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Background 1. The International Life Sciences Institute (ILSI) and the Health and Environmental Science Institute (HESI) have co-ordinated a multinational research programme from 1996 - 2001 to more fully characterise the responsiveness of several alternative cancer models. The research has focused predominantly on a number of proposed short-term in-vivo test models for assessment of potential carcinogenic activity carcinogenicity in mice (in particular ras H2, Tg.AC, p53 +/-,Xpa-/-, Xpa-/-/p53+/- double knockout, neonatal mouse) but also included the in-vitro cell transformation assay using Syrian hamster embryo cells (SHE cell transformation assay) The overall objective of the work was to evaluate the ability of these models to provide useful information for human cancer risk assessment. The research involved input from over 50 industrial, governmental (USA, Denmark, Netherlands and Japan) and academic laboratories and cost around $35m.(1) 2. The Committee on Carcinogenicity of Chemicals on Food, Consumer Products and the Environment (COC) has considered all of the data submitted by ILSI/HESI except for the results of the investigations using the SHE cell transformation assay. These data have been considered by Committee on Mutagenicity on Chemicals in Food, Consumer Products and the Environment (COM) whose conclusions are given below. Previous considerations of the SHE cell transformation assay by COM 3. The assessment of the SHE in-vitro test system was considered by the COM in 1994 and in 1996. Full details of these considerations can be found in the relevant Annual reports.(2,3) 4. In 1994 the COM considered a number of cell transformation assays including the modified SHE method, and had agreed that transformation assays using Syrian Hamster cells were not yet ready for routine use, but warranted further work on both their validation and providing an understanding of the underlying mechanisms. The Committee considered a draft proposal for an OECD guideline for cell transformation using the modified SHE method (low pH culture). A number of supporting publications and papers claiming a predictive correlation between cell transformation in this test system and carcinogenicity were also reviewed. 5. In 1996, the Committee agreed that there were no mechanistic data available which gave an insight into the relationship between cell transformation in the SHE assay and the carcinogenic process. Although apparently high correlations had been reported in trials using a range of animal carcinogens including non-genotoxic carcinogens, only a very limited number of laboratories were involved and little value could be attributed to these results in the absence of appropriate supporting mechanistic data. 6. The COM concluded at the end of its 1996 review that it was not possible to support the proposed OECD guideline for the reasons given below:
ILSI/HESI Alternative Cancer Test research 7. The Committee considered a draft paper by Mauthe RJ and colleagues which had been submitted to Toxicologic Pathology. The Committee had considerable reservations on several aspects of the ILSI study relating to the assay system, the study design, the data obtained and the data analyses. There is still no mechanistic basis to underpin the assay and the endpoint (transformed foci) remains subjective as there is a lack of a clear, objective definition of transformed foci. There were reservations about the selection and categorisation of test chemicals, concentration ranges were considered to be too narrow for reasonable analysis of dose-response and, concerns about the extent of repeat testing. Control values were unsatisfactorily low (0 to 6) and in some tests there was a lack of consistency of result between closely spaced doses. Finally, the definition of positive and negative results relied on statistical analyses that seemed inappropriate to the data. Most importantly, there was no correction for multiple comparisons. Also methods had been used (Fisher exact and binomial-based) that assume uniformity of sampling circumstance (ie that all cells have an equal chance of transformation under fixed experimental conditions). This is unlikely to be true for the mixture of cell types derived from embryos. No tables of historical negative and positive control ranges (and/or confidence limits) were given to enable general variability, test validity and biological importance to be assessed. Some members of the committee considered that two-sided (-tailed) statistical analyses would be more appropriate as it is probable that compounds may have negative as well as positive effects on transformation Thus the Committee felt that the results of some tests reported as positive were most likely to be either equivocal or negative. Members also considered that the difficulties in objectively identifying transformed cells combined with the limited number of repeat trials suggested that interpretation of the ILSI/HESI data was particularly problematic. The results for a number of the chemicals tested are considered in detail below. 