This document discusses various screening methods for detecting teratogenicity or the capacity of drugs to cause fetal abnormalities. It describes animal models like rat and rabbit studies that are used to test drugs during key stages of pregnancy. In vitro techniques like whole embryo culture, micromass assays, and stem cell tests are summarized as alternatives that can reduce animal use. The mechanisms by which certain proven human teratogens cause defects are outlined. Regulatory guidelines for preclinical teratogenicity studies and testing categories for drug use in pregnancy are also summarized.

1. Screening methods for
Teratogenicity
Capt. Htet Wai Moe
6th July 2017
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2. 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
• High demand for a rapid, reliable and cost-
effective method for detection of teratogenic
toxicity 2

3. 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)
Teratogenicity
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4. Teratogenic mechanisms
Folate antagonism
• Inhibition of the folate methylation cycle
• E.g. many antiepileptic drugs, metformin,
methotrexate, sulfasalazine, trimethoprim
Neural crest cell disruption
• Interference in molecular pathways involved
in migration, differentiation, and proliferation
of neural crest cells
• E.g. Bosentan, isotretinoin, ketoconazole
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5. Teratogenic mechanisms
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, phthalates in coatings
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6. Teratogenic mechanisms
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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7. Teratogenic mechanisms
Vascular disruption
• Disturbances in blood circulation in the
uterine–placental unit, placental–fetal unit or
the fetus itself, interfering with prenatal
development of vasculature
• E.g. Antihypertensive medication, aspirin,
ephedrine, NSAIDs
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8. Teratogenic mechanisms
Specific receptor- or enzyme-mediated
teratogenesis
ACE and AT II receptors
• Disruption of fetal renin–angiotensin system,
producing fetal hypotension, vascular
disruption and decrease in fetal renal vascular
tone
• E.g. ACE inhibitors, AT II receptor inhibitors
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9. Teratogenic mechanisms
HMG-CoA reductase
• Down-regulation of Hedgehog proteins, which
regulate embryonic growth, patterning and
morphogenesis of many structures
• E.g. Statins
Histone deacetylases (HDACs)
• Interruption of cell proliferation, differentiation
and apoptosis through disregulation of gene
expression by chromatin remodelling
• E.g. Boric acid as inactive ingredient, salicylates,
valproic acid
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10. Teratogenic mechanisms
Cyclooxygenase-1
• Inhibition of COX-1, which catalyzes the
conversion of arachidonic acid to prostaglandins
• E.g. NSAIDs
NMDA receptors
• Errors in migration of neuronal and glial elements
in the developing brain
• E.g. Amantadine, dextromethorphan, ketamine
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11. Teratogenic mechanisms
5-HT receptors and transporters
• Increased stimulation or suppression of 5-HT
receptors; inhibition of 5-HT uptake by 5-HT
transporters
• E.g. Risperidone, SSRIs, sumatriptan
GABA receptors
• Enhancements of the effects of GABA, the major
inhibitory neurotransmitter
• E.g. Barbiturates, benzodiazepines
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12. Teratogenic mechanisms
Carbonic anhydrase
• Reduction in embryonic intracellular pH
• E.g. Acetazolamide, topiramate
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13. Proven Human Teratogens and Abnormality
Drug Abnormality
Thalidomide Phocomelia, multiple defects
Anti-neoplastic drugs Multiple defects, foetal death
Androgens Virilization, esophageal, 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, hypoplastic
phalanges

14. 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
Proven Human Teratogens and Abnormality

15. 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

16. 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
FDA category of drugs used in
pregnancy

17. 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
FDA category of drugs used in
pregnancy

18. Schedule Y
• Requirements and guidelines for permission
to import and/or manufacture of new drugs
for sale or to undertake clinical trials
• Female reproduction and Developmental
Toxicity Studies
– Appendix I, Item 4.4
– Segment I – Female Fertility Study
– Segment II – Teratogenicity Study
– Segment III – Perinatal Study
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19. Animal toxicity requirements for clinical trails
and marketing of a new drug
Toxicity study Human phase for which study is
proposed to be conducted
Segment II
(Teratogenicity Study)
Phase II, III involving female
patients of child-bearing age
Segment III
(Perinatal Study)
Phase III
• Drugs to be given to pregnant
or nursing mothers for long
periods
• Drugs with indications of
possible adverse effects on
foetal development

20. One rodent (preferably rat)
One non-rodent (rabbit)
Drug administered throughout the period of
organogenesis
Three graded doses
• Highest dose – minimum maternal toxicity
• Lowest dose – proportional to the proposed dose
for clinical use in humans
• Intermediate dose – logarithmically between two
other doses
Teratogenicity Study (Segment II)

21. Route of administration same as intended for human
therapeutic use
Control and treated groups
At least 20 pregnant rats (or mice) and 12 rabbits, on
each dose level
Teratogenicity Study (Segment II)
All foetuses subjected to gross examination
Skeletal and visceral abnormalities

22. Foetuses
• Total number
• Gender
• Body length
• Weight
• Gross/ visceral/ skeletal
abnormalities
Teratogenicity Study (Segment II)
Observation parameters
Dams
• Signs of intoxication
• Effect on body weight
• Effect on food intake
• Examination of uterus,
ovaries & uterine
contents
• Number of corpora
lutea
• Implantation sites
• Resorption

