Investigating genotoxic and hematotoxic effects of N-nitrosodimethylamine, N-nitrosodiethylamine and N-nitrosodiethanolamine in the hen's egg-micronucleus test (HET-MN).

The hen's egg test for micronucleus induction (HET-MN) combines the use of the commonly accepted genetic endpoint "formation of micronuclei" with the well-characterized and complex model of the incubated hen's egg, which enables metabolic activation, elimination and excretion of xenobiotics including mutagens and promutagens and does not conflict with animal protection regulations and ethical aspects. N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) produced clearly positive, dose-dependent and reproducible results in this assay. NDMA revealed, in accordance with literature data, a much higher mutagenicity than NDEA. For both compounds the sensitivity of HETMN was to a large extent higher than published for the rodent micronucleus test, which is not capable of unequivocally identifying NDEA as positive. Additionally, NDEA induced severe anemia without obscuring the formation of micronucleated cells. N-nitrosodiethanolamine (NDELA), which in the literature is described as a non-mutagenic animal carcinogen, could clearly be confirmed as non-mutagenic in the HETMN without showing any disturbing effects on the formation of erythrocytes. The micronucleus frequencies of the concurrent negative controls of all experiments was in agreement with the historic negative control from 302 eggs and 412,532 cells. The same is true for the historic control of proliferation marker from 61 eggs and 13,020 cells. We interpret these results, which correspond well to published data from animal tests, as being further support for using the HET-MN as a reliable alternative genotoxicity assay system, which is physiologically closer to in vivo conditions than in vitro genotoxicity tests, and allows the observation of further local and systemic effects.

Evidence for toxic effects of alkylphenols from Ginkgo biloba in the hen's egg test (HET).

Extracts from the leaves of the Gingko tree (Ginkgo biloba L.) are therapeutically used for the treatment of peripheral and cerebral vascular disorders as well as multi-infarct or Alzheimer-type dementia. As constituents with potential contact allergenic and toxic properties in crude Ginkgo extracts a group of alkylphenols (e.g., ginkgolic acids, ginkgol, bilobol) has been described. Thus, for reasons of drug safety a maximal concentration (< or = 5 ppm) of ginkgolic acids is requested by the Monograph of the Commission E of the former German Federal Health Agency (Bundesgesundheitsamt, BGA). During production of the standardized Ginkgo extract EGb 761, alkylphenols are largely eliminated as water insoluble compounds (decanter sludge) from the primary acetone extract. To further assess the adverse properties of alkylphenols, different fractions derived from the decanter sludge were evaluated for their embryotoxic effects in the hen's egg test (HET). A fraction enriched for ginkgolic acids (16%) and biflavones (6.7%) was found to induce death of 50% of the chick embryos (LD50) at a dose of 1.8 mg/egg (approximately/= 33 ppm). A similar strong lethal effect (LD50: 3.5 mg/egg; 64 ppm) was oberserved for a fraction which contained 58% ginkgolic acids but less than 0.02% biflavones. In contrast, an extreme low toxic potential (LD50: 250 mg/egg or 4540 ppm) was established for a fraction containing 16% biflavones and 1% ginkgolic acids. Thus, the present investigations confirm the high toxic potential of ginkgolic acids, although it can not be excluded that biflavones or some other constituents in the different fractions may amplify the adverse effect of these substances. Since no contribution of alkylphenols to the therapeutic efficacy of Ginkgo extracts has been confirmed and their elimination during the manufacturing process does not cause technical problems, these results further support the requirement for the completest possible removal of these compounds under toxicological considerations.

Formation of micronuclei in incubated hen's eggs as a measure of genotoxicity.

The formation of micronuclei (MN) is a widely used and accepted endpoint of genotoxicity testing. The micronucleus assay provides a simple and rapid indirect measure of the induction of structural or numerical chromosome aberrations. In this work we describe hen's eggs, incubated for 11 days, as ex vivo assay system for the detection of micronucleus formation in young erythrocytes (Hen's Egg Test for Micronucleus Induction, HET-MN). At this stage of development the chick embryo presents a high metabolic competency which allows an adequate activation of several types of promutagens, as previously reported by several authors. As all stages of maturing erythrocytes are present in the bloodstream of the chick embryo, we could conveniently use samples of peripheral blood for scoring micronuclei as well as for determining the ratio between mature and immature erythrocytes as a measure of an undisturbed erythropoiesis. The obtained blood smears were stained by a modified May-Gruenwald-Giemsa procedure and scored microscopically. The examinations were facilitated by using a semiautomatic image analysis system. We could demonstrate a strong increase of the micronucleus frequency after the administration of the promutagens diethylnitrosamine (DENA), 7,12-dimethyl-benz[a]anthracene (DMBA), cyclophosphamide (CP), ifosphamide (IF), mitomycin C (MMC), and the direct-acting mutagen methanesulfonic acid methyl ester (MMS) compared to the concomitant negative controls. CP was used to demonstrate a dose-response relation and the effect of using two different routes of application (air cell and albumen). Nuclear aberrations, other than MN, were demonstrated after application of high doses of CP or IF. Expanded exposure times revealed a similar effect. The HET-MN, as an ex vivo assay, is a simple, inexpensive, and rapid assay system for genotoxicity testing, positioned between pure in vitro and in vivo assays, strictly in line with animal protection regulations and ethical aspects.

Environmental Specimen Banking: The Selection, Collection, Transport, and Storage or Biomedical Samples.

In order to adequately ensure the protection of human health and the environment from the thousands of presently suspected hazardous substances and the new compounds added to those by new industrial processes, sophisticated approaches to hazard assessment and monitoring are being established. Environmental specimen banking (ESB) is necessary, useful, and important for environmental monitoring currently, and in the future for monitoring the past. ESB has already proved a good lool for recording inorganic and/or organic pollution trends over the years. Moreover, ESB offers the possibilities and potentials for retrospective analysis of authentic samples from the past by improved future analytical procedures, including the detection of presently unnoticed environmental chemicals of biological interest. Among the specimens representing the environment, specimens of human origin play a key role. The selection criteria for human specimens include ethical and legal considerations together with the appropriate scientific approaches and epidemiological criteria. Technical considerations for sampling, preparation, transportation, and storage of the specimens include the selection and development of specific materials and implements, cold storage, and clean room technology in order not to compromise the original composition of the sample.

Hen's egg chorioallantoic membrane test for irritation potential.

The increasingly large number of chemicals introduced onto the market and into the environment has necessitated the monitoring of environmental materials and specimen banking, as well as the development of rapid and reliable methods for the evaluation of toxicity. The Hen's Egg Test, or Hühner-Embryonen-Test (HET) is a rapid, sensitive and inexpensive toxicity test and can give information on embryotoxicity, teratogenicity, systemic and immunopathological effects, metabolic pathways and now, in developed form, on mucous-membrane irritation potencies of chemical substances. Testing with incubated hen's eggs is a borderline case between in vivo and in vitro systems and does not conflict with ethical and legal obligations especially animal protection laws. In the special field of mucous-membrane irritation testing, a specific score and classification scheme was developed for the HET, which allows risk assessments analogous to the Draize scheme. There is a good correlation between the results for HET tests on a variety of pyrithiones, phenols and isothiazolinones, and the corresponding data based on Draize tests. HET chorioallantoic membrane testing should and could not entirely replace current irritation tests in mammals, but it can diminish the number of investigations with mammals, as well as limit or eliminate pain and injury during animal experiments and allow regulators to set priority and toxicity categories.