Use Of Animals In Testing Research Articles

QSAR development and bioavailability determination: The toxicity of chloroanilines to the soil dwelling springtail Folsomia candida

Quantitative structure–activity relationships (QSARs) are an established tool in environmental risk assessment and a valuable alternative to the exhaustive use of test animals under REACH. In this study a QSAR was developed for the toxicity of a series of six chloroanilines to the soil-dwelling collembolan Folsomia candida in standardized natural LUFA2.2 soil. Toxicity endpoints incorporated in the QSAR were the concentrations causing 10% (EC10) and 50% (EC50) reduction in reproduction of F. candida. Toxicity was based on concentrations in interstitial water estimated from nominal concentrations in the soil and published soil–water partition coefficients. Estimated effect concentrations were negatively correlated with the lipophilicity of the compounds. Interstitial water concentrations for both the EC10 and EC50 for four compounds were determined by using solid-phase microextraction (SPME). Measured and estimated concentrations were comparable only for tetra- and pentachloroaniline. With decreasing chlorination the disparity between modelled and actual concentrations increased. Optimisation of the QSAR therefore could not be accomplished, showing the necessity to move from total soil to (bio)available concentration measurements.

Studying the effects of genistein on gene expression of fish embryos as an alternative testing approach for endocrine disruption

Assessment of endocrine disruption currently relies on testing strategies involving adult vertebrates. In order to minimize the use of animal tests according to the 3Rs principle of replacement, reduction and refinement, we propose a transcriptomics and fish embryo based approach as an alternative to identify and analyze an estrogenic activity of environmental chemicals. For this purpose, the suitability of 48h and 7days post-fertilization zebrafish and medaka embryos to test for estrogenic disruption was evaluated. The embryos were exposed to the phytoestrogen genistein and subsequently analyzed by microarrays and quantitative real-time PCR. The functional analysis showed that the genes affected related to multiple metabolic and signaling pathways in the early fish embryo, which reflect the known components of genistein's mode of actions, like apoptosis, estrogenic response, hox gene expression and steroid hormone synthesis. Moreover, the transcriptomic data also suggested a thyroidal mode of action and disruption of the nervous system development. The parallel testing of two fish species provided complementary data on the effects of genistein at gene expression level and facilitated the separation of common from species-dependent effects. Overall, the study demonstrated that combining fish embryo testing with transcriptomics can deliver abundant information about the mechanistic effects of endocrine disrupting chemicals, rendering this strategy a promising alternative approach to test for endocrine disruption in a whole organism in-vitro scale system.

Ethical and scientific considerations regarding animal testing and research.

