low-dose, 26/50; and high-dose, 31/50. Chronic tubular
nephropathy was observed in all treated rats. ln males, the incidence of mainly
microscopic renal tubular adenomas was 3/50 controls, 11/50 low-dose ip = 0.02,
Fisher's exact test) and 19/50 fp ~ 0.001; Fisher's exact test) high-dose animaIs
fp ~ 0.001, Cochran-Armitage test for trend). Renal tubular carcinomas were seen
in two high-dose males. Osteosarcomas were seen in one low-dose and two
high-dose males. Reductions in the incidences of a number of neoplasias were
observed in males: preputial gland adenomas-control, 6/48; low-dose, 5/50; and
high-dose, 0/47 (p = 0.018, incidental tumour test); preputial gland carcinomas-
control, 6/48; low-dose, 6/50; and high-dose, 0/47 (p = 0.028, incidental tumour test);
and interstitial-cell adenomas of the testis-control, 47/50; low-dose, 45/50; and
high-dose, 21/50 (p ~ 0.001, incidental tumour test). No change in tumour
incidence was observed in females (National Toxicology Program, 1989). (The
Working Group was not convinced of the neoplastic nature of the microscopic
kidney lesions.)
(b) Transplacental administration
Mouse: A group of 10 pregnant ICR/Jcl mice, 10- 12 weeks of age, received
three subcutaneous injections of nitrofurantoin (purity unspecified) at 75 llglg bw
suspended in a 1% gelatin solution on days 13, 15 and 17 of gestation. Groups of 22
gelatin-treated and 76 untreated dams served as controls. Offspring were
foster-nursed by untreated dams and were sacrificed 32 weeks after birth. Survival
was comparable in treated and untreated mice at 32 weeks. The incidence of
papilary adenomas of the lung in the offspring of nitrofurantoin-treated dams was
10/78, that in gelatine controls, 5/203, and that in untreated controls, 29/478
(Nomura et al., 1984). (The Working Group noted that the distribution of tumours
among litters was not given, that the sex of the offspring was not given and that the
experiment was short.)

The pharmacokinetics of nitrofurantoin have been reviewed (Conklin, 1978).
After oral or parenteral administration, nitrofurantoin is rapidly absorbed

and is excreted primarly unchanged in the urne and bile of rats (Paul et al., 196;
Buzard et al., 1961; Veronese et al., 1974; Wierzba et al., 1982) and mice (Maiti &
Banerjee, 1978). Afer intravenous administration of nitrofurantoin to dogs at
1.5-24 mglg bw, up to 23% was recvered from the bile, while urinary excretion
accunted for up to 36% (Conklin & Wagner, 1971). ln male Sprague-Dawley rats,
16-30% of a total dose of nitrofurantoin was recvered in the urine (Olivard et al.,
1976). After a single administration of nitrofurantoin at 25 mglg bw by gavage to
female albino rats, 52% and 2.0% of the total dose were recvered in the urine and
faeces, respectively (Paul et al., 196). Excretion of nitrofurantoin in the urine of rats
has been reported to be age-dependent (Braunlich et al., 1978; Wierzba et al., 1982).
Intravenous administration of nitrofurantoin at 1.5-24 mglg bw to adult male
beagle dogs weighing 10-16 kg stimulated bile excretion, and nitrofurantoin was
found in bile (at 6 mglg bw, 22.6 :: 4.7% total dose) and urine (24.1 :: 4.7%)
(Conklin & Wagner, 1971). Nitrofurantoin is excreted in bile, reabsorbed and
recirculated enterohepatically (Conklin et al., 1973). Afer intravenous adminis-
tration of nitrofurantoin to rats, the plasma half-time was 25 min, and 50% was
recovered in the urine as unchanged compound (Buzard et al., 1961). The small
intestine was considered to be the main site of absorption (Maiti & Banerjee, 1978).
4-Hydroxyurantoin has ben isolated from the urine of rats treated with
nitrofurantoin (Olivard et al., 1976; Streeter et al., 1988). Reductive metabolism of
nitrofurantoin under anaerobic conditions has been described in both rodent tissue
and bacteria. ln the absence of oxygen, nitrofurantoin appears to be reduced
irreversibly via nitroso and/or hydroxylamine forms (Mason & Holtzman, 1975a;
Biaglow et al., 1977; Leskovac & Popovic, 1980).
Un der aerobic conditions in vitro, reduction of nitrofurantoin stimulates
consumption of oxygen and production of superoxide anion, free radicals and
hydrogen peroxide in avian liver and in mammalian liver, lung, small intestine,
kidney and gastrointestinal contents (Mason & Holtzan, 1975b; Biaglow et al.,
1977; Aufrere et al., 1978; Sasame & Boyd, 1979; Leskovac & Popovic, 1980; Peterson
et al., 1982).
Under anaerobic conditions, microsomal and soluble fractions from rat lung
and liver mediated the covalent binding of 14c-nitrofurantoin-derived radioactivity
to macromolecules. Covalent binding of 14C-nitrofurantoin activity was greatest in
the kidney, liver, ileum, lung and heart of rats (Boyd et al., 1979).
(ii) Toxic effects
The LDso of nitrofurantoin in mIce was 150 mglg bw by intraperitoneal

injection and 30 mglg bw by gavage (Ákerblom & Campbell, 1973).
NITOFURAOIN 217
Subcutaneous administration of nitrofurantoin to male rats caused severe

pulmonary damage characterized by oedema, congestion and haemorrhage (Boyd
et al., 1979). Male and female rats administered nitrofurantoin orally at 20, 50 or 100
mg!g bw twice a day were reported to develop structural and functional changes in
the sciatic nerve (Behar et al., 1965).
When nitrofurantoin was administered to female mice in the diet at 350 and
500 mg/kg bw and animaIs were examined after 4-64 weeks of treatment, a
dose-related effect on body weight gain was seen as well as a reduction in
uterus:brain and ovary:brain weight ratios. Histological examination revealed a
dose-related decrease in the ocurrence of old corpora lutea and an increase in the
occurrence of intermediate and atretic follcles. The effects were more pronounced
with higher dose and longer treatments. Oestrous cycles were lengthened in a
dose-dependent fashion. Ovaries were atrophic and non-functioning at 43 weeks
(Stitzl et al., 1989).
ln a 9O-day toxicity study involving the administration of nitrofurantoin in the

diets of rats and mice, necrosis of ovarian follcular epithelial cells was the principal
pathological finding (Maron pot, 1987).
Four of five male and four of fIve female mice fed nitrofurantoin at 10 00
mg/kg of di et died within 14 days. No rats receiving up to 20 00 mg/kg of di
et for 14
days died; treatment-related signs included inactivity, rough coats, sunken eyes,
bright yellow urine and/or yellow fur. Feeding of nitrofurantoin at 10 00 mg/kg of
diet to female rats for 13 weeks caused normal-to-mild necrosis of ovarian follicles;
the effect was seen in a smaller proportion of animaIs receiving lower doses.
Minimal-to-mild degeneration of the germinal epithelium of the testis was observed
in male mice fed nitrofurantoin at up to 500 mg!g of di
et for 13 weeks. Similar
treatment of male mice caused minimal-to-mild necrosis of the kidney epithelium
ln two-year studies (sec section 3.1), ovarian atrophywas observed in low- and
high-dose female mice, and testIcular aspermatogenesis, degeneration of. the
germinal epithelium and atypical cells and depletion of the epididymis were
observed at increased incidences in high-dose male mice. Spindle-cell hyperplasia
of the adrenal cortex ocurred in treated female mice, and mineralization of the
renal medulla and dilatation of the renal tubules were observed in high-dose mice.
Ovarian abscesses were observed in control but not in treated mice. ln the two-year
study in rats, fibrous osteodystrophy and mineralization of the glandular stomach
ocurred in treated animaIs. Atypical cells of the epididymis and degeneration of
the testis were observed in high-dose animaIs; and fibrinoid necrosis of arterioles
and perivascular infiltration of mononuclear cells were observed in the testis

Nitrofurantoin has similar toxic effects on the testis as other nitrofurans
(Cohen, 1978). Rats treated with nitrofurantoin at 10 or 85 mglg bw by gastric
intubation daily for one month showed depression of spermatogenesis, mainly at
the stage of primary spermatoces; sorne effect on spermatogonia was also
observed. Partial regeneration had ocurred by 48 days after cessation of treatment.
The gonadotoxic effects could be prevented by simultaneous administration of
'cystine' (Yunda et al., 1974). (The Working Group assumed that cysteine was
meant.)
Testicular and ovarian degeneration was observed in F344/N rats given
nitrofurantoin in the diet at a dose equivalent to 110 mglg bw (males) and 60 mg/kg
bw (females) for 13 weeks. Testicular degeneration was observed in B6C3F1 mice
given nitrofurantoin in the di et at a dose equivalent to 285 mglg bw for 13 weeks
ln routine safety evaluations of nitrofurantoin macrocrystals, including studies
of fertilty and perinatal-postnatal effects in rats and teratogenicity in rats and
rabbits, no adverse effect was observed with daily doses of 10, 20 and 30 mg/kg bw
administered orally. ln the fertilty test, however, male rats were treated with only
10 mglg bw; at this dose, no adverse effect on fertility or testicular histology was
observed (Pryherch et al., 1984).
After subcutaneous injection of nitrofurantoin to ICR/Jcl mice at 100 or 250
mglg bwon days 9-11 of gestation, no increase in embryo- or fetomortality was
observed, but a decrease in fetal weight occurred in the high-dose group only. A
significant (p .: 0.001) increase in the incidence of malformations (cleft palate and
syndactyly) was observed in the high-dose group only (Nomura et al., 1984).
The mutagenicity of nitrofurans has been reviewed (Klemencic & Wang, 1978;
McCalla, 1983).
Nitrofurantoin inhibited DNA synthesis in Escherichia coli (Lu & McCalla,
1978). It induced DNA single-strand breaks in a nitroreductase-proficient but not
in a nitroreductase-deficient strain of E. coli (McCalla et al., 1971). It induced
differential toxicity in E. coli, Salmonella tyhimurium and Bacillus subtilis in the
presence and absence of an exogenous metabolic system (McCalla & Voutsinos,
1974; Yahagi et al., 1974; Ebringer & Bencova, 1980; McCarroll et al., 1981a,b; Suter
& Jaeger, 1982; De Flora et al., 1984).
Nitrofurantoin was weakly mutagenic to E. coli in the presence and absence of
an exogenous metabolic system (McCalla & Voutsinos, 1974; Yahagi et al., 1974;
Setnikar et al., 1976; Lu et al., 1979; Ebringer & Bencova, 1980; Obaseiki-Ebor &
Akerele, 1986). It was mutagenic to S. tyhimurium TA100 and TA98, in the
NITOFURAOIN 219
presence and absence of an exogenous metabolic system (Rosenkranz & Speck,

1976; Wang & Le, 1976; Gooman et al., 1977; Chin et al.,. 1978; De Flora, 1979;
Shirai & Wang, 1980; Haworth et al., 1983; De Flora et al., 1984; Ni et al., 1987), and to
TA97 (Obaseiki-Ebor & Akerele, 1986) but not to TA1535, TA1536, TA1537 or
TA1538 (Yahagi et al., 1974; Haworth et al., 1983; De Flora et al., 1984). The strong
responses in the Salmonella mutagenicity tests are due to the high activity of
bacterial nitroreductases (Rosenkranz & Spek, 1976; Wang & Lee, 1976;
Rosenkranz & Mermelstein, 1983; Ni et al., 1987).
Urine of rats fed a diet containing 0.5% nitrofurantoin was mutagenic to S.
tyhimurium (Wang & Le, 1976).
The urine of rats treated orally with nitrofurantoin at 500 or 100 mglg bw
induced mitotic gene conversion in S. cerevisiae D4-RDII (Siebert et al., 1979). ln a
host-mediated assay with mice treated orally with nitrofurantoin at 0.3 mM/kg (72
mglg), no increase in the frequency of gene conversion was found in S. cerevisiae
D4 (Setnikar et al., 1976). Oral treatment of rats with nitrofurantoin at 500 mg/kg
bw led to an increase in the frequency of gene conversion in S. cerevisiae D4-RDII
(Siebert et al., 1979).
Nitrofurantoin did not induce gene conversion in Saccharomyces cerevisiae D4
(Setnikar et al., 1976). ln strains D4-RDII and D7, it induced mitotic gene
conversion (Siebert et al., 1979; Callen, 1981). It induced non-disjunction and
mitotic crossing-over in spot tests with diploid strains of Aspergillus nidulans
(Bignami et al., 1974).
Nitrofurantoin fed or injected to adult Drosophila melanogaster gave
ambiguous results in the sex-linked recssive lethal test (Kramers, 1982; Zimmering
et al., 1985). It gave positive results in the wing spot test in Drosophila, producing
large single spots (Graf et al., 1989).
Nitrofurantoin inhibited DNA synthesis in mouse L-929 cells (Olive, 1979) and
in diploid human fibroblasts (Hirsch-Kauffmann et al., 1978). ln Chinese hamster
ovary (CHO Ki-BHi and CHO UV-5) cells, it induced mutations to 6-thioguanine
resistance in the presence, but not in the absence, of an exogenous metabolic system
(Gao et al., 1989). Nitrofurantoin induced DNA strand breaks in mou se L cells
(Olive & McCalla, 1977), in purified rat liver nuclei and in the human cell line HuF22
(Parodi et al., 1983). It did not induce unscheduled DNA synthesis in human
fibroblasts (Tonomura & Sasaki, 1973) or in rat primary hepatoces (Willams et
al., 1989).
Nitrofurantoin induced sister chromatid exchange in Chinese hamster CHO

cells (Shi rai & Wang, 1980) but not in the human fibroblastic cellline HE 2144
(Sasak et al., 1980). It did not induce chromosomal aberrations in human
lymphocytes in vitro (Tonomura & Sasaki, 1973) or in the human cell line HE 2144
(Sasaki et al., 1980). It induced chromosomal aberrations in Chinese hamster lung

(CHL) cells (Ishidate, 1988).
Intraperitoneal injection of n.itrofurantoin at up to 112 mglg bw induced
DNA strand breaks in liver (Russo et al., 1982), kidney, lung and spleen cells of rats
and in mouse bone-marrow cells (Parodi et al., 1983). Intraperitoneal treatment at
up to 64 mglg bw induced sister chromatid exchange in mouse bone-marrow cells
in vivo (Parodi et al., 1983).
Nitrofurantoin at 80 mglg bw intraperitoneally gave negative results in the
mouse spot test (Gocke et al., 1983) and, at up to 20 mglg intraperitoneally or 40
mglg orally, in the rat micronucleus test (Setnikar et al., 1976; Goodman et al.,
1977). At five daily intraperitoneal doses of 8 or 40 mglg bw, nitrofurantoin did not
induce chromosomal aberrations in spermatoces of mice (Fonatsch, 1977). It also
gave negative results in the dominant lethal test in mice after intraperitoneal
administration of 16 and 80 mglg bw (Epstein et al., 1972) and equivocal results
after five daily oral doses of 17.5 mg/kg bw (Setnikar et al., 1976).
(h) Humans
(i) Pharmcokinetics
Nitrofurantoin is readily absorbed from the gastrointestinal tract (Reynolds,
1989). The macrocrystallne form is dissolved and absorbed more slowly and
produces lower serum concentrations than the microcrystallne form, and peak
concentrations in the urine are achieved more slowy (Cunha, 1988; Reynolds, 1989).
After oral administration of nitrofurantoin at 50 mg to six healthy men, the
bioavailabilty was 94 :l 13% on a full stomach and 87:l 13% on a fasting stomach
(Hoener & Patterson, 1981). About 60% of the nitrofurantoin was bound to plasma
proteins. After a 45-min intravenous infusion, the plasma distribution followed an
open two-compartment model, with a terminal half-time of approximately 1 h.
Afer oral and intravenous infusion, 34 and 47% of the dose was excreted
unchanged in the urine, respectively, and 1.2-1.4% was recovered as the reduced
metabolite aminofurantoin.
us following pathways simIlar
Nitrofurantoin is reduced to aminofurantoin, th
to those known for other nitrofurans (Hoener & Patterson, 1981). Hydroxylation of
the furan ring of nitrofurantoin has also been shown (Olivard et al., 1976).
Recovery of the drug in the urine is related linearly to creatinine clearance
(Sachs et al., 1968).
After parenteral administration, nitrofurantoin crosses the human placenta
(Perry & Leblanc, 1967; Kobyletzki, 1968).
NfIOFUOIN 221

ln a study of 757 courses of nitrofurantoin in hospitalized patients, the overall
frequency of adverse reactions was 9.2%. Toxic reactions constituted 5.1% of
adverse effects; the remainder were allergic (Koch-Weser et al., 1971).
The most common gastrointestinal side-effects of nitrofurantoin are nausea,
vomiting and anorexia. These symptoms usually ocur during the first week of
treatment and are dose-related. Abdominal pain, gastrointestinal bleeding and
diarrhoea occur less frequently and without a clear dose-response (Koch-Weser et
al., 1971; Gleckman et al., 1979).
Pulmonary infiltration may be caused by sensitivity to nitrofurantoin (Israel &
Diamond, 1962). Acute pulmonary sensitivity reactions are manifested by fever,
chils, cough, dyspnoea, and possible bronchospasm and chest pain associated with
eosinophila (Glueck & Janower, 1969). Subacute pulmonary reactions have been
considered to be a separate syndrome (Gleckman et al., 1979; D'Arcy, 1985),
developing after one month of treatment with nitrofurantoin, and are characterized
by persistent and progressive cough, dyspnoea and fever, together with interstitial
pneumonitis (Sollaccio et al., 196; Sovijärv et al., 1977). The chronic nitrofurantoin
pulmonary reaction is characterized histologically by nonspecific, diffuse
interstitial pneumonitis and fibrosis (Rosenow et al., 1968; Ruikka et al., 1971;
Castleman, 1974; Holmberg et al., 1980).
Nitrofurantoin has been associated with adverse effects on the liver, including
acute hepatocllular and cholestatic injury (Goldstein et al., 1974), as weIl as rare
cases resembling chronic active hepatitis (Klemola et al., 1975; Black et al., 1980;
Sharp et al., 1980).
Peripheral polyneuropathy is the most common neurological side-effect,
although dizzness, vertigo, diplopia and cerebellar disturbance have also been
reported (Graebner et al., 1973).
Haemolytic anaemia is a well-documented complication of nitrofurantoin
therapy in patients with glucose-6-phosphate dehydrogenase deficiency (Swanson
& Cook, 1977). Haemolysis has also been reported in patients deficient in enolase
and glutathione peroxidases (Steinberg et al., 1970; Stefanini, 1972). ln addition,
there have been case reports of megaloblastic anaemia (Bass, 1963),
agranulocytosis (Palva & Lehmola, 1973; Böttiger & Westerholm, 1977) and aplastic
anaemia (Böttiger & Westerholm, 1977).

exposed to nitrofurantoin during the first trimester of pregnancy. Six malformed
children were born in the expsed group, giving a stadardized nonsignificant

relative risk of 1.07 (Heinonen et al. 1977).
Hailey et al. (1983) reported on the use of nitrofurantoin during 91 pregnancies
in 81 women in one practicc in 1972-80. ln 36% of women, treatment was given
during the first trimester. ln the 91 pregnancies, one feta death and two malformed
babies (all with expsure during the send or third trimester) were observed.
There was no significant differencc in the incidence of mortality, malformation,
prematurity or low birth weight compared with the general population.
ln a brief review of the management of urnar-tract infections in pregnancy, it
was stated that, in over 500 pregnancies treate with nitrofuantoin macrocrystals
at 100 mg daily for ten days, the treatment did not producc adverse feta or neonatal
effects and there was no rccrded case of neonatal haemolytic anaemia (Whalley,
1985).

Urine of 12 patients was collected before and after treatment with nitro-
furantoin at 100 mg. Increased mutagenic activity in S. tyhimurium TAl00 was
found in samples taken after treatment (Wang et al., 1977).

A single case report has ben published of focal nodular hyperplasia of the
liver in association with nitrofurantoin treatment in a six-year-old girl who had been
treated for seven months (Anttinen et al., 1982).
ln a hypothesis-generating cohort study designed to screen a large number of
ampicilin), 135 persons to whom at least one prescription for nitrofurantoin had
ben dispensed during 1969-73 were followed for up to 15 years (Sclby et al., 1989).
Increased risks were noted for cancer of the uterine corpus (six cases observed, 2.1
expected; p .: 0.05) and for cancer of other female genital organs (three cases
observed, 0.3 expted; p .: 0.05) during folIow-up of up to nine years (Friedman &
Ury, 1980, 1983), and for cancers of the nervous system (other than brain) (three
cases observed, 0.6 expected; p .: 0.05) during follow-up of up to 15 years (Selby et
al., 1989). (Te Working Group noted, as did the authors, that, sincc sorne 12 00
comparisons were made in this study, the associations should be verified
independently. Data on duration of use were not provided.)
NITOFURAOIN 223
4.1 Exposure data
Nitrofurantoin has ben used since 1972 in the treatment of urinary-tract

infections.
4.2 Experimental c8rcinogenicity data

Nitrofurantoin was tested byoral administration to mice in four studies and to
rats in three studies and by transplacental administration to mi
ce in one study. Two
of the studies in mice, including the transplacental study, were inadequate for
evaluation. ln one study in mice, an increase in the incidence of ovarian tubular
adenomas and benign mixed tumours was observed. ln two studies in other strains
of mice, no such increase was observed, although in one study there was an increase
in the incidence of malignant lymphomas in males. One study in rats was
inadequate for evaluation. A further study in female rats demonstrated an increase
in the incidence of mammary fibroadenomas. ln the third study in rats, although a
few rare tumours were observed, there was no significant increase in the incidence
of malignant neoplasms.
4.3 Ruman carcinogenicity data

ln a hypothesis-generating cohort study, use of nitrofurantoin was associated
with the occurrence of cancers of the female genital tract and nervous system, but
these findings could have been due to chance.
4.4. Other relevant data
Use of nitrofurantoin during pregnancy has not been associated with birth
defects. The drug has gonadotoxic effects in male and female rats and mice and
teratogenic effects in mice at high doses.
ln humans, use of nitrofurantoin has been associated with pulmonary fibrosis,
hepatocellular injury, aplastic anaemia and other blood dyscrasias.
Nitrofurantoin gave riegative results in the mouse spot test and in the rat
micronucleus test. It did Dot induce chromosomal aberrations in male germ cells or
dominant lethal effects in mice. It induced DNA strand breaks in rats and mice and
sister chromatid exchange and unscheduled DNA synthesis in bone-marrow cells of
mice. Nitrofurantoin induced DNA strand breaks in mouse, rat and human cells in
vitro and increased the frequency of sister chromatid exchange in Chinese hamster
cens but not in human cells in vitro. Nitrofurantoin induced chromos omal
aberrations in Chinese hamster cells but not in human cens in vitro. It did not
induce unscheduled DNA synthesis in human fibroblasts or rat hepatocytes in vitro.
It induced gene mutations in Chinese hamster cens. Nitrofurantoin gave
ambiguous results in Drosophila in the sex-linked recssive lethal test but positive
results in the wing spot test. It gave contradictory results in tests for mitotic gene
conversion in Saccharomyces cerevisiae. Nitrofurantoin induced differential toxicity
in Escherichia coli, Salmonella tyhimurium and Bacillus subtilis and mutations in
E. coli and S. tyhimurium. (Se Appendix 1.)
4.5 Evaluation!
There is inadequate evidence for the carcinogenicity of nitrofurantoin in

humans.
There is limited evidence for the carcinogenicity of nitrofurantoin in experi-
mental animaIs.
Overall evaluation
Nitrofurantoin is not classifable as to ifs carcinogenicity to humans (Group 3).
5. References
Akerblom, E.B. & Campbell, D.E.S. (1973) Nitrofuiyltriole derivatives as potential

uriaiy tract antibacterial agents. J med. Chem., 16,312-317
Antinnen, H., Ahonen, A., Leinonen, A., Kallioinen, M. & Heiken, E.S. (1982)
Diagnostic imaging of focl nodular hyperplasia of the liver developing durig
nitrofurantoin therapy. Acta med. scand., 211, 227-232
Apteksbolaget (1988) Svensk Läemedelsstatistik (Swedish Drugs Statistics), Stockholm,

Apteksbolaget (1989) Outprint of the Drug Data Bae (17 October 1989), Stockholm,
Aufrere, M.B., Hoener, B.-A. & Vore, M. (1978) Reductive metabolism of nitrofurantoin in
the rat. Drug Metab. Dispos., 6, 403-411
Barnhart, E. (1989) Physician' Desk Refererue, 43rd ed., Oradell, NJ, Medical Economies, pp.
1485-1486

NITOFURAOIN 225
Bass, B.H. (1963) Megaloblastic anemia due to nitrofurantoin. Laet, i, 530-531

Behar, A., Rachmilewitz, E., Rahamimoff, R. & Denman, M. (1965) Expenmental
nitrofurantoin polyneuropathy in rats. Arch. Neurol., 13; 160-163
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NITOFURAOIN 227
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NITOFURAOIN 229
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OTHER DRUGS
elMETIDINE

Cimetiine
1.1 Synonyms

ehem. Ahstr. Name: Guanidine N-cyano-N'-methyl-N" -(2-H(5-methyl-lH-
imidazl-4-yl)methyl)thio ìethyl)-
Synnym: 2-Cyano- 1 -methyl-3-(2-(5-methylimidazo-4-ylmethylthio )ethyl)gua-
nidine
H
1
DeN H3)L~
CH3NHCNHCH2CHßCH2
C wH 16N6S MoL. wt: 252.34
1.3 ChemÎCal and physical properties of the pure substance

From WIndholz (1983) and Bavin et al. (1984)
(a) Description: White to off-white crystallne powder
(b) Meltingpoint: 141-143°C (base), 193°C dec (hydrochloride)
(c) Solubility Soluble (1.14%) in water at 37°C; soluble in ethanol; very
slightly soluble in chloroform; insoluble in diethyl ether. The hydrochlo-
ride is freely soluble in water; soluble in ethanol; very slightly soluble in
chloroform; and practically insoluble in diethyl ether.
-235-
236 !AC MONOGRAHS VOLUME 50
(d) Spectroscopy data: Ultraviolet, infrared, nuc1ear magnetic resonance and

mass spetral data have ben reported.
(e) Stability Dry compound, stored in a c10sed container at room tempe-
rature, showed no decmposition after five years. Cimetidine hydrochlo-
ride is stable for 48 h at normal room temperature when diluted with most
commonly used solutions for intravenous injection.
Trade names: Acibilin; Aciloc; Acinil; Altramet; Cianosel; 'Cim'; Cimal;

Cimegan; Cimet; Cimetid; Cimetidina; Cimetin; Cimetum; Cinamet; Cinulcus;
Citimid; Citius; Climatidine; Dina; Duncamet; Duogastril; Duractin; Dyspamet;
Edalene; Etidine; Eurecptor; Evicer; Fisiol; Fremet; Gasmetin; Gastrobitan;
Gastro H2; Gastromet; Himetin; Itacem; Lucimet; Lucomet; Mansal; Nimus
(Udine) Gadol; Notul; Novocimetine; Peptol; Prometidine; Regastric; SKF 92334;
Stomakon; Tagacid; Tagama; Tagagel; Tagamet; Tametin; Temic; Tratul; Tratul
Retard (SR); Ulcedine; Ulcenon; Ulcerdine; Ulccnen; Ulcestop; Ulcidin; Ulcimet;
Ulcodina; Ulcomedina; Ulhys; Vagolisal; Valmagen
Hydrochlorie: Biomag; Brumetidina; Cimet; Notul
Cimetidine is available for oral administration as 20 or 3O-mg tablets. The
hydrochloride is available for oral administration as a 30 mg(5 ml solution and for
parenteral administration as a 150 mg/ml liquide
Impurities in tablets available in the USA include cellulose, D&C yellow #0,
FD&C Blue #2, FD&C Red #40, FD&C yellow #6, hydroxypropyl methylcellulose,
iron oxides (sec IAC, 1987a), magnesium stearate, povidone, propylene glycol,
sodium lauryl sulfate, sodium starch glycolate, starch and titanium dioxide (see
IAC, 1989a). Impurities in the liquid (oral) preparation are ethanol(2.8%),
FD&C Yellow #6, methylparaben, polyoxyethylene polyoxypropylene glycol, pro-
pylene glycol, propylparaben, saccharin sodium (sec IAC, 1987b), sodium chlo-
ride, sodium phosphate, sorbitol and water. Solutions for intramuscular or intra-
venous injections contain phenol (0.5%; sec IAC, 1989b) (Barnhart, 1989).
Single-dose, premixed plastic containers for intravenous administration are
available (30 mg cimetidine, 0.45 g sodium chloride and no preservative)
(Barnhart, 1989).
N-Nitrosocimetiine
1.1 Synonyms
Chem. Abstr. Seivices Reg. No.: 73785-50-7

CIMETIDINE 237
ehem. Abstr. Name: Guanidine, N-cyano-N'-methyl-N'-nitroso-N" -(2-H(5-

methyl-lH-imidazl-4-yl)methyl)thio )ethyl)-
NO
1
ÜeN H3XÌl
CH3NHCNHCH2CHiSCH2
ClOH1SN7ÛS Mol wt: 281.33
From Bavin et al. (1980) and Foster et al. (1980)
(a) Description: Pale-yellow crystals
(b) Melting-point: 112-113°C
(c) Solubility: Soluble in dimethylsulfoxide
(d) Spectroscopy data: Ultraviolet, nuclear magnetIc resonance and field
desorption mass spectra have been recorded.
(e) Stability: Unstable in alkaline solution
N-Nitrosocimetidine has been synthesized for research purposes (Bavin et al.,

1980; Foster et aL., 1980). The N-methyl-N' -cyanoguanidine moiety of cimetidine
can be converted to the corresponding N-nitroso derivative (N-nitrosocimetidine)
by the action of acidic solutions of nitrite (Bavin et al., 1980).
Cimetidine is prepared from 2-methyl-3-hydroxymethyl-lH-imidazole via a

multistep synthesis involving sequential additions of 2-mercaptoethylamine, di-
methylcyanodithioimidocarbonate and methylamine and variations of this method
(Durant et al., 1974; Bavin et al., 1984). The hydrochloride is prepared by addition of
hydrochloric acid and ethyl acetate as an ethanolic suspension of cimetidine (Bavin
et al., 1984).
Cimetidine is synthesized in Brazl, Hungary, India, Italy, Mexico, the
Republic of Korea, Spain, Taiwan and Yugoslavia (Chemical Information Services,
1989-90).
ln Sweden, cimetidine sales in 1988 were 2.32 defined daily doses (1 g) per 100
Finland, cimetidine sales in 1987were
inhabitants (Apoteksbolaget, 1988, 1989). ln
0.15 defined daily doses per 100 inhabitants (Finnish Committee on Drug
Information and Statistics, 1987). ln the USA, cimetidine was the sixh ranking
prescription drug in 1988 (La Piana Simonsen, 1989).
Cimetidine is not known to ocur as a natural product.
The intragastric formation of N-nitrosocimetidine has been proposed via
reaction of cimetidine with nitrous acid (EIder et al., 1979a,b).
2.2 Use
As a histamine Hi-receptor antagonist, cimetidine inhibits gastric acid

secretion and reduces pepsin output; it may also inhibit other actions of histamine
that are mediated via Hi-receptors. Its clinical indications include duodenal and
gastric ulcers, oesophageal reflux, selected cases of persistent dyspepsia and
pathological hypersecretory states such as the Zollnger-Ellson syndrome. Due to
its capacity to inhibit acid secretion, it is also indicated for the prophylaxs of
gastrointestinal haemorrhage in stress ulceration and in patients at risk of acid
aspiration during general anaesthesia. Cimetidine may also be used to reduce mal-
absorption and fluid loss in patients with the short-bowel syndrome and to reduce
the degradation of enzyme supplements given to patients with pancreatic
insufficiency (Reynolds, 1989). Treatment of damage to the gastric mucosa by non-
steroidal anti-inflammatory drugs (Friedman et al., 1989) is a minor indication.
Cimetidine may be given orally (40 mg two to four times daily), by the
nasogastric route or parenterally by intramuscular or slow intravenous injections
(20 mg) as well as by intraveilous infusion (40 mg in 1 h repeated every 4-6 h)
(Reynolds, 1989). ln maintenance therapy of duodenal ulcer, cimetidine has been
administered daily for up to five years (Barnhart, 1989).
The use of cimetidine in children, in particular in neonates, is limited. ln
full-term neonates, the dosage adjustments are based on renal function, and the
dose of 15-20 mg/kg daily is reduced in premature infants (Ziemniak et al. 1984).
The dosage regimen in children aged 4-13 years is 30 mglg bw daily, divided into
three or more doses (see Reynolds, 1989).
ln elderly people, the standard dose of cimetidine can be reduced by about
30-50% without loss of effectiveness (sec p. 248) (Redolfi et al., 1979).
2.3 Analysis
Analysis of cimetidine and its metabolites in biological fluids by

high-performance liquid chromatography has hen described (Randolph et al.,
1977; Kunitani et al., 1981; Ziemniak et al., 1981; Lloyd & Martin, 1985; Chiou et al.,
CIMETIDlNE 239
1986; Kaneniwa et al., 1986; Strong & Spino, 1987; Rustum & Hoffman, 1988).
Cimetidine can be analysed in pharmaceutical preparations by high-performance
liquid chromatography, thin-Iayer chromatography and spectrophotometric
methods (Bavin et al., 1984; Lovering & Curran, 1985).
A method for the analysis of N-nitrosocimetidine in hum
an gastric juice
samples using reverse-phase high-performance liquid chromatography with an
N-nitroso compound speific detector has ben reported (Shuker & Tannenbaum,
1983).

Carcinogenic Risk to Humans
Cimetuine

Mouse: ln a two-generation carcinogenicity study, groups of male BALB/c and
female C57BI/6 mice (numbers unspeified), seven to eight weeks of age, were given
pharmaceutical-grade cimetidine at either what was stated to be a corn
mon human
dose-0.113 mg/ml-or at 1.13 mg/ml in their drinking-water for two weeks.
Treated mice were mated, and Fo females were treated throughout gestation, lacta-
tion and the remainder of their lives. The hybrid progeny (F 1) were weaned at four
weeks and were also dosed throughout their life. A group of 20 untreated female
C57BL/6 mice served as controls for the treated dams and were mated with
untreated BALB/c males. A group of 51 male and 66 female untreated hybrid
progeny served as controls for the F 1 generation. The average daily doses of
cimetidine were 18.8 mglg bwand 190 mglg bw, and the average total doses were
15.5 glg bw and 155 glg bw in the low-dose and high-dose groups, respectively. AlI
moribund mice were killed and subjected to complete necropsy, and all major
organs, tissues and lesions were examined histologically. Among the treated
C57BI/6 dams, the effective numbers of animaIs were 15 at the low dose and 16 at the
high dose, with mean survival times of 21-24 months; no significant difference in
either survival rates or tumour incidence was observed in comparison with the
control groupe Among the progeny, the effective numbers of females ranged from
39 to 66 among the different groups, with mean survval times of 28.0-30.8 months;
the effective numbers of males ranged from 50 to 79 3;mong the different groups,
with mean survval times of 23.5-27 months. ln the cImetidine-treated progeny, a
significant dose-related increase in the incidence of lymphoid neoplasms (site and
histology unspecified) was observed in females (31/66, 30/65 and 41/59 in the
control, low- and high-dose groups, respetively; p = 0.008, Fisher's exact test)
(Anderson et al., 1985). (Te Working Group noted the high incidence of this
neoplasm in control animaIs.)
Rat: Groups of 65, 70 and 100 male and 65, 70 and 99 female SPF Wistar rats,
5.5 weeks of age, recived clinical-grade cimetidine at 150, 378 or 950 mglg bw
(which represent 30, 75 and 190 times the dose required for 50% inhibition of basal
gastric acid secretion in the rat and are equivalent to 9, 22.6 and 57 times the
recommended daily dose for a 6Okg human) in distiled water by gavage daily for
two years. One control group of 84 males and 85 females recived distilled water by
gavage daily, and another group of 107 males and 108 females served as untreated
controls. Interim kills were carred out at 6, 10 and 12 months after the start of the
experiment, during which a total of 54/235 and 55/23 treated males and females
and 32/191 and 32/193 control males and females were killed. The experiment was
terminated at 105-106 weeks, at which time survival was: males-untreated
controls, 58/107; water controls, 34/84; low-dose, 24/65; mid-dose, 14/70; and
high-dose, 34/100; females-untreated controls, 71/108; water controls, 32/85;
low-dose, 26/65; mid-dose, 18/70; and high-dose, 34/99. During the first year of the
experiment, those rats that died did so mainly as a result of either reflux or direct
administration of the dose into the trachea. AlI rats were necropsied, and major
organs and tissues were examined histologically. An increased incidence ofbenign
Leydig-cell tumours of the testis was observed among treated animaIs (low-dose,
15/65; mid-dose, 14/68; high-dose, 23/98) as compared to combined controls
(35/191). The increase was significant in the low- and high-dose groups (p c( 0.025
for both groups; Peto test: Peto et al., 1980). A slightly greater incidence of
follcular-cell tumours (benign and malignant) of the thyroid gland was observed in
high-dose males (4/98) as compared to control males (2/191) (p = 0.049, Peto exact
test) (Leslie et al., 1981).
Dog: Eight male and four female beagle dogs, 7-9.5 months of age, received a
daily oral administration of cimetidine in film-coated tablets at 144 mg/kg bw for
385 weeks. Four male and two female controls received placebo tablets. Multiple
biopsies of gastric mucosa were taken at intervals of about six months froID week
177 to week 363. Two cimetidine-treated and three control dogs died during the
experiment. AlI animaIs were necropsied, and numerous samples from the stomach
and other major organs and tissues were examined histologically. No increase in the
incidence of either neoplasms or preneoplastic lesions was observed among the
treated animaIs (Walker et al., 1987a). (The Working Group noted the small number
of animaIs used and the high mortality).
CIMETIDINE 241
(b) Administration with other compounds

Mouse: ln the study described on p. 239, groups of male BALB/c and female
C57Bl/6 mice were given sodium nitrite at either 0.184 or 1.84 mg/ml or cimetidine
at 0.113 or 1.13 mg/ml with sodium nitrite at either 0.184 or 1.84 mg/ml in the
drinking-water for two weeks. Treated mice were mated, and females were treated
throughout gestation, lactation and for the remainder of their lives. The hybrid
progeny were weaned at four weeks of age and dosed from that time throughout
their lives. No increase in tumour incidence was seen in the dams. Among the
progeny, there was a dose-related increase in the incidence of lung tumours in
males: 30/52, 36/50 and 71/79 in the untreated control, low- and high-dose groups,
respectively (p ~ 0.01, Cox exact test for trend) (Anderson et al., 1985).
Rat: Two groups of 20 male Sprague- Dawley rats, weighing 250 g, were
wounded surgically in the antro-fundic gastric mucosa. Seven days later, the groups
received 1-1.4 ml of either sodium nitrate at 3.75 mg/ml and sodium nitrite at 0.75
mg/ml in deionized water (nitrate-nitrite solution) or commercial-grade cimetidine
at 25 mg/kg bw in nitrate-nitrite solution, by gavage daily on six days per week for six
months. A group of 50 males served as untreated con troIs. An interim kil of five
rats from each group was carried out at six mon s, and all surviving rats were killed
th
at 14-15 months. Including the five animaIs per group from the interim kill, 19 and
20 animaIs from the nitrate-nitrite and treated groups were necropsied, but samples
for histological examination were taken only from the stomach and grossly visible
lesions. No neoplasm was found (EIder et al., 1982). (The Working Group noted the
small number of animaIs used, the short duration of the study and the limited
histological examination.)
Two groups of 25 Sprague-Dawley rats received weekly subcutaneous
injections of either 1,2-dimethylhydrazne alone at 20 mg/kg bw for 16 weeks or
concurrently with cimetidine at 100 mg/kg bw daily in the drinking-water for 26
weeks, at which time the experiment was terminated. One group of ten rats received
cimetidine treatment only. No increase in the incidence of colonie tumours was
observed in the combined cimetidine plus 1,2-dimethylhydrazine-treated group
(15/22) over that in the group receiving 1,2-dimethylhydrazine alone (14/22); no such
tumour occurred in rats given cimetidine alone (Nee et al., 1984). (The Working
Group noted the small number of animaIs used and the short duration of
treatment. )
Two groups of 15 weanling male Sprague- Dawley rats received 1,2-dimethyl-
hydrazne at 30 mg/kg bw in saline by gavage once a week for five weeks. Four days
after the last treatment, the groups received either cimetidine at 500 mg/ml in the
drinking-water or drinking-water alone, until the animaIs were killed, seven months
after the beginning of the experiment. AlI animaIs were necropsied,.and samples
from the gastrointestinal tract and lymph nodes from the peritoneal cavity and
242 !AC MONOGRAHS VOLUM 50
lungs were examIned histologically. The incidence of colonie carcinomas among

survivors was significantly different (p .: 0.05; Mann-Whitney non-parametric test)
for the group reciving 1,2-dimethylhydrazne (4/13) compared with that reciving
1,2-dimethylhydrazne and cimetidine together (10/14) (Caignard et al., 1984). (Te
Working Group noted the absence of a group treated with cimetidine only.)
N-Nitrosocimetiine
Beause of the suspicion that N-nitrosocimetidine might be a carcinogenic
derivative of cimetidine, it was tested in a number of studies. N-Nitroso compounds
are often potent carcinogens, and so, in these studies, smal numbers of animaIs
were used. Since the studies would have detected a potent carcinogen, they are
included to support the interpretation thafN-nitrosocimetidine is, at least, not a
strong carcinogen.

