The tenth anniversary of the Björn Ekwall memorial foundation

The Bj?rn Ekwall Memorial Foundation (BEMF) was initiated by the Scandinavian Society for Cell Toxicology in 2001, to honour the memory of Dr Bj?rn Ekwall (1940-2000) and to establish a prize, the Bj?rn Ekwall Memorial Award. The prize is awarded to scientists who have significantly contributed to the field of cell toxicology, and whose work is contributing toward the replacement of animal experiments by alternative toxicity tests. Over the past 10 years, the Bj?rn Ekwall Memorial Award has been presented annually. Bj?rn Ekwall, an outstanding Swedish cell toxicologist, was one of the pioneers in the development and application of alternative methods to animal tests in toxicology. All his scientific work was devoted to in vitro toxicology, and in particular, to the use of cultured human cells for the screening of toxic chemicals. In the middle of the 1980s, he initiated the international Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to evaluate the usefulness of in vitro tests for the estimation of human acute systemic toxicity. To prove his "basal cytotoxicity concept", he established the MEMO database, in which data on the acutely toxic human blood concentrations of drugs and chemicals were collated from the literature and from clinical studies. He also initiated another project, Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT). The ideas from the EDIT project, together with those from the MEIC project, became the basis for today's international EU projects, e.g. ACuteTox, Sens-it-iv and ReProTect. In this article, 10 years after the start of the BEMF, the scientific achievements of each of the award winners in the field of in vitro toxicology are presented, together with a brief synopsis of their careers.     

The prediction of human acute systemic toxicity by the EDIT/MEIC in vitro test battery: the importance of protein binding and of partitioning into lipids

The aim of the two studies presented in this paper was to further improve the predictability of the original Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) in vitro test battery for acute systemic toxicity. In the first study, whether a protein-free cytotoxicity assay could improve the prediction of human acute systemic toxicity was investigated. The cytotoxicity of 39 MEIC reference chemicals was measured by the neutral red uptake inhibition test after 30 minutes in phosphate-buffered saline (PBS), with hepatoma-derived Fa32 cells. The results were compared with the corresponding values obtained in complete culture medium, including 10% fetal calf serum. Mercuric chloride and hexachlorophene were much more cytotoxic in PBS, as was the case, to a lesser extent, for seven other chemicals. Potassium cyanide and eight other chemicals were less cytotoxic in PBS than in complete culture medium, probably because of poor physiological conditions. The correlation between the cytotoxicity measured in PBS and human acute toxicity was rather low, but became of the same order as for other assays, when mercuric chloride and hexachlorophene were withdrawn from the comparison. In the second study, modelling of human lethal blood concentrations by using the results of the three cell line tests of the original MEIC test battery were complemented by logP (octanol-water partition coefficient) values. The introduction of logP into the modelling did not improve the correlations, but some improvement of both R(2) and Q(2) was obtained by expanding the logP values with logP(2) values. The highest R(2) (0.84) and Q(2) (0.80) values were obtained for a model in which both experimental and calculated (ambiguous) logP values were used. When only experimental logP values were used, the corresponding values were 0.80 and 0.78. These two studies showed that including protein binding and the partition of chemicals in the MEIC in vitro test battery is important, in order to improve the predictability of the results obtained.     

Björn Ekwall and his contribution to modern cell toxicology

Dr. Bj?rn Ekwall (1940-2000) was a prominent Swedish scientist--cell toxicologist, who made an outstanding contribution in the field of in vitro toxicology. In the early 80-ties Ekwall formulated so called basal cytotoxicity concept, which served as a basis for modern orientation in the field of cell toxicology: the use of tests on cells in culture for prediction of acute systemic toxicity in humans, instead of the use of tests on experimental animals. To be able to verify his theories, Ekwall organized and led the international toxicological project called MEIC: Multicentre Evaluation of In Vitro Cytotoxicity Programme (1989-1999). In this project, 50 selected chemicals were tested in 100 laboratories worldwide with more than 60 different in vitro tests (laboratories have chosen tests themselves). MEIC project was unique not only because its large scale, but, in particular, because, for the first time, the human peak blood concentrations after acute poisoning with chemicals were used as references, aiming to check predictability of the in vitro assays. The results of the MEIC project have clearly demonstrated a possibility to use in vitro tests for prediction of toxicity of chemicals in humans.     