One of the most influential attempts to examine and affect the use of animals in research can be traced back to1959, with the publication of The Principles of Humane Experimental Technique [1]. William Russell and Rex Burch published this seminal book in response to marked growth in medical and veterinary research and the concomitant increase in the numbers of animals used. Russell and Burch's text emphasized reduction, refinement, and replacement of animal use, principles which have since been referred to as the “3 Rs”. These principles encouraged researchers to work to reduce the number of animals used in experiments to the minimum considered necessary, refine or limit the pain and distress to which animals are exposed, and replace the use of animals with non-animal alternatives when possible. Despite the attention brought to this issue by Russell and Burch, the number of animals used in research and testing has continued to increase. Recent estimates suggest that at least 100 million animals are used each year worldwide [2]. However, this is likely an underestimate, and it is impossible to accurately quantify the number of animals used in or for experimentation. Full reporting of all animal use is not required or made public in most countries. Nevertheless, based on available information, it is clear that the number of animals used in research has not significantly declined over the past several decades. The “3 Rs” serve as the cornerstone for current animal research guidelines, but questions remain about the adequacy of existing guidelines and whether researchers, review boards, and funders have fully and adequately implemented the “3 Rs”. Further, while the “3 Rs” capture crucially important concepts, they do not adequately reflect the substantial developments in our new knowledge about the cognitive and emotional capabilities of animals; an updated understanding of the harms inherent in animal research; and the changing cultural perspectives about the place of animals in society [3], [4]. In addition, serious questions have been raised about the effectiveness of animal testing and research in predicting anticipated outcomes [5]–[13]. In August 2010, the Georgetown University Kennedy Institute of Ethics, the Johns Hopkins University Center for Alternatives to Animal Testing, the Institute for In Vitro Sciences, The George Washington University, and the Physicians Committee for Responsible Medicine jointly held a two day multi-disciplinary, international conference in Washington, DC, to address the scientific, legal, and political opportunities and challenges to implementing alternatives to animal research. This two-day symposium aimed to advance the study of the ethical and scientific issues surrounding the use of animals in testing and research, with particular emphasis on the adequacy of current protections and the promise and challenges of developing alternatives to the use of animals in basic research, pharmaceutical research and development, and regulatory toxicology. Speakers who contributed to the conference reviewed and contributed new knowledge regarding the cognitive and affective capabilities of animals, revealed through ethology, cognitive psychology, neuroscience, and related disciplines. Speakers also explored the dimensions of harm associated with animal research, touching on the ethical implications regarding the use of animals in research. Finally, several contributors presented the latest scientific advances in developing alternatives to the use of animals in pharmaceutical research and development and regulatory toxicity testing. This Collection combines some papers that were written following this conference with an aim to highlight relevant progress and research. This Overview provides a brief summary of the ethical and scientific considerations regarding the use of animals in research and testing, some of which are highlighted in the accompanying Collection.

Application of the consistency approach to reduce animal use in vaccine potency testing

Biologics are usually produced from live organisms, and the manufacturing process often involves a degree of natural variability. Characterization of biologics such as vaccines is inherently difficult due to the complex molecular structure of the antigens they contain and the presence of excipients such as preservatives and adjuvants that can interfere with testing. Therefore, each batch, lot, or serial produced must be tested before market release to ensure that the product complies with regulatory standards. This batch release testing emphasizes quality control of the final product and may be characterized by an extensive use of laboratory animals. The consistency approach is based upon the principle that the quality of a biologic is the result of the strict application of a quality system and consistent production. Subsequent batches are determined to be similar to clinically evaluated batches and therefore acceptable for release through the in-process testing that comprises this quality system. The European Centre for Validation of Alternative Methods (ECVAM) organized international workshops in 2006 and 2010 to discuss the consistency approach and its potential to reduce the number of animals used in testing of biological products. This paper provides an overview of these workshops.

In vitro cytokine release from human peripheral blood mononuclear cells in the assessment of the immunotoxic potential of chemicals

Alternative methods to the use of animals in testing of chemicals are needed. We investigated if the immunotoxic potential of 12 dietary toxicants could be predicted from effects on cytokine release from human peripheral blood mononuclear cells (PBMC) after in vitro exposure. Nine cytokines were selected to reflect different types of immune responses. The toxicants were classified as immunotoxic or non-immunotoxic substances according to the published in vivo data.Isolated human PBMC were exposed for 20h to three concentrations of each of the 12 substances in the presence of human liver S9 fraction. After further incubation of PBMC in fresh medium containing the mitogen phytohemagglutinin (PHA, 10μg/ml) for 48h, release of the nine selected cytokines into the supernatant as well as cell proliferation were measured by Luminex technology™ and the BrdU incorporation assay, respectively.All 12 substances investigated affected the release of one or more cytokines, and each of the substances showed different cytokine release patterns. Within the limitations of the study design, the present study suggests that the effect of the substances on mitogen-induced cytokine release from PBMC cannot predict their immunotoxic potential, but may be useful in mechanistic studies.

