Reproductive toxicity studies are conducted according to OECD Guideline 422 to evaluate effects on fertility and development. The study involves administering graduated doses of a test chemical to male and female rats for at least 4 weeks prior to mating. Males continue dosing until sacrifice, while females are dosed throughout mating, pregnancy, and lactation until sacrifice on postnatal day 13. Offspring are observed for signs of toxicity. Clinical observations and pathology examinations are conducted to identify any reproductive or developmental effects.

1. REPRODUCTIVE
TOXICOLOGY STUDIES
OECD GUIDELINES :422
Prepared by: SONAL. VIJAY. PANDE
M Pharm sem 2
Department : pharmacology

2. WHAT IS REPRODUCTIVE TOXICOLOGY?
 Reproductive toxicity refers to structural and functional alterations that affect
reproductive system in sexually mature males and females.
 Reproductive toxicity includes effects on male fertility and female fertility and
lactation.

3. Two major class:
• 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

4. INTRODUCTION
ESTROUS CYCLE: Reproductive cycle of female, generally defined as
period from one estrus to the next.
ESTRUS: Period of sexual receptivity.
• Commonly referred to as “Heat”
ANESTRUS: Period when female does not experience cycles
• Occurs during pregnancy.

5. Polyestrus: Uniform distribution of estrous cycles which occur
regularly throughout the year eg: rodents, cattle
Monoestrus: – One cycle per year
Eg: dogs, wolf,bears

6. Estrous cycle
Follicular
phase
Luteal phase
Begins during
luteolysis
Ends at ovulation
Proestrus
Estrus
Begins during
ovulation
Ends at luteolysis
Metestrus
Diestrus

7. The oestrous cycle consists of four stages:
1.Prooestrus
2.oestrus
3.metestrus (or dioestrus 1)
4.dioestrus (or dioestrus 2).
• Because rats are continuously polyoestrous (i.e., cycle constantly throughout the year)
dioestrus is immediately followed by the prooestrus phase of the next cycle
• Anoestrus, a period of reproductive quiescence between oestrous cycles, is thus not
usually observed in healthy, cycling female rats.
• Oestrous cyclicity only ceases during pseudopregnancy, pregnancy, and lactation,
although a fertile postpartum oestrus does occur within 24 hours after birth.

8. Proestrus
• Begins when progesterone declines(luteolysis:regression of corpus luteum ) and ends
at onset of estrus
• Lasts 2 to 5 days
Estrus
• Period of sexual receptivity • Large increase in estrogen
Metestrus:
↑Increase in progesterone, ↆ decrease in estrogen • Endometrial lining thickens and
uterine muscles show increased development.
Diestrus:
Relatively short period of time between estrous cycles during the breeding season of
polyestrous animals – No reproductive activity

9. • Sexual maturity in female rats usually occurs between 30 and 50 days of age.
• The occurrence of vaginal opening (VO) and first oestrus, as 38 days.
• The first oestrous cycle begins within approximately one week after vaginal
opening and recurs regularly every 4 or 5 days for a variable proportion of the
animal’s lifespan, depending on the strain of rat.

11. duration of an ovarian cycle can vary:
4 days: mice, rats and hamsters
16 days: guinea pigs
28 days: humans

14. Combined repeated dose toxicity study with the
reproduction/developmental toxicity screening test
OECD guideline for testing of chemicals on reproductive
toxicology

15. PRINCIPLE OF THE TEST:
 The test chemical is administered in graduated doses to several groups
of males and females.
 Males should be dosed for a minimum of four weeks and up to and
including the day before scheduled kill
 pre-mating dosing period in males, fertility may not be a particular
sensitive indicator of testicular toxicity.

16.  Therefore, a detailed histological examination of the testes is essential.
 Histopathology of the male gonads, is considered sufficient to enable
detection of the majority of effects on male fertility and spermatogenesis.
 Females should be dosed throughout the study.
 This includes mating, the duration of pregnancy and at least thirteen days
after delivery, up to and including the day before scheduled kill.

17.  Duration of study, following acclimatisation and pre-dosing oestrous
cycle evaluation, is dependent on the female performance and is
approximately 63 day. [at least 14 days pre-mating, (up to) 14 days mating, 22 days gestation, 13 days lactation].

18. DESCRIPTION OF THE METHOD
Selection of animal species
Guideline is designed for use with the rat. The rat was the only species used.
The test animals should be characterized as to species, strain, sex, weight and
age.
Weight variation of animals used should be minimal and not exceed 20% of the
mean weight of each sex.
 Animals from the same strain and source are used in both studies.

19. HOUSING AND FEEDING
 The temperature in the experimental animal room should be 22º C (±
3). relative humidity should be at least 30%
 Lighting should be artificial, the photoperiod being 12 hours light, 12
hours dark.
 For feeding, laboratory diets used with an unlimited supply of drinking
water.

20.  no more than five animals should be housed per cage.
 Pregnant females should be caged individually and provided with nesting
materials.
 Lactating females will be caged individually with their offspring.

