Biological Assays and Tests

Methods of assay described as Suggested methods are not obligatory, but when another method is used
its precision must be not less than that required for the Suggested method.
For those antibiotics for which the monograph specifies a microbiological assay the potency requirement
is expressed in the monograph in International Units (IU) per milligram. The material is not of
pharmacopoeial quality if the upper fiducial limit of error is less than the stated potency. For such
antibiotics the required precision of the assay is stated in the monograph in terms of the fiducial limits of
error about the estimated potency.
For other substances and preparations for which the monograph specifies a biological assay, unless
otherwise stated, the precision of the assay is such that the fiducial limits of error, expressed as a
percentage of the estimated potency, are within a range not wider than that obtained by multiplying by a
factor of 10 the square roots of the limits given in the monograph for the fiducial limits of error about the
stated potency.
In all cases fiducial limits of error are based on a probability of 95% (P = 0.95).
Where the biological assay is being used to ascertain the purity of the material, the stated potency
means the potency stated on the label in terms of International Units (IU) or other Units per gram, per
milligram or per millilitre. When no such statement appears on the label, the stated potency means the
fixed or minimum potency required in the monograph. This interpretation of stated potency applies in all
cases except where the monograph specifically directs otherwise.
Where the biological assay is being used to determine the total activity in the container, the stated
potency means the total number of International Units (IU) or other Units stated on the label or, if no such
statement appears, the total activity calculated in accordance with the instructions in the monograph.
Wherever possible the primary standard used in an assay or test is the respective International Standard
or Reference Preparation established by the World Health Organization for international use and the
biological activity is expressed in International Units (IU).
In other cases, where Units are referred to in an assay or test, the Unit for a particular substance or
preparation is, for the United Kingdom, the specific biological activity contained in such an amount of the
respective primary standard as the appropriate international or national organisation indicates. The
necessary information is provided with the primary standard.
Unless otherwise directed, animals used in an assay or a test are healthy animals, drawn from a uniform
stock, that have not previously been treated with any material that will interfere with the assay or test.
Unless otherwise stated, guinea-pigs weigh not less than 250 g or, when used in systemic toxicity tests,
not less than 350 g. When used in skin tests they are white or light coloured. Unless otherwise stated,
mice weigh not less than 17 g and not more than 22 g.
Certain of the biological assays and tests of the Pharmacopoeia are such that in the United Kingdom
they may be carried out only in accordance with the Animals (Scientific Procedures) Act 1986.
Instructions included in such assays and tests in the Pharmacopoeia, with respect to the handling of
animals, are therefore confined to those concerned with the accuracy and reproducibility of the assay or
test.

Reference Substances and Reference Preparations

Certain monographs require the use of a reference substance, a reference preparation or a reference
spectrum. These are chosen with regard to their intended use as prescribed in the monographs of the
Pharmacopoeia and are not necessarily suitable in other circumstances.
Any information necessary for proper use of the reference substance or reference preparation is given
on the label or in the accompanying leaflet or brochure. Where no drying conditions are stated in the
leaflet or on the label, the substance is to be used as received. No certificate of analysis or other data not
relevant to the prescribed use of the product are provided. The products are guaranteed to be suitable
for use for a period of three months from dispatch when stored under the appropriate conditions. The
stability of the contents of opened containers cannot be guaranteed. The current lot is listed in the BP
Laboratory website catalogue. Additional information is provided in Supplementary Chapter III E.

Chemical Reference Substances The abbreviation BPCRS indicates a Chemical Reference

Substance established by the British Pharmacopoeia Commission. The abbreviation CRS or EPCRS
indicates a Chemical Reference Substance established by the European Pharmacopoeia Commission.
Some Chemical Reference Substances are used for the microbiological assay of antibiotics and their
activity is stated, in International Units, on the label or on the accompanying leaflet and defined in the
same manner as for Biological Reference Preparations.