8. Phenacetin was included in the study as a definite human carcinogen, but was considered by the World Health Organisations International Agency for Research on Cancer (IARC) to be a probable human carcinogen. The IARC conclusions indicated that phenacetin was carcinogenic in rodents inducing malignant tumours of the urinary tract in rats and mice and nasal tumours in rats. IARC had noted that Phenacetin was mutagenic in-vitro in Chinese hamster cells but gave equivocal results in in-vivo tests in rodents for chromosomal aberrations and micronuclei. Overall the data on phenacetin suggested that a positive response would have been expected in the SHE cell transformation assay whereas a negative response had been obtained with phenacetin. There was a need for further consideration of this result. 9. The COM's evaluation suggested that the positive responses obtained with melphalan (included as a human carcinogen) and sulphamethoxazole (rodent carcinogen) were equivocal and needed further trials to confirm the response obtained with this assay. Members noted that ampicillin had given a statistically significant increased response at the highest dose tested (13 transformed foci at 3000 m g/ml) whereas a dose level of 2450 m g/ml had not yielded any transformed cells. Overall ampicillin was classified by the authors as giving a positive response in the SHE assay. However, members considered that the outcome was equivocal or negative and that further testing was required to elucidate the response of ampicillin in this assay. 10. Members felt that the critical problem preventing the use of the SHE assay in a regulatory context was the difficulty in the morphological identification of transformed foci. Use of SHE assay for regulatory screening of chemicals 11. The Committee considered on the basis of all the available information that some important generic comments could be made on the SHE cell transformation assay. 12. Members felt there was little prospect in obtaining reproducible results with the SHE cell transformation assay as a screen for the identification of chemical carcinogens in the absence of an objective molecular marker to identify cell transformation. Members stressed that there was a need to explain on a mechanistic basis why chemical carcinogens with disparate mechanisms of action should be expected to produce cell transformation in an embryonic cell line. 13. Members considered that whilst positive results could be obtained for benzo(a)pyrene indicating that SHE cells (and or its feeder layer) could activate carcinogens there was very little information available to define the metabolic competency of SHE cells and the feeder layer used. 14. Members noted that there was a tendency to report positive results for a diverse range of chemicals in the SHE cell transformation assay thereby demonstrating good sensitivity. However, there was a lack of chemicals reported to give negative data so that neither the specificity nor the accuracy of the test can be properly assessed. This problem had been illustrated by the data set from the ILSI/HESI trial. However Members felt that no particular value could be currently ascribed to either positive or negative results obtained in the SHE cell transformation assay in the absence of an objective endpoint to measure and an understanding of the mechanism underlying the process of cell transformation. Many of the chemicals selected for this trial are rodent carcinogens that are not human carcinogens. There are concerns that this assay has poor specificity for the detection of human carcinogens. Conclusion 15. The Committee agreed that the SHE cell transformation assay should not be used for regulatory screening of chemicals for potential carcinogenicity. 16. There are insufficient date on validation of the SHE cell transformation assay to justify the development of an OECD test guideline for this assay. 17. The Committee has considerable reservations regarding the mechanistic basis underpinning the rationale for using the SHE cell transformation assay to screen for chemical carcinogenesis. 18. The Committee has considerable reservations regarding the validity of using morphological assessment alone to define transformed foci in the SHE cell transformation assay . The development of an objective molecular marker is essential before validation work can proceed. References 1. Robinson DE and MacDonald J (2001). Background and Framework for ILSI's collaborative evaluation program on alternative models for carcinogenicity assessment. Toxicologic Pathology, 29 (suppl), 13-19. 2. Department of Health (1994). 1994 Annual Report of the Committees on Toxicity, Mutagenicity and Carcinogenicity of Chemicals in Food, Consumer Products and the Environment. Published The Stationary Office, London. 3. Department of Health (1998). 1996 Annual Report of the Committees on Toxicity, Mutagenicity and Carcinogenicity of Chemicals in Food, Consumer Products and the Environment. Published The Stationary Office, London. J36818 C6 01/98. 4. Mauthe RJ, Gibson DP, Bunch RT, and Custer L (2001). The syrian Hamster Embryo (SHE) cell transformation assay: Review of the methods and results from ILSI/HESI program on alternative carcinogenicity testing. Toxiologic Pathology 29, 138-146.
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