ECVAM prevalidation study on in vitro cell transformation assays: general outline and conclusions of the study

The potential for a compound to induce carcinogenicity is a key consideration when ascertaining hazard and risk assessment of chemicals. Among the in vitro alternatives that have been developed for predicting carcinogenicity, in vitro cell transformation assays (CTAs) have been shown to involve a multistage process that closely models important stages of in vivo carcinogenesis and have the potential to detect both genotoxic and non-genotoxic carcinogens. These assays have been in use for decades and a substantial amount of data demonstrating their performance is available in the literature. However, for the standardised use of these assays for regulatory purposes, a formal evaluation of the assays, in particular focusing on development of standardised transferable protocols and further information on assay reproducibility, was considered important to serve as a basis for the drafting of generally accepted OECD test guidelines. To address this issue, a prevalidation study of the CTAs using the BALB/c 3T3 cell line, SHE cells at pH 6.7, and SHE cells at pH 7.0 was coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and focused on issues of standardisation of protocols, test method transferability and within- and between-laboratory reproducibility. The study resulted in the availability of standardised protocols that had undergone prevalidation [1,2]. The results of the ECVAM study demonstrated that for the BALB/c 3T3 method, some modifications to the protocol were needed to obtain reproducible results between laboratories, while the SHE pH 6.7 and the SHE pH 7.0 protocols are transferable between laboratories, and results are reproducible within- and between-laboratories. It is recommended that the BALB/c 3T3 and SHE protocols as instituted in this prevalidation study should be used in future applications of these respective transformation assays. To support their harmonised use and regulatory application, the development of an OECD test guideline for the SHE CTAs, based on the protocol published in this issue, is recommended. The development of an OECD test guideline for the BALB/c 3T3 CTA should likewise be further pursued upon the availability of additional supportive data and improvement of the statistical analysis.     

Photo catalogue for the classification of cell colonies in the Syrian hamster embryo (SHE) cell transformation assay at pH 6.7

This catalogue is a display of Syrian hamster embryo (SHE) cell colony photos representative of the cell transformation assay (CTA) carried out at pH 6.7. It is intended as a visual aid for the identification and the scoring of cell colonies 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.     

Photo catalogue for the classification of cell colonies in the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0

This catalogue is a display of Syrian hamster embryo (SHE) cell colony photos representative of the cell transformation assay (CTA) carried out at pH 7.0. It is intended as a visual aid for the identification and the scoring of cell colonies 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.     

Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 6.7 for assessment of carcinogenic potential of chemicals

The Syrian hamster embryo (SHE) cell transformation assay (CTA) is an important in vitro method that is highly predictive of rodent carcinogenicity. It is a key method for reducing animal usage for carcinogenicity prediction. The SHE assay has been used for many years primarily to investigate and identify potential rodent carcinogens thereby reducing the number of 2-year bioassays performed in rodents. As for other assays with a long history of use, the SHE CTA has not undergone formal validation. To address this, the European Centre for the Validation of Alternative Methods (ECVAM) coordinated a prevalidation study. The aim of this study was to evaluate the within-laboratory reproducibility, test method transferability, and between-laboratory reproducibility and to develop a standardised state-of-the-art protocol for the SHE CTA at pH 6.7. Formal ECVAM principles for criteria on reproducibility (including the within-laboratory reproducibility, the transferability and the between-laboratories reproducibility) were applied. In addition to the assessment of reproducibility, this study helped define a standard protocol for use in developing an Organisation for Economic Co-operation and Development (OECD) test guideline for the SHE CTA. Six compounds were evaluated in this study: benzo(a)pyrene, 3-methylcholanthrene, o-toluidine HCl, 2,4-diaminotoluene, phthalic anhydride and anthracene. Results of this study demonstrate that a protocol is available that is transferable between laboratories, and that the SHE CTA at pH 6.7 is reproducible within- and between-laboratories.     

Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0 for assessment of carcinogenic potential of chemicals

The European Centre for the Validation of Alternative Methods (ECVAM) has organised an interlaboratory prevalidation study on the Syrian hamster embryo (SHE) cell transformation assay (CTA) at pH 7.0 for the detection of rodent carcinogens. The SHE CTA at pH 7.0 has been evaluated for its within-laboratory reproducibility, transferability and between-laboratory reproducibility. Four laboratories using the same basic protocol with minor modifications participated in this study and tested a series of six coded-chemicals: four rodent carcinogens (benzo(a)pyrene, 3-methylcholanthrene, 2,4-diaminotoluene and o-toluidine HCl) and two non-carcinogens (anthracene and phthalic anhydride). All the laboratories found the expected results with coded chemicals except for phthalic anhydride which resulted in a different call in only one laboratory. Based on the outcome of this study, it can be concluded that a standardised protocol is available that should be the basis for future use. This protocol and the assay system itself are transferable between laboratories and the SHE CTA at pH 7.0 is reproducible within- and between-laboratories.     

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.     