Mouse: ln the study described on p. 239, groups of male BALB/c and female
C57Bl/6 mice were given N':nitrosocimetidine (purity, 98%) at either 0.113 or 1.13
mg/ml in the drinking-water for two weeks. Treated mice were mated, and females
were treated throughout gestation, lactation and for the remainder of their lives.
The hybrid progeny were weaned at four weeks of age and dosed from that time
throughout their life. The average daily doses of N-nitrosocimetidine were 18.8
mg/g bwand 190.0 mg/g bw, and the average total doses were 15.5 glg bwand 155
g/g bw in the low-dose and high-dose groups, respectively. No significant
difference in either survval rates or the incidence of tumours was observed between
treated and control groups (Anderson et al., 1985).
Two groups of 20 female hybrid B6D2F 1 mice, eight weeks of age, were given
four weekly intragastric administrations of olive oH, which was used as the vehicle
for other compounds; one week after the last dose, they received either
N-nitrosocimetidine at 1.13 mg/ml in the drinking-water or deionized water alone.
AlI survivors were killed 14 months after the beginning of treatment. Papilomas of
the forestomach ocurred in 2/20 mice receiving N-nitrosocimetidine and in 0/19
controls (Anderson et al., 1988). (Te Working Group noted the small number of
animaIs used.)
Groups of male BALB/CanNCr mice, six weeks of age, received intra-
peritoneal injections of saline solution once a week for ten weeks, after which time
theywere given N-nitrosocimetidine at 0, 1.0 or 1.8 mg/ml in the drinking-water. AIl
survvors were killed at 14 months. The effective numbers of animaIs were 13 in the
group reciving N-nitrosocimetidine and 15 in the control groupe Lung neoplasms
(type unspecified) were observed in 3/13 animaIs in each treated group and in 6/15
CIMEllDINE 243
control mice (Anderson et al., 1988). (The Working Group noted the small number
of animaIs used and the short duration of the study.)
Rat: Groups of 20 male and 20 female outbred Sprague-Dawley rats,
approximately 100 days old, recived N-nitrosocimetidine at 50 or 500 mg/kg bw by
gavage twce a week for one year. A group of 50 male and 50 female rats served as
untreated con troIs. AlI animaIs were observed for life or were killed when
moribund, and were necropsied. Samples from the forestomach, glandular
stomach, duodenum and all other organs with gross lesions were examined
histologically. Mean survival was 393 days in high-dose animaIs, 400 days in
low-dose animaIs and 630 days in controls. No increase in the incidence of tumours
and no gastric neoplasm were found in treated animaIs (Habs et al., 1982a,b). (The
Working Group noted the small number of animaIs used and the poor survival of
treated animaIs.)
Two groups of 20 male Sprague- Dawley rats, weighing 250 g, were wounded
surgically in the antro-fundic gastric mucosa. Seven days later, the groups received
1-1.4 ml of either sodium nitrate at 3.75 mg/ml and sodium nitrite at 0.75 mg/ml in
deonized water (nitrate-nitrite solution) or N-nitrosocimetidine at 2.80 mg/ml in
nitrate-nitrite solution, by gavage daily on six days per week for six months. A group .
of 50 males served as untreated controls. An interim kill of five rats from each group
was carried out at six months, and all surviving rats were killed at 14-15 months.
Including the five animaIs per group froID the interim kil, 19, 16 and 9 animaIs from
the respective groups were necropsied, but samples for histological examination
were taken only from the stomach and grossly visible lesions. A gastric carcinoma
at the site ofwoundingwas found in one rat treated with N-nitrosocimetidine. No
other neoplasm was found (EIder et al., 1982). (The Working Group noted the small
number of animaIs used.)
Groups of 20 male and 20 female Fischer 344 rats (age unspecified) received
N-nitrosocimetidine at 20 ml (0.15 mg/ml) in deionized water daily as drinking fluid
on five days per week for 106 weeks (total dose, 1.6 g/rat). Groups of 20 males and 20
females were untreated and served as controls. AlI survivors were killed at week
131, and all animaIs were necropsied and major organs and lesions examined
histologically. At 90 weeks, survval was 19/20 males and 20/20 females in the
treated group and 15/20 males and 17/20 females in controls. No increase in the
incidence of tumours was observed (LijinkSy & Reuber, 1984). (The Working Group
noted the small number of animaIs used.)
(h) Skin application

Mouse: Groups of 20 female Swiss mice (age unspecified) received
twce-weekly 25-l11 skin applications on the shaved interscapular skin of either
N-nitrosocimetidine (reagent grade) at 2.2 or 5.6 mg/ml in acetone (total doses, 12 or
31 mg/mouse), or acetone alone for 110 weeks or were left untreated. Skin was the
only tissue examined grossly and histologically. At week 100, survival among
treated animaIs was 18/20 at the low dose and 13/20 at the high dose. A malignant
lymphoma of the skin (site unspecified) was observed in one high-dose mouse
(Lijinsky, 1982). (The Working Group noted that the tumour incidence among
control groups was not specified and that survival was por in the group given the
high dose.)

(i) Absorption, distriution, e.retion and metabolism

The kinetics, absorption, distribution, metabolism and elimination of
cimetidine in humans and experimental animaIs have been reviewed (Griffiths et al.,
1977).
ln rats and dogs, cimetidine is rapidly absorbed; the concentration of
unchanged cimetidine in plasma is greater than that of any metabolite, and the
plasma half-time is about 1 h. The drug is excreted mainly unchanged in the urine.
The principal metabolite in both rats and dogs is formed by oxidation of the
side-chain sulfur to give the sulfoxide (Taylor, D.C. et al., 1978).
ln rats, detoxication of N-nitrosocimetidine involves denitrosation, primarily
(but not exclusively) by haemoglobin sulfhydryl residues. The rates of degradation
of N-nitrosocimetidine by isolated whole blood decreased in the order: rat ~ mouse
t: guinea-pig ~ hamster ~ human; the half-time of N-nitrosocimetidine at 37° C
was ,.2 min in hamster blood and 27 min in human bloo. After intravenous admi-
nistration to hamsters in vivo, the half-time of N-nitrosocimetidine was ~5 min and
degradation via denitrosation reached 100%. Additional denitrosating activity was
found in the cytosol of several organs from rats and hamsters; this activity required
reduced glutathione (Jensen, 1983; Jensen et al., 1987).
The metabolic fate of N-nitrosocimetidine has been investigated, although it
has not been shown to be formed in animaIs in vivo.
Radiolabelled N-nitrosocimetidine, but not cimetidine, methylated DNA in a
variety of tissues in rats after oral administration (Gombar & Magee, 1982). ln
studies in which cimetidine was administered with an excess of nitrite by stomach
tube to rats, no evidence could be obtained for the presence of 06-methylguanine in
DNA isolated from stomach, liver or intestine (large and small pooled)
(Kyrtopoulos et al., 1982). N-Nitrosocimetidine produced a low level of DNA
alkylation (determined as 7-methylguanine) in the liver and other organs of
hamsters after intravenous administration (Jensen et al., 1987).
CIMETIDINE 245
(ii) Toxic effects

The oral LD5( of cimetidine were approximately 2.6 g/kg bw in mice, 5 g/kg bw
in rats, 4 g/kg bw in hamsters and 2.60 glg bw in dogs. The intraperitoneal LD5()
were 470 mglg bw in mi ce, 650 mglg bw in rats and 88 mglg bw in hamsters
(Crean et al., 1981).
Daily oral administration of 160 mglg bw cimetidine to female
Sprague-Dawley rats for two months increased total gastrin-cell numbers,
gastrin-cell density of antral mucosa and parietal-cell density of fundic mucosa, as
compared with controls (Del Tacca et al., 1987). ln contras t, in male WistarlLwis
rats reciving cimetidine orally at 150-20 mglg bw daily for up to 12 months, it was
not possible to demonstrate by autoradiography epithelial proliferation in either
fundus or antrum as a consequence of treatment (Eastwood & Quimby, 1983).
ln studies of up to 24 months' duration, rats receiving repeated doses of
cimetidine at up to 950 mglg bw pcr day showed few adverse effects. Liver weight
was consistently increased at the highest dose, and testis, prostate and seminal
vesicle weights were reduced in a dose- and time-related manner (Brimblecombe et
al., 1985).
ln a study of up to 12 months' duration, two dogs reciving cimetidine at 504

mg/kg bw per day orally exhibited degenerative changes in the liver, renal tubular
nephrosis and elevated levels of serum transaminases. No such change was seen
with doses of 36 mglg bw per day or less. Prostate weights were reduced in a dose-
and time-related manner. ln beagle dogs administered cimetidine at 144 mg/kg bw.
per day orally, no treatment-related effect was seen after four years, on the basis of
haematology, clinical biochemistry, urinalysis, electrocardiography or clinical
condition, and no treatment-associated change was observed in biopsies of gastric
mucosa. After seven years of follow-up, no change of the stomach mucosa was seen
during regular biopsy-(Crean et al., 1981; Brimblecmbe et al., 1985).
ln-vivo and in-vitro studies suggest that cimetidine inhibits gastric acid
secretion in rats and dogs by blocking histalf'ine Hi-recptors in the gastric mucosa
(Brimblecmbe et al., 1978). Cimetidine administered intraperitoneally to male
Wistar rats at 20 mglg bw twce daily for seven days reduced the gastric mucosal
concentrations of prostaglandin Ei and 6-keto-prostaglandin F la, both 30 min and
24 h after the last injection (Arakawa et al., 1988).
ln rats, the oral LDSO of N-nitrosocimetidine did not differ from that of
cimetidine itself. No tissue-specific toxic lesion could be attributed to the nitroso
derivative (Ogiu et al., 1986).
ln routine safety evaluation studies on cimetidine, including fertility and peri-
and postnatal studies in rats and teratology studies in mice, rats and rabbits, no
adverse effect was reported with oral doses of up to 950 mglg bw (Brimblecmbe et
al., 1978). (The Working Group noted the lack of detaIled reporting.)
Cimetidine has ben shown to possess weak antiandrogenic activity in rats, as
shown by reduced weights of testis, prostate and seminal vesicles (Brimblecmbe et
al., 1978; Pereira, 1987). Inhibition of dihydrotestosterone binding to the prostatic
androgen receptor has ben demonstrated (Sivelle et al., 1982).
Differentiation of the genital organs of male fetuses is influenced by
endogenous testosterone produced during prenatal development, and gonadal and
sexual dysfunction have ben reported in adult male rats afer prenatal and
neonatal exposure to cimetidine at daily doses of 17.1 and 137 mglg bw in
drinking-water from day 12 of pregnancy untIl weaning on postnatal day 21 (Anand
& Van Thiel, 1982; Parker et aI., 1984a,b). These results were not confirmed in other
studies. Rats were administered cimetidine at 180 mglg bw daIly in the drinking-
water from day 12 of pregnancy untIl the end of lactation or during late lactation
only, or a combination of drinking-water during pregnancy and early lactation and
gavage treatment during late lactation. Several end-points, such as anogenital
distance, serum testosterone, mating performance and sexual organ weights, were
evaluated soon after littering or up to 148 days postnatally. Maternally admi-
nistered cimetidine had no effect in male offspring on any parameter measured
(Walker et al., 1987b).
ln another study, cimetidine was administered to rats in drinking-water from
day 17 of gestation through day 7 of lactation. With the highest drug concentration
tested (4 mg/ml), the daily dose ingested ranged from about 40 mglg bw before
parturition to approximately 850 mglg bwafterwards. The developmental profile
of serum dehydroepiandrosterone, androstenedione, testosterone and 5-Q!-di-
hydrotestosterone, when measured at 1, 4 and 18 weeks of age, was unaffected by
perinatal exposure to cimetidine (Shapiro et al., 1988).
The effects of maternally administered cimetidine during lactation on the
development of drug metabolizing enzymes in BALB/c mouse pups have been
investigated. When dams were administered cimetidine at 50 mg/kg bw per day
intraperitoneally for six weeks after delivery, microsomal enzme activity was
inhibited in pups. Dams were less affected than pups (Kwanashie, 1989).

Cimetidine did not induce differential toxicity in Escherichia coli (Pool et al.,
1979; De Flora, 1981).
Cimetidine was reported not to be mutagenic to Salmonella tyhimurium (De

Flora & Picciotto, 1980; O'Connor et aL., 1987). Cimetidine alone or in combination
with sodium nitrite gave negative results in S. tyhimurium in a host-mediated assay
in mice (Baumeister, 1982).
CIMETIINE 247
No single-strand breakage, measured by the alkalIne elution assay, was found

in the DNA of a transformed mouse epithelial cell line after treatment with
cimetidine at concentrations of up to 5 mM (Schwarz et al., 1980). ln an alkaline
elution assay with rat primary hepatoces, the highest concentration of cimetidine
hydrochloride (3 mM) induced a significant increase in the frequency of DNA
strand breaks (Martell et al., 1983). No such effect was observed in human
hepatoces (Martell et al., 1986).
Cimetidine hydrochloride induced unscheduled DNA synthesis in cultures of
rat primary hepatocytes (Martell et al., 1983; Lefevre & Ashby, 1985), while the base
was inactive (Lefevre & Ashby, 1985). ln another study, cimetidine (form not
speified) did not induce unscheduled DNA synthesis in rat hepatocytes (Williams
et al., 1989). Human hepatocytes from four donors did not exhibit unscheduled
DNA synthesis after treatment with cimetidine hydrochloride (Martell et al., 1986).
ln an abstract, it was reported that cimetidine did not induce mutation to
6-thioguanine resistance, to trifluorothymidine resistance or to ouabain resistance
in a human lymphoblastoid cell line (TK6) after 1 -h treatment with concentrations
of up to 1.2 mM, with a cell survival of 6% (Tatsumi et al., 1987).
Cimetidine did not induce sister chromatid exchange in human lymphocytes in
vitro (Inoue et al., 1985).
When rats were given cimetidine orally at 250 mglg bw, no DNA damage was
detected in cells of the gastric mucosa (Pino & Robbiano, 1983) or in liver cells
(Brambila et al., 1982); combination treatment with sodium nitrite also gave
negative results.
N-Nitrosocimetidine and cimetidine treated with sodium nItrite in an acid
environment like that of human gastric juice have consistently been shown to be
directly mutagenic in vitro: either treatment induced differential toxicity and
mutation in bacteria, inhibited DNA synthesis in human cells and induced DNA
single-strand breaks, mutation, sister chromatid exchange, chromosomal damage
and morphological transformation in mammalian cells. For a comprehensive
tabulation of these data and their references, see Appendix 2.
(h) Humans
(i) PharmcokInetics
The pharmacokinetics of cimetidine have been reviewed (Reynolds, 1989).
Cimetidine is readily absorbed from the gastrointestinal tract, and peak
plasma concentrations are obtained about 1 h after administration on an empty
stomach and about 2 h after administration with food (Somogyi & Gugler, 1983).
The bioavailabilty of cimetidine is 60-70%, as a result of moderate first-pass
metabolism. Twenty percent of cimetidine is bound to plasma proteins, and its
elimination half-time from plasma is about 2 h; it is partially metabolized in the liver
to the sulfoxide and to hydroxymethylcimetidine. Afer intravenous adminis-

tration, 50-80% of the dose was excreted unchanged in the urine. Afer an oral dose,
the corresponding figure was 40%. Cimetidine penetrates the bloo-brain barrier
with difficulty but easily crosses the placental barer and is excreted into milk,
where the concentrations may be higher than those in plasma. Dose-dependent
kinetics of cimetidine have ben observed in neonates (Lloyd et al., 1985).
A markedly reduced plasma clearance of cImetidine has ben reported in
elderly patients (Gugler & Somogy, 1979; Redolfi et al., 1979). A decrease in
non-renal clearance of cimetidine was reported in patients with lIver cirrhosis
(Gugler et al., 1982; Cello & Oie, 1983); in one study, this effect was limited to
cirrhosis patients with a history of hepatic encephalopathy (Ziemniak et al., 1983).
Renal failure may lead to elevated plasma levels of cimetidine (Las
son et al., 1982).
The toxicity of cimetidine has ben reviewed (Penston & Wormsley, 1986).
Adverse effects are infrequent and are usually reversible following reduction of
dosage or withdrawal of therapy. Diarrhoea, rashes and other allergic phenomena
have been reported. Various symptoms of the central nervous system have been
reported frequently, particularly at greater than therapeutic doses (Nelson, 1977;
Illngworth & J arve, 1979; Schentag et al., 1979). Adverse haematologicaleffects
possibly associated with cImetidine are rare and include granulocytopenia or
agranulocytosis, thromboopenia and pancytopenia (Penston & Wormsley, 1986).
Strongly reduced gastric acid secretion favours colonization of the stomach by

bacteria, sorne of which can reduce nitrate to nitrite and catalyse nitrosation of
amino precursors at neutral pH (Hil, 1986; Leaf et al., 1989). Since these conditions
could lead to intragastric formation of nitroso compounds, gastric juice samples
from patients before and after treatment with cImetidine have ben analysed for
total nitroso compounds or nitrite in several studies. Fasting gastric juice from 140
patients taking cimetidine for a variety of gastric or duodenal disorders ( daily doses
of 0.2- 1.6 g for periods of one week to 45 months) and 267 subjectswho had not
taken the drug were analysed. Significantly higher mean levels of total nitroso
compounds were found in the former group (Reed et al., 1981).
ln a study of a group of 23 peptic ulcer patients after a six-week course of 1 g
cimetidine per day, a statistically significant increase in nitrite and nitroso
compound concentrations was found (Stockbrugger et al., 1982). ln six volunteers
who took 20 mg cimetidine three times a day and 40 mg a night for at least three
weeks, increases in the level of gastric juice nitrite were found only rarely (Muscroft
et al., 1981). ln eight healthy subjects studied half-hourlyor hourly for 24-h periods
before, during and after cimetidine treatment (two weeks with 1 g per day), no
CIMETIDINE 249
significant difference in the level of intragastric nitrite or total nitroso compounds

was found following cimetidine treatment (Milton-Thompson et al., 1982).
Gastric juice and urine collected from 17 duodenal ulcer patients receïving
0.8 g cimetidine per day for four to six weeks were analysed for nitrosation capacity
before, during and after treatment using the N-nitrosoproline test (Ohshima &
Bartsch,1981). Cimetidine treatment did not lead to a uniform or pronounced ri se
in gastric levels of total nitroso compounds or urinary N-nitrosoproline levels
(Bartsch et al., 1984).
The methods used for determining total nitroso compounds in gastric juice in
all these studies have been criticized beause oftheir lack of specificity (Pignatell et
al., 1987); moreover, in none of the studies was N-nitrosocimetidine itself measured
in the gastric juice samples.
Cimetidine inhibits cytochrome P450-dependent microsomal mixed-funciton
oxidase (Pelkonen & Puuronen, 1980), elevating the plasma levels of other drugs,
such as lignocaine, phenytoin, theophyllne, warfarin and ciclosporin (Somogyi &
Muirhead, 1987; Rodighiero, 1989).
A number of case reports have been published of ci meti di ne use in pregnancy,
indicating no adverse effect (Coraza et al., 1982; Meggs et al., 1984) or ab normal
outcome (Gladeet al., 1980; Say et al., 1985); the significance ofthese reports cannot
be assessed.
ln a UK postmarketing surveilance study, 9928 patients given cimetidine in
general practices were compared with 9351 age- and sex-matched unexposed people
from the same practices; 98.8% of takers and 97.7% of con troIs were successfully
followed up for at least one year (Colin-Jones et al., 1983, 1985a,b). ln the 20 exposed
women and the 22 controls who became pregnant during the study year, therewas
no evidence of an adverse effect of cimetidine treatment.
Impotence, reduced sperm count and gynaecomastia have been reported in
men treated with cimetidine. ln one study, gynaecmastia was reported in 20/6240
men (3.21100), but in only 13 of these was cimetidine thought to be the likely cause.
Impotence was reported in 121624 men (21100) taking cimetidine and in 3/5868
controls (0.5/100); however, impotence and gynaecmastia were not reported in
the same individuals (Colin-Jones et al., 1985a,b). (The Working Group considered
that, because of the method of ascertainment, underreporting was likely.) ln other
studies in which cimetidine was administered at doses larger than those normally
used in ulcer therapy, these adverse effects were more common. ln a study by
Jensen et al. (1983), of patients with gastric hypersecretion (mostly Zollnger-Ellison
syndrome) who were treated with high doses of cimetidine, 11/22 subjects
developed one or more signs or symptoms of impotence, breast tenderness or
gynaecomastia. The Il affected subjects had recived a mean daily dose of 5.3 g,
compared with 3.0 g in the unaffected groupe Spence and Celestin (1979), however,
reported gynaecmastia in 5/25 (20%) male patients treated with 1.6 g cimetidine
daily. ln all the reported cases, the condition reversed rapidly after cessation of and
sometimes during treatment.
Several well-controlled studies have shown no effect of cimetidine on sperff
count (Wang et al., 1982; Paulsen et al., 1983; Bianchi Porro et al., 1985), but one
showed a smaH but significant reduction (Van Thiel et al., 1979). No consistent
effect on plasma levels of gonadotrophins or sex hormones was demonstrated in
these studies.

When gastric juice from patients who had recived cimetidine at 20 mg 2 h
earlier was incubated with sodium nitrite at 10 iiglml, a significant increase in the
inumber of revertants was seen in S. tyhimurium TA100 in the absence of an
exogenous metabolic system (DeFlora & Picciotto, 1980).
Gastric juice taken from 49 patients 1-3 h after intake of cimetidine at 40 mg
was mutagenic when tested in S. tyhimurium TA100; no mutagenic activity was
seen in samples taken 12 h later (Morris et al., 1984). When the mutagenicity of
gastric juice from eight fasting patients under cimetidine treatment (40 mg twice
daily) was tested before and after administration of the drug, using S. tyhimurium
TA98 and TA100, considerable variation in mutagenicity was seen between sub-
jects, all samples being mutagenic even before therapy. No significant change in
mutagenic activity was detected after therapy and there was no relation to duration
of therapy, changes in gastric pH or ulcer healing (O'Connor et al., 1987)
3.3 Case reports and epidemiological studies in humans

(a) ease reports
Numerous case reports of neoplasms following cimetidine use have been
published (Welsh et al., 1977; Murray et al., 1978; Taylor, T~ et al., 1978; Buck et al.,
1979; EIder et al., 1979a,b; Reed et al., 1979; Taylor et al., 1979; Hawker et al., 1980;
Kjærgaard et al., 1980; Knigge et al., 1980; Stoddard et al., 1980; Sctcher et al., 1981;
Taylor et al., 1981; Kaplinsky et al., 1982; Eschar et al., 1983; Porter et al., 1984;
Stockley & Kiff, 1987). The stomach has been the most frequently reported cancer
site, followed by lymphomas of the gastrointestinal tract; less commonly, benign
and malignant tumours at other sites have been reported (Kaplinsky et al., 1982;
Eschar et al., 1983). Most of the cases were diagnosed within one year; the longest
interval was five years (Stockley & Kiff, 1987). ln certain of the reports, gastric
carcinoma had arisen after oesophagitis or duodenal ulcer-conditions not thought
to be associated with gastric carcinoma. ln other report, gastric ulcers diagnosed
CIMETIDINE 251
as benign on the basis of biopsy samples and appearance on gastroscopy have been
followed by malignant changes.
(h) eohort studies
A cohort of (initially) 99 individuals who recived cimetidine during 1977-79

in the UK and who were followed up for four years has been the subject of four
papers (Colin-Jones et al., 1982, 1983, 1985a,b). AlSO studied were (initially) 9366
controls matched for age, sex and general practice, who did not receive cimetidine,
but these were followed up for only one year. For this reason, national rates were
used as the standard for evaluation of mortality (incident cancers were evaluated
only in the first year). A total of 330 deaths were due to neoplasms in cimetidine
users (compared to 65.6 expected), most of the excess being gastric and lung
cancers. The relative risks (RRs) declined markedly over the study period: for
gastric cancer, the approximative RRs for men and women combined (and
observed numbers) were 12.9 (45 deaths), 3.4 (12), 1.8 (6) and 2.3 (8) after one, two,
three and four years follow-up, respectively. Some of the patients were known to
have had cancer before cimetidine use was started, and, even among deaths after
the first year, there were three such deaths from gastric cancer. For lung cancer, the
RRs in the four periods were 2.8 (35 deaths), 2.0 (25), 1.4 (17) and 1.8 (22). ln an
approximately 5% sample of study participants, 45% of the exposed and 28% of
unexposed subjects had smoked ;: 15 cigarettes per day five years previously. (The
Working Group noted that cigarette smoking is associated with peptic ulcer as well
as lung cancer and that smoking habits were not accounted for in the evaluation of
lung cancer risk.)
A secnd cohort study concerned cancer incidence among 16 739 patients
treated with cimetidine in Denmark in the period 1977-81 who were followed for up
to eight years by linkage procdures with the central population and national cancer
registers (Møller et al., 1989). A total of 105 cases of gastric cancer were recorded,
compared with 39.5 expected (RR, 2.6). The RR was greatest early in the follow-up
period, declining markedly later. For sites with positive associations and for all
other sites combined, the RRs and observed numbers of cases during the first year,
during the secnd and third years and for more than three years of follow-up are
shown in Table 1. The increased risks for gastric cancer were greater in persons with
a diagnosis of gastric ulcer (19.3 (45 observed cases), 3.5 (16) and 3.2 (13) for the
three time periods) than in those with duodenal ulcer (2.2, 1.1 and 1.1, with seven
observed cases each). Increased incidences of gastric and certain other
gastrointestinal malignancies were noted particularly in the first year of follow-up.
The increased incidences of lymphatic and haematopoietic malignaIlcies were due
mainly to non-Hodgkin's lymphoma of the stomach and small intestine. The risk
Table 1. Relative risks (RRs) and numbers of cases observed after

different periods in patients treated with cimetidinea
Site -: 1 year 1-3 years ~ 3 year
RR Obs RR Obs RR Obs
Stomach 9.4 57 2.3 27 1.7 21

Pancreas 4.7 21 1.5 15 1.7 14
Colon 2.1 23 1.8 38 1.3 26
Small intestine 10.5 4 3.9 3 3.4 2
Lymphatic and haematopoietic 3.0 22 0.9 13 1.5 20
tissue
Lung 1.4 34 1.7 83 1.6 73
Other sites 1.1 91 1.4 218 1.1 169
a:rom Møller et al. (1989). RRs calculated by the Working Group for the two sexes
combined
for lung cancer did not change appreciably over time. (The Working Group noted
that no data on smoking status were provided.)
By record linkage, 3802 cimetidine users and an equal number of non-users
matched byage, sex and general practitioner in Tayside, Scotland, were followed up
for mortality during a period of four years (Beardon et al., 1988). Mortality due to
neoplasms of the digestive organs and peritoneum was markedly increased in the
cimetidine takers, with a RR of 2.7 (95% confidence interval, 1.6-4.7) based on 49
deaths. The largest excesses were seen for neoplasms of the oesophagus, stomach
and pancreas. Mortality from all causes among cImetidine users was increased only
during the first two years of follow-up (corresponding data by duration of follow-up
were not presented for cancer). There was no significant increase in mortality due
to neoplasms of respiratory and intrathoracic organs (32 deaths; RR, 1.1; 95%
confidence interval, 0.67-1.8) or other neoplasms.
(The Working Group noted, as did the authors of the relevant studies, that in
the case reports and studies undiagnosed gastric and intra-abdominal neoplasms
could have been responsible for the symptoms that led to use of cimetidine. This
possibility is supported by the short interval between exposure and observation of
increased RRs and by the decreasing risks for intra-abdominal cancer, particularly
gastric cancer, with time. The maxmal interval that follow-up studies have so far
covered is only eight years.)
CIMETIDINE 253
4. Summaryof Data Reported and Evaluation

4.1 Exposure data
Cimetidine is a histamine Hi-receptor antagonist which inhibits gastric acid

secretion. Since its introduction in the mid- 1970s, it has been used widely by oral
administration for the treatment of duodenal and gastric ulcers.
Although cimetidine can be nitrosated in vitro in the presence of nitrite under
acidic conditions to form N-nitrosocimetidine, no study in experimental animaIs or
in humans has demonstrated that this reaction occurs in vivo.

Cimetidine was tested for carcinogenicity by oral administration in single
studies in mice, rats and dogs. ln the experiment in mice, dams were treated
throughout life beginning two weeks prior to pregnancy, with no increase in tumour
incidence. ln female progeny that were exposed throughout life from conception,
there was an increase in the incidence of lymphomas, although these tumours also
occurred at relatively high rates in control animaIs. ln rats, an increase in the
incidence of benign Leydig-cell tumours of the testis was observed in the low- and
, high-dose groups but not in the mid-dose groupe The study in dogs was inadequate
for evaluation.
ln a study in which mice were exposed from conception throughout life to a
combination of cimetidine and sodium nitrite, males had an increased incidence of
lung neoplasms, although these tumours also occurred at a high frequency in
control animaIs.
N-Nitrosocimetidine was tested for carcinogenicity byoral administration in
mice and rats and by skin application in mice. The experiments in rats and three of
the studies in mice were inadequate for evaluation. ln one study by oral
administration in mice, there was no increase in the incidence of tumours.

ln a large number of case reports, cancer, particularly gastric cancer, was
diagnosed at various intervals after the start of cimetidine therapy. These reports
are difficult to interpret because gastric cancer Is a common malignancy and .
cimetidine is a commonly used drug, and coincidence cannot be ruled out.
Three cohort studies showed increased incidences of gastric cancer but also of
other gastrointestinal cancers among cimetidine users; however, as for the case
reports, the association could weIl have been due to the drug being given for
symptoms of pre-existing cancers. This interpretation is supported by a diminution
of the association with increasing duration of follow-up. Two of the studies also
showed an association between cimetidine use and lung cancer, but confounding
with cigarette smoking could well have been the explanation.

Cimetidine has been associated with reversible impotence and other anti-
androgenic effects in men.
N-Nitrosocimetidine is rapidly converted to cimetidine in vivo in experimental
animaIs.
Cimetidine did not induce single-strand breaks in DNA from rats treated in
vivo, nor did it methylate DNA in a variety of tissues of rats in vivo. It did not induce
single-strand breaks in the DNA of rat cells treated in vitro. Cimetidine was not
mutagenic to and did not cause DNA damage in Salmonella tyhimurium or
Escherichia coli. Cimetidine hydrochloride induced single-strand breaks and
unscheduled DNA synthesis in rat but not human cells in vitro. It did not cause
sister chromatid exchange in human cells in vitro.
Cimetidine in combination with sodium nitrite did not induce DNA damage in
vivo or methylate DNA in a variety of tissues of rats in vivo. Gastric juice from
cimetidine-treated patients was mutagenic to bacteria when enriched with nitrite.
N-Nitrosocimetidine has not been demonstrated in gastric juice of humans;
however, increased gastric concentrations of nitrite and total N-nitroso compounds
have been reported in some studies of patients taking cimetidine. N-Nitroso-
cimetidine induced DNA damage, sister chromatid exchange, chromosomal
aberrations and morphological transformation in mammalian cells in vitro and
caused DNA damage and mutation in bacteria. Radiolabelled N-nitrosocimetidine
methylated DNA in a variety of tissues of rats in vivo. (See Appendix 1.)
4.5 Evaluationl
There is inadequate evidence for the carcinogenicity of cImetidine in humans.

There is inadequate evidence for the carcinogenicIty of cImetidine in
Overall evaluation
Cimetidine is not classifible as to ifs carcinogenicity to humans (Group 3).
IFor description of the italicize terms, se Preamble, pp. 2629.

CIMETIDIN 255
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Some Organi,e Solvents, Resin Monomers an Related Compounds, Pigmnts an
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CIMETIDINE 259
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o
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CIMETIDINE 261
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CIMETIDINE 263
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30-38
DANRON (eHRYSAZIN;
1,8-DIHYROXYANTHRQUINONE)
1.1 Synonyis
ehem. Abstr. Services Reg. No.: 117-10-2 (replaces CAS Reg. No. 32073-07-7)
ehem.Abstr. Name: 9,10-Anthracenedione, 1,8-dihydroxy-
Synnym: Antrapurol; danthron; dianthon; dihydroxyanthraquinone; 1,8-di-
hydroxy-9,10-anthraquinone; dioxyanthrachinonum; 1,8-dioxyanthraquinone
HO o OH
o
Cl~804 MoL. wt: 240.23

(a) Description: Red or reddish-yellow needles or leaves (froID ethanol)
(Weast, 1985); orange crystallne powder (Anon., 1981)
(h) Me/ting-point: 193°C (Weast, 1985); 195°C (Anon., 1981)
(c) Spectroscopy dataI: Infrared (Coblenz (5147); Aldrich, prism (90D);
Aldrich, prism-Fl (87D)), ultraviolet (Sadtler (4318)), proton nuclear
lBracketed numbers are spectrum numbers in the relevant compilation.
-265-
magnetic resonance (Aldrich (91B)) and mass (Aldermaston (195))
spectral data have been reported (Sadtler Research Laboratories, 1980;
Pouchert, 1981, 1983, 1985; Weast & Astle, 1985).
(d) Solubility Very soluble in aqueous alkali hydroxides; soluble in acetone,
chloroform, diethyl ether and ethanol; almost insoluble in water (Enviro
Control, 1981; Weast, 1985)
i.4 Technical products and impurities
Trade Names: Altan; Antrapurol; Bancon; Benno; DanSunate D; Danthron;

Diaquone; Dionone; Dorban; Dorbane; Duolax; Fructines-Vichy; Istin; Istizin;
Julax; Laxanorm; Laxans; Laxanthreen; Laxenta; Laxpur; Laxpurin; Modane;
Neokutin S; Pastomin; Prugol; Roydan; Scatron D; Solven; Unilax; Zwitsalax
The following trade names are those of multi-ingredient preparations
containing dantron: Agarol Capsules; Coloxyl; Dorbanate; Dorbanex; Dorbantyl;
Doss; Doxidan; Normax (Reynolds, 1989).
Dantron is available commercially at a purity of 95-99% (Aldrich Chemical
Co., 1988; Lancaster Synthesis Ltd, 1988; Sigma Chemical Co., 1988).
(a) Production
Dantron has been prepared by several processes, including the alkaline
hydrolysis of 1,8-dinitroanthraquinone, the caustic fusion of 1,8-anthraquinone-
disulfonic acid, the diazotization of 1,8-diaminoanthraquinone followed by hydro-
lysis of the bisdiazo compound, the acid hydrolysis of 1,8-dimethoxyanthraquinone
in glacial acetic acid-sulfuric acid, the alkaline hydrolysis of 1,8-anthraquinone-
disulfonic acid using calcium oxide, and the reaction of 1,8-dinitroanthraquinone
with sodium formate or potassium formate (Michalowicz, 1981).
ln 1987, US manufacturers voluntarily withdrew production of all hum
an drug
products containing dantron (Anon., 1987).
Dantron is synthesized in the Federal Republic of Germany, India, J apan,
Poland, the UK and the USA (Chemical Information Services Ltd, 1989-90).
(b) Natural occurrence

Dantron has been isolated from dried leaves and stems of Xyr semifcata
harvested in Madagascar (Fournier et aL., 1975). Dantron is the basic structure of
DANON 267
the aglycones of naturally occurring laxative glycosides, in, e.g., eassia (senna), Aloe,
Rheum and Rhamnus (cascara) species (Baars et al., 1976; Reynolds, 1989).
Dantron has been identified in larvae of the elm-Ieaf beetle, Pyrhalta luteola.
The presence of a mixure of anthraquinones and anthrones was suggested to be a
means of protection from predators, and these compounds appear to be
biosynthesized by the insect (Howard et al., 1982).
2.2 Use
Dantron has been widely used since the beginning of this century as a laxative
and as an intermediate for dyes (Enviro Control, 1981; Michalowicz, 1981).
2.3 Analysis
Dantron can be determined in pharmaceutical preparations by high-perfor-

mance liquid chromatography with ultraviolet detection (Wurster & Upadrashta,
1986) and by fluorimetry (Miler & Danielson, 1987). It has been determined in
urine and faeces by gas chromatography with flame ionization detection (BaafS et
al., 1976) and in urine by gas chromatography with mass spectrometry (Kok &
Faber, 1981) and high-performance liquid chromatography with fluorimetry (Miler
& Danielson, 1987).

3.1 CarciDogenicity studies iD animaIs

Mouse: A group of 20 male C3H/HeN mi ce, eight weeks of age, was fed
dantron (commercial grade; no impurity detected on thin-Iayer chromatography) at
20 mg/kg diet for 540 days, at which time the experiment was terminated. A group
of 20 untreated male mice served as controls. Hepatocellular adenomas were found
in 9/17 treated . and 5/19 control mice. Hepatocellular carcInomas were found in
4/17 treated mice (all also had adenomas), an incidence that was significantly
different (p c: 0.05; Fisher exact test) from that in controls (0/19). Adenomatous
rpolypoid) hyperplasia, occasionally associated with dysplastic changes, was
observed in the caecum of 17/17 treated mice and in the remainder of the colon of
5/17 treated mice, but not in controls (Mori et al., 1986).
Rat: A group of 18 male ACI rats, eight weeks of age, was fed dantron rpurity
unspecified) at 10 00 mg/kg diet for 16 months. A group of 15 untreated males
served as controls. Twelve treated and 14 untreated rats survived more than one
year. Nine tumours of the large intestine were found in 7112 treated rats (three
adenomas and four adenocarcinomas (p .c 0.02) of the colon and two adenomas of
the caecum). ln addition, focal epithelial hyperplasia was observed frequently in
the mucosa of the colon and caecum of treated rats with and without intestinal
tumours. No intestinal tumour or hyperplastic les ion was found in the 14 controls
(Mori et al., 1985).
(b) Administration with knwn carcinogens
Mouse: ln a two-stage carcinogenesis experiment, a group of 20 female
ICRIHa Swiss mice, seven weeks of age, received a single skin application of
7,12-dimethylbenz( a )anthracene at 20 iig in 0.1 ml acetone, followed two weeks later
by applications three times a week of commercial-grade dantron at 170 iig in 0.1 ml
acetone. A control group of 20 female mice received only skin applications of.
dantron at 170 iig in 0.1 ml acetone three times a week. Median survival time of
animaIs in both groups was greater th an 490 days, when the experiment was
terminated. No skin tumour was found in either group (Segal et al., 1971).
Rat: ln a two-stage carcinogenicity study, groups of 30 male Sprague-Dawley
rats, 50 days of age, received a single subcutaneous injection of 1,2-dimethyl-
hydrazine (DMH) at 150 mg/kg bw. After one week they were fed dantron (purity,
~ 97%) at 0, 60 or 240 mglg diet; the average daily intakes were approximately 30
and 60 mg/kg bw. After 26 weeks, all animaIs were killed. Two additional groups of
30 male rats received either no treatment or were given the di et with the higher
concentration of dantron alone. There was no significant difference in mean body
weight gain between treated and control animaIs. ln the rats treated with DMH
plus dantron, the combined incidences of intestinal adenomas and adeno-
carcinomas were 4/30 in the low-dose and 2/30 in the high-dose groupe The
incidences of intestinal adenocarcinomas were 0/30 in untreated controls, 0/30 in
the group treated with dantron alone and 2/30 in the group treated with DMH alone.
The difference in tumour incidence between animaIs treated with DMH alone and
DMH plus dantron was not significant (Sjöberg et al., 1988)

Male Wistar rats were given the sodium salt of dantron intravenously at 4.8, 22
or 58 iimol/kg (1.2, 5.3 or 14 mg/kg) bwor at 12 iimol/g (28.8 mglg) bw by gastric
tube. Metabolites identified in the bile and urine following administration byeither
DANON 269
route included the monosulfate, ß-glucuronide and other unidentified metabolites.

Following intravenous administration, about 80% of the dantron con jugates in bile
were excreted after 1 h; the dose fractions found after 5 h represented about 20%,
30% and 40% of the low-; intermediate- and high-dose levels, respectively. The
corresponding fractions in urine were 16%, 12% and 10%, giving rise to bile:urine
excretion ratios of 1.3, 2.7 and 4.0, respectively. Only 30-50% of the dose could be
accounted for by conjugates (Sund, 1987). Earlier studies also showed that after
oral administration of dantron only 30-40% of the total dose administered could be
recovered in faeces and urine, mostly during the first 24 h (Breimer & Baars, 1976).
ln vitro, rat jejunum and colon transformed dantron into its monoglucuronide
and monosulfate, the monoglucuronide being the major metabolite (Sund &
Elvegård, 1988).
(ii) Toxic ejfects

The oral LDso for dantron in male ARS/ICR mice was ~ 7 g/kg bw. Groups of
four male and four female beagle dogs received either a vehicle capsule or a capsule
containing dantron at 5 or 15 mg/kg bw daily for one year. No adverse effect was
observed. The doses employed were reported to be several-fold higher than the
usual clinical dose (Case et al., 1977-78).
Apoptosis together with accumulation of lipofuscin pigment in gut wall was
noted in guinea-pigs given dantron orally at 25 mg/kg bw (Walker et al., 1988).
Male rats given dantron at 60 or 240 mg/kg diet for 26 weeks (Sjöberg et al.,
1988) had enlarged lymph nodes in the mesocolon, which were brownish due to
pigmentation of the accumulated mononuclear phagocytes. ln kidney, pigment
deposition was seen in the cortical region.
(iii) Ejfects on reproduction and prenatal toxicity
Dantron was mutagenic to Salmonella tyhimurium TA1537 in the presence
and absence of an exogenous metabolic system (Brown & Brown, 1976; Liberman et
al., 1982). It was also mutagenic to TA237 (Tikkanen et al., 1983), TA102 (Levin et
al., 1984) and TA104 (Chesis et al., 1984) in the presence of an exogenous metabolic
system. ln TA104, the results were not significantly changed by the addition of
superoxide dismutase and catalase (Chesis et aL., 1984). ln S. tyhimurium TA100,
TA1535, TA1538 and TA98, dantron was not mutagenIc in the presence or absence
of an exogenous metabolic system (Brown & Brown, 1976; Liberman et al., .1982;
Tikkanen et al., 1983).
Dantron induced respiration-deficient mutants in. yeast (Zetterberg &
Swanbeck, 1971).
It induced unscheduled DNA synthesis in hepatoces from mice (Mori et al.,

1984) and rats (Mori et al., 1984; Kawai et al., 1986) but not in another studywith rat
hepatocytes (Probst et al., 1981). Dantron induced chromosomal aberrations in
human peripheral lymphocytes in vitro in the absence of an exogenous metabolic
system (Carballo et al., 1981). ln some studies, dantron inhibited gap-junctional
intercellular communication in Chinese hamster V79 cells (Ume
da et al., 1980 (The
Working Group noted that the way in which the data were presented precluded
statistical analysis.); Trosko et al., 1982 (one dose)), but in other studies no such
effect was found in Chinese hamster V79 cells (Kinsella, 1982; Zeilmaker &
Yamasaki, 1986) or in human fibroblasts (Si et al., 1988).
(h) Humans
(i) Pharmcokinetics
Following its administration within 24 h of the induction of labour in 12
women, dantron was found in maternaI urine, neonatal urine and amniotic fluide
Most of the drug appeared as a glucuronide in both mothers and babies (Blair et al.,
1977).

