"Dive into the intricate world of reproductive toxicity studies with our comprehensive seminar on TG 421. Explore the latest research, methodologies, and implications in assessing the effects of substances on reproduction. Gain insights into regulatory requirements, experimental design, and emerging trends in this critical field. Whether you're a seasoned researcher or a curious academic, this presentation offers valuable knowledge and insights into understanding reproductive toxicity and its implications."

1. REPRODUCTIVE
TOXICITY STUDIES
TG 421
Presented By
Mr. Rahul Tipare
M pharm (Pharmacology)
Guided By
Mr. Mukul Tambe
Assistant professor Dept. of
Pharmacology 1

2. CONTENT
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• Introduction to reproductive toxicity
• Introduction to OECD Guideline 421
• Principle
• Description of method
• Procedure
• Experimental design
• Observations
• Pathology
• Data Reporting
• Evaluation of Result
• Test Report
• Test Result
• References

3. Introduction
• Reproductive toxicity studies aim to assess the potential adverse
effects of substances on the reproductive system.
• These studies evaluate the impact on fertility, pregnancy
outcomes, and offspring development.
• Reproductive toxicities can include disruptions to reproductive
organs, hormone levels, fertility, miscarriages, birth defects, and
developmental abnormalities in offspring.
• Common substances studied for reproductive toxicity include
pharmaceuticals, industrial chemicals, pesticides, and
environmental pollutants.
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4. • Reproduction/Developmental Toxicity
Screening Test
• TG : 421
Segment 1
• Prenatal Developmental Toxicity Study
• TG : 414
Segment 2
• Extended One-Generation Reproductive
Toxicity Study
• TG : 443
Segment 3
OECD : Organization for Economic
Cooperation and Development
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5. Segment I :
Reproduction/Developmental
Toxicity Screening Test
TG : 421 •Segment I of reproductive toxicity testing
typically refers to the OECD test guideline 421
•This guideline is designed to generate limited
information concerning the effects of a test
chemical on male and female reproductive
performance such as gonadal function, mating
behavior, conception development of the
conceptus and parturition.
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6. 1. Chemical administration to Males
• Dosed for 4 weeks, 2 weeks prior to mating & during mating
and other 2 weeks after mating.
• Fertility may not be particular sensitive indicator of
testicular toxicity, so detailed histological examination of
testes is required.
2. Chemical administration to Females
• Females should be dosed throughout the study.
• 2 weeks prior to mating (where 2 oestrous cycles are
covered).
• During mating period.
• Throughout gestation period.
• 13 days after delivery.
• Total study Duration: 77 days (14 pre-exposure +14
premating + 14 during mating +22 gestation period + 13
lactation period)
Principle
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7. Description of method
1. Selection of animal species
• Use rats for this test guideline, Justify if using a different species.
• Only healthy nulliparous animals should be used.
• Ensure minimal weight variation (within 20% of mean weight).
• Use same strain and source for preliminary and long-term studies.
• Strains with low fecundity or well known for reproductive defects should not be used.
2. Housing & Feeding
• Follow local animal care standards;
a. Maintain room temperature at 22°C
b. 50-60% humidity
c. 12-hour light/dark cycle;
• Group house animals unless justified; limit group cages to five animals .
• Provide suitable cages for mating and individual housing for pregnant and lactating females
with nesting materials.
• Regularly analyze feed for contaminants and retain a sample until the report is finalized.
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8. • 10 Males + 12-13 Females.
• Females will be evaluated pre-exposure for oestrous
cyclicity.
• Animals that fail to exhibit typical 4–5-day cycles will not
be included in study.
• Extra females recommended to yield 10 females out of at
least 8 should obtained pregnant.
• Observe potential of test chemical to affect fertility,
pregnancy, growth and development of F1 offsprings.
1.1. Number &
sex of animals
• 3 test group + control group.
• Dose levels based on acute toxicity tests.
• It is also taken in consideration that sensitivity between
pregnant and non-pregnant animals.
• Highest dose level is chosen with aim of inducing toxicity
but not death.
• Descending dose level procedure is followed.
2. Dosage
Procedure
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9. • If study finds no harmful effects at 100 mg/kg/day i.e.
suggest no toxicity then no need of further testing with
different dose levels.
• If human exposure suggests higher dose is necessary then
additional testing will be require.
1.3. Limit test
• Dosed daily for 7 days a week.
• Administration by oral gavage.
• Volume: 1ml/100 g body weight.
• If administering test chemical via diet or drinking water it
is important to ensure that quantities of test chemical
should not interfere with normal nutrition.
• Dose adjustment is required weekly according to body
weight.
4.
Administration
of doses
Procedure (contd.)
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10. • Dosing of both sexes should start at least 2 weeks prior to
mating.
• Dosing should be continued during mating period.
• Sacrifice the dams with offsprings on day 13th of post
partum.
• Males should continue dosing after mating until 28 days
completed & then sacrificed.
• Dosing in parental females should continue throughout
pregnancy.
1.5. Experimental
schedule
• Normally 1:1 male female should be used, placed together
till the mating confirmation or for 2 weeks.
• Every morning females should be checked for signs of
mating like presence of sperms or vaginal plug.
• When mating is confirmed it is considered as day 0 of
pregnancy.
6. Mating
procedure
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Procedure (contd.)