A refined protocol for conducting the low pH 6.7 Syrian hamster embryo (SHE) cell transformation assay

The Syrian hamster embryo (SHE) cell transformation assay evaluates the potential of chemicals to induce morphological transformation in karyotypically normal primary cells. Induction of transformation has been shown to correlate well with the carcinogenicity of many compounds in the rodent bioassay. Historically the assay has not received wide-spread use due to technical difficulty. An improved protocol for a low pH 6.7 assay was developed by LeBoeuf et al. [R.A. LeBoeuf, G.A. Kerckaert, M.J. Aardema, D.P. Gibson, R. Brauninger, R.J. Isfort, Mutat. Res., 356 (1996) 85-127], that greatly reduced many of the technical difficulties associated with the SHE assay. The purpose of this paper is to describe the most current execution of the pH 6.70 protocol including protocol refinements made since the publication of a comprehensive protocol for this assay in Kerckaert et al. [G.A. Kerckaert, R.J. Isfort, G.J. Carr, M.J. Aardema, Mutat. Res., 356 (1996) 65-84].     

Application of a two-stage Syrian hamster embryo cell transformation assay to cigarette smoke particulate matter

The induction of transformation in Syrian hamster embryo (SHE) cells is a multifactorial process, in comparison to endpoints induced in in vitro genotoxicity assays such as Ames, mouse lymphoma and cytogenetics [Y. Berwald, L. Sachs, In vitro cell transformation with chemical carcinogens, Nature (London) 200 (1963) 1182-1184]. Furthermore, a number of non-genotoxic carcinogens and promoters such as clofibrate and diethylhexylphthalate, have been positively identified in this assay, while giving false negative results in traditional genotoxicity assays [H. Yamasaki, J. Ashby, M. Bignami, W. Jongen, K. Linnainmaa, R.F. Newbold, G. Nguyen-Ba, S. Parodi, E. Rivedal, D. Schiffmann, J.W.I.M. Simons, P. Vasseur, Nongenotoxic carcinogens: development of detection methods based on mechanisms: a European project, Mutat. Res. 353 (1996) 47-63]. A high concordance between results obtained in this assay when compared with rodent carcinogenesis bioassays has also been noted [R.J. Isfort, G.A. Kerckaert, R.A. LeBoeuf, Comparison of the standard and reduced pH Syrian hamster embryo (SHE) in vitro cell transformation assays to predict the carcinogenic potential of chemicals, Mutat. Res. 356 (1996) 11-63]. Carcinogenesis is known to be a multistage process, with agents potentially acting at each stage. Specifically, mouse skin painting experiments established that tumour induction could be mechanistically divided into two distinct phases, termed initiation and promotion. Initiation, is defined as the stage at which a normal cell is converted to a latent tumour cell, followed by promotion where the latent tumour cell progresses to a tumour [W.F. Friedwald, P. Rous, The initiating and promoting elements in tumour production: analysis of the effects of tar, benzpyrene and methylcholanthrene on rabbit skin, J. Exp. Med. 80 (1944) 101-125]. A protocol for the pH 6.7 SHE transformation assay has been developed which allows separation of cell transformation process into two phases, potentially analogous to initiation and promotion in vivo. This allows chemicals found to be positive in the traditional SHE cell transformation assay to be further classified as initiators or promoters. Following validation with known initiators, benzo(a)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine and promoters, 12-O-tetradecanoyl-phorbol-13-acetate and phenobarbitone, the two-stage model was applied to cigarette smoke particulates which was found to act both at the initiation and promotion stage of cell transformation.     

Prevalidation study of the BALB/c 3T3 cell transformation assay for assessment of carcinogenic potential of chemicals

The cell transformation assays (CTAs) have attracted attention within the field of alternative methods due to their potential to reduce the number of animal experiments in the field of carcinogenicity. The CTA using BALB/c 3T3 cells has proved to be able to respond to chemical carcinogens by inducing morphologically transformed foci. Although a considerable amount of data on the performance of the assay has been collected, a formal evaluation focusing particularly on reproducibility, and a standardised protocol were considered important. Therefore the European Centre for the Validation of Alternative Methods (ECVAM) decided to coordinate a prevalidation study of the BALB/c 3T3 CTA. Three different laboratories from Japan and Europe participated. In the study the following modules were assessed stepwise: test definition (Module 1) consisted of the standardisation of the protocol, the selection of the cell lineage, and the preparation of a photo catalogue on the transformed foci. The within-laboratory reproducibility (Module 2) and the transferability (Module 3) were assessed using non-coded and coded 3-methylcholanthrene. Then, five coded chemicals were tested for the assessment of between-laboratory reproducibility (Module 4). All three laboratories obtained positive results with benzo[a]pyrene, phenanthrene and o-toluidine HCl. 2-Acetylaminofluorene was positive in two laboratories and equivocal in one laboratory. Anthracene was negative in all three laboratories. The chemicals except phenanthrene, which is classified by IARC (http://monographs.iarc.fr) as group 3 "not classifiable as to its carcinogenicity to human", were correctly predicted as carcinogens. Further studies on phenanthrene will clarify this discrepancy