Animal toxicity studies

ANIMAL TOXICITY STUDIES
Dr.Swaroopa,
1st year PG,
MD Pharmacology,
Rangaraya Medical College.
1

CONTENTS
• INTRODUCTION
• OBJECTIVES OF THE TOXICITY STUDIES
• GENERAL PRINCIPLES
• TYPES OF TOXICITY STUDIES
• REFERENCES
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OBJECTIVES OF THE TOXICITY STUDY
 To identify any toxic substance prior to clinical use.
 Qualitative and Quantitative assessment of drug use.
 Mostly used to examine specific adverse events or specific end points such as cancer, cardiotoxicity,
skin/eye irritation.
 Different types of dose identification can be done. (MLD, LD50, MTD, ED50, NOAEL)
 Prediction of therapeutic index (LD50 / ED50 )
 Benefit-risk ratio can be calculated.
 To relate the toxicological findings to clinical safety.
 To support in selecting species, treatment regimen and designing subsequent non clinical toxicity
studies. 3
• SK Gupta, New Drug Development, Drug Discovery and Clinical Research,1st edition, pg :
18 to 21.
• Bikash Medhi, Ajay Prakash, Practical Manual of Experimental and Clinical Pharmacology, 2nd
edition, pg 136 to 140.

GENERAL PRINCIPLES
In a large set-up, conducting toxicity studies should comply the following important
requirements:
 Should follow the Good Laboratory Practices(GLP).
 Studies should be performed by suitably trained and qualified staff.
 Instruments should be calibrated and standardized properly and of adequate capacity.
 Standard operating procedures(SOPs)should be followed.
 Documentation must be proper and anticipate the results.
 All documents, histology slides and paraffin tissue blocks should be preserved for a
minimum of 5years after marketing of the drug.
4
• Bikash Medhi, Ajay Prakash, Practical Manual of Experimental and Clinical Pharmacology, 2nd
edition, pg 136 to 140.
• SK Gupta, New Drug Development, Drug Discovery and Clinical Research,1st edition, pg :
18 to 21.

TYPES OF TOXICITY STUDIES:
Systemic Toxicity studies.
Reproduction and Developmental Toxicity Studies.
Local toxicity studies
Carcinogenicity studies.
Geno toxicity studies.
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OECD- guidelines for the testing of chemicals
(Organisation for Economic Cooperation and Development)
.
 Is a collection of about 150 of the most relevant internationally agreed testing
methods used by the government industry and independent laboratories to identify
and characterise potential hazard of chemicals.
 They are a set of tools for professionals, used primarily in regulatory safety testing
and subsequent chemicals & chemical product notification, chemical registration &
in chemical evaluation.
 They can also be used for selection and ranking of candidate chemicals during the
development of new chemicals & products and in toxicology research.
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OECD guidelines for testing chemicals are a unique tool for assessing the
potential effects of chemicals on human Health & the environment.
 They are split into 5 sections.
1. Physical-chemical properties.
2. Effects on Biotic system.
3. Environmental fate and Behaviour.
4. Health effects.
5. Other test guidelines.

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REPRODUCTIVE
AND
DEVELOPMENTAL TOXICITY

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REPRODUCTIVE TOXICITY:-
Is defined by Globally Harmonized system(GHS) as “adverse effects of
chemicals on sexual function & fertility in adult males and females as well as
developmental toxicity in the offspring”.
For reproductive toxicity, a variety of tests are available.
Screening level test is ‘The Reproductive Developmental Toxicity Screening
Assay’
Which uses male and female rats dosed with test substances for 2 weeks prior to
mating, during mating & gestation(3 weeks) and parturition up until postnatal
day 4.
The definitive test for reproductive toxicity is the two generation reproduction
toxicity study – involves continuous dosing of parental rats & their offspring
with the test substance for two entire generations up through weaning of F2 pups
at 3 weeks of age.

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• A number of retrospective analyses have indicated that the data from the 2nd
generation has had little impact on the final outcome of the study in terms of
regulation.
• Therefore, a New test design called Extended One Generation Reproductive Toxicity
Study(EOGRTS) was developed.
• EOGRTS – this test omits the second mating ,but in its place makes much more
complete use of F1 offspring by carrying them out for extended periods in order to
assess development of immune system function, developmental toxicity as well as
reproductive function & additional endocrine sensitive end points.

