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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.

Dominant lethal (DL) effects cause embryonic or foetal death. Induction of a dominant lethal event after exposure to a test substance (liquid, solid, vapour or gas, …) indicates that the substance has affected germinal tissue of the test species. Dominant lethals are generally accepted to be the result of chromosomal aberrations (structural and numerical anomalies), but gene mutations and toxic effects cannot be excluded.

This Test Guideline recommends rats or mice as the test species. Generally, male animals are exposed to the test substance and mated to untreated virgin females. The most widely used is the single administration of the test substance by oral or by intraperitoneal injection. Normally, three dose levels should be used. The various germ cell stages can be tested separately by the use of sequential mating intervals. The females are sacrificed after an appropriate period of time, and the contents of the uteri are examined to determine the numbers of implants and live and dead embryos. The calculation of the dominant lethal effect is based on comparison of the live implants per female in the treated group to the live implants per female in the control group.

This Test Guideline includes two methods: a paper contact toxicity test and an artificial soil test. The recommended specie is Eisenia foetida (Michaelsen).

The initial screening test (filter paper contact test) involves exposing earthworms to test substances on moist filter paper in order to identify potentially toxic chemicals to earthworms in soil. Five or more treatment levels in a geometric series and, at least, ten replicates (one worm per vial) for each treatment should be used. Tests are done in the dark and for a period of 48 hours. The artificial soil test gives toxicity data more representative of natural exposure of earthworms to chemicals. It involves keeping earthworms in samples of a precisely defined artificial soil. Five concentrations, in a geometric series, of the test substance have been applied. One concentration resulting in no mortality and one resulting in total mortality should be used. Four replicates for each treatment are recommended. Mortality is assessed 7 and 14 days after application.

The purpose of this Test Guideline is to determine the effects of a substance administered with food to birds.

Birds are fed a diet containing the test substance at a range of concentrations for a period of five days. Two control groups and one treatment group for each of the, at least, five dietary levels of the test substance should be used. Each group consists of 10 birds. The minimum duration of the test is eight days: five days on the test diet followed by a minimum of three days on normal diet. Suitable facilities for holding birds indoors are necessary. These include mechanisms for temperature, humidity and light control as required, as well as pens of suitable capacity for rearing the birds. Mortalities and signs of toxicity are recorded daily. The following observations should be made during the test: signs of intoxication and other abnormal behaviour; mortality; body weights; food consumption.

The purpose of this Test Guideline is to determine the effects on the reproduction of a substance administered with food to birds.

Birds are fed a diet containing the test substance in various concentrations for a period of not less than 20 weeks. A minimum of three dietary concentrations of the test substance is required. The maximum recommended test concentration is 1000 ppm. Birds may be kept in pens as pairs (at least 12 pens per test group) or as groups of one male and two or three females (at least 8-12 pens per group). Birds are induced, by photoperiod manipulation, to lay eggs. Eggs are collected over a ten-week period, artificially incubated and hatched, and the young maintained for 14 days. Suitable facilities for rearing birds, preferably indoors, are necessary. Mortality of adults, egg production, cracked eggs, egg shell thickness (at least two eggs from each pen), viability, hatchability and effects on young birds are observed during the study.

The mouse heritable translocation test detects structural and numerical chromosome changes in mammalian germ cells as recovered in first generation progeny.

The types of chromosome changes detected in this test system are reciprocal translocations. Carriers of translocations and XO-females show reduced fertility which is used to select first generation progeny for cytogenetic analysis. Translocations are cytogenetically observed in meiotic cells at diakinesis metaphase I of male individuals. The test is usually performed by analysis of male first generation progeny. About 500 first generation males per dose level are required. One dose level is tested, usually the highest dose associated with the production of minimal toxic effects, and administered by oral intubation or intraperitoneal injection. A single administration of the test substance or the administration of the test substance on 7 days/week for 35 days, are possible. The test substance can be solid, liquid, vapour or gaseous. For translocation heterozygosity one of two possible methods is used: fertility testing of first generation progeny; or cytogenetic analysis of all male first generation progeny are possible. A test substance producing neither a statistically significant increase in the number of translocations observed for at least one test point, nor a statistically significant, dose-related, increase in the number of translocations observed, is considered non-mutagenic in this system.