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OECD GUIDELINE FOR TESTING OF CHEMICALS Two-Generation Reproduction Toxicity Study:

OECD GUIDELINE FOR TESTING OF CHEMICALS Two-Generation Reproduction Toxicity Study By:- Yashveer Singh Rathore M pharm Sem III ,Pharmacology K.B.I.P.E.R


INTRODUCTION In Copenhagen in June 1995, an OECD Working Group on Reproduction and Developmental Toxicity discussed and recommended that the Guideline for the Two-generation Reproduction Toxicity Study should be revised, based on proposals received from US and Germany.


INITIAL CONSIDERATIONS This Guideline for two-generation reproduction testing provides information concerning the effects of a test substance on the integrity and performance of the male and female reproductive systems, gonadal function,oestrus cycle, mating behaviour , conception,gestation , parturition, lactation,weaning , growth and development of the offspring,neonatal morbidity, mortality, and preliminary data on prenatal and postnatal developmental toxicity

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In addition to studying growth and development of the F1 generation, this Guideline is also intended to assess the integrity and performance of the male and female reproductive systems as well as growth and development of the F2 generation.


PRINCIPLE OF THE TEST The test substance is given in graduated doses to several groups of males and females.Males of the P generation should be dosed during growth and for at least one complete spermatogenic cycle (56 days in the mouse and 70 days in the rat) to produce adverse effects on spermatogenesis . Effects on sperm are determined by parameters ( e.g sperm morphology and motility) and histopathology.

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If data on spermatogenesis are available from a previous repeated dose study of sufficient duration, e.g. a 90 day study, males of the P generation need not be included in the evaluation. Samples or digital recordings of sperm of the P generation are saved for later evaluation.

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Females of the P generation should be dosed during growth and for several complete oestrus cycles in order to detect any adverse effects on oestrus cycle normality by the test substance. The test substance is administered to parental (P) animals during their mating, pregnancies, and weaning of their F1 offspring.

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At weaning the administration of the substance is continued to Fl offspring during their growth into adulthood, mating and production of an F2 generation, until the F2 generation is weaned. Clinical observations and pathological examinations are performed on all animals for signs of toxicity on the integrity and performance of the male and female reproductive systems and on the growth and development of the offspring


DESCRIPTION OF THE METHOD / PREPARATIONS FOR THE TEST Selection of animal species:- The rat is the preferred species. If other species are used, justification should be given. Strains with low fecundity or high incidence of developmental defects should not be used. At the commencement of the study, the weight variation of animals used should not exceed 20% of the mean weight of each sex.

Housing and feeding conditions :

Housing and feeding conditions The temp :- 22 °C (+/- 3 °). relative humidity :-50-60 %. Lighting :- 12 hours light, 12 hours dark. feeding:- laboratory diets with unlimited supply of drinking water.

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Animals may be housed individually or be caged in small groups of the same sex . Mating is carried out in cages suitable for the purpose . After copulation , mated females shall be single-caged in delivery or maternity cages.

Preparation of animals :

Preparation of animals Healthy young animals, which have been acclimated to laboratory conditions for 5 days should be used. The test animals should be characterised as to species, strain, source , sex, weight and/or age. sibling relationships should be known so that mating of siblings is avoided. The animals should be randomly assigned to the control and treated groups (stratification by body weight is recommended

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Each animal should be assigned a unique identification number. Records indicating the litter of origin should be maintained for F1 animals. In addition, individual identification of pups as soon after birth as possible is recommended when individual weighing of pups or functional tests are considered.Parental (P) animals shall be about 5 to 9 weeks old at the start of dosing. The animals of all test groups shall be of uniform weight and age.


PROCEDURE Number and sex of animals Each test and control group should contain not less than 20 pregnant females. For substances that cause undesirable effects (e.g. sterility, excessive toxicity at the high dose), this may not be possible. The objective is to produce enough pregnancies to assure a meaningful evaluation of the potential of the substance to affect fertility , pregnancy and maternal behaviour and suckling, growth and development of the F1 and (F2) offsprings .