Protein precipitation In Vitro as a measure of chemical-induced cytotoxicity: an EDIT sub-programme

As a priority area of the Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT) programme, an in vitro protein precipitation (PP) assay was used on the 50 reference chemicals of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to confirm and extend the MEIC results. Dose-response curves were generated for only 30 of the chemicals, and the concentrations causing 10% (EC10) and 50% (EC50) protein precipitation versus the positive control were chosen as endpoints. The number of chemicals with a positive response increased to 46 when a new endpoint, the minimum effect concentration (MEC) that induces protein precipitation with respect to the negative control, was used. When the results were correlated with in vitro cytotoxicity in human cell lines, a similarly good correlation was found between the various endpoints of the PP assay at 5 hours and the 24-hour IC50 average cytotoxicity in human cell lines, even though the number of chemicals included in the correlation was larger for the MEC. Using the prediction error, the endpoint that gave the best correlation between the PP assay and human cell cytotoxicity was once more found to be the 5-hour MEC, and this was chosen for the PP assay. The sensitivity of the PP assay is lower than that of the in vitro cell-line cytotoxicity assay, possibly due to its shorter exposure period and because precipitation is the ultimate event in the sequence of a protein disturbance. It is expected that earlier denaturation steps would give better sensitivity. However, this simple, inexpensive and rapid assay could be useful in the early stages of testing chemicals.     

MEIC—A new international multicenter project to evaluate the relevance to human toxicity of in vitro cytotoxicity tests

A new international project to evaluate the relevance for human systemic and local toxicity of in vitro tests of general toxicity of chemicals has been organized by the Scandinavian Society of Cell Toxicology under the title Multicenter Evaluation of In Vitro Cytotoxicity (MEIC). The basic assumptions underlying the project, as well as the practical goals and the design of the program are outlined. The list of the first 50 reference chemicals is presented. The chemicals are an otherwise unbiased selection of compounds with known human acutely lethal dosage and blood concentrations, including LD50-values in the rat or mouse. Most agents also have other data on human toxicity and toxicokinetics, including more extensive animal toxicity data. International laboratories already using or developing in vitro tests of various partial aspects of general toxicity are invited to test the substances, the results of which will be evaluated by us. The predictivity of the in vitro results for both partial and gross human toxicity data will be determined with combined use of univariate regression analysis and soft multivariate modeling. The predictivity of the in vitro results will be compared with the predictivity of conventional animal tests for the same chemicals. Finally, batteries of tests with optimal prediction power for various types of human toxicity will be selected. The need for and possible uses of such batteries are discussed.     

Conformational changes in proteins in vitro as a means of predicting the acute toxicities of chemicals

Experimental data are presented on ovalbumin denaturation (OD, EC10) and human acetylcholine esterase (AChE) inhibition (IC50) in vitro, following exposure to the chemicals used in the international Multicentre Evaluation of In vitro Cytotoxicity (MEIC) programme. Data were obtained for 40 (OD test) and 43 (AChE test) of the 50 MEIC chemicals. These data were compared with similar data from other methods used in the MEIC programme, and good correlations (R2) were obtained with data from MEIC studies on cell lines: 0.80 for human, 0.81 for other animal, and 0.78 for fish cell line IC50 values and AChE values, and 0.76 for human, 0.69 other animal and 0.75 for fish cell line IC50 values and OD values. The correlation increased substantially, if chemicals which freely cross the blood-brain barrier were solely considered, with R2 = 0.90 for human, 0.90 for other animal, and 0.82 for fish cell line IC50 values and AchE values, and 0.87 for human, 0.86 for other animal, and 0.92 for fish cell line IC50 values and OD values, in this case. Such chemicals are the main cause of non-specific depression of the central nervous system (CNS). The AChE IC50 permits a good prediction of human acute toxicity, similar to the IC50 values obtained with human cell lines and the same MEIC chemicals. These results confirm the basal toxicity hypothesis formulated by Bj?rn Ekwall. It is concluded that in vitro methods based on the disruption of the functions of the proteins vital for body operation can be used as an alternative to the cell culture methods, when non-specific toxic effects of chemicals on humans and animals are evaluated.     

