Acute Toxicity Testing

Acute Toxicity Testing
Florencio V. Arce, Jr., RPh, MSc, PhD, SRPharmS

Jose Roel P. Tagalog, RPh
USC Department of Pharmacy
Objectives
Materials
Methods
Results: General observations
Adopted from Saleem, U., Amin, S., Ahmad, B., Azeem, H., Anwar, F., & Mary, S. (2017). Acute oral toxicity
evaluation of aqueous ethanolic extract of Saccharum munja Roxb. roots in albino mice as per OECD 425
TG. Toxicology reports, 4, 580–585. https://doi.org/10.1016/j.toxrep.2017.10.005
Discussion
• All chemical entities are toxic at sufficiently high doses.
• The therapeutic index is defined as the ratio of the toxic
dose to the therapeutic dose (TI). It has been used in
defining ‘safety margins’ in clinical studies. For
nonclinical toxicology evaluations, designed to provide
data supporting safety in a clinical trial, the safety term
used most often is the margin of safety (MOS).
• The MOS relates the No Observed Adverse Effect Level
(a dose that produces no relevant adverse effect) to the
maximum targeted dose in a clinical trial or a
therapeutically effective dose in a nonclinical model.
Discussion
• No Observed Adverse Effect Level
• It may be considered to be the highest
dose/exposure that does not cause biologically
important increases in the frequency or severity of
adverse effects between the expose population and
the appropriate control.
• While minimal toxic effects may be observed at the
NOAEL, they are not thought to endanger human
health or be precursors of serious adverse events.
Discussion
• Acute oral toxicity data are used to satisfy hazard
classification and labelling requirements, for risk
assessment for human health and the environment,
and when estimating the toxicity of mixtures. The
provision of either a point estimate of the LD50 value
or range estimate of the LD50 generally meets the
acute oral toxicity data requirements for classification
for all regulatory authorities.
• This procedure involves the administration of a single
bolus dose of test substance to fasted healthy young
adult rodents by oral gavage, observation for up to 14
days after dosing, recording of body weight and the
necropsy of all animals.
Discussion
• Sighting study (observing signs of evident toxicity or
death)
• Careful clinical observations should be made at least
twice on the day of dosing or more frequently when
indicated by the response of the animals to the
treatment, and at least once daily thereafter. Additional
observations are made if the animals continue to
display signs of toxicity. Observations include changes
in skin and fur, eyes and mucous membranes, and also
respiratory, circulatory, autonomic and central nervous
systems, and somatomotor activity and behavior
pattern.
Discussion
• Acute toxicity testing is conducted using a single
sex in order to reduce variability and as a means of
minimizing the number of animals used.
• Normally females are used. This is because
literature surveys of conventional LD50 tests show
that usually there is little difference in sensitivity
between the sexes but, in those cases where
differences were observed, females were generally
slightly more sensitive
Discussion
• Toxicity evaluation of most pharmaceuticals
includes test in each of these three categories.
• Acute toxicology testing entails single-dose studies
with a 14-day observation period.
• Generally conducted in two species, bot of which can be
rodents, prior to the first human dose.
• Sub chronic toxicology testing involves a multiple-
dose studies usually lasting from 2 weeks to 6
months.
• Chronic toxicology involves multiple-dose studies of
more than 6 months.
Discussion: Animal models
Assessment Animal model Comment
General toxicity rat, mouse, dog, Rat and dog are preferred species; other species may
monkey be selected based on a closer similarity to humans
Ocular irritation Rabbit Draize model
Dermal toxicity / Rabbit, rat Draize model
irritation
Dermal Guinea pig -
sensitization
Phototoxicity Guinea pig, -
mouse
Immunotoxicity Mouse, rat For antigenicity studies, rabbit and guinea pigs are
used
Developmental Rat, rabbit Mouse is alternative to rabbits (antibacterial), dogs
toxicity (neonatal studies)
Carcinogenicity Rat, mouse Rodents (maintained over a lifetime, 1.5 – 2 years)
Environmental Earthworm, Effect on target species evaluated directly
toxicity Daphnia, rainbow
trout
Discussion: In vitro models of toxicity
Assessment In vitro system Specific observations
Lethality Culture cell systems (mouse Cell viability, membrane
lymphoma, hepatocytes permeability, metabolic competence
Ocular irritation Cell systems Altered morphology, cytotoxicity,
release of inflammatory mediators
Dermal irritation Skin organ cultures or cultured Altered morphology, cytotoxicity,
cells (human keratinocytes) release of inflammatory mediators
Toxicity or Cultured rat skeletal muscle Medium creatinine kinase levels
irritation cause Erythrocytes Hemolysis
by IM or IV adm Limulus amebocyte lysate test Pyrogenicity
Developmental Drosophila, brine shrimp, medaka, Anatomical, functional, biochemical,
toxicity cell or organ cultures, and molecular alterations
sub/mammalian embryos
Targe-organ Isolated organ preparation, Morphologic, observational,
toxicity tissue/organ culture, cultured cells functional parameters
Carcinogenicity Bacteria, cultured cells Genetic damage
Cell lines, human tumor cell lines Neoplastic transformation
Discussion: Standard Genetic
Toxicology Test Battery
Genetic toxicology test Purpose

Ames bacterial mutation assay Gene mutation in bacteria
Mouse lymphoma assay, In vitro evaluation of chromosomal
Chinese hamster ovary, chromosomal damage
aberration assay
Micronucleus test Evaluation of in vivo chromosomal
damage in bone marrow polychromatic
erythrocytes
Discussion
• Genotoxicity
• Defined as the ability of an agent to damage DNA or alter
DNA sequence in such a way as to cause mutation.
• The most serious effects of these mutations are
neoplasms, inheritable neoplasms, or birth defects.
This is generally conducted prior to the first human
dose.
• Genotoxicity analysis entails in vitro and in vivo tests
designed to detect compounds that induce direct or
indirect genetic damage by various mechanism.
• The suspicion that a compound may induce heritable
defects is considered to be just as serious as the
possibility that it may cause cancer.
Discussion: Reproductive
toxicology
• Studies of potential adverse effects on fertility, fetal
development and behavior, and fetal toxicity should
be conducted to support the population chosen for
a clinical trial.
• This is considered in evaluating the potential to
increase risk of an adverse developmental or
reproductive outcomes in humans.
Discussion: Carcinogenicity
studies
• Carcinogenicity studies are not generally required in
advance of clinical trials unless there is a concern about
a class effect relevant to humans, evidence of
preneoplastic lesion in repeated-dose toxicity studies.
• Carcinogenicity studies should be conducted for any
NCE with an expected clinical use of at least 6 months.
• Selection of maximum dose in carcinogenicity study is
based on maximum tolerated dose, dose expected to
produce minimal toxicity over the course of the
carcinogenicity study, 25-fold AUC rodent/human ratio,
saturation of absorption, dose-limiting
pharmacodynamics, or maximum feasible dose (5% of
the diet).

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Florencio V. Arce, Jr., RPh, MSc, PhD, SRPharmS

Genetic toxicology test Purpose

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