TG 440 is an OECD validated in vivo short-term screening test based on the increase in uterine weight or uterotrophic response. The Uterotrophic Bioassay relies for its sensitivity on an animal test system in which the hypothalamic-pituitary-ovarian axis is not functional. Two oestrogen sensitive states in the female rodent meet this requirement: i) immature females after weaning and prior to puberty and ii) young adult females after ovariectomy with adequate time for uterine tissues to regress. The test substance is administered daily by oral gavage or subcutaneous injection. Each treated and control group should include at least 6 animals. Graduated test substance doses are administered to a minimum of two treatment groups of experimental animals using one dose level per group and an administration period of three consecutive days for immature method and a minimum administration period of three consecutive days for ovx-adult method. The animals are necropsied approximately 24 hours after the last dose. For oestrogen agonists, the mean uterine weight of the treated animal groups relative to the vehicle group is assessed for a statistically significant increase. A statistically significant increase in the mean uterine weight of a test group indicates a positive response in this bioassay. The report should include: the daily body weights, the daily record of status of animal, the wet and blotted uterine weight, the daily food consumption.

TG 441 is a OECD validated in vivo short–term screening test to evaluate the ability of a chemical to elicit biological activities consistent with androgen agonists, antagonists or 5 alpha-reductase inhibition. Weights of five androgen-dependent tissues are measured in the castrate-peripubertal male rat: the ventral prostate, seminal vesicle (plus fluids and coagulating glands), levator ani-bulbocavernosus muscle, paired Cowper’s glands and the glans penis. The test substance is administered by gavage or subcutaneous injection daily for 10 consecutive days. To test for antiandrogens, the test substance is administered together with a reference androgen agonist. Each treated and control group should include a minimum of 6 animals. The animals are necropsied approximately 24 hours after the last administration of the test substance. The tissues are excised and their fresh weights determined. A statistically significant increase (androgenic) or decrease (antiandrogenic) in the weights of two of the five tissues indicates a positive response in this assay.

TG 407 is an OECD validated method providing information on health hazard likely to arise from exposure to test chemical via oral administration. The method is based on the repeated oral administration of the chemical of interest during one limited period (one dose level daily during 28 days). This Guideline is intended primarily for use with rodents (rat preferably). At least 10 animals (5 female and 5 male) should be used for each dose level. Three tests groups, at least, should be used. The test chemical is administered by gavage or via the diet or drinking water. A limit test may be performed if no effects would be expected at a dose of 1000 mg/kg bw/d. The report of this study will include results from clinical and functional observations, body weight and food/water consumption measurements, hematology and clinical biochemistry; as well as gross necropsy and histopathology.

TG 408 is an OECD validated method providing information on health hazard likely to arise from exposure to test substance via oral administration. The determination of sub-chronic oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained from acute or repeated dose 28-day toxicity tests. The method is based on the repeated oral administration of the substance of interest over a prolonged period (one dose level daily during 90 days). This Test Guideline is intended primarily for use with rodents (rat preferably). At least 20 animals (10 female and 10 male) should be used for each test group. Three concentrations, at least, should be used.

TG 451-3 are OECD validated in vivo long term studies with the objectives of identifying carcinogenic and chronic effects, and to determining dose-response relationships following prolonged and repeated exposure. The rat is typically used for this study. For rodents, each dose group and concurrent control group intended for the carcinogenicity phase of the study should contain at least 50 animals of each sex, while for the chronic toxicity phase of the study should contain at least 10 animals of each sex. At least three dose levels should be used, in addition to the concurrent control group for both the chronic toxicity phase and the carcinogenicity phase of the study. The three main routes of administration are oral, dermal, and inhalation. The Test Guideline focuses on the oral route of administration. The period of dosing and duration of the study is normally 12 months for the chronic phase, and 24 months for the carcinogenicity phase. The study report should include: measurements (weighing) and regular detailed observations (haematological examination, urinalysis, clinical chemistry), as well as necropsy procedures and histopathology. All these observations permit the detection of neoplastic effects and a determination of carcinogenic potential as well as the general toxicity.

TG421/422 are OECD validated methods for evaluting effects of a test chemical on male and female reproductive performance. The TG is designed to be used with the rat and has been updated with endocrine disruptor endpoints, in particular measure of anogenital distance and male nipple retention in pups and thyroid examination. Males are dosed for a minimum of four weeks and females should be dosed throughout the study (~ 63 d). At least three test groups and a control group should be used, and each group should be started with >10 animals of each sex. Dose levels may be based on information from acute toxicity tests or on results from repeated dose studies. The test substance is administered orally and daily. The results of this study include clinical observations, body weight and food/water consumption, oestrous cycle monitoring, offspring parameters observation/measurement, thyroid hormone measurement, as well as gross necropsy and histopathology. The findings of this toxicity study should be evaluated in terms of the observed effects, necropsy and microscopic findings. Because of the short period of treatment of the male, the histopathology of the testis and epididymus should be considered along with the fertility data, when assessing male reproductive effects.