Biological Reference Preparations The majority of the primary biological reference preparations
referred to are the appropriate International Standards and Reference Preparations established by the
World Health Organisation. Because these reference materials are usually available only in limited
quantities, the European Pharmacopoiea has established Biological Reference Preparations (indicated
by the abbreviation BRP or EPBRP) where appropriate. Where applicable, the potency of the Biological
Reference Preparations is expressed in International Units. For some Biological Reference Preparations,
where an international standard or reference preparation does not exist, the potency is expressed in
European Pharmacopoeia Units.

Labelling

The labelling requirements of the Pharmacopoeia are not comprehensive and laws governing the
statements to be declared on labels of official articles should also be met. In the United Kingdom the
provisions of regulations issued in accordance with the Medicines Act 1968, together with those of
regulations for the labelling of hazardous materials, should be met.
Only those statements in monographs given under the heading Labelling that are necessary to
demonstrate compliance or otherwise with the monograph are mandatory. Any other statements are
included as recommendations.
Such matters as the exact form of wording to be used and whether a particular item of information
should appear on the primary label and additionally, or alternatively, on the package or exceptionally in a
leaflet are, in general, outside the scope of the Pharmacopoeia. When the term 'label' is used in Labelling
statements of the Pharmacopoeia, decisions as to where the particular statement should appear should
therefore be made in accordance with relevant legislation.
The label of every official article states (i) the name at the head of the monograph and (ii) a reference
consisting of either figures or letters, or a combination of figures and letters, by which the history of the
article may be traced.
The label of every official formulated preparation other than those of fixed strength also states the
content of the active ingredient or ingredients expressed in the terms required by the monograph. Where
the content of active ingredient is required to be expressed in terms other than the weight of the official
medicinal substance used in making the formulation, this is specifically stated under the heading
Labelling. Thus, where no specific requirement is included under the heading Labelling, it is implied that
the content of active ingredient is expressed in terms of the weight of the official medicinal substance
used in making the formulation. For example, for Ampicillin Injection, which contains Ampicillin Sodium
but for which the content is expressed in terms of the equivalent amount of ampicillin, a specific
requirement to this effect is included under the heading Labelling. For Amitriptyline Tablets which contain
Amitriptyline Hydrochloride and for which the result of the assay is expressed in terms of amitriptyline
hydrochloride no specific statement is included under the heading Labelling; these Tablets are thus
labelled with the nominal weight of Amitriptyline Hydrochloride.
These requirements do not necessarily apply to the labelling of articles supplied in compliance with a
prescription.

1.5. Abbreviations and symbols

A Absorbance

Specific absorbance
Ar Relative atomic mass

Specific optical rotation

bp Boiling point
BRP Biological Reference Preparation
CRS Chemical Reference Substance
Relative density
IU International Unit
l Wavelength
M Molarity
Mr Relative molecular mass
mp Melting point
Refractive index
Ph. Eur. U. European Pharmacopoeia Unit
Ppm Parts per million
R Substance or solution defined under 4. Reagents
Rf Used in chromatography to indicate the ratio of the distance travelled by a substance to the
distance travelled by the solvent front
Rst Used in chromatography to indicate the ratio of the distance travelled by a substance to the
distance travelled by a reference substance
RV Substance used as a primary standard in volumetric analysis (chapter 4.2.1)

