Noriho Tanaka

Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs

Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting.

A Bhas 42 cell transformation assay on 98 chemicals: The characteristics and performance for the prediction of chemical carcinogenicity

The Bhas 42 cell transformation assay is a short-term system using a clone of the BALB/c 3T3 cells transfected with an oncogenic murine ras gene (v-Ha-ras). The assay has previously been reported to be capable of detecting the tumor-initiating and tumor-promoting activities of chemical carcinogens according to the different protocols, an initiation assay and a promotion assay, respectively. We applied this short-term assay to 98 chemicals to characterize the assay and evaluate its performance for the detection of chemical carcinogenicity. When the assay results were compared with the existing genotoxicity data, the Bhas 42 cell transformation assay could detect a considerable number of Ames-negative and Ames-discordant carcinogens: and the promotion assay detected most of those Ames-negative and -discordant carcinogens. This fact suggested that the Bhas 42 cells behaved as initiated cells in the transformation assay. The performance indices were calculated from the assay results of 52 carcinogens and 37 non-carcinogens. The concordance was 78%, sensitivity 73%, specificity 84%, positive predictivity 86%, negative predictivity 69%, false negative 27% and false positive 16%. Of these values, the concordance, specificity, negative predictivity and false positive were superior and the other performance indices were equivalent to those of conventional genotoxicity tests. From overall results, we concluded that the accuracy of prediction of chemical carcinogenicity would be improved by introducing the Bhas 42 cell transformation assay into the battery of in vitro assays.

Photo catalogue for the classification of foci in the BALB/c 3T3 cell transformation assay.

This catalogue is a display of focus photos representative of the BALB/c 3T3 cell transformation assay (CTA). It is intended as a visual aid for the identification and the scoring of foci in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.

An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity.

The Bhas 42 cell transformation assay is a sensitive short-term system for predicting chemical carcinogenicity. Bhas 42 cells were established from BALB/c 3T3 cells by the transfection of v-Ha-ras gene and postulated to have acquired an initiated state in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay is capable of detecting both tumor-initiating and tumor-promoting activities of chemical carcinogens. The full assay protocol consists of two components, the initiation assay and the promotion assay, to detect the initiating activity and the promoting activity, respectively. An international study was carried out to validate this cell transformation assay in which six laboratories from three countries participated. Twelve coded chemicals were examined in total and each chemical was tested by three laboratories. In the initiation assay, concordant results were obtained by three laboratories for eight out of ten chemicals and in the promotion assay, concordant results were achieved for ten of twelve chemicals. The positive results were obtained in all three laboratories with the following chemicals: 2-acetylaminofluorene was positive in both initiation and promotion assays; dibenz[a,h]anthracene was positive in the initiation assay; sodium arsenite, lithocholic acid, cadmium chloride, mezerein and methapyrilene hydrochloride were positive in the promotion assay. o-Toluidin hydrochloride was positive in the both assays in two of the three laboratories. d-Mannitol, caffeine and l-ascorbic acid were negative in both assays in all the laboratories, and anthracene was negative in both assays in two of the three laboratories except one laboratory obtaining positive result in the promotion assay. Consequently, the Bhas 42 cell transformation assay correctly discriminated all six carcinogens and two tumor promoters from four non-carcinogens. Thus, the present study demonstrated that the Bhas 42 cell transformation assay is transferable and reproducible between laboratories and applicable to the prediction of chemical carcinogenicity. In addition, by comparison of the present results with intra-laboratory data previously published, within-laboratory reproducibility using the Bhas 42 cell transformation assay was also confirmed.

Recommended protocol for the BALB/c 3T3 cell transformation assay

The present protocol has been developed for the BALB/c 3T3 cell transformation assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes cell culture maintenance, the dose-range finding (DRF) experiment and the transformation assay, including cytotoxicity and morphological transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA.

Relevance of chemical structure and cytotoxicity to the induction of chromosome aberrations based on the testing results of 98 high production volume industrial chemicals.

Over a 6-year period (1991-1996), the chromosomal aberration testing of high production volume (HPV) industrial chemicals had been conducted using Chinese hamster lung (CHL/IU) cells according to OECD HPV testing program and the national program in Japan. A total of 98 chemicals were tested for the induction of chromosome aberration (CA), consisting of structural CA and polyploidy. Of the 98 chemicals, structural CA and/or polyploidy were induced by 39 chemicals (40%). Anilines and phenols tended to induce only structural CA. p-tert-Butylphenol had a peculiar feature in inducing not only structural CA but also polyploidy at considerably high frequency (93.2%) after continuous treatment for 48 h, posing an aneugenic potential. Not all, but six of 11 carboxylic acids or esters also showed the simultaneous induction of structural CA and polyploidy. The majority of organic phosphates, alcohols or ethers, alkyl benzenes and non-cyclic alkanes had no CA induction activity. For chemicals which were negative in the bacterial reverse mutation assay (Ames test), the proportion of the chemicals that induced CA at a severely cytotoxic dose (doses manifesting more than 50% cytotoxicity) was similar to that of the CA-negative chemicals manifesting severe cytotoxicity, suggesting that severely cytotoxic chemicals do not always induce CA.