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Reproductive toxicity studies :- 1)male fertility studies
2) female reproduction and developmental toxicity studies.
MALE FERTILITY :
 One Test species used – Rat (rodent)
 3 dose groups taken (each with 6 adult males and 1 control)
 Treated with test drug for 28 days by intended route for use.
 Paired with female rats in 1:2 ratio
 Drug treatment of male animals continues during pairing
 Pairing continued till the detection of sperm in vagina, or in 10 days whichever is earlier.
 Pregnant females examined after day 13 of gestation
 Males sacrificed at the end of the study
 Weight of each testis & epididymis recorded
 Sperms from one epididymis examined for mortality & morphology.
 Other epididymis & both testis examined for histology.

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FEMALE REPRODUCTION AND DEVELOPMENTAL TOXICITY STUDIES
1. Female fertility
2. Teratogenicity
3. Perinatal toxicity
• In view of complexity of female reproductive system, chemical –induced disruptions in
fertility & health can be caused at a wide variety of sites & developmental stages.
• And also there is difficulty in collecting human information & specimens for the
assessment of risk by a given chemical or class of compounds.
• Much of the information available regarding female reproductive toxicities is based on
results from animal studies in controlled laboratory environments.
we can have basic information related to effects that can be potentially be produced by
a variety of chemicals in women can be generated effectively.
REPRODUCTIVE & DEVELOPMENTAL
TOXICITY
Last updated: May 21, 2014
http://alttox.org/

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• In many cases, mouse & rat have demonstrated different degrees or levels of
responsiveness to reproductive toxicants.
• Species specificity is of particular importance in using information generated in
animals to predict human risk.
• Animals used: for female fertility and perinatal toxicity studies – mice or rats are
used.
for teratogenic toxicity studies also albino rabbits are used as 2nd species.
Female fertility toxicity studies :-
 Drug administration: 28 days(males) and 14 days(female) rats before mating.
 Route of administration same as intended for therapeutic use.
 Drug treatment continued during mating & gestation period.
 Female rats allowed to litter, medication continued till weaning of pups.

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 Check for Body weight, food intake
Clinical signs of intoxication
Reproduction & parturition
Pathological changes (gross
and microscopic)
PUPS: physiology, behaviour, pathology(sex wise distribution noted)

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Example: Of using animal studies to predict human exposure outcomes was seen with
dosing of female mice with the PAH(polycyclic Aromatic hydrocarbons) DMBA (9,10
dimethyl benza anthracene.
 By single high dose exposure ovotoxic effects are seen within 1- 2 days.
 But repeated low dose exposure is a more likely source of toxicity in women.
 In another study, female mice were exposed repeatedly with much lower daily doses of
DMBA for 15 days,
 After comparing the doses required to cause 50% loss of primordial follicles in both
studies, it was determined that relative to single high dose exposure, repeated low dose
exposure to DMBA was ovotoxic to a 250 times greater extent.
Thus, these results demonstrate that animal studies designed to more closely mimic human
types of exposures may reveal surprising & disturbing insights as to relative risk.
Reproductive and endocrine toxicology
P.B. Hoyer, in Comprehensive Toxicology (Third Edition), 2018

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Site of action Function Mechanism of action of Toxicant- examples
Anterior pituitary • Synthesis & secretion of
FSH, LH & Prolactin.
• Have receptors for GnRH,
FSH, LH & Steroids.
1. Modification of pituitary harmone
secretion-- DES
2. Acting on pituitary membrane
receptors- Bromocriptine inhibition of
prolactin release.
Ovaries • Development of
follicles(ovum, granulosa
cells)
• Development of corpus
luteum
• Synthesis & secretion of
oestrogen & progesterone
1. Hormone analogs –
medroxyprogesterone acetate,
mifepristone, tamoxifen
2. Primordial follicle damaging agents – 4-
vinylcyclohexene diepoxide, busulfan,
cisplatin, cyclophosphamide.
3. Metabolite imbalance inducers –
anastrozole, di-(2-ethylhexyl)phthalate,
ethylene glycol monomethyl ether,
endomethacin
Toxicologic Pathology of the Reproductive System
Moges Woldemeskel, in Reproductive and Developmental Toxicology (Second Edition), 2017

