Reproductive toxicology studies ACCORDING TO OECD guidlines 422

reproduction toxicity studies according to OECD 422
FEMALE REPRODUCTION STUDIES

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. 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. D iestrus : 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.

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

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

12. 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.

13. 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.

14. 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].

15. 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.

16. Housing and feeding The temperature in the experimental animal room should be 22 º 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.

17. 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.

18. 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.

19. 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.

20. 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.

21. 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.

22. 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.

23. Experimental schedule

24. 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

25. 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.

26. 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.

27. 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

28. 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.

29. 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.

30. 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.

31. 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.

32. 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.

33. 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.

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

35. 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.

36. 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

37. 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. Results: 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;

38. Female reproductive toxicity study ICH S5(R3) SCHEDULE Y

39. Female fertility test Drugs administered to both males (28days)and female (14 days) before mating

40. 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.

41. 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

42. 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.

43. 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.

44. Study design

45. SEGMENT II:Teratogenicity

46. 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.

47. 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.

48. 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.

49. 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.

50. Study design

51. 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

52. 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

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