Bioaccessibility studies of ferro‐chromium alloy particles for a simulated inhalation scenario: A comparative study with the pure metals and stainless steel

The European product safety legislation, REACH, requires that companies that manufacture, import, or use chemicals demonstrate safe use and high level of protection of their products placed on the market from a human health and environmental perspective. This process involves detailed assessment of potential hazards for various toxicity endpoints induced by the use of chemicals with a minimum use of animal testing. Such an assessment requires thorough understanding of relevant exposure scenarios including material characteristics and intrinsic properties and how, for instance, physical and chemical properties change from the manufacturing phase, throughout use, to final disposal. Temporary or permanent adverse health effects induced by particles depend either on their shape or physical characteristics, and/or on chemical interactions with the particle surface upon human exposure. Potential adverse effects caused by the exposure of metal particles through the gastrointestinal system, the pulmonary system, or the skin, and their subsequent potential for particle dissolution and metal release in contact with biological media, show significant gaps of knowledge. In vitro bioaccessibility testing at conditions of relevance for different exposure scenarios, combined with the generation of a detailed understanding of intrinsic material properties and surface characteristics, are in this context a useful approach to address aspects of relevance for accurate risk and hazard assessment of chemicals, including metals and alloys and to avoid the use of in vivo testing. Alloys are essential engineering materials in all kinds of applications in society, but their potential adverse effects on human health and the environment are very seldom assessed. Alloys are treated in REACH as mixtures of their constituent elements, an approach highly inappropriate because intrinsic properties of alloys generally are totally different compared with their pure metal components. A large research effort was therefore conducted to generate quantitative bioaccessibility data for particles of ferro-chromium alloys compared with particles of the pure metals and stainless steel exposed at in vitro conditions in synthetic biological media of relevance for particle inhalation and ingestion. All results are presented combining bioaccessibility data with aspects of particle characteristics, surface composition, and barrier properties of surface oxides. Iron and chromium were the main elements released from ferro-chromium alloys upon exposure in synthetic biological media. Both elements revealed time-dependent release processes. One week exposures resulted in very small released particle fractions being less than 0.3% of the particle mass at acidic conditions and less than 0.001% in near pH-neutral media. The extent of Fe released from ferro-chromium alloy particles was significantly lower compared with particles of pure Fe, whereas Cr was released to a very low and similar extent as from particles of pure Cr and stainless steel. Low release rates are a result of a surface oxide with passive properties predominantly composed of chromium(III)-rich oxides and silica and, to a lesser extent, of iron(II,III)oxides. Neither the relative bulk alloy composition nor the surface composition can be used to predict or assess the extent of metals released in different synthetic biological media. Ferro-chromium alloys cannot be assessed from the behavior of their pure metal constituents.

Animal Use in the Chemical and Product Manufacturing Sectors — Can the Downtrend Continue?

During the 1990s and early 2000s, a number of manufacturing companies in the cosmetic, personal care and household product industries were able to substantially reduce their use of animals for testing (or to not use animals in the first place). These reductions were almost always the result of significant financial contributions to either direct, in-house alternatives research, or to support personnel whose duties were to understand and apply the current state-of-the-art for in vitro testing. They occurred almost exclusively in non-regulatory areas, and primarily involved acute topical toxicities. Over the last few years, the reduction in animal use has been much less dramatic, because some companies are still reluctant to change from the traditional animal studies, because systemic, repeat-dose toxicity is more difficult to model in vitro, and because many products still require animal testing for regulatory approval. Encouragingly, we are now observing an increased acceptance of non-animal methods by regulatory agencies. This is due to mounting scientific evidence from larger databases, agreement by companies to share data and testing strategies with regulatory agencies, and a focus on smaller domains of applicability. These changes, along with new emphasis and financial support for addressing systemic toxicities, promise to provide additional possibilities for industry to replace animals with in vitro methods, alone or in combination with in silico methods. However, the largest advance will not occur until more companies commit to using the non-animal test strategies that are currently available.

