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The mammalian in vivo micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts, by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents (mice or rats).

The purpose of the micronucleus test is to identify substances (liquid or solid) that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. Animals are exposed to the test substance by an appropriate route (usually by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection). Bone marrow and/or blood cells are collected, prepared and stained. Preparations are analyzed for the presence of micronuclei. Each treated and control group must include at least 5 analysable animals per sex. Administration of the treatments consists of a single dose of test substance or two daily doses (or more). The limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days.

The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian somatic cells. Structural aberrations may be of two types: chromosome or chromatid.

The in vitro chromosome aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. Cell cultures are exposed to the test substance (liquid or solid) both with and without metabolic activation during about 1.5 normal cell cycle lengths. At least three analysable concentrations of the test substance should be used. At each concentration duplicate cultures should normally be used. At predetermined intervals after exposure of cell cultures to the test substance, the cells are treated with a metaphase-arresting substance, harvested, stained. Metaphase cells are analysed microscopically for the presence of chromosome aberrations.

The Uterotrophic Bioassay is an in vivo short-term screening test. It is 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.

This Test Guideline is for an in vitro membrane barrier test method that can be used to identify corrosive substances.

The test method utilizes an artificial membrane designed to respond to corrosive substances in a manner similar to animal skin in situ. The in vitro membrane barrier test method may be used to test solids, liquids (aqueous substances with a pH in the range of 4.5 to 8.5 often do not qualify for testing) and emulsions. The test described in this Test Guideline allows the identification of corrosive chemical substances and mixtures and allows the subcategorisation of corrosive substances as permitted in the GHS. This classification is based on the substance penetration time through the membrane barrier. The test system is composed of two components, a synthetic macromolecular bio-barrier and a Chemical Detection System (which one detect the test substance). An appropriate number of replicates is prepared for each test substance and its corresponding controls. The times of applying the test substance to the membrane barrier are recorded and staggered. The time (in minutes) elapsed between application of the test substance to the membrane barrier and barrier penetration is used to predict the corrosivity of the test substance.

The test described in this Test Guideline allows the identification of corrosive chemical substances and mixtures and it enables the identification of non-corrosive substances and mixtures when supported by a weight of evidence determination using other existing information. The test protocol may also provide an indication of the distinction between severe and less severe skin corrosives. This Test Guideline does not require the use of live animals or animal tissue for the assessment of skin corrosivity.

The test material (solid or liquid) is applied uniformly and topically to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum. Two tissue replicates are used for each treatment (exposure time), and for controls. Corrosive materials are identified by their ability to produce a decrease in cell viability below defined threshold levels at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.

The Globally Harmonised System for the Classification and Labelling of Chemicals defines skin corrosion as the production of irreversible tissue damage in the skin following the application of a test material.

The test material (150 µL for liquids or solid with 150 µL of deionised water added on the top) is applied for up to 24 hours to the epidermal surfaces of skin discs (three skin discs are used for each test and control substance) in a two-compartment test system in which the skin discs function as the separation between the compartments. The skin discs are taken from humanely killed rats aged 28-30 days. Corrosive materials are identified by their ability to produce a loss of normal stratum corneum integrity and barrier function, which is measured as a reduction in the TER below a threshold level (5kΩ for rat). A dye-binding step incorporated into the test procedure permits to determine if the increase in ionic permeability is due to physical destruction of the stratum corneum.

This Test method has been designed to provide information on absorption of a test substance, (ideally radiolabelled), applied to the surface of a skin sample separating the two chambers (a donor chamber and a receptor chamber) of a diffusion cell. Static and flow-through diffusion cells are both acceptable.

Skin from human or animal sources can be used. Although viable skin is preferred, non-viable skin can also be used. The skin has been shown to have the capability to metabolise some chemicals during percutaneous absorption. In this case, metabolites of the test chemical may be analysed by appropriate methods. Normally more than one concentration of the test substance is used in typical formulations, spanning the realistic range of potential human exposures. The application should mimic human exposure, normally 1-5 mg/cm2 of skin for a solid and up to 10 µl/cm2 for liquids. The temperature must be constant because it affects the passive diffusion of chemicals. The absorption of a test substance during a given time period (normally 24h) is measured by analysis of the receptor fluid and the distribution of the test substance chemical in the test system and the absorption profile with time should be presented.

