2. 2
PRESENTATION OUTLINE
INTRODUCTION
AIM OF REPRODUCTIVE TOXICOLOGY STUDIES
STAGES OF A REPRODUCTIVE CYCLE
ESTROUS CYCLE
FEMALE FERTILITY TESTS
◦ SEGMENT 1
◦ SEGMENT 2
◦ SEGMENT 3
REFERENCES
3. INTRODUCTION
•Reproductive toxicity is a hazard associated with chemical substances, which interfere in some way with
normal reproduction; such substances are called reprotoxic.
•They may adversely affect sexual function and fertility in adult males and females, as well as causing
developmental toxicity in the offspring.
•Reproductive toxicities include structural and functional alterations that may affect reproductive competence
(fertility, parturition, and lactation). Evaluations of fertility, pregnancy, lactation, and maternal and paternal
behaviors provide measures of the consequences of reproductive injury. These evaluations provide
information concerning gonadal function, estrous or menstrual cyclicity, mating behavior, conception,
parturition, lactation, weaning, and the growth and development of the offspring.
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4. AIM OF REPRODUCTIVE TOXICOLOGY STUDIES
•To reveal any effect of one or more active substances on mammalian reproduction.
•To explore the possible effects of the drug on fertility and reproductive performance
•The studies conducted should allow exposure of mature adults and all stages of development
to sexual maturity.
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5. 5
STAGES OFA REPRODUCTIVE CYCLE
Stage A: Pre-Mating to conception
Stage B: Conception to implantation
Stage C: Implantation to organ formation
Stage D: Organ formation to end of pregnancy
Stage E: Birth to weaning
Stage F: Weaning to sexual maturity.
6. ESTROUS CYCLE
•The estrous cycle refers to the reproductive cycle in rodents.
•It is similar to human reproductive cycle.
•The estrous cycle consists of four stages:
1.Proestrus
2.Estrus
3.Metestrus
4.Diestrus. Appearance of the vagina in different phases
of estrous cycle of a Swiss albino strain
mouse. a-Proestrus, b-Estrus, c- Metestrus, d-
Diestrus
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7. Proestrus:
• Begins when progesterone declines (luteolysis: regression of corpus luteum) and ends at onset of estrus.
Estrus:
• Period of sexual receptivity
• Large increase in estrogen.
Metestrus:
• Increase in progesterone, decrease in estrogen.
• Endometrial lining thickens and uterine muscles show increased development.
Diestrus:
• Relatively short period of time between estrous cycle during the breeding season of polyestrous animals
• No reproductive activity.
• Female is no longer receptive to male.
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8. FEMALE FERTILITY TEST
1. The fertility and reproductive toxicity study: Segment I
◦ covers the period of premating, cohabitation and mating, and early
pregnancy through implantation (Gestation Day [GD] 6 in the rat)
2. The prenatal developmental toxicity study: Segment II
◦ covers pregnancy from implantation through major organogenesis (closure
of the hard palate; GD 15 in the rat) up to the day before delivery (GD
20/21)
3. The prenatal and postnatal study: Segment III
◦ covers late pregnancy and postnatal development (usually until weaning at
Postnatal Day [PND] 21)
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10. PURPOSE:
It provides information on the changes occurring in the reproductive performances and the fertility
patterns following test item administration at different dose levels in several groups of male and female
rats
PRINCIPLE:
Male and female rats are used for the segment-I study
In females, the test item is administered 2 weeks prior to mating (covering a minimum of two oestrous
cycles), 2 weeks during mating days, 21/22 days gestation and 13 days lactation.
During test item administration period, the animals are observed for the development of clinical signs, if
any.
At the end of the dosing period, all the survived animals are euthanized and necropsied to determine
female specific endpoints, clinical chemistry and histopathological changes
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11. Species Rodent (rat)
Strain Sprague Dawley, Wistar Albino
Sex Male and female (nulliparous, nonpregnant with normal oestrous cycle)
Age at dosing 8–10 weeks
Housing Animals should be housed at a temperature of 22±3 C, relative humidity between 30–70%
and 12/12 h light–dark cycle. Animals are housed in grouped cages. Pregnant and
lactating females should be housed individually in cages
Acclimatization Minimum 5 days before start of the experiment
Randomization Healthy males and females tests are selected and randomized based on stratified
bodyweight or by random distribution. In any case, the mean differences in body weights
between the groups should not be statistically significant.
Dose selection Administered to both sexes, beginning with a sufficient number of days before mating
Groups 1 control and 3 test groups; 15 males and 15 females per dose
Route of administration Same as intended for therapeutic use
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12. STUDY STATE EXPERIMENT
1. The study includes control and three graded doses, the highest dose ( usually the MTD obtained from
previous systemic toxicity studies) should not effect general health of the parent animals.
2. The drug is administered to both males (2 weeks before mating, during mating and 2 weeks after mating,
followed by scheduled necropsy) and females (2 weeks prior to mating (ensure to cover minimum two
complete estrous cycles) and continues up to day 21/22 of the gestation period, and at least 2 weeks after
delivery)
3. Females showing no evidence of copulation should be euthanized between 24 and 26 days after
introducing to mating, up to and as well as the day before necropsy.