Occludin gene expression as an early in vitro sign for mild eye irritation assessment

Purpose To test a new multiple endpoint analysis (MEA) including occludin gene expression for screening the ocular irritation potential of tear substitutes on human corneal epithelium (HCE), an in vitro model proposed to limit the use of animal testing in pre-clinical studies. Methods Four chemically-preserved and two non chemically-preserved tear substitutes were tested after acute (24 h, 24 h + 24 h post incubation) and repeated applications (for 72 h) and compared to the positive control, benzalkonium chloride (BAK) at 0.1% and 0.01%, by assessing complementary parameters. Cellular viability was evaluated using MTT, histomorphologic analysis was performed on H&E stained vertical sections, IL-8 release was measured by ELISA, and occludin gene expression was quantified using qRT-PCR. Results Cellular viability was moderately reduced by Perborate and Polyquad-preserved tear substitutes and dramatically reduced by BAK and by Thiomersal ® and Oxyd ® preserved tear substitutes. Thiomersal ® also increased IL-8 release. Occludin expression profiles were modified by the four chemically-preserved tear substitutes and by the mechanically-preserved Comod ®, but not by the mechanically-preserved Abak ®. The behavior of BAK and tear substitutes led us to propose a prediction model for the classification of different levels of irritants, mainly based on the occludin transcriptional study. Conclusion The versatility and sensitivity of the HCE model allowed the modeling of cumulative effects that may approach conditions obtained after long term application of tear substitutes. Thus, the modified MEA proposed in this study represents a valuable tool for in vitro eye irritation assessment with the power to detect mild irritants and subclinical eye irritant potential.

The human skin/chick chorioallantoic membrane model accurately predicts the potency of cosmetic allergens

The current standard method for predicting contact allergenicity is the murine local lymph node assay (LLNA). Public objection to the use of animals in testing of cosmetics makes the development of a system that does not use sentient animals highly desirable. The chorioallantoic membrane (CAM) of the chick egg has been extensively used for the growth of normal and transformed mammalian tissues. The CAM is not innervated, and embryos are sacrificed before the development of pain perception. The aim of this study was to determine whether the sensitization phase of contact dermatitis to known cosmetic allergens can be quantified using CAM-engrafted human skin and how these results compare with published EC3 data obtained with the LLNA. We studied six common molecules used in allergen testing and quantified migration of epidermal Langerhans cells (LC) as a measure of their allergic potency. All agents with known allergic potential induced statistically significant migration of LC. The data obtained correlated well with published data for these allergens generated using the LLNA test. The human-skin CAM model therefore has great potential as an inexpensive, non-radioactive, in vivo alternative to the LLNA, which does not require the use of sentient animals. In addition, this system has the advantage of testing the allergic response of human, rather than animal skin.

Cosmetic Safety: Proposal for the Replacement of In Vivo (Draize) by In Vitro Test

The procedures described by Draize have been criticized for ethical reasons. Thus, a comparative study was performed between ocular and cutaneous irritation tests, using rabbits and in vitro test through agar diffusion with the use of NCTC clone 929, FPC-IAL and SIRC cell lines. The results obtained revealed that the agar diffusion test positive sam- ples, which presented up to degree 3 reactivity rate, according to USP 31, did not provoke ocular or cutaneous irritation. The samples which presented reactivity grade 4 also showed different degrees of ocular and cutaneous irritation, with the exception of two units of liquid soap for children use. According to the data, the diffusion agar method, using the Ameri- can Pharmacopeia graduation, can be adopted as a sorting procedure in the evaluation of cosmetics. This is a result of its capacity of predicting irritation, what largely contributes for a decrease in the use of animals in tests.