Abbreviations used in the monographs on immunoglobulins, immunosera and vaccines

LD50 The statistically determined quantity of a substance that, when administered by the specified
route, may be expected to cause the death of 50 per cent of the test animals within a given period
MLD Minimum lethal dose
L+/10 dose The smallest quantity of a toxin that, in the conditions of the test, when mixed with 0.1 IU
of antitoxin and administered by the specified route, causes the death of the test animals within a given
period
L+ dose The smallest quantity of a toxin that, in the conditions of the test, when mixed with 1 IU of
antitoxin and administered by the specified route, causes the death of the test animals within a given
period
lr/100 dose The smallest quantity of a toxin that, in the conditions of the test, when mixed with 0.01
IU of antitoxin and injected intracutaneously causes a characteristic reaction at the site of injection within
a given period
Lp/10 dose The smallest quantity of toxin that, in the conditions of the test, when mixed with 0.1 IU of
antitoxin and administered by the specified route, causes paralysis in the test animals within a given
period
Lo/10 dose The largest quantity of a toxin that, in the conditions of the test, when mixed with 0.1 IU of
antitoxin and administered by the specified route, does not cause symptoms of toxicity in the test animals
within a given period
Lf dose The quantity of toxin or toxoid that flocculates in the shortest time with 1 IU of antitoxin
CCID50 The statistically determined quantity of virus that may be expected to infect 50 per cent of
the cell cultures to which it is added
EID50 The statistically determined quantity of virus that may be expected to infect 50 per cent of
fertilised eggs into which it is inoculated
ID50 The statistically determined quantity of a virus that may be expected to infect 50 per cent
of the animals into which it is inoculated
PD50 The statistically determined dose of a vaccine that, in the conditions of the tests, may be
expected to protect 50 per cent of the animals against a challenge dose of the micro-organisms or toxins
against which it is active
ED50 The statistically determined dose of a vaccine that, in the conditions of the tests, may be
expected to induce specific antibodies in 50 per cent of the animals for the relevant vaccine antigens
PFU Pock-forming units or plaque-forming units
SPF Specified-pathogen-free.

Collections of micro-organisms

ATCC American Type Culture Collection

C.I.P. Collection de Bactéries de l'Institut Pasteur
IMI International Mycological Institute
I.P. Collection Nationale de Culture de Microorganismes (C.N.C.M.)
Institut Pasteur
NCIMB National Collection of Industrial and Marine Bacteria Ltd
NCPF National Collection of Pathogenic Fungi
London School of Hygiene and Tropical Medicine
NCTC National Collection of Type Cultures
Central Public Health Laboratory
NCYC National Collection of Yeast Cultures
AFRC Food Research Institute
S.S.I. Statens Serum Institut
80 Amager Boulevard, Copenhagen, Denmark

1.6. Units of the international system (SI) used in the Pharmacopoeia and equivalence
with other units

International System Of Units (SI)

The International System of Units comprises 3 classes of units, namely base units, derived units and
supplementary units1. The base units and their definitions are set out in Table 1.6-1.
 The derived units may be formed by combining the base units according to the algebraic relationships
linking the corresponding quantities. Some of these derived units have special names and symbols. The
SI units used in the European Pharmacopoeia are shown in Table 1.6-2.
Some important and widely used units outside the International System are shown in Table 1.6-3.
The prefixes shown in Table 1.6-4 are used to form the names and symbols of the decimal multiples and
submultiples of SI units.

(IMMUNOSERA FOR HUMAN USE, ANIMAL, PH EUR MONOGRAPH 0084)

An antiserum for human use that is the subject of an individual monograph in the European
Pharmacopoeia or in the British Pharmacopoeia complies with the requirements of the European
Pharmacopoeia monograph for Immunosera for Human Use, Animal. These requirements are
reproduced below.
The provisions of this monograph apply to the following antisera:

Botulinum Antitoxin*
Diphtheria Antitoxin*
European Viper Venom Antiserum*
Gas-gangrene Antitoxin (Novyi)*
Gas-gangrene Antitoxin (Perfringens)*
Gas-gangrene Antitoxin (Septicum)*
Mixed Gas-gangrene Antitoxin*
Tetanus Antitoxin*
*Monograph of the European Pharmacopoeia
Ph Eur

DEFINITION
 Animal immunosera for human use are liquid or freeze-dried preparations containing purified
immunoglobulins or immunoglobulin fragments obtained from serum or plasma of immunised animals of
different species.
The immunoglobulins or immunoglobulin fragments have the power of specifically neutralising or binding
to the antigen used for immunisation. The antigens include microbial or other toxins, human antigens,
suspensions of bacterial and viral antigens and venoms of snakes, scorpions and spiders. The
preparation is intended for intravenous or intramuscular administration, after dilution where applicable.

