Assignment on Reproductive toxicology studies, Male reproductive toxicity studies, female reproductive studies (segment I and segment III), teratogenecity studies (segment II)

1. Reproductive toxicity
Any adverse effect on any aspect of male or female sexual
structure or function, or on the conceptus or on lactation, which
would interfere with the production of development of normal
offspring which could be reared to sexual maturity, capable in
turn of reproducing the species.

2. Reproductive toxicity
 Two major classes:
– Reproductive toxicity -Effects on sexual behavior and
fertility in males and non-pregnant females
– Developmental toxicity-Abnormal structure or functional
development following exposure of pregnant or lactating
females

3. Reproductive cycle
 Within this cycle, we divide it up into an integrated sequence…….for
convenience
 Pre-Mating to conception
 Conception to implantation
 Implantation and organ formation
 Organ formation to end of pregnancy
 Birth to Weaning
 Weaning to sexual maturity

4. Aim of Reproduction Toxicology
Studies
 ICHS5 Guideline:
“The studies conducted should allow exposure of mature
adults and all stages of development to sexual maturity”
OR
The aim of reproduction toxicity studies is to reveal any effect of one or
more active substance(s) on mammalian reproduction

5. ICH Guidelines
The main guideline for reproduction studies was adopted in1993:
 ICHS5(R2)- DETECTION OF TOXICITY TO REPRODUCTION FOR MEDICINAL
PRODUCTS & TOXICITY TO MALE FERTILITY
 Current Step 2 draft version dated 5 July 2017
DETECTION OF TOXICITY TO REPRODUCTION FOR HUMAN PHARMACEUTICALS
S5(R3)

6. Testing Strategy
The most probable option of study designs :
 Studies for effects on fertility and early embryonic development
 Studies for effects on pre and postnatal development
 Studies for effects on embryo-fetal development

7. Study of Fertility and Early Embryonic
Development (Segment I)
 This comprises evaluation of stages A and B of the reproductive process.
 For females this should detect effects on the estrous cycle, tubal transport,
implantation, and development of preimplantation stages of the embryo.
 For males it will permit detection of functional effects (e.g. on libido,
epididymal sperm maturation) that may not be detected by histological
examinations of the male reproductive organs

8. FEED Study Design: Rats, combined male
and female study
Parameter - Male and Female
 Typical Group size - 20 + 20
 Number of dose groups - 4
 Administration period - M: ≥ 2 weeks prior to cohabitation through at least confirmation of mating
F: ≥ 2 weeks prior to cohabitation through implantation (GD6)
 Mating ratio - 1 male:1 female
 Mating period - ≥ 2 weeks
 Estrous cycle evaluation - Daily, commencing 2 weeks before cohabitation and until confirmation of
mating
 Clinical observations/mortality -At least once daily
 Body weight -At least twice weekly
 Food consumption - At least once weekly (except during mating)
 Male euthanasia- Perform macroscopic examination and preserve macroscopic findings, testes and
epididymides for possible microscopic examination

9. Continued..
 Sperm analysis - Optional
 Mated female euthanasia - Perform macroscopic examination and cesarean section;
preserve macroscopic findings, ovaries and uteri for possible microscopic examination
 Scheduled cesarean section: uterine implantation data
Corpora lutea counts, number of implantation sites, live and dead embryos

10. Study for effects on pre- and postnatal
development(Segment III)
To detect adverse effects on the pregnant/lactating
female and on development of the conceptus and the
offspring following exposure of the female from
implantation through weaning.

11. Pre- and Postnatal Developmental (PPND)
toxicity study
 Parameter
 Typical Group size- Approximately 20 females
 Number of dose groups - 4
 Administration period - From implantation (GD 6/7) through weaning (PND 20/21)
 F0 Females
 Clinical observations/mortality - At least once daily
 Body weight - At least twice weekly
 Food consumption - At least once weekly at least until delivery
 Parturition observations - GD 21 until complete
 Necropsy - PND 21,At necropsy, preserve and retain tissues with macroscopic findings and
corresponding control tissues for possible histological evaluation

12. Continued…
F1 Pre-weaning
 Clinical observations/mortality - Daily from PND 0
 Litter size, live and dead - Daily from PND 0
 Body weights and sex - PND 1, 4, 7, 14, and 21
 Optional Standardization of litter size - ≥ PND 4, to 4 or 5 pups per sex
 Physical development and reflex ontogeny - Depending on landmark
F1 Post-weaning
 Selection for post-weaning evaluation and group size - PND 21, at least 1 male and 1 female/litter
where possible to achieve 20 animals per group/sex
 Clinical observations/mortality - Daily
 Body weight - Weekly
 Optional Food consumption - Weekly
 Maturation (puberty) Females: vaginal opening, from PND 30 until complete
Males: preputial separation, from Day 40 until complete