In vitro toxicity: mechanisms,alternatives and validation - a report from the 19th annual scientific meeting of the Scandinavian Society for Cell Toxicology

The Scandinavian Society for Cell Toxicology (SSCT) has arranged annual scientific meetings since 1983. These workshops were the forum for the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. Along with the MEIC programme, which was completed in 1998, a wide range of topics relating to cytotoxicity have been discussed. The meetings have also given an opportunity for graduate students and young scientists to present their work to an international audience. At the same time, experts in the fields of in vitro toxicity have been invited as speakers. The 19th SSCT scientific meeting, which was held at S?rup Manor in Ringsted, Denmark, was no exception. The meeting consisted of four sessions: mechanisms of toxicity; environmental toxicological testing; alternatives to animal experiments; and validation of in vitro tests.     

Use of the PFGE assay for studies of DNA breakage induced by toxic chemicals

The relevance of the pulsed field gel electrophoresis (PFGE) assay for the estimation of the DNA damaging effects of chemicals was studied. Four chemicals were randomly chosen from the list of 50 Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) reference chemicals with known human acute systemic toxicity: acetylsalicylic acid, paracetamol, ethylene glycol and sodium chloride. Human fibroblasts (VH-10) were used as a model system. For the estimation of cytotoxic effect, cell monolayers were treated with chemicals for 24 hours. Cloning efficiency (colony-forming ability) at different concentrations of the test chemicals was estimated, and the 50% inhibitory concentration (IC50) was determined. The IC50 values obtained demonstrated a correlation with human lethal blood concentrations. The induction of DNA double-strand breaks, measured by PFGE as the fraction of activity released, was detected after treatment with paracetamol. However, the other three chemicals tested mainly induced DNA degradation.     

Prediction of human acute toxicity by the hep G2/24-hour/total protein assay, with protein measurement by the CBQCA method

In our previously described Hep G2/24-hour/total protein assay, protein levels were measured by using the Lowry method. This assay was the best acute in vitro assay for the prediction of human toxicity within the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) study. In order to increase the MEIC data-base with a wider range of chemicals, we were interested in introducing the more practical 3-(4-carboxybenzoyl)- quinoline-2-carboxaldehyde (CBQCA) method for the quantification of the total protein content. Therefore, we investigated whether the same good results for the prediction of acute human toxicity would be obtained with the CBQCA method. The cells were treated for 24 hours, then cytotoxicity was determined by measuring the total protein content with CBQCA. The results were quantified by using the PI50c: the concentration (in mM) of test compound required to reduce the total protein content measured with the CBQCA-method by 50% as compared to the control cells. The results were compared with the PI50, the corresponding value when the Lowry method was used. A relatively low correlation was observed between PI50 and PI50c, reflecting the large and unexpected, differences when using the two protein assays. However, when comparing the log PI50c with the human toxicity, a correlation coefficient of r(2) = 0.761 (n = 44) was obtained for exactly the same series of MEIC chemicals. This value is clearly higher than that for the Lowry method (r(2) = 0.695). Compared to the Lowry method originally used, the Hep G2/24-hour/CBQCA total protein assay has the additional important advantage that it can be very easily adapted for large-scale analyses with robotic systems, including the on-line calculation of the results.     

Morphology of cell injury: An approach to the EDIT programme by the use of tobacco pollen tubes. Evaluation-guided Development of New In Vitro Test Batteries

Tobacco pollen tubes were used as a standard in vitro system to investigate cell growth aberrations caused by some of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme chemicals and other toxic compounds. Changes in cytoskeletal pattern were observed in the tube cells by using tubulin immunofluorescence and rhodamin-phalloidin fluorescence for the localisation of microtubules and actin filaments, respectively. Four different types of cell malformation were found: screw-like growth, isodiametric tip swelling, hook formation, and pollen grain enlargement. We suggest that these malformations resulted from an interference by the chemicals with the cytosolic calcium gradient which controls tip growth and the orientation of the pollen tube. The results may contribute to a general understanding of toxicity-based cell malformations.     