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• Metabolic processes 4. Endocrine imbalance inducers – Atrazine,
Bromocriptine
Uterus • Have oestrogen &
progesterone receptors
• Prostaglandin, protein &
glycoprotein secretion
• Luminal fluid & sperm
transport
1. Agents as tamoxifen, toremifene &
butyrophenones cause uterine atrophy by
loss or supressionof ovarian sex harmone
2. Xenobiotics with oestrogenic activity
cause endometrial hyperplasia eg., oral
contraceptives
3. Endometrial metaplasia or endometriosis
interfere with sperm transport.
Vagina & cervix • Multiple cell layers &
cyclical changes
• Responds to hormones
• Sperm transport
1. Oestrogen compounds cause
hyperkeratosis & hyperplasia
2. Progestational compounds cause
increased mucous secretion by
endocervical epithelium.

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Examples of some ovarian toxicants and their mechanism of action
1. Medroxyprogesterone
acetate
Inhibits the gonadotropin release from pituitary, also
possesses antioestrogenic, antiandrogenic &
glucocorticoid like effects
2. Mifepristone progesterone receptor antagonist
3. Tamoxifen Selective oestrogen receptor modulator
4. 4 – venylcyclohexene
diepoxide
Occupational chemical, metabolite of 4-
venylcyclohexene. Directly toxicant to small follicles by
accelerating the natural process of apoptosis.
5. Busulfan Antineoplastic alkylating agent.
Depletion of primordial & primary follicles.

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6. Cisplatin Platinum – based antitumor agent. Depletion of primordial & small
follicles.
7. Cyclophosphamide Alkylating agent. Depletion of medium to large follicles.
8. Anastrazole Potent inhibitor of aromatase, a rate-limiting enzyme in transforming
androgen to oestrogen. Essential for maturation of follicles
9. Di(2-
ethylhexyl)phthalate
Plasticizer, inhibits biosynthesis of estrdiol through peroxisome
proliferator-activated receptor (PPAR)activation and inhibits follicular
maturation
10. Indomethacin Non steroidal anti-inflammatory drug. Directly & specifically acts on
preovulatory follicles

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11. PPARα/γ dual
agonist
Inhibits the activation of & expression of aromatase,
apoptosis of granulosa cells and decrease in corpora lutea
(CL)
12. Atrazine Herbicide, inhibits the estrogen binding to its receptors &
supresses the secretion of luteinizing hormone(LH).
13. Bromocritine Dopamine agonist, hypoprolactinemia and alters CL
formation.
14. Chlorpromazine
hydrochloride
Decreased secretion of LH and follicle stimulating
hormone.

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 Developmental toxicity means “ adverse effects induced during pregnancy or as a result of
parental exposure, manifested at any point in the life span of the organism.
 The definitive animal test for evaluating potential developmental toxicity is the prenatal
developmental toxicity test.
 Usually performed with female rats & rabbits.
 Routes of administration: test substance can be administered by a variety of routes
depending upon the routes of human exposure.
 Drug dosing : usually beginning at implantation & continuing through the end of
organogenesis or to full term.
Teratogenic toxicity
REPRODUCTIVE & DEVELOPMENTAL TOXICITY
http://alttox.org/

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 3 dose levels & a control group
 Highest dose – minimum maternal toxicity, lowest dose- as proposed clinical dose or
its multiples.
 Atleast 20 pregnant rats or mice / 12 rabbits for each dose
 Observational parameters :
FEMALES
General : signs of intoxication,
body weight
food intake
Reproductive :
uterus, ovaries
products of conception
FOETUSES
General : number
gender
length
weight
Pathology : gross
visceral
skeletal

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LOCAL TOXICITY STUDIES
These studies are required when a new drug is proposed to be used by some special
route(other than oral) in humans.
TYPES:
type of study animal Observational parameters
Dermal toxicity studies Rats and rabbits Local signs of erythema, oedema
Dermal photo toxicity studies Guinea pig Used in treatment of leukoderma, erythema &
oedema
Vaginal toxicity studies Rabbit or Dog Observation of swelling, histopathology of vaginal
wall
Rectal tolerance studies Rabbit or Dog Signs of pain, blood or mucous, histopathology of
rectal mucosa
Ocular toxicity studies Albino Rabbit Changes in cornea, iris & aqueous humor,
histological examination of eye.
Parenteral drugs For IV/IM/SC/ID injections, site of injection
examined grossly & microscopically.
Inhalational toxicity One rodent & one non
rodent
Acute, subacute & chronic studies performed
Observation of respiratory rate, histological
examination of respiratory passages , lung tissues.