PRODUCTION

GENERAL PROVISIONS

The production method shall have been shown to yield consistently immunosera of acceptable safety,
potency in man and stability.
Any reagent of biological origin used in the production of immunosera shall be free of contamination with
bacteria, fungi and viruses. The method of preparation includes a step or steps that have been shown to
remove or inactivate known agents of infection.
Methods used for production are validated, effective, reproducible and do not impair biological activity of
the product.
The production method is validated to demonstrate that the product, if tested, would comply with the test
for abnormal toxicity for immunosera and vaccines for human use (2.6.9).
Reference preparation A batch shown to be suitable in clinical trials, or a batch representative thereof,
is used as the reference preparation for the tests for high molecular mass proteins and purity.

ANIMALS

The animals used are of a species approved by the competent authority, are healthy and exclusively
reserved for production of immunoserum. They are tested and shown to be free from a defined list of
infectious agents. The introduction of animals into a closed herd follows specified procedures, including
definition of quarantine measures. Where appropriate, additional specific agents are considered
depending on the geographical localisation of the establishment used for the breeding and production of
the animals. The feed originates from a controlled source and no animal proteins are added. The
suppliers of animals are certified by the competent authority.
If the animals are treated with antibiotics, a suitable withdrawal period is allowed before collection of
blood or plasma. The animals are not treated with penicillin antibiotics. If a live vaccine is administered, a
suitable waiting period is imposed between vaccination and collection of serum or plasma for
immunoserum production.

IMMUNISATION

The antigens used are identified and characterised, where appropriate; where relevant, they are shown
to be free from extraneous infectious agents. They are identified by their names and a batch number;
information on the source and preparation are recorded.
The selected animals are isolated for at least 1 week before being immunised according to a defined
schedule with booster injections at suitable intervals. Adjuvants may be used.
Animals are kept under general health surveillance and specific antibody production is controlled at each
cycle of immunisation.
Animals are thoroughly examined before collection of blood or plasma. If an animal shows any
pathological lesion not related to the immunisation process, it is not used, nor are any other of the
animals in the group concerned, unless it is evident that their use will not impair the safety of the product.
COLLECTION OF BLOOD OR PLASMA

Collection of blood is made by venepuncture or plasmapheresis. The puncture area is shaved, cleaned
and disinfected. The animals may be anaesthetised under conditions that do not influence the quality of
the product. Unless otherwise prescribed, an antimicrobial preservative may be added. The blood or
plasma is collected in such a manner as to maintain sterility of the product. The blood or plasma
collection is conducted at a site separate from the area where the animals are kept or bred and the area
where the immunoserum is purified. If the serum or plasma is stored before further processing,
precautions are taken to avoid microbial contamination.
Several single plasma or serum samples may be pooled before purification. The single or pooled
samples are tested before purification for the following tests.

Tests for contaminating viruses

If an antimicrobial preservative is added, it must be neutralised before carrying out the tests or the tests
carried out on a sample taken before addition of the antimicrobial preservative. Each pool is tested for
contaminating viruses by suitable in vitro tests.
Each pool is tested for viruses by inoculation to cell cultures capable of detecting a wide range of viruses
relevant for the particular product.

Potency

Carry out a biological assay as indicated in the monograph and express the result in International Units
per millilitre, where applicable. A validated in vitro method may also be used.

