Rodents standard toxicity tests

Studies investigating hormonal activity revealed the oestrogenic activity of short-chain NPE in a number of test systems using either recombinant yeast, oestrogen-sensitive MCF-7 cells [98] or a rodent uterotrophic assay response. None of these assays have yet been validated as an internationally accepted toxicity test method, although the MCF-7 and uterotrophic assays have been established for a number of years as standard assays for oestrogenic activity. 

The standard repeated dose toxicity guidehne studies include a number of parameters relevant for the evaluation of a substance s immunotoxic potential. While some information on potential immunotoxic effects may be obtained from the evaluation of hematology, lymphoid organ weights, and histopathology in these studies, there are data which demonstrate that these endpoints alone are not sufficient to predict immunotoxicity. In addition to these standard studies, the US-EPA has developed a specific test guideline for immunotoxicity testing in rodents (OPPTS 870.7800). This... 

Animals, usually rodents, are exposed to the test substance by an appropriate route, usually by gavage or by intraperitoneal injection. Each treated and control group must include at least five animals per sex. Positive controls should produce micronuclei in vivo at exposure levels expected to give a detectable increase over background. No standard treatment schedule (i.e., 1, 2, or more treatments at 24-h intervals) has been recommended. Three dose levels are generally used these should cover a range from the maximum to little or no toxicity. The erythrocytes are sampled from the bone marrow and/or peripheral blood of the animals. If bone marrow is used, the animals are sacrificed at appropriate times after treatment, the bone marrow extracted, and preparations made and stained. When peripheral blood is used, the blood is collected at appropriate times after treatment and smear preparations are made and stained. Preparations are analyzed for the presence of micronuclei. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. 

These segments can be tested separately or in a combined manner. All stages of development from conception to maturity and the detection of acute and delayed effects of exposure through one complete life cycle should be examined. The standard species are rodents, rats as the preferred rodent species for all study types and, the rabbit as the second nonrodent species for the embryo-toxicity studies. In some rare cases mice or monkeys are used too, if special conditions - usually kinetic data - justify such species. 

In all reproduction toxicity studies at least three doses (minimally toxic, intermediate, non-toxic) should be given, preferrably by the proposed clinical route of application. Fertility and peri- and postnatal toxicity studies require tests in only one species (usually rats or mice), the teratology segment must be tested in two species (e.g. rodents and rabbits). Pharmacokinetic data and differences in the placentation may suggest other modes of application and/or other test species, e.g. primates. Of course the minimum animal numbers for the standard tests do not apply to studies in primates. 

It is conceivable that some excipients may not require the standard 2-year, two rodent species carcinogenicity studies. Such excipients include those that are not absorbed (or are rapidly metabolized and/or rapidly excreted), that do not exhibit toxicity in 90-day studies, and those that are negative for genotoxi-city. This is the approach taken by the IPEC-Americas Safety Committee and one of the reasons that the 1996 peer-reviewed journal publication indicates that the conduct of rodent carcinogenicity studies is conditional. The carcinogenicity studies that are conditional are the traditional 50 animals/sex/group rodent studies conducted for 18 or 24 months or variations thereof. The decision to make these tests conditional was also... 

The EPA and PDA state that there is no standard treatment schedule therefore, one, two, or more doses every 24 h is acceptable as long as toxicity has been demonstrated, or that a limit dose has been achieved. In addition, if a large volume of material needs to be administered to the rodents, it may be administered as a divided dose, as long as the doses are not separated by more than a few hours. Doses may be selected from the results of a range finding study, or any preliminary tests done with the rodents after administration via the same route. The highest dose selected for the main assay would produce some bone marrow toxicity, including, in the bone marrow or peripheral blood, a decreased number of immature erythrocytes to total erythrocytes. In the case of a nontoxic test article, the limit dose is defined as 2000 mg kg... 

---