Liver damage was reported in a woman who had used a laxative containing
dantron and dioctyl calcium sulfosuccinate for one year. The symptoms dis-
appeared after discontinuation of the medication but reappeared upon resumption;
none of the compounds given alone had any effect on the results of hepatic function
tests (Tolman et al., 1976).
A woman developed deep discoloration of the skin following ingestion of large
amounts of a laxative containing dantron (Darke & Cooper, 1978). Such staining
was also found in other studies, predominantly in elderly subjects, and was localized
to the buttocks and thighs, with minor inflammatory symptoms (Bunney & Noble,
1974; Cox & Vickers, 1984). Contact of skin with faeces or urine containing the drug
seems to be a prerequisite for discoloration. Inflammation, when present, may
result from reduction of the parent compound in the colon to the diol derivative,
which irritates both the gut and skin (puschmann, 1983; Ippen, 1974), while the
parent compound does not (Green et al., 1988).
Melanosis coli, astate involving apoptosis and lipofuscin pigment accumu-
lation in macrophages in colonic lamina propria, has been described in pers
ons
using anthraquinone laxatives (Bokus et al., 1933; Speare, 1951; Wittoesch et al.,
1958; Steer & Colin-Jones, 1975; Badiali et al., 1985; Walker et al., 1988).

DANON 271
4.1 Exposure data
Dantron ocurs naturally in several species of plants and in insects. It has been
produced and widely used since the beginning of the century as a laxative and, to a
lesser extent, as an intermediate for dyes. No data on occupational exposure levels
were available.

Dantron was tested for carcinogenicity byoral administration in single studies
in male mice of one strain and in male rats of one strain. ln mi
ce, a small increase iD
the incidence of hepatocllular carcinomas and a large increase in adenomatous
polypoid hyperplasia of the colon were observed; there was also an increased
combined incidence of adenomas and adenocarcinomas of the colon and caecum.
ln rats, dantron increased the incidence of adenocarcinomas of the colon.

ln one study, dantron caused chromosomal aberrations in human lymphocytes

in vitro. It gave contradictory results with respect to the induction of unscheduled
DNA synthesis in rodent cells and was mutagenic to yeast in one study and to
Salmonella tyhimurium. Dantron did not inhibit gap-junctional intercellular
communication in human cells, but conflicting results were obtained in Chinese
hamster cells. (See Appendix 1.)
4.5 Evaluationl
There is suffâent evidence for the carcinogenicity of dantron in experimental

animaIs.
dantron.
1For definition of the italicized terrs, se Preamble, pp. 2629.

Overall evaluation
Dantron is possibly carcinogenic 10 humans (Group 2B).
5. References
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Baars, A.J., Vermeulen, R.J. & Breimer, D.D. (1976) Gas chromatographie determination
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Blair, A.VV., Burdon, M., Powell, J., Gerrard, M. & Smith, R. (1977) Fetal expsure to 1:8
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Bockus, H.L., VVilard, J.H. & Bank, J. (1933) Melanosis coli. The etiologic significance of
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Brown, J."P & Brown R.J. (1976) Mutagenesis by 9,10-anthraquinone derivatives and related
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Case, M.l:, Smith, J.K. & Nelson, R.A. (1977-78) Acute mouse and chronic dog toxicity
studies of danthron, diocl soium sulfosuccinate, foloxalkol and combinations. Drug. .
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Oceanside, NY
Chesis, "PL., Levi, D.E., Smith, M.l:, Emster, L. & Ames, B.N. (1984) Mutagenicity of
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Cox, N.H. & Vickers, C.F.H. (1984) A cutaneous complication of Dorbanex therapy. Clin.
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Darke, C.S. & Cooper, R.G. (1978) Unusual case of ski disloration. Br. med. J., ii,
1188-1189
DANON 273
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NTS PB83- 16633), Rockvile, MD
Fournier, G., Ludwig Bercht, C.A., Pari, R.R. & Paris, M.R. (1975) 3-Methoxychiysazin, a
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Howard, D.E, Philips, D.W., Jones, 'fH. & Blum, M.S. (1982) Anthraquinones and
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Levi, D.E., Hollstein, M., Chrit
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Liberman, D.E, Fink, R.C., Schaefer, EL., Mulcahy, R.J. & Stark, A.-A. (1982) Muta-
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pharmaceutical tablets and in urie~ Anal. chim. Acta, 192, 293-299
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G.M. (1984) Genotoxicity of a variety of mycotoxis in the hepatoce priaiy
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Mori, H., Sugie, S., Niwa, K., Thkahashi, M. & Kawai, K. (1985) Induction of intestinal
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Mori H., Sugie, S., Niwa, K., Yoshimi, N., Thnaka, 'f & Hirono, I. (1986) Carcinogenicity of
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Pouchert, C.J., 00. (1983) Th Aldrich Lirar of NMR Spectra, 2nd ed., VoL. 2, Milwaukee,
WI, Aldrich Chemical Co., p. 91
Pouchert, C.J., 00. (1985) The Aldrich librar of FTIR Spectra, VoL. 2, Milwaukee, WI,
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DANON 275
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Acta dermovenerol.~ 51,45-49
FUROSEMIDE (FRUSEMIDE)
1.1 Synonyms
Chem. Abstr. Services Reg. No.: 54-31-9

ehem. Abstr. Name: 5-( Aminosulfonyl)-4-chloro-2-( (2- furanylmethyl)-amino J-
benzoic acid
Synnym: Sulfamoylanthranilc acid, 4-chloro-N-furfuryl-5
COOH
NH - CH2 'i
Ci
CiiH Il CINiOsS MoL. wt: 330.77

(a) Description: White, microcrystallne powder; crystals from aqueous
ethanol (Reynolds, 1989)
(b) Me/ting-point: 20°C dec (Windholz, 1983)

(c) So/ubi/ity Slightly soluble in water; soluble in aqueous solutions ab ove
pH 8; slightly soluble in chloroform, ethanol and diethyl ether; soluble in
acetone, methanol and dimethylformamide (Windholz, 1983)
(d) Spectroscopy data: Ultraviolet and infrared spectra have been reported
(Anon., 1979).
-277-
(e) Stability Discolours on eXPsure to light (Barnhart, 1989); precipitates

with calcium gluconate, ascorbic acid, tetracyclines, urea and adrenalIne
(Windholz, 1983)
if Dissociation constant: pKa = 3.9 (Anon., 1979)
Trade names: Aluzine; Aquamide; Aquasin; Arasemide; Discoid; Diural;

Diuresal; Diurolasa; Drytal; Durafurid; Errolon; Franyl; Frusetic; Furetic; FurIx
Furo-basan; Fur-O-Ims; Furo-Puren; Furose; Furoside; Fusid; Hydrex;
Hydro-rapid; Impugan; Lasiletten; Lasilix Lasix Laxr; Min-I-Jet Frusemide;
Moilarorin; Neo- Renal; Nicorol; Novosemide; Odemase; Oedemex; Promedes;
Puresis; Seguril; Sigasalur; SK-Furosemide; Uremide; Urex; Urex-M; Uritol
The following names have ben used for multi-ingredient preparations
containing furosemide: Diumide-K; Frumil; Frusene; Lasikal; Lasilactone;
Lasipressin; Lasix + K; Lasoride
Furosemide is available as tablets (20 mg, 40 mg, 80 mg) with lactose,
magnesium stearate, starch and talc (see IAC, 1987), and for injection in 2-,4- and
10-ml ampoules containing furosemide at 10 mg/ml sterile solution in amber vials
(water and sodium hydroxide). It is also available as an oral solution containing
furosemide at 10 mg/ml and Il.5% alcohol, D & C Yellow #10, FD & C Yellow #6,
glycerine, parabens, sodium hydroxide and sorbitol (Barnhart, 1989).
Furosemide is prepared from 4,6-dichlorobenzoic acid-3-sulfonylchloride via

a multistep synthesis involving the sequential addition of ammonia and 6-furfuryl-
amine (Sturm et al., 1962). It is synthesized in Brazl, Bulgaria, China, Hungary,
Israel, Italy, Poland, Switzerland and the USA (Chemical Information Servces,
1989-90).
Specific data on production of furosemide are not available, but the number of
prescriptions for this drug in the USA increased from 16 milion in 1973 to 23
millon in 1981. The oral form (Lasix) alone was the eighth most frequently
prescribed drug in the USA in 1985 (La Piana Simonsen, 1989). ln Sweden, furo-
semide was sold at a level of 44.08 defined daily doses per 100 inhabitants in 1988
(Apoteksbolaget, 1988, 1989). ln Finland, furosemide sales were 13.87 defined daily
doses (40 mg) per 100 inhabitants in 1987 (Finnish Committee on Drug Infor-
mation and Statistics, 1987).
FUROSEMIDE 279
Furosemide is not known to occur naturally.
2.2 Use
Furosemide is a potent, short-acting diuretic (Weiner & Mudge, 1985). It is

used for the treatment of oedema of cardiac, hepatic or renal origin and in a variety
of situationsranging from the control of hypertension to the symptomatic treatment
of hypercalcaemia.
Furosemide has a steep dose-effect curve, and therapeutic doses range from 40
to 20 mg daily in adults (Weiner & Mudge, 1985). Treatment of oedema is usually
started with an initial oral dose of 40 mg daily; in severe cases, a graduaI increase up
to 60 mg dailymay be required. Intramuscular or slow intravenous injections of
furosemide are also used, although the oral route is preferred. ln the management
of oliguria in acute or chronic renal failure, doses up to 6 g have been given in slow
(less than 4 mg per min) intravenous infusions (see Reynolds, 1989).
The usual dose for children is 1-3 mg/kg bw daily given orally and 0.5-1.5 mg/kg
bw by injection (Reynolds, 1989).
2.3 Analysis
Furosemide has been determined in biological fluids by high-performance

liquid chromatography with detection by spectrofluorimetry (Uchino et al. ~ 1984;
Sood et al., 1987) and ultraviolet (Andreasen et al., 1981) and mass spectrometry
(Uchino et al., 1984). Analysis of furosemide in pharmaceutical preparations by
high-performance liquid chromatography and colorimetric complexation with
copper has been reported (Mishra et al., 1989; US Pharmacopeial Convention, Inc.,
1989).


Mouse: Groups of 50 male and 50 female B6C3F1 mice, eight weeks old, were
fed furosemide (99% pure, USP grade) at 0, 700 or 140 mglg of diet for 104 weeks.
The average amounts of furosemide consumed per day were approximately 100 and
20 mgJg bw for low- and high-dose groups, respectively. Mean body weights of
treated and control mice were comparable. Final survival rates in males were:
control, 31/50; low-dose, 24/50; and high-dose, 26/50; and those in females were:
control, 36/50; low-dose, 29/50; and high-dose, 18/50. Survival in high-dose females
was significantly lower than that in controls (p = 0.(03). AlI survivors were killed at
weeks 105- 107 then necropsied, and about 40 different tissues were examined
microscopically. ln female mi ce, a small but statistically significant increase ifi the
incidence of mammary gland carcinomas was observed: control, 0/50; low-dose,
2/50; and high-dose, 5/48 (p = 0.01, logistic regression test for trend taking account
of survival) (National Toxicology Program, 1989).
Rat: Groups of 50 male and 50 female F344/N rats, seven weeks old, were fed
furosemide (99% pure; USP grade) at 0, 350 or 700 mg/kg of diet for 104 weeks. The
average amounts of furosemide consumed per day were approximately 15 and 30
mg/kg bw for low- and high-dose groups, respectively. Mean body weights of
treated and control mice were comparable. Survivors at 104-106 weeks in males
were: controls, 17/50; low-dose, 17/50; and high-dose, 20/50; those in females were:
controls, 35/50; low-dose, 31/50; and high-dose, 34/50. About 40 different tissues
were examined microscopically. No statistically significant increase in the inci-
dence of tumours at any site was reported; however, in males, meningiomas of the
troIs. The
brain were observed in 3/50 low-dose rats versus 2/1928 in historical con
authors noted that these rare tumours occurred early in the study in low-dose
animaIs (National Toxicology Program, 1989).
(h) Administration with known carcinogens

Rat: Four groups of 25 male Fischer 344 rats, five weeks of age, were given
drinking water containing 0.01% or 0.05% N-nitrosobutyl-N-( 4-hydroxybutyl)-
amine (NBHBA) for four weeks, followed by no further treatment or administration
of furosemide (purity unspecified) dissolved in 0.5% carboxymethyl cellulose by
gavage three times per week for 32 weeks (total dose, 250 mg/kg bw). The
experiment was terminated at 36 weeks. One group of 25 male rats was treated with
furosemide alone. No treatment-related mortality was observed in any group, but
body weights of furosemide-treated groups were significantly lower; almost all rats
survived to the end of the experiment. Following sacrifice, aU bladders were
examined histologically. No significant difference in the incidence of bladder
lesions (simple, papilary or nodular hyperplasia, papilomas or carcinomas) was
seen in furosemide-treated versus other groups. Treatment with furosemide alone
did not induce any lesion in the bladder (Shibata et al., 1989). (The Working Group
noted the short duration of the study and the limited pathological examination.)
FUROSEMIDE 281

After oral administration of furosemide to dogs, about 50% of the dose was
absorbed (Yakatan et al., 1979). ln one study in male Sprague-Dawley rats, the
bioavailability of oral furosemide was estimated to be 30% (Lee & Chiou, 1983).
The pharmacokinetics of the disappearance of furosemide from the blood is
best described by two- or three-cmpartment open models, with dose-dependent
variations in plasma protein binding (Hammarlund & Paalzow, 1982). ln rats,
furosemide is cleared from the plasma by the kidneys, is biotransformed by the liver
or is excreted unchanged in the bile, with subsequent intestinal reabsorption (Kitani
et al., 1988). About 4% of furosemide administered intravenously to rats was
recovered from the gut (Le & Chiou, 1983). ln contrast, bilary excretion of
furosemide has been reported to be as high as 30% of doses of 50-100 mg/kg bw
given to male Swiss mice (Spitzagle et al., 1977). Glucuronidation of furosemide
appears to take place in the kidney; removal of the liver did not affect clearance of
furosemide in dogs (Le & Chiou, 1983; Verbeeck et al., 1981).
Covalent binding of furosemide to mouse liver proteins has been shown, and
this was enhanced by administration of an inhibitor of epoxide hydrase, suggesting
formation of an arene oxide intermediate involving the furan ring (Wirth et al.,
1976). ln vitro, human liver microsomes can convert furosemide to metabolites that
bind irreversibly to microsomal proteins (Dybing, 1977).
Formation of unidentified metabolites was demonstrated after incubation of
furosemide with a 90 x g supernatant fraction of washed stomach homogenates
from rats. The apparent metabolism per gram of tissue was greater in the stomach
or liver (Lee & Chiou, 1983).
than in the small intestine, large intestine
(ii) Toxic effects

The oral LDSO for furosemide was approximately 2700 mglg bw in 6O-day-old
rats (Goldenthal, 1971),220 mglg bw in mice (Romanova & Rudzit, 1985) and 80
mglg bw in rabbits (Horioka et al., 1982). The intravenous LDso in rabbits was 80
mglg (Horioka et al., 1982). Intraperitoneal injection of 40 mglg bw into male
mice produced massive necrosis in both the midzonal and centrilobular areas of the
liver; this damage was prevented by prior administration of cyochrome P450
inhibitors (Mitchell et al., 1974).
Two of five male and three of five female rats that were fed diets containing
furosemide at up to 46 glg for 14 days died before the end of the studies.
Minimal-to-mild nephrosis was found in all rats that received furosemide at 1.3 or
46 glkg and in one male receiving 5.1 glg. Microscopically, the toxic lesionwas
subcapsular or cortical and was characterized by tubular-cell regeneration;
mineralization was present at the corticomedullary junction. Dose-related

nephrosis was also observed in mice in a14-day study. ln a 13-week study, male rats
given a diet containing furosemide at 12.5 glg or more and females given a diet
containing 15 g/kg had increased liver:body weight ratios; dose-related diuresis was
also observed. Compound-related minimal-to-moderate nephrosis ocurred in
male rats given 5 or 10 glg and in females given 7.5 or 15 glg. Minerálization was
observed at the corticomedullary junction in male rats given 0.625 glg or more. ln
mice, dose-related minimal-to-mild nephrosis was also observed in a 13-week study
ln a two-year study (sec section 3.1), nephropathy ocurred with greater
severity in dosed male rats than in non-dosed rats. Inmice, compound-related
nephropathy and dilatation of the renal pelvis ocurred in males and females; and
tubular cysts, suppurative inflammation and epithelial hyperplasia of the renal
pelvis were observed. Epithelial hyperplasia and inflammation of the urinary
bladder and suppurative inflammation of the prostate were seen in dosed male
mice; and suppurative inflammation of the ovary, uterus and adrenal cortex was
observed at increased incidence in high-dose female mice (National Toxicology
Program, 1989).
Subcutaneous doses of furosemide at 5 or 15 mg/kg bw per day were given to
Sprague-Dawley pups from day 4 to day 28 after birth. Increased urinary calcium
and magnesium excretion was observed, and the total concentration of calcium and
magnesium in bone was lower. The growth of the pups was inhibited in a dose-
dependent manner, and bone mineraI content was appropriate for the smaller bone
mass (Koo et al., 1986).
Furosemide at 0.5 mM reduced the viabilty of isolated mouse hepatocytes and
induced ultrastructural changes related to toxicity (Massey et al., 1987).
Haemodynamic effects Include an increase in renal blood flow (Hook et al.,
1965) and decreases in mesenteric (Gaffney et al., 1978), hepatic (Gaffney et al.,
1979) and splenic (Gaffney & Willamson, 1979) blood flow.
When CRCD rats were administered furosemide at 37.5, 75, 150 or 30 mg/kg
bw twce daily on days 6- 17 of gestation (route of administration unspecified), the
two highest dose levels, which caused maternaI deaths, resulted in increased
resorption rates and decreased fetal weights. Dose-related. increases in the
frequency of wavy ribs occurred in all treatment groups. ln addition, five of 176
fetuses in the group receiving 150 mglg bw had malformations of the scapula
(Robertson et al., 1981).
FUROSEMIDE 283

Furosemide was not mutagenic to Salmonella tyhimurium in plate
incorporation tests in the presence or absence of an exogenous metabolic system
The urine of rats treated in vivo with furosemide at 45 mg!kg bw did not induce
gene conversion in growing cells of Saccharomyces cerevisiae D4-RDII (Marquardt
& Siebert, 1971). .
Furosemide was reported to induce mutations to trifluorothymidine resistance
in L5178Y mouse lymphoma cells in the presence of an exogenous metabolic system
onlyat the highest concentration tested (1500 llglml). It was also reported to induce
sister chromatid exchange and chromosomal aberrations in Chinese hamster CHÛ
cells at 3750 and 500 Jlglml in the presence and absence of an exogenous metabolic
system (National Toxicology Program, 1989). (Te Working Group noted the excep-
tionally high concentrations used in these studies, surpassing the solubilty limits of
the test substance, which preclude an assessment of the observed effects.) No sister
chromatid exchange was induced in a diploid human fibroblast cell line (HE2144)
by concentrations of up to 0.33 mg/ml (Sasaki et al., 1980). Furosemide induced
chromosomal damage in Chinese hamster lung fibroblasts in vitro, but only in the
absence of an exogenous metabolic system (Matsuoka et al., 1979; Ishidate, 1988). A
concentration-dependent increase in the frequency of chromosomal aberrations
was observed in human lymphocytes exposed in vitro to furosemide for 24 and 72 h
(Jameela et aL., 1979). No such effect was detected in the human fibroblast cell line
HE2144 (Sasaki et al., 1980).
ln male C3H/HE mice treated intraperitoneally with furosemide at 0.3-50
mglg bw, a non-dose-dependent increase in the percentage of meiotic cens with
chromosomal aberrations was observed during the whole spermatogenic cycle, Le.,
in weeks 1-5 after treatment (Subramanyam & Jameela, 1977). (The Working
Group noted that only one mouse per dose per week was apparently used.)
(h) Humons
ln healthy subjects, the bioavailabilty of furosemide ranges from 60 to 69%
(Kelly et al., 1973; Rupp, 1974; Tilstone & Fine, 1978); but in end-stage renal failure
its availabilty is reduced to 43-46% (Rane et al., 1978; Tilstone & Fine, 1978).
According to early report, foo does not alter bioavailability, although the rate of
absorption is decreased (Kelly et aL., 1973). ln a recent study, however, a reduction
of approximately 30% in bioavailability, accompanied by a reduced diuretic effect,
was observed when furosemide was given at 40 mg to ten healthy volunteers with
breakfast as compared to when it was given in the fasting state (Beermann &
Midskov, 1986).
About 99% of furosemide is bound to plasma proteins (Smith et al., 1980),

almost exclusively to albumin (Andreasen & Jacobsen, 1974; Prandota & Pruitt,
1975; Branch, 1983).
Two-compartment models are most often used to describe the kInetics of

furosemide (Rupp, 1974; Bermann et al., 1975). The half-time of the ~-phase
averages 10-15 min and that of the ß-phase, 47-90 min (Bermann et al., 1977;
Mikkelsen & Andreasen, 1977; Rane et al., 1978; Andreasen et al., 1982). The
apparent volume of distribution at steady state is approximately 190 mVkg
(Mikkelsen & Andreasen, 1977; Andreasen et al., 1978). The plasma clearance of
furosemide is 2.2-3.0 ml/min per kg (Mikkelsen & Andreasen, 1977; Andreasen et
al.,1978). A higher non-renal clearance ratio is seen after oral dosing (15.7:l 4.8%)
than after intravenous administration (11.2 :l 4.0%) (Zhu & Koizumi, 1987).
Glucuronide conjugate is the only weIl documented metabolite of furosemide in
man (Bermann et al., 1975; Andreasen & Mikkelsen, 1977; Verbek et al., 1982).
About 20% of furosemide is eliminated by renal glucuronidation (Smith et al.,
1980); it has been suggested that the remaining 25-30% may be secreted into the gut
in unchanged and/or conjugated form (Branch, 1983). However, gastrointestinal
elimination amounted to only 2% of renal clearance, and active secretion into the
intestinal lumen did not ocur in six healthy volunteers given furosemide as a 4O-mg
bolus followed by a continuous infusion of 0.55 mglg per h. Plasma clearance was
22 :l 15, renal clearance, 93.1 :f 21.2 and total clearance by the gastrointestinal
tract, 2.1 :f 0.2 ml/min. There was no change in the intestinal clearance of furo-
semide after administration of probenecid, but plasma and renal clearance
decreased by 48 and 70%, respectively. It was also shown that incubation of urine
samples with ß-glucuronidase increased furosemide levels (Valentine et al., 1986).
The disposition of furosemide during renal insuffciency, nephrotic syndrome,
cIrrhosis and congestive heart failure has been reviewed (Brater, 1986). The mean
plasma half-time of furosemide in patients with nephrosis does not differ from that
in normal subjects but is prolonged about three fold in patients with uraemia (Rane
et al., 1978). A positive relationship between the renal clearance of creatinine and of
furosemide has been shown (Bermann et al., 1977). Liver disease may prolong
plasma half-time by up to 4.3 h, depending on the degree of liver failure
(AlIgulander et al., 1980; Fuller et aL., 1981; Verbeeck et al., 1982).
The most common adverse effects of furosemide are fluid and electrolyte
imbalance, including hypnatraemia, hypokalaemia and hypochloraemic alkalosis.
Hyperuricaemia is relatively common, and a variety of uncommon adverse
reactions have been reported (see Reynolds, 1989).
FUROSEMIDE 285
Signs of volume depletionand hypokalaemia have been reported in several

studies (Greenblatt et al., 1977; Naranjo et al., 1978; Spino et al., 1978; Lowe et al.,
1979). Rare adverse effects reported in patients receiving furosemide include skin
rash, thrombocytopenia (Lowe et al., 1979), gynaecomastia (Tuzel, 1981), temporary
hearing impairment (Naranjo et al., 1978; Spi no et al., 1978) and hepatic coma in
cirrhotic patients (Naranjo et al., 1978). Elevated serum concentrations of
parathyroid hormone and alkaline phosphatase, together with decreased calcium
concentration, were shown in 36 patients with congestive heart failure (Elmgreen et
al., 1980).
Renal calcification was documented in ten premature infants who had received
furosemide in a dose of at least 2 mg/kg bw per day for at least 12 days (Htifnagle et
al., 1982).

No report of pregnancy outcomes following first-trimester use of furosemide
has been found. Furosemide has been used extensively for treatment of oedema,
hypertension and heart failure in the later stages of pregnancy, with no apparent
adverse effect on the fetus or newborn (see review by Briggs et al., 1986).


ampicilin), 232 persons to whom at least one prescription for furosemide had been
dispensed during 1969-73 were followed up for up to 15 years (Selby et al., 1989).
Increased risks were noted for cancer of the lung (50 observed, 25.4 expected; p -c
0.(02) and for cancers at all sites combined (233 observed, 164.5 expected; p -c
0.(02). (The Working Group noted that heart failure and cirrhosis of the liver, both
of which are associated directly or indirectly with cigarette smoking, are frequent
indications for prescribing furosemide, and confounding by cigarette smoking
(which was not analysed in the study) may explain the observed associations.) ln an
earlier report with up to nine years of follow-up (Friedman & Ury, 1983), there was
also an association with cancer of the lIver (5 observed, 1.6 expected cases; p -c 0.05).
The medical records indicated that this association was due to underlying lIver
disease for which furosemide was prescribed. (Te Working Group noted, as did
the authors, that, since sorne 12 00 comparisons were made in this study, the
associations should be verified independently. Data on duration of use were not
provided.)
4. Summary of Data Repôrted and Evaluation
4.1 Exposure data
Furosemide is a diuretic. It has been used extensively since 1964 in the

treatment of oedema and hypertension.

Furosemide was tested for carcinogenicity by oral administration in one strain
of mice and one strain of rats. A small increase in the incidence of mammary gland
carcinomas was observed in female mice. No increase in the incidence of tumours
was seen in rats.

ln one hypothesis-generating study in which many drugs were screened for
possible carcinogenicity, associations with furosemide use were observed for
cancers of the lung and of all sites combined, which could have been accounted for
by smoking and/or chance.

The data are inadequate to assess the effects of furosemide on human
reproduction. ln rats, the drug induces skeletal anomalies.
Furosemide is metabolized by m9use and human liver microsomes and binds
covalently to proteins. Renal tubular hyperplasia and hepatic centrilobular necro-
sis have been observed after administration of large doses of furosemide to mice.
Studies on the induction by furosemide of chromosomal aberrations in mIce
were inconclusive. Reports of studies on chromosomal aberrations in human cells
in vitro gave conflicting results; it induced chromosomal damage in hamster cells.
Furosemide did not induce sister chromatid exchange in human cells in vitro; one
study gave questionably positive results for sister chromatid exchange in Chinese
hamster cells and for gene mutation in mouse lymphoma cells. The urine of rats
treated with this drug did not induce gene conversion in Saccharomyces cerevisiae.
It was not mutagenic to Salnwnella tyhimurium. (See Appendix 1.)
FUROSEMIDE 287
4.5 Evaluation 1
There is inaequate evidence for the carcinogenicity of furosemide in humans.

There is inaequate evidence for the carcinogenicity of furosemide in experi-
mental animaIs.
Overall evaluation
Furosemide is not classifle as to its carcinogenicity to humans (Group 3).
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FUROSEMIDE 289
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HYROeHLOROTHMZIDE
1.1 Synonyms

ehem. Ahstr. Name: 2H-1,2,4-Benzothiadiazine-7-sulfonamide,6-chloro-3,4_
dihydro-l,l-dioxide
Synnym: 6-Chloro-3,4-dihydro- 7 -sulfamoyl-2H -1,2,4-benzothiadiazine 1,1-
dioxide; 6-chloro- 7-sulfamyl- 3,4-dihydro- 1,2,4-benzothiadiazine 1,I-dioxide-
3,4-dihydrochlorothiazide; chlorosulfonamidodihydrobenzothiadiazine dio-
xide; chlorosulthiadil
o 0
NH,5yYS(N_H
CI~N) 1
H
C7H8CIN304S MoL. wt: 297.72
1.3 ChemIcal and physical properties of the pure substance

From Deppeler (1981), unless otherwse specified
(a) Description: White, fluffy, microcrystallne powder
(b) Me/ting-point: 273-275°C (Windholz, 1983)
(c) So/ubility Practically insoluble in water; soluble in dilute ammonia and
sodium hydroxide; soluble in methanol, ethanol, acetone and acetonitrile
(d) Spectroscopy data: Infrared, ultraviolet, nuclearmagnetic resonance and
(e) Stabi/ity data: Stable in bulk for five years at room temperature; at
extremes of pH in aqueous solution, hydrolysed to formaldehyde and
6-chloro-2,4-disulfamoylanilne
-293-
(f Dissociation constant: pKa 7.2, 9.2 (Windholz 1983)
Trade names: Apo-Hydro; Aquarius; Atenadon; Bremil; Caturida; Chlorthia;

Chlorzide; Cidrex; Cloredema; Delco-Retic; Dichlorosal; Dichlortride; Dichlotride;
Dic1otride; Didral; Diidrotiazde; Direma; Disalunil; Diu 25; Diucen-H;Diurex;
Diursana-H; Dixdrasi; Edemex; Esidrex; EsidrIx Fluvin; Hidrenox; Hidroronol;
Hidrosaluretil; Hydril; Hydro-Aquil; Hydro-Diuril; Hydro-MURIL; Hydrosaluric;
Hydrothide; Hydro-Z; Hydrozide; Hypothiazde; Idrodiuvis; Idrofluin; Idrolisin;
Ivaugan; Jen-diral; Lexor; Loqua; Maschitt; Mietrin; Natrimax NefrIx
Neo-Codema; Neoflumen; NeoFlumen; Neo Minzil; Newtolide; Novohydrazde;
Oretic; Pantemon; Panurin; Ridaq; Ro-Hydrazde; Salupres; Serapres; SK-
Hydrochlorothiazde; Tandiur; Thiaretic; Thiuretic; Urirex; Urodiazn; Urozide;
Vetidrex
Hydrochlorothiazde is also contained in numerous multi-ingredient
preparations.
Hydrochlorothiazde is available as tablets for oral use (25, 50 or 100 mg)
containing calcium phosphate, D & C Yellow #6, gelatin, lactose, magnesium
stearate, starch and talc (see IAC, 1987) (Barnhart, 1989).
Hydrochlorothiazde in synthesized by either the reaction of para-

formaldehyde with 5-chloro-2,4-disulfamoylaniline in nonaqueous media, or the
reaction of formaldehyde with 6-chloro-7-sulfamoyl-2H- 1,2,4-benzothiadiazne-
1,1-dioxide in aqueous alkalIne solution (Deppeler, 1981). It is,--synthesized in
China, Hungary, India, Italy, Japan, Romania, Switzrland, the UK, the USA and
Yugoslavia (Chemical Information Servces, 1989-90).
Hydrochlorothiazde has ben used as a diuretic and antihypertensive agent
since 1957 (Reynolds, 1989). More prescriptions were written for hydrochlor-
thiazde/triamterene combination than for any other prescription drug product in
the USA in 1984 and 1985 (Chappell, 1985), and hydrochlorothiazde was the sixh
most frequently prescribed generic drug in 1987 and 1988 in the USA (La Piana
Simonsen, 1989). ln 1988, this drug was sold in Sweden at a level of 0.14 defined
daily doses per 100 inhabitants (Apoteksbolaget, 1988, 1989). ln 1987, it was sold
in Finland at a level of 2.06 defined daily doses per 100 inhabitants (Finnish
Committee on Drug Information and Statistics, 1987).
HYDROCHLOROlHIAZIDE 295
Hydrochlorothiazde is not known to ocur as a natural product.

2.2 Use
Hydrochlorothiazde is a thiazde diuretic (Reynolds, 1989). It is used to
reduce oedema associated with heart failure; as an antihypertensive agent, and for
special indications such as Ménière's disease (Roydhouse, 1974) and reduction of
the formation of renal calculi in patients with hypercalciuria (Yendt et aL., 1970;
Baggio et al., 1986). The daily dose of hydrochlorothiazde in treating oedema is
25-50 mg after an initial dose of twice this amount. The daily dose for children is 2.5
mglg bw and that for infants under six months, 3.5 mglg bw. The antihyper-
tensive doses of hydrochlorothiazde vary between 25 and 20 mg daily (Reynolds,
1989).
2.3 Analysis
Hydrochlorothiazde can be analysed in urine and plasma by colorimetry,

thin-Iayer chromatography and high-performance liquid chromatography
(Sheppard et al., 196; Redalieu et aL., 1978; Suria, 1978; Koopmans et al., 1984; AIton
et al., 1986; Fullnfaw et al., 1987; van der Meer & Brown, 1987). Analysis of hydro-
chlorothiazde in pharmaceutical preparations has also been reported (Cieri, 1988;
US Pharmacopieal Convention, Inc., 1989).


Mouse: Groups of 50 male and 50 female B6C3Fl mice, seven to eight weeks of
age, were fed hydrochlorothiazde (:: 98% pure) at 0, 2500 or 500 mg/kg of diet for
103-104 weeks (average daily intake, 28 or 575 mg/kg bw), and all survivors were
killed at weeks 113-114. Mean body weights were similar in control and treated
mice. Survival in males was: control, 43/50; low-dose, 42/50 and high-dose, 43/50;
that in females was: control, 38/50; low-dose, 40/50 and high-dose, 35/50. All
animaIs were necropsied, and samples taken from all major organs, tissues and
gross lesions were examined histologically. A significant increase in the incidence
of hepatocellular adenomas and of combined adenomas and carcinomas (control,
7/48; low-dose, 10/49; high-dose, 21/50 (p = 0.00, incidental tumour test)) but not
of carcinoma alone was observed in males. No increase in the incidenceof any other
neoplasm was observed (National Toxicology Program, 1989).
2% IARC MONOGRAHS VOLUME 50
Rat: A group of 24 male and 24 female Fischer 34 rats, six to eight weeks of
age, were fed hydrochlorothiazde (purity unspecified) at 100 mglg of diet for 104
weeks (total intake: males, 21 g; females, 14 g). A control group of 24 male and 24
. female rats remained untreated. Over 70% of the rats survived more than twoyears,
with similar survival rates in all groups. AlI survivors were killed after 130 weeks;
complete necropsies were performed on aIl animaIs, and major organs were
examined histologicaIly. No difference in overall tumour incidence or in the
incidence of tumours at any site was observed between treated and control rats
(Lijinsky & Reuber, 1987).
Four groups each of 50 male and 50 female Fischer 344/N rats, seven to eight
weeks of age, were fed hydrochlorothiazde (:; 98% pure) at 0, 250, 500 or 20
mg/kg of diet for 105- 106 weeks (average daily intake, Il,23 or 89 mg!g bw), and aU
survivors were killed at weeks 113-114. Survval was-males: control, 18/50;
low-dose, 16/50; mid-dose, 9/50; high-dose, 11150; females: control, 31/50; low-dose,
25/50; mid-dose, 30/50; high-dose, 27/50. AlI animaIs were necropsied, and samples
from all major organs, tissues and gross les ions were examined histologically. No
increase in either overall tumour incidence or in the incidence of tumours at any site
was observed (National Toxicology Program, 1989).
(b) Administration in combination with other compounds

Rat: ln the experiment by Lijinsky and Reuber (1987), described above, three
groups each of 24 male and 24 female Fischer 344 rats, six to eight weeks of age, were
fed diets containing hydrochlorothiazde (purity unspecified) at 100 mg/kg,
sodium nitrite at 20 mg!g or hydrochlorothiazde at 100 mg/kg plus sodium
nitrite at 20 mglkg for 104 weeks. Over 70% of the rats survived more than two
years, with similar survival rates in all groups. AlI survivors were killed after 130
weeks; complete necropsies were performed on all animaIs, and major organs were
examined histologicaIly. No difference in overaU tumour incidence or in the
incidence of tumours at any site was observed between treated and control rats.

. (i) Absorption, distriution, exretion and metabolism
(ii) Toxic effects
The oral LDso for hydrochlorothiazde in mice was 3080 mglg bw (Barnes &
Eltherington, 1%5).
20 dogs receiving hydrochlorothiazide at daily doses of 50-20 mg for up to
AlI
nine months had enlarged, hyperactive parathyroid glands (Pickleman et al., 1969).
HYDROCHLOROTHIAZIDE 297
ln male (but not female) Syrian golden hamsters reciving hydrochlorothiazde at 1

or 2 mg/g bw by gavage for six months, increased total cholesterol and high-density
lipoprotein cholesterol levels were observed. When a dose of 4 mg/g bw was
administered, a similar increase was seen in animaIs of each sex (Sarva et al., 1985).
AlI male and female rats fed dietscontaining 3.12-50 glg (five dose levels)
hydrochlorothiazde survved for 15 days. Thymic haemorrhage of slIght to
moderate severity was observed in animaIs recivIng the highest doses, but no other
toxic effect was observed (National Toxicology Program, 1989).
ln groups of 24 male and 24 female rats fed hydrochlorotriazde at 100 mg/g
of dietfor two years, the incidence and severity of chronic progressive nephropathy
and of lesions secondary to chronic renal disease, polyarteritis and mural
thrombosis were increased (Lijinsky & Reuber, 1987).
ln a two-year study (see section 3.1), there was a uniform reduction in the body
weight of treated rats (male and female) at all doses. Chronic renal disease (cysts of
the parenchyma and epithelial hyperplasia of the renal pelvis) was present in aIl
groups of male and female rats, but it was more severe in dosed groups. Secondary
signs of chronic renal disease, including parathyroid hyperplasia, mineralization in
multiple organs and fibrous osteodystrophy, also occurred at increased frequency
in dosed groups. No other lesion in rats appeared to be related to exposure to
hydrochlorothiazde. ln mice, a two-year exposure hadonly negligible effects on
body weight. No increase in the frequency of non-neoplastic lesions in the kidney,
urinary bladder or any other organ was attributed to hydrochlorothiazde
administration (National Toxicology Program, 1989).

Hydrochlorothiazde was administered by gavage to pregnant CD rats at 100,
30 or 100 mg/kg bw per day and to CD-1 mIce at 30, 100 or 30 mglg bw per
dayon gestation al days 6-15. No dose-related fetal toxiCity or significant increase in
the incidence of malformations was observed (National Toxicology Program, 1989).
Hydrochlorothiozide did not induce reversion in an arg- strain of Escherichia
coli (Hs30R) (Fujita, 1985). lt was not mutagenic to Salmonella tyhimurium in the
presence or absence of an exogenous metabolIc system (Waskell, 1978; Andrews et
al., 1984). (The Working Group noted that onlyone concentration was used in both
studies.) ln strain TA98, but not in TA1535, TA1537 or TA1oo, a smaIl, repro-
ducible, concentration-dependent increase in the mean number of revertants was
observed in the absence, but not in the presence, of an exogenous metabolic system
(Mortelmans et al., 1986).
ln a spot test, hydrochlorothiazde induced nondisjunction and mitotic
crossing-over in Aspergillus nidulans (Bignami et al., 1974).
Hydrochlorothiazde did not induce sex-linked recessive lethal mutations in

Drosophila melanogaster either fed or injected with solutions of 10 mg/ml (Valencia
et al., 1985).
At concentrations above 500 J.g/ml, hydrochlorothiazde produced cytotoxic
effects and induced mutations to trifluorothymidine resistance in L5178Y mouse
lymphoma cells in the absence of an exogenous metabolic system (National
Toxicology Program, 1989). Significant, but not concentration-dependent, increases
in the frequency of sister chromatid exchange were observed in Chinese hamster
CHO cells in the presence and absence of an exogenous metabolic system
(Galloway et al., 1987). Chromosomal aberrations were not found in Chinese
hamster lung CHL cells, but polyploidy was observed after 48 h treatment (Ishidate,
1988). Chromos omal aberrations were also not detected in Chinese hamster CHO
cells in the presence or absence of an exogenous metabolic system at concentrations
of up to 26 J.g/ml (Galloway et al., 1987).
(h) Humans
The pharmacokinetics of hydrochlorothiazide have been reviewed (Wellng,
1986).
Hydrochlorothiazide is incompletely absorbed from the duodenum and upper
jejunum (Beermann et al., 1976), and plasma concentrations, peaking at about 2-3 h
after intake, are proportional to the dose within the range 25-100 mg (Patel et al.,
1984). Administration with food either enhances (Beermann & Groschinsky-
Grind, 1978) or reduces (Barbhaiya et al., 1982) the absorption of hydro-
chlorothi de, as compared with fastingconditions. The discrepancy is partly
azi
attributable to differences in fasting states in these experiments. Food might delay

passage through the small intestine; patients with intestinal shunt surgery and
accelerated intestinal passage have shown reduced absorption of hydrochloro-
thiazide (Backmanet al., 1979).
Hydrochlorothiazde is concentrated in red blood cells (Beermann et al., 1976;
Redalieu et al., 1985). It is excreted almost entirely unchanged in urine; its renal
clearance rate (about 30 ml/min) indicates combined glomerular filtration and
tubular secretion (Barbhaiya et al., 1982). Its plasma elimination half-time is about
6 h initially but up to 15 h terminally (Patel et al., 1984). ln patients with decreased
renal function, the plasma half-time of hydrochlorothiazde is prolonged to 20 h
(Niemeyer et al., 1983).
Concentrations of hydrochlorothiazde in maternaI plasma and umbilcal cord
plasma were similar (Beermann et al., 1980) and were lower than those in amniotic
fluid (Mulley et al., 1978). The drug was detected in the milk of nursing mothers
treated with it, but no measurable concentration was found in nursing infants
(detection Ii mit, 20 nglml) (Miler et al., 1982).
Administration of large doses of hydrochlorothiazide often leads to electrolyte
imbalance~ including hypohloraemic alkalosis, hyponatraemia, hypokalaemia and
hypercalcaemia (Porter et al., 1978; Zalin et al., 1984; Bayer et al., 1986; Reynolds,
1989).
Like other thiazde diuretics, hydrochlorotriazide is known to produee
metabolie effeets, sueh as hyperglycaemia and glyeosuria, in diabetie and other
susceptible patients (Flamenbaum, 1983; Freis, 1986). It produces asymptornatie
mon
hyperuricaemia in many patients, although aetual attaeks of gout are not corn
(Anon., 1987).
Hyperparathyroidism associated with prolonged intake of thiazdes, inc1uding
hydrochlorothiazde, has been reported (Paloyan & Picklernan, 1969; Christensson
et al., 1977; Klimiuk et al., 1981).
A number of skin diseases of an allergie and idiosyneratic nature have been
reported among patients treated with thiazide diuretics (Ebstein & Wintroub, 1985;
Reedet al., 1985; Hardwiek & Saxe, 1986).
Interstitial nephritis (Linton et al., 1980; Seully et al., 1983), idiosyneratie
pneumonitis (Piper et al., 1983; Parfrey & Herlong, 1984), thrombocytopenia
(Eisner & Crowell, 1971), intravaseular haemolysis (Beek et al., 1984) and
pancreatitis ,(Cornish et al., 1961) have been reported in patients treated with
thiazde diuretics.
ln the eollaborative Perinatal Project, in which drug intake and pregnaney
outcome were studied in a series of 50 282 wornen in 1959-65, 107 women had been
exposed to hydrochlorothiazde during the first trimester of pregnaney. There were
nine malformed children in the exposed group, giving a nonsignificant standardized
relative risk of 1.2 (Heinonen et al., 1977).

drugs for possible carcinogenicity (described in detail in the rnonograph on
ampicilln), 12 799 persons to whom at least one prescription for a thiazde diuretic
had been dispensed during 1969-73 were followed up for up to 15 years (Selby et al.,
1989). Hydrochlorothiazde was the predominant drug used in this groupe
Increased risks were noted for cancer of the prostate (53 cases observed, 38.2
expected; p ~ 0.05) during follow-up of up to seven years (Friedman & Ury, 1980)
and for cancers at all sites combined (120 observed, 1132.9 expected; p ~ 0.05)
during follow-up of up to 15 years (Sclby et al., 1989). The association with prostatic
cancer diminished in later follow-up. (Te Working Group noted that prostatic
cancer may be diagnosed more readily in patients under more intensive medical
care. ln addition, as also noted by the authors, since some 12 () comparisons were
made in this hypothesis-generating study, the associations should be verified
independently. Data on duration of use were not provided.)
4.1 Exposure data
Hydrochlorothiazide has been used extensively since 1957 as a diuretic and

antihypertensive agent.