21. PREPARATION OF THE ANIMALS
 Healthy young adult animals are randomly assigned to the control and
treatment groups.
 Cages should be arranged in such a way that possible effects due to cage
placement are minimized.
 The animals are uniquely identified and kept in their cages for at least five
days prior to the start of the study to allow for acclimatisation to the laboratory
conditions.

22. PREPARATION OF DOSES
The test chemical be administered orally unless other routes of administration are
considered more appropriate. When the oral route is selected, the test chemical is
usually administered by gavage.
The test chemical is dissolved or suspended in a suitable vehicle.
PROCEDURE:
 Number and sex of animals
• It is recommended that each group be started with at least 10 males and 12-13
females.

23.  Dosage
• Generally, at least three test groups and a control group should be used.
• Dose levels may be based on information from acute toxicity tests or on
results from repeated dose studies.
• If a vehicle is used in administering the test chemical, the control group
should receive the vehicle in the highest volume used.
• Dose levels should be selected taking into account any existing toxicity
and (toxico-) kinetic data available.

24. • It should also be taken into account that there may be differences in sensitivity
between pregnant and non-pregnant animals.
• The highest dose level should be chosen with the aim of inducing toxic effects but
not death nor obvious suffering.
• Thereafter, a descending sequence of dose levels should be selected with a view to
demonstrating any dosage related response and no adverse effects at the lowest dose
level.
• Two- to four- fold intervals are frequently optimum and addition of a fourth test
group is often preferable to using very large intervals (e.g. more than a factor of 10)
between dosages.

25. Administration of doses
• The animals are dosed with the test chemical daily for 7 days a week.
• The volume should not exceed 1 ml/100 g body weight, except in the case of aqueous
solutions where 2 ml/100 g body weight may be used.
• Except for irritating or corrosive test chemicals which will normally reveal
exacerbated effects with higher concentrations, variability in test volume should be
minimized by adjusting the concentration to ensure a constant volume at all dose
levels.
• For test chemical administered via the diet or drinking water, it is important to ensure
that the quantities of the test chemical involved do not interfere with normal nutrition
or water balance.

26. EXPERIMENTAL SCHEDULE

27. EXPERIMENTAL SCHEDULE
 Dosing of both sexes should begin at least 2 weeks prior to mating, after they
have been acclimatised for at least five days and females have been screened for
normal oestrous cycles.
 The study should be scheduled in such a way that oestrous cycle evaluation
begins soon after the animals have attained full sexual maturity.
 This may vary slightly for different strains of rats in different laboratories, e.g.
Sprague Dawley rats 10 weeks of age, Wistar rats about 12 weeks of age

28.  Dams with offspring should be killed on day 13 post-partum, or shortly
thereafter. In order to allow for overnight fasting of dams prior to blood
collection (if this option is preferred), dams and their offspring need not
necessarily be killed on the same day.
 The day of birth (viz. when parturition is complete) is defined as day 0 post-
partum
 Dosing is continued in both sexes during the mating period.

29.  Males should further be dosed after the mating period at least until the
minimum total dosing period of 28 days has been completed
 They are then killed, or, alternatively, are retained and continued to be dosed
for the possible conduction of a second mating if considered appropriate.
 Daily dosing of the parental females should continue throughout pregnancy.

30. Mating procedure: Normally, 1:1 (one male to one female) matings should be
used in this study.
 Each morning the females should be examined for the presence of sperm or
a vaginal plug.
 Day 0 of pregnancy is defined as the day on which mating evidence is
confirmed

31. IN LIFE OBSERVATIONS
Clinical observation:
 Throughout the test period, general clinical observations should be made at
least once a day, and more frequently when signs of toxicity are observed.
 They should be made preferably at the same time(s) each day. all signs of
toxicity, including mortality, should be recorded.
 These records should include time of onset, degree and duration of toxicity
signs.

32. Body weight and food/water consumption
 Males and females should be weighed on the first day of dosing, at least weekly
thereafter, and at termination.
 During pregnancy, females should be weighed on days 0, 7, 14 and 20.

33. Oestrous cycles
 Oestrous cycles should be monitored before treatment starts to select for the
study females with regular cycle.
 Vaginal smears should also be monitored daily from the beginning of the
treatment period until evidence of mating. If there is concern about acute stress
effects that could alter oestrous cycles.

34. Offspring parameters:
 The duration of gestation should be recorded and is calculated
from day 0 of pregnancy.
 Any abnormal behaviour of the offspring should be recorded.

35. Clinical biochemistry
 Blood samples from a defined site are taken.
 Plasma samples specifically for hormone determination should be
obtained at a comparable time of the day.
 The numerical value obtained when analysing hormone
concentration.

36. Pathology
Gross necropsy :
 At the time of sacrifice or death during the study, the adult animals
should be examined macroscopically for any abnormalities or
pathological changes.
 Vaginal smears should be examined in the morning on the day of
necropsy to determine the stage of the oestrous cycle and
histopathology of ovaries.
 The testes and epididymides of all male adult animals should be
weighed.