Protein content

Dilute the product to be examined with a 9 g/l solution of sodium chloride R to obtain a solution
containing about 15 mg of protein in 2 ml. To 2 ml of this solution in a round-bottomed centrifuge tube
add 2 ml of a 75 g/l solution of sodium molybdate R and 2 ml of a mixture of 1 volume of nitrogen-free
sulphuric acid R and 30 volumes of water R. Shake, centrifuge for 5 min, decant the supernatant liquid
and allow the inverted tube to drain on filter paper. Determine the nitrogen in the residue by the method
of sulphuric acid digestion (2.5.9) and calculate the content of protein by multiplying by 6.25. The protein
content is within approved limits.

PURIFICATION AND VIRAL INACTIVATION

The immunoglobulins are concentrated and purified by fractional precipitation, chromatography,

immunoadsorption or by other chemical or physical methods. They may be processed further by enzyme
treatment. The methods are selected and validated to avoid contamination at all steps of processing and
to avoid formation of protein aggregates that effect immunobiological characteristics of the product. For
products intended to consist of immunoglobulin fragments, the methods are validated to guarantee total
fragmentation. The methods of purification used are such that they do not generate additional
components that compromise the quality and the safety of the product.
Unless otherwise justified and authorised, validated procedures are applied for removal and/or
inactivation of viruses. The procedures are selected to avoid the formation of polymers or aggregates
and, unless the product is intended to consist of Fab fragments, to minimise the splitting of F(ab)2 into
Fab fragments.
After purification and treatment for removal and/or inactivation of viruses, a stabiliser may be added to
the intermediate product, which may be stored for a period defined in the light of stability data.
Only an intermediate product that complies with the following requirements may be used in the
preparation of the final bulk.

Purity

Examine by non-reducing polyacrylamide gel electrophoresis (2.2.31), by comparison with the reference
preparation, the bands are compared in intensity and no additional bands are found.

FINAL BULK

The final bulk is prepared from a single intermediate product or from a pool of intermediate products
obtained from animals of the same species. Intermediate products with different specificities may be
pooled.
An antimicrobial preservative and a stabiliser may be added. If an antimicrobial preservative has been
added to the blood or plasma, the same substance is used as antimicrobial preservative in the final bulk.
Only a final bulk that complies with the following requirements may be used in the preparation of the final
lot.

Antimicrobial preservative

Where applicable, determine the amount of antimicrobial preservative by a suitable physico-chemical

method. It contains not less than 85 per cent and not more than 115 per cent of the amount stated on the
label.

Sterility (2.6.1)

It complies with the test for sterility.

FINAL LOT

The final bulk of immunoserum is distributed aseptically into sterile, tamper-proof containers. The
containers are closed so as to prevent contamination.
Only a final lot that complies with the requirements prescribed below under Identification, Tests and
Assay may be released for use. Provided that the tests for osmolality, protein content, molecular-size
distribution, antimicrobial preservative, stabiliser, purity, foreign proteins and albumin and the assay have
been carried out with satisfactory results on the final bulk, they may be omitted on the final lot.
Reconstitute the preparation to be examined as stated on the label immediately before carrying out the
identification, tests (except those for solubility and water ) and assay.
IDENTIFICATION

The identity is established by immunological tests and, where necessary, by determination of biological
activity. The assay may also serve for identification.
CHARACTERS

Immunosera are clear to opalescent and colourless to very faintly yellow liquids. They are free from
turbidity. Freeze-dried products are white or slightly yellow powders or solid friable masses. After
reconstitution they show the same characteristics as liquid preparations.

TESTS

Solubility
To a container of the preparation to be examined, add the volume of the liquid for reconstitution stated
on the label. The preparation dissolves completely within the time stated on the label.