TG 414 is an OECD validated developmental toxicity test designed to provide general information concerning the effects of prenatal exposure on the pregnant test animal and on the developing organism. The test substance is normally administered to pregnant animals at least from implantation to one day prior to the day of scheduled kill, which should be as close as possible to the normal day of delivery. This Test Guideline is intended for use with rodent (rat preferably) and non-rodent (rabbit preferably). Each test and control group should contain a sufficient number of females to result in approximately 20 female animals with implantation sites at necropsy. Three concentrations, at least, should be used. The test substance or vehicle is usually administered orally by intubation. A limit test may be performed if no effects would be expected at a dose of 1000 mg/kg bw/d. The results of this study include measurements (weighing) and clinical daily observations, each day preferably at the same time. Shortly before caesarean section, the females are killed (one day prior to the expected day of delivery), the uterine contents are examined, and the foetuses are evaluated for soft tissue and skeletal changes. A number of endocrine-related measurements in the dams and in the fetuses have been added in 2018. In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered.

TG 426 is an OECD validated developmental neurotoxicity study providing information on the effects of repeated exposure to a substance during in utero and early postnatal development. The test substance is administered daily, generally orally, to mated females (rats are preferred) from the time of implantation (GD 6) throughout lactation (PND 21). At least three dose levels and a concurrent control should be used and a total of 20 litters are recommended at each dose level. Dams are tested to assess effects in pregnant and lactating females and may also provide comparative information. Offspring are randomly selected from within litters for neurotoxicity evaluation. All dams and all offspring should be carefully observed at least once daily with respect to their health condition, including morbidity and mortality. The evaluation consists of observations to detect gross neurologic and behavioural abnormalities, and the evaluation of brain weights and neuropathology during postnatal development and adulthood. The report should include the body weight, the food/water consumption; the detailed clinical observations, the necropsy findings, a detailed description of all behavioural, the number of animals at the start and at the end of the study and the toxic response data by sex and dose level.

TG 410 is an OECD valiated test to identify the health hazard of solid or liquid test substance following dermal application This method is composed of two tests: the main test and the limit test. This Test Guideline is intended for use with the adult rat, rabbit or guinea pig. At least 10 animals (5 female and 5 male) with healthy skin should be used for each of at least three dose levels. The highest dose level should result in toxic effects but not be fatal. The limit test corresponds to one dose level of at least 1000 mg/kg body weight. Test chemical is repeatedly applied for a limited time each day (several hours daily for 21/28 days). The test chemical should be applied over not less than 10 per cent of the body surface area. The results of this study include haematology, clinical biochemistry and urinalysis, gross necropsy and histopathology. A properly conducted 21-day or 28-day study should provide information on the effects of repeated inhalation exposure and can indicate the need for further longer term studies and provide information on the dose levels of the latter.

TG 411 is an OECD validated test to identify the health hazards of solid or liquid test substance following dermal application. It may be carried out after initial information obtained by acute testing. This method is composed of the main test and the limit test. This Test Guideline is intended for use with the adult rat, rabbit or guinea pig. At least 20 animals (10 female and 10 male) with healthy skin should be used at each dose level (at least three). The highest dose level should result in toxic effects but not produce an incidence of fatalities. The limit test corresponds to one dose level of at least 1000 mg/kg body weight. The method is based on the repeated application of the chemical of interest during a limited time each day (several hours daily during 90 days). The test chemical should be applied over not less than 10 per cent of the body surface area. The results of this study include: measurements and daily and detailed observations (ophthalmological examination, haematology, clinical biochemistry and urinalysis), as well as gross necropsy and histopathology.

TG 412 is an OECD validated test designed to fully characterize test chemical toxicity by the inhalation route following repeated exposure for 28 days, and to provide data for quantitative inhalation risk assessments. Groups of at least five male and five female rodents are exposed six hours per day for 28 days to a) the test chemical at three or more concentration levels, b) filtered air (negative control), and/or c) the vehicle (vehicle control). Animals are generally exposed five days per week but exposure for seven days per week is also allowed. Males and females are always tested, but they may be exposed at different concentration levels if it is known that one sex is more susceptible to a given test chemical. This guideline allows the study director the flexibility to include satellite (reversibility) groups, bronchoalveolar lavage (BAL), lung burden (LB) for particles, neurologic tests, and additional clinical pathology and histopathological evaluations in order to better characterize the toxicity of a test chemical.

