Reproductive Toxicity Studies and Male and Female toxicity testing

1. REPRODUCTIVE TOXICITY STUDIES
Prepared by:
Loveinder Bhardwaj
MPharma(Cology)2 Sem

2. WHAT IS REPRODUCTIVE TOXICITY ?
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. OECD GUIDELINE FOR TESTING OF CHEMICALS
ON REPRODUCTIVE TOXICOLOGY
Principle of test :
• The test chemical is administered in graduated doses to severalgroups of males and
females.
• Males should be dosed for a minimum of four weeks and up toand including the day
before scheduled kill
• Pre-mating dosing period in males, fertility may not be a particular sensitive indicator
of testicular toxicity.
• Therefore, a detailed histological examination of the testesis essential.

4. • Histopathology of the male gonads, is considered sufficient to enable detection of the
majority of effectson male fertility and spermatogenesis.
• 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 acclimatization and pre-dosing oestrous cycle evaluation,
is dependent on the female performance and is approximately 63 day.

5. CON….
 Test No. 422: Combined Repeated Dose Toxicity Study with the
Reproduction/ DevelopmentalToxicity Screening Test
 Test No. 421: Reproduction/ Developmental Toxicity ScreeningTest
 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 ToxicityStudy

6. ICH GUIDELINES
ICHS5(R2)- DETECTION OF TOXICITY TO REPRODUCTION FOR
MEDICINAL PRODUCTS & TOXICITYTOMALE FERTILITY
Current Step 2 draft version dated 5 July 2017
DETECTIONOF TOXICITYTOREPRODUCTION FOR HUMAN
PHARMACEUTICALS S5(R3)

7. OBJECTIVE
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

8. DESCRIPTIONOFTHEMETHOD
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.
• Healthy young adult animals are randomly assigned to the control and treatment group
• 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 atleast five days prior to
the start of the study to allow for acclimatization to the laboratory conditions
• Each animal should be assign with each identical number.

9. HOUSING AND FEEDING
1) The temperature in the experimental animal room should be 22 C (± 3). Relative
humidity should be at least 60%
2) 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
3) No more than five animals should be housed per cage.
4) Pregnant females should be caged individually and provided with nesting materials
and when there is a parturition is near.
5) Lactating females will be caged individually with their offspring.

10. DOSAGE
• Generally, at least three test groups and a control group should be used. the highest
dose level should be chosen with the aim to induce toxicity but not death or severe
suffering.
• Dose levels may be based on information from acute toxicity testsor on results
from repeated dose studies.
• If a vehicle is used in administering the test chemical, the controlgroup should receive
the vehicle in the highest volume used. If a test substance is administered in the diet,
and causes reduced dietary intake or utilization, then the use of a paired control group
may be considered necessary.
• Any available information on metabolism and kinetics of the test compound or related
materials should also be considered

11. ADMINISTRATION OFDOSES
• Each test and control group should contain a sufficient number of animals to yield
preferably not less than 20 pregnant females at or near parturition The animals are
dosed with the test chemical daily for 7 days a week.
• It is recommended that the test substance be administered orally (by diet, drinking
water or gavage) unless another route of administration (e.g. dermal or inhalation) is
considered more appropriate.
• Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It
is recommended that, wherever possible, the use of an aqueous solution/suspension
be considered first
• For vehicles other than water, the toxic characteristics of the vehicle must be known.
The stability of the test substance in the vehicle should be determined.

12. • The volume should not exceed 1ml/100g body weight, except in the case of aqueous
solutions where 2ml/100g body weight may be used.
• 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 waterbalance.
When the test
• In gavage studies, the pups will normally only receive test substance indirectly through the
milk, until direct dosing commences for them at weaning.
• In diet or drinking water studies, the pups will additionally receive test substance directly
when they commence eating for themselves during the last week of the lactation period.
• For a substance administered by gavage, the dose should be given at similar times each day,
and adjusted at least weekly to maintain a constant dose level in terms of animal body
weight.

14. EXPERIMENTAL SCHEDULE
• Daily dosing of the males and females shall begin when they are 5 to 9 weeks old.
• For both sexes dosing shall be continued for at least 4 weeks before the mating period.
Dosing is continued in both sexes during the 2 week mating period.
• Males should be humanely killed and examined when they are no longer needed for
assessment of reproductive effects.
• Daily dosing of the parental females should continuethroughout pregnancy and up to
the weaning of the offspring.
• The dose to each animal should normally be based on the most recent individual
bodyweight determination.

15. MATING PROCEDURE
• For each mating, each female shall be placed with a single male from the same dose level
(1:1 mating) until copulation occurs or 2 weeks have elapsed.
• Mating of siblings should be avoided.
• In certain instances, such as treatment-related alterations in litter size , it is recommended
that the adults be remated to produce a second litter which have not produced a litter with
proven breeders of the opposite sex.

16. CLINICAL OBSERVATION
Throughout the test period, general clinical observations should be made at least once a
day, behavioural changes, signs of difficult or prolonged parturition and more frequently
when signs of toxicityare observed.
They should be made preferably at the same time(s) each day. allsigns
of toxicity, including mortality, should be recorded.
These records should include time of onset, degree and duration of toxicity signs.

17. 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.
Offspring parameters
The duration of gestation should be recorded and is calculatedfrom day 0of pregnancy any
abnormal behaviour of the offspring should be recorded.

18. Clinical biochemistry
Blood samples from a defined site aretaken.
Plasma samples specifically for hormone determination shouldbe obtained at a
comparable time of theday.
The numerical value obtained when analysing hormone concentration.
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.

19. Vaginal smears should be examined in the morning on the dayof necropsy to determine
the stage of the oestrous cycle and histopathology of ovaries.
The testes and epididymitis of all male adult animals shouldbe weighed.
Histopathology
Histological examination should be performed on the ovaries, testes and epididymites
of the animals of the highest dosegroup and the control group.

20. DATAAND REPORTING
• Individual animal data should be provided.
• Additionally, all data should be summarized 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 orkilled 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 and its description
• Time of onset, duration, and severity of any toxic effects
• The types of histopathological changes, and all relevant litter data.

21. REPRODUCTIVE TOXICOLOGY STUDIES
Male fertility
Method:
One rodent species(rat)
3dose group taken (each 6 adult males)
Drug treatment by clinical route for 28-72day

22. Mixed with female in 1:2ratio
Female getting pregnant should be examined after 14days of
gestation
All male animals sacrificed weights of testis, epididymis recorded and examined
for their histology
sperms examined for motility and morphology

23. B) female fertility
Drugs administered to both males (28days)andfemale (14 days)
before mating
Segment I : fertility and general reproductive performance
study
Segment II: Teratogenicity
Segment III: Perinatal and post-natal study
perinatal : fertility and early embryonic development.
Post-natal development (rat) (post natal survival of offspring) growth parameter,
vital senses, behavioral effect

24. EVALUATION OF RESULT
The findings of this reproduction toxicity study should be evaluated in terms of the observed effects
including necropsy and microscopic findings. The evaluation will including gross lesions, identified target
organs, affected fertility, clinical abnormalities, affected reproductive and litter performance, body weight
changes, effects on mortality and any other toxic effects.
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

25. TEST ANIMALS: -
• Species/strain used
• Number, age and sex of animals
• Source, housing conditions, diet, nesting materials
• Individual weights of animals at the start of the test.
-
- RESULT
• Food consumption, and water consumption if available
• Absorption data (if available)
• Body weight at sacrifice and absolute and relative organ weight data for the parental
animals
• Toxic response data by sex and dose, including indices of mating, fertility, gestation, birth,
viability, and lactation;