An Integrated Decision-tree Testing Strategy for Skin Sensitisation with Respect to the Requirements of the EU REACH Legislation

This report presents some of the results of a joint research project, sponsored by Defra and conducted by FRAME and Liverpool John Moores University, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This report focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for skin sensitisation testing. The manuscript reviews in vitro tests based on protein-ligand binding, dendritic/Langerhans cells and T-lymphocyte activation, and also the QSAR models and expert systems available for this endpoint. These tests are then incorporated into an integrated, decision-tree testing strategy, which also includes the Local Lymph Node Assay (in its original and new reduced protocols) and the traditional guinea-pig tests (which should only be used as a last resort). The aim of the strategy is to minimise the use of animals in testing for skin sensitisation, while satisfying the scientific and logistical demands of the EU REACH legislation.

REACH and the HSE Case for Formate Brines

This article, written by Assistant Technology Editor Karen Bybee, contains highlights of paper IPTC 11222, "REACH and the HSE case for Formate Brines," by Ylva Gilbert, SPE, Gaia Consulting; Adrian Nordone, Cabot Corporation; John Downs, Cabot Speciality Fluids; and Anu Vaahtera, Piia Pessala, and Tuomas Raivio, Gaia Consulting, originally prepared for the 2007 International Petroleum Technology Conference, Dubai, UAE, 4-6 December. The paper has not been peer reviewed. Europe's largest single piece of legislation, Registration, Evaluation, and Authorization of Chemicals (REACH), went into effect on 1 June 2007. REACH requires manufacturers, importers, and users of chemicals to demonstrate that their products are safe to use for humans and the environment. There will be increasing pressure to substitute less-hazardous materials for chemicals perceived as potentially harmful. The legislation is expected to have a significant effect on the management and application of chemicals used by the oil industry in Europe. Introduction The new European chemical legislation, REACH, represents a major revision of European chemicals management, replacing some 40 existing European Union (EU) Directives and Regulations. An objective of REACH is to place the responsibility of demonstrating the safety of chemicals on the manufacturers, importers, and users of the chemical. At the same time, the aim is to minimize the use of animal testing and encourage the substitution principle. The legislation encompasses all chemicals or chemical substances, including oilfield chemicals such as drilling and completion fluids. The full-length paper details the effect that REACH will have on management decisions and compliance procedures related to ensuring safe use of chemicals. Methodology The work presented in the full-length paper is based on a review of the literature and legislation as it applies to oilfield well-construction operations using formate brines as example chemicals throughout the discussion. Where comparisons with traditional brines are made, the data mainly have been sourced from open literature, and there are many gaps. For the formates, the authors also have had access to previously unpublished test results from the manufacturers. The findings from the literature have been complemented with information on the practical challenges of meeting REACH requirements, gleaned from discussions with four major operators. To set the scene, the following section introduces the case-study chemicals. This is followed by a brief overview of two means of ranking chemicals on the basis of regulatory data. REACH and its processes as well as the time frame of implementation are then presented, followed by an overview of data requirements to support REACH. The findings then are discussed from a management point of view, as illustrated with formate brines.

An Integrated Decision-tree Testing Strategy for Skin Sensitisation with Respect to the Requirements of the EU REACH Legislation

This report presents some of the results of a joint research project, sponsored by Defra and conducted by FRAME and Liverpool John Moores University, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This report focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for skin sensitisation testing. The manuscript reviews in vitro tests based on protein-ligand binding, dendritic/Langerhans cells and T-lymphocyte activation, and also the QSAR models and expert systems available for this endpoint. These tests are then incorporated into an integrated, decision-tree testing strategy, which also includes the Local Lymph Node Assay (in its original and new reduced protocols) and the traditional guinea-pig tests (which should only be used as a last resort). The aim of the strategy is to minimise the use of animals in testing for skin sensitisation, while satisfying the scientific and logistical demands of the EU REACH legislation.