Hydrochlorothiazde was tested for carcinogenicity by oral administration in
one strain of mice and one strain of rats. An increase in the incidence of
hepatocellular adenomas was observed in male mice. No increase in the incidence.
of tumours at any site was observed in two studies in rats.

ln one hypothesis-generating study in which many drugs were screened for
possible carcinogenicity, associations with hydrochlorothiazde use were observed
for cancers of the prostate and of all sites combined, which could be accounted for
by chance.

One study provided no evidence that use of hydrochlorothiazde in the first
trimester of pregnancy is associated with the induction of birth defects. ln rats, no
teratogenic, embryotoxic or fetotoxic effect was observed.
Hydrochlorothiazde induced gene mutations in mouse lymphoma cells and
sister chromatid exchange in Chinese hamster cells. It did not induce chromosomal
aberrations in Chinese hamster cells in vitro or sex-linked recssive lethal mutations
in Drosophila. Hydrochlorothiazde induced mitotic recmbination and nondis-
junction inAspergilus niduJans. Itwas not mutagenic toSalmonella tyhimurium or

Escherichia coli. (Se Appendix 1.)
4.5 Evaluation 1
There is inaequote evidence for the carcinogenicity of hydrochlorothiazide in

humans.
There is inaequote evidence for the carcinogenicity of hydrochlorothiazide in
Overall evaluation
Hydrochlorothiazde is no! classifle as to its carcinogenicity to humans
(Group 3).
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1
Suria, D. (1978) Quantitative determination of thiazides in urie by a sensitive colorietric

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US Pharmacopeial Convention, Inc. (1989) US Phaopeia, 22nd rev., Easton, PA, pp.
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Welling, P.G. (1986) Pharmacokietics of the thiazide diuretics. Biophar. Drug Dispos., 7,
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Br. med. J., 289, 659
PAReETAMOL (AeETAMINOPHEN)
1.1 Synonyms and trade names

ehem. Ahstr. Name: Acetamide, N-( 4-hydroxyphenyl)-
Synonym: 4' - Hydroxy-acetanilde; para-acetaminophenol; acetophenum;
para-acetylamidophenol; N-acetyl-para-aminophenol; para-acetylamino-
phenol; para-hydroxyacetanilde; N-para-hydroxyphenylacetainide
A large number of fixed combinations containing paracetamol are available.
CH3CONH-Q OH
CsH9NOi MoL. wt: 151.16

From Fairbrother (1974) and EI-Obeid and Al-Badr (1985)
(a) Description: White odourless crystallne powder; large monoclinic prisms
from water
(h) Me/ting-point: 169-170.5°C
(c) So/ubility Soluble in water (1:70, 1:20 at 100°C), ethanol (1:7), acetone
(1:13), chloroform (1:50), glycerol (1:40), methanol (1:10), propylene glycol
(1:9) and solutions of alkali hydroxides; insoluble iu diethyl ether. A
saturated aqueous solution has a pH of ",6.
(d) Spectroscopy data: Infrared, ultraviolet, nuclear magnetic resonance,
fluorescence and mass spectra have ben reported.
-307-
(e) Stability Dry, pure paracetamol is stable to 45°C. Contamination with

traces of para-aminophenol, and humid conditions that cause hydrolysis
to para-aminophenol, result in further degradation and discoloration.
Slightly light-sensitive in solution, and degradation is catalysed byacids
or bases.
(l Dissociation constant: pKa = 9.0-9.5

(g) Partition coeffcient: Pc = 6.237 (octanol: pH 7.2 buffer)
i.4 TechnIcal products and impurities
Paracetamol is available in pure form as numerous trade-name preparations

for oral use. It is also found combined in over 20 preparations with other drugs.
Trade names: Abensanil; Acamol; Acephen; Acetalgin; Acetamol; Aferadol;
Alba-Temp; Alpiny; Alvedon; Amadil; Anacin-3; Anaflon; Anhiba; Anuphen;
Apamide; APAP; Atasol; Ben-u-ron; Bickie-mol; Bramcetamol; Calip; Calpol;
Calpon; Campain; Capital; Captin; Ceetamol; Cetadol; Cetamol; Cetapon;
Claradol; Claratal; Custodial; Dafalgan; Datril; Dial-a-gesix; Dirox; Disprol
Paediatric; Dolamin; Dolanex; Doliprane; Doloral; Dolorol; Dolprone; Dorcol
Children's Fever and Pain Reducer; Doregrippin; Dymadon; Efferalgan; Enelfa;
Eneril; Ennagesic; Eu-Med; Exdol; Fanalgic; Febrigesic; Febrilix; Fendon; Fevamol;
Finimal; Fonafor; Gelocatil; Glenpar; Gynospasmine; Hedex; Homoolan;
Kinderfinimal; Kinder-Finiweh; Korum; Liquiprin; Lyteca; Malgis; Melabon;
Momentum; Napamol; Naprinol; Nebs; Neuridal; Nevral; Nina 120; Nobedon;
Ophinal; Oraphen; Pacemo; Pacemol; Painamol; Painaway; Paldesic; Pamol;
Panado; Panadol; Panaleve; Panamax; Panasorb; Panets; Panex; Panodil; Panofen;
Pantalgin; Paracet; Paracetamolum; Paraclear; Paralgin; Parapain; Paraprom;
Parasin; Paraspen; Para toI; Parmol; Pasolind; Phendex; Pinex; Placemol;
Praecimed; Proval; Puernol; Pyragesic; Pyralen; Reliv; Repamol; Resolve;
Robigesic; Rounox; Salzone; Schmerzex; Sedapyren; Servgesic; Setamol; SK-APAP;
Summadol; Tabalgin; Tachipirina; Tapar; Temlo; Tempra; Tenasfen; Ticelgesic;
Tralgon; Treupel; Treuphadol; Tricocetamol; Tylenol; Tymol; Valadol; Zolben
Paracetamol is available as 325-mg or 5OO-mg tablets, which may include
calcium stearate or magnesium stearate, cellulose, docusate sodium and sodium
benzoate or sodium lauryl sulfate, starch, hydroxypropyl methylcellulose, propylene
glycol, sodium starch glycolate, polyethylene glycol and Red #40.
It is also available as 5OO-mg gelatin capsules and as a mint-flavoured liquid
containing 500 mg/15 ml solution, which can include 7% ethanol, citric acid,
glycerine, polyethylene glycol, sodium benzoate, sorbitol, sucrose, Yellow #6, #10
and Blue #1. For children, drops (80 mg/0.8 ml), chewable tablets (80 mg), elixr (160
mg/5 ml) and coated capsules (160 mg/capsule) are available (Barn
hart, 1989).
PARCETAMOL 30
Characteristic impurities may inc1ude para-nitrophenol, para-aminophenol,
para-chloroanilne, ortho-acetyl paracetamol, azbenzcne (see IAC, 1975),
azxybenzene, quinone (sec IAC, 1977), quinonimine, inorganic chloride,
inorganic sulfate, inorganic sulfide andwater (Fairbrother, 1974).
Paracetamol may be made by acetylation of para-aminophenol (obtained by

reduction of para-nitrophenol) with acetic acid or acetic anhydride. A number of
other synthetic routes have ben described (Fairbrother, 1974).
Paracetamol is synthesized in Argentina, Brazl, China, Colombia, France, the
Federal Republic of Germany, India, Japan, Mexico, Poland, Republic of Korea,
Romania, Taiwan, Turkey, the UK and the USA (Chemical Information Services
Ltd, 1989-90).
ln Sweden, paracetamol sales in 1988 were 20.02 defined daily doses per 100
inhabitants (Apoteksbolaget, 1988, 1989).
Paracetamol is not known to occur naturally, but it is the major metabolite of
phenacetin (see IAC, 1980, 1987).
2.2 Use
Paracetamol is used as an analgesic and antipyretic drug. It is the preferred

alternative analgesic-antipyretic to aspirin (acetylsalicylic acid), particularly in
patients with coagulation disorders, individuals with a history of peptIc ulcer or who
cannot tolerate aspirin, as well as in children (American Medical Association,
1986). Paracetamol was first used in clinical medicine in 1893. Following initial use
as a prescription product in the USA in 1951, it subsequently became availabJe
without prescription in 1955 (Ameer & Greenblatt, 1977). ln many countries, it is
widely available without prescription.
The conventional oral dose for adults is 500- 100 mg. Dosing may be repeated
every 4 h as necessary, but the total daily dose should flot exceed 40 mg. For
children, the recommended dose is 10- 15 mg/kg bw; no more than five doses should
be administered over 24 h. Prolonged use (for more th an ten days) and use for
young children is not recommended (Flower et al., 1985).
The usual dose for rectal administration is equal to that for oral administration
(American Medical Association, 1986).
2.3 Analysis
Methods for the analysis of paracetamol have ben reviewed (El-Obeid and
Al-Badr, 1985).
Paracetamol and its metabolites can be analysed in biologicaI fluids by
high-performance liquid chromatography (HPLC; Manno et al., 1981; Kinney &
Kelly, 1987; Aguilar et al., 1988; MeatheraU & Ford, 1988), HPLC-mass
spectrometry (Betowski et al., 1987) and fluorescence polarization immunoassay
(Koizumi et al., 1988). It can be analysed in pharmaceutical preparations by HPLC
(Biemer, 1987) and spetrophotometric (US Pharmacopeial Convention, Inc., 1989)
methods.

Since paracetamol is a metabolite of phenacetin (Reynolds, 1989), carcino-

genicity studies of phenacetin result in exposure of animaIs to paracetamol. For the
results of studies on phenacetin, see IAC (1987).

Mouse: Groups of 60 male and 60 female young adult IF strain mice were fed
paracetamol ( ~ 98% pure; dissolved in acetone then evaporated) at 500 or 10 00
mglg of diet for 18 months (approximate daily intake, 250 or 500 mglg bw,
respectively). A group of 52 males and 52 females fed basal diet served as controls.
Shortly after the beginning of treatment, 33 males and seven females in the
higher-dose group died from liver necrosis. Subsequent survivaI in all groups was
high. AlI survors were killed at 18 months after the beginning of the experiment,
and complete necropsy was carried out with histological examination of the liver;
lungs, pancreas, kidneys, spleen, bladder andadrenal glands. The effective
numbers of animaIs were 50 male and 48 female controls, 54 males and 57 females in
the lower-dose group and 23 males and 47 females in the higher-dose groupe The
incidences of large, often multiple liver neoplasms (adenomas and carcinomas
combined) were 20/23 (87%: 15 adenomas, 5 carcinomas) in higher-dose males, 9/47
(7 adenomas, 2 carcinomas) in higher-dose females, 1/54 (adenoma) in lower-dose
males, 0/57 in lower-dose females, 1/50 (adenoma) in control males and 0/48 in
control females (Flaks ~ Flaks, 1983). (The Working Group noted that the high
dose produced early lethal hepatotoxicity in half the males.)
PARCETAMOL 311
Groups of 50 and 55 male or female (C57BI/6 X C3H/He)F1 (B6C3Fl) mice,

eight to nine weeks of age, were fed paracetamol (:: 98% pure) at 300 and 60
mglg of diet, respectively. The total intake of paracetamol in the high-dose groups
was 863 g/kg bw for males and 675 glg bw for females. Two groups of 50 males and
females were maintained on basal diet. AlI survvors were killed at 134 weeks.
Survival among males was 43/50 (controls), 39/50 (low-dose) and 45/55 (high-dose),
and that among females was 49/50
( controls), 46/50 (low-dose) and 50/55
(high-dose). The numbers of mice scored for tumours were 27/43 control males,
32/49 control females, 21/39 low-dose males, 33/46 low-dose females, 23/45
high-dose males and 33/50 high-dose females. No difference was found in the
incidence of tumours at any site between treated and control mice (Aro &
Matsuyama, 1985).
Groups of 60 and 120 male B6C3F1 mice, six weeks of age, received
paracetamol at 500 or 10 00 mglg of diet, respectively, for up to 70 weeks, at
which time the remaining animaIs were killed. A group of 30 mice served as
controls. Survival in the high-dose group was less than 50% at 24 weeks and 16% at
72 weeks; in the low-dose group, the survival was greater th an 90%. Severe.
ce that died. No increased incidence of
hepatotoxicity was a common finding in mi
neoplasms was observed (Hagiwara & Ward, 1986). (The Working Group noted the
poor survival in the high-dose group.)
Rat: Groups of 30 male SPF Sprague- Dawley rats, six weeks of age, were fed
paracetamol (99.5-99.7% pure) at 0 or 5350 mg/kg of diet for 117 weeks (total
paracetamol intake, 86.5 g per rat). AlI animaIs were necropsied, andkidneys,
urinary bladder, adrenal glands, liver, stomach, spleen, lungs, heart and anygrossly
ab normal organs or tissues were examined histologically. No significant difference
in survival rates was observed. ln the treated group, 4/30 rats developed bladder
papilomatosis or tumours versus 2/30 controls (Johansson, 1981). (The Working
Group noted the relatively small number of animaIs used in the study.)
Groups of 50 male and 50 female Fischer 34/DuCrj rats, five weeks of age
were fed pharmacopoeial-grade paracetamol at 0, 4500 or 90 mg/kg (males) and 0,
6500 or 13 00 mg/kg (females) of diet for 104 weeks and were then observed for a
further26 weeks (average daily intakes: lower-dose males, 195 mg/kg hw; lower-dose
females, 336 mglg bw; higher-dose males, 402 mglg bw; higher-dose females, 688
mglg bw), at which time all survivors were killed. Survival rates at 104 weeks varied
between 86 and 90% in males and 80 and 82% in females, with no significant
difference between treated and control rats. AlI rats were necropsied, and major
organs, tissues and gross abnormalities were examined histologically. No
differencewas seen in tumour incidence between the groups (Hiraga & Fujii, 1985).
Groups of 50 male and 50 female young adult Leeds inbred rats were fed
paracetamol ( :: 98% pure) at 500 or 1000 mg/kg of diet for up to 18 months (mean
daily intake, 300 and 60 mg/kg bw, respectively), at which time all survivors were
killed. A group of 40 males and 40 females fed basal diet alone served as controls.
Survival was high: male controls, 40/40; female controls, 40/40; lower-dose males,
48/50; lower-dose females, 49/50; higher-dose males, 45/50; and higher-dose
females, 49/50. AlI animaIs were necropsied, and samples from each liver lobe,
lungs, kidneys, pancreas, mammary glands, spleen, adrenal glands and from grossly
visible lesions were examined histologically. No tumour was observed among
controls. ln treated animaIs, no hepatocellular carcinoma was observed, but
hepatocellular neoplastic nodules occurred in 0/40, 1/48 and 9/45 control,
lower-dose and higher-dose males and 0/40, 0/49 and 10/49 control, lower-dose and
higher-dose females; and 20-25% of rats in each treated group developed
hyperplasia of the bladder epithelium. Bladder calculi were present in about 30%
of all treated male animaIs and in 6% of females; no clear association was seen
between hyperplasia and the presence of bladder calculI. Bladder papillomas were
observed in 5/49 higher-dose males and bladder carcinomas in 1/49 higher-dose
males; the total bladder tumour incidence was significantly higher (p = 0.02,
Fisher's exact test) among high-dose males. ln the low-dose group, 4/49 females
developed bladder papilomas and 1/49 females developed bladder carcinoma.
Total bladder tumour incidence was significantly higher in low-dose female rats fp
= 0.045, Fisher's exact test) (Flaks et al., 1985). (The Working Group noted that
there were increased incidences of calculi, hyperplasia and tumours of the bladder
in treated animaIs but there was no relationship between the presence of calculI and
the presence of either hyperplasia or tumours.)
(h) Administration with knwn carcinogens

Mouse: Groups of 30 and 60 male B6C3F1 mice, six weeks of age, received
paracetamol at 500 or 1000 mg/kg of diet, respectively, continuously for up to 70
weeks following a single intraperitoneal injection of 40 mg/kg bw N-nitroso-
diethylamine at four weeks of age. A group of 30 mice that received N-nitrosodi-
ethylamine alone served as controls. Mice were sacrified at either 24 or 72 weeks
after injection of the nitrosamine. Survival in the higher-dose group was very poor;
severe hepatotoxicity was a common finding in mice that died. No increased
incidence of neoplasms was found (Hagiwara & Ward, 1986).
Rat: Two groups of 25 or 30 male Fisher 34 rats weighing 150 g were
administered N-nitrosoethyl-N-hydroxyethylamine (NEHEA) at 0 or 0.1% (v/v)
in drinking-water for two weeks and one week later were fed diets containing
paracetamol (purity unspccified) at 1.3% for 29 weeks. One group of 25 rats
received NEHEA in the drinking-water followed by no further treatment.
Ali animaIs were killed at the end of week 32, and samples from liver, kidneys
and other organs with gross abnormalities were examined histologically.
PARCETAMOL 313
)'-Glutamyltranspeptidase foci, hyperplastic nodules, hepatocllular carcinomas,

renal-cell carcinomas, as well as 'atypical cell foci' and adenomas were measured.
Paracetamol inhibited the formation of NEHEA-induced )'-glutamyltrans-
peptidase foci, hyperplastic nodules and carcinomas in comparison with animaIs
treated with NEHEA only. No liver lesion was found in any animal treated with
paracetamolonly. ln contras t, the incidence and multiplicity of preneoplastic renal
les il adenomas were significantly increased in NEHEA-initiated
ions and renal-ce
animaIs treated with paracetamol in comparison with animaIs treated with

NEHEA only. No such renal lesion was observed in groups treated with
paracetamol alone (Tsuda et al., 1984). (The Working Group noted that the
ions described as preneoplastic to neoplasms was not
progression of the les
documented. )
Groups of 25 male Fischer 34 rats, seven weeks old, were administered
N-nitrosobutyl-N-(4-hydroxybutyl)amine at 0 or 0.05% (v/v) in the drinking-water
for four weeks to initiate bladder carcinogenesis and were then fed paracetamol
(purity unspecified) at 1300 mglg of diet for a further 32 weeks, at which time all
rats were killed. One group received treatment with the nitrosamine only. Urinary
bladders, livers and kidneys were examined histologically. No significant difference
in the incidence ofbladder tumours was observed between the groups (Kurata et al.,
1986).
Groups of male Fischer 34 rats (numbers unspecified), six weeks of age, were
subjected to a two-thirds partial hepatectomy and 24 h later received either
intragastric intubations of paracetamol (purity, :: 99%) at 0 or 100 mglg bw in
0.2% tragacanth gum twice a week for five weeks, or a single intragastric instillation
of paracetamol at 500 mglg bw. Two weeks after the end of paracetamol treatment,
the animaIs were administered phenobarbital (pharmacopoeial grade) at 0 or 1
mg/ml drinking-water for 12 weeks. The experiment was terminated at the end of
phenobarbital treatment (weeks 13 and 18). Livers, kidneys, thyroid glands and any
gross lesions were examined histologically. The tumour-initiating activity of
paracetamol was evaluated by the formation of placental-type glutathione
S-transferase-positive foci in liver cells; treatment with paracetamol did not result
in the induction of such foci (Hasegawa et al., 1988). (The Working Group noted
that the rate of absorption of paracetamol from the tragacanth suspension was not
measured, and the limited reporting of the experiment.)
To examine possible intenerence with the activation of 2-acetylaminofluorene,
groups of 20 female SPF CD rats were given diets containing acetylaminofluorene at
250 mglg alone or with paracetamol at Il 00 mglg for 20 weeks and were
observed for an additional ten weeks. Mammary tumours were seen in 14/20
females given acetylaminofluorene and in 7/20 (p = 0.028, Fisher's exact test)
animaIs given acetylaminofluorene and paracetamol (Weisburger et al., 1973).
Hamster. Groups of 30 male and 30 female Syrian golden hamsters, six weeks
old, were given N-hydroxyacetylaminofluorene at 430 mglg alone or with
paracetamol at 1100 mglg of diet for 39 weeks. The experiment was terminated at
47 weeks. The incidences of liver cholangiomas in animaIs treated with N-hydroxy-
acetylaminofluorene were 13/26 in males and 2225 in females; in the group treated
with N-hydroxyacetylaminofluorene and paracetamol, no liver tumour was seen in
24 males but two occurred in 24 females. Similar results were found in groups given
acetylaminofluorene at 400 mglg alone or with paracetamol at 11 00 mglg: with
acetylaminofluorene, the incidence of liver cholangiomas was 6/30 males and 28/30
females; in the group treated with acetylaminofluorene and paracetamol, the
incidence was 0/29 males (p = 0.013, Fisher's exact test) and 4/28 females (p ~
0.001, Fisher's exact test) (Weisburger et al., 1973).
(a) Experimental systems

Dogs receiving a single oral administration of a wide range of doses of
paracetamol excreted about 85% of the administered dose within the first 24 h
(Savides et al., 1984).
A summary of the proposed metabolic pathways of paracetamol is shown in
Figure 1. The major urinary metabolites (the glucuronide, sulfate and 3-mercapto
derivatives) are observed in most species, although there is much species variation
regarding the percentages of these conjugates excreted in the urine (Davis et al.,
1976). Each of the other metabolites shown in Figure 1 has been identified in one
species (see Gemborys & Mudge, 1981, for details). ln rats, biliary excretion of
the
various metabolites of paracetamol increased from 20 to 49% as doses were
increased from 37.5 to 60 mg/kg bw. The glucuronide conjugate was the major
metabolite recovered in the bile at all doses (Hjelle & Klaassen, 1984). The putative
reactive intermediates are not known but
are thought to include benzoquinone
(Hinson et al., 1977).
A minor but important metabolic pathway involves the conversion of
paracetamol to a reactive metabolite by the hepatic cytochrome P450-dependent
mixed-function oxidase system (Mitchell et al., 1973; Potter et al., 1973). The reactive
metabolite is thought to be either N-acetyl-para-benzoquinoneimine (Corcoran et
al., 1980) or the corresponding semiquinone free radical (De Vries, 1981; Nelson et
al., 1981). With low doses of paracetamol, a conjugate of reduced glutathione with
the reactive metabolite is further transformed to cysteine and mercapturic acid
con jugates, which are excreted. As the dose of paracetamol increases, hepatic
glutathione stores are diminished and the glucuronidation and sulfation pathways
PARCETAMOL 315
Fig. 1. Summary or metabolism of paracetamol based on data for different

speciesa
o
)~
o
~~'5 OH
o
)~ )~
Q 0803-
o
)~
r
~Q O-Glucronide
QOHNH,
--0
OH
REACTIVE INTERMEDlATE(S)
o
~~NH /
0'
VGlutthion o
l
OH )~
CHQ~NH
Jl/
CQH3 NH
~ 1
OH
SCH3
o
)~
~ 1
. SCHiCHCOO-
OH NH3 + 1
"6
VOH OH
J
CH~~NH
J J
)~
Q~ 1
)~
~
SOCH3
CH3 NH
OH
VSCHfHCOO-
OH NHCOCH3 Q~ 1
OH
OCH3
tlrom Jollow et al. (1974), Wong et al. (1976) and Gemboiys & Mudge (1981)
becme saturated (Galinsky & Levy, 1981). A correlation has ben demonstrated
between speies sensitivity to the hepatotoxicity of paracetamol and the balance
between two pathways: (i) formation of glutathione conjugates and the
corresponding hydrolysis products (indicative of the 'toxic' pathway) and (ii)
metabolism via formation of glucuronide and sulfate esters (the 'detoxification
pathway') (Gregus et al., 1988). Paracetamol-induced liver toxicity and depletion of
glutathione may be partially prevented by provision of dietary methionine (Reicks
et al., 1988; McLean et al., 1989). At suffciently high doses of paracetamol,
glutathione is depleted and the reactive metabolite binds covalently to cell
macromolecules. It has also ren noted that paracetamol and N-acetyl-pra-benz
quinoneimine may exert their cyotoxic effects via disruption of Ca2+ homeostasis
secondary to the depletion of soluble and protein-bound thiols (Moore et al., 1985).
These data indicate that oxidative or free-radical reactions initiated by paracetamol
have a role in the hepatotoxicity of this drug (Birge et al., 1988).
Radiolabel was bound covalently to hepatocllular proteins following
incubation of mou se, rat, hamster, rabbit or guinea-pig liver microsomes with
3H -paracetamol; the degree of binding was correlated with the susceptibilty of the
species to hepatotoxicity in vivo (Davis et al., 1974). Similar covalent binding of

radiolabel to liver proteins of rats 48 h after administration of (ring-l4c)-
paracetamol was proportional to the extent of liver damage (Davis et al., 1976).
Covalent binding of radiolabel to liver plasma membranes and microsomes was
demonstrated 2.5 h after oral administration of 3H -paracetamol at 2.5 glkg bw to
rats (Tsokos- Kuhn et al., 1988).
Paracetamol is activated in the kidney by an NADPH-dependent cytochrome
P450 mechanism to an arylating agent which can bind covalently to cellular
macromolecules (McMurt et al., 1978). Studies in several species have suggested
that formation of para-aminophenol may be of importance with respect to
paracetamol nephrotoxicity. para-Aminophenol was identified as a urinaiy
metabolite in hamsters (Gemborys & Mudge, 1981); the deacetylation of
paracetamol to para-aminophenol has also been demonstrated in mouse renal
cortical slices (Carpenter & Mudge, 1981). ln comparison to acetyl-Iabelled
paracetamol, ring-labelled paracetamol was preferentially bound to renal
macromolecules in Fischer rats,which are sensitive to paracetamol nephrotoxicity,
whereas binding of ring- and acetyl-Iabelled paracetamol to renal macromolecules
was similar in non-susceptible Sprague-Dawley rats (Newton et al., 1985). This
suggests that para-aminophenol may be responsible for paracetamol-induced renal
necrosis in Fischer 34 rats (Newton et al., 1982).
PARCETAMOL 317
(ii) Taxie effects
The single-dose oral LDso of paracetamol in male rats was 3.7 glkg bw (Boyd &
Bereczky, 196); the 100-day LDso in rats was 40 mg per day (Boyd & Hogan, 1968).
Hepatic necrosis following administration of paracetamol was first reported in
rats (Boyd & Bereczky, 196). The main signs are hydropic vacuolation,
centrilobular necrosis, macrophage infiltration and regenerative activity (Dixon et
al., 1971). Paracetamol-induced hepatotoxicity varies considerably among species:
hamsters and mice are most sensitive, whereas rats, rabbits and guinea-pigs are
resistant to paracetamol-induced liver injury (Davis et al., 1974; Siegers et al., 1978).
Toxic effects in dogs and cats given a single oral dose of paracetamol (maxmal
doses, 500 and 120 mg/kg bw, respectively) included hepatic centrilobular pathology
in dogs, while cats, which do not glucuronidate exogenous compounds, had more
diffuse liver pathological changes (Savides et al., 1984).
The hepatotoxic effects of paracetamol administered in the diet to mice have
been examined histologically. After continuous exposure at 1000 mg/kg diet for 72
weeks (Hagiwara & Ward, 1986), severe chronic hepatotoxicity was obseived, with
centrilobular hepatocytomegaly, cirrhosis, lipofuscin deposition and hepatocyte
necrosis varying from focal to massive. WIth the same dose, Ham and Calder (1984)
observed macroscopically and microscopically deformed livers with extensive
lobular collapse, foci of hepatic necrosis and lymphoid aggregatiòn in portal tracts
after 32 weeks. At a lower dose (500 mg/kg bw) and a shorter exposure time (24
weeks), histological changes were mild. Ultrastructural changes in the livers of rats
administered paracetamol at 10 00 mg/kg diet for up to 18 months have been
described (Flaks et al., 1985).
Histopathological review of liver sections from B6C3F1 mice of each sex fed
paracetamol at 300, 60 or 12 500 mglg diet for 41 weeks and from NIH
general-purpose mice of each sex fed paracetamol at Il 00 mglg diet for 48 weeks
indicated severe liver injury, characterized by centrilobular necrosis in animaIs
receiving more th an 10 00 mglg diet (Maruyama & Willams, 1988).
A single subcutaneous dose of paracetamol at 750 mglg bw to male Fischer
34 rats produced renal tubular necrosis restricted to the upper part of the proximal
tubule (McMurt et al., 1978). Chronic cortical and medullary damage has been
produced in uninephrectomized homozygous Gunn rats by single doses of various
analgesic preparations containing paracetamol (Henry & Tange, 1984).
ln fasted adult male mice given paracetamol at 60 mglg bw orally and killed
within 48 h after treatment, degenerative and necrotic changes were detected in the
bronchial epithelium and in testicular and lymphoid tissue, in addition to renal and
hepatic effects (Placke et al., 1987).
When male rats were given paracetamol at 500 mglg bw per day orally for 70
days, a significant decrease in testicular weight was observed (Jacqueson et al.,
1984).

ln Sprague- Dawley rats administered paracetamol at 250 mg/kg bw orally on
days 8 through 19 of gestation, embryo- and fetotoxic effects were not seen (Lubawy
& Burriss Garret, 1977).
(iv) . Genetic and related effects

Paracetamol was not mutagenic to Salmonella tyhimurium at concentrations
of up to 50 mg/plate in the presence or absence of an exogenous metabolic system
(King et al., 1979; Wirth et al., 1980; Imamuraet al., 1983; Dybing et al., 1984; Oldham
et al., 1986; Jasiewicz & Richardson, 1987). It did not induce mutations in a lIquid
pre-incubation test with Escherichia coli in the presence or absence of an exogenous
metabolic system (King et al., 1979). As reported in an abstract, paracetamol
exhibited mutagenic activity towards S. tyhimurium TA100 in the presence of an
exogenous metabolic system (Tamura et al., 1980).
Feeding of male Drosophila melanogaster with a 4O-mM solution of
paracetamol did not induce sex-linked recessive mutations (King et al., 1979).
Treatment of Chinese hamster V79 cells with low concentrations (0.1-3.0 mM)
of paracetamol inhibited DNA synthesis (Holme et al., 1988; Hongslo et al., 1988).
Paracetamol at 10 mM had no effect on Reuber H4-II-E rat hepatoma cell DNA, as
assayed by alkaline elution, but the toxic metabolite of paracetamol,
N-acetyl-para-benzoquinoneiminè, induced DNA strand breaks (Dybing et òl.,
1984). Treatment of Chinese hamster V79 cells induced DNA strand breaks at 3
and 10 mM but not at 1 mM (Hongslo et al., 1988). Analogous results were obtained
with Chinese hamster ovary cells (Sasaki, 1986). Speies specificitywas observed in
assays for unscheduled DNA synthesis in vitro. No unscheduled DNA synthesis
was detected in Chinese hamster V79 cells (Hongslo et al., 1988), in Syrian hamster
or guinea-pig primary hepatoces (Holme & Soderlund, 1986) or in rat hepatocytes
(Milam & Byard, 1985; Sasaki, 1986; Willams et al., 1989); however, a small but
significant increase in unscheduled DNA synthesis was seen in rat primary
hepatocytes and a marked increase in unscheduled DNA synthesis was observed in
mouse hepatocytes (Holme & Soerlund, 1986).
Paracetamol did not induce mutations to ouabain-resistance in C3H/10Fh
clone 8 mouse embryo cells (Patiemo et al., 1989). It was reported in an abstract that
paracetamol did not induce mutations at the hprt locus in Chinese hamster V79
cells (Sawada et al., 1985). It induced sister chromatid exchange in Chinese hamster
V79 (Holme et al., 1988; Hongslo et al., 1988) and CHO cells (Sasaki, 1986).
Micronuclei were induced by paracetamol in a r~t kidney cell line (NR-49F) at
PARCETAMOL 319
concentrations above 10 mM (Dunn et al., 1987). Paracetamol induced

chromosomal aberrations in three different Chinese hamster celllines (Sasaki et al.,
1980; Sasaki, 1986; Ishidate, 1988) and in human lymphocytes (Watanabe, 1982). It
weakly transformed C3H/10T% clone 8 mouse embryo cells (Patierno et al., 1989).
Paracetamol given twce at a dose of 3 mM (450 mglg bw) either intra-
peritoneally or orally to NMRI mice did not induce micronuclei (King et al., 1979).
Oral treatment of female Sprague-Dawley rats with paracetamol at 500 and 100
mglg bw induced aneuploidy in 12-day embryos (Tsuruzaki et al., 1982). Oral
treatment of Swiss mice with single or three consecutive daily doses of aqueous
solutions of up to 2.5 mglO.5 ml did not lead to chromatid breaks in bone-marrow
cells (Reddy, 1984) or meiotic cells of male Swiss mice (Reddy & Subramanyam,
1985). (Te Working Group noted that the description of the doses used in the two
last studies was unclear.)
(h) Humans
(i) Pharmcokinetics
Following an oral dose, paracetamol is absorbed rapidly from the small
intestine. The rate of absorption depends on the rate of gastric emptying (Clements
et al., 1978). First-pass metabolism of paracetamol is dose-dependent: systemic
availabilty ranges from 90% (with 1-2 g) to 68% (with 0.5 g). Plasma concentrations
of paracetamol in fasting healthy subjects peaked within 1 h after treatment with 0.5
or 1.0 g but continued to rise up to 2 h after treatment with 2.0 g (Rawlings et al.,
1977).
Paracetamol is rapidly and relatively uniformly distributed throughout the
body fluids (GwIlt et al., 1963). Binding to plasma proteins is considered
insignificant (Gazrd et al., 1973). The apparent volume of distribution of
paracetamol in man is about 0.9 l/kg bw (Forrest et al., 1982). The decrease in
paracetamol concentrations in plasma is multiphasic both after intravenous
injections and after oral dosing with 500 and 100 mg. When the data from six
healthy volunteers were interpreted accrding to a two-mpartment open model,
the half-time of the first exponential ranged from 0.15 to 0.53 h and that of the
secnd expnential from 2.24 to 3.30 h. The latter value was in agreement with that
found after oral dosing. Mean clearance (:1 SEM) after intravenous administration
of 100 mg was 352 (:1 40) mlÎmin (Rawlings et al., 1977). Renal excretion of
paracetamol involves glomerular filtration and passive reabsorption, and the
sulfate conjugate is subject to active renal tubular secretion (Morris & Levy, 1984).
Both these metabolites have ben shown 10 accumulate in plasma in patients with
renal faIlure who are taking paracetamol (Lowenthal et al., 1976).
Paracetamol crosses the placenta in unconjugated form, and excretion in the

urine of an exposed neonate was similar to that of a two- to three-day-old infant
(Collns, 1981).
Paracetamol passes rapidly into milk, and the milk:plasma concentration ratio
ranges from 0.7 to 1.1 (Berlin et al., 1980; Notarianni et al., 1987).
Paracetamol is metabolized predominantly to the glucuronide and sulfate
conjugates in the human liver. A minor fraction is converted by cytochrome
P450-dependent hepatic mIxed-function oxidase to a highly reactive arylating
metabolite, which is postulated to be N-acetyl-para-benwquinoneimine (Miner &
Kissenger, 1979). This metabolite is rapidly inactivated by conjugation with
reduced gIutathione and eventually excreted in the urine as acetyl cysteine and
mercapturic acid conjugates. Large doses of paracetamol can deplete glutathione
stores, and the excess of highly reactive intermediate binds covalently with vital cell
elements, which may result in acute hepatic necrosis (Mitchell et al., 1973, 1974).
Only 2-5% of a therapeutic dose was excreted unchanged in the urine. ln young
healthy subjects, about 55, 30, 4 and 4% of a therapeutic dose was excreteclafter
hepatic conjugation with glucuronic acid, sulfuric acid, cysteine and mercapturic
acid, respectively (Forrest et al., 1982).
The fractional recovery of mercapturic acid and cysteine conjugates after
ingestion of paracetamol at 1500 mg was 9.3% in Caucasians compared with only
4.4-5.2% in Africans(Critchley et al., 1986). This may reflect different susceptibilty
to paracetamol hepatotoxicity.
The toxic effects of paracetamol have been reviewed (Flower et al., 1985).
Reports on the acute toxicity, and in particular hepatotoxicity, of paracetamol
have continued to appear since the reporting of the first two cases in 196 (Davidson
& Eastham, 196). Initial symptoms of overdose are nausea, vomiting, diarrhoea
and abdominal pain. Clinical indications of hepatic damage beme manifest
within two to four days after ingestion of toxic doses; in adults, a single dose of 10-15
g (20250 mglg bw) is toxic. Serum transaminases, lactic dehydrogenase and
bilrubin concentrations are elevated, and prothrombin time is prolonged
(Koch-Weser, 1976). The severity of hepatIc injury increases with the ingested dose
and with previous consumption of other drugs that induce liver cytochrome P450
enzymes (Wright & Prescott, 1973). Biopsy of the liver reveals centrilobular
necrosis with sparing of the peri portal area (James et al., 1975). ln nonfatal cases,
the hepatic lesions are reversible over a period of months, without development of
cirrhosis (Hamlyn et al., 1977).
Heavy alcohol consumption has been stated in several case reports to be
related to more severe paracetamol hepatotoxicity th an in non- or moderate
PARCETAMOL 321
drinkers (for review, see Black, 1984). Five cases of combined hepatocellular injury
and renal tubular nccrosis have been reported among patients with a history of
chronic alcohol use who were reciving therapeutic doses of paracetamol (Kaysen et
al., 1985).
No association of paracetamol use with congenital abnormalities or stilbirths
was observed in a studyon drug use in approximately 1000 pregnancies in the UK
(Crombie et al., 1970). ln a case-cntrol study of 458 mothers of malformed babies
and 911 controls, there was no association of abnormalities with use of paracetamol
during the first trimester (Nelson & Forfar, 1971). ln the Collaborative Perinatal
Project, in which drug intake and pregnancy outcome were studied in a series of
50 282 women in 1959-65, 22 women had ben expsed to paracetamol during the
first trimester of pregnancy. There were 17 malformed children in the exposed
group, giving a nonsignificant standardized relative risk (RR) of 1.05 (Heinonen et
al., 1977).
ln a study of 28 00 women belonging to a prepaid health plan in Seattle, WA
(USA), all drug prescriptions and aIl pregnancy outcomes were monitored between
July 1977 and Dccember 1979. Among the liveborn babies of 6837women, 80(1.2%)
had major congenital malformations. Three of the infants born to 493 women for
whom paracetamol had been prescribed in the first trimester had major
malformations (types not specified), giving a prevalence of 6 per 100, which was not
significantly different from the overall prevalence in the total population studied (12
per 100). A second group of 328 women were exposed to paracetamol with codeine
in the first trimester. Five of these had malformed babies, giving a prevalence of 15
per 100, which was not significantly different from that in controls (Jick et al.,
1981).
ln a secnd study of the same population, covering the period J anuary 1980 to
June 1982, 6509 women had pregnancies ending in livebirths; 105 (1.5%) of the
infants had major congenital malformations. 'Io of the infants born to 350 women
for whom paracetamol had been prescribed in the first trimester had major
malformations (types not specified), giving a prevalence of 6 per 100 compared
with an overall prevalence in the entire group of 16 per 100. Three of 347 women
exposed to paracetamol with codeine had malformed babies, giving a prevalence of
9 per 100 (not signific;int) (Aselton et al., 1985).
(iv)
Genetic and related effects
Eleven healthy volunteers were given paracetamol at 100 mg three times over
a period of 8 h. The frequency of chromatid breaks in peripheral bloo lymphocytes
was significantly increased after one day but returned to normal one week later
(Kocisova et al., 1988).
322 lAe MONOGRAHS VOLUM 50
3.3 Case report and epidemiological studies or carcinogenicity to humans
The Working Group considered only studies in which paracetamol was taen
directly, either alone or in mixures. Paracetamol may be taen by analgesic users
who previously took phenacetin. Analgesic mixures containing phenacetin are
carcinogenic to humans; and phenacetin is probably carcinogenic to hum ans
(IAC, 1980, 1987).
A population-based case-cntrol study was conducted in Minnesota, USA,
involving 495 cases of cancer of the renal parenchyma and 74 cases of cancer of the
renal pelvis, diagnosed in 1974-79, and 697 controls (McLaugln et al., 1983, 1984,
1985). An association between cancer of the renal pelvis and intensity and duration
of use of paracetamol-cntaining drugs was seen in women (p for trend, -c 0.05; RR
in the highest expsure category, based on three expsed cass and eight expsed
controls, 5.8; 95% confidence interval (CI), 0.8-). (Te Working Group noted that
the trend test included unexpsed cases and controls; if the unexpsed are excluded,
the trend is not statistically significant.) No other significant association was
observed. Four of the five cases in the highest expsure category (two men, three
women) who developed renal pelvic cancer had also taken phenacetin-cntaining
analgesics; in the entire study, only two cases of cancer of the renal pelvis and seven
con troIs had taken paracetamol alone.
Another population-based case control study was conducted among women
aged 2049 years in the state of New York (USA) involving 173 cases of bladder
cancer diagnosed in 1975-79 and an equal number of controls matched for age and
telephone area coe (Piper et al., 1985). A history of regular use of analgesics
containing paracetamol (and not phenacetin) at least one year before diagnosis
yielded a smoking-adjusted RR of 1.5 (95% CI, 0.4-7.2). ln contrast, the risk for
regular users of phenacetin-containing analgesics was significantly elevated
whether they also regularly took paracetamol (RR, 3.8; 95% CI, 1.4-13.0) or not (RR,
6.5; 95% CI, 1.5-59.2).
,
A series of population-based case-control studies of urinary-tract cancer were
conducted in New South Wales, Australia, involving cases identified In 1977-82
(McCredie et al., 1983a,b, 1988; McCredie & Stewart, 1988). Ultimately, therewere
36 cases of renal parenchymal cancer, 73 cases of renal pelvic cancer, 55 cases of
ureteral cancer and 162 cases (women only) of bladder cancer. Controls (985 for
renal parenchymal cancer and 689 for the other sites) were derived from electoral
roUs. The only significant increase in risk for regular use of paracetamol
(cumulative consumption of at least 0.1 kg) was with ureteral cancer (RR, 2.5; 95%
CI, 1. 1-5.9); this association was not further elevated in the subgroup with higher
exposure (at least 1 kg; RR, 2.0; 95% CI, 0.8-4.5). The RR for cancer of the renal
PARCETAMOL 323
pelvis was 1.2 (95% CI, 0.6-2.3). These analyses were ad justed for cigarette smoking
and the presence of urological disease.
A further population-based case-cntrol study was conducted in Los Angeles
County, USA, based on 187 cases of cancer of the renal pelvis or ureter diagnosed in
1978-82 and an equal number of neighbourhoo controls (Ross et al., 1989). An
association was found with use of nonprescription analgesics in general. The risks
for use of analgesics containing paracetamol were nonsignificantly elevated, at 1.3
for use more th an 30 days/year (p = 0.34) and 2.0 for use more than 30 consecutive
days/year (p = 0.08). The analyses were controlled for cigarette smoking and
history of urinary-tract stones. The authors noted that it was difficult to distinguish
the effects of individual compounds in this study.
ampicilln), 3238 persons to whom at least one prescription for paracetamol alone
and 2612 to whom at least one prescription for paracetamol with codeine had been
dispensed during 1969-73 were followed up for up to 15 years (Selby et al., 1989). No
significant association with cancer at any site was seen for use of paracetamol with
codeine. For paracetamol alone, a positive association was noted for melanoma
(seven cases observed, 1.7 expected; RR, 4.1; 95% Ci, 1.7-8.5), and negative
associations for cancer of the colon (four observed, 12.1 expected; RR, 0.33; 95% CI,
0.1-0.85) and cancer of the uterine corpus (one observed, 6.5 expected; RR, 0.15;
95% Ci, 0-0.86); but no association was seen for any cancer of the urinary tract or for
all cancers combined (Friedman & Ury, 1980, 1983; Selby et aL., 1989). (The
Working Group noted that there was no information on non-prescription
dispensing of paracetamol, which is the most common way that it is obtained.
Since, as also noted by the authors, sorne 12 00 comparisons were made in this
study, the associations should be verified independently. Data on duration of use
were not provided.)
4.1 Exposure data
Paracetamol has been used extensivelyas an analgesic and antipyretic since

1946.