Biokinetic and toxicodynamic modelling and its role in toxicological research and risk assessment

Toxicological risk assessment for chemicals is still mainly based on highly standardised protocols for animal experimentation and exposure assessment. However, developments in our knowledge of general physiology, in chemicobiological interactions and in (computer-supported) modelling, have resulted in a tremendous change in our understanding of the molecular mechanisms underlying the toxicity of chemicals. This permits the development of biologically based models, in which the biokinetics as well as the toxicodynamics of compounds can be described. In this paper, the possibilities are discussed of developing systems in which the systemic (acute and chronic) toxicities of chemicals can be quantified without the heavy reliance on animal experiments. By integrating data derived from different sources, predictions of toxicity can be made. Key elements in this integrated approach are the evaluation of chemical functionalities representing structural alerts for toxic actions, the construction of biokinetic models on the basis of non-animal data (for example, tissue-blood partition coefficients, in vitro biotransformation parameters), tests or batteries of tests for determining basal cytotoxicity, and more-specific tests for evaluating tissue or organ toxicity. It is concluded that this approach is a useful tool for various steps in toxicological hazard and risk assessment, especially for those forms of toxicity for which validated in vitro and other non-animal tests have already been developed.     

Cytotoxicity of the MEIC reference chemicals in antioxidant-enriched, rat hepatoma-derived Fa32 cells

Since vitamin E increases the antioxidant status of cells, its influence on cytotoxicity was investigated. The neutral red uptake (NRU) inhibition effects of 39 MEIC reference chemicals were measured after treatment of rat hepatoma-derived Fa32 cells in the presence of vitamin E for 30 minutes. The results were quantified in terms of the NI50, the concentration of test compound required to reduce the NRU by 50%. Sodium chloride was the only chemical that was more toxic in the presence of vitamin E. This effect was related to the concentration of vitamin E in the cell culture medium. A vitamin E dose-related response was also observed for the decreased toxicity of paracetamol and caffeine. Glutathione levels were slightly increased in the presence of vitamin E, which could contribute to the protective effect of vitamin E. Of the remaining chemicals, 50% were less toxic in the presence of vitamin E, but the correlation with the acute human toxicity data of the MEIC study was not improved. The results imply that reactive oxygen species interfere with the toxicity of a high proportion of toxic chemicals. The assay described provides a quick and easy method for checking whether reactive oxygen species contribute to the toxicity of a chemical.     

Prediction of acute toxicity in HPCT-1E3 hepatocytoma cells with liver-like transport activities

A battery of in vitro methods has been developed for the prediction of acute oral toxicity, to reduce the number of animals used for this purpose. However, the results of these tests correlate more closely with lethal serum concentrations than with lethal doses. To address this issue, we have further evaluated the HPCT-1E3 model, which may be better able to emulate toxicokinetic factors that occur in vivo, due to the presence in these hepatocytoma cells of endogenous transmembrane carriers and a basal activity of xenobiotic metabolism. IC50 values produced by using the MTT test after a 48-hour incubation with 20 randomly-selected MEIC substances, correlated better with human oral LD50 values than with LC50 data, supporting this hypothesis. As with other models, the toxicity of receptor-specific rather than cytotoxic substances, for example digoxin, was underpredicted. When digoxin was removed from the correlation analysis, the coefficient of determination (r(2)) improved to 0.81, and none of remaining chemicals were wrongly predicted by more than one order of magnitude. IC50 values obtained with HepG2 cells under similar conditions (MEIC Test No. 3, 24 hours, MTT) correlated with human LD50 data with a r(2) value of 0.55. A direct comparison of HPCT-1E3 and HepG2 cells further suggested that the differences between them may be due to transport processes. In conclusion, the HPCT-1E3 model may be valuable in improving the prediction of lethal doses, rather than lethal serum concentrations.     

Integrated decision-tree testing strategies for acute systemic toxicity and toxicokinetics with respect to the requirements of the EU REACH legislation

Liverpool John Moores University and FRAME conducted a joint research project, sponsored by Defra, 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 REACH. This paper focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for acute systemic toxicity and toxicokinetic testing. The paper reviews in vitro tests based on basal cytotoxicity and target organ toxicity, along with QSAR models and expert systems available for this endpoint. The use of PBPK modelling for the prediction of ADME properties is also discussed. These tests are then incorporated into a decision-tree style, integrated testing strategy, which also includes the use of refined in vivo acute toxicity tests, as a last resort. The implementation of the strategy is intended to minimise the use of animals in the testing of acute systemic toxicity and toxicokinetics, whilst satisfying the scientific and logistical demands of the EU REACH legislation.     

