1. The document summarizes studies on teratogenicity (the ability of substances to cause birth defects) including principles, mechanisms, testing protocols, and experimental design. 2. Teratogenicity testing aims to identify substances that should be avoided during pregnancy due to their potential to cause fetal abnormalities. It became important after the thalidomide tragedy of 1961. 3. Drugs can affect the fetus at different developmental stages, with the period of organogenesis from 18-55 days being the most vulnerable. Testing follows FDA and ICH guidelines and involves dosing animals during pre-conception, pregnancy and lactation to assess effects on fertility and offspring development.

1. TERATOGENICITY
STUDIES(SEGMENT II)
Presented by:
Md Asif Ansari
M. Pharma, Pharmacology
2nd Semester
Department of Pharmacology ,SPER
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2. Presentation outline
• Introduction
• Principles of teratogenicity
• Teratogenicity mechanism
• Teratogen drugs
• FDA category of drugs used in pregnancy
• Testing and Experimentation
• Conclusion
• References
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3. Teratogenicity
• Capacity of a drug to cause foetal abnormalities when administered
to the pregnant mother
• Teratogenicity testing came into being since the thalidomide tragedy
of 1961.
• Less than 2% of congenital malformations are caused by drugs or
chemicals.
• Teratogenic drugs should be avoided either during or prior to
conception.
• Women to avoid all medications in the first 8 weeks after conception.
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4. Drugs can affect the foetus at 3 stages:-
1. Fertilization and implantation- conception to 17 days (unnoticed
failure of pregnancy)
2. Organogenesis- 18-55 days of gestation (most vulnerable period)
3. Growth and development- 56 days onward(developmental and
functional abnormalities can occur)
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5. Principles of Teratogenicity
1. Teratogenic susceptibility is determined by the genotype of the
conceptus.
2. Susceptibility to teratogenic agents depends on the Developmental
stage of the embryo or fetus at the time of exposure.
3. These agents work by specific mechanisms on developing cells and
tissues to initiate pathogenesis.
4. Perturbations of developmental processes can result in death,
malformation, growth retardation.
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6. Teratogenicity mechanisms
• Folate antagonism
• Neural crest cell disruption
• Endocrine disruption
• Oxidative stress
• Vascular disruption
• 5-HT receptors and transporters
• Enzyme mediated teratogenesis
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7. Teratogenicity mechanisms
1. Folate antagonism
• Inhibition of the folate methylation cycle
• E.g. many antiepileptic drugs, metformin, methotrexate, sulfasalazine,
trimethoprim
2. Neural crest cell disruption
• Interference in molecular pathways involved in migration,
differentiation, and proliferation of neural crest cells
• E.g. Bosentan, ketoconazole
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8. 3. Endocrine disruption (sex hormones)
• Affecting the release, binding or metabolism of endogenous
sex hormones, particularly in male development
• E.g. Drugs used in fertility treatment, oral contraceptives.
4. Oxidative stress
• Imbalance between generation of reactive oxygen species
and antioxidant defence mechanisms, causing irreversible
oxidation of DNA, proteins and lipids and affecting gene
expression.
• E.g. Class III antiarrhythmic drugs, iron supplements,
phenytoin, terbutaline, tetracyclines, thalidomide, valproic
acid
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9. Proven Human Teratogens
Drugs Abnormality
Thalidomide Phocomelia, multiple defects
Anti-neoplastic drugs Multiple defects, foetal death
Androgens Virilization, esophageal and cardiac defects
Progestins Virilization of female foetus
Stilboestrol Vaginal carcinoma
Tetracyclines Discoloured teeth, bone defects
Warfarin Nose, Eye, Hand defects, Growth retardation.