37. Histopathology:
 Histological examination should be performed on the ovaries, testes and
epididymides of the animals of the highest dose group and the control
group.

38. DATA AND REPORTING
• Individual animal data should be provided.
• Additionally, all data should be summarised in tabular form, showing for each
test group the number of animals at the start of the test, the number of animals
found dead during the test or killed
• The time of any death or humane kill, the number of fertile animals, the number
of pregnant females,
• the number of animals showing signs of toxicity, a description of the signs of
toxicity observed.

39. Evaluation of results:
• The findings of this toxicity study should be evaluated in terms of
the observed effects, necropsy and microscopic findings.
Test report :
 Test chemical:
- source, limit date for use, if available stability of the test chemical
physical appearance, water solubility, and additional relevant
physicochemical properties; Vehicle

40.  Test animals:
o species/strain used;
o number, age and sex of animals;
o source, housing conditions, diet, etc.;
o individual weights of animals at the start of the test.
 Results:
o body weight/body weight changes;
o food consumption, and water consumption if available;
o toxic response data by sex and dose, including fertility, gestation, and any
other signs of toxicity;

41. FEMALE REPRODUCTIVE
TOXICITY STUDY
ICH S5(R3)
SCHEDULE Y

42. FEMALE FERTILITY TEST
• Drugs administered to both males (28days)and female (14 days) before mating
Segment I : Fertility and general reproductive performance study
Segment II: Teratogenicity
Segment III: Prenatal and post-natal study perinatal : fertility and early
embryonic development

43. FEMALE FERTILITY STUDY (SEGMENT I):
• The study should be done in one rodent species (rat preferred).
• The drug should be administered to both males and females, beginning a
sufficient number of days (28 days in males and 14 days in females) before
mating.
• Drug treatment should continue during mating and, subsequently, during the
gestation period.

44. • Three graded doses should be used, the highest dose (usually the MTD
obtained from previous systemic toxicity studies) should not affect general
health of the parent animals, At least 15 males and 15 females should be used
per dose group.
• Control and the treated groups should be of similar size.
• The route of administration should be the same as intended for therapeutic use

45. •Dams should be allowed to litter and their medication should be
continued till the weaning of pups.
• Observations on body weight, food intake, clinical signs of
intoxication, mating behaviour, progress of gestation! parturition
periods, length of gestation, parturition, post-partum health and gross
pathology (and histopathology of affected organs) of dams should be
recorded.

46. •The pups from both treated and control groups should be
observed for general signs of intoxication, sex-wise
distribution in different treatment groups, body weight, growth
parameters, survival, gross examination, and autopsy.
•Histopathology of affected organs should be done.

47. STUDY DESIGN

48. SEGMENT II:Teratogenicity

49. PERINATAL STUDY (SEGMENT III):
• This study is specially recommended if the drug is to be given to pregnant or
nursing mothers for long periods or where there are indications of possible
adverse effects on foetal development.
• One rodent species (preferably rat) is needed. Dosing at levels comparable to
multiples of human dose should be done by the intended clinical route.
• At least 4 groups (including control), each consisting of 15 dams should be
used.

50. • The drug should be administered throughout the last trimester of pregnancy
(from day 15 of gestation) and then the dose that causes low foetal loss should
be continued throughout lactation and weaning.
• Dams should then be sacrificed and examined as described below.

51. • One male and one female from each litter of Fl generation (total 15 males and 15
females in each group) should be selected at weaning and treated with vehicle or
test substance (at the dose levels described above) throughout their periods of
growth to sexual maturity, pairing, gestation, parturition and lactation.
• Mating performance and fertility of FI generation should thus be. evaluated to
obtain the F2 generation whose growth parameters should be monitored till
weaning.
• The criteria of evaluation should be the same as described earlier.

52. • Animals should be sacrificed at the end of the study and the observation
parameters should include (Dams) body weight, food intake, general signs of
intoxication, progress of gestation / parturition periods and gross pathology (if
any);andfor pups, the clinical signs, sex-wise distribution in dose groups, body
weight, growth parameters, gross examination, survival and autopsy (if
needed) and where necessary, histopathology.

53. Study design

55. • Estrous female rats at 10 to 11 weeks of age were cohabited overnight with a
single male.
• The next morning, females with sperm in their vaginal smears were regarded
as pregnant, and this day was designated as day 0 of gestation.
• Once insemination was confirmed, the females were weighed and randomly
allocated to six experimental groups.
• The dams were allowed to deliver naturally and nurse their pups until
postnatal day (PND) 21.
• On PND 0 (the day of birth), all pups were weighed and their sex was
determined, and the litters were culled randomly to eight (four pups/sex/ litter
when possible)
PROCEDURE

56. • In the present study, three to five males and three to five females per litter were
used.
• All neonates were given daily gavage administration of 12.5, 25, 50, or 100
mg/kg genistein.
• The measurement of sexual organ weight and histopathologic observation of the
reproductive tracts were performed

57. THANK YOU