Local lymph node assay - validation, conduct and use in practice.

The validation of alternative methods is a relatively new activity in toxicology. The local lymph node assay (LLNA), a novel method for the identification of chemicals that have the potential to cause skin sensitization, was the first test to pass through the formal regulatory validation process established in the USA under the auspices of ICCVAM, the Interagency Coordinating Committee on the Validation of Alternative Methods. ICCVAM approved the LLNA as an alternative to guinea pig tests for the identification of skin sensitisation hazards. In this report, we explore the nine recommendations made by ICCVAM and discuss their interpretation in relation to the new OECD Guideline 429, which describes the LLNA. In particular, the value and limitations of the use of statistical evaluation of data and of the inclusion of routine positive controls is examined. It is concluded that the OECD Guideline as currently written embodies the necessary flexibility to permit conduct of the LLNA in a manner necessary to meet the varying needs of regulatory agencies and toxicologists around the world.

Mouse-specific carcinogens: an assessment of hazard and significance for validation of short-term carcinogenicity bioassays in transgenic mice.

1. The International Conference on the Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for human use (ICH) has agreed that bioassay data from only one species, the rat, supported by appropriate mutagenicity and pharmacokinetic data and also information from new (unvalidated) short term in vivo screening tests for potential carcinogenicity, could be used for the licensing of human medicines. This proposal has been supported by reviews of the utility of testing pharmaceuticals in the mouse which have concluded that the mouse bioassay contributes little to regulatory decisions. The current review was undertaken to identify 'genuine' mouse-specific carcinogens using the Gold Carcinogenicity Potency Database (CPD) for the initial identification of potential mouse-specific carcinogens from published literature. Hazard assessments were completed for these chemicals with particular attention focused on the 'genuine' mouse-specific carcinogens. The significance of such chemicals has been discussed together with consideration of on-going work on the validation of short-term carcinogenicity bioassays using transgenic mice. 2. Seventy-six potential mouse specific carcinogens were identified through the Gold Carcinogenicity Potency Database. Following more detailed consideration a total of ten chemicals were excluded from further consideration (three were multispecies carcinogens, five were considered to be non-carcinogenic in the mouse, and the data for two were uninterpretable). The review focused on the remaining 66 chemicals. There was equivocal evidence of carcinogenicity to the rat for 28 chemicals and inadequate data for a further 23 chemicals. Fifteen 'genuine' mouse-specific carcinogens were identified. These 15 chemicals comprise two genotoxic mouse-specific carcinogens (N-methylolacrylamide (924-42-5), 2,6-Dichloro-p-phenylenediamine (609-20-1); five non-genotoxic mouse-specific carcinogens 2-Aminobiphenyl.HCl (2185-92-4), Captan (133-06-2), Dieldrin (60-57-7), Diethylhexyladipate (103-23-1), and Probenicid (57-66-9); five mouse-specific carcinogens with equivocal evidence of mutagenicity were identified; (2,4-diaminophenol.2HCl (137-09-7), Dipyrone (68-89-3), Ozone (10028-15-6), Vinylidene chloride (75-35-4), and Zearalenone (17924-92-4)), and three mouse-specific carcinogens with inadequate mutagenicity data (Benzaldehyde (100-52-7), Piperonyl sulphoxide (120-62-7), Ripazepam (26308-28-1)). 3. It is suggested that the two genotoxic mouse carcinogens would have been considered as potential carcinogens in the absence of a mouse bioassay. Of the five non-genotoxic mouse-specific carcinogens; three induced tumours in mouse liver only and are considered as being of low potential hazard to human health. The remaining two chemicals would have been missed in the absence of a mouse bioassay (2-aminobiphenyl (2185-92-4) and captan (133-06-2)) and thus are good candidates for evaluation in the short term bioassays in transgenic mice currently being validated. 4. The hardest group of mouse-specific carcinogens to evaluate are those for which there is equivocal or inadequate mutagenicity data. The difficulty in evaluating these particular chemicals emphasises the need for adequate mutagenicity data in addition to adequate carcinogenicity data in order to assess potential hazards to human health. Hazard assessments and a consideration of the potential role for short-term bioassays in transgenic mice for the eight chemicals in this subgroup are presented. 5. A number of general conclusions have been derived from this review. Firstly, there are insufficient published genotoxicity data to allow a full assessment fo mutagenic potential for 57/76 of the potential mouse-specific carcinogens identified from the CPD. This is surprising given the clear value of such data in interpreting bioassay results and the much greater resources required for carcinogenicity bioassays. (ABSTRACT TRUNCATED)

Risk assessment of butadiene in ambient air; the approach used in the UK.

The UK Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment (COC) and the related Committee on Mutagenicity provided advice on 1,3-butadiene in 1992. This followed detailed consideration of the available mutagenicity, animal carcinogenicity and epidemiology data plus information on toxicokinetics. They concluded that 1,3-butadiene was an in vivo mutagen, a potent genotoxic animal carcinogen and should be regarded as a probable human carcinogen. The Department of Health is not aware of more recent data warranting reconsideration of these conclusions. General advice on setting air quality standards for carcinogenic air pollutants was given by the COC. Although the prudent assumption of the absence of any safe level for genotoxic carcinogens was preferred, a pragmatic approach based essentially on assessment of the exposure at which no increased risk would be detected, plus a safety factor, was considered reasonable for compounds like butadiene where exposure cannot be totally avoided. This approach, plus recognition that it is unadvisable to allow ambient levels of genotoxic carcinogens to rise, is used in the UK. The procedure by which the Department of Environment's Expert Panel on Air Quality Standards recommended a value of 1 ppb for butadiene based on these principles is described.