Working with other Partners — the Retail Sector

Animal testing to demonstrate the safety of cosmetic and toiletries has been used for many years. However, such testing, now viewed by many consumers as an emotive issue, has been banned within the UK, and is severely restricted in the European Union. Consumer knowledge and understanding have been challenged by the use of focus group studies, which showed that consumers were often confused and potentially misled by claims on labels and in leaflets. Despite the fact that some pressure groups had taken positive action, even holding store demonstrations, in attempts to stop such testing or claims, there was still a legal requirement to demonstrate the safety of products, and this involved the use of animal testing. Nevertheless, there was great pressure to move away from animal testing, clarify marketing and pack claims, and provide general transparency to the consumer. Therefore, the decision was taken to actively seek out and work with organisations which were working toward the validation of alternative testing. A number of potential organisations were approached and, after careful consideration of their respective aims and aspirations, it was decided that, for the retailer in question, FRAME was the most appropriate organisation with which to work.

The case for mandatory inclusion of ethics within the zoological sciences curriculum

Traditionally, undergraduate science curricula include little or no "ethics," either as theory or practice. However, zoologists are currently enjoying considerable media exposure: some of it positive (as in conservation practice), but more often negative (pertaining to issues such as the use of animals for testing of drugs, and genetic engineering). More than ever before, zoologists are being asked to make value judgments, and many of these involve moral assessment; if we accept that zoologists (along with other scientists) are professionals, then we must accept that they are responsible for any decisions they make, and it then follows that they are accountable, which can have serious ramifications in cases of malpractice. Ethics involves the application of morality in a professional setting. In light of this, teaching ethics is mandatory in degree programs such as engineering and medicine. This paper contends that a key output of zoological education is the undergraduate who is cognizant of the ethical framework and constructs within which he/she must function. The paper concludes with comment on the nature and style of delivery of ethics education.

Assessing the aquatic hazard of commercial hydrocarbon resins

Hydrocarbon resins are used to modify polymer products to achieve desired functional properties for a diverse range of products. These complex hydrocarbon-based mixtures are typically poorly soluble in water. However, resins may leach lower-molecular-weight monomers or impurities upon contact with water, thus posing a potential hazard to the aquatic environment. The bioavailability and toxicity of leachable constituents of four solid and three liquid resins were evaluated by analyzing water-accommodated fractions prepared with each resin, using biomimetic solid phase microextraction (SPME) techniques. Liquid resins exhibited concentrations of bioavailable constituents that were sufficiently elevated to cause acute toxicity to the aquatic organism Daphnia magna. All solid resins exhibited lower bioavailable concentrations of leachable constituents that were unlikely to pose an aquatic toxicity concern. Since observed toxicity of both resin types was generally consistent with bioavailable concentrations determined using SPME fiber measurements, it is concluded that this approach provides a convenient in vitro screening tool that can help reduce the use of animal testing in environmental hazard assessment of complex hydrocarbon-based substances.

Chlorosilane Acute Inhalation Toxicity and Development of an LC50 Prediction Model

The acute inhalation toxicity of 10 chlorosilanes was investigated in Fischer 344 rats using a 1-h whole-body vapor inhalation exposure and a 14-day recovery period. The median lethal concentration (LC501) for each material was calculated from the nominal exposure concentrations and mortality. Experimentally derived LC501 values for monochlorosilanes (4257–4478 ppm) were greater than those for dichlorosilanes (1785–2092 ppm), which were greater than those for trichlorosilanes (1257–1611 ppm). Apparent was a strong structure-activity relationship (r2 = .97) between chlorine content and LC501 value. Estimated LC501 values for mono-, di-, and trichlorosilanes were determined to be 3262, 1639, and 1066 ppm, respectively, utilizing this relationship and the lower limit of the 95% prediction interval. The LC501 values determined in this series of studies were greater than that reported for hydrogen chloride (3124 ppm), when expressed on a chlorine equivalence basis (3570–5248 ppm), demonstrating that the acute toxicity of these chlorosilanes is similar to or less than that for hydrogen chloride. The good correlation between chlorine content and LC501 provides a sound basis for estimation of LC501 for chlorosilanes not already evaluated. The use of structure–activity relationships is consistent with the chemical industry and federal agency initiatives to reduce, refine, and/or replace the use of animals in testing without compromising the quality of health and safety assessments.