Preparation of Doses :

Preparation of Doses 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 in a suitable vehicle . T he use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles.

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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.

Dosage :

Dosage Three dose levels and a control is used. The highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering. A descending sequence of dose levels should be selected with a view to demonstrating any dosage related effect and no-observed-adverse-effects level (NOAEL) .

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Dose levels is selected on the basis of any existing toxicity data i.e.( repeated dose studies). Any available information on metabolism and kinetics of the test compound or related materials should be considered for preparing dosing regimen .

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The control group is an untreated group or a vehicle-control group .The control group shall receive the vehicle in the highest volume used. If a test substance is administered in the diet, and causes reduced dietary intake or utilisation , then the use of a paired control group may be considered necessary .

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Characteristics of vehicle and other additives to be considered are :effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.

Limit test :

Limit test The limit test applies except when human exposure indicates the need for a higher oral dose level to be used.

Administration of doses :

Administration of doses The animals are dosed with the test substance on a 7-days-a-week basis. The oral route of administration (diet, drinking water, or gavage ) is preferred. If another route of administration is used,justification shall be provided. the test substance is administered by gavage using a stomach tube. The volume of liquid administered at one time should not exceed 1 ml/l00 g body weight (0.4 ml/100 g body weight is the maximum for corn oil), except in the case of aqueous solutions where 2 ml/100 g body weight may be used

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In gavage studies, the pups will receive test substance indirectly through the mothers 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 start eating. For a substance administered by gavage , the dose should be given at similar times each day

Experimental schedules :

Experimental schedules Daily dosing of the parental (P) males and females shall begin when they are 5 to 9 weeks old . Daily dosing of the F1 males and females shall begin at weaning. For both sexes (P and F1), dosing shall be continued for at least 10 weeks before the mating period. Dosing is continued in both sexes during the 2 week mating period. For parental (P) females, dosing should continue throughout pregnancy and up to the weaning of the F1 offspring.

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weight determination. However, caution should be exercised when adjusting the dose during the last trimester of pregnancy. 20. Treatment of the P and F1 males and females shall continue until termination. All P and F1 adult males and females should be humanely killed when they are no longer needed for assessment of reproductive effects. F1 offspring not selected for mating and all F2 offspring should be humanely killed after weaning.

Mating procedure :

Mating procedure Parental (P) mating For each mating, each female shall be placed with a single male from the same dose level for 2 weeks. Each day, the females shall be examined for presence of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal plug or sperm are found . In case pairing is unsuccessful, re-mating of females with proven males of the same group could be considered. Mating pairs should be clearly identified in the data. Mating of siblings should be avoided

F1 mating :

F1 mating One male and one female should be selected at weaning from each litter for mating with other pups of the same dose level but different litter, to produce the F2 generation. The F1 offspring should not be mated until they have attained full sexual maturity. Pairs without progeny should be evaluated. Microscopic examination of the reproductive organs, and examination of the oestrous cycles or Spermatogenesis is done .


OBSERVATIONS Clinical observations G avage dose timing should be noted. Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity should be recorded . Examination of each animal should be conducted once daily when appropriate, all animals should be observed for morbidity and mortality.

Body weight and food/water consumption of parent animals :

Body weight and food/water consumption of parent animals Parental animals (P and Fl) shall be weighed on the first day of dosing and at least weekly thereafter . Parental females (P and F1) shall be weighed at a minimum on gestation days 0, 7, 14, and 20 or 21, and during lactation on the same days as the weighing of litters and on the day the animals are killed. These observations should be reported individually for each adult animal. During the premating and gestation periods food consumption shall be measured weekly at a minimum. Water consumption shall be measured weekly at a minimum if the test substance is administered in the water.