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DRAIZE TEST :- it is type of acute toxicity test
Test subject : albino rabbit
Procedure : applying 0.5 ml or 0.5 gm of test substance to the eye or skin of a
restrained, conscious animal and leaving it for 4 hrs.
Observational parameters : redness,
swelling,
discharge,
ulceration,
haemorrhage,
cloudiness or blindness in the tested eye.

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CARCINOGENIC/ONCOGENIC TOXICITY STUDIES
 Carcinogenic substances are ones that induce tumors(benign or malignant), increase
their incidence or malignancy or shorten the time of tumor occurrence when they are
inhaled, injected, dermally applied or ingested.
 Carcinogens are classified according to their mode of action as
1. genotoxic carcinogens
2. non genotoxic carcinogens.
 Genotoxic carcinogens :- initiate carcinogenesis by direct interaction with DNA,
resulting in DNA damage or chromosomal aberrations that can be detected by
genotoxicity test.
 Non Genotoxic carcinogens :- are agents that do not directly interact with DNA and
are believed to enhance tumor development by affecting signal transduction, cell
proliferation. TOXICITY ENDPOINTS & TESTS
CARCINOGENICITY
Last updated: September 8, 2011
http://alttox.org/

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Animal tests:- The conventional test for carcinogenicity is long term rodent
carcinogenicity bioassay in OECD.
 The objective of this test is ‘to observe test animals for a major portion of their life
span for the development of neoplastic lesions during or after exposure to various
doses of a test substance by an appropriate route of administration.
 Study is usually conducted in two species rats and mice of both sexes.
 Drug administration – by oral, dermal or inhalation exposure ( based upon the
expected type of human exposure)
 Dosing typically lasts around 2 years.
 Animal health features are monitored throughout the study.
 But key assessment resides in the full pathological analysis of the animal tissues &
organs when the study is terminated.
http://alttox.org/
TOXICITY ENDPOINTS & TESTS
CARCINOGENICITY

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REGULATORY REQUIREMENTS & TEST GUIDELINES
1) The UN Globally Harmonized System(GHS) :-
classifies carcinogens under two categories based on the strength of the evidence.
category 1 :- chemicals are known or presumed human carcinogens.
1A – based on human data
2A – based on Animal data
category 2 :- chemicals suspected human carcinogens.
GHS describes important factors to be taken into consideration in carcinogen hazard
classification such as location & number of tumor, tumor type & characteristics, responses
in both sexes & or multiple species relevance of the mode of action to humans.
2) OECD Guidance Notes for Analysis & Evaluation of Chronic Toxicity &
Carcinogenicity Studies (2002 to 2019) ---- provides broad guidance on approaches to
hazard assessment & on some of the problems & pitfalls that may arise during an
assessment. TOXICITY ENDPOINTS & TESTS
CARCINOGENICITY
http://alttox.org/

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3) The US Environmental Protection Agency’s(EPA) guidelines for carcinogen risk
assessment (2005).
4) The International conference on Harmonisation of Technical Requirements for
Registration of pharmaceuticals for Human use(ICH) & Drug Regulatory authorities
provide guidance on testing for the carcinogenic potential of new drugs.
5) The International Agency for Research on Cancer (IARC) part of World Health
Organisation(WHO), provides monographs on the evaluation of carcinogenic risks to
humans (evaluated carcinogenic risk of >900 substances.
the IARC Monographs are recognised as an authoritative source of information &
used by national and international authorities in making risk assessments.
http://alttox.org/
CARCINOGENICITY

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GENOTOXICITY / MUTAGENICITY
Germ cell mutagens/genotoxins are substances that cause heritable (passed on to
progeny) changes in the genetic material in germ cells, namely spermatocytes or
oocytes.
Genotoxins are a broader category of substances that induce changes to the structure
or number of genes via chemical interaction with DNA and/or non-DNA targets
The term mutagen refers to a substance that induces transmissible changes in DNA
structure involving a single gene or a group of genes.
http://alttox.org/
GENOTOXICITY
Last updated: June 20, 2011