Practical issues on the application of the GHS classification criteria for germ cell mutagens

The Globally Harmonized System of Classification and Labeling of Chemicals (GHS) requires classification of chemicals on germ cell mutagenicity. The Japanese government has conducted GHS classification on about 1400 chemicals in a 2-year project (J-GHS) for implementing GHS domestically. Prior to the classification work, the technical guidance for classification of germ cell mutagens was prepared. This guidance introduces the concept of heritable mutagenicity, and presents detailed criteria for germ cell mutagens, test data to be used, and a practical decision tree for classification. These practical guidance and supporting explanations are useful for non-expert Classifiers (scientists applying the classification criteria). Several issues, however, were identified during the course of J-GHS and in re-evaluating the classification results. These include: (1) the information sources when available data are limited; (2) lack of understanding GHS classification criteria or insufficient review of the information by Classifiers; (3) varying opinions of experts on data quality and weight of evidence, and; (4) decision tree approaches, e.g., inadequacy for use in overall evaluation in some cases. Ideally, classification should be performed by Classifiers with high expertise using high quality information sources. Genetic toxicologists as experts should consider data quality and reliability, and give a critical review of all available information for support of classification. A weight of evidence approach is also required to assess mutagenic potential of chemicals. Critical points for suitable classification for GHS are discussed.

Cytotoxicity study of 32 MEIC chemicals by colony formation and ATP assays

The cytotoxicity of the first 32 of the 50 chemicals listed in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme was evaluated by colony formation (BALB 3T3 cells) and adenosine triphosphate (ATP) assays (HL-60 cells and mouse erythrocytes). Significant correlations (r = 0.9-0.95) were obtained with ID(50) values (50% inhibition dose in comparison with the control) of the 23-30 chemicals from which such values could be obtained, in erythrocytes v. HL-60 cells, BALB 3T3 v. HL-60 cells and BALB 3T3 cells v. erythrocytes, respectively. When ID(50) values from colony formation and ATP assays of nine or 10 chemicals were compared with human acute oral lethal dose, human acute lethal blood concentration and mouse oral LD(50), close correlations (r = 0.80-0.97) were seen between data from in vitro and in vivo tests. These results suggest that colony formation and ATP assays are useful for screening chemicals.

Comparison of cytotoxic effects of chemicals in four different cell types

Cytotoxic effects were compared using a colony-formation assay in three established cell lines (Balb 3T3, mouse whole embryo, ARLJ301-3, rat liver and FRSK, rat keratinocytes) and one primary cell culture (RC-1, rabbit cornea) with the Draize eye irritancy score in vivo. The cells were treated with 52 chemicals on the day after plating, then cultured for 7 or 8 days. The 50% inhibition dose (ID(50)) for each chemical was calculated based on the colony number. With a few exceptions, the cytotoxicities of the chemicals were in the following order in all four cells: cationic detergents anionic detergents non-ionic detergents glycol or oil. These results were almost the same as the data in vivo. The correlation coefficients of the ID(50) to the Draize score of 20 in vivo were 0.57 (Balb 3T3), 0.61 (ARLJ301-3), 0.71 (FRSK) and 0.65 (RC-1). Balb 3T3 and ARLJ301-3 cells were slightly more sensitive to chemicals than FRSK and RC-1 cells. These results suggest that the colony-formation assay using established cell lines is an attractive method for the screening of chemicals in that large differences among cell types in their response to direct-acting chemicals, were not observed.

Validation study on five different cytotoxicity assays in Japan : an intermediate report

In October 1992, the Japanese Society of Alternatives to Animal Experiments (JSAAE) organized a first-step inter-laboratory validation study on five Cytotoxicity assays: crystal-violet staining assay, neutral red uptake, MTT assay, colony formation, and lactate dehydrogenase release assay. This study is to clarify problems in organizing system of validation, protocols and intra- and inter-laboratory variation of ED(50) values on six representative chemicals determined with five cell lines in 45 laboratories, and thereby to evaluate practicability of the Cytotoxicity assays for further validation targetting to the Draize test. Two samples of one of the six chemicals were used to test intra-laboratory variation, so seven chemical preparations were sent to each laboratory after double-blind coding. More than 2300 data files were submitted to the organizing committee in 1993 and now they are under data cleaning and comprehensive analysis. Preliminary results calculated from hand-plotted figures were presented on a typical severe irritant, cetylpyridium chloride monohydrate.