Extractable volume (2.9.17)

It complies with the requirement for extractable volume.
pH (2.2.3)
The pH is within the limits approved for the particular product.
Osmolality (2.2.35)
Minimum 240 mosmol/kg after dilution, where applicable.
Protein content
90 per cent to 110 per cent of the amount stated on the label, and not more than 100 g/l.
Dilute the preparation to be examined with a 9 g/l solution of sodium chloride R to obtain a solution
containing about 15 mg of protein in 2 ml. To 2 ml of this solution in a round-bottomed centrifuge tube
add 2 ml of 75 g/l solution of sodium molybdate R and 2 ml of a mixture of 1 volume of nitrogen-free
sulphuric acid R and 30 volumes of water R. Shake, centrifuge for 5 min, decant the supernatant liquid
and allow the inverted tube to drain on filter paper. Determine the nitrogen in the residue by the method
of sulphuric acid digestion (2.5.9) and calculate the content of protein by multiplying by 6.25.
Molecular-size distribution
Examine by liquid chromatography (2.2.29 or 2.2.30). It complies with the specification approved for the
particular product.
Antimicrobial preservative
Where applicable, determine the amount of antimicrobial preservative by a suitable physicochemical
method. The amount is not less than the minimum amount shown to be effective and is not greater than
115 per cent of that stated on the label.
Phenol (2.5.15)
Maximum 2.5 g/l for preparations containing phenol.
Stabiliser
Determine the amount of stabiliser by a suitable physico-chemical method. The preparation contains not
less than 80 per cent and not more than 120 per cent of the quantity stated on the label.
Purity

Examine by non-reducing polyacrylamide gel electrophoresis (2.2.31), by comparison with the reference
preparation. No additional bands are found for the preparation to be examined.

Foreign proteins

When examined by precipitation tests with specific antisera, only protein from the declared animals
species is shown to be present, unless otherwise prescribed, for example where material of human origin
is used during production.

Albumin

Unless otherwise prescribed in the monograph, when examined electrophoretically, the content of
albumin is not greater than the limit approved for the particular product and, in any case, not greater than
3 per cent.

Water (2.5.12) Maximum 3 per cent.

Sterility (2.6.1) It complies with the test for sterility.
Pyrogens (2.6.8)
Unless otherwise justified and authorised, it complies with the test for pyrogens. Unless otherwise
prescribed, inject 1 ml per kilogram of the rabbit's body mass.

ASSAY
Carry out a biological assay as indicated in the monograph and express the result in International Units
per millilitre, where appropriate. A validated in vitro method may also be used.

STORAGE

Store protected from light at the temperature stated on the label. Do not allow liquid preparations to
freeze.
Expiry date The expiry date is calculated from the beginning of the assay.

LABELLING

The label states:

—the number of International Units per millilitre, where applicable,

—the amount of protein per container,

—for freeze-dried preparations,

—the name and volume of the reconstituting liquid to be added,

—that the immunoserum is to be used immediately after reconstitution,

—the time required for complete dissolution,

—the route of administration,

—the storage conditions,

—the expiry date, except for containers of less than 1 ml which are individually packed. The expiry
date may be omitted from the label on the container, provided it is shown on the package and the label
on the package states that the container must be kept in the package until required for use,

—the animal species of origin,

—the name and amount of any antimicrobial preservative, any stabiliser and any other substance
added to the immunoserum.

Ph Eur
European Viper Venom Antiserum
(Ph Eur monograph 0145)

The only poisonous snake native to the British Isles is the adder or common viper, Vipera berus. In a
geographical region where other species of snake (including elapids) are found, antisera able to
neutralise the venoms of the species of snake indigenous to the region should be used. When the
preparation is intended to neutralise the venom or venoms of one or more snakes other than vipers, the
title Snake Venom Antiserum is used.

DEFINITION
European viper venom antiserum is a preparation containing antitoxic globulins that have the power of
neutralising the venom of one or more species of viper. The globulins are obtained by fractionation of the
serum of animals that have been immunised against the venom or venoms.

IDENTIFICATION

It neutralises the venom of Vipera ammodytes, or Vipera aspis, or Vipera berus, or Vipera ursinii or the
mixture of these venoms stated on the label, rendering them harmless to susceptible animals.