Phenytoin Cleft lip/palate, microcephaly, hypoplasticphalanges
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10. Drugs Abnormality
Alcohol Low IQ, Growth Retardation, Foetal alcohol syndrome
ACE Inhibitors Hypoplasia of organs, growth retardation, foetal loss
Lithium Foetal goiter, Cardiac abnormality
Anti – Thyroid drugs Foetal goiter, hypothyroidism
Indomethacin Premature closure of ductus arterious
Isotretinoin Craniofacial, Heart & CNS defects
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11. FDA category of drugs used in pregnancy
Category A:
• Adequate studies in human demonstrate no risk
Category B:
• Animal studies indicate no risk, but there are no adequate
studies in human
• Animal studies show adverse effects, but adequate studies in
human have not demonstrated a risk
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12. Category C:
• A potential risk, when:-
Animal studies have been performed, or
Animal studies indicated adverse effects and
There are no data from human studies
• These drugs may be used when potential benefits outweigh the
potential risks
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13. Category D:
• There is evidence of human fetal risk, but the potential benefits to the
mother may be acceptable
Category X:
• Studies in animals or humans show adverse reaction reports or both
have demonstrated fetal abnormalities
• The risk of use in a pregnant woman clearly outweighs any possible
benefit
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14. Testing Protocols:
1) Under the guidelines of FDA
2) Under the guidelines of ICH (International Conference on
Harmonisation)
3) Under the guidelines of Schedule-Y1)
Test under FDA:
• Multigenerational studies
• Single generational studies.
a) Segment I:Evaluation of Fertility and Reproductive Performance.
b) Segment II: Assessment of Developmental Toxicity.
c) Segment III: Postnatal Evaluation.
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15. Single Generation Studies
• Test Guideline(TG no. 415) for reproduction testing is designed to
provide general information.
• Concerning the effects of a test substance on male and female
reproductive performance, such as gonadal function, oestrous cycle,
mating behaviour, conception, parturition, lactation and weaning.
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16. Principle
• The test substance is administered in graduated doses to several
groups of males and females.
• Males should be dosed during growth.
• Females should be dosed for at least two complete oestrous cycles.
• The animals are then mated.
• The test substance is administered to both sexes during the mating
period.
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17. Description
Preparation
• Healthy young adult animals are randomised and assigned to the
treatment groups.
• The animals are kept in cages for at least fivedays to allow for
acclimatisation.
• Dosing should be on a seven-day per week basis.
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18. Experimental animals
Selection of species
• This Test Guideline is designed for use with the rat or mouse.
• The test animals should be characterized as to species, strain, sex,
weight and/or age.
• Healthy animals, not subjected to previous experimental procedures,
should be used.
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19. Number and Sex
• Each test and control group should contain a sufficient number of
animals to yield about 20 pregnant females.
• The objective is to produce enough pregnancies and offspring.
Test conditions
a) Housing and feeding conditions
b) Dose levels
• At least three treatment and a control group should be used.
1) Lowest dose level
2) Intermediate dose level
3) Highest dose level
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20. Limit test
• If a dose of at least 1000 mg/kg produces no evidence of interference
with reproductive performance, studies at other dose levels may not
be considered necessary.
• If a preliminary study at the high dose level, with definite evidence of
maternal toxicity, shows no adverse effects on fertility, studies at
other dose levels may not be considered necessary.
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21. Experimental schedules
• Daily dosing of the parental (P) males should begin when they are
about five to nine weeks old.
• In rats dosing is continued for ten weeks prior to the mating period.
• Males should be killed and examined either at the end of the mating
period or some time before the end of the experiment.
• For parental (P) females, dosing should begin after at least five days of
acclimatization and continue for at least two weeks prior to mating.
• Daily dosing of the P females should continue throughout the 3-week
mating period, pregnancy and up to the weaning of the F 1 offspring.
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22. Mating procedure
• Based on 1: 1 mating, one female placed with the same male until
pregnancy occurs.
• Each morning the females should be examined for presence of sperm
or vaginal plugs.
• Day 0 of pregnancy is defined as the day a vaginal plug or sperm are
found.
• Those pairs that fail to mate should be evaluatedto determine the
cause of the apparent infertility
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23. Observations
• Each animal should be observed at least once daily.
• Pertinent behavioral changes, signs of difficult or prolonged
parturition and all signs of toxicity, including mortality, should be
recorded.
• During pre-mating and mating periods, food consumption should be
measured weekly.
• Each litter should be examined to establish the number and sex of
pups, stillbirths, live births and the presence of gross anomalies.
• Dead pups and pups killed should be preserved and studied for
possible defects.
• Physical or behavioural abnormalities observed in the dams or
offspring should be recorded.
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24. Pathology
a) Gross necropsy
• At the time of sacrifice during the study the animals of the P generation
should be examined macroscopically for any structural abnormalities with
special attention paid to the organs of the reproductive system.
• Dead or moribund pups should be examined for defects.
b) Histopathology
• The ovaries, uterus, cervix, vagina, testes, epididymides, seminal vesicles,
prostate, coagulating gland, pituitary gland and target organ or organs of all
parent animals should be preserved for microscopic examination.
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25. Data and Reporting
Treatment of results
• Data may be summarized in tabular form.
• Showing for each test group the number of animals at the start of the
test, the number of fertile males, the number of pregnant females,
the types of changes and the percentage of animals displaying each
type of change.
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26. Evaluation and Interpretation of results
• This study should be evaluated in terms of the observed effects,
necropsy and microscopic findings.
• Abnormalities including fertility, clinical abnormalities, body weight
changes, effects on mortality and any other toxic effects.
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27. Test report
• Species/strain used.
• Fertility, gestation, and viability indices.
• Time of death during the study or whether animals survived to
termination.
• Table presenting the weights of each litter, the mean pup weights and
the individual weights of the pups at termination.
• Toxic or other effects on reproduction, offspring.
• The day of observation of each abnormal signs.
• Body weight data for P animals.
• Necropsy findings. 27

28. Segment II : Teratological study:
• Species :Rats
• Number of animals/group : 20
• Route :Oral
• Duration of treatment : Day 6 through Day 15 of pregnancy
• Autopsy :Day 20 of gestation
• Dosage Control :Treated with Vehicle
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29. Procedure
• Rats are sacrificed on gestation day 20.
• Foetus are removed by cesarean section after noting the number of
resorptions, implantations, and normal foetus.
• The size, weight, and any abnormality of each fetus are noted along
with parent parameters observation.
• Two thirds of the fetuses are eviscerated.
• Then preserved in absolute alcohol for staining with Alizarin Red S for
skeletal assessment.
• The other one-third of the fetuses is fixed in Allen's modification of
Bouin's fluid for slicing with a razor blade (Wilson's Technique) to
evaluate visceral anomalies.
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30. Observation parameters
Parent
• Signs of intoxication
• Effect on body weight
• Effect on food intake
• Examination of uterus,
ovaries & uterine
contents
• Number of corpora lutea
Implantation sites
• Resorption
Foetuses
• Total number
• Gender
• Body length
• Weight
• Gross/ visceral/
skeletal
abnormalities
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31. In vitro study
Features
• Simple, easy to perform, yield of interpretable results
• Rapid, usage of large numbers of samples
• Giving few false negative
• Having relevance to mechanisms of teratogenesis
• Involving aspects of progressive development
• Usable with various types of agents
• Usage of intact organisms capable to absorb, circulate and excrete
chemicals
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32. In vitro techniques
1) Whole embryo culture test
a) Rodent embryo culture
b) Zebrafish embryo
2) Micromass teratogen test
3) Embryonic stem cell test
4) Dicto-stelium discoideum
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33. Whole embryo culture test
Rodent embryo culture (rat or mouse)
• Culturing of whole embryos at early stage of organogenesis
• Exposing of these to a potential teratogen
• Endpoints generally used
-Mortality
- Malformation
- Growth inhibition
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34. Whole embryo culture test
Zebrafish embryo
• Cover the whole period of organogenesis
• Easy to maintain in large stocks due to their high fertility
• Develop rapidly ex utero, with most organs becoming functional
between 3 and 5 days- post-fertilization.
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35. Conclusion
• Both in vivo and in vitro tests have the possibility of
generating false negative or positive data
• Several types of test systems are required to securely
identify different classes of teratogenic agents
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36. References
• ESSENTIALS OF MEDICAL PHARMACOLOGY BY KD TRIPATHI.,8TH
EDITION , JAYPEE BROTHERS MEDICAL PUBLISHERS PVT. LTD.,2019
• https://www.slideshare.net/RajeshYadav405/teratogenicity-studies
• https://www.slideshare.net/hwrain/screening-methods-for-
teratogenicity
• Principles of toxicology 3rd Edition by Karen E. Stine , Thomas M.
Brown (Pg. 138 – 147)
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558154/
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