Paracetamol was tested for carcinogenicity by oral administration in mice and
rats. ln one strain of mice, a significant increase in the incidence of multiple liver
carcinomas and adenomas was observed in animaIs of each sex at a markedly toxic
dose; in two studies on another strain, no increase in the incidence of any tumour
was observed at a well-tolerated dose that was approximately half that in the
preceding study. Administration of paracetamol to two different strains of rats did
not increase tumour incidence. ln a further strain of rats, the incidence of neo-
plastic liver nodules was increased in animaIs of each sex given the higher dose; the
combined incidence of bladder papilomas and carcinomas (mostly papilomas)
was significantly greater in high-dose male and in low-dose female rats. Although
treatment increased the incidence of bladder calculi in treated rats, there was no
relationship between the presence of calculi and of either hyperplasia or tumours in
the bladder.
Oral administration of paracetamol to rats enhanced the incidence of renal
adenomas induced by N-nitrosoethyl-N-hydroxyethylamine.

A positive association between use of paracetamol and cancer of the ureter
(but not of other sites in the urinary tract) was observed in an Australian
case-control study. None of three other population-based case-control studies
showed an association between paracetamol use and cancer in the urinary tract.
One study provided no evidence that use of paracetamol in the first trimester
of pregnancy is associated with an increase in the incidence of malformations.
Paracetamol induced testicular atrophy in rats.
Hepatotoxicity has been reported repeatedly in people taking high doses of
paracetamol; chronic alcohol users are particularly sensitive. Paracetamol is meta-
bolized in humans and animaIs to reactive intermediates that bind to proteins. It is
hepatotoxic to experimental animaIs and causes renal tubular necrosis in rats.
Paracetamol induced chromatid breaks in peripheral human lymphocytes in
vivo. It induced aneuploidy in rat embryos treated transplacentally. It gave negative
results in the micronucleus test in mice in vivo. It did not induce chromosomal
aberrations in bone-marrow cells or spermatocytes of mice.
Paracetamol induced sister chromatid exchange and chromosomal aberra-
tions in Chinese hamster cells, micronuclei in. rat kidney cells and chromosomal
aberrations in human lymphoces in vitro. It did not induce point mutations in
mouse or Chinese hamster cells. Paracetamol gave positive results in a transfor-
mation test in mouse cells in vitro. It induced unscheduled DNA synthesis in mouse
and rat cells but not in Chinese or Syrian hamster or guinea-pig cells. Paracetamol
did not induce sex-lInked recessive lethal mutations in Drosophila and was not
mutagenic to Salmonella tyhimurium or Escherichia coli. (Se Appendix 1.)
PARCETAMOL 325
4.5 Evaluation 1
There is inaequate evidence for the carcinogenicity of paracetamol in humans.

There is limited evidence for the carcinogenicity of paracetamol in experi-
mental animaIs.
Overall evaluation
Paracetamol is not classifile as 10 ifs carcinogenicity to humans (Group 3).
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Genotoxic
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PARCETAMOL 329
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PARCETAMOL 331
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SUMMAY OF FINAL EVALUATIONS
Agent Evidence for cacinogenicity Overall evaluation

Hum ans AnimaIs
Ampicilln Inadequate Limited Not c1asifiable (3)

Azacitidine No data Suffcient Probably cacinogenic (2A)
Chloramphenicol Limited Inadequate Probably cacinogenic (2A)
Chlorowtocin No data Suffcient Probably cacinogenic (2A)
Cic1osporin Suffcient Limited Carcinogenic (1)
Cimetidine Inadequate Inadequate Not c1asifiable (3)
Dantron (Chryazn; 1,8-Dihydroxy- No data Suffcient Posibly cacinogenic (2B)
anthraquinone)
Furosmide (Frumide) Inadequate Inadequate Not c1asifiable (3)
Hydrochlorothiazde Inadequate Inadequate Not c1asifiable (3)
Nitrofural (Nitrofurawne) Inadequate Limited Not c1asifiable (3)
Nitrofurantoin Inadequate Limited Not c1asifiable (3)
Paracetamol (Actaminophen) Inadequate Limited Not c1asifiable (3)
Prednimustine No data Inadequate Not c1asifiable (3)
Thiotepa Suffcient Suffcient Carcinogenic (1)
Trichlormethine (frimustine hydro- No data Suffcient Posibly cacinogenic (2B)
chloride)
-333-
Appendix 1. Summary table of genetic and related efTects
Nonmammalan systems Mammalan systems
Prka- Lor
otes eukaotes
Plants Ins ln vitro ln viv
Animal cells Human cells Animais Humans

D G D H G A D G C R G C A D A
Antineoplastic and immunosuppressive agnts

Aztidine
+ + +1 +1 _1 +1 +1 +1 + + +1 + +1 +1 ? ? _1
Chlorozotocn
1 +1
v.
v.
+ +1 + +1 + +1 +1
Ciclosporin
V\ _1 _1 - _1 _1
1 + ?
Thiotepa
+ +1 + + + + + +1 +1 +1 +1 +1 +1
+ + + + +
1ìchlonnethine
+1 +1
AntimicrobiaI agents
Ampicilin
+1 _1 _1 _1 _1
Chloramphenicol
_1 + - - + +1 - ? _1
Nitrfura
+ + ? _1 ? ? +1 + ? _1
Nitrofurantoin
+ + ? ? ? +1 +1 +1 ? _1 _1 + _1 +1 - _1
w
W
0'
Appendix 1 (contd)
Nonmammalian systems Mammalian systems
Prokar- Lower Plants Insets ln vitro
otes eukarotes ln vivo
Animal cells Human cells AnimaIs Humans

D G D R G A D G C R G C A D
A
Other drugs
~
(ì
Cimetidine
a:
-a - _1 o
N-Nitroimetidine z
+ + +1 +1 + + +1 + +1 o
Dantron
+ +1 -,
o
? +1
Furosmide
_1 ~
Hydrochlorothiazde
.1 .1 +
:i
en
+1 +1 _1 +1 +1
~
Parcetamol
_1 ? _1 + +1 + +1 +1
o
- - +1 +1
B
A, aneuploidy; C, ehromosmal aberrtions; D, DNA damage; DL, dominant lethal mutation; G, gene mutation; l, inhibition of intercellular communication; M, mieronuclei; R, mitotie recmbination and gene a:
conversion; S, sister ehromatid exehange; T, cell transformation t'
ln completing the tables, th following ~mbols indicate th consens of the Worling Group withregard to the reults for each end
point:
~
+ considere to be poitive for the speifie endpoint and level of biological complexity
+ 1 considere to be poitive, but only one valid study wa avalable to the Working Group
considered to be negative
considered to be negative, but only one valid study wa avalable to the Working Group
considered to be equivocl or inconc1usive (e.g., there were contradictory results from different laboratories; there were confounding expures; the results were equivocl)
a Cimetidine hydrochloride gave a poitive result
APPENDIX 2
AeTIVTY PROFILES
FOR GENETie AND RELATED EFFEeTS
Methods
The x-axs of the activity profile (Waters et al., 1987, 1988) represents the
bioassays in phylogenetic sequence by end point, and the values on the y-axs
represent the logarithmically transformed lowest effective doses (LED) and highest
ineffective doses (HID) tested. The term 'dose', as used in this report, does not take
into consideration length of treatment or exposure and may therefore be considered
synonymous with concentration. ln practice, the concentrations used in all the
in-vitro tests were converted to lLglml, and those for in-vivo tests were expressed
as mglg bw. Because dose units are plotted on a log scale, differences in molecular
weights of compounds do not, in most cases, greatly influence comparisons of their
activity profiles. Conventions for dose conversions are given below.
Profile-line height (the magnitude of each bar) is a function of the LED or
HID, which is associated with the characteristics of each individual test system -
such as population size, cell-cycle kinetics and metabolic competence. Thus, the
detection lImit of each test system is different, and, across a given activity profie,
responses wil vary substantially. No attempt is made to adjust or relate responses
in one test system to those of another.
Line heights are derived as follows: for negative test results, the highest dose
tested without appreciable toxicity is defined as the HID. If there was evidence of
extreme toxicity, the next highest dose is used. A single dose tested with a negative
result is considered to be equivalent to the HID. Similarly, for positive results, the
LED is recrded. If the original data were analysed statistically by the author, the
dose recorded is that at which the response was significant (p .c 0.05). If the
available data were not analysed statistically, the dose required to produce an effect
is estimated as follows: when a dose-related positive response is observed with two
or more doses, the lower of the doses is taken as the LED; a single dose resulting
in a positive response is considered to be equivalent to the LED.
ln order to accommodate both the wide. range of doses encountered and
positive and negative responses on a continuous scale, doses are transformed
-337-
logarithmically, so that effective (LED) and ineffective (HI) doses are represented
by positive and negative numbers, respetively.The response, or logarithmic dose
unit (LDUij), for a given test system i and chemical j is represented by the
expressions
~d (ij
LDUij = -log1o (dose), for HID values; LDU -c0
LDUij = -log1o (dose x 10-), for LED values; LDU ~O.

These simple relationships define a dose range of 0 to -5 logarithmic units for
ineffective doses (1-100 () J.g/ml or mg/kg bw) and 0 to + 8 logarithmic units for
effective doses (100 00.001 J.g/ml or mg/g bw). A scale ilustrating the LDU
values is shown in Figure 1. Negative responses at doses less than 1 J.g/ml (mglg
bw) are set equal to 1. Effectively, ~ LED value ~100 00 oran HID value -c1
produces an LDU = 0; no quantitative information is gained from such extreme
values. The dotted lInes at the levels of log dose units 1 and -1 define a 'zone of
uncertainty' iD which positive results are reported at such high doses (between
10 () and 100 00 J.g/ml or mg/g bw) or negative results are reported at such low
dose levels (1 to 10 J.g/ml or mg/kg bw) as to call into question the adequacy of the
test.
Fig. 1. Scale of log dose units used on the y-axs of activity profiles
Positive Log dose
(J.g/ml or mg/g bw) units
0.001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
0.01 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
0.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
10 00 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
100 00 . . . . . . . . . . . . . . . .. 1 . . . . . . . . . . . . . . . 0
. . . . . . . . . . . ~ . . .. 10 . . . . . . . . . . . . . . . -1
. . . . . . . . . . . . . .. 100 . . . . . . . . . . . . . .' . -2
. . . . . . . . . . . . .. 100 ... . . . . . . . . . . . . -3
. . . . . . . . . . . . . 10 () . . . . . . . . . . . . . . . ..
. . -. . . . . . . . . . 100 () . . . . . . . . . . . . . . . -5
Negative
(J.g/ml or mg/g bw)
LED and HID are expressed as J.g/ml or mglg bw.
APPENDIX 2 339
ln practice, an activity profile is computer generated. A data entry programme

is used to store abstracted data from published reports. A sequential file (in ASCII)
is created for each compound, and a record within that file consists of the name
and Chemical Abstracts Service number of the compound, a three-Ietter code for
the test system (see below), the qualitative test result (with and without an
exogenous metabolic system), dose (LED or HID), citation number and additional
source information. An abbreviated citation for each publication is stored in a
segment of a record accessing both the test data file and the citation fie. During
processing of the data file, an average of the logarithmic values of the data subset
is calculated, and the length of the profile line represents this average value. AlI dose
values are plotted for each profile line, regardless of whether results are positive
or negative. Results obtained in the absence of an exogenous metabolic system are
indicated by a bar (-), and results obtained in the presence of an exogenous
metabolic system are indicated by an upward-directed arrow ( t). When all results
for a given assay are either positive or negative, the mean of the LDU values is
plotted as a solid line; when conflicting data are reported for the same assay (Le.,
both positive and negative results), the majority data are shown by a solid line and
the minority data by a dashed line (drawn to the extreme conflicting response). ln
the few cases in which the numbers of positive and negative results are equal, the
solid line is drawn in the positive direction and the maximal negative response is
indicated with a dashed line.
Profile lines are identified by three-Ietter code words representing the
commonly used tests. Code words for most of the test systems in current use in
genetic toxicology were defined for the US Environmental Protection Agency's
GENE-TOX Program (Waters, 1979; Waters & Auletta, 1981). For !AC
Monographs Supplement 6, Volume 44 and subsequent volumes, including this
publication, codes were redefined in a manner that should facilitate inclusion of
additional tests. If a test system is not defined precisely, a general code is used that
best defines the category of the test. Naming conventions are described below.
Data listings are preseQted with each activity profile and include endpoint and
test codes, a short test code definition, results (either with (M) or without (NM) an
exogenous activation system), the associated LED or HID value and a short
citation. Test codes areorganized phylogenetically and by endpoint from left to
right across each activity profile and from top to bottom of the corresponding data
listing. Endpoints are defined as follows: A, aneuploidy; C, chromos omal
aberrations; D, DNA damage; F: assays of body fluids; G, gene mutation; H,
host-mediated assays; 1, inhibition of intercellular communication; M, micronuclei;
E sperm morphology; R, mitotic recombination or gene conversion; S, sister
chromatid exchange; and 'l cell transformation.
Dose conversions for activity profiles

Doses are converted to iig/ml for in-vitro tests and to mg/kg bw per day for
in-vivo experiments.
1. ln-vitro test systems
(a) Weight/volume converts directly to iig/ml.

(b) Molar (M) concentration X molecular weight = mg/ml = 1ü3 iig/ml; mM
concentration X molecular weight = iig/ml.
(c) Soluble solids expressed as % concentration are assumed to be in units
of mass per volume (Le., 1% = 0.01 g/ml = 10 00 l1g/ml; also, 1 ppm
= 1 iig/ml).
(d) Liquids and gases expressed as % concentration are assumed to be given
in units of volume per volume. Liquids are converted to weight per volume
using the density (D) of the solution (D = g/ml). Gases are converted
from volume to mass using the ideal gas law, PV = nRT For exposure
at 2O37°C at standard atmospheric pressure, 1% (v/v) = 0.4 iig/ml X
molecular weight of the gas. Also, 1 ppm (v/v) = 4 x 10-5 l1g/ml x
molecular weight.
(e) ln microbial plate tests, it is usual for the doses to be reported as
weight/plate, whereas concentrations are required to enter data on the
activity profile chart. While remaining cognisant of the errors involved
in the process, it is assumed that a 2-ml volume of top agar is delivered
to each plate and that the test substance remains in solution within it;
concentrations are derived from the reported weight/plate values by
dividing by this arbitrary volume. For spot tests, al-ml volume is used
in the calculation.
(f Conversion of particulate concentrations given in l1g1cm2 are based on

the area (A) of the dish and the volume of medium per dish; Le., for a
100mm dish: A = irR2 = 'T x (5 cmf .. 78.5 cm2. If the volume of
. medium is 10 ml, then 78.5 cm2 = 10 ml and 1 cm2 = 0.13 mL.
2. ln-vitro systems using in-vivo activation
For the body fluid-urine (BF-) test, the concentration used is the dose (in
mg/kg bw) of the compound administered to test animaIs or patients.
3. ln-vivo test systems
(a) Doses are converted to mg/kg hw per day of exposure, assuming 100%
absorption. Standard values are used for each sex and species of rodent,
including body weight and average intake per day, as reported by Gold
APPENDIX 2 341
et al. (1984). For example, in a test using male mice fed 50 ppm of the agent
in the diet, the standard foo intake per day is 12% of boy weight, and
the conversion is dose = 50 ppm x 12% = 6 mg!g bw per day.
Standard values used for humans are: weight - males, 70 kg; females,
55 kg; surface area, 1.7 ml; inhalation rate, 20 Umin for light work, 30 Umin
for mi Id exercIse.
(b) When reporte
d, the dose at the target site is used. For example, doses
given in studies. of lymphocytes of humans exposed in vivo are the
measured bloo concentrations in J-glml.
eodes for test sytems
For specific nonmammalian test systems, the first two letters of the
three-symbol code word define the test organism (e.g., SA- for Salmonella
tyhimurium, EC- for Escherichia coli). If the species is not known, the convention
used is -S-. The third symbol may be used to define the tester strain (e.g., SA8 for
S. tyhimurium TA1538, ECW for E. coli WP2uvrA). When strain designation is not
indicated, the third letter is used to define the specific genetic endpoint under
investigation (e.g., -D for differential toxicity, -F for forward mutation, -G for
gene conversion or genetic crossing-over, -N for aneuploidy, -R for reverse
mutation, -U for unscheduled DNA synthesis). The third letter may also be used
to define the general endpoint under investigation when a more complete definition
is not possible or relevant (e.g., -M for mutation, -C for chromos omal
aberration).
For mammalian test systems, the first letter of the three-Ietter code word
der investigation: A- for aneuploidy, B- for
defines the genetic endpoint un
binding, C- for chromosomal aberration, D- for DNA strand breaks, G- for

gene mutation, 1- for inhibition of intercellular communication, M- for
micronucleus formation, R- for DNA repair, S- for sister chromatid exchange,
T- for cell transformation and U- for unscheduled DNA synthesis.
For animal (Le., non-human) test systems in vitro, when the cell type is not
specified, the code letters -lA are used. For such assays in vivo, when the animal
species is not specified, the code letters ~ V A are used. Commonly used animal
species are identified by the third letter (e.g., -C for Chinese hamster, -M for
mouse, -R for rat, -S for Syrian hamster)~
For test systems using humancells in vitro, when the cell type is not specified,
the code letters -IR are used. For assays on humans in vivo, when the cell type is
not specified, the code letters - VH are used. Otherwise, the second letter specifies
the cell type under investigation (e.g., -BH for bone marrow, -LH for lymphocytes).
Some other specific coding conventions used for mammalian systems are as
follows: BF- for body fluids, HM- for host-mediated, -L for leucocytes or
lymphocytes in vitro (-AL animaIs; -HL, hum ans), -L- for leucocytes in vivo (-LA,
animaIs; -LH, humans), - T for tran~formed cells.
Note that these are examples of major conventions used to define the assay
code words. The alphabetized listing of codes must be examined to confirm a
specific coe word. As might be expected from the limitation to three symbols,
sorne codes do not fit the naming conventions precisely. ln a few cases, test systems
are defined by first-Ietter code words, for example: MS'f mouse spot test; SLp,
mouse specific locus test, postspermatogonia; SLO, mouse specific locus test, other
stages; DLM, dominant lethal test in mice; DLR, dominant le th al test in rats; MHl;
mou se heritable translocation test.
. The geneticactivity profiles and listings that follow were prepared in
collaboration with Environmental Health Research and Testing Inc. (EHRT) under
contract to the US Environmental Protection Agency; EHRT also determined the
doses used. The references cited in each genetic activity profile listing can be found
in the list of references in the appropriate monograph.
References
Garrett, N.E., Stack, H.F., Gross, M.R. & Waters, M.D. (1984) An analysis of the spectra
of genetic activity produce by known or suspected human carcinogens. Mutat. Re.,
134, 89-111
Gold, L.S., Sawyer, C.B., Magaw, R., Backman, G.M., de Veciana, M., Levison, R.,
Hooper, N.K., Havender, W.R., Bernstein, L., Peto, R., Pie, M.C. &Ames, RN.
(1984) A carciogenic potency database of the standardized results of animal bioassays.
Environ. Bea/th Perspect., 58,9-319
Waters, M.D. (1979) Th GENE-TOX pro
gram. ln: Hsie, A W., O'Neil, l.P. & McElheny,
\ZK., cds, Mamian Cell Mutagenes: Th Maturation of Tes Systems (Baury Report
2), Cold Sprig Harbr, NY, CHS Press, pp. 449-47
Waters, M.D. & Auletta, A. (1981) The GENE-TOX program: genetic activity evaluation.
L chem. ln! comput. Sei., 21,35-38
Waters M.D., Stade, H.F., Brady, AL., Lohman, P.H.M., Haroun, L. & Vainio, H. (1987)
Appendix 1: Acivity profies for genetic and related tests. In: /AC Monographs on the
Eva/uation of the Careinogec Rik of Chemical to Buma, Suppl. 6, Genetic an
IARC, pp. 687-696 .
Related Effects: An Update of Selected IARC Monographs from Volumes 1 to 42, Lyon,
Waters, M.D., Stade, H.F., Brady, A.L., Lohman, P.H.M., Haroun, L. & Vainio, H. (1988)
Use of computeried data listings and activity profiles of genetic and related effects
in the reviewof 195 compounds. Mutat. Re., 205,295-312
AZCITIDINE
_Test system Result Dose Reference

LED/HID
wi thout With
exogenous exogenous
metabolic metabolic
system system
RN
PRB, virus, mutation
Prophage induction +
+ o 5 t/g/ml
20 t/g/ml
Halle (1968)
ECB, Escherichia coli, DR damage (dcm+/recA6) + o
o 2 . 0 t/g/ml
Barbe et al. (1986)
Bhagwat , Roberts (1987)
SA, Salmonella typhimurium TM677, forward mutation + o o . 24 t/g¡ml
SAO, Salmonella typhimurium TAlOO, reverse mutation + CalI et al. (1986)
o 10 t/g/ml Marquardt , Marquardt (1977)
SA, Salmonella tì~himurium TAlOO, reverse mutation _ o 5 t/g/ml
; SAC, Salmonella typhimurium TAlOO, reverse mutation + Podger (1983)
o 6 t/g/ml
: SA2, Salmonella typhimuriumTAl02, reverse mutation + Schmck et al. (1986)
SA4, Salmo.iella typhimurium TAl04, reverse mutation + o
o
2 .4 t/g/ml
2 . 4 t/g/ml
Schmck et al. (1986) ~
~
SA, Salmonella typhimurium TAl535, reverse mutation + o 24 t/g/ml
Schmck
Schmck
et
et
al.
al.
(1986)
(1986) t'
SA8, Salmonella typhimurium TAl538 , reverse mutation
SA9, Salmonella typhimurium TA98, reverse mutation
o 25 jJg/ml Schmck et al. (1986) Z
o
o
o
5 t/g/ml
25 t/g/ml
12.5 t/g/ml
Podger (1983)
Schmck et al. (1986)
Levin & Ams (1986)
-:xo
SA, Salmonella typhimurium miscellaneous strains, reverse mutation +
SA, Salmonella typhimurium TA2638, reverse mutation +
o o . 5 t/g/ml Podger (1983) N
o 2 . 4 jJg/ml Schmck et al. (1986)
SA, Salmonella typhimurium TA92, reverse mutation + o 12 t/g/ml
SA, Salmonella typhimurium TA2640, reverse mutation Schmck et al. (1986)
o 25 t/g/ml Schmck et al. (1986)
SA, Salmonella typhimurium TA96, TA97, hisG428, hisG46, hisG1775,
o 12 . 5 t/g¡ml Levin , Ames (1986)
reverse mutation
SAS, Salmonella typhimurium TA2661, reverse mutation + 12.5 t/g/ml
SAS, Salmonella typhimurium TA4006, reverse mutation +
o Levin & Ams (1986)
o 12 . 5 jJg¡ml Levin & Ames (1986)
EC2, Escherichia coli WP2, reverse mutation
ECF, Escherichia coli exclusive o'f strain K12, forward mutation +
o 4 t/g/ml Fucik et al. (1965)
o o .1 t/g/ml LaI et al. (1988)
ECR, Escherichia coli other miscellaneous strains, reverse mutation +
o o . 4 t/g/ml
SCH, Saccharomyces cerevisiae, mitotic recombination + o 2500 t/g/ml
Fucik et al. (1965)
Zimmermnn & Scheel (1984)
SCG, Saccharomyces cerevisiae, mi totic gene conversion + o 1000 t/g/ml Zimmerma & Scheel (1984)
SeR, Saccharomyces cerevisiae, reverse mutation + o 1000 t/g/ml Zimmrmann & Scheel (1984)
SCN, Saccharomyces cerevisiae, aneuploidy o 5000 jJg/ml zimmermnn & Scheel (1984)
VFC, vicia faba, chromosomal aberrations o 24 jJg¡ml Fucik et al. (1970)
OM, Drosophila melanogaster, wing-spot assay (somatic mutation + o 244 jJg/ml Katz (1985)
and recombination)
G9H, Gene mutation, Chinese hamster lung V79 cells, hprt locus
o o . 7 t/g/ml Landolph & Jones (1982)
w
.i
w
t
w
AZCITIDlNE (contd)
Test system Resul t Dose Reference

LED¡1ID
wi thout With
exogenous exogenous
metabolic metabolic
system system
-
G90, Gene mutation, Chinese hamster lung V79 cells, ouabain +
resistance
o 1 pg/ml Marquart ¡ Marquart (1977)
~
G90, Gene mutation, Chinese hamster lung V79 cells, ouabain o .7 pg/ml Landolph ¡ Jones (1982)
resistance
o a:
o
G5T, Gene mutation, mouse lymphoma L5178Y cells in vitro, tk locus +
G5T, Gene mutation, mouse lymhoma L5178Y cells in vitro, tk locus + o
0.02 pg/ml
0.01 pg/ml
Amacher ¡ Turner (1987)
McGregor et al. (1989)
z
o
GIA, Gene mutation, mouse C3H/10 T1/2 cells, ouabain resistance
GIA, Gene mutation, BHK cells, hprt locus
o
o
2 . 4 pg/ml
2 . 4 pgjml
Landolph ¡ Jones (1982)
Bouck et al. (1984)
o
GIA, Gene mutation, BHK_ cells, ouabain resistance o 2 . 4 pg/ml Bouck,et al. (1984)
GIA, Gene mutation, primary rat tracheal epithelial cells, ouabin
resistancejhprt locus
o 1 pg/ml Walker ¡ Nettesheim (1986) ~
GIA, Gene mutation, mouse lymphoma L5178Y cells in vitro, hprt locus - :=
o 0.33 pg/ml McGregor et al. (1989) en
SIT, Sister chromatid ex change , hamster cells in vitro + o 1.00 pg/ml Banerjee ¡ Benedict (1979)
SIC, sister chromatid exchange, hamster cells in vitro +
CIC, Chromosomal aberratións, Chinese hamster Don cells in vitro +
o
o
0.24 pg/ml
10 pgj
Hori (1983)
Karon , Benedict (1972) â
CIC, Chromosomal aberrations, Chinese hamster embryo fibroblasts +
(CHF/l8) in vitro
o 0.73 pg/ml Harrison et al. (1983)
8
CIT, Chromosomal aberrations, hamster cells in vitro (+) o 2 . 5 pgl1 Benedict et al. (1977) a:
TB, Cell transformation, BA/c 3T3 mouse cells + o 1 . 2 pgj Yasutake et al. (1987) tT
TCM, Cell transformation, C3H 10T1/2 mouse cells' + o . 25 pgl1
TCL, Cell transformation, Chinese hamster embryo fibroblasts +
o
o 0.73 pgj
Benedict
Harrison
et
et
al.
al.
(1977)
(1983)
~
(CHF/l8 )
TCL, Cell transformation, primary rat tracheal epithelial cells + o o . 24 pgjml Walker , Nettesheim (1986)
DIH, DN strand breaks, Hela cells + o 48 pg/ml Snyder , Lachmnn (1989)
GIH, Gene mutation, human cells in vitro, hprt locus + o o . 12 pgjml Ca11 et al. (1986)
GIH, Gene mutation, human cells in vitro, tk locus + o 0.024 pgjml CalI et al. (1986)
SHL, Sister chromatid exchange, humn lymphocytes in vitro + o 1. 95 pgjml Lavia et al. (1985)
CH, Chromosomal aberrations, humn lymhocytes in vitro + o 1. 95 pg/i Lavia et al. (1985)
CH, Chromosomal aberrations, transformed humn cells in vitro o 2 . 4 pgj Call et al. (1986)
OLM, Dominant lethal test, mice o 10 mg!kg x 1, i.p. Epstein et al. (1972)
APPENDIX 2 345
0
rn
1
0. lJ a
W
U1
z~-
a:
L ~
i
:: _
:i z
..
CO
a
1
=-i:: ::;:
1
1
1
=
1 lJ
1
1
-.
a:
1 L
1 L
1 a:
1
1
L
1
1
1
:i
"-
1 IL
1
1
1
LJ=-J 1
1
LJ :% l-
lJ a
1 - Z a:
1 a: t:
Ln=-J 1 L
:: ~
i-_= 1
1 :i =
ru =-=
1
l" 1- LJ -J a
a:
lD l= b: l: 1-
:;
01
ru
LJ _ 1-
LJ _ '- =
/' lJ
Ln _ 1- -.
a:
Ln _ '- L
i-_= L
i-c:=
i- Ln l-
a:
L
1-
U
W
=:::: lJ
=
=- i. '-
lJ
1-
Z
a:
-.
a.
t. LJ :z
ci
a:
~
::
w
Ln LJ 0: a:
W
~~a; ~
a
W -.
Z
0 u. LJ 0:
u. LJCU lJ
i- 1:b:1: W
() ~EE19
~æEB a1-
)-
a:
tf ¡fU~~ --i a:
~ a~
a:
a.
i: i: æ
co lD "t ru o ru
1
51INn 3500 ::Oi
~
~
OlROZOIN

LED/HD
wi thout
exogenous
iatablic
With
exogenous
metablic
-
system system
~
PR, Strand breaks in PM2-CCC DR
1570 pq ~
PRB, Plasmid pBR 322 DR strand breaks
+
+
0
0 2857 .ug,ml
Lowr & McLaughlin (1979)
Vadi & Reed (1983)
0
Plasmid pBR 322 DR alkylation + Z
PRB, Plasmid pBR 322 DR interstrand cross-links +
0 2857 .uo/ Vadi & Reed (1983)
Vadi & Reed (1983) 0
DR cross-links, calf thyms
SAO, Salmonella typhimurium TAlOO, reverse mutation
+
0
0
2857 .uq/
1570 .uq/ Alexander et al. (1986) 0
+ + 31 .uq/ Franza et al. (1980)
SA, Salmonella typhimurium TAl535, reverse mutation + +
SA, Salmonella typhimurium TAl535, reverse mutation + +
0
50 .u
Franza et al. (1980)
~
SA8, Salmonella typhimurium TAl538, reverse mutation 62 .uq/
Suling et
Franza et
al. (1983)
al. (1980)
::
en
SA9, Salmonella typhimurium TA98, reverse mutation 62 .uq/
SA, Salmonella typhirnurium hisG46, reverse mutation Franza et al. (1980)
+ 0 100 .u Zimmr & Bhuyan (1976)
SCG, Saccharomyces cerevisiae, gene conversion
DH, Drosophila melanogaster, sex-linked recessive lethl mutation
+ 0 314 .u Siebert & Eisenbrand (1977) â
+ 0 31. 4 pq
DIA, DR cross-links and strand breaks, mouse leukaeDda L1210 cells
DIA, DR strand breaks, Chinese hamster V79 cells
+ 0 7 . 85 .ug,ml
Kortselius (1978)
Ewig & Kohn (1977) 8
DIA, DR strand breaks, rnouse leukaernia L1210 cel1s
+ 0 4 .u9/
15 . 7 .ug/
Erickson et al. (1978) ~
G9H, Gene mutation, Chinese hamster lung V79 cells, bprt locus
+ 0 Alexander et al. (1986) m
+ 0
SIM, Sister chromatid ex
SIT, Sister chromatid ex
change , mouse leukaemia L1210 cells
change , 9L rat brain tumur cells
+ 0
6.28 .ug,ml
o . 1 .ug/
Bradley et al. (1980)
Siddiqui et al. (1988) ~
DIH, DR cross-links, huran cells in vitro
+ 0 o . 3 .uo/ Tofilon et al. (1983)
+ 0 15. 7 .ug,ml Erickson et al. (1980)
DVA, DR interstrand cross-links, strand breaks, rat bone-marrow
cells in vivo
+ 0 31.4 mgg x l, i.p. Bedford & Eisenbrand (1984)
BV, DR binding in vitro + 0 24 .ug¡ Panasci et al. (1979)
BV, DR binding in vitro + 0 24 .ug/ Ahlgren et al. (1982)
APPENDIX 2 347
0
(J
i
0.
W i. a
z::
lJ a:
L ::-
i-
1
~z
:x _
0
:;
s:
~
i.
.J
a:
L
L
a:
L
= =- a:
::
"-
lL
i. a
z a:
a: t:
L
~ ::
i- :x =
01
=-=
(J
1
(J 0
CI
~ l-
(' s:
~ ~
i- ui_i- i.
ui_x .J
a:
¡
'-== a:
L
=-a:
l-
U
W
=X'" i.
~
i.
zl-
a:
.J
a.
ci
a:
~
~
w
CI
w
..Z ui '- '- 0~
.J
aU
l-
N
a tn a: ui
i.
w
l"
0 ui a: Ln tß lE li 0
;.
ci ui a: = cr
0
-.
a:
0~.
:i
U
CI
a.
aJ tD ~ ru o ru a-
1 1
51INn 3500 :JOL
~
00
CICLSPORIN
'lest syt_ Resul t Dose Reference

LED/HID
wi thout with ~
exoçenous exogenous (J
_tablic aetablic ~
systea systea
o
- -
z
o
SA, Sai-ella typiauriua TAlOO, reverse autiition 15000 )/g/1I Matter et al. (1982 )
SA, Salinella typiauriua TAl535, reverse autiition - - 15000 )/9/ Matter et al. (1982) Cì
SA7, Salinella tyiaurium TAl537, reverse autiition - - 15000 )/9/ Matter et al. (1982)
SA, Sai-.lla týphiauriua TAl538, reverse autiition - - 15000 )/9/ Matter et al. (1982)
SA, Salinella tyiauriua miscellaneous striiins, reverse autiition - - 15000 p9/ Matter et al. (1982 ) ~
G9B, Gee autation, Chinese hater lung V79 cells, bprt loc - - 250 pg/ii Zwanenburg et al. (1988) :i
en
SB, Sister chroatid exchange, hua lyaocyes 10 vitro (+) 0 1 pgli Yuzawa et al. (1986)
SB, Sister chroaatid exchage, hum lyaocyes 10 vitro
-(+)
0 1 pg/ml Yuzawa et al. (1987)
UV, thcheded mA synthesis, aouse cells in vivo
J1, Micronucleus test,. mee in vivo -
0
0
o.
1500 mgg x 1, p.o.
Matter et al. (1982)
â
MVC, Micronucleus test, hamters in vivo -
-
0 1500 mqg x 1, p.o. Matter et al. (1982) 8
CB, Chroaosoaal abrrations, animal bone-marrow cells 10 vivo
DL,Donat lethal test, aice -
0
0
1500 mgjkg x 1, p.o.
1000 mgjkg x l, p.o.
Matter et al. (1982 )
~
tT
uv, Unchedued mA synthesis, hum lymhocyes in vivo + 0 0 Peti tjean et al. (1986)
CL, Chroasoaal abrrations, hum lymhocyes in vivo (+) 0 9.5 mg/šg
a
Fuuda et al. (1987) ~
CL, Chroaosomal abrrations, hum lymhocyes in vivo (+) 9 mgjkg Fuuda et al. (1988 )
~iipering to 5-6 mgjkg per day; prednisolone was also given at 10 mg/person per day.
Tiipering to 4 mgjkg per day after one year; predisolone was also given at 50 aqperson/day and tapering to 10 mg/person per day.
CICLOSPORIN 59865-13-3 5-SEP-90
5 u c
H V L
L H H
8
4
Ul
..
1- ~
~
Z tr
:: 2 Z
w
Ul
o - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ~- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -. - -- -
U
~
o N
L)
o
..o - - - - - - ~ - ~~ ~ - - - - - - - - - -"- - - - - - - - - - - - - - - - - - - - - - - - - - - ~- - - - - - - - - - - - - --- - - - - - - - - - - - - - - - - - - - - - - - - - --
-2
-4
S !HS G HH C 0
R IHR 9 VVBL
o fY5 H He R H
PROKARYOTES 1 LOWER EUKAR. MAMMALS lN VITRO HUMANS 1 F /HI MAMMALS lN ViVO
lN VITRO w
~
\0
v.
THIOTEPA
~
Test systeii Resul t Dose Reference