11. • On day 4th after birth litter size is adjusted by eliminating
surplus pups.
• 4-5 pups per sex per litter.
• Blood samples are taken from surplus pups and used for
determination of serum T4 levels.
1.7. Litter size
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11
Procedure (contd.)

12. Males
Females
Males
Females
Males
+
Females
Males/Sires Optional exposure
Pregnant Females
Pups
Dams
Non-Pregnant Females
Mating
Max.
14 days
Pre-
exposure
14 days
Pre-
mating
14 days
Gestation
Approx
22 days
Lactation
Approx
13 days
Experimental Schedule : Total 77 days
Without dose
With dose
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13. Observations
1. Clinical observations
• General observations at least once a day & more frequently if signs of toxicity.
2. Body weight & food – water consumption
• Body weight measurement on day of dosing and weekly later.
• During pregnancy, females should weigh on days 0,7,14 & 20th day.
• 24 hours before parturition.
• Day 4 &13 of post partum.
3. Oestrous Cycle
• Monitor before study starts for animal selection.
• Vaginal smears also checked daily until evidence of mating.
• Females having irregular Oestrous cycles are directly eliminated from the study.
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Stages of Oestrous Cycle in rats

15. Observations (contd.)
4. Offspring parameters
• Duration of gestation should be recorded and calculated from day 0 of pregnancy.
• Examine each litter ASAP to establish number and sex of pups along with stillbirth, livebirth,
runts
5. Clinical biochemistry
• Blood samples taken from surplus pups.
• All dams and at least 2 pups per litter are at termination on day 13 of post partum.
• Blood from males taken to check thyroid hormone levels.
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16. • Gross necropsy
• At time of sacrifice, adult animal should be
examine macroscopically for any abnormalities
and pathological changes.
• No. of implantation sites should be recorded .
• Vaginal smears should be examine to determine
stages of oestrous cycle.
• In males: Testes, epididymis, prostate, seminal
vesicles, coagulating glands.
• In females: Paired ovaries & uterus.
• Change in weight will suggest toxicity occurs at
specific site.
• Histopathology
• Stages of spermatogenesis are observed.
• Oestrous cycle stages are observed.
Pathology
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17. • No. of animals at start of test.
• No. of animals found dead during test or
sacrificed for human reason.
• No. of fertile animals.
• No. of pregnant females.
• No. of animals showing sign of toxicity.
• Time of onset, duration, severity of any toxic
effect.
• Types of histopathological changes.
• Statistical analysis of AGD & Nipple Retention.
Data & Reporting
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18. 1. Evaluation include relationship
between dose of test chemical and
presence or absence,
incidence and severity of abnormalities
like
• Weight change
• Reproductive and litter performance
• Mortality
Evaluation of Result
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19. Test Report
1. Test chemical:
• Source, lot number, limit date for use, if available
• Stability of the test chemical, if known.
• Mono-constituent substance
• physical appearance, water solubility, and additional relevant physicochemical properties
• Chemical identification, such as IUPAC or CAS name, CAS number, SMILES or inchl code,
structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc.
• Multi-constituent substance, uvbcs and mixtures , characterised as far as possible by chemical
identity. Quantitative occurrence and relevant physicochemical properties of the constituents.
• Vehicle (justification for choice of vehicle if other than water)
2. Test animals:
• Species/strain used
• Number, age and sex of animals
• Source, housing conditions, diet, etc.
• Individual weights of animals at the start of the test.
• Justification for species if not rat.
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20. 3. Test conditions:
• Rationale for dose level selection
• Details of test chemical formulation/diet preparation, achieved concentrations, stability and
homogeneity of the preparation
• Details of the administration of the test chemical
• Conversion from diet/drinking water test chemical concentration (ppm) to the actual
dose(mg/kg body weight/day), if applicable
• Details of food and water quality
• detailed description of the randomization procedure to select pups for culling. If culled
Test Report contd.
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21. • Body weight/body weight changes
• Food consumption, and water consumption if available
• Toxic response data by sex and dose, including fertility, gestation, and any other signs of
toxicity.
• Gestation length
• Toxic or other effects on reproduction, offspring, post-natal growth, etc.
• Nature, severity and duration of clinical observations (whether reversible or not)
• Number of adult females with normal or abnormal oestrous cycle and cycle duration
• Number of live births and post-implantation loss
• pup body weight data.
• AGD of all pups (and body weight on day of AGD measurement)
• Nipple retention in male pups.
Result
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22. • Thyroid hormone levels, day 13 pups and adult males (and dams and day 4 pups if
measured) .
• Number pups with grossly visible abnormalities, gross evaluation of external
genitalia, number of runts.
• Time of death during the study or whether animals survived to termination.
• Number of implantations, litter size and litter weights at the time of recording.
• Body weight at sacrifice and organ weight data for the parental animals.
• Necropsy findings.
• Detailed description of histopathological findings.
• Absorption data (if available).
• Statistical treatment of results, where appropriate.
Result
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Reference
1.Hubscher, Charles & Brooks, D & Johnson, J. (2005). A quantitative method for assessing
stages of rat estrous cycle. Biotechnic & histochemistry : official publication of the
Biological Stain Commission. 80. 79-87.
http://www.doi.10.1080/10520290500138422
2. OECD (2016), Test No. 421: Reproduction/Developmental Toxicity Screening Test, OECD
Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
https://doi.org/10.1787/9789264264380-en.
3. http://ratfanclub.org/autopsy.html

24. THANK
YOU
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