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The classification of germ cell mutagenicity is hazard based, taking into account a
chemical’s intrinsic ability to induce genotoxicity in germ cells, and is not meant for
quantitative risk assessment
Category 1 chemicals are “known to induce heritable mutations or to be regarded as
if they induce heritable mutations in the germ cells of humans,” and
Category 2 chemicals are those that “cause concern for humans owing to the
possibility that they may induce heritable mutations in the germ cells of humans”
GENOTOXICITY
http://alttox.org/

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THE ANIMAL TEST(S)
In vivo tests for assessing potential heritable genotoxicity include:
1. Heritable germ cell mutagenicity tests that include a component that measures
damage passed onto progeny are: the mouse heritable translocation test
(OECD Test Guideline (TG) 485), the mouse specific locus test, and the
rodent dominant lethal test (OECD TG 478)
2. Assays for measuring genotoxicity induction in germ cells that are used to predict
chemicals that might induce heritable damage include the mammalian
spermatogonial chromosome aberration test (OECD TG 483), the
spermatid micronucleus assay, the mammalian oocyte chromosome
aberration/aneuploidy test, and unscheduled DNA synthesis test in
testicular cells.
GENOTOXICITY
http://alttox.org/

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1. Assays for measuring genotoxicity induction in somatic cells that are used to
predict whether a chemical has the potential to induce germ cell genotoxicity
(these are also used in predicting potential carcinogenicity) include the
mammalian erythrocyte micronucleus test, the mammalian bone
marrow chromosome aberration test, the liver unscheduled DNA
synthesis (UDS), and the mouse spot test which measures genotoxicity in
fetal somatic cells
GENOTOXICITY
http://alttox.org/

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in vitro genotoxicity test methods :-
four are commonly used, have been adopted at the EU level with OECD guidelines
These four in vitro assays include two mutagenicity test methods based on bacterial
cells
• the bacterial reverse mutation test [Ames test]; and
• the Escehrichia coli reverse mutation assay,
as well as two methods based on mammalian cells
• the in vitro mammalian chromosome aberration test,; and
• the in vitro mammalian cell gene mutation test.
CARCINOGENICITY
http://alttox.org/
AMES TEST

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Commonly used laboratory
animals for research purpose

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RODENTS ANIMALS PURPOSE
Laboratory Mouse
(Mus musculus domesticus)
• Cancer research
• Diabetes mellitus
• Arthrosclerosis
• Epilepsy
• Autoimmune diseases
Laboratory rat
(Rattus norvegicus)
• Biochemistry
• Nutrition
• Toxicology
• Oncology
• Pharmacology
• Endocrinology
• Neurophysiology
Mastomys
(pramyscoucha)
• Routine tests of plague
suspect material
• Protozological and
helminthological research

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NONRODENTS Monkeys • Virology
• Toxicology
• Reproductive physiology
• Contraceptive biology
• Endocrinology
• Immunology
Dogs
(Canisfamiliaris)
• Chronic arterial disease
• Congestive heart failure
• Diabetes mellitus
• Ulcerative colitis
• Lymphocytic leukemia
• Glaucoma
Cats
(feliscatus)
• Neurophysiology
• Reflexes
• Synaptic transmission
• Perception of light & sound

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Gerbil
(Merioneshurrianae)
• Behavioural studies
• Cancer
• Epilepsy
• Neurological experiments
Hamster
(Mesocricetusauratus)
• Cancer
Guinea pig
(Caviaporcellus)
• Delayed hypersensitivity
• Biochemistry
• Toxicology
• Physiology
• Pharmacology
• Oncology
• Vit C metabolism

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Rabbit
(oryctolaguscuniculus)
• Immunology
• Hypertension
• Infectious diseases
• Virology
• Embryology
• Toxicology
• Teratology
• Atherosclerosis

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REFERENCES
• SK Gupta, New Drug Development, Drug Discovery and Clinical
Research,1st edition, pg : 18 to 21.
• Bikash Medhi, Ajay Prakash, Practical Manual of Experimental and
Clinical Pharmacology, 2nd edition, pg 136 to 140.
• REPRODUCTIVE & DEVELOPMENTAL TOXICITY
• Reproductive and endocrine toxicology
P.B. Hoyer, in Comprehensive Toxicology (Third Edition), 2018
• Toxicologic Pathology of the Reproductive System
• TOXICITY ENDPOINTS & TESTS
GENOTOXICITY
http://alttox.org/
http://alttox.org/
http://alttox.org

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• TOXICITY ENDPOINTS & TESTS
CARCINOGENICITY
http://alttox.org/

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