LED/HID
wi thout with
exogenous exogenous
intablic intabolic
system system
SAO, Salmonella typhimurium TAlOO, reverse mutation + o 250 pg/ml Pak et al. (1979)
SAS, Salmonella typhimurium TAl535, reverse mutation +
n~
o 50 pg/ml Benedict et al. (1977a)
SA, Salmonella typhimurium TA98, reverse mutation + o 250 pg/ml Bruce " Heddle (1979)
SA, Salmonella typhimurium TA98, reverse mutation 500 pg/ml
AN, Aspergillus nidulans, forward mutation + o Pak et al. (1979)
VFS, Vicia faba, sister chromatid ex
VFC, Vicia faba, chromosomal aberrations +
change + o
o
o
12.5 pg/ml
37.8 pg/ml
37.8 pg/ml
Bignami et al. (1982)
Kihlma (1975)
Kihlman (1975)
o
z
~
VFC, Vicia faba, chromosomal abrrations + o 95 pg/ml Sturelid " Kihlma (1975) o
VFC, Vicia faba, chromosomal aberrations +
DM, Drosophila melanogaster, sex-linked recessive lethal mutations +
o
o
19 pg/ml
0.23 pg/ml
Popa et al. (1976)
LÜers " Röhrborn (1965)
o
DM, Drosophila inlanogaster, sex-linked recessive lethal mutations + o 1. 9 pg/ml Fah " Fah (1970)
G9H, Gene mutation, Chinese hamster V79 lung cells, hprt locus + o 2 pg/IiI Paschin " Kozachenko (1982) ~
sic, Sis ter chromatid ex
SiC, Sister chromatid ex
change , Chinese
change , Chinese
hamster cells in
hamster cells in
vitro
vitro
+
+
o
o
2 . 5 pg/IiI
o .05 pgjml
Chebotarev " Selezneva (1979)
Chebotarev et al. (1980)
::
en
sic, Sister chromatid exchange, Chinese hamster cells ln vitro + o o . 06 pg/ml Selezneva et al. (1982)
change , mouse cells in vitro +
SIT, sister chromatid ex
SIT, Sister chromatid exchange, cloned hamster cells iD vitro +
o
o
o . 2 pgjml
0.01 pg/ml
Andersen (1983)
Baerjee " Benedict (1979)
d
change , rhesus monkey cells in vitro + 2 . 5 pgf
SIA, Sister chromatid ex
CIC, Chromosomal aberrations, Chinese hamster cells in vitro +
o
o 2 pgf
Kuzin et al. (1987) 8
CIC, Chromosomal aberrations, Chinese hamster cells in vitro + o 10 pg/ml
Sturelid (1976)
Maier " Schmd (1976) ~
CIC, Chromosomal aberrations, Chinese hamster cells in vitro + o 3.78 pg/ml Sturelid " Kihlma (1975)
tr
CIT, Chromosomal aberrations, cloned hamster cells in vitro + o . 5 pq/
CI, Chromosomal aberrations, rabbit cells in vitro +
o
o 5 pq/
Benedict et al. (1977b)
Bochkov et al. (1982)
~
CIA, Chromosomal aberrations, rhesus monkey cells in vitro + o 2 . 5 pg/ml Kuzin et al. (1987)
TC, Cell transformtion, C3H lOT1/2 mouse ce11s + o o . 1 pgjml Benedict et aL (1977a)
UH, uncheduled DN synthesis, humn lymphocyes in vitro + o 1 pg/ml Ti tenko (1983)
SHL, Sister chromatid exchange, humn lymphocyes in vitro + o 2.5 pgjml Li ttlefield et al. (1979)
SH, sister chromatid exchange, hum lymhocyes in vitro + o 0.03 pgj Mourelatos (1979)
SHL, Sister chromatid exchange, humn lymphocyes in vitro + o 5 pg/m Chebotarev " Listopad (1980)
SHL, Sister chromatid exchange, humn lymphocyes in vitro + o 1 pgjml Listopad " Chebotarev (1982)
SHL, Sister chromatid exchange, humn lymhocyes in vitro + o 2 . 8 pg/ml Shcheglova , Chebotarev (1983a)
CH, Chromosomal abrrations, humn lymhocyes iD vitro + o 3 pgjml Haml et al. (1966)
CH, Chromosomal abrrations, hum lymhocyes in vitro + o 1 pg/ml Bohkov " Kuleshov (1972)
CH, Chromosoma1 abrrations, humn lymhocyes in vitro + o 10 pg/iù Bochkov et al. (1972)
THIOTEPA (contd)
Test system
Result Dose Reference
LED/HID
wi thout With
exogenous exogenous
metablic metablic
system system
CH, Chromosoaal abrrations, humn lymhocyes in vitro +

o 8 ,ug/ml Chebotarev (1974)
CH, Chromosomal abrrations, humn lymhocyes in vitro +
CH, Chro.osoaal abrrations, hum lymhocytes in vitro + o 20 ,ug/ml Kirichenko (1974)
CH, Chro.osomal abrrations, hum lymhocyes in vitro + o IO ,ug/ml Kirichenko & Chebotarev (1976)
o 6 ,ug/ml Yakovenko & Nazarenko (1977)
CH, Chro.osomal abrrations, hum lymhocyes in vitro +
o 6 ,ug/ml
CH, Chro.osoaal aberrations, humn lymhocytes in vitro + Bochkov et al. (1979)
CH, Chro.osoaal abrrations, humn lymhocyes in vitro + o 200 ,ug/ml WOlff & Artyuyan (1979)
CH, Chromosomal abrrations, humn lymhócyes -in vitro + o 10 ,ug/ml Yakovenko & Kagramanyan (1982)
HM, Host--.ated assay, mouse leukaemia LS187Y cells in mice + o 6 . 6 ,ug/ml Shcheglova & Chebotarev (1983a)
~, Host__diated assay, Salmonella typhimuriui in IIce +" o 7 . 5 mg/kg X 1, s. c . Lee (1973) ~
"'
o 12.4 mg/kg x 3, i.p.
HM, Host-_diated assay, Salmonella typhimuriui in IIce +
o 2.5 mg/kg x 2, p.o.
Ami et al. (1977)
Devi & Reddy (1980)
tr
_SVA, Sister chromatid exchange, mouse bone-marrow cells in vivo +
Z
MV, Micronucleus test,
SVA, Sister chroaatid ex
MV, Micronucleus test, mice

iace in vivo
in vivo +
+
change , rhesus monkey lymocyes in vivo +
o
o
o
2 mg/kg X 1, i.p.
3 mg/kg x 1, i.v.
1 mg/kg X 2, i.p.
Shcheglova & Chebotarev (1983b)
Kuzin et al. (1987)
Maier & Schmid (1976)
-~Ü
MV,
Mv, Micronucleus test,
Micronucleus test, mice
mice in
in vivo
vivo +
+ o
o
20 mgg x 1, i.p.
2.5 mqg x S, i.p.
Ioan et al. (1977)
Bruce & Heddle (1979)
N
MV, Micronucleus test, rat in vivo +
CB, .Chro.osoaal abrrations, .ouse bone-marrow cells in vivo +
o
o
2.5 mg/kg x 1, i.p.
4 mg/kg X l, i.p.
Leonard et al. (1979)
Setnikar et al. (1976)
CB, Chro.osoaal abrrations, .ouse bone-marrow cells in vivo + o 0.32 agg x 1, i.p. Malashenko & Surkova (1974b)
CB, Chro.osoaal abrrations, .ouse bone-aarrow cells in vivo + o 2 agg x 1, i.p. Malashenko & Surkova (1975)
CB, Chro.osoaal abrrations, mouse bone-marrow cells in vivo + o 1.25 agg x 1, i.p. Leonard et al. (1979)
CCC, Chro.osoaal abrrations, iiuse spermatocyes in vivo + o 1 agg X l, i.p. Shcheglova & Chebotarev (1983b)
CCC, Chro.osoaal abrrations, .ouse spermtocyes in vivo + o 1.66 mqg x 2, p.o. Devi & Reddy (1980)
CG, Chro.osomal abrrations, lIuse spermtogonia in vivo + o 20 mg/kg X 1, i.p.
1 mgg x 1, i.p.
Meistrich et al. (1982)
CV, Chro.osoaal abrrations, BOuse liver cells in vivo + o
o 8 mg!kg X 1, i.p.
Malashenko & Beskova (1988)
OOE, Chrollsomal abrrations, preimplantation .ouse ~ryos in vivo + Korogodina & Lil'p (1978)
CVA, Chro.osoaal abrrations, embryonal mouse Ii ver in vivo + o 1.25 mqg x 1, i.p. Malashenko et al. (1978)
OOE, Chro.osoaal abrrations, pre implantation .ouse eBryos in vivo +
o 2.5 mgg x 1, i.p. Korogodina i et al. (1979)
o
OOE, Chro.osoaal abrrations, embryonal mouse Ii ver in vivo + 1.25 mg/kg x 1, i.p. Semenov & Malashenko (1979)
CL, Chrollsomal abrrations, rabi t lymhocyes in vivo + o 2.5 mg/kg x 1, i.p.
3 mqg X 1, i.v.
Korogodina & S 'yakste (1981)
Dut,
OUI,Doainat lethaltest,
test,mice
iùce +
+
CVA, Chro.osoaal abrrations, rhesus monkey lymoces in vivo +
Doait lethal
o
o
o
3 mqg x 1, i.v.
5 mg/kg X 1, i.p.
Bochkov et al. (1982)
Kuzin et al. (1987)
Machemer & Hess (1971)
o 5 mg/kg X l, i.p. Epstein et al. (1972)
w
VI
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THIOTEPA 52-24-4 5-5EP-90
5 5 5 v V 0 G 5$CII T U 5 C
A A A
o 5 9
A
N
F
F F H
5 C X
9IIIIIIC
H ClAClAH
H H H
L L L
HH
HH
AH
51l aI am H
VW B.IIH
A Hl fi imH
C
L
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=
6 ~
- . - -
- -
- . - - - - -
4 ; - :
(J
-
i-
Z
~
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:: 2 tT
w r ------------- ~
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00
---~ ~
N
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0
0
..
1
1
1
1
.----- ----T-- - - -- ------ - - - - -------- - - - - - - - - - - - --- - - - -- - - - - - - - - - - - - --- - - - - - - - - - - - - - - - - - - - - - _ _ _ _ _ _ __ _ _ _ _ __ _ _ _ _ _ __
1
1
-2 1
1
1
1
~
-4
PROKARYOTES 1 LO'iER EUKAR. MAMMALS lN VITRO HUMANS 1 F /HI MAMMALS lN ViVO

lN VITRO w
~
w
~
-
TRCHRMlNE
~
3:
Test syste. o
Reaul t Dose
LED/HID
Reference
z
o
Wi thout With o
exogenous exogenous
_tablic metablic
~
system system
::
CI
C9H, Gee -itation, Chinese haater lung V79 cells, hprt locu + o 3 JlWii slamenova et al. (1983)
cr, Chro~soaal abrrations, walker 256 cells
cr, Chroaosoaal abrrations, Walker 256 cells
+
+
o 1 mqg x 4 - x 10, i.p. Boyland et al. (1948) â
o o Koller (1969)
OL, Damnat lethl test, mce + o 5 mg/kg x 1, i.p. Sykora & Gandalovicova (1978) B
3:
tT
~
TRICHLORMETHINE 817-09-4 5-5EP-90
G o
9 L
H
H
8
4
Ul
..Z
1- ~
'"
:: 2 tT
Z
w
Ul
0
-~o
- - - - - - - -- - - - - - - - - -- - - -- - - - -- - - - - - - - - - - - - - - - - - - - - -1- _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
0 N
LJ
0
0
-i - - - - - - - - - - - - --- - - - - - - -- ---- - - - - -- - - - - - - - - - -- - - - - - - - - --- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - --
-2
-4
PROKARYOTES 1 LOWER EUKAR. MAMMALS lN VITRO HUMANS 1 F ¡HI MAMMALS lN ViVO

lN VITRO W
LI
LI
VJ
~
1\ICILLN
Test system Result Dose Reference

LED/HID
Wi thout
exogenous
metabolic
with
exogenous
metabolic
-
system system ~
(j
a:
PRB, Staphylococcus aureus, prophage induction +
-
0 5 ~g/ml Manthey et al. (1975) 0
PRB, Escherichia coli PQ37, sos induction
ER, Eseherichia coli, DN repair -
0 41 7 ~g/m1 Venier et al. (1989) Z
0
-
1PO ~g/ml Green & Tweats (1981) 0
ECB,
ECB,
Escherichia coli, DN repair
Escherichia coli, DN repair
0
- -
100 t.g/m1
2 ~g/ml
Twats et al. (1981 )
De Flora et al. (1984)
0
SAO, Salmonella typhimurium TAlOO, reverse mutation - 0 0.00 De Flora et al. (1984 )
SAO, Salmonella typhimurium TAlOO, reverse mutation - - 167 ~g/ml Mortelmas et al. (1986 ) ~
SAS, Salmonella typhimurium TAl535, reverse mutation - 0 0.00 De Flora et al. (1984 ) ::
SA, Salmonella typhimurium TAl535, reverse mutation - - 1 ~g/ml Mortelmas et al. (1986) en
SA7, Salmonella typhimurium TAl537, reverse mutation - 0 0.00 De Flora et al. (1984)
SA7, Salmonella typhimurium TAl537, reverse mutation - -
SA8, Salmonella typhimurium TAl538, reverse mutation - 0
1 t.g/ml
0.00
Mortelmans et al. (1986)
De Flora et al. (1984)
ã
SA9, Salmonella typhimurium TA98, reverse mutation - 0.00
SA9, Salmonella typhimurium TA98, reverse mutation -
0
- 167 ~g/ml
De Flora et al. (1984 )
Mortelmas et al. (1986) E
SAS, Salmonella typhimurium TA97, reverse mutation - 0 0.00 De Flora et al. a:
VFC, Vicia faba, aberrant cell division + 0 5000 t.g/ml Prasad (1977)
(1984 )
tr
- -
G5T,
sic,
Gene mutation, mouse lymhoma L5l78Y cells, TK locus
Sister chromatid exchange, Chinese hamster cells in vitro - -
5000 t.g/ml
1500 pg/ml
National Toxicology Program (1987)
National Toxicolog Program (1987)
~
CIC, Chromosomal aberrations, Chinese hamster cells in vitro - - 1500 pg/ml National Toxicology Program (1987)
SHL, sister chromatid ex change , humn lymhocyes in vitro - 0 28 pgli Jaju et aL. (1984)
CH, Chromosomal aberrations, humn fibroblasts in vitro - 0 4000 t.g/1i1 Byarugaba et al. (1975)
CH, Chromosoma1 aberrations, humn lymhocyes in vitro + 0 28 ~g/ml Jaju et aL. (1984)
AMPICILLIN 69-53-4 5-SEP-90
P V C
R F H
B C L
-z
U1
i-
~
tr
:: 2 Z
w o~
-- - -- - - - - - - --- -- - - - - - - - - - - - - - - - - - - - - ~ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -~ -- - - - - - - - - - - - - - - - - - - - - -- - -- - - - ---
U1
oo ~
N
o 1
LJ
o
-l
1
i
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1
1
1
1
-2 -l
l
¡l
-4
t
E E S S G S C S C
C R A A 5 1 1
BOO 9 Tee H H
Lr
PROKARYOTES 1 im/ER EUKAR. MAMMAlS lN VITRO HUMANS 1 F /HI MAMMAlS lN vivo
lN VITRO w
~
W
01
CHRACOL 00
Test system Result Dose Reference

LED/HID
Without With
exogenous exogenous
metalic iitab li c
system system
PR, Prophage induction, sos repair test, DN strand breaks

PR, Escherichia coli, DN dage
-
-
0
-
10 .ugj
30 ,U9/
Mathey et al. (1975)
Mar et al.
-
Sal~nella typmurium, DN breaks (1986)
ECB, Escherichia coli, DN breaks
i:
+
0
0
0
1615 .ug/ii
Jackson et al. (1977)
~
SA, Salllnella typhillrium, differential toxicity - 0 15 .ug/ii Russell et al. (1980) ~
SA, Sal~nella typimurium, differential toxicity - 0 5 .u9/al Nader et aL. (1981) 0
ECO, Escherichia coli pol A, differential toxicity (spot test) - - 30 .ug/a Longnecker et al. (1974) Z
- Nestma et al.
ECO,
ECO,
Escherichia coli pol A, differentia1 toxicity (spot test)
Escherichia co~i pol A, differential toxicity (spot test) -
0 30 .ugj
30 .u
(1979) 0
ECO, Escherichia coli pol A, differential toxicity (spot test) -
0
- 30 .ug/ii
Boyle&. Simpson (1980)
Leifer et al. (1981 )
0
ECL, ESchericha coli pol A, differential toxicity (liqud) - 0 20 .ug/ii Leifer et al. (1981 )
ECO, Eschericha coli, differential toxicity - Slater et al.
ECO, Eschericha coli, differential toxicity -
0 30 .ugli (1971) ~
ER, Eschericha coli, differential toxici ty +
0
0
0
0
Venturini &. Monti-Bragadin (1978)
suter &. Jaeger (1982)
:i
en
ER, Eschericha coli rec, differential toxicity - 0 o . 6 .ugj Shiiizu &. Rosenbrg (1973)
BSD, Bacillus subtilis, differential toxicity - 0 1000 pg/ii
BSD, Bacillus subtilis, differential toxicity
BSO, Bacillus sutilis, differential toxicity
-
-
0 2.5 pgj
Kada et al. (1972)
Sekizawa &. Shibamto (1982) â
0
BR, Bacteria (other), differential toxicity
BR, Bacteria (other), differential toxicity
-
-
0
0
500 p9/
Suter &. Jaeger (1982)
Ader et al. (1976) 8
SA, Sal~nella typhimurium TAlOO, reverse mutation -
0 20 ,Ugj Leifer et al. (1981) ~
0 0 Jackson et al.(1977) tT
SA, Sal~nella tyhimurium TAlOO, reverse mutation 0 - 2 . 5 pgj McCan et al. (1975)
SA, Sai-nella tyimurium TAlOO, reverse mutation - - 333 p9/ Mortelmas et al. (1986) ~
SA, Sal~n.lla typhimurium TAl530, reverse mutation - 0 30 pgj Brem et al. (1974)
SA, Sal~nella typimurium TAl02, reverse mutation - 0 5 pgj Albertini &. Gocke (1988)
SA, Sai-nella tyhimurium TAl535, reverse mutation - 0 30 pgj Breil et al. (1974)
SA, Sal~nella tyhimurium TAl535 , reverse mutation 0 - 2 . 5 .u McCan et al. (1975)
SA, Sal~nella tyillrium TAl53S, reverse mutation - 0 0 Jackson et al. (1977)
SA, sal~n.lla tyillriua TAl535, reverse mutation - - 333 pgj Mortelma et al. (1986)
SA7, Salllnella tyillriua TAl537, reverse mutation 0 - 2 .5 p9/ii McCan et al. (1975)
SA7, sai-nella tyillrium TAS37, reverse Iltation - - 333 pg Mortelmas et al. (1986)
SA, Sal~nella tyillriua TAl538 , reverse mutation - 0 30 .u9/al Brem et al. (1974)
SA, sai-nella typillrium TA9!, reverse mutation 0 - 2 . 5 ,Ugj McCan et al. (1975)
SA, ,Sal~nella tyillriua TA98, reverse mutation (+) (+) 9 ,Ugj Mi tchell et al. (1980)
CHRAHECOL (contd)

LEO/HIO
Wi thout With
exogenous exogenous
IItablic IItab li c
system system
SA9, Salmonella typhimurium TA98, reverse mutation 333 pgji Mortelaas et al. (1986)
ECF, Escherichia coli, forward mutation +
ECF, Escherichia coli WP2, forward mutation +
o
+
o
10 .ug/1I
Mitchell et al. (1980)
o 27 .ugl Mitchell & Gilbert (1985)
ECF, Escherichia coli CM89l, forward mutation (+) o 27 .19/11 Mitchell & Gilbert (1985)
EC2, Escherichia coli WP2, reverse mutation o 200 .u9/ Hemmrly & Deaerec (1955)
EC2, Escherichia coli WP2, reverse mutation o 27 p9/al Mitchell & Gilbert (1985)
ECR, Escherichia coli CM89l, reverse mutation Hemmrly & Deaerec (1955)
ECR, Escherichia coli CM89l, reverse mutation + o
o
200 p9/al
27 pg/ia Mi tchell & Gilbert (1985)
~
"'
SCF, Saccharomyces cerevisiae 01121, petite mutations (+) o 4000 .u9/ weislogel & Butow (1970)
SCF, Saccharomyces cerevisiae 035 and 44, petite mutations (-) tT
SCF, Saccharomyces cerevisiae, petite mutations (+) o 3000 .19/11 Carnevali et al. (1971) Z
AS, Arabidopsis species, mutation
o
o
3000 .ugl
1620 p9/
Williamon et al. (1971)
Miller (1965)
t:
~
TSI, Tradescantia paludosa, micronuclei
HSC, Hordeum species, chromosomal aberrations +
o 1615 p9/al Ma et aL. (1984) ~
o 300 .u Yoshida et al. (1972) N
VFC, Vicia faba, chromosomal aberrations + o 5000 pg/a Prasad (1977)
DM, Drosophila melanogaster, sex-linked recessive lethal mutation o 2500 .u Clark (1963)
DM, Drosophila melanogaster, sex-linked recessive lethl mutation o 100000 .u9/ Nasrat et al. (1977)
DM, Drosophila melanogaster, dominant lethal test o 100000 p9/ia Nasrat et al. (1977)
ut, Unscheduled DR synthesis, Syrian hamster cells in vitro 1000 .u Suzuki (1987)
G5T, Gene mutation, mouse lymhoma L5178Y cells, TK locus + + 3000 pq/ Mitchell et al. (1988)
G5T, Gene mutation, mouse lymhoma L5178Y cells, TK locus + + 2000 .u ~hr & Caspary (1988)
sis, Sister chromatid exchange, Syrian hamster cells in vitro + o 30 .ugl Suzuld (1987)
CIA, Chromosoma1 abrrations, other animal ce11s in vitro + o 500 .u9/ Quéinnec et al. (1975)
TCS, Cell transformtion, Syrian hamster embryo cells o 1000 .u Suzuki (1987)
T7S, Cell transformtion, SA7/Syrian hamster embryo cells (-) o 3490 p9/ Hatch et al. (1986)
DIH, DR strand breaks, humn lymhocyes in vitro (+) o 646 p9/ Yunis et al. (1987)
DIH, DR strand breaks, humn lymhocyes in vitro o 258 p9/ Isildar et al. (1988)'
DIH, DR strand breaks, hum lymhoblastoid cells in vitro o 258 .u Isildar et al. (1988)
DIH, DR strand breaks, humn bone-marrow cells in VI o 258 .u Isildar et al. (1988)
SHL, Sister chromatid exchange, humn lymhocyes in vitro o 200 .u Pant et al. (1976)
CH, Chromosomal aberrations, humn fibroblasts in-v o 625 P9lia Byarugara et al. (1975)
CH, Chromosomal aberrations, humn lymhocyes in vitro + o 10 p9/ Mitus & Colema (1970)
CH, Chromosomal aberrations, humn lymhocyes in vitro o 500 pq/ Jensen (1972)
tH
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LED/HIO
wi thout With
exogenous exogenous
metablic aetablic
system system
SA, Salmonella typhi.urium TA98NR, reverse .utation + + 1 pg/ml Ni et al. (1987)

SA, Salmonella typhimuriua TA98/1, 8-DN6, reverse mutation + + 1 pg/ml Ni et al. (1987)
SA, salmonella typhi.urium TA1536, reverse mutation o 59.4 pg/ml Yahagi et al. (1974)
SA, Sal-nella typi.urium TA97, reverse .utation (f1uct. test) + o 0.32 pg/ml
ECF, Escherichia coli 343/113/R-9, forward mutation _ + 40 pg/ml
Obaseiki-Ebr , Akere1e (1986)
Baars et al. (1980)
EeH, Escherichia coli WP2uvrA, reverse mutation (spot test) + o 50 pgll McCa1la , Voutsinos (1974)
EeH, Bscherichia coli WP2uvrA, reverse .utation + o 8 pg/ml McCa11a , Voutsinos (1974)
EeH,' Escherichia coli WP2uvrA, reverse mutation (fluctuation test) + o
EeH, Eschericha coli WP2uvrA, reverse mutation + o
0.01 pg/ml
0.25, pg/ml
Green et al. (1977)
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Haveland-Smi th et al. (1979)
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o
McCalla et al. (1975)
tr
EeH, Escherichia coli WP2uvrA, reverse mutation + o
59.4 ,ug/ml
10 ,ug/m
Yahagi et al. (1974)
Lu et al. (1979) Z
EC2, Bscherichia coli WP2, reverse mutation (spot test) (+)
EC2, BScherichia coli wp2, reverse mutation + o 100 ,ug/m McCa1la , Voutsinos (1974) U
~
o 16 ,ug/Il
EC2, Escherichia coli WP2, reverse mutation (fluctuation test) (+) o 0.04 pgl
McCalla , Voutsinos (1974)
Obseiki-Ebr , Akere1e (1986) ~
ECR, Escherichia coli nfe 343, reverse mutation o 10 pg/m McCa1la , Voutsinos (1974) N
EC, Escherichia coli nfr 343, reverse mutation o 2 . 5 t/g/m McCal1a et al. (1975)
BCR, Bscbericha coli nfr 345, reverse mutation o 10 t/g/ml McCa1la , Voutsinos (1974)
EeR, Escbericbia coli CM61, reverse mutation o 100 t/g/ McCa1la , Voutsinos (1974)
EeR, Bscbericha coli CM71, reverse mutation o 100 ,ugl KcCalla , Voutsinos (1974)
ECR, Escbericbia coli Ol611,- reverse mutation o 50 ,ug/ml
BCR, Bscbericha coli S, Lac, reverse mutation + o 50 ,ug/ml
McCa1la , Voutsinos (1974)
Zamieri , Greenbrg (1964)
ECR, Bscherichia coli CH1l, reverse mutation (fluctuation test) + o 0.01 ,ug/ml Green et al. (1977)
EeR, Eschericha coli EE97, reverse mutation (fluctuation test) + o 0.02 t/g/m Obaseiki~Ebr , Akere1e (1986)
BeR, gscbericha coli 343/113/R-9, reverse mutation + 40 t/g/m Baars et al. (1980)
1i, Asperqi1lus nidu1ans, forward mutation
Re, Reurospora crassa, reverse mutation + o
o
1000 t/gl
198 pg/m
Bign et al. (1982)
One¡ (1977)
DM, Drosophla aelanogaster, sex-linked recessi ve lethal .utation o 990 pgl Kramrs (1982)
DIA, !B single strand breaks, mouse L929 cells in vitro + o 49.5 ,ug/ml Olive' McCalla (1975)
DIA, !B single strand breaks, hamter BH-21 cells in vitro + o 49.5 pg/ml Olive' McCa1la (1975)
DIA, !B sinqle strand breaks, aouse L929 cells in vIt + 39.6 t/g/ml Olive (1978)
Ul, Ubcbeded DR synthesis, rat primary hepatoces o 0.011 pg/m Meri et al. (1987)
OL, Ubchedled !B synthesis, mouse hepatocyes in vitro o 0.011 pç/ml Meri et al. (1987)
GC, Gee .utation, Chinese liter ovary cells in VI 200 .u9' Anderson , Phillips (1985)
GIA, Gee .utation, Chinese hamter ~79 cells, 6-thiogue res. + o 150 ,ug/ 01i ve (1981)
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NITRFUIN (contd)
-
'lest syste. Resul t Dose Reference ~
LED/HID
~
Wi thout With 0Z
exogenous exogenous
_tablic metablic 0
systeii system 0
HM, saccharo~ces cerevisiae D4-RDII, aitotic gene conversion + 0 500 mg/kg x 1, p.o. 8h Siebert et al. (1979) ~
rN, t' dage, Spragu_Dawley rats in vivo + 0 56 aq/kg x 1, i.p. Russo et al. (1982) ==
rN, t' dage, Spragu_Dawley rats in vivo + 0 14 aq/kg x 1, i.p. Parodi et al. (1983)
en
rN, t' dage, ~use bon_iarrow cells in vivo + 0 64 mg/kg x 1, i.p. Parodi et al. (1983)
MS, Mouse spot test
SVA, Sister chroiatid exchange, .ouse bon_iarrow cells in vivo +
0
0
80 aq/kg x 1, i.p.
32 B9g x 1, i.p.
Gocke et al. (1983) d
Parodi et al. (1983)
MV, Micronucleus test, Spragu_Dawley rats. in vivo 0 400 mg/kg x 1, p.o. Setnikar et al. (1976) B
MV, Micronucleus test, Spragu_Dawley rats in vivo 0 200 mg/kg x 1, i.p. Goodmn et al. (1977)
cec, Chroaoso..l abrrations, iale NM mice meiotic cells ? 0 40 B9g x 2, i.p. Fonatsch (1977) æ
OL, Doainant lethal test, ICRa swiss mice 0 80 B9g x 1, i.p. Epstein et al. (1972)
DL, Doait lethal test, NM mice 0 17.5 l1/kg x 1, p.o. Setnikar et al. (1976) ~
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Test system
Result Dose Reference
LED/HID
wi thout with
n~
exogenous exogenous
_tablic metablic
system system
~
o
ER, Escherichia coli, differential toxicity
ER, Escherichia coli, differential toxici ty o 60 .ug/ii Pool et al. (1979
zo
o 1250 .uq/ De Flora (1981)
SAO, Salmonella typhimurium TAlOO, reverse mutation
10000 .ug/ De Flora , Picciotto (1980) Cì
SA, Salmonella typhimurium TAl535 , reverse mutation 10000 ,ug¡ De Flora , Picciotto (1980)
SA7, Salmonella typhimurium TAl537, reverse mutation
10000 .ug/D
SA~, Salmonella typhimurium TAl538 , reverse mutation 10000 .ug/D
De Flora , Picciotto (1980)
~
DIA, DR strand breaks, transformed mouse epithelial cells in vitro - 10000 .uq/
De Flora , picciotto (1980)
De Flora , Picciotto (1980) ::
CI
DIA, DR damage, rat hepatocytes in vitro
0 1260 .ug/D Schwarz et al. (1980)
+ 0 756 .ug/
VR, Unscheduled DN synthesis, rat primary hepatocyes Martel1i et al. (1983)
VR, Unscheduled DN synthesis, rat primary hepatocyes
+ 0
0
83 ,ug/ a
2520 .ug/ii a
Martelli et al. (1983) â
VR, Unscheduled DR synthesis, rat primary hepatocyes Lefevre , Ashb (1985)
DIH, DR damage, humn hepatocytes in vitro
+ 0 25.2 ,uq/Da Lefevre , Ashb (1985) B
UIH, Unscheduled DN synthesis, humn hepatocytes in vitro
0 2268 .ug/ml Martelli et al. (1986) ~
0 2268 .ug/ a Martelli et al. (1986) tr
SHL, Sister chromatid ex change , human lymhocyes in vitro 0 252 .ug/D b Inoue et al. (1985)
OVA, DR damage, rat liver cells in vivo
OVA, DR damage, rat gastric mucosa in vivo
0 250 mg/kq xl - x20bP.o. Bramilla et al. (1982) ~
0 250 aq/kq x 1 p.o. Pino' Robbiano (1983)
~Cimetidine hydrochloride
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LED/HID
wi thout With
exogenous exogenous
metabolic metablic ~
system system "'
tr
f Z
DVA, DR damage, rat liver cells in vivo
DVA, DR damage, rat gastric mucosa in vivo
0 250 mg/kgf xl - x20
250 mg/kg x 1 p.o.
p.o. Brambilla et al. (1982 ) 01-
0 Pino & Robbiano (1983)
~
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;With 60/120 mg/kg NaNo2
With 80 mg/kg NaN02
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~
-2 tr
~
-4
HD
HV
HA
PROKARYOTES 1 LOWER EUKAR. MAMMALS lN VITRO HUMANS 1 F /HI MAMMALS lN ViVO

lN VITRO
APPENDIX 2 375
l'.. ID
..'" co
'"
N~ N N N MM M~ ~ ..
-~ø -ø-ø-W-øø-ø u- m~
~mm-~m~m~~~mm~~ø" ø.~
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~.~rl-
...-.~._.~..-
~ rl ~ ~rl.~-",..",.El
rl~ M-Mrl~
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co
co
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Gl
o
i:
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M~~Ø .. 0.. 0....
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.. ~ ~ Gl .. ~ .. ~ .. ~ .. ~ ~ II ~ t .. ~ Gl ~ ~ ~
..II
Gl
.. ~..Ili:~..~..~..~..Il...IlGlll... il~ ..Gl
..GlGl oJ~t ol ol ol oJ~ ~~~~t ~tt~
P: ~j~~~j~j~j~j~õ~~~~~~~~ ...
tI
o
.. .. ..
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.c tJll..
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ln UJ UJ ..
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U
Gl GO oI .... ..
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inini-i-coco l'..
ggg~~~~~~~cococo~~~ ~~~~ o~ ~
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a a a a a a a a a a a a a a a ~ l l ~ Iii ~
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.c ...
.c ...
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... ...- II
... II
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II ..II..II_
tUlfuitninrn.. -l
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II II II II II II II II II II II II II II 1I.i.i.c.cOGl
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u
~~~~~~~~~~~~~~~ ~ ~~~~~ ~ ~
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i: i: i: i: i: i: i: i: i: i: i: i: i: i: Gl .. i: i:..
~~~~~~~~~~~~~~~ ~~~~~~: :
II II II II II II II II II II II II II II II 1 0 i:
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tItltltltltltltltltltltltltltlU~~~~..i:
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El
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w
DANTRON (DIHYDROXYANTHRAQUINONE, 1,8-) 117-10-2 5-SEP-90 ~
5 5 5 W C
A A A RI H
2 7 5 RI L
6 -
- A
~
a:
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0 1
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8
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r -----
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PROKARYDTES 1 LmJER EUKAR. MAMMALS lN VITRO HUMANS 1 F /HI MAMMLS lN vivo

lN VITRO
FUOSEMDE

LED/HID
without With
exogenous exogenous
iitabolic metabolic
system system
?;
""
SAO, Salmonella typhimurium TAlOO, reverse mutation - - 5000 .ug/ml National Toxicology Program (1989) tr
SA, Salmonella typhimurium TAl535, reverse mutation - - 5000 .ug/ml National Toxicology Program (1989) Z
SA7, Salmonella typhimurium TAl537, reverse mutation
SA, Salmonella typhimurium TA98, reverse mutation
-
-
-
-
5000 .ug/ml National Toxicology Program (1989) 0~
G5l Gene mutation mouse lymhoma L 5178Y cells - (+)
5000 .ug/ml
1500 .ug/ml
National Toxicology Program
National Toxicology Program
(1989)
(1989)
~
sic Sister chromatid exchange, Chinese hamster ovary cells (+) (+) 750 .uglil National Toxicology Program (1989)
N
CIC, Chromosomal abrrations, Chinese hamter lung cells in vitro (+) 0 2000 .ug/ml Ishidate (1988)
CiC, Chromosomal aberrations, Chinese hamster lung ce1ls in vitro (+) - 500 .ug/m Matsuoka et al. (1979)
CIC, Chromosomal aberrations, Chinese hamster ovary ce1ls (+) (+) 3750 .uglil National Toxico1ogy Program (1989)
SHF, Sister chromatid exchange, humn fibrob1asts in vitro - 0 1654 .ug/ml Sasaki et al. (1980)
CH, Chromosomal aberrations, humn fibroblasts in vitro - 0 1654 .ug/ml Sasaki et al. (1980)
CIH, Chromosomal aberrations, humn leukocyes in vitro + 0 200 .uo/ml Jamela et al. (1979)
CCC, Chromosomal aberrations, C3H/He mouse germ cells in vivo + 0 50 mgg x 1, i.p. Subramyam ¡ Jamela (1977)
BFA, Saccharomyces cerevisiae D4-RDII, gene conversion mouse urine - 0 45 mg/kg x 1, i.p. Marquardt ¡ siebert (1971)
W
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a:
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W
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51INn 3500 :JOL
HYROOROTIAIDE