In vitro cytotoxicity testing for prediction of acute human toxicity

This study was designed to compare the cytotoxic concentrations of chemicals, determined with three independentin vitro cytotoxicity testing protocols, with each other and with established animal LD₅₀ values, and against human toxic concentrations for the same chemicals. Ultimately, these comparisons allow us to evaluate the potential ofin vitro cell culture methods for the ability to screen a variety of chemicals for prediction of human toxicity. Each laboratory independently tested 50 chemicals with known human lethal plasma concentrations and LD₅₀ values. Two of the methods used monolayer cell cultures to measure the incorporation of radiolabeled amino acids into newly synthesized proteins and cellular protein content, while the third technique used the pollen tube growth test. The latter is based on the photometric quantification of pollen tube mass production in suspension culture. Experiments were performed in the absence or presence of increasing doses of the test chemical, during an 18- to 24-h incubation. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxic concentrations confirms previous independent findings that the experimental IC₅₀ values are more accurate predictors of human toxicity than equivalent toxic blood concentrations (HETC values) derived from rodent LD₅₀s. In addition, there were no conclusive statistical differences among the methods. It is anticipated that, together, these procedures can be used as a battery of tests to supplement or replace currently used animal protocols for human risk assessment.Abbreviations DCP dichlorophenoxyacetic acid - DMEM Dulbecco's modified Eagles' medium - DMSO dimethylsulfoxide - IC inhibitory concentration - LD₅₀ lethal dose 50% - MEIC Multicenter Evaluation forIn Vitro Cytotoxicity - PI₅₀ protein inhibition 50% - PTG pollen tube growth - TCA trichloroacetic acid - TCE trichloroethane     

Estimation of the acute inhalation hazards of chemicals based on route-to-route and local endpoint extrapolation: experience from bulk maritime transport

Data on acute lethal inhalation toxicity from animal studies are commonly required for assessing the hazards to human health of volatile, gaseous and dusty chemicals or their mixtures. The International Maritime Organisation (IMO) made the provision of acute inhalation toxicity data a mandatory requirement for the carriage of bulk liquid chemicals transported by sea in tank ships, thereby creating the need for inhalation data on many hundreds of chemicals in bulk maritime transport. Taking note of previously published proposals for estimating acute inhalation toxicity hazards for chemicals, and the paucity of measured experimental data, an extrapolation method has been developed by the Group of Experts on the Scientific Aspects of Marine Environmental Protection (GESAMP) to partly fulfil this need. This method should be seen as a pragmatic approach to the challenge of missing measured experimental test data, with the added benefit of reducing tests in experimental animals. The method is based on a route-to-route (i.e. between-route) extrapolation of information on acute oral and/or dermal toxicity, in combination with data on the potential for irritation and/or corrosion to skin and eyes. The validation of this method was based on the individual evaluation of inhalation toxicity studies for 330 chemicals, including mixtures and many important chemical groups, for which the IMO holds public and industry-confidential data. The authors contend that this extrapolation method offers a reliable basis for hazard evaluation in the context of bulk maritime transport, and the 'GESAMP inhalation toxicity extrapolation method' has become part of the IMO regulatory system for the carriage of bulk liquids (i.e. noxious liquid substances) on board tank ships.     

The influence of biochemical species differences on acute fish toxicity of organic chemicals.

1. Reported LC50s of hundreds of chemicals and data of many enzymes in many fishes have been reviewed in order to find the hypothesized influence of biotransformation on acute toxicity. 2. Biotransformation of carbaryl was correlated and biotransformation of endrin was inversely correlated with acute toxicity. 3. For most compounds no correlation was found which indicates that either biotransformation is not important in acute toxicity or enzyme activities are not representative parameters to quantify biotransformation reactions.     

Testing strategies in mutagenicity and genetic toxicology: an appraisal of the guidelines of the European Scientific Committee for Cosmetics and Non-Food Products for the evaluation of hair dyes