13. Continued….
 Other functional tests - According to standard procedures
 Reproductive performance - At least 10 weeks old, paired for mating (1M:1F) within
the same group (not siblings)
 Terminal procedures of males and females -Preserve organs with macroscopic
findings for possible histological evaluation; keep corresponding organs of sufficient
controls for comparison Cesarean section: uterine implantation data, corpora lutea
counts, number of implantation sites, live and dead embryos

14. Study for effects on embryo-fetal
development(Segment II or Teratology
study)
 To detect adverse effects on the pregnant female and development of
the embryo and fetus consequent to exposure of the female from
implantation to closure of the hard palate
 Females should be killed and examined about one day prior to parturition.
All fetuses should be examined for viability and abnormalities.
 50% of fetuses from each litter be allocated for skeletal examination. A
minimum of 50% rat fetuses should be examined for visceral alterations,
regardless of the technique used
 Usually two species: rabbits (12 per group) and rats (20per group)
 Mated animals are treated during the period of organogenesis (days 6-18
in rabbits, 6-15 in rats)
 Pups delivered by Caesarean one day before expected parturition (21
days rat, 31 days rabbit

15. Study for effects on embryo-fetal
development
 Parameter - Rat
 Minimum number of litters - 16
 Number of dose groups - 4
 Administration period GD6-17
 Antemortem endpoints
 Clinical observations/mortality - At least once daily
 Body weight- At least twice weekly
 Food consumption - At least once weekly
 Toxicokinetics - Yes
 Postmortem endpoints
 Cesarean section - GD20/21 Uterine weight - Optional
 Corpora lutea Optional ,Fetal external evaluations - Yes
 Fetal soft tissue evaluations - Yes
 Fetal skeletal evaluations - Yes

16. Why 2 Species?
 Genotype influences response to exogenous agents
 Two species better than one at detecting hazard
 No species is intrinsically best at predicting for man
 Aim to have at least one pharmacologically relevant species

17. Species – factors for consideration
 Maternal weight
 Litter size
 Maternal basic metabolic rate
 Size and constitution of placenta
 Hormones/vitamins etc

18. Species selection
 Similar physiology and toxicokinetic profile to humans
 Rat is predominant rodent species
 Rabbit is predominant non-rodent species
 An alternative animal species should also be considered

19. Species - continued
 RAT
 For:
 good size
 Highly fertile
 Genetic stability
 (background data)
 RABBIT
 For:
 ‘non-rodent’
 Optimal foetal size
 Malformation rate approx.,
equal to human

20. Species - continued
 Against:
 Low spontaneous malformation
 rate
 Low sensitivity to teratogens
 Sensitive to sex hormones
 Susceptible to NSAIDs in late
pregnancy
 Against:
 Absence of ‘pure’ strains
 Lack of kinetic/toxicity data
 Susceptible to antibiotics
 Gastric disturbances

21. Dose And Route of Administration
 Generally ,at least 3 doses should be studied in main studies.
 Lowest dose-dose which is active in test animal at which no toxic effects
are expected
 Higest dose-signs of toxicity are expected
 Intermediate dose-geometric mean between high and low dose
 Route of administration- similar to humans
 Upper limit dose-1000mg/kg/day

22. Evaluation of Data
 The key to good reporting is the tabulation of individual values in a clear
concise manner to account for all animals that are being assessed
 Group summary values should be presented with significant figures that
avoid false precision and that reflect the distribution of the variable
 For the presentation of data on structural changes (e.g., fetal
abnormalities) the primary listing (tabulation) should clearly identify the
litters containing abnormal fetuses, identify the affected fetuses in the litter
and report all the changes observed in the affected fetus.
 Secondary listings by type of change can be derived from this.
 Where data from non-pregnant animals have been excluded from
summary tables, this should be clearly indicated.
 Any biologically meaningful difference in treated animals compared with
concurrent controls should be discussed.

23. OECD Guidelines
 Test No. 422: Combined Repeated Dose Toxicity Study with the
Reproduction/Developmental Toxicity Screening Test
 Test No. 421: Reproduction/Developmental Toxicity Screening Test
 Test No. 416: Two-Generation Reproduction Toxicity
 Test No. 415: One-Generation Reproduction Toxicity Study
 Test No. 443: Extended One-Generation Reproductive Toxicity Study
 Test No. 414: Prenatal Development Toxicity Study

24. References
 Vogel H G, et al ,Drug discovery and evaluation: safty and
pharmacokinetic assays vol. 2, editon 2nd,springer publication,page no.
1317-1328.
 S5a – Note for Guidance on Reproductive Toxicology: Detection of Toxicity
to Reproduction for Medicinal Products(CPMP adopted Sept 1993)
 S5b – Note for Guidance on Reproductive Toxicology: Toxicity on Male
Fertility (CPMP adopted Dec, 1995)