LED/HID
Without With
exogenous exogenous
metabolic metabolic
system system
ECR,
SAO,
Escherichia coli Hs30R, reverse mutation
Salmonella typhimurium TAlOO, reverse mutation
-
-
0
- 77 . 5 .ug/ml Fujita (1985) ~
'"
5000 .u9/ml Mortelmans et al. (1986 )
SAO, Salmonella typhimurium TAlOO, reverse mutation - - 2500 .ug/ml tT
SAO, Salmonella typhimurium TAlOO, reverse mutation - - 500 .ug/m1
Waskell (1978)
Andrews et al. (1984)
Z
SA, Salmonella typhimurium TAl535, reverse mutation -
SA, Salmonella tyhimurium TAl535, reverse mutation -
-
-
5000 .u9/ml Mortelmans et al. (1986 ) ..U
SA7, Salmonella typhimurium TAl537 , reverse mutation - -
500 .ug/ml
5000 .ug/ml
Andrews et al. (1984) ~
SA, Salmonella typhimurium TAl538, reverse mutation - - Mortelmans et al. (1986) N
500 .u9/ml Andrews et al. (1984 )
SA, Salmonella typhimurium TA98, reverse mutation ? - 5000 .u9/ml Mortelmans et al. (1986 )
SA9, Salmonella typhirnurium TA98, reverse mutation - - 2500 .ug/ml Waskell (1978)
SA, Salmonella typhirnurium TA98, reverse mutation - - 500 .ug/ml Andrews et al. (1984 )
AN, Aspergillus nidulans, non-dis junction and mi totic crossing-ove r + 0 0.00
DM, Drosophila melanogaster, sex-linked recessive lethal mutation - Bignarni et al. (1974)
0 10000 .ug/ml Valencia et al. (1985)
G51, Gene mutation, mouse lyrhoma L5178Y cells + 0 500 .u9/ml National Toxicology Program (1989)
sic, Sister chromatid exchange, Chinese hamster ovary cells in vitro + + 500 .ug/m Galloway et al. (1987)
cie, Chromosomal aberrations, Chinese hamster ovary cel1s in vitro - - 2600 .ug¡' National Toxico1ogy Program (1989)
CIC, Chromosomal aberrations, Chinese hamster lung cell - 0 500 .u9/ Ishidate (1988)
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51INn 3500 ~OL
APPENDIX 2
381
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Z
CI -..
ø; ~ ~~~ 1 101 10 1 1 1 1 1 1 1 1 1 1 10 1 1 1 1 + + +
1 - + + 1 1
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ø~~øøø~W~~~~~~M
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e. 8 e. e. e. 8 e. e. e. 8 e. e. 8 e. 8 e. e. e.e. ~ e. M _.. II;;;;;;;.-
~~~~~~~~
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9 3 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 ~~ :i ~ ~ ~ ~ ~ ~ ~ ~ ~ U
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:i.. ..:i H
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0' II.. i:II~::::::::::::;::i
i: ~ i: i: i: II
~ei;i;i;~ei;i;i;i;e~~ei;ei;i;~e OQlII il il ~i~i~i il IIQ
lllll~ ~ ll~ llll~ ll~ ~ l~ i ~ ~ ~ ~ ~ g ~ ~ ~ g g ::
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~~~~~~M~~rl~M~~~~M~MMM~ ø~ ~~rlrlMMMrl~ ~
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i: i: i: i: i: i: i: i: i: i: i: i: i: i: i: i: i: i: i: i: i: .. 3 o.~ a ev
aiiiiii~~
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::
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II
.. ~ ~ ~~~~ ~ ~ ~~~~ ~ ~~ ~~~~ ~~ ~~~~~ 55 5555 5 ~
II
CI
e. ~ ~~iiiiii~~~i
w ~. ~. ~ ~ ~ ~ ~ ~ ~ ~ii
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w
00
N
PARAML (contd)
-
Test system Dose Reference
Resul t
LED/HID ~
~
Wi thout With
o
exogenous
metablic
exogenous
metablic z
system system o
o
sic, Sister chromatid exchange, Chinese hamter V79 cells in vitro 453 p9/ml
SiC, Sister chromatid exchange, Chinese hamster V79 cells in vitro
+
+
+
0 151 pglm
Hongslo et al. (1988)
Holme et al. (1988)
~
MI, Micronucleus test, rat kidney ce11s in vitro + 0 1510 p9/ml Du et al. (1987)
:=
en
CIC, Chromosomal aberrations, Chinese hamster lung cells in vitro (+) 0 60 .uglm Ishidate et aL. (1978)
CIC, Chromosomal aberrations, Chinese hamster Don-6 cells in vitro + 0 75 .ug/ml
TCM, Cell transformation, C3H 10T1/2 clone 8 cells in vitro
CH, Chromosomal aberrations, humn lymphocyes in vitro
(+) + 1000 pg/ml
Sasaki et al. (1980)
Patierno et al. (1989) â
+ 0 200 pg/ml Watanab (1982)
MV, Micronucleus test, NMI mice in vivo - 0 894 mgjkg x 2, i.p. King et al. (1979)
CB, Chromosomal aberrations, Swiss mice bone-marrow cells in vivo (+) 0 100 mgjkg x 3, p.o. Reddy (1984) ~
CCC, Chromosomal aberrations, male Swiss mice germ ce1ls in vivo
AVA, Aneuploidy, rat embryos in vivo
? 0 100 mgjkg x 3, p.o. Reddy , Subramnyam (1985) tr
+ 0 500 mg/kg x 25, p.o.
CL, Chromosomal aberrations, human lymphocytes in vivo + 0 50 mgjkg x l, p.o.
Tsuruzaki et al. (1982)
KocisoVil et al. (1988) ~
PARACETAMOL 103-90-2 5-SEP-90
A DU 5 He T C C A C
C IR i II C H B V L
C AP CAC H L A A H
-ZlJt- ~
'i
:: 2 tr
Z
W U
lJ - - -- - - - - -- -- - - - - - - - - - - - - - -- - - - - - - - - - ~ - -- - - - -- ""
o
o 1
- - ~ ~- - - - ~- - - - - - - - -- - - - - ~ - - - - - - - - - - - - - - - - -- - - - - ~- - - -- - ~ - --
~
1
1 N
t. o ¿,
o.. "
, 1
II
- - - - - - - - -- - - - - - - - - - - - -- - - - - - - - - - - - -- - - - - - - - - - -- - - - - - - - - - --
-- -- - - - - --- ---- - - - - - - - - - --- - -~ - - -: 1
-2 , 1
II
L,
"
, 1
II
1
~
-4
!i!mE o U G H
fl fH e H i i V
æ !Y K X A A H
PROKARYOTES LOWER EUKAR. MAMMALS lN VITRO HUMANS 1 F ¡HI MAMMALS lN vivo

lN VITRO w
00
w
SUPPLEMENTARY eORRGENDA TO VOLUMES 1-49
Corrigenda to Volumes 1-43 are lIsted in Volume 42, pp. 251-26; additional
corrigenda are given in Volume 43, p. 261, in Volume 45, p. 283, in Volume 46, p. 419
and in Volume 47, p. 505.
Volume 47
p.499 G9H Paschin & Bahitova (1982) Replace + 0 by 0 +

SHL Jansson et al. (1986) Replace 2.QO by 188.~~
SHL Erexson et al. (1985) Replace 0.0050 by 0.50
SHL Morioto et al. (1983) Replace 3.~~ by 282.~~
-385-
eUMULATIV eROSS INDEX TO IARC MONOGRAHS ON
THE EVALUATION OF CARCINOGENIC RISKS TO HUMANS
The volume, page and year are given. References to corrigenda are given in
parentheses.
A
A-~-C 40,245 (1986); Suppl. 7,56 (1987)
Acetaldehyde 36, 101 (1985) (con: 42, 263);
Suppl. 7, TI (1987)
Actaldehyde formylmethylhydrazone (see Gyromitrin)
Actamide 7, 197 (1974); Suppl. 7,389 (1987)
Acetaminophen (see Paracetamol)
Acridine orange 16, 145 (1978); Suppl. 7,56 (1987)
Acritlavinium chloride 13, 31 (1977); Suppl. 7, 56 (1987)
Acrolein 19,479 (1979); 36,133 (1985);
Suppl. 7, 78 (1987);
Acrylamide 39, 41 (1986); Suppl. 7,56 (1987)
Acrylic acid 19,47 (1979); Suppl. 7,56 (1987)
Acrylic fibres 19,86 (1979); Suppl. 7,56 (1987)
Acrylonitrile 19, 73 (1979); Suppl. 7, 79 (1987)
Acrylonitrile-butadiene-styene copolymers 19, 91 (1979); Suppl. 7,56(1987)
Acinolite (se Asbetos)
Actinomycins 10,29 (1976) (con: 42,255);
Suppl. 7, 80 (1987)
Adriamycin 10, 43 (1976); Suppl. 7,82 (1987)
AF-2 31,47 (1983); Suppl. 7,56 (1987)
Atatoxins 1, 145 (1972) (con: 42, 251);
10, 51 (1976); Suppl. 7,83 (1987)
Atatoxin Bi (se Atatoxins)
Atatoxin Bi (se Atatoxins)
Atatoxn 01 (see Afatoxins)
Atatoxin O2 (se Afatoxins)
Atatoxin Mi (se Afatoxins)
Agartine 31,63 (1983); Suppl. 7,56 (1987)
-387-
Alcohol drinking 44
Aldrin 5, 25 (1974); Suppl. 7, 88 (1987)
Allyl chloride 36, 39 (1985); Suppl. 7, 56 (1987)
Allyl isthiocanate 36, 55 (1985); Suppl. 7, 56 (1987)
Allyl isvalerate 36, 69 (1985); Suppl. 7, 56 (1987)
Aluminium production 34, 37 (1984); Suppl. 7, 89 (1987)
Amaranth 8, 41 (1975); Suppl. 7, 56 (1987)
5-Aminoacenaphthene 16,243 (1978); Suppl. 7,56(1987)
2-Aminoanthraquinone 27, 191 (1982); Suppl. 7, 56 (1987)
pa-Aminoazbenzene 8, 53 (1975); Suppl. 7, 390 (1987)
orlho-Aminoaztoluene 8, 61 (1975) (con: 42, 254);.
Suppl. 7, 56 (1987)
pa-Aminobenwic acid 16,249 (1978); Suppl. 7,56(1987)

4-Aminobiphenyl 1, 74 (197)(con: 42, 251);
Suppl. 7,91 (1987)
2-Amino-3,4-imethylimidaz(4,5-f)quinoline (se MeIQ)
2-Amino-3,~imethylimidaz(4,5-f)quinoxaline (se MeIQx)
3-Amino-1,4-imethyl-5H -pyrdo( 4,3-b )indole (se TrpP-1)
2-Aminodipyrdo(1,2-a:3' ,2' -d)imidazle (se Olu-P-2)
1-Amino-2-methylanthrauinone 27, 199 (1982); Suppl. 7, 57 (1987)
2-Amino-3-methylimidaz(4,5-f)quinoline (se IQ)
2-Amin0-methyldipyrdo(1,2-a:3' ,2' -d)-imidazle (se Olu-P-1)
2-Amino-3-methyl-9H-pyrdo(2,3-b)indole (se MeA-a-C)
3-Amino-1-methyl-5H-pyrdo(4,3-b )indole (se TrpP-2)
2-Amino-5-(5-nitro-2-furyl)-1,3,4-thiadiazle 7, 143 (1974); Suppl. 7,57 (1987)
4-Amino-2-nitrophenol 16,43 (1978); Suppl.7, 57 (1987)
2-Amino-5-nitrothiazle 31, 71 (1983); Suppl. 7, 57 (1987)
2-Amino-9H-pyrdo(2,3-b)indole (se A-a-C)
11-Aminoundecnoic acid 39, 239 (1986); Suppl. 7, 57 (1987)
Amitrole 7,31 (1974); 41, 293 (1986)
Suppl. 7, 92 (1987)
Ammonium potasium selenide (se Selenium and selenium
compounds)
Amorphous silca (se aJ Silca) Suppl. 7,341 (1987)
Amosite (se Asbetos)
Ampicilin 50, 153 (199)
Anabolic steroids (se Androgenic (an abolie) steroids)
Anaethetics, volatile 11,285 (1976); Suppl. 7,93 (1987)
Analgesie mixres contaning phenactin (se aJ Phenactin) Suppl. 7,310 (1987)
Androgenic (anabolic) steroids Suppl. 7, 96 (1987)
Angelicin and sorne synthetic derivatives (se aJ Angelicins) 40, 291 (1986)
Angelicin plus ultrviolet raiation (se aJ Angelicin and sorne Suppl. 7, 57 (1987)
synthetic derivatives)
Angelicins Suppl. 7, 57 (1987)
CUMULATI CROSS INDEX 389
Anilne 4, 27 (1974) (con: 42, 252);

27,39 (1982); Suppl. 7, 99 (1987)
orto-Anisidine 27, 63 (1982); Suppl. 7, 57 (1987)
pa-Anisidine 27, 65 (1982); Suppl. 7, 57 (1987)
Anthanthrene 32, 95 (1983); Suppl. 7, 57 (1987)
Anthophyllte (se Asbetos)
Anthrane 32, 105 (1983); Suppl. 7, 57 (1987)
Anthranilc acid 16,265 (1978); Suppl. 7,57 (1987)
Antimony trioxide 47, 291 (1989)
Antimony triulfide 47,291 (1989)
ANU (se 1-NaphthyIthiourea)
Apholate 9,31 (1975); Suppl. 7,57 (1987)

ArmitelP 5, 39 (1974); Suppl. 7, 57 (1987)
Arec nut (se Betel quid)
Aranilic acd (se Arnic and arnic compounds)
Arnic and arnic compounds 1,41 (1972); 2, 48 (1973);
23, 39 (1980); Suppl. 7, 100 (1987)
Arnic pentoxide (se Arnic and arnic compounds)
Arnic sulphide (se Arnic and arnic compounds)
Arnic trioxide (see Arnic and arnic compounds)
Arine (se Arnic and arnic compounds)
Asbetos 2, 17 (1973) (con: 42,252);
14 (1977 (con: 42, 256~ Suppl. 7,
106 (1987) (con: 45, 283)
Attapulgite 42, 159 (1987) Suppl. 7,117 (1987)
Auramine (technical-grade) 1,69 (1972) (con: 42,251); Suppl.
7, 118 (1987
Auramine, manufacture of (se also Auramine, technical-grde) Suppl. 7, 118 (1987)
Aurothiogluco 13,39 (1977 Suppl. 7,57 (1987)
Azcitidine 26,37 (1981); Suppl. 7,57 (1987);
50, 47 (199)
5-Azcyidine (see Azacitidine)
Azrine 10, 73 (1976) (con: 42,255);
Suppl. 7, 57 (1987)
Azathioprine 26, 47 (1981); Suppl. 7, 119 (1987)
Azridine 9, 37 (1975~ Suppl. 7, 58 (1987)
2-(1-Azridinyl)ethanol 9, 47 (1975~ Suppl. 7, 58 (1987)
Azridyl benzouinone 9, 51 (1975~ Suppl. 7, 58 (1987)
Azbenzene 8, 75 (1975~ Suppl. 7, 58 (1987)
B
Baum chromate (se Chromium and chromium compounds)
Basic chromic sulphate (se Chromium and chromium compounds)
BCNU (se Bishloroethyl nitrosurea)

Benz(a )acridine 32, 123 (1983); Suppl. 7, 58 (1987)
Benz(c )acrdine 3, 241 (1973); 32, 129 (1983);
Suppl. 7, 58 (1987)
Benzal chloride (se aJ Q-Chlorinated toluenes) 29, 65 (1982); Supp/. 7, 148 (1987)
Benz(a )anthrane 3, 45 (1973); 32, 135 (1983);
Supp/. 7, 58 (1987)
Benzene 7,203 (1974) (con: 42, 254); 29, 93,
391 (1982); Supp/. 7, 12 (1987)
Benzidine 1,80 (1972); 29, 149,391 (1982);
Supp/. 7, 123 (1987)
Benzidine-bas dyes Supp/. 7, 125 (1987)
Benzo(b )fluoranthene 3, 69 (1973); 32, 147 (1983);
Supp/. 7, 58 (1987)
Benzoúlfluoranthene 3, 82 (1973); 32, 155 (1983); Suppl.
7,58(1987)
Benzo(k )fluoranthene 32, 163 (1983); Suppl. 7, 58 (1987)
Benzo(gilfluoranthene 32, 171 (1983); Suppl. 7, 58 (1987)
Benzo(a )fluorene 32, in (1983); Supp/. 7,58 (1987)
Benzo(b )fluorene 32, 183 (1983); Suppl. 7, 58 (1987)
Benzo(c )fluorene 32, 189 (1983); Suppl. 7, 58 (1987)
Benzo(gilperylene 32, 195 (1983); Suppl. 7, 58 (1987)
Benzo(c )phenanthrene 32, 205 (1983); Suppl. 7, 58 (1987)
Benzo(a)pyrene 3, 91 (1973); 32, 211 (1983);
Supp/. 7, 58 (1987)
Benzo(e )pyrene 3, 137 (1973); 32, 22 (1983);
Supp/. 7, 58 (1987)
pa-Benzouinone dioxime 29, 185 (1982); Suppl; 7, 58 (1987)
Benzotrichloride (se a/so Q-Chlorinated toluenes) 29, 73 (1982); Suppl. 7, 148 (1987)
Benzoyl chloride 29,83 (1982) (con: 42,261); Suppl.
7, 126 (1987)
Benzoyl peroxide 36, 267 (1985); Suppl. 7, 58 (1987)
Benzyl actate 40, 109 (1986); Suppl. 7,58 (1987)
Benzyl chloride (se aJ Q-Chlorinated toluenes) 11, 217 (1976) (con: 42, 25); 29,
49 (1982); Suppl. 7, 148 (1987)
Benzyl violet 4B 16, 153 (1978); Supp/. 7,58(1987)
Bertrandite (seBeryllum and beryllum compounds)
Beryllum and beryllum compounds 1, 17 (1972); 23, 143 (1980) (con:
42, 26); Suppl. 7, 127 (1987)
Beryllum actate (se Beryllum and beryllum compounds)
Beryllum acetate, basic (see Beryllum and beryllum compounds)
Beryllum-aluminium alloy (see Beryllum and beryllum
compounds)
Beryllum cabonate (see Beryllum and beryllum compounds)
Beryllum chloride (see Beryllum and beryllum compounds)
Beryllum~pper alloy (se Beryllum and beryllum compounds)

Beryllum~pper~ba1t alloy (se Beryllum and beryllum compounds)
Beryllum fluoride (se Beryllum and beryllum compounds)
Beryllum hydroe (se Beryllum and beryllum compounds)
Beryllum-nickel alloy (se Beryllum and beryllum componds)
Beryllum oxde (se Beryllum and beryllum compounds)
Beryllum phophate (se Beryllum and beryllum compounds)
Beryllum silicate (se Beryllum and beryllum compounds)
Beryllum sulphate (se Beryllum and beryllum compounds)
Beryl ore (se Beryllum and beryllum compounds)
Betel quid 37,141 (1985); Suppl. 7,12(1987)
Betel-quid chewing (se Betel quid)
BHA (se Butylated hydroxanisle)
BHT (se Butylated hydroxyoluene)
BisI-azridinyl)morpholinophosphine sulphide 9, 55 (1975); Suppl. 7, 58 (1987)
Bis2-chlorothyl)ether 9, 117 (1975); Suppl. 7, 58 (1987)
N,N-Bis2-chloroethyl~2-naphthylamine 4, 119 (1974) (con: 42, 253);
Suppl. 7, 13 (1987)
Bishloroethyl nitrosurea (se alo Chloroethyl nitrosureas) 26, 79 (1981); Suppl. 7, 15 (1987)
1,2-Bis chloromethox)ethane 15, 31 (19n Suppl. 7, 58 (1987)
1,4-Bis chloromethoxyethyl)bnzene 15, 37 (1977 Suppl. 7, 58 (1987)
Bis chloromethyl)ether 4,231 (1974) (con: 42,253);
Suppl. 7, 131 (1987)
Bis2-chloro-l-methylethyl)ether 41, 149 (1986); Suppl. 7,59 (1987)
Bis2,3-epoxycyclopentyl)ether 47,231 (1989)
Bitumens 35,39 (1985); Suppl. 7, 133 (1987)
Bleomycins 26, 97 (1981); Suppl. 7, 134 (1987)
Blue VRS 16, 163 (1978); Suppl. 7, 59 (1987)
Bot and shoe manufacture and repair 25,249 (1981); Suppl. 7,232 (1987)
Braken fem 40,47 (1986); Suppl. 7, 135 (1987)
Brillant Blue FCF 16, 171 (1978) (con: 42, 257)
Suppl. 7, 59 (1987)
1,3-Butadiene 39, 155 (1986) (con: 42, 26);
Suppl. 7, 136 (1987)
1,4-Butanediol dimethanesulphonate 4, 247 (1974); Suppl. 7, 137 (1987)
n-Butyl acrlate 39,67 (1986); Suppl. 7,59 (1987)
Butylated hydroxyanisle 40, 12 (1986); Suppl. 7, 59 (1987)
Butylated hydroxoluene 40, 161 (1986); Suppl. 7,59 (1987)
Butyl benzyl phthalate 29, 193 (1982) (con: 42, 261);
Suppl. 7, 59 (1987)
ß-Butyolactone 11, 22 (1976); Suppl. 7, 59 (1987)
)'-Butyolactone 11, 231 (1976); Suppl. 7, 59 (1987)
c
Cabinet-makng (se Fumiture and cabinet-makng)
Cadmium acetate (see Cadmium and camium compounds)

Cadmium and cadmium compounds 2, 74 (1973); Il, 39 (1976)
(con: 42, 255);
Supp/. 7, 139 (1987)
Cadmium chloride (see Cadmium and cadmium compounds)
Cadmium oxide (see Cadmium and camium compounds)
Cadmium sulphate (see Cadmium and cadmium compounds)
Cadmium sulphide (see Cadmium and cadmium compounds)
Calcium arnate (see Arnic and arnic compounds)
Calcium chromate (see Chromium and chromium compounds)
Calcium cyclamate (see Cyclamates)
Calcium saccharn (se Saccharn)
Canthardin 10, 79 (1976); Supp/. 7, 59 (1987)
Caprolactam 19, 115 (1979) (con: 42, 258);
39, 247 (1986) (con: 42, 26);
Suppl. 7, 390 (1987)
Captan 30, 295 (1983); Supp/. 7, 59 (1987)
Carbail 12, 37 (1976); Suppl. 7, 59 (1987)
Carbazle 32, 239 (1983); Suppl. 7, 59 (1987)
3-Carbethoxyralen 40,317 (1986); Suppl. 7,59 (1987)
Carbon blacks 3, 22 (1973); 33, 35 (1984); Suppl.
7, 142 (1987)
Carbon tetrachloride 1, 53 (1972); 20, 371 (1979);
Suppl. 7, 143 (1987)
Caroisine 8, 83 (1975); Supp/. 7, 59 (1987)
Carntiy and joineiy 25,139 (1981); Suppl. 7,378 (1987)
Cargeenan 10, 181 (1976) (con: 42, 255); 31,
79 (1983); Suppl. 7,59 (1987)
Catehol 15, 155 (1977 Suppl. 7,59 (1987)
CCU (se 1-(2-Chloroethyl)-3-cclohexyl-1-nitrosurea)
Ceramic fibres (se Man-made mineraI fibres)
Chemotherapy, combined, including alkylating agents
(se MOPP and other combined chemotherapy including
aIkyIating agents)
Chlorambucil 9, 12 (1975); 26, 115 (1981);
Suppl. 7, 144 (1987)
Chloramphenicol 10, 85 (1976); Supp/. 7, 145 (1987)
50, 169 (199)
Chlorendic acid 48, 45 (199)
Chlordane (see aI ChlordanelHeptachlor) 20, 45 (1979) (con: 42, 258)
ChlordanelHeptachlor Suppl. 7, 146 (1987)
Chlordecne 20, 67 (1979); Supp/. 7, 59 (1987)
Chlordimeform 30, 61 (1983); Suppl. 7, 59 (1987)
Chlorinate dibenzoioxins (other than TCDD) 15,41 (1977 Suppl. 7,59 (1987)
Chlorinate parns 48, 55 (199)
n-Chlorinated toluenes Suppl. 7, 148 (1987)

Chlonnadinone actate (see aJ Progestins; Combined oral 6, 149 (1974); 21, 365 (1979)
contraceptives)
Chlomaphazne (see N;N-Bis(2-chloroethyl)-2-naphthylamine)
Chlorobenzilate 5, 75 (1974); 30, 73 (1983);
Suppl. 7, 60 (1987)
Chlorodifluoromethane 41,237 (1986); Supp/. 7, 149 (1987)
1-(2-Chloroethyl)-3-cclohexyl-1-nitrosurea (se also 26, 137 (1981) (con: 42, 26);
Chloroethyl nitrureas) Suppl. 7, 150 (1987)
1-(2-Chloroethyl)-3-( 4-methylcyclohexyl)-l-nitrosurea (se also Supp/. 7, 150 (1987)
Chloroethyl nitrosureas)
Chloroethyl nitrosureas Supp/. 7, 150 (1987)
Chlorofluoromethane 41,229 (1986); Supp/. 7,60(1987)
Chlorofonn 1, 61 (1972); 20, 401 (1979);
Supp/. 7, 152 (1987)
Chloromethyl methyl ether (technical-grde) (se aJ
Bis chloromethyl)ether) 4, 239 (1974)
(4-hloro-2-methylphenoxy)acetic acid (see MCPA)
Chlorophenols Suppl. 7, 154 (1987)
Chlorophenols (ocpation al expures to) 41,319 (1986)
Chlorophenoxy herbicides Suppl. 7, 156 (1987)
Chlorophenoxy herbicides (ocpation al expures to) 41,357 (1986)
4-hloro-rlo-phenylenediamine 27, 81 (1982); Suppl. 7, 60 (1987)
4-hloro-meta-phenylenediamine 27,82 (1982); Suppl. 7,60(1987)
Chloroprene 19,131 (1979); Suppl. 7,160 (1987)
Chloropropham 12, 55 (1976); Suppl. 7, 60 (1987)
Chloroquine 13,47 (1977; Suppl. 7,60(1987)
Chlorothalonil 30, 319 (1983); Suppl. 7, 60 (1987)
pa-ChlorO-rlho-toluidine and its strong acid salts 16,277 (1978); 30, 65 (1983);
(se aJ Chlordimefonn) Suppl. 7, 60 (1987); 48, 123 (199)
Chlorotranisne (see aJ Nonsteroidal oetrogens) 21, 139 (1979)
2-Chloro-1,1,1-trfluoroethane 41,253 (1986); Suppl. 7,60(1987)
Chlorozotocin 50, 65 (199)
Cholesterol 10,99 (1976); 31, 95 (1983);
Supp/. 7, 161 (1987)
Chromic actate (se Chromium and chromium compounds)
Chromic chloride (se Chromium and chromium compounds)
Chromic oxide (se Chromium and chromium compounds)
Chromic phosphate (se Chromium and chromium compounds)
Chromite ore (se Chromium and chromium compounds)
Chromium and chromium compounds 2, 100 (1973); 23, 205 (1980);
Supp/. 7, 165 (1987); 49, 49 (199)
Chromium cabonyJ (se Chromium and chromium compounds)
Chromium potasium suJphate (se Chromium and chromium
compounds)
Chromium sulphate (se Chromium and chromium compounds)

Chromium trioxde (se Chromium and chromium compounds)
Chryazn (se Dantron)
Chryne 3, 159 (1973); 32, 247 (1983);
Suppl. 7,60(1987)
Chryidine 8, 91 (1975); Suppl. 7, 169 (1987)
Chrytile (se Asbetos)
Cic1osporin 50, n (199)
CI Dispers Yellow 3 8, 97 (1975); Suppl. 7, 60 (1987)
Cimetidine 50, 235 (199)
Cinnamyl anthranilate . 16, 'l7 (1978); 31, 133 (1983);
Suppl. 7, 60 (1987)
Cisplatin 26, 151 (1981); Suppl. 7, 170 (1987)
Citrinin 40,67 (1986); Suppl. 7,60(1987)
Citru Red No. 2 8, 101 (1975) (con: 42, 25);
Suppl. 7, 60 (1987)
Clofibrate 24,39 (1980); Suppl. 7, 171 (1987)
Clomiphene citrate 21,551 (197); Suppl. 7,172(1987)
Coal gasification 34,65 (1984); Suppl. 7, 173 (1987)
Coal-tar pitches (se aI Coal-tar) Suppl. 7, 174 (1987)
Coal-tar 35,83 (1985); Suppl. 7, 175 (1987)
Cobalt-chromium alloy (se Chromium and chromium
compounds)
Coke production 34, 101 (1984); Suppl. 7, 176 (1987)
Combined oral contraceptives (se a1 Oestrogens, progestins Suppl. 7,297 (1987)
and combinations)
Conjugated oetrogens (se aI Steroidal oetrogens) 21, 147 (1979)
Contraptives, oral (se Combined oral contraceptives;
Seuential oral contraptives)
Copper 8-hydroxquinoline 15, 103 (19n Suppl. 7, 61 (1987)
Coronene 32, 26 (1983); Suppl. 7, 61 (1987
Coumarn JO, 113 (1976); Suppl. 7, 61 (1987)
Creotes (see aI Coal-ta) Suppl. 7, in (1987)
meta-Cesidine 27,91 (1982); Suppl. 7,61 (1987)
pa-Cesidine . 27, 92 (1982); Suppl. 7, 61 (1987)
Crocdolite (se Asbetos)
Crude oH 45, 119 (1989)
Crytallne silca (se aI Silica) Suppl. 7,341 (1987)
Cycain 1, 157 (im) (con: 42,251); 10,
121 (1976); Suppl. 7, 61 (1987)
Cyclamate 22,55 (1980); Suppl. 7, 178 (1987)
Cyclamic acd (se Cyclamates)
Cyclochlorotine 10, 139 (1976); Suppl. 7, 61 (1987)
Cyclohexnone 47, 157 (198)
Cyclohexlamine (se Cyc1amates)
Cyc1opentarcd)pyrene 32, 269 (1983); Suppl. 7, 61 (1987)

Cyc1opropane (se Anaethetics, volatile)
Cyc1ophosphamide 9, 135 (1975); 26, 165 (1981);
Suppl. 7, 182 (1987)
o
2,4-D (se al Chlorophenox herbicides; Chlorophenoxy 15, 111 (1977
herbicides, ocpational expures to)
Dacabazne 26,203 (1981); Suppl. 7, 184 (1987)
Dantron 50, 265 (199)
D & C Red No. 9 8, 107 (1975); Suppl. 7, 61 (1987)
Dapsne 24,59 (1980); Suppl. 7,185 (1987)
Daunomycin 10, 145 (1976); Suppl. 7, 61 (1987)
DDD (se DDT)
DDE (se DDT)
DDT 5, 83 (1974) (con: 42, 253);
Suppl. 7, 186 (1987)
Decbromodiphenyl oxide 48, 73 (199)
Diacetylaminoaztoluene 8, 113 (1975); Suppl. 7, 61 (1987)
N,N' -Diacetylbenzidine 16, 293 (1978); Suppl. 7, 61 (1987)
Diallate 12, 69 (1976); 30, 235 (1983);
Suppl. 7, 61 (1987)
2,4-Diaminoanisole 16, 51 (1978); 27, 103 (1982);
Suppl 7,61 (1987)
4,4' -Diaminodiphenyl ether 16,301 (1978); 29, 203 (1982);
Suppl. 7,61 (1987)
1,2-Diamin0-nitrobenzene 16,63 (1978); Suppl. 7,61 (1987)
1,4- Diamino-2-nitrobenzene 16, 73 (1978); Suppl. 7,61 (1987)
2,6-Diamino-3-(henylaz )pyrdine (see Phenazpyrdine
hydrochloride)
2,4-Diaminotoluene (see al Toluene diisoanates) 16,83 (1978); Suppl. 7,61 (1987)
2,5-Diaminotoluene (se al Toluene diisoanates) 16,97 (1978); Suppl. 7,61 (1987)
onho-Dianisidine (se 3,3' - Dimethoxybenzidine)
Diazpam 13,57 (1977 Suppl. 7,189 (1987)
Diazmethane 7, 223 (1974); Suppl. 7, 61 (1987)
Dibenzra,h )acridine 3, 247 (1973); 32, 2n (1983);
Suppl. 7, 61 (1987)
Dibenzra,Jlacridine 3, 254 (1973); 32, 283 (1983);
Suppl. 7,61 (1987)
Dibenzra,c )arithracene 32, 289 (1983) (con: 42, 262);
Suppl. 7,61 (1987)
Dibenzra,h )anthracene 3, 178 (1973) (con: 43,261);
32, 299 (1983); Suppl. 7, 61 (1987)
Dibenz(a,lanthracene 32, 30 (1983); Suppl. 7, 61 (1987)

7H-Dibenzo(c,g)cabazle 3,26 (1973); 32,315 (1983);
Suppl. 7,61 (1987)
Dibenzoioxins, chlorinate (other than TCDD)
(se Chlorinated dibenzoioxins (other than TCDD))
Dibenzo(a,e )fluoranthene 32, 321 (1983); Suppl. 7, 61 (1987)
Dibenzo(h,m)pentaphene 3, 197 (1973); Suppl. 7, 62 (1987)
Dibenzo(a,e)pyrene 3, 201 (1973); 32, 327 (1983);
Suppl. 7, 62 (1987)
Dibenzo(a,h )pyrene 3,207 (1973); 32,331 (1983);
Suppl. 7, 62 (1987)
Dibenz(a,ilpyrne 3, 215 (1973); 32, 337 (1983);
Suppl. 7, 62 (1987)
Dibenzo(a,/)pyrene 3, 22 (1973); 32, 343 (1983);
Suppl. 7, 62 (1987)
1,2-Dibromo-3-chloropropane 15, 139 (19n) 20, 83 (1979);
Suppl. 7, 191 (1987)
Dichloroacetylene 39,369 (1986); Suppl. 7,62 (1987)
ortho-Dichlorobenzene 7, 231 (1974); 29, 213 (1982);
Suppl. 7, 192 (1987)
pa-Dichlorobenzene 7, 231 (1974); 29,215 (1982);
Suppl. 7, 192 (1987)
3,3' -Dichlorobenzidine 4, 49 (1974); 29, 239 (1982);
Suppl. 7, 193 (1987)
tra-1,4-Dichlorobutene 15, 149 (19n) Suppl. 7, 62 (1987)
3,3' -Dichlor0-,4' 4iiaminodiphenyl ether 16, 30 (1978); Suppl. 7, 62 (1987)
1,2-Dichloroethane 20, 429 (1979); Suppl. 7, 62 (1987)
Dichloromethane 20,449 (1979); 41, 43 (1986);
Suppl. 7, 194 (1987)
2,4-Dichlorophenol (see Chlorophenols; Chlorophenols,
ocpational expures to)
(2,4-Dichlorophenoxy)acetic acid (se 2,4-D)
2,6-Dichloro-pa-phenylenediamine 39,325 (1986); Suppl. 7,62 (1987)
1,2-Dichloropropane 41, 131 (1986); Suppl. 7,62 (1987)
1,3-Dichloropropene (technical-grade) 41,113 (1986); Suppl. 7,195 (1987)
Dichlorvos 20, 97 (1979); Suppl. 7, 62 (1987)
Dicofol 30, 87 (1983); Suppl. 7, 62 (1987)
Dicyclohexylamine (se Cyclamates)
Dieldrin 5, 125 (1974); Suppl. 7, 196 (1987)
Dienoetrol (see a/so Nonsteroidal oetrogens) 21, 161 (1979)
Diepoxybutane Il, 115 (1976)
(COlT 42,255); Suppl.
7, 62 (1987)
Diesl and gasline engine exhausts 46,41 (1989)
Diesl fuels 45, 219 (1989) (COlT 47, 505)
Diethyl ether (se Anaesthetics, volatile)
CUMULTI CROSS INEX 397
Di(2-ethylhexyl)aipate 29, 257 (1982); Suppl. 7,62 (1987)

Di(2-ethylhexyl)phthalate 29, 269
(1982) (con: 42,261); Suppl.
7, 62 (1987)
l,2-Diethylhydrane 4, 153 (1974); Suppl. 7,62 (1987)
Diethylstilbotrol 6,55 (1974); 21, 173 (197)
(con: 42, 259); Suppl. 7, 273 (1987)
Diethylstilbotrl dipropionate (se Diethylstilbotrl)
Diethyl sulphate 4, 2n (1974); Suppl. 7, 198 (1987)
Diglycidyl resrcinol ether Il, 12 (1976); 36, 181 (1985);
Suppl. 7,62(1987)
Dihydroafle l, 170 (197); 10, 233 (1976);
Suppl. 7, 62 (1987)
l,8Dihydroxanthraquinone (se Dantrn)
Dihydroxbenzene (se Catechol; Hydroquinone; Resrcinol)
Dihydroxethylfuratrizine 24, n (198); Suppl. 7, 62 (1987)
Dimethisterone (se aJ Progestins; Seuential oral 6, 167 (1974); 21, 3n (197)
contraptives)
Dimethoxane 15, in (19' Suppl. 7, 62 (1987)
3,3' -Dimethoxybenzidine 4,41 (1974); Suppl. 7,198 (1987)
3,3' -Dimethoxbenzidine-4,4' -diisoanate 39,279 (1986); Suppl. 7,62 (1987)
pa-Dimethylaminoabenzene 8, 12 (1975); Suppl. 7, 62 (1987)
pa-Dimethylaminoazbenzenediaz soium sulphonate 8, 147 (1975); Suppl. 7, 62 (1987)
tra-2-((Dimethylamino )methylimino )-5-(2-(5-nitro-2-furyl)- 7, 147 (1974) (con: 42, 253);
vinyl)-l,3,~xaiazle Suppl. 7, 62 (1987)
4,4' -Dimethylangelicin plus ultrviolet radiation (se a/sa Suppl. 7, 57 (1987)
Angelicin and sorne synthe tic derivatives)
4,5' -Dimethylangelicin plus ultraviolet radiation (see aI Suppl. 7, 57 (1987)
Angelicin and sorne synthetic derivatives)
Dimethylarinic acd (se Arnic and arnic compounds)
3,3' -Dimethylbenzidine l, 87 (1972); Suppl. 7, 62 (1987)
Dimethylcabamoyl chloride 12, n (1976); Suppl. 7, 199 (1987)
Dimethylformamide 47, 171 (1989)
1,1-Dimethylhydrane 4, 137 (1974); Suppl.7, 62 (1987)
1,2-Dimethylhydrane 4, 145 (1974) (con: 42, 253);
Suppl. 7, 62 (1987)
Dimethyl hydrogen phosphite 48, 85 (199)
1,4-Dimethylphenanthrene 32, 349 (1983); Suppl. 7, 62 (1987)
Dimethyl sulphate 4, 271 (1974); Suppl. 7, 20 (1987)
3,7-Dinitrotluoranthene 46, 189 (1989)
3,9-Dinitrotluoranthene 46, 195 (1989)
l,3Dinitropyrene 46, 201 (1989)
1,6- Dinitropyrene 46, 215 (1989)
1,8-Dinitropyrene 33, 171 (1984); Suppl. 7,63 (1987);
46,231 (1989)
Dinitrospentamethylenetetramine 11, 241 (1976); Supp/. 7, 63 (1987)
1,4-Dioxane 11, 247 (1976); Suppl. 7, 201 (1987)

2,4' -Diphenyldiamine 16,313 (1978); Suppl. 7,63(1987)
Direct Black 38 (see aIo Benzidine-bas dyes) 29, 295 (1982) (con: 42, 261)
Direct Blue 6 (see a/so Benzidine-bas dyes) 29, 311 (1982)
Direct Brown 95 (se a/so Benzidine-bas dyes) 29, 321 (1982)
Dispers Blue 1 48, 139 (199)
Dispers Yellow 3 48, 149 (199)
Disulfiram 12, 85 (1976); Suppl. 7, 63 (1987)
Dithranol 13, 75 (197; Suppl. 7, 63 (1987)
Divinyl ether (see Anaesthetics, volatile)
Dulcin 12, 97 (1976); Suppl. 7, 63 (1987)
E
Endrin 5, 157 (1974); Suppl. 7,63 (1987)
Enflurane (see Anaesthetics, volatile)
Boin 15, 183 (1977; Suppl. 7,63 (1987)
Epichlorohydrin 11, 131 (1976)(con: 42,256);
Suppl. 7,20(1987)
1,2- Epoxybutane 47,217 (1989)
1-Epoxyethyl-3,4-poxycyc1ohexane 11, 141 (1976); Suppl. 7,63 (1987)
3,4-Epoxy-fmethylcyc1ohexylmethyl-3,4-poxy-fmethyl- 11, 147 (1976); Suppl. 7, 63 (1987)
cyc10hexane caboxylate
cis-9,10-Epoxytearc acid 11, 153 (1976); Suppl. 7, 63 (1987)
Erionite 42,22 (1987) Suppl. 7, 203 (1987)
Ethinyloetrdiol (se a/so Steroidal oetrogens) 6, TI (1974); 21, 233 (1979)
Ethionamide 13,83 (1977 Suppl. 7,63(1987)
Ethyl acrylate 19, 57 (1979); 39, 81 (1986);
Suppl. 7, 63 (1987)
Ethylene 19, 157 (1979); Suppl. 7,63(1987)
Ethylene dibromide 15, 195 (1977; Suppl. 7,20(1987)
Ethylene oxde 11, 157 (1976); 36, 189 (1985)
(con: 42, 26); Suppl. 7, 205 (1987)
Ethylene sulphide 11, 257 (1976); Suppl. 7, 63 (1987)
Ethylene thiourea 7, 45 (1974); Suppl. 7, 207 (1987)
Ethyl methanesulphonate 7,245 (1974); Suppl. 7,63 (1987)
N-Ethyl-N-nitrûsurea 1, 135 (1972); 17, 191 (1978);
Suppl. 7, 63 (1987)
Ethyl selenac (se a/so Selenium and selenium compounds) 12, 107 (1976); Suppl. 7, 63 (1987)
~thyl tellurac. . 12, 115 (1976); Suppl. 7, 63 (1987)
Ethynodiol diactate (se a/so Progestins; Combined oral 6, 173 (1974); 21, 387 (1979)
contrceptives)
Eugenol 36, 75 (1985); Suppl. 7, 63 (1987)
Eva blue 8, 151 (1975); Suppl. 7, 63 (1987)
F
Fat Green FCF 16, 187 (1978); Suppl. 7,63 (1987)
Ferbam 12, 121 (1976) (con: 42, 256);
Supp/. 7,63 (1987)
Ferrc oxide 1, 29 (1972); Suppl. 7, 216 (1987)
Ferrochromium (se Chromium and chromium compounds)
Fluometuron 30, 245 (1983); Supp/. 7, 63 (1987)
Fluoranthene 32, 355 (1983); Suppl. 7, 63 (1987)
Fluorene 32, 365 (1983); Supp/. 7,63 (1987)
Fluorides (inorganic, us in drinking-water) 27, 237 (1982); Supp/. 7,20 (1987)
5-Fluorouracil 26,217 (1981); Supp/. 7,210 (1987)
Fluorspar (se Fluorides)
Fluosilicic acid (se Fluorides)
Fluroxene (se Anaesthetics, volatile)
Formaldehyde 29,345 (1982); Suppl. 7,211 (1987)
2-(2- Formylhydrano )-5-nitro-2-furyl)thiazle 7, 151 (1974) (con: 42, 253);
Suppl. 7, 63 (1987)
Frumide (see Furosmide)
Fuel oils (heating oils) 45, 239 (1989) (con: 47, 505)
Furazlidone 31, 141 (1983); Suppl. 7,63 (1987)
Furniture and cabinet-making 25, 99 (1981); Suppl. 7, 380 (1987)
Furosmide 50, 2n (199)
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (see AF-2)
Fusarenon-X 11, 169 (1976); 31, 153 (1983);
Suppl. 7, 64 (1987)
G
Gasline 45, 159 (1989) (con: 47,505)
Gasline engine exhaust (se Diesl and gasline engine exhausts)
Glas fibres (se Man-made minerai fibres)
Glaswol (se Man-made mineraI fibres)
Glas fiaments (see Man-made minerai fibres)
Glu-P-1 40,22 (1986);SupP/. 7,64(1987)
Glu-P-2 40,235 (1986); Supp/. 7,64(1987)
L-Glutamic acid, 5-(2-( 4-hydroxyethyl)phenylhydrazde)
(se Agartine)
GlycidaJdehyde 11, 175 (1976); Supp/. 7, 64 (1987)
Sorne glycidyl ethers 47,237 (1989)
Glycidyl oleate
11, 183 (1976); Supp/. 7, 64 (1987)
Glycidyl stearte 11, 187 (1976); Supp/. 7, 64 (1987)
Grifulvin 10,153 (1976); Supp/. 7,391 (1987)
Guinea Green B 16, 199 (1978); Supp/. 7, 64 (1987)
Gyrmitrn 31, 163 (1983); Suppl. 7,391 (1987)
H
Haematite 1, 29 (1972); Suppl. 7, 216 (1987)
Haematite and ferrc oxde Suppl. 7,216 (1987)
Haematite mining, underground, with exure to raon 1, 29 (im); Suppl. 7, 216 (1987)
Hair dyes, epidemiology of 16, 29 (1978); 27, 307 (1982)
Halothane (se Anaesthetics, volatile)
(I-HCH (se Hexchlorocclohexanes)
ß-HCH (see Hexachlorocclohexnes)
'Y-HCH (see Hexachlorocclohexanes)
Heating oils (se Fuel oils)
Heptachlor (see a/sa ChlordanelHeptahlor) 5, 173 (1974); 20, 12 (1979)
Hexahlorobenzene 20, 155 (197); Suppl. 7, 219 (1987)
Hexachlorobutadiene 20, 179 (1979); Suppl. 7,64(1987)
Hexachlorocclohexanes 5, 47 (1974); 20, 195 (1979)
(con: 42, 25); Suppl. 7, 22 (1987)
Hexachlorocclohexane, technical-gre (se Hexachloro-
cyclohexanes )
Hexachloroethane 20, 467 (1979); Suppl. 7, 64 (1987)
Hexachlorophene 20, 241 (1979); Suppl. 7, 64 (1987)
Hexamethylphosphoramide 15,211 (1977 Suppl. 7,64(1987)
Hexoetrol (se Nonsteroidal oetrogens)
Hycanthone mesylate 13, 91 (1977 Suppl. 7, 64 (1987)
Hydralazne 24, 85 (1980); Suppl. 7, 22 (1987)
Hydrazne 4, 127 (1974); Suppl. 7,22(1987)
Hydrochlorothiazde 50, 293 (199)
Hydrogen peroxide 36, 285 (1985); Suppl. 7, 64 (1987)
Hydroquinone 15, 155 (1977 Suppl. 7,64(1987)