ASSAY

Each millilitre of the preparation to be examined contains sufficient antitoxic globulins to neutralise not
less than 100 mouse LD50 of Vipera ammodytes venom or Vipera aspis venom and not less than 50
mouse LD50 of the venoms of other species of viper.
The potency of European viper venom antiserum is determined by estimating the dose necessary to
protect mice against the lethal effects of a fixed dose of venom of the relevant species of viper.

Selection of test venoms

Use venoms which have the normal physicochemical, toxicological and immunological characteristics of
venoms from the particular species of vipers. They are preferably freeze-dried and stored in the dark at 5
± 3 °C.
Select a venom for use as a test venom by determining the LD50 for mice, the observation period being
48 h.

Determination of the test dose of venom

Prepare graded dilutions of the reconstituted venom in a 9 g/l solution of sodium chloride R or other
isotonic diluent in such a manner that the middle dilution contains in 0.25 ml the dose expected to be the
LD50. Dilute with an equal volume of the same diluent. Using at least four mice, each weighing 18 g to 20
g, for each dilution, inject 0.5 ml intravenously into each mouse. Observe the mice for 48 h and record
the number of deaths. Calculate the LD50 using the usual statistical methods.

Determination of the potency of the antiserum to be examined

Dilute the reconstituted test venom so that 0.25 ml contains the test dose of 5 LD50 (test venom solution).
Prepare serial dilutions of the antiserum to be examined in a 9 g/l solution of sodium chloride R or other
isotonic diluent, the dilution factor being 1.5 to 2.5. Use a sufficient number and range of dilutions to
enable a mortality curve between 20 per cent and 80 per cent mortality to be established and to permit
an estimation of the statistical variation.
Prepare mixtures such that 5 ml of each mixture contains 2.5 ml of one of the dilutions of the antiserum
to be examined and 2.5 ml of the test venom solution. Allow the mixtures to stand in a water-bath at 37
°C for 30 min. Using not fewer than six mice, each weighing 18 g to 20 g, for each mixture, inject 0.5 ml
intravenously into each mouse. Observe the mice for 48 h and record the number of deaths. Calculate
the PD50, using the usual statistical methods. At the same time verify the number of LD50 in the test dose
of venom, using the method described above. Calculate the potency of the antiserum from the
expression:
Tv = number of LD50 in the test dose of venom.
In each mouse dose of the venom-antiserum mixture at the end point there is one LD50 of venom
remaining unneutralised by the antiserum and it is this unneutralised venom that is responsible for the
deaths of 50 per cent of the mice inoculated with the mixture. The amount of venom neutralised by the
antiserum is thus one LD50 less than the total amount contained in each mouse dose. Therefore, as the
potency of the antiserum is defined in terms of the number of LD50 of venom that are neutralised. rather
than the number of LD50 in each mouse dose, the expression required in the calculation of potency is Tv -
1 rather than Tv.
Alternatively, the quantity of test venom in milligrams that is neutralised by 1 ml or some other defined
volume of the antiserum to be examined may be calculated.

LABELLING

The label states the venom or venoms against which the antiserum is effective.
Warning: Because of the allergenic properties of viper venoms, inhalation of venom dust should be
avoided by suitable precautions.

Ph Eur
Gas-gangrene Antitoxin (Perfringens)
General Notices

The label may state 'Perf/Ser'.

Preparation

Mixed Gas-gangrene Antitoxin

DEFINITION

Gas-gangrene antitoxin (perfringens) is a preparation containing antitoxic globulins that have the power
of specifically neutralising the alpha toxin formed by Clostridium perfringens. It is obtained by
fractionation from the serum of horses, or other mammals, that have been immunised against Cl.
perfringens alpha toxin.

IDENTIFICATION

It specifically neutralises the alpha toxin formed by Cl. perfringens, rendering it harmless to susceptible
animals.