Oestrus cycle :

Oestrus cycle Oestrous cycle length and normality are evaluated in P and F1 females by vaginal smears prior to mating. When obtaining vaginal/cervical cells , care should be taken to avoid disturbance of mucosa and subsequently , the induction of pseudopregnancy

Sperm parameters :

Sperm parameters Testis and epididymis weight shall be recorded and one of each organ reserved for histopathological examination .For this same subset of males, sperm from the cauda epididymides or vas deferens should be collected for evaluation of sperm motility and sperm morphology. A morphological evaluation of an epididymal (or vas deferens) sperm sample should be performed .

Offspring :

Offspring Each litter should be examined as soon as possible after delivery (lactation day 0) to establish the number and sex of pups, stillbirths, live births, and the presence of gross anomalies. Pups found dead on day 0, if not macerated, should preferably be examined for possible defects and cause of death and preserved . Live pups should be counted and weighed individually at birth (lactation day 0) or on day 1, and on regular weigh days thereafter, e.g., on days 4, 7, 14, and 21 of lactation. Physical or behavioural abnormalities observed in the dams or offspring should be recorded.

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Other physical parameters (e.g. ear and eye opening, tooth eruption, hair growth). Functional investigations(e.g . motor activity, sensory function, reflex ontogeny)

Gross necropsy:

Gross necropsy At the time of termination or death during the study, all parental animals (P and F1), all pups with external abnormalities or clinical signs, as well as at least one randomly selected pup/sex/litter from both the F1 and F2 generation, shall be examined macroscopically for any structural abnormalities or pathological changes.

Organ weights :

Organ weights At the time of termination, body weight and the weight of the following organs of all P and F1 parental animals shall be determined: • Uterus, ovaries; • Testes, epididymides (total and cauda ); • Prostate; • Seminal vesicles with coagulating glands and their fluids (as one unit); • Brain, liver, kidneys, spleen, pituitary, thyroid and adrenal glands and known target organs.

Histopathology :

Histopathology Parental Animals H istopathological examination of following organs and tissues is recommended. Vagina , uterus with cervix, and ovaries , One testis , one epididymis , seminal vesicles, prostate,and coagulating gland;Previously identified target organ(s) from all P and F1 animals selected for mating.


DATA and REPORTING Data Data shall be reported individually and summarised in tabular form, showing for each test group and each generation the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons , 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, including time of onset, duration, and severity of any toxic effects, the types ofhistopathological changes, and all relevant litter data.

Evaluation of results :

Evaluation of results The evaluation will include the :- incidence and severity of abnormalities , gross lesions, identified target organs , affected fertility , clinical abnormalities , affected reproductive and litter performance, body weight changes , effects on mortality other toxic effects.

Test Report:

Test Report Test substance: - physical nature, purity and physicochemical properties; - identification data. Vehicle : justification for choice of vehicle, if other than water. Test animals : species/strain used; No., age and sex of animals; source, housing conditions, diet, etc.; individual weights of animals at the start of the test. justification for species if not rat


Cont…. Test conditions : rationale for dose level selection; details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation; details of administration of test substance; details of food and water quality. Optional endpoints investigated : list of optional endpoints investigated.


Cont…. Results : food consumption, and water consumption i absorption data (if available); body weight data for P and F1 animals selected for mating; litter and pup weight data; body weight at sacrifice and absolute and relative organ weight data for the parental animals; nature, severity and duration of clinical observations (whether reversible or not); time of death during the study or whether animals survived to termination;

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-toxic or other effects on reproduction, offspring, postnatal growth . - necropsy findings - detailed description of all histopathological findings; - number of P and F1 females cycling normally and cycle length; - total cauda epididymal sperm number, percent progressively motile sperm, percent morphologically normal sperm, and percent of sperm with each identified abnormality; - time-to-mating, including the number of days until mating - gestation length; - Number data on functional observations in pups and adults, as applicable; - statistical treatment of results, where appropriate.

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