Reproductive toxicology studies

Male and Female reproductive toxicology studies and stages

1. REPRODUCTIVE TOXICOLOGY STUDIES BY G.KUSUMA KUMARI I.M.PHARMACY DEPARTMENT OF PHARMACOLOGY

2. DEFINTION:- Reproductive toxicology is the subject area dealing with the cause , mechanisms, effects and prevention of disturbances through out the entire reproductive cycle ,including fertility induced by chemicals . Any adverse effect on any aspect of male or female sexual structure or function(or) on the conceptu’s 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.

3. REPRODUCTIVE SYSTEM: The reproductive system is the system in which we reproduce. The male reproductive system consists of a adrenal gland, kidney, bladder, ureter, seminal vesicle, prostate gland ,vas deferens, uriogenitial orfice , epididymis, penis, testis, primord , scrotal sac . This allows the male to produce sperm cells which gets the female pregnant. The female reproductive system consists of a adrenal gland ,kidney, ovary, oviduct, uterine hormone ,urinary bladder, urethra, vagina, anus . This allows the female to make eggs and to get pregnant to reproduce babies with the male.

4. Male reproductive system and female reproductive system:

5. Neuroendocrine targets for reproductive toxicology :

6. Schematic representation of hypothalamic pituitary gondal axes:

7. REPRODUCTIVE TOXICITY: Reproductive toxicity is a hazard associated with some chemical substances that they will interfere in some way with normal reproduction; such substance are called reprotoxic . It includes adverse effects on sexual function and fertility in adult males and females as well as developmental toxicity in the offspring”. They are two types of reproductive toxicity ,they are: 1.Male reproductive toxicological study. 2.Female reproductive toxicological study:- Segment-I. Segment-II. Segment-III.

8. MALE REPRODUCTIVE TOXICOLOGICAL STUDY

9. MALE FERTILITY STUDY : DOSE GROUPS DOSING INTERVAL PARMETERS

10. FEMALE REPRODUCTIVE TOXICOLOGICAL STUDY

11. FEMALE FERTILITY STUDY: Drug administered to both males (28 days) and females (14 days) before mating Segment I,II and III studies in albino mice or rats. Segment II study also in albino rabbits as a second test species.

12. SEGMENT-I ( FERTILITY AND GENERAL REPRODUCTIVE PERFORMANCE STUDY)

13. FERTILITY AND GENERAL REPRODUCTIVE PERFORMANCE STUDY

14. Drug administration:28 days (males)& 14 (females)before mating Drug treatment continued during mating and gestation period. Females allowed to litter medication continued till weaning of pups Body weight food intake Clinical signs of intoxication Reproduction and parturition Pathology gross & micro Pups: Physiology, Behaviour, pathology(sex-wise distribution noted)

15. SEGMENT I DESIGN 10 week PBE F0 MALES Q Q N N 2 WEEK PBE males 1/2females at midterm for dead/resorbing foetuses Q = Quarantine F1 M = Mating G = Gestation period of direct exposure to chemical L = Lactation possible indirect exposure from transplacental W = wean and/or translactational transfer N = Necroscopy M G L M M G G L L

16. SEGMENT –I END POINTS : Fertility index = % matings that result in pregnancy gestation index = % pregnancies yielding live litters viability index = % animals surviving 4 days lactation index = % of animals alive at 4 days that survive the 21 day lactation period Pup body weights pnd 4, 7, 14, and 21 – Gross necropsy and histopathology on some parents (both reproductive and non- reproductiveorgans ) – Many other possible endpoints

17. Segment -II ( Teratogenicity)

18. Teratogenicity Study (Segment II) Species : One rodent(Rats)-12 per group & a non-rodent (rabbit)-20 per group. Dose selection : Three graded doses. Drug administered throughout the period of organogenesis(6-18 days in rabbits,6-15 in rats) . The route of administration should be the same as intended for human therapeutic use. Pups delivered by casesarean one day before expected parturition (21 days rat,31 days rabbit). Groups : The control and the treated groups should consist of at least 20 pregnant rats (or mice) and 12 rabbits , on each dose level . All foetuses should to be subjected to gross examination, Skeletal abnormalities and visceral abnormalities.

19. SEGMENT ll DESIGN gd6 gd15 N gd 20 Q M Mated female rats gd 6-7 gd 18-19 N gd 30 Q M Mated female rabbits N = Necropsy gd = gestational day. Direct exposure to pregnant dams

20. OBSERVATION PARAMETERS Dams Foetuses sign of intoxication Total number Effects on body weight Gender Effect on food intake. Body length Examination of uterus , ovaries weight & uterine contents. Gross/Visceral/Skeletal Number of corpora lutea abnormalities Implantation sites Resorption

21. SEGMENT -III ( PREi -POST/NATAL SEGMENTS)

22. SEGMENT –III ( PREI – POST/ NATAL SEGMENTS) Usually one species (rats) Pregnant females (20 per group) 2-3 dosages administered from end of organogenesis period through delivery and lactation Endpoints: birth weight, survival, growth during first 3 weeks of life, many others