The European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) guideline for testing of hair dyes for genotoxic/mutagenic/carcinogenic potential has been reviewed. The battery of six in vitro tests recommended therein differs substantially from the batteries of two or three in vitro tests recommended in other guidelines. Our evaluation of the chemical types used in hair dyes and comparison with other guidelines for testing a wide range of chemical substances, lead to the conclusion that potential genotoxic activity may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced gene mutations and structural and numerical chromosomal changes. We conclude that highly effective screening for genotoxicity of hair dyes can be achieved by the use of three assays, namely the bacterial gene mutation assay, the mammalian cell gene mutation assay (mouse lymphoma tk assay preferred) and the in vitro micronucleus assay. These need to be combined with metabolic activation systems optimised for the individual chemical types. Recent published evidence [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggests that our recommended three tests will detect all known genotoxic carcinogens, and that increasing the number of in vitro assays further would merely reduce specificity (increase false positives). Of course there may be occasions when standard tests need to be modified to take account of special situations such as a specific pathway of biotransformation, but this should be considered as part of routine testing. It is clear that individual dyes and any other novel ingredients should be tested in this three-test battery. However, new products are formed on the scalp by reaction between the chemicals present in hair-dye formulations. Ideally, these should also be tested for genotoxicity, but at present such experiences are very limited. There is also the possibility that one component could mask the genotoxicity of another (e.g. by being more toxic), and so it is not practical at this time to recommend routine testing of complete hair-dye formulations as well. The most sensible approach would be to establish whether any reaction products within the hair-dye formulation penetrate the skin under normal conditions of use and test only those that penetrate at toxicologically relevant levels in the three-test in vitro battery. Recently published data [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggest the three-test battery will produce a significant number of false as well as real positives. Whilst we are aware of the desire to reduce animal experiments, determining the relevance of positive results in any of the three recommended in vitro assays will most likely have to be determined by use of in vivo assays. The bone marrow micronucleus test using routes of administration such as oral or intraperitoneal may be used where the objective is extended hazard identification. If negative results are obtained in this test, then a second in vivo test should be conducted. This could be an in vivo UDS in rat liver or a Comet assay in a relevant tissue. However, for hazard characterisation, tests using topical application with measurement of genotoxicity in the skin would be more appropriate. Such specific site-of-contact in vivo tests would minimise animal toxicity burden and invasiveness, and, especially for hair dyes, be more relevant to human routes of exposure, but there are not sufficient scientific data available to allow recommendations to be made. The generation of such data is encouraged.     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles

One of the consequences of the low specificity of the in vitro mammalian cell genotoxicity assays reported in our previous paper [D. Kirkland, M. Aardema, L. Henderson, L. Muller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] is industry and regulatory agencies dealing with a large number of false-positive results during the safety assessment of new chemicals and drugs. Addressing positive results from in vitro genotoxicity assays to determine which are "false" requires extensive resources, including the conduct of additional animal studies. In order to reduce animal usage, and to conserve industry and regulatory agency resources, we thought it was important to raise the question as to whether the protocol requirements for a valid in vitro assay or the criteria for a positive result could be changed in order to increase specificity without a significant loss in sensitivity of these tests. We therefore analysed some results of the mouse lymphoma assay (MLA) and the chromosomal aberration (CA) test obtained for rodent carcinogens and non-carcinogens in more detail. For a number of chemicals that are positive only in either of these mammalian cell tests (i.e. negative in the Ames test) there was no correlation between rodent carcinogenicity and level of toxicity (we could not analyse this for the CA test as insufficient data were available in publications), magnitude of response or lowest effective positive concentration. On the basis of very limited in vitro and in vivo data, we could also find no correlation between the above parameters and formation of DNA adducts. Therefore, a change to the current criteria for required level of toxicity in the MLA, to limit positive calls to certain magnitudes of response, or to certain concentration ranges would not improve the specificity of the tests without significantly reducing the sensitivity. We also investigated a possible correlation between tumour profile (trans-species, trans-sex and multi-site versus single-species, single-sex and single-site) and pattern of genotoxicity results. Carcinogens showing the combination of trans-species, trans-sex and multi-site tumour profile were much more prevalent (70% more) in the group of chemicals giving positive results in all three in vitro assays than amongst those giving all negative results. However, single-species, single-sex, single-site carcinogens were not very prevalent even amongst those chemicals giving three negative results in vitro. Surprisingly, when mixed positive and negative results were compared, multi-site carcinogens were highly prevalent amongst chemicals giving only a single positive result in the battery of three in vitro tests. Finally we extended our relative predictivity (RP) calculations to combinations of positive and negative results in the genotoxicity battery. For two out of three tests positive, the RP for carcinogenicity was no higher than 1.0 and for 2/3 tests negative the RP for non-carcinogenicity was either zero (for Ames+MLA+MN) or 1.7 (for Ames+MLA+CA). Thus, all values were less than a meaningful RP of two, and indicate that it is not possible to predict outcome of the rodent carcinogenicity study when only 2/3 genotoxicity results are in agreement.