The in vivo percutaneous absorption study set out in this Test Guideline provides the linkage necessary to extrapolate from oral studies when making safety assessments following dermal exposure. The in vivo method, described in this guideline, allows the determination of the penetration of the test substance through the skin into the systemic compartment.

The test substance, preferably radiolabelled, is applied, for a fixed period of time, to the clipped skin of animals at one or more appropriate dose levels in the form of a representative in-use preparation. The rat is the most commonly used species. At least four animals of one sex should be used for each test preparation and each scheduled termination time. A known amount of the test preparation is evenly applied to the site. This amount should normally mimic potential human exposure, typically 1-5 mg/cm² for a solid or up to 10 µl/cm² for liquids. A relevant exposure period (typically 6 or 24 hours) should be used, based on the expected human exposure duration. The animals should be observed for signs of toxicity/abnormal reactions at intervals for the entire duration of the study. This study includes: daily measurements (excreta), regular detailed observations, as well as sacrifice at the scheduled time and blood collected for analysis.

A developmental neurotoxicity study will provide 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.

This Test Guideline has been designed to obtain the information necessary to confirm or to further characterise the potential neurotoxicity of chemicals in adult animals.

This Test Guideline is designed for use with the rat. It specifically addresses the daily oral administration, by gavage, (in the diet, in drinking water or by capsules) of the test substance. When the study is conducted as a separate study, at least 20 animals (10 females and 10 males) should be used in each dose. At least three dose groups and a control group should generally be used. Dose levels should be selected by taking into account any previously observed toxicity and kinetic data available for the test compound or related materials. The dosing regimen may be 28 days, subchronic (90 days) or chronic (1 year or longer). The procedures set out in this Test Guideline may also be used for an acute neurotoxicity study. The limit test corresponds to one dose level of at least 1000 mg/kg body weight. The results of this study include measurements (weighing, food /water consumption), functional tests, and, at least, daily detailed observations (Ophthalmology, haematology, clinical biochemistry and histopathology). At least five males and five females, selected from test group, should be perfused in situ and used for detailed neurohistopathology at the end of the study. The findings of the study should be evaluated in terms of the incidence, severity and correlation of neurobehavioural and neuropathological effects (neurochemical or electrophysiological effects as well if supplementary examinations are included) and any other adverse effects observed.

The test substance is administered in graduated doses to several groups of males and females. Males should be dosed for a minimum of four weeks; females should be dosed throughout the study (approximately 54 days). Normally, matings “one male to one female” should be used in this study.

This Test Guideline is designed for use with the rat. It is recommended that the test substance be administered orally by gavage. This should be done in a single dose daily to the animals using a stomach tube or a suitable intubation cannula. Each group should be started with at least 10 animals of each sex. Generally, at least three test groups and a control group should be used. Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available. The limit test corresponds to one dose level of at least 1000 mg/kg body weight. The results of this study include measurements (weighing, food/water consumption) and daily detailed observations (including sensory reactivity to stimuli), preferably each day at the same time, 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. The evaluation will include the relationship between the dose of the test substance and the presence or absence of observations. Because of the short period of treatment of the male, the histopathology of the testis and epididymus must be considered along with the fertility data, when assessing male reproduction effects.

The test substance is administered in graduated doses to several groups of males and females.

Males should be dosed for a minimum of four weeks. Females should be dosed throughout the study, so approximately 54 days. This Test Guideline is designed for use with the rat. It is recommended that each group be started with at least 10 animals of each sex. Generally, at least three test groups and a control group should be used. 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 limit test corresponds to one dose level of at least 1000 mg/kg body weight. The results of this study include measurements (weighing, food/water consumption) and daily and detailed observations, preferably each day at the same time, 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 must be considered along with the fertility data, when assessing male reproductive effects.

A principle of the method is that in the main study only moderately toxic doses are used, and the administration of doses that are expected to be lethal should be avoided.