4. Pregnancy is confirmed by examining (morning time) the sperm-positive vaginal lavage or copulatory
plugs in the vagina or cage pan. Confirmation of sperm or copulatory plug is declared as gestation day 0
and the pregnant are housed in individual cages.
5. Dams should be allowed to litter and their medication should be continued till the weaning of pups
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13. OBSERVATIONS:
1. During the test item administration, the animals should be observed once daily for mortality and
routine clinical signs
2. Males and females should be monitored for bodyweight changes, feed intake and functional battery
observation on weekly basis. Pregnant rats should be weighted on days 0, 7, 14, 20, 24 h postpartum
and 4 and 13 PND
3. Post-treatment, both male and female subjects are subjected to necropsy
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14. FEMALE-SPECIFIC ENDPOINTS
1. In case the female test subject(s) is found in moribund condition or dead after mating, they should be
necropsied in order to determine the cause. Signs of pregnancy or development of embryo should also be
recorded at this stage.
2. Female sex organs uterus, cervix, the vagina should be examined for gross and histopathological changes, if
any.
3. The uterine contents should be checked for evidences of implantation. The number of corpus lutea for each
ovary should be counted.
4. Dead pups or pups euthanized on PND 13 should also be necropsied and subjected to gross and
histopathological examination
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16. DEVELOPMENTAL TOXICOLOGY
It is the study of adverse effects on the developing organism that may result from exposure prior to conception,
during prenatal development, or postnatally to the time of sexual maturation.
The major manifestations of developmental toxicity include:
(1) death of the organism,
(2) structural abnormality,
(3) altered growth and
(4) functional deficiency.
Developmental toxicology was formerly often referred to as teratology
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17. • Usually conducted in 2 species: Rat and Rabbit.
• In rats and rabbits 4 groups are taken and 16 to 20 litters are collected.
• Mated animals are treated during the period of organogenesis ( days 6-18 in rabbits, 6-15 in rats).
• Pups are delivered by caesarean one day before expected parturition (21 days rat, 31 days rabbit).
• Uterus removed , weighed and examined for dead or resorbed foetuses.
• Live pups are weighed , ½ examined for skeletal abnormalities, other half for soft tissue abnormalities,
histology.
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18. 18
OBSERVATIONS
1. Pregnant rats should be examined twice daily for mortality and the clinical signs of toxicity, if any.
2. Body weight should be recorded on GD 0, and every 3 days during the dosing period, and once before the
scheduled caesarean section.
3. Feed intake should be measured every third day during the dosing period
4. Female rats showing signs of morbidity or abortion should be sacrificed humanely and subjected to
macroscopic evaluation.
5. Dams die during the treatment period should be subjected to gross pathological examination. Organs showing
gross pathological changes should be subjected to histopathologic examination
6. At the end of the treatment duration, the below-mentioned observations should be recorded in the dam and
foetus
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MATERNAL END-POINTS
(a) Gestation length
(b) Before the scheduled caesarean section, the blood samples should be collected and measured for T3, T4 and
thyroid-stimulating hormones levels to ascertain the effects of the test items on endocrine functions.
(c) The gravid uterus should be collected and weighed separately and subjected to histopathology.
(d) Detailed examination of the uterus should be carried out to check the number of implant sites and corpora
lutea per dam
(e) Number and percentage of living, dead and resorbed foetus.
(f) The number and percentage of affected implants (non-live + malformed), total resorptions and stillbirths per
litter
(g) Number of live foetuses
(h) The absolute weight of all components of reproductive organs should be recorded
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FOETAL ENDPOINTS
The uterine contents should be collected and observed for signs of toxicity.
Each foetus is separated from the uterus and the umbilical cords are cut off and blotted on absorbent paper in
order to remove the traces of blood and amniotic fluid.
The following foetal specific endpoints should be recorded
(a) Number of live and dead foetuses
(b) Live foetus body weight
(c) Sex ratio per litter
(d) The number of foetus with external and visceral malformations per litter
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PURPOSE
• Segment III study gives information about the health hazards likely to arise in various life stages
following the exposure to the test item prior to conceptus [in parent males and females], during in-
utero development of embryo/ foetus, preweaning (lactation) (in pregnant and dams), adolescence
and adulthood (first generation offspring (F1)).
• The effects of the test item in terms of F1 generation reproductive functions, development of
nervous and immune systems is investigated to establish the NOAEL, LOAEL and benchmark
dose
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• 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.
• 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.
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• One male and one female from each litter of F1 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 F1 generation should thus be evaluated to obtain the F2 generation
whose growth parameters should be monitored till weaning.
• 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);and for 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.
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REFERENCES
1. Introduction to Toxicological Screening Methods and Good Laboratory Practice by
Saravana Babu Chidambaram, M. Mohamed Essa and M. Walid Qoronfleh. Page 114-125
(https://doi.org/10.1007/978-981-16-6092-4 )
2. Developmental and Reproductive Toxicology A Practical Approach, Third Edition, Edited by
Ronald D. Hood
(https://doi.org/10.3109/9781841848211 )
3. Staging of the estrous cycle and induction of estrus in experimental rodents: an update
(https://fertilityresearchandpractice.biomedcentral.com/articles/10.1186/s40738-020-00074-3 )
4. OECD (2016) guideline number 421: reproduction/ developmental toxicity screening test