23. SEGMENT –III DESIGN F o rats females gd 15 pnd o pnd 21 Q M NF0 F1 M G L Pnd4 N NF1 females F1males & F2 pups Q =Quarantine , N= Necropsy , M = Mating, gd = gestational day , L = Lactation Direct exposure to adults Direct exposure to offspring if test material is administered via feed or water Possible indirect exposure from transplacental and/or translactational transfer

24. Observation parameters should include: (Dams) signs of intoxication, effect on body weight, effect on food intake, examination of uterus, ovaries and uterine contents, number of corpora lutea , implantation sites The foetuses, the total number, gender, body length, weight and gross/ visceral/ skeletal abnormalities, if any.

25. OECD GUIDE LINE -421 ( Reproduction and developmental toxicity screening test )

26. OECD GUIDELINES 421 : ( Reproduction and developmental toxicity screening test ): INTRODUCTION:- This Guideline is designed to generate limited information concerning the effects of a test substance on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. INITIAL CONSIDERATION : It can also be used as part of a set of initial screening tests for existing chemicals for which little or no toxicological information is available, as a dose range finding study for more extensive reproduction/developmental studies, or when otherwise considered relevant.

27. PRINCIPLES : The test chemical is administered in graduated dose to several groups of males and females. Males reproductive system principle :- It should be dosed for minimum of four weaks & up to & including the day before scheduled ( this includes minimum of 2 weeks prior to mating ,during the mating period & approximate,2 weeks post mating ). In view of the limited pre mating dosing period in males fertility may not be a particular sensitive indicator of testicular toxicity i.e detalis histological examination of testes is essential The combination of pre mating dosing period of 2 weeks & sub sequenting mating /fertility . Observation with an overall.

28. Principle for female reproductive system:- Dose through out the study 4 weeks 2 weeks before mating consider 2 oestrous cycle. duration of pregnancy at least 13 days of delivery up to & including the day before schedule. Duration of study following acclimation & pre dosing osterous cycle evaluation It depend of female performance & in approximately 63 days ( at least 14 days premating 14 th day mating ,22 days gestation period ,13 days lactation. During the period of administration animals observed closely each days for sign of toxicity end of study all animals sacrified at the end of study.

29. DESCRIPTION OF METHOD : 1 . Selection of animals species : RATS 2 . Housing and feeding conditions : The temperature in the experimental animal room should be 22 °C (± 3°). Although the relative humidity should be at least 30% and preferably not exceed 70% other than during room cleaning, the aim should be 50-60% . 3 . Preparation of animals : 4 . Preparation of doses: PROCEDURE : Number and sex of animals dosage limit test administration of dose experimental schedule

30. EXPERIMENTAL SCHEDULE: Premating mating gestation ( 14 days) ( maxi.14 days) (app. 22 days) lactation 1 7 14 21 28 35 42 50 54 MALES FEMALES MALES + FEMALES MALES/SERIES OPTIONAL EXTENDED EXPOSURE PREGNANT FEMALES DAMS PUPS NON PREGNANT FEMALES Start of study necropsy

31. MATING PROCEDURE : Males : females-1:1 ratio OSERVATION : Throughout the test period, general clinical observations should be made at least once a day, and more frequently when signs of toxicity are observed. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, should be recorded. The duration of gestation should be recorded and is calculated from day 0 of pregnancy. Live pups should be counted and sexed and litters weighed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum, any abnormal behaviour of the offspring should be recorded.

32. BODY WEIGHT ,FOOD/WATER CONSUMPTION :- PATHOLOGY : 1. Gross necropsy. 2. Histopathology. DATA AND REPORTING : INTERPRETATION OF RESULTS : The study will provide evaluations of reproduction/developmental toxicity associated with administration of repeated doses. It could provide an indication of the need to conduct further investigations and provides guidance in the design of subsequent studies.

33. CONCLUSION:- Reproductive toxicology aims to predict effects of medicines and chemicals on the ability of man to reproduce by assessing effects in animals. Study designs divide the total process of reproduction in to series of manageable units which can provide answers to specific challenges to the reproductive processes. No real prospects of replacing animals in these studies.

34. REFERENCE:- Ruddon , R. W. (1990) Chemical Teratogenesis in Principles of Drug Action-The Basis of Pharmacology, Third Ed. Chapter 13, p. 775-796 Eds. W.B. Pratt and P.Taylor , Churchill Livingstone Eaton, D. L., and Klaassen , C.D., (1996) Section on Developmental and Reproductive Toxicity, p. 31 in Chapter 2, Principles of Toxicology, in Cassarett and Doull’s Toxicology, Fifth Ed., McGraw Hill Koren , G., Pastuszak , A., and Ito, S. (1998) Drugs in Pregnancy N. Engl. J. Med.338: 1128-1137. Collins, M.D., and Mao, M.D. (1999) Teratology of retinoids . Ann. Rev. Pharmacol.Toxicol . 39: 399-430.