This Guideline is intended primarily for use with rat. Groups of animals of a single sex (normally females) are dosed in a stepwise procedure using the fixed doses of 5, 50, 300 and 2000 mg/kg (exceptionally 5000 mg/kg). The initial dose level is selected on the basis of a sighting study as the dose expected to produce some signs of toxicity without causing severe toxic effects or mortality. Further groups of animals may be dosed at higher or lower fixed doses, depending on the presence or absence of signs of toxicity or mortality. This procedure continues until the dose causing evident toxicity or death is identified, or when no effects are seen at the highest dose or when deaths occur at the lowest dose. The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation canula. Animals should be fasted prior to dosing. A total of five animals of one sex will normally be used for each dose level investigated. The results of this study include: measurements (weighing at least weekly) and daily detailed observations, as well as gross necropsy. The method provides information on the hazardous properties and allows the substance to be classified for acute toxicity according to the Globally Harmonised System of classification and labelling of chemicals.

This Test Guideline is used in the assessment and evaluation of the toxic effects of organophosporus substances.

Daily doses of the test substance are administered orally (preferably by gavage or administration of gelatin capsules) to domestic laying hens (aged 8 to 12 months) for 28 days. The animals are observed at least daily until 14 days after the last dose. Biochemical measurements are undertaken on hens randomly selected from each group after the last dose. Two weeks after the last dose, the remainder of the hens are killed and histopathological examination is undertaken. The treatment group should contain at least 12 hens. Generally, at least three treatment groups should be used. The highest dose level should be chosen with the aim of inducing toxic effects; thereafter a descending sequence of dose levels should be selected. 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 weighing at least once a week, biochemistry (neuropathy target esterase, acetylcholinesterase) and, at least, and detailed observations, as well as gross necropsy and histopathology. The findings of this study should be evaluated in terms of the incidence, severity, and correlation of behavioral, biochemical and histopathological effects and any other observed effects in each of the treated and control groups.

The test substance is administered orally in a single dose to domestic hens. The animals are observed for 21 days, then the remainder of the hens are killed and histopathological examination is undertaken.

The young adult domestic laying hen (Gallus gallus domesticus), aged 8 to 12 months, is recommended. The single dosing with the test substance should normally be by the oral route using gavage, gelatine capsules, or a comparable method. The treatment group should contain, at least 12 hens, and the positive control group at least 6 hens. The objective of the preliminary study is to maximize the main study dose. The limit test corresponds to one dose level of at least 2000 mg/kg body weight/day. The dose level of the main study should be high as possible taking into account the results of preliminary study and the maximum dose level (2000 mg/kg bw/d). The results of this study include measurements (weighing), biochemistry (neuropathy target esterase) and, at least, daily and detailed observations, as well as gross necropsy and histopathology. The findings of this study should be evaluated in terms of the incidence, severity, and correlation of behavioral, biochemical and histopathological effects and any other observed effects in the treated and control groups.

This Test Guideline for two-generation reproduction testing is 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.

This Test Guideline for reproduction testing is designed to provide general information concerning the effects of a test substance (Solid, liquid, gas or vapour) on male and female reproductive performance. The test substance is administered orally in graduated doses to several groups of males and females.

Males should be dosed during growth and for at least one complete spermatogenic cycle; females of the Parent generation should be dosed for at least two complete oestrous cycles. The animals are then mated. The test substance is administered to both sexes during the mating period and thereafter only to females during pregnancy and for the duration of the nursing period. This Test Guideline is intended primarily for use with the rat or mouse. Each test and control group should contain a sufficient number of animals to yield about 20 pregnant females at or near term. Three test groups, at least, should be used. It is recommended that the test substance be administered in 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 results of this study include measurements (weighing, food consumption) and daily and detailed observations, each day preferably at the same time, as well as gross necropsy and histopathology. The findings of a reproduction toxicity study should be evaluated in terms of the observed effects, necropsy and microscopic findings. A properly conducted reproduction test should provide a satisfactory estimation of a no-effect level and an understanding of adverse effects on reproduction, parturition, lactation and postnatal growth.

This study relates to the analysis, via dermal application, of the health hazards of solid or liquid test substance. 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 substance of interest during one limited period (several hours daily during 90 days). The test substance 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. A properly conducted subchronic test should provide a satisfactory estimation of a non effect level.

This study relates to the analysis, via dermal application, of the health hazard of solid or liquid test substance.

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 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 substance of interest during one limited period (several hours daily during 21/28 days). The test substance 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 (haematology, clinical biochemistry and urinalysis), as well as 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.