ASSAY

Not less than 1500 IU of antitoxin per millilitre.

The potency of gas-gangrene antitoxin (perfringens) is determined by comparing the dose necessary to
protect mice or other suitable animals against the lethal effects of a fixed dose of Cl. perfringens toxin
with the quantity of the standard preparation of gas-gangrene antitoxin (perfringens) necessary to give
the same protection. For this comparison a reference preparation of gas-gangrene antitoxin
(perfringens), calibrated in International Units, and a suitable preparation of Cl. perfringens toxin for use
as a test toxin are required. The potency of the test toxin is determined in relation to the reference
preparation; the potency of the gas-gangrene antitoxin (perfringens) to be examined is determined in
relation to the potency of the test toxin by the same method.
The International Unit of antitoxin is the specific neutralising activity for Cl. perfringens toxin contained in
a stated amount of the International Standard, which consists of a quantity of dried immune horse serum.
The equivalence in International Units of the International Standard is stated by the World Health
Organisation.

Selection of animals

Use mice having body masses such that the difference between the lightest and the heaviest does not
exceed 5 g.

Preparation of test toxin

Prepare the test toxin from a sterile filtrate of an approximately 5-day culture in liquid medium of Cl.
perfringens. Treat the filtrate with ammonium sulphate R, collect the precipitate, which contains the toxin,
dry in vacuo over diphosphorus pentoxide R, powder and store dry.

Selection of test toxin

Select a toxin for use as a test toxin by determining for mice the L+ dose and the LD50, the observation
period being 48 h. The test toxin has an L+ dose of 4 mg or less and contains not less than 20 LD50 in
each L+ dose.

Determination of test dose of toxin (L+ dose)

Prepare a solution of the reference preparation in a suitable liquid such that it contains 5 IU of antitoxin
per millilitre.
Prepare a solution of the test toxin in a suitable liquid such that 1 ml contains a precisely known amount
such as 10 mg.
Prepare mixtures of the solution of the reference preparation and the solution of the test toxin such that
each contains 2.0 ml of the solution of the reference preparation, one of a graded series of volumes of
the solution of the test toxin and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Allow the
mixtures to stand at room temperature, protected from light, for 60 min. Using six mice for each mixture,
inject a dose of 0.5 ml intravenously into each mouse. Observe the mice for 48 h.
The test dose of toxin is the quantity in 0.5 ml of the mixture made with the smallest amount of toxin
capable of causing, despite partial neutralisation by the reference preparation, the death of all six mice
injected with the mixture within the observation period.

Determination of potency of the antitoxin

Prepare a solution of the reference preparation in a suitable liquid such that it contains 5 IU of antitoxin
per millilitre.
Prepare a solution of the test toxin in a suitable liquid such that it contains five test doses per millilitre.
Prepare mixtures of the solution of the test toxin and the antitoxin to be examined such that each
contains 2.0 ml of the solution of the test toxin, one of a graded series of volumes of the antitoxin to be
examined and sufficient of a suitable liquid to bring the total volume to 5.0 ml. Also prepare mixtures of
the solution of the test toxin and the solution of the reference preparation such that each contains 2.0 ml
of the solution of the test toxin, one of a graded series of volumes of the solution of the reference
preparation centred on that volume (2.0 ml) that contains 10 IU and sufficient of a suitable liquid to bring
the total volume to 5.0 ml. Allow the mixtures to stand at room temperature, protected from light, for 60
min. Using six mice for each mixture, inject a dose of 0.5 ml intravenously into each mouse. Observe the
mice for 48 h.
The mixture that contains the largest volume of antitoxin that fails to protect the mice from death contains
10 IU. This quantity is used to calculate the potency of the antitoxin in International Units per millilitre.
The test is not valid unless all the mice injected with mixtures containing 2.0 ml or less of the solution of
the reference preparation die and all those injected with mixtures containing a larger volume survive.

Ph Eur