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.

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.

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.

During 1988–1992, a validation study was carried out in Germany on the capacity of two in vitro tests to replace the Draize eye test for severely eye irritating chemicals, namely, the hen's egg chorio-allantoic membrane (HET-CAM) test and the 3T3 cell neutral red uptake (NRU) cytotoxicity test, which had shown promising results in an earlier test development project. The formal validation study, which was coordinated by Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET) and funded by the German Department of Research and Technology (BMBF), was conducted in two phases: Phase I consisted of a prevalidation study and a blind trial (1988–1990); and Phase II was the database development phase (1991/1992). During prevalidation, the two in vitro tests were established in 13 laboratories, standard protocols were developed, including PC-based software programs for data recording, and 34 chemicals backed by high quality literature data were selected for the ring trial. In the 1-year ring trial, the two in vitro tests were validated with 34 coded chemicals under blind conditions in 13 laboratories, to evaluate the reproducibility of the two tests within and among laboratories. In the blind trial, the 3T3 NRU cytotoxicity test showed a better reproducibility than the HET-CAM test, but compared to the cytotoxicity test, the HET-CAM test permitted a significantly better classification of severely eye irritating chemicals, which are labelled R41 according to EU regulations. Since it was recommended in 1990 by the first Amden validation workshop that a database of around 200 chemicals is required for the assessment of test performance to reach regulatory acceptance at the international level, a 2-year database development was conducted as Phase II, during which 166 coded chemicals were tested in the two in vitro tests, each of them in two laboratories. Test chemicals backed by high-quality Draize eye test data were provided by industry and selected to represent a wide spectrum of chemical classes and eye irritation properties. Independent quality control of in vitro and in vivo data and biostatistical evaluation were performed during an additional BMBF project on biostatistics. In the quality assurance step, which is an essential prerequisite for biostatistics, the number of chemicals was reduced to 143, and these data were entered into an MS-EXCEL database to facilitate determination of in vitro/in vivo correlations. Unexpectedly, the evaluation of the study had to take into account a change of criteria within the EU for classifying severely eye irritating chemicals as R41, since irreversible damage within a 21-day observation period was introduced as a new criterion for R41 chemicals. The results of the 3T3 NRU cytotoxicity test showed an insufficient in vitro/in vivo correlation for classifying R41 chemicals. Classification of HET-CAM data was also insufficient in the Bundesgesundhütsamt (BGA) scoring system, which uses an empirically developed weighted scoring of the three endpoints, namely, haemorrhage, lysis and coagulation. Discriminant analysis of ten endpoints routinely determined in the HET-CAM test and in the 3T3 NRU cytotoxicity test revealed that the detection time of coagulation, the most severe reaction on the CAM, was significantly better suited to identifying severely eye irritating properties than any other endpoint, and better than the BGA score for the HET-CAM test. For water-soluble chemicals (mean time for detection of coagulation [mtc]10), the detection time for coagulation of a 10% solution had the highest discriminant power, and for less water-soluble chemicals (mtc100), the detection time of coagulation of the undiluted chemical was more appropriate. Discriminant analysis of the combination of mtc10 and mtc100 with other endpoints of the two in vitro tests revealed that classification of water-soluble chemicals is significantly improved by combining mtc10 and lgfg50m (logarithm of IC50 value calculated with the Fit-Graph program), the endpoint of the 3T3 NRU cytotoxicity test. Further analysis of data from Phase I and Phase II of the study demonstrated that chemicals characterised by an mtc10 of < 50 seconds can be labelled R41 without any false positive classifications. By using this cut-off point, around 25% of R41 chemicals can be classified without further testing in vitro or in vivo. Classification was further improved when solubility in water and oil was taken into account. The best classification of water-soluble R41 chemicals (> 10%) was obtained when the mtc10 of the HET-CAM test and the lgfg50m of the 3T3 NRU cytotoxicity test were combined. For chemicals soluble in oil (> 10%) and for insoluble chemicals, the mtc100 provided the best classification. The in vitro classification results were confirmed by cross-validation. These promising results allowed a sequential approach to be developed for classifying severely eye irritating chemicals as R41 according to EU regulations by combining the HET-CAM test and the 3T3 NRU cytotoxicity test results. The present study suggests that severely eye irritating chemicals can be classified as R41 with a sufficiently high level of confidence with the two in vitro tests, since the percentage of false positive and false negative results are kept within an acceptably low range. Thus, the combined use of the HET-CAM test and the 3T3 NRU cytotoxicity test meets the requirements for “well-validated” tests, as defined in the escape clause of OECD Guideline 405 for eye irritation testing.

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.

The Hen's Egg Test (HET) is well known as a basic test for embryotoxicity and for special aspects of systemic toxicity and immunopathology. The HET embryotoxicity test has been extended and standardized as the HET-chorioallantoic membrane (CAM) test for membrane irritation. The CAM of fertile White Leghorn eggs incubated for 10 days is a vital vascular membrane, in which irritant effects can be observed following exposure to liquid or solid test materials. The HET-CAM effects are scored and classified to give risk assessments analogous to those of the Draize rabbit-eye test. There is good correlation between classifications based on HET-CAM observations (including electron microscopy) and reported data from Draize tests.

Ever since that industrial revolution, man has been subjecting the earth’s biosphere to an increasing variety of chemical insults [5]. The increasingly large number of chemicals introduced in the market and also in environment each year has necessitated the monitoring of environmental materials and specimen banking as well as the development of rapid and reliable methods for the evaluation of their toxicity [7]. Furthermore it must be realized that many new agents have been and are now being introduced without adequate or any toxicological evaluation. Toxicologic studies of all these chemicals by the “usual” methods using laboratory animals, especially small rodents, are very difficult and expensive in time and money, and such studies sometimes give inconclusive results; ethical and legal issues (e.g. animal protection laws) must also be taken into account [1].

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 (≤ 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 (≅ 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.

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.

A chemical pollutant is a chemical substance (inorganic or organic) inserted in ecosystems which cause adverse effects, e. g. disturbance, damage or even fatality to the system or for single elements of it. The same chemical could have also beneficial effects in other circumstances. Physical agents, e. g. heat, are considered only insofar here as they modify the effect of chemical pollutants. Chemical pollutants are frequently anthropogenic but sometimes are also natural, or both.

This chapter covers the various types of programmes that can be initiated, the concepts central to the design of a programme, practical organizational aspects, and the advantages of collaborative programmes at international level.