Regulatory requirements and ICH guidelines on carcinogenicity testing of pharmaceuticals: A review on current status

International regulatory guidelines require that new chemical entities (NCEs) be tested first on animals (preclinical studies) for safety and efficacy before the start of clinical development. Long-term nonclinical studies such as carcinogenicity testing, the subject of the present review, is conducted in tandem with on-going clinical studies. Over the last few years, a number of regulatory changes have occurred in the regulatory requirements of carcinogenicity testing and the use of such data in human risk assessment process. The efforts are to provide a better mechanistic basis in interpretation of standard lifetime and short-term rodent bioassays. The major areas of focus of this review include the basic considerations for the lifetime rodent bioassay, the recommendations of the International Conference on Harmonization (ICH), and the development and validation of new short-term or alternative models for carcinogenicity testing. The objectives of these guidelines are to avoid the unnecessary use of animals in testing and to provide consistency in world wide regulatory assessments of applications. It will ultimately improve the precision in carcinogenicity testing, minimize the time requirement for such testing, and help to conserve the resources in the process of drug discovery and development.

げっ歯類を用いる小核試験の基礎研究ならびにその行政面への応用

Genotoxicity is an important consideration in the safety evaluation of chemicals. It is well known that there are in vitro and in vivo assay systems with different endpoints for evaluating chemical genotoxicity. Bacterial gene mutation test and chromosomal aberrations test using mammalian cultured cells are representative examples. It is apparent that there are limitations of in vitro assay systems for chemical safety evaluation and risk assessment for human health, and in vivo assay systems are becoming more important from the viewpoint of weight of evidence. There are several in vivo assay systems that have been developed and which are based on various endpoints. Among these, the rodent micronucleus test using hematopoietic cells has been most widely and frequently used to detect induction of chromosomal aberration. It is evident that there are chemicals that gave a positive result in the in vitro chromosome aberration test but were negative in the rodent micronucleus test. In such case, as a rule, the in vivo negativity is considered dominant to the in vitro positivity.It is important and necessary to reduce use of test animals without any loss of evaluation accuracy. In the micronucleus test, development of the method using peripheral blood instead of bone marrow cells succeeded in reducing the total number of animals required for chromosomal aberration evaluation in vivo. Sampling of very small amounts of blood can be done without killing animals, which is one of the most important advantages of the method; it also permits combining with other assays for different endpoints that require different optimal sampling times. Based on this development, in vivo multiple endpoint assay systems will be realized and will lead to further reduction of animal use for the evaluation of chemical genotoxicity. In this manuscript, I describe the history of development and applications of the peripheral blood micronucleus assay.

Tiered testing strategies--acute local toxicity.

More work has been done to develop alternatives to animal use in the areas of eye and skin irritation than in any area other than carcinogenicity. There has long been a belief both in the scientific community and among the public that the development of nonanimal tests in these areas should be simple and straightforward. After more than 20 yr of research, we can identify materials corrosive to the skin without using animals, but the assessment of irritation using in vitro methods alone is still an illusive goal. This review of current recommendations and industry practices that reduce the number of animals needed for these two tests concludes that animal use for skin irritation testing is not necessary today, with currently available and accepted methodology, except for regulatory reasons. Scientifically sound improvements in current eye irritation methods are also available. Advances in the understanding of the mechanisms of eye irritation that have been made in the last 5 yr should lead to improved in vitro methods for this endpoint. In the meantime, changes should be made in the current animal protocol to reduce pain and distress. This paper provides an overview of the progress that has been made toward discontinuing the use of animals in tests to determine the potential of materials to cause skin or eye damage after a single acute exposure. It also discusses some additional changes that could be made now to reduce animal use further or to reduce pain and distress in the testing that must still be done until such time as we can meet the ultimate goal--validated and accepted nonanimal methods for these endpoints.