reproductive studies of males and females

1. Kumaun University, bhimtal
Department of pharmaceutical sciences
Presented by Submitted to :
Manisha Jyala Dr. Tirath Kumar
M.Pharm 1st year(Pharmacology)

2. Reproductive Toxicology
 Reproductive toxicity refers to structural and functional alternations that
affect reproductive system in sexually mature males and females.
 Reproductive toxicity includes effects on males fertility , female fertility
and lactation.
 it involves a broad range of target and mechanism such as direct effects on
the semniferous epithelium or leydig cells and sertoli cells supporting
spermatogenesis, epididymal sperm maturation as well as endocrine
disruption.

3. OECD guidelines for reproductive
toxicity studies
Principle of the test
The test chemical is administered in graduated doses of males and females.
Males should be dosed for minimum of four weeks and including the day
before scheduled kill.
Therefore, a detailed histological examination of the test is essential.
Histopathology of male gonads, is considered sufficient to enable detection
of the majority of effects on male fertility and spermatogenesis.

4. Description of the method
 Selection of animal species guidelines is designed for use with the rat.
 The rat was the only species used.
 The test animals should be characterised 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.

5. Females should be dosed throughout the study.
This includes mating, the duration of pregnancy and including the day
before scheduled kill.
Duration of study,following acclimatisation and pre-dosing oestrous cycle
evaluation, is dependent on the female performance and is approximately
63 days.

6. Housing and feeding
Temperature 20 degree celsius (+_3).
Relative humidity 30%.
Lightning should be artificial.
The photoperiod being 12 hours light and 12 hours dark.
Laboratory diets used with an unlimited supply of drinking water.
No more than five animals shuld be housed per cage.
Pregnant females should be caged invidually and provided with nesting materials.

7. Number and sex of animals: 10 males and 12-13 females.
Dosage at least three test groups and control group.
Dose level maybe 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 kevel should be selected taking into account any existing toxicity
and toxicokinetic data available.

8.  Administration of doses, the animals are dosed with the test chemical daily
for 7 days a week.
 The volume should not exceed 1ml/100 g body weight, except in the case
of aqueous solutions where 2ml/100 g body weight may be used.
 For test chemical administerd 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 on water balance.

9. Procedure
 Number and sex of animals:
 It is recommended that each group be started with at least 10 males and 12
– 13 females .
 Dosage
 At least three test groups and a control group should be used.
 Dose levels may be based on information from acute toxicity test por on
results from repeated dose studies.

10.  If the vehicle is used in administering the test chemicals , the control group
should receive the vehicle in the highest volume used.
 Dose levels should be selected taking into account any existing toxicity and
kinetic data available.

11. Administration of doses
 The animals are dosed with the test chemicals daily for 7 days a week.
 The volume should not exceed 1 ml/100g body weight , except in the case
of aqueous solutions where 2ml/100g body weight may be used.
 For test chemical administered via at the diet or drinking water, it is
important to ensure that the quantities of the test chemical involved do not
interfere with normal nutrition of water balance .

13. Experimental schedule
 Dosing of both sexes should begin at least 2 weeks prior to mating, females have
been screened for normal oestrous cycle.
 Dosing is continued in both the sexes during the mating period.
 Males should further be dosed after 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 parentral females should continue throughout pregnancy.

14. 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
vaginal plug.

15. In life observations
Clinical observations :
 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 each day. All signs of
toxicity, including mortality should be recorded.
 These records should include time of onset, degree and duration of toxicity
signs.

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

17. Oestrous cycle
 Oestrous cycle 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 intil evidence of mating.
 If there is concern about acute stress effects that could alter oestrous cycles.

18. Offspring parameter
 The duration of gestation should be recorded and is calculated from
day of pregnancy.
 Any abnormal behavior of the offspring should be recorded.
 Clinical biochemistry
 Blood samples from a defined site are taken.
 Plasma samples specifically for hormone determination should be
obtaained at a comparable time of the day.
 The numerical value obtained when analysing hormone
concentration.

19. Pathology
 Gross necrosy:
 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
necrosy to determine the stage of oestrous cycle and histopathology
of ovaries.
 The testies and epididymis of all male adult animals should be
weighed.

20. Histopathology
Histopathological examination should be performed on the ovaries, testes and
epididymis of the animals of the highest dose group and the control group.

21. Data and reporting
 Individual animal data should be provided.
 Additionally, all the 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 test or killed.
 The time of any death or human kill, the number of fertile animals, the
number of pregnant females,
 The number of animals showing the signs of toxicity, a description of signs
of toxicity observed.

22. Evaluation of results
 The finding of this toxicity study should be evaluated in terms of the
observed effects, necrosy and microscopic findings.

23. 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.

24. Results
 Body weight/ body weight changes.
 Food consumption, water consumption.
 Toxic response data by sex and dose, including fertility gestation, and any
other signs of toxicity.

25. Male fertility
 One rodent species(rat)
 3 dose group taken ( each 6 adult males)
 Drug treatment by clinical route for 28- 72 days
 Mixed with female in 1:2 ratio
 Female getting pregnant should be examined after 13 days of gestation
 All male animals sacrificed weight of testies, epididymis recorded and
examined for their histology.
 Sperms examined for motility and morphology .

26. Female reproductive toxicity study
 Female fertility test
 Drugs administered to males (28 days) and female (14 female) before
mating
 Segment 1 :fertility and general reproductive performance study
 Segment 2: teratogenicity
 Segment 3: prenatal and post natal study perinatal: fertility and early
embryonic development

27. Female fertility study (segment 1)
 Study should be done in one rodent species.
 The drug should be administerd 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.
 Three grade doses should be used , the highest dose 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 group should be of similar size .

28.  The route of administration should be the same as in intended for
therapeutic use.
 Observations on the body weight ,food intake , clinical signs intoxication,
mating behaviour, progress of gestation partution periods, length of
gestation , post- partum health and gross pathology of dams should be
recorded.
 The pups from both treated and control group should be observed for
general signs of intoxication, sex-wise distribution in different treatment
groups, body weight , growth parameters.
 Histopathology of affected organs should be done

29. 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 fetal development .
 One rodent species is needed. Dosing at levels comparable to multiples of
human dose should be done by intended clinical route.

30.  At least 4 groups each consisting 15 dams should be used.
 The drug should be administered throughout the last trimester of pregnancy
and then dose that cause low fetal loss should be continued through out
lactation and weaning.
 One male and one female from each litter of FI generation should be
selected at weaning and treated with vehicle or test substance throughout
their period of growth to sexual maturity pairing gestation , parturition and
lactation.

31.  Mating performence and fertility of FI generation should thus be evaluated
to obtain the F2 generation whose growth parameters should be monitered
till weaning.
 the criteria of evaluation should be same as described earlier.
 Animals should be sacrificed at the end of the study and observation
parameters should include body weight, food intake , general signs.

32.  Of intoxication, progess of gestation /parturiton periods and
gross pathology (if any) and for pups: the clinical signs, sex
wise distribution in dose group, body weight , growth
parameters , gross examination , survival and autopsy ( if
needed) and where neccesary , histopathology.

33. Procedure
 Estrous female rats at 10to 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 female were weighed and randomly
allocated to six experimental groups.

34.  The dams were allowed to deliver naturally and nurse their pups until
postnatal day 21.
 On PND 0,all pups were weighed and their sex was determined, and their
litters were culled randomly to eight .
 In the present study, threee to five males and three to five females per litter
were used.
 All neonates were given administration of of12.5,25,50, or 100 mg/kg
genistein.
 The measurement of sexual organ weight and histopathological
observations of the reproductive tracts were performed.