4-Hydroxyazbenzene 8, 157 (1975); Suppl. 7, 64 (1987)
17(I-Hydroxyrogesterone caproate (se aJ Progestins) 21, 399 (197) (con: 42, 259)
8-Hydroxyquinoline 13, 101 (1977 Suppl. 7, 64 (1987)
8-Hydroxynkirkine 10, 26 (1976); Suppl. 7, 64 (1987)
Indeno(l,2,-cd)pyrene 3, 'l (1973); 32, 373 (1983);

Suppl. 7, 64 (1987)
IQ 40, 261 (1986); Suppl. 7, 64 (1987
Iron and stel founding 34, 133 (1984); Suppl. 7,22(1987)
Iron-dexn complex 2, 161 (1973); Suppl. 7, 22 (1987)
Iron-dexrin complex 2, 161 (1973) (con: 42, 252);
Suppl. 7, 64 (1987
Iron oxde (se Ferrc oxde)
Irn oxde, sacharte (se Sacharte iron oxde)
Iron sorbitol-ctric acd complex 2, 161 (1973); Suppl. 7, 64 (1987)
Isatidine 10,269 (1976); Suppl. 7,65 (1987)

Isflurane (se Anaesthetics, volatile)
Isniazd (se Isnicotinic acid hydrazde)
Isnicotinic acid hydrade 4, 159 (1974); Suppl. 7, 227 (1987)
Isphosphamide 26,137 (1981); Suppl. 7,65 (1987)
Ispropyl alcohol 15,22 (1977 Suppl. 7,229 (1987)
Ispropyl alcohol manufacture (strong-acid proc) Suppl. 7,229 (1987)
(se al Ispropyl alcohol)

Ispropyl oils 15,22 (1977 Suppl. 7,229 (1987)
Isafole 1, 169 (1972); 10, 232 (1976);
Suppl. 7,65 (1987)
J
Jacbine 10,275 (1976); Suppl. 7,65(1987)
Jet fuel 45, 203 (1989)
Joinery (se Carntry and joinery)
K
Kaempferol 31, 171 (1983); Suppl. 7, 65 (1987)
Kepone (see Chlordecne)
L
Laiocine 10,281 (1976); Suppl. 7, 65 (1987)
Lauroyl peroxide 36,315 (1985); Suppl. 7, 65 (1987)
Lead acetate (see Lead and lead compounds)
Lead and lead compounds 1,40 (1972) (con: 42,251); 2, 52,
150 (1973); 12, 131 (1976);
23,40,20,20,325 (1980);
Suppl. 7, 23 (1987)
Lead arnate (see Arnic and arnic compounds)
Lead cabonate (see Lead and lead compounds)
Lead chloride (se Lead and lead compounds)
Lead chromate (see Chromium and chromium compounds)
Lead chromate oxide (see Chromium and chromium èompounds)
Lead naphthenate (se Lead and lead compounds)
Lead nitrate (se Lead and lead compounds)
Lead oxide (see Lead and lead compounds)
Lead phosphate (se Lead and lead compounds)
Lead subacetate (see Lead and lead compounds)
Lead tetroxide (se Lead and lead compounds)
Leather goo manufacture 25,279 (1981); Suppl. 7,235 (1987)
Leather industries 25, 199 (1981); Suppl. 7,232 (1987)

Leather tanning and procing 25,201 (1981); Suppl. 7,23(1987)
Leate (see aI Lead and lead compounds) 12, 131 (1976)
Ught Green SF 16,20 (1978); Suppl. 7, 65 (1987)
Undane (se Hexachlorocclohexnes)
The lumber and sawmil industries (including loggng) 25, 49 (1981); Suppl. 7, 383 (1987)
Luteokyn 10, 163 (1976); Suppl. 7,65(1987)
Lynoetrenol (se aI Progestins; Combine oral contrceptives) 21, 40 (197)
M
Magenta 4, 57 (1974) (con: 42, 252);
Suppl. 7,23(1987)
Magenta manufacture of (se a/ Magenta) Suppl. 7, 23 (1987)
Malathion 30, 103 (1983); Suppl. 7, 65 (1987)
Maleic hydrade 4, 173 (1974) (con: 42, 253);
Suppl. ' 7, 65 (1987)
Malonaldehyde 36, 163 (1985); Suppl. 7, 65 (1987)
Maneb 12, 137 (1976); Suppl. 7, 65 (1987)
Man-made mineraI fibres 43, 39 (1988)
Mannomustine 9, 157 (1975); Suppl. 7, 65 (1987)
MCPA (se aI Chlorophenoxy herbicides; Chlorophenoxy 30,255 (1983)
herbicides, ocpational expures to)
MeA-~-C 40,253 (1986); Suppl. 7,65 (1987)
Medphalan 9, 168 (1975); Suppl. 7,65 (1987)
Medroxyrogesterone acetate 6, 157 (1974); 21, 417 (1979)
(con: 42, 259); Suppl. 7, 289 (1987)
Megestrol acetate (se aOO Progestins; Combined oral
contraceptives)
MelQ 40,275 (1986); Suppl. 7,65(1987)
MelQx 40, 283 (1986); Supp/. 7, 65 (1987)
Melamine. 39,333 (1986); Suppl. 7,65 (1987)
Melphalan 9, 167 (1975); Supp/. 7,239 (1987)
6-Mercaptopurine 26,249 (1981); Supp/. 7,240 (1987)
Merphalan 9, 169 (1975); Suppl. 7, 65 (1987)
Mestranol (see aIo Steroidal oetrogens) 6, 87 (1974); 21, 257 (1979)
(con: 42, 259)
Methanearnic acd, disoium salt (see Arnic and arnic
compounds )
Methanearnic acid, monosium salt (see Arnic and arnic
compounds
Methotrexate 26,267 (1981); Suppl. 7,241 (1987)
M:ethoxsalen (se 8-Methoxyralen)
Methoxychlor 5, 193 (1974); 20, 259 (1979);
Suppl. 7, 66 (1987)
CUMULTI CROSS INEX 403
Methoxurane (se Anaethetics, volatile)

5-Methoxralen 40, 327 (1986); Suppl. 7,242 (1987)
8-Methoxraen (se a/ 8-Methoxyraen plus ultrvilet 24, 101 (1980)
raiation)
8-Methoxralen plus ultrvilet raiation Suppl. 7,243 (1987)
Methyl aclate 19,52 (197); 39, 99 (1986);
Suppl. 7, 66 (1987)
S-Methylangelicin plus ultraviolet radiation (se a/ Angelicin
and sorne synthetic derivative) Suppl. 7,57 (1987)
2-Methylazridine 9,61 (1975); Suppl. 7,66(1987)
Methylazethanol actate 1, 164 (1972); 10, 131 (1976);
Suppl. 7, 66 (1987)
Methyl brornide 41, 187 (1986) (con: 45, 283);
Suppl. 7, 245 (1987)
Methyl caarnate 12, 151 (1976); Suppl. 7, 66 (1987)
Methyl-C (se 1-(2-Chloroethyl)-3- 4-rnethylcyclohexl)-
1-nitrosurea)
Methyl chloride 41, 161 (1986); Suppl. 7, 24 (1987)
1-,2-,3-,4-,5- and 6-Methylchrynes 32, 379 (1983); Suppl. 7, 66 (1987)
N-Methyl-N,4-nitrosanilne 1, 141 (1972); Suppl. 7,66(1987)
4,4' -Methylene bis2-chloroanilne) 4, 6S (1974) (con: 42, 252); .
Suppl. 7, 24 (1987)
4,4'-Methylene bisN;N-dirnethyl)bnzenarnine 27, 119 (1982); Suppl. 7, 66 (1987)
4,4' -Methylene bis2-methylanilne) 4, 73 (1974); Suppl. 7, 24 (1987)
4,4' -Methylenedianiline 4, 79 (1974) (con: 42, 252);
39,347 (1986); Suppl. 7,66(1987)
4,4' -Methylenediphenyl diisanate 19,314 (1979); Suppl. 7,66(1987)
2-Methylfluoranthene 32,399 (1983); Suppl. 7,66(1987)
3- Methylfuoranthene 32, 399 (1983); Suppl. 7, 66 (1987)
Methyl iodide 15,245 (1977 41, 213 (1986);
Suppl. 7,66(1987)
Methyl Methacrylate 19, 187 (1979); Suppl. 7, l) (1987)
Methyl methanesulphonate 7, 253 (1974); Suppl. 7, 66 (1987)
2-Methyl-l-nitroanthraquinone 27, 205 (1982); Suppl. 7, 66 (1987)
N-Methyl-N' -nitro-N-nitrosguanidine 4, 183 (1974); Suppl. 7, 24 (1987)
3-Methylnitrosrninopropionaldehyde (se 3-(N-Nitros
methylamino )propionaldehyde)
3-Methylnitrosarninopropionitrile (see 3-N-Nitrosrnethyl-
arnino )propionitrle)
4-ethylnitrosarnino )-3-pyrdyl)-l-butanal (se 4-N-Nitros

rnethylamino )-3-pyrdyl)-l-butanal)
4-ethylnitrosrnino )-1-(3-pyrdyl)-l-butanone (se 4-N-Nitros
rnethylamino )-1-(3-pyrdyl)-l-butanone)
N-Methyl-N-nitrosurea 1, 12 (1972); 17, 227 (1978);
Suppl. 7, 66 (1987)
N-Methyl-N-nitrosurethane 4, 211 (1974); Suppl. 7, 66 (1987)

Methyl parthion 30, 131 (1983); Suppl. 7,392 (1987)
1-Methylphenanthrene 32, 405 (1983); Suppl. 7, 66 (1987)
7- Methylpyrdo(3,4- )psralen 40,349 (1986); Suppl. 7,71(1987)
Methyl red 8, 161 (1975); Suppl. 7, 66 (1987)
Methyl selenac (se also Selenium and selenium compounds) 12, 161 (1976); Suppl. 7, 66 (1987)
Methylthiouracil 7, 53 (1974); Suppl. 7, 66 (1987)
Metronidazle 13, 113 (1977 Suppl. 7, 25 (1987)
Mineral oils 3, 30 (1973); 33, 87 (1984) (con: 42,
262); Suppl. 7, 252 (1987)
Mirex 5, 203 (1974); 20, 283 (1979) (con:
42, 258); Suppl. 7, 66 (1987)
Mitomycin C 10, 171 (1976); Suppl. 7,67 (1987)
MNG (se N-Methyl-N' -nitro-N-nitrosguanidine)
MOCA (se 4,4' -Methylene bis2-chloroanilne))
Modacrylic fibres 19, 86 (1979); Suppl. 7, 67 (1987)
Monocrotaline 10,291 (1976); Suppl. 7,67 (1987)
Monuron 12, 167 (1976); Suppl. 7, 67 (1987)
MOPP and other combined chemotherapy inc1uding Suppl. 7, 254 (1987)
alkylating agents
Morpholine 47, 199 (1989)
5-(orpholinomethylt-3-( (5-nitrofurfrylidene )amino )-2- 7, 161 (1974); Suppl. 7, 67 (1987)
oxawlidinone
Mustard gas 9, 181 (1975) (con: 42, 254);
Suppl. 7, 259 (1987)
Myleran (se 1,4-Butanediol dimethanesulphonate)
N
Nafenopin 24, 12 (1980); Suppl. 7, 67 (1987)
l,5-Naphthalenediamine 27, 127 (1982); Suppl. 7,67 (1987)
1,5-Naphthalene diisoanate 19,311 (1979); Suppl. 7,67 (1987)
1-Naphthylamine 4, 87 (1974) (con: 42, 253);
Suppl. 7, 26 (1987)
2-Naphthylamine 4,97 (1974); Suppl. 7,261 (1987)
1-Naphthylthiourea 30,347 (1983); Suppl. 7,263 (1987)
Nickel actate (se Nickel and nickel compounds)
Nickel ammonium sulphate (se Nickel and nickel compounds)
Nickel and nickel compounds 2, 126 (1973) (con: 42, 252); 11, 75
(1976); Suppl. 7, 26 (1987)
(con: 45, 283); 49, 257 (199)
Nickel cabonate (se Nickel and nickel compounds)
Nickel canyl (se Nickel and nickel compounds)
NiclCel chloride (se Nickel and nickel compounds)
Nickel-gallum alloy (se Nickel and nickel compounds)
Nickel hydroxide (see Nickel and nickel compounds)

Nickelocne (se Nickel and nickel compounds)
Nickel oxide (se Nickel and nickel compounds)
Nickel subsulphide (see Nickel and nickel compounds)
Nickel sulphate (se Nickel and nickel compounds)
Niridazle 13, 123 (1977); Suppl. 7, 67 (1987)
Nithiazde 31, 179 (1983); Suppl. 7, 67 (1987)
Nitrilotriacetic acid and its salts 48, 181 (199)
5-Nitroacenaphthene 16,319 (1978); Suppl. 7, 67 (1987)
5-Nitro-nho-anisidine 27, 133 (1982); Suppl. 7, 67 (1987)
9-Nitroanthracene 33, 179 (1984); Suppl. 7,67 (1987)
7-Nitrobenz(a )anthracene 46, 247 (1989)
6-Nitrobenzo(a )pyrene 33, 187 (1984); Suppl. 7,67 (1987);
46, 255 (1989)
4-Nitrobiphenyl 4, 113 (1974); Suppl. 7, 67 (1987)
6-Nitrochryne 33, 195 (1984); Suppl. 7,67 (1987);
46, 2f7 (1989)
Nitrofen (technical-grade) 30, 271 (1983); Suppl. 7, 67 (1987)
3-Nitrofluoranthene 33, 201 (1984); Suppl. 7, 67 (1987)
2-Nitrofluorene 46, 277 (1989)
Nitrofural 7, 171 (1974); Suppl. 7,67 (1987)
50, 195 (199)
5-Nitro-2-furaldehyde semicabazne (see Nitrofural)
Nitrofurantoin 50, 211 (199)
Nitrofurazne (see Nitrofural)
1-( (5-Nitrofurfrylidene )amino )-2-imidazlidinone 7, 181 (1974); Suppl. 7, 67 (1987)
N-( 4-5-Nitro-2-furyl)-2-thiazolyl)acetamide 1, 181 (1972); 7, 185 (1974);
Suppl. 7, 67 (1987)
Nitrogen mustard 9, 193 (1975); Suppl. 7,269 (1987)
Nitrogen mustard N-oxide 9,20 (1975); Suppl. 7,67 (1987)
1-Nitronaphthalene 46,291 (1989)
2-Nitronaphthalene 46, 303 (1989)
3-Nitroperylene 46, 313 (1989)
2-Nitropropane 29, 331 (1982); Suppl. 7, 67 (1987)
1-Nitropyrene 33,20 (1984); Suppl. 7,67 (1987)
46, 321 (1989)
2-Nitropyrene 46, 359 (1989)
4-Nitropyrene 46, 367 (1989)
N-Nitrosatable drugs 24,297 (1980) (con: 42,26)
N-Nitrosatable peticides 30, 359 (1983)
N' -Nitrosanabasine 37, 22 (1985); Suppl. 7, 67 (1987)
N' -Nitrosanatabine 37, 233 (1985); Suppl. 7, 67 (1987)
N-Nitrosi-n-butylamine 4, 197 (1974); 17, 51 (1978);
Suppl. 7, 67 (1987)
N-Nitrosiethanolamine 17, TI (1978); Suppl. 7, 67 (1987)
N-Nitrosiethylamine 1, 107 (1972) (con: 42, 251);
17,83 (1978) (con: 42,257)

Suppl. 7, 67 (1987)
N-Nitrosimethylamine 1, 95 (1972); 17, 12 (1978)
(con: 42, 257) Suppl. 7, 67 (1987)
N-Nitrosiphenylamine 27, 213 (1982); Suppl. 7, 67 (1987)
pa-Nitrosiphenylamine 27, '17 (1982) (con: 42, 261);
Suppl. 7, 68 (1987)
N-Nitrosi-n-propylamine 17, in (1978); Suppl. 7,68(1987)
N-NitrosN-ethylurea (se N-Ethyl-N-nitrosurea)
N-Nitrosfolic acid 17,217 (1978); Suppl. 7,68(1987)
N-Nitrosguvacine 37,26 (1985); Suppl. 7,68(1987)
N-Nitrosguvaline 37, 26 (1985); Suppl. 7, 68 (1987)
N-Nitroshydroxyroline 17,30 (1978); Suppl. 7,68(1987)
3-(N-Nitrosmethylamino )propionaldehyde 37, 26 (1985); Suppl. 7, 68 (1987)
3-(N-Nitrosmethylamino )propionitrile 37, 263 (1985); Suppl. 7, 68 (1987)
4-N-Nitrosmethylamino )-3-pyrdyl~ 1-butanal 37,205.(1985); Suppl. 7,68(1987)
4-N-Nitrosmethylamino ~ 1-(3-pyrdyl~ I-butanone 37,20 (1985); Suppl. 7,68(1987)
N-Nitrosmethylethylamine 17, '11 (1978); Suppl. 7, 68 (1987)
N-NitrosN-methylurea (see N-Methyl-N-nitrosurea)
N-NitrosN-methylurethane (se N-Methyl-N-methylurethane)
N-Nitrosmethylvinylamine 17, 257 (1978); Suppl. 7, 68 (1987)
N-Nitrosmorpholine 17, 263 (1978); Suppl. 7, 68 (1987)
N'-Nitrosnomicotine 17,281 (1978); 37,241 (1985);
Suppl. 7, 68 (1987)
N-Nitrospiperidine 17,287 (1978); Suppl. 7,68(1987)
N-Nitrosproline 17, 303 (1978); Suppl. 7, 68 (1987)
N-Nitrospyrolidine 17,313 (1978); Suppl. 7,68(1987)
N-Nitrosarcoine 17, 327 (1978); Suppl. 7, 68 (1987)
Nitrosureas, chloroethyl (se Chloroethyl nitrosureas)
5-NitrO-nho-toluidine 48, 169 (199)
Nitrous oxde (se Anaethetics, volatile)
Nitrovin 31, 185 (1983); Suppl. 7, 68 (1987)
NNA (se 4-N-Nitrosmethylamino )-3-pyrdyl~ 1-butanal)
NN (see 4-N-Nitrosmethylamino ~ l-(3-pyrdyl~ 1-butanone)
Nonsteroidal oetrogens (se aJ Oetrogens, progestins and Suppl. 7, 272 (1987)
combinations )
Norethisterone (se aJ Progestins; Combined oral 6, 179 (1974); 21, 461 (1979)
contraptives)
Norethynodrel (se aJ Progestins; Combined oral 6, 191 (1974); 21, 461 (197)
contraptives (con: 42, 259)
Norgestrel (se aJ Progestins, Combined oral contraceptives) 6, 201 (1974); 21, 479 (197)
Nylon 6 19, 12 (197); Suppl. 7, 68 (1987)
CUMUTI CROSS INDEX 407
o
Ochratoxn A 10, 191 (1976); 31,191 (1983) (con:
42,262); Suppl. 7,271 (1987)
Oetradiol-17ß (se a/so Steroidal oetrogens) 6, 99 (1974); 21, 279 (1979)
Oetradiol3-benzoate (see Oetradiol-17ß)
Oetradiol dipropionate (se Oetradiol-17ß)
Oetraiol mustard 9, 217 (1975)
Oetradiol-17ß-valerate (se Oetradiol-17ß)
Oetriol (see a/so Steroidal oetrogens) 6, 117 (1974); 21, 327 (1979)
Oetrogen-progestin combinations (se Oetrogens, progestins
and combinations)
Oetrogen-progestin replacement therapy (se aI Oetrogens, Suppl. 7, 30 (1987)
progestins and combinations)
Oetrogen replacement therapy (se a/so Oestrogens, progestins Suppl. 7,28(1987)
and combinations)
Oestrogens (se Oetrogens, progestins and combinations)
Oetrogens, conjugated (se Conjugated oetrogens)
Oestrogens, nonsteroidal (se Nonsteroidal oetrogens)
Oestrogens, progestins and combinations 6 (1974); 21 (1979);
Suppl; 7, 272 (1987)
Oetrogens, steroidal (se Steroidal oetrogens)
Oestrone (see a/so Steroidal oetrogens) 6, 123 (1974); 21, 343 (1979)
(con: 42, 259)
Oestrone benzoate (see Oestrone)
Oil Orange SS 8, 165 (1975); Suppl. 7, 69 (1987)
Oral contraceptives, combined (se Combined oral contraptives)
Oral contraceptives, investigational (see Combined oral
contraceptives)
Oral contraptives, sequential (see Seuential oral contraceptives)
Orange 1 8, 173 (1975); Suppl. 7, 69 (1987)
Orange G 8, 181 (1975); Suppl. 7,69 (1987)
Organolead compounds (see a/so Lead and lead compounds) Suppl. 7,23(1987)
Oxazpam 13, 58 (197 Supp/. . 7, 69 (1987)
Oxetholone (see a/so Androgenic (anabolic) steroids) 13, 131 (1977
Oxhenbutazne 13, 185 (1977 Suppl. 7,69 (1987)
p
Paint manufacture and painting (ocpation al expures in) 47, 329 (1989)
Panfuran S (see a/so Dihydroxyethylfuratrizine) 24, TI (1980); Supp/. 7, 69 (1987)
Paper manufacture (se Pulp and paper manufacture)
Parcetamol 50, 307 (199)
Parasrbic acid 10, 199 (1976) (con: 42, 255);
Suppl. 7,69 (1987)
Parthion 30, 153 (1983); Suppl. 7, 69 (1987)

Patulin 10, 205 (1976); 40, 83 (1986);
Suppl. 7, 69 (1987)
Penicilic acd 10,211 (1976); Suppl. 7,69 (1987)
Pentachloroethane 41, 99 (1986); Suppl. 7, 69 (1987)
Pentachloronitrobenzene (se Quintozene)
Pentachlorophenol (se a1 Chlorophenols; Chlorophenols, 20, 303 (1979)
ocpational exure to)
Perylene 32,411 (1983); Suppl. 7,69 (1987)
Petasitenine 31, 207 (1983); Suppl. 7, 69 (1987)
Petasites janicus (se Pyolizidine alkaloids)
Petroleurn refining (ocpational exures in) 45,39 (1989)
Sorne petroleurn solvents 47, 43 (1989)
Phenactin 13, 141 (1977 24, 135 (1980);
Suppl. 7,310 (1987)
Phenanthrene 32,419 (1983); Suppl. 7,69 (1987)
Phenazpyrdine hydrochloride 8, 117 (1975); 24, 163 (1980)
(con: 42, 26); Suppl. 7,312 (1987)
Phenelzine sulphate 24, 175 (1980); Suppl. 7,312 (1987)

Phenicabazde 12, in (1976); Suppl. 7, 70 (1987)
Phenobarbital 13,157 (1977 Suppl. 7,313 (1987)
Phenol 47, 263 (1989)
Phenoxyactic acid herbicides (se Chlorophenoxy herbicides)
Phenoxybenzarnine hydrochloride 9, 22 (1975); 24, 185 (1980);
Suppl. 7, 70 (1987)
Phenylbutazne 13, 183 (197 Suppl. 7,316 (1987)
meta-Phenylenediarnine 16, 111 (1978); Suppl. 7, 70 (1987)
pa-Phenylenediarnine 16, 12 (1978); Suppl. 7, 70 (1987)
N-Phenyl-2-naphthylarnine 16, 325 (1978) (con: 42, 257)
Suppl. 7, 318 (1987)
ortPhenylphenol 30, 329 (1983); Suppl. 7, 70 (1987)
Phenytoin 13,201 (1977 Suppl. 7,319 (1987)
Piperane oetrone sulphate (se Conjugated oetrogens)
Piperonyl butoxide 30,183 (1983); Suppl. 7, 70 (1987)
Pitches, coal-tar (se Coal-ta pitches)
Polyacrylic acid 19,62 (197); Suppl. 7, 70 (1987)
Polybrorninated biphenyls 18, 107 (1978); 41,261 (1986);
Suppl. 7, 321 (1987)
Polychlorinated biphenyls 7, 261 (1974); 18, 43 (1978) (con:
42,25);
Suppl. 7, 322 (1987)
Polychlorinated carnphenes (se Toxaphene)
Polychloroprene 19, 141 (1979); Suppl. 7, 70 (1987)
Polyethylene 19,164 (1979); Suppl. 7, 70 (1987)
CUMUL~ CROSS mDEX 40
Polymethylene polyphenyl isnate 19, 314 (197); Suppl. 7, 70 (1987)
Polymethyl methaclate 19, 195 (197); Suppl. 7, 70 (1987)
Polyoetraiol phosphate (se Oetraiol-17ß)

Polypropylene 19,218 (1979); Suppl. 7,70(1987)
Polystyne 19,245 (1979); Suppl. 7, 70 (1987)
Polytetrauorothylene 19,28 (197); Suppl. 7, 70 (1987)
Polyurethane foams 19,320 (1979); Suppl. 7, 70 (1987)
Polyvnyl acetate 19,34 (1979); Suppl. 7, 70 (1987)
Polyvnyl alcohol 19,351 (1979); Suppl. 7, 70 (1987)
Polyvnyl chloride 7, 30 (1974); 19, 40 (1979);
Suppl. 7, 70 (1987)
Polyvnyl pyrlidone 19, 46 (197); Suppl. 7, 70 (1987)
Ponceau MX 8, 189 (1975); Suppl. 7, 70 (1987)

Ponceau 3R 8, 199 (1975); Suppl. 7, 70 (1987)
Ponceau SX 8, 117 (1975); Suppl. 7, 70 (1987)
Potasium arnate (se Arnic and arnic compounds)
Potasium arnite (se Arnic and arnic compounds)
Potasium bis2-hydroxethyl)dithiocbamate 12, 183 (1976); Suppl. 7, 70 (1987)
Potasium bromate 40, 207 (1986); Suppl. 7, 70 (1987)
Potasium chromate (se Chromium and chromium compounds)
Prednimustine 50, 115 (199)

Potaium dichromate (se Chromium and chromium compounds)
Prednisne 26,293 (1981); Suppl. 7,326 (1987)

Prazne hydrochloride 26,311 (1981); Suppl. 7,327 (1987)
Proflavine salts 24, 195 (1980); Suppl. 7, 70 (1987)
Progesterone (se a/so Progestins; Combined oral contraceptives) 6, 135 (1974); 21,491 (1979)
(con: 42, 259)
Progestins (se aJ Oetrogens, progestins and combinations) Suppl. 7, 289 (1987)
Pronetalol hydrochloride 13, 227 (197 (con: 42, 256);
Suppl. 7, 70 (1987)
l,3-Propane sultone 4, 253 (1974) (con: 42, 253);
Suppl. 7, 70 (1987)
Prpham 12, 189 (1976); Suppl. 7, 70 (1987)
ß-Propiolacone 4, 259 (1974) (con: 42, 253);
Suppl. 7, 70 (1987)
n-Propyl cabamate 12, 201 (1976); Suppl. 7, 70 (1987)
Propylene 19,213 (1979); Suppl. 7, 71 (1987)
Propylene oxde Il, 191 (1976); 36, 227 (1985) (con:
42, 26); Suppl. 7, 328 (1987)
Propylthiouracil 7, 67 (1974); Suppl. 7, 329 (1987)

Ptaquiloside (se aJ Bracken fem) 40, 55 (1986); Suppl. 7, 71 (1987)
Pulp and paper manufacture 25, 157 (1981); Suppl. 7,385 (1987)
Pyene 32,431 (1983); Suppl. 7, 71 (1987)
Pydo(3,4- )psralen 40, 349 (1986); Suppl. 7, 71 (1987)
Pymethamine 13,233 (1977 Suppl. 7, 71 (1987)
Pyrolizidine alkaloids (see Hydroxynkirkine; Isatidine;

Jacobine; Laiocine; Monocrotaline; Retrorsine; Riddelline;
Seneciphyllne; Senkirkine)
Q
Quercetin (see alo Bracken fem) 31,213 (1983); Suppl. 7,71 (1987)
pa-Quinone 15,255 (1977; Suppl. 7,71(1987)
Quintozene 5, 211 (1974); Suppl. 7, 71 (1987)
R
Radon 43, 173 (1988) (con: 45,283)
Reserpine 10,217 (1976); 24,211 (1980)
(con: 42, 26); Suppl. 7, 330 (1987)
Resorcinol 15, 155 (1977; Suppl. 7, 11 (1987)
Retrorsine 10, 303 (1976); Suppl. 7, 71 (1987)
Rhodamine B 16, 221 (1978); Suppl. 7, 71 (1987)
Rhodamine 60 16,233 (1978); Suppl. 7, 71 (1987)
Riddelline JO, 313 (1976); SuppI. 7, 71 (1987)
Rifampicin . 24,243 (1980); Suppl. 7,71(1987)
Rockwl (see Man-made mineraI fibres)
The rubber industry
28 (1982) (con: 42, 261); Suppl. 7,
332 (1987)
Rugulosin 40,99 (1986); Suppl. 7,71(1987)
s
Saccharted iron oxide 2, 161 (1973); Suppl. 7, 71 (1987)
Saccharn 22, 111 (1980) (con: 42, 259);
Suppl. 7,334 (1987)
Safole 1, 169 (1972); JO, 231 (1976);
Suppl. 7, 71 (1987)
The sawmiJ industry (inc1uding loggng) (se The lum ber and
sawmiJ industry (inc1uding logng))
Sealet Red
8,217 (1975); Suppl. 7, 71 (1987)
Selenium and selenium compounds 9, 245 (1975) (con: 42, 255);
Suppl. 7, 71 (1987)
Selenium dioxide (se Selenium and selenium compounds)
Selenium oxide (se Selenium and selenium compounds)
Semieabazde hydrochloride 12, 20 (1976) (con: 42, 256);
Suppl. 7, 71 (1987)
Senecio jaoba L. (se Pyolizidine alkaloids)
Senecio longiobus (se Pylizidine alkaloids)
Seneciphyllne 10,319,335 (1976); Suppl. 7, 71

(1987)
Senkirkine 10,327 (1976); 31, 231 (1983);
Suppl. 7, 71 (1987)
Sepiolite 42, 175 (1987); Suppl. 7, 71 (1987)
Seuential oral contraceptives (se also Oestrogens, progestins Suppl. 7,296 (1987)
and combinations)
Shale-oils 35,161 (1985); Suppl. 7,339 (1987)
Shikimic acid (see also Bracken fem) 40, 55 (1986); Suppl. 7, 71 (1987)
Shoe manufacture and repair (see Bot and shoe manufacture
and repair)
Silca (see also Amorphous silca Crytallne silca) 42, 39 (1987)
Slagwl (see Man-made mineraI fibres)
Soium arnate (see Arnic and arnic compounds)
Soium arnite (se Arnic and arnic compounds)
Soium cacoylate (se Arnic and arnic compounds)
Soium chromate (see Chromium and chromium compounds)
Soium cyc1amate (see Cyc1amates)
Soium dichromate (see Chromium and chromium compounds)
Soium diethyldithiocbamate 12,217 (1976); Suppl. 7, 71 (1987)
Soium equiln sulphate (see Conjugated oestrogens)
Soium fluoride (se Fluorides)
Soium monofluorophosphate (see Fluorides)
Soium oetrone sulphate (see Conjugated oetrogens)
Soium ortho-phenylphenate (see al ortho-Phenylphenol) 30,329 (1983); Suppl. 7,392 (1987)
Soium saccharn (see Saccharn)
Soium selenate (se Selenium and selenium compounds)
Soium selenite (see Selenium and selenium compounds)
Soium silicofluoride (see Fluorides)
Sots 3, 22 (1973); 35, 219 (1985);
Suppl. 7, 343 (1987)
Spironolactone 24, 259 (1980); Suppl. 7,344 (1987)
Stannous fluoride (se Fluorides)
Steel founding (se Iron and stel founding)
Sterigmatoctin 1, 175 (1972); 10, 245 (1976);
Suppl. 7, 72 (1987)
Steroidaloetrogens (se al Oetrogens, progestins and Suppl. 7, 28 (1987)
combinations )
Streptozotocin 4, 221 (1974); 17, 337 (1978);
Suppl. 7, 72 (1987)
Strobane~ (se Terpne polychlorinates)
Strontium chromate (see Chromium and chromium compounds)
Styene 19, 231 (1979) (con: 42, 258);
Suppl. 7, 345 (1987)
Styene-acrylonitrile copolymers 19, 97 (1979); Suppl. 7, 72 (1987)
Styene-butadiene copolymers
19, 252 (1979); SupjJ. 7, 72 (1987)
Styene oxide 11,201 (1976); 19, 275 (1979);
36, 245 (1985); Suppl. 7, 72 (1987)
Succinic anhydride 15, 26 (1977 Suppl. 7, 72 (1987)
Sudan 1
8, 22 (1975); Suppl. 7, 72 (1987)
Sudan II 8,233 (1975); Suppl. 7,72(1987)
Sudan III 8,241 (1975); Suppl. 7,72(1987)
Sudan Brow RR 8, 249 (1975); Suppl. 7, 72 (1987)
Sudan Red 7B 8, 253 (1975); Suppl. 7, 72 (1987)
Sulfafrazle 24,275 (1980); Suppl. 7, 347 (1987)
Sulfallate 30, 283 (1983); Suppl. 7, 72 (1987)
Sulfamethoxazle 24,285 (1980); Suppl. 7, 34 (1987)
Sulphisxale (se Sulfafrale)
Sulphur mustard (se Mustard gas)
Sunst Yellow FCF 8, 257 (1975); Suppl. 7, 72 (1987)
Symphytine 31, 239 (1983); Suppl. 7, 72 (1987)
T
2,4,5- T (se a/so Chlorophenox herbicides; Chlorophenox
herbicides, ocpation al expures to) 15, 273 (1977
Talc 42, 185 (1987) Suppl. 7,349 (1987)
Tannic acid 10, 253 (1976) (con: 42, 255);
Suppl. 7, 72 (1987)
Tannins (se aJ Tannic acid) 10, 25 (1976); Suppl. 7, 72 (1987)
TCDD (see 2,3,7,8-Tetrachlorodibenzopa-dioxin)
IDE (see DDT)
Terpne polychlorinates 5, 219 (1974); Suppl. 7, 72 (1987)
Testosterone (se a1 Androgenic (anabolic) steroids) 6,20 (1974); 21, 519 (1979)
Testosterone oenanthate (se Testosterone)
Testosterone propionate (se Testosterone)
2,2' ,S,S' - Tetrachlorobenzidine 27, 141 (1982); Suppl. 7, 72 (1987)
2,3,7,8- Tetrahlorodibenzopa-dioxn 15,41 (191 Suppl. 7,350 (1987)
1,1,1,2- Tetrahloroethane 41,87 (1986); Suppl. 7,72(1987)
1,1,2,2- Tetrachlorothane 20, 4TI (1979); Suppl. 7,354 (1987)
Tetrachloroethylene 20,491 (1979); Suppl. 7, 355 (1987)
2,,4,6- Tetrahlorophenol (se Chlorophenols; Chlorophenols,
ocpation al exures to)
Tetrachlorvnphos 30, 197 (1983); Suppl. 7, 72 (1987)
Tetraethyllead (se Lead and lead compounds)
TetrauoroethyIene 19,285 (1979); Suppl. 7,72(1987)
Tetraydroxyethyl) phosphonium salts
48, 95 (199)
Tetramethyllead (se Lead and lead compounds)
Texile manufacring industry, exures in 48, 215 (199)
Thioatamide 7, TI (1974); Suppl. 7, 72 (1987)
4,4' - Thiodianilne 16,343 (1978); 27, 147 (1982);

Suppl. 7, 72 (1987)
Thiotepa 9,85 (1975); Suppl. 7,36(1987);
50, 12 (199)
Thiouracil 7,85 (1974); Suppl. 7,72(1987)
Thiourea 7, 95 (1974); Suppl. 7, 72 (1987)
Thiram 12, 22(1976); Suppl. 7, 72 (1987)
Titanium dioxde 47, 307 (1989)
Tobacc habits other than smoking (se Tobac products,
smokeles )
Tobacc products, smokeles 37 (1985) (con: 42, 263); Suppl. 7,
357 (1987)
Tobac smoke 38 (1986) (con: 42, 26~ Suppl. 7,
357 (1987)
Tobacc smoking (se Tobacc smoke)
orlho- Tolidine (se 3,3' -Dimethylbenzidine)
2,4- Toluene diisanate (see also Toluene diisanates) 19,303 (1979); 39, '27 (1986)
2,6- Toluene diisanate (se alo Toluene diisoanates) 19,303 (1979); 39, '29 (1986)
Toluene 47, 79 (1989)
Toluene diisoanates 39, U,7 (1986) (corr 42, 26);
Suppl. 7, 72 (1987)
Toluenes, a-chlorinated (see a-Chlorinated toluenes)
orlho- Toluenesulphonamide (see Saccharin)
orlho- Toluidine 16,349 (1978); 27, 155 (1982);
Siippl. 7,362 (1987)
Toxaphene 20,327 (1979); Suppl. 7, 72 (1987)
Tremolite (se Asbetos)
Treoulphan 26,341 (1981); Suppl. 7,363 (1987)
Triazquone (see Tri azridinyl)-pa-benzouinone)
Trichlorfon 30, ')7 (1983); Suppl. 7, 73 (1987)
Trichlormethine 9, 229 (1975); Suppl. 7, 73 (1987)
50, 143 (199)
1,1,1- Trichloroethane 20,515 (1979); Suppl. 7, 73 (1987)
1,1,2- Trichloroethane 20, 533 (1979); Suppl. 7, 73 (1987)
Trichloroethylene 11, 263 (1976); 20, 545 (1979);
Suppl. 7, 364 (1987)
2,4,5- Trichlorophenol (se also Chlorophenols; Chlorophenols 20, 349 (1979)
2,4,6- Trichlorophenol (see also Chloropheno1s; Chlorophenols, 20, 349 (1979)
(2,4,5- Trichlorophenoxy)acetic acid (se 2,4,5- n
Trichlorotriethylamine hydrochloride (see Trichlormethine)
T 2- Trichothecne 31,265 (1983); Suppl. 7,73 (1987)
Triethylene glycol diglycidyl ether 11, 20 (1976); Suppl. 7, 73 (1987)
4,4' ,6- Trirnethylangelicin plus ultraviolet radiation (se aI Suppl. 7, 57 (1987)

Angelicin and sorne synthetic derivatives)
2,4,5- Trirnethylanilne 27, 177 (1982); Suppl. 7, 73 (1987)
2,4,6- Trirnethylanilne 27,178 (1982); Suppl. 7,73 (1'987)
4,5' ,8- Trirnethylpsralen 40,357 (1986); Suppl. 7,36(1987)
Trirnustine hydrohloride (se Trichlormethine)
Triphenylene 32, 447 (1983); Suppl. 7, 73 (1987)
Tris( azridinyl)-pa-benzuinone 9,67 (1975); Suppl. 7,367 (1987)
Tril-azridinyl)phosphine oxide 9, 75 (1975); Suppl. 7, 73 (1987)
Tril-azridinyl)phosphine sulphide (se Thiotepa)
2,4,6- Tril-azridinyl)-s-trazne 9, 95 (1975); Suppl. 7, 73 (1987)
Tri2~hloroethyl) phosphate 48, 109 (199)
1,2,3- Tri chloromethox)propane 15, 301 (197 Suppl. 7, 73 (1987)
Tri2,-dibrornopropyl)phophate 20,575 (1979); Suppl. 7,369 (1987)
Tri2-rnethyl-1-azridinyl)phosphine oxde 9, 107 (1975); Suppl. 7, 73 (1987)
TrpP-1 31,247 (1983); Suppl. 7, 73 (1987)
TrpP-2 31,255 (1983); Suppl. 7, 73 (1987)
Tryan blue 8, 267 (1975); Suppl. 7, 73 (1987)
Tusüago farar L (se Pyrolizidine alkaloids)
u
Ultraviolet radiation 40,379 (1986)
Underground haernatite rnining with expure to radon 1, 29 (1972); Suppl. 7, 216 (1987)
Urail rnustad 9, 235 (1975); Suppl. 7, 370 (1987)
Urethane 7, 111 (1974); Suppl. 7, 73 (1987)
v
Vat Yellow 4 48, 161 (199)
Vinblastine sulphate 26, 349 (1981) (con: 42, 261);
Suppl. 7, 371 (1987)
Vincrstine sulphate 26,365 (1981); Suppl. 7,372 (1987)
Vinyl acetate 19, 341 (1979); 39, 113 (1986);
Suppl. 7, 73 (1987)
Vinyl brornide 19, 367 (1979); 39, 133 (1986);
Suppl. 7, 73 (1987)
Vinyl chloride 7, 291 (1974); 19, 377 (1979)
(con: 42, 258); Suppl. 7, 373 (1987)
Vinyl chloride-vinyl acetate copolymers 7,311 (1976); 19,412 (197) (con:
42,258); Suppl. 7, 73 (1987)
4- Vinylcyc10hexene Il,277 (1976); 39, 181 (1986);
Suppl. 7, 73 (1987)
Vinyl fluoride 39, 147 (1986); Suppl. 7, 73 (1987)
CUMTI CROSS INEX 415
VinyIidene chloride 19, 439 (1979); 39, 195 (1986);

Suppl. 7, 376 (1987)
VinyIidene chloride-vinyl chloride copolymers 19,44 (197) (con: 42, 258);
Suppl. 7, 73 (1987)
Vinylidene fluoride 39, 22 (1986); Suppl. 7, 73 (1987)
N- Vinyl-2-pyrIidone 19,461 (197); Suppl. 7, 73 (1987)
w
Welding 49, 447 (199)
Wollastonite 42, 145 (1987) Suppl. 7,377 (1987)
Woo industr 25 (1981); Suppl. 7,378 (1987)
x
Xylene 47, 12 (1989)
2,4-Xylidine 16,367 (1978); Suppl. 7, 74 (1987)
2,-XyIidine 16, 377 (1978); Suppl. 7, 74 (1987)
y
Yellow AB 8, 279 (1975); Suppl. 7, 74 (1987)
Yellow OB 8, 2ß7 (1975); Suppl. 7, 74 (1987)
z
Zearlenone 31,279 (1983); Suppl. 7, 74 (1987)
Zetran 12, '17 (1976); Suppl. 7, 74 (1987)
Zinc beryllum silcate (se Beryllum and beryllum compounds)
Zinc chromate (se Chromium.and chromium compounds)
Zinc chromate hydroxde (se Chromium and chromium
compounds )
Zinc potasium chromate (see Chromium and chromium
compounds)
Zinc yellow (se Chromium and chromium compounds)
Zineb 12, 245 (1976); Suppl. 7, 74 (1987)
Ziram 12,259 (1976); Suppl. 7, 74 (1987)
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WORLD HEAL1H ORGANZATION

INTERNATONAL AGENCY FOR RESEARCH ON CANCER

This publication represents the views and expert opinions

17-24 October 1989

ln 1969, the International Agency for Research on Cancer (IAC) initiated a

The objective of the programme is to elaborate and publish in the form of

C9lnternational Agency for Research on Cancer 1990

ISBN 92 832 1250 9

AlI rights reserved. Application for rights of reproduction or translation, in part

GENERA REMAKS ............................................... 33

SUMMAY OF FINAL EVALUATIONS 000000000.0000..0.000000000000 333

APPENDIX 2. ACTIVI PROFILES FOR GENETIC AN RELATED

SUPPLEMENTARY CORRIGENDA TO VOLUMES 1-49. 0 0 . 0 . . . 0 0 . . 0 0 385

CUMULATIVE INDEX TO THE MONOGRAHS SERIES 0 0 0 0 0 . 0 . 0 0 0 0 387

Representatives and observers2

reviews and evaluations of evidence on the carcinogenicity of a wide range of human

3. SELECTION OF TOPICS FOR MONOGRAPHS

The scientific literature is surveyed for published data relevant to an

lARe Monographs series(11,12).

4. DATA FOR MONOGRAPHS

5. THE WORKING GROUP

as: historical perspectives, description of an industry or habit, exposures in the work

or toxicological interactions of the components of mixures may result in nonlinear

reported. Experiments on the carcinogenicity of known metabolites and derivatives

(a) Qualitative aspects

(c) Statistical analysis of long-term exeriments in animaIs

(a) Structure-activity considerations

(b) Absorption, distribution, excretwn and metabolism

metabolic fate of the agent in experimental animaIs and hum

(h) Quality of studies considered

response is not necessarily evidence against a causal relationship. Demonstration

12. SUMMARY OF DATA REPORTED

(h) Exerimental carcinogenicity data

(c) Human carcinogenicity data

(d) Other relevant data

(a) Degrees of evidence for carcinogenicity in humans and in exerimental

(i) Human carcinogenicity data

Suffcient evidence of carcinogenicity The Working Group considers that a

exposure and length of observation covered by the available studies. ln addition,

include data on tumour pathology, genetic and related effects, structure-activity

(h) Overall evaluation

Group lA-The agent (mixure) is probably carcinogenic to humans.

3. IARC (1978) Chemicals with Sufficient Evidence of Carcinogenicity in Exprimental

This fiftieth volume of the lARe Monographs comprises monographs on five

Agent Vol. no. Year Evaluationa

Ruman Animal Overall

Agent Vol. no. Year Evaluationa

Ruman Animal Ove raIl

Analgesies and anti-injlammatory agents lcontd)

Agent Vol. no. Year Evaluationa

Antineoplastic dmn (contd)

Agent Vol. no. Year Evaluationa

Antineoplastic drugr (contd)

pa-Aminobenzoic acid 16 1978 ND 1 3

Agent Vol. no. Year EvaluationQ

Human Animal Overall

Dennatologial agents (contd)

Agent Vol. no. Year Evaluationa