TG 413 is an OECD validated test designed to characterize test chemical toxicity by the inhalation route following repeated exposure for a period of 90 days, and to provide data for quantitative inhalation risk assessments. Groups of at least 10 male and 10 female rodents are exposed six hours per day for 90 days to a) the test chemical at three or more concentration levels, b) filtered air (negative control), and/or c) the vehicle (vehicle control). Animals are generally exposed five days per week but exposure for seven days per week is also allowed. Males and females are always tested, but they may be exposed at different concentration levels if it is known that one sex is more susceptible to a given test chemical. The results of the study include haematology, clinical chemistry, ophthalmology, gross pathology, organ weights, and histopathology. This Test Guideline allows the flexibility to include satellite (reversibility) groups, interim sacrifices, bronchoalveolar lavage (BAL), lung burden (LB) for particles, neurologic tests, and additional clinical pathology and histopathological evaluations in order to better characterize the toxicity of a test chemical.

TG 409 is an OECD validated test providing information on health hazard likely to arise from short-term exposure to test chemical. The determination of sub-chronic oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained from acute or repeated dose 28-day toxicity tests. The method is based on the repeated oral administration of the chemical of interest over a prolonged period (one dose level daily during 90 days). This Test Guideline is intended primarily for use with non-rodents species. The commonly used non-rodent species is the dog (the beagle is frequently used). At least 8 animals (4 female and 4 male) should be used for each test group. At least three concentrations of test chemical should be used. The test chemical is administered in the diet or in the drinking water, by gavage or in capsules. A limit test may be performed if no effects would be expected at a dose of 1000 mg/kg bw/d. The results of this study include: measurements (body weight measured at least once a week, food/water consumption) and daily (preferably at the same time) and detailed observations (ophtalmological examination, haematology, clinical biochemistry and urinalysis), as well as gross necropsy and histopathology.

TG 416 is an OECD validated two-generation reproduction test designed to provide general information concerning the effects of a test substance on the integrity and performance of the male and female reproductive systems, and on the growth and development of the offspring. The test substance is administered daily in graduated doses to several groups of males and females. Males and females of the Parent generation (5-9 weeks old) should be dosed during growth, during their mating, during the resulting pregnancies, and through the weaning of their first generation offspring. The administration of the substance is continued to first generation offspring during their growth into adulthood, mating and production of a second generation (until the weaning). The rat is the preferred species for testing. 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. At least three dose levels and a concurrent control shall be used. It is recommended that the test substance be administered orally (by diet, drinking water or gavage). A limit test may be performed if no effects would be expected at a dose of 1000 mg/kg bw/d. The results of this study include: measurements (weighing, sperm parameters, oestrus cycle parameters and offspring parameters), clinical daily observations, as well as gross necropsy and histopathology. The findings of this two-generation reproduction toxicity study should be evaluated in terms of the observed effects including necropsy and microscopic findings. A properly conducted reproductive toxicity test should provide a satisfactory estimation of a no-effect level and an understanding of adverse effects on reproduction, parturition, lactation, postnatal development including growth and sexual development.

TG 443 is an OECD validated method designed to provide an evaluation of reproductive and developmental effects that may occur as a result of pre- and postnatal chemical exposure as well as an evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring. In the assay, sexually-mature males and females rodents (parental (P) generation) are exposed to graduated doses of the test substance starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups are selected and assigned to cohorts of animals for reproductive/developmental toxicity testing (cohort 1), developmental neurotoxicity testing (cohort 2) and developmental immunotoxicity testing (cohort 3). The F1 offspring receive further treatment with the test substance from weaning to adulthood. Clinical observations and pathology examinations are performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems and the health, growth, development and function of the offspring. Part of cohort 1 (cohort 1B) may be extended to include an F2 generation; in this case, procedures for F1 animals will be similar to those for the P animals.

Specific guidance is provided for in vivo screening assays validated at the national level by the US Environmental Protection Agency (the Pubertal Development and Thyroid Function Assay in Male Rats and the Pubertal Development and Thyroid Function Assay in Female Rats). These relatively short (31 days in the male, 21 days in the female) in vivo assays provide information on test chemical interaction with the sex steroid and thyroid hormone signalling pathways following administration of test chemical by oral gavage during the sensitive peri-pubertal period. Assay endpoints include the timing of reproductive advance (preputial separation or vaginal opening), organ weights, histopathology of reproductive and thyroid tissues, and thyroid hormone levels.