Carcinogens in food: priorities for regulatory action.

A pragmatic possible approach to the prioritization of chemical carcinogens occurring as food contaminants is described, based on the carcinogenic risk to the population. This should be of value in ensuring that resources for assessment and management of carcinogens in food are directed to the most important areas with regard to carcinogenic risk to the population. Key components of this approach are an assessment of the carcinogenic hazard to humans combined with estimations of intakes per person and of the proportion of the population exposed. These are used to derive an index referred to as the Population Carcinogenic Index. Concerning the hazard assessment expert judgement is used to place the chemical in one of five categories. The highest category is for chemical carcinogens that are believed to act by a genotoxic mechanism. It is recognised that such compounds may vary enormously with respect to their potency and various approaches to ranking carcinogens on the basis of potency are reviewed. The approach adopted is to subdivide the genotoxic carcinogens category into high, medium and low potency based on the TD50 value. Methods of estimating intakes and exposed populations are considered and an approach which groups these into broad categories is developed. The hazard and exposure assessments are then combined to derive the Population Carcinogenicity Index.

Acceptance of in vitro studies by regulatory authorities.

There has been relatively little progress regarding the acceptance of in vitro tests by regulatory authorities since the second Practical In Vitro Toxicology Conference in 1989. Advances have been made in the international acceptance of the use of in vitro methods to identify compounds with mutagenic potential. The recognition, in international guidelines on skin and eye irritancy studies, of the need for a hierarchical approach, including the use of in vitro methods to screen out severe irritants, has also been welcome. The reasons for the relatively slow progress are considered. In the case of repeated-dose animal toxicity studies, these represent an effective broad-spectrum approach to identifying the general toxic effects and target organs. It will be difficult to design in vitro methods capable of mirroring the complex interactions seen in the whole animal or the multitudes of potential targets for toxic effects. In vitro studies may, however, be valuable in characterizing such effects once identified from the animal studies. Similar considerations apply to teratogenicity, where possible mechanisms include disturbances in placental function or in maternal metabolism. These examples illustrate the fairly substantial scientific obstacles that exist in some areas. The relatively rapid acceptance of mutagenicity studies was, to some extent, due to their single, underlying mechanism, namely, damage to DNA. In view of these problems, effort has concentrated on local effects such as skin and eye irritancy. Even here, the in vivo response is a complex series of reactions, and there is the assumption that a battery of in vitro tests will be needed. There have been difficulties in identifying the most promising combination of tests to subject to detailed validation. In addition, the number and identity of the 'reference' chemicals to be used and the comparative data (in vivo animal data or human data) needed has proved to be a matter of much debate. In addition to these difficulties, the need for international acceptance of the validation data has necessitated the adoption of a more international perspective. However, a large international study is now underway which, it is hoped, will lead to some real progress in the eye irritancy area.

The international validation of a fixed-dose procedure as an alternative to the classical LD50 test.

In an international study involving 33 laboratories in 11 countries, the acute oral toxicity to the rat of 20 substances and preparations was evaluated using a fixed-dose procedure and the results compared with those obtained for the test materials using the classical LD50 test. The study has shown that the fixed-dose approach to acute oral toxicity testing: (1) produces consistent results that are not substantially affected by inter-laboratory variations; (2) provides adequate information for risk assessment purposes on signs of toxicity, including their nature, time to onset, duration and outcome; (3) uses fewer animals than the current internationally agreed OECD procedure (Guideline 401-revised); (4) subjects animals to less pain and distress than the classical LD50 test and causes less compound-related mortality; and (5) enables substances and preparations to be ranked according to the EEC classification system on the basis of their acute oral toxicity, such ranking being compatible with that allocated by the results of classical LD50 studies.

Acceptance of in vitro testing by regulatory authorities.

National regulatory authorities have responsibility for taking decisions that possibly affect the health of whole populations and it is therefore to be expected that they will be reluctant to substitute alternative in vitro toxicity test methods for conventional animal studies unless the new procedures have been demonstrated to be reliable and have been adequately validated. Validation of in vitro methods presents particularly difficult problems because whilst they tend to produce consistent and objective results, the test systems used are incapable of mirroring the complexity of the biochemical processes seen in animals. As a result, a single animal study would need a large battery of in vitro studies to replace it to cover the various endpoints that are involved in the in vivo study; each of these in vitro tests would need to be validated with regard to the specific endpoint that it is investigating. Although great advances have been made in recent years in the development of alternative methods, few have been validated to an extent that makes them acceptable to regulatory authorities as replacements for in vivo studies. Rather, they are largely seen and used as screening techniques whereby decisions can be taken on the value of further development of newly discovered compounds, and as aids in the interpretation of animal studies and in their extrapolation to man, that is, they are of value in 'mechanism of action' studies. Nevertheless, certain in vitro procedures are already accepted by regulatory authorities and their use, for example, in 'screening out' compounds that have severe irritant properties, and in identifying compounds with potential mutagenic and carcinogenic activity, has had a profound effect on both the number of animal studies carried out and on the welfare of those animals still used.