23. One rodent species (preferably rat)
Dosing at levels comparable to multiples of human
dose by intended clinical route
At least 4 groups (including control) each consisting
15 dams
Perinatal study (Segment III)

24. Dams sacrificed and examined
Then, dose that causes low foetal loss should be
continued throughout lactation and weaning
Perinatal study (Segment III)
Drug administered throughout the last trimester of
pregnancy

25. Treated with vehicle or test substance throughout
their periods of growth to
• Sexual maturity
• Pairing
• Gestation
• Parturition
• Lactation
Perinatal study (Segment III)
One male and one female from each litter of
F1 generation (total 15 males and 15 females in each
group) selected at weaning

26. Mating performance and fertility of FI generation
should be evaluated
Perinatal study (Segment III)
Obtain F2 generation whose growth parameters should
be monitored till weaning

27. Pups
• Clinical signs
• Sex-wise distribution
in dose groups,
• Body weight
• Growth parameters
• Gross examination
• Survival
• Autopsy
Perinatal Study (Segment III)
Observation parameters
Dams
• Body weight
• Food intake
• General signs of
intoxication
• Progress of gestation/
parturition periods
• Gross pathology

28. In vitro techniques
• Simple and cost-efficient
• Can reduce pressure imposed by society to
decrease or replace the number of animals
used in research and testing strategies
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29. Features of an ideal in vitro
teratogenicity test system
• 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 some 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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30. In vitro techniques
• Whole embryo culture test
• Micromass teratogen test
• Embryonic stem cell test
• Dictyostelium discoideum based assay
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31. 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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32. Whole embryo culture test
Zebrafish embryo
• Cover the whole period of organogenesis
• Easy to maintain in large stocks due to their
high fecundity
• Develop rapidly ex utero, with most organs
becoming functional between 3 and 5 days-
post-fertilization (dpf)
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33. Whole embryo culture test
Zebrafish embryo
• Transparency of larval zebrafish
• Reasonably tolerant to concentration of
dimethylsulphoxide (DMSO) generally used as
a solvent at drug screening
• High concordance with mammalian data
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34. Fig. Anomalies induced by retinoic acid treatment. The
notochord and tail were normally formed in control group
(A). The treated group (A’) exhibited kinked notochord
(arrow) and kinked tail (arrowhead)
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35. Fig. Anomalies induced by retinoic acid treatment. The eyes
and otic vesicles were normally formed in control group (B).
The treated group (B’) exhibited deficiencies of eyes (arrow)
and deficiencies of otic vesicles (arrowhead)
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36. Fig. Anomalies induced by retinoic acid treatment. The
treated group (C’) exhibited facial and abdominal edema
(arrowhead)
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37. Micromass Teratogen Test
• Chick, mouse and rat embryo midbrain or limb
cell culture
• Exposed to test compounds for varying times
and concentrations
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38. In vitro culturing of embryo limb or rather central
neural cells
Differentiation of the cells into chondrocytes or
neurons starting from numerous small aggregates or
foci of cells
Micromass Teratogen Test
Observation of cell adhesion, movement,
communication, division and differentiation
involving the new synthesis of tissue specific
patterns of enzymes and structural proteins

39. Micromass Teratogen Test
• A phase of in vivo embryo exposure will act
as a control for the effects of drug metabolism
and pharmacokinetics
• Using cells from different organs and species
to reproduce the in vivo sensitivity of
particular embryonic tissues or species to
teratogenic agents
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40. • Pluripotent embryonic stem cells (ESC) isolated
from mouse blastocysts
• ESC are able to differentiate into a variety of
embryonal tissues, retain the euploid
chromosome constitution, and proliferate very
rapidly
Embryonic stem cell test
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41. Embryonic Stem Cell Test
• Based on the resemblance between early
embryonic stages and the differentiation of
embryonic stem cells in vitro
• Cell lines are used to identify cytotoxic,
mutagenic, embryo toxic and teratogenic
effects of chemical compounds
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42. Embryonic Stem Cell Test
• Evaluation of cell differentiation is
performed both morphological and via
molecular techniques
• Identification of predictive marker genes for
the major target tissues during organogenesis
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43. Dictyostelium discoideum
• Single-cell, eukaryotic microorganism competent
to undergo both vegetative growth and
development (fruiting body formation)
• Cell differentiation leading to the formation of
stalk and spore cells has been shown to be
similar, to some extent, to the development
of mammals
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44. Dictyostelium discoideum
• Reporter genes serve as highly specific indicators
for the developmental fate of a certain cell at a
given time point
• Easier to handle and less expensive
• Rapid large-scale screening of chemicals
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45. Conclusions
• 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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46. • Anital Kotwani et al., 1995. Methods for teratogenicity
testing – Existing and future models. IJP; 27: 204-213
• Van Gelder MMHJ et al., 2010. Teratogenic mechanisms
of medical drugs. Hum Reprod; 29: 168-183
• Julia Schumann. 2010. Teratogen Screening: State of the
Art. Avicenna J Med Biotech; 2(3): 115-121
• Wayne R Cohen, Phyllis August. 2013. Obstetric medicine,
Management of Medical Disorders in Pregnancy. 6th Ed.
Shelton: PMPH-USA Ltd; p.51-86
• Akhito Yamashita et al., 2014. Improvement of the
evaluation method for teratogenicity using zebrafish
embryos. J. Toxicol. Sci. 39(3), 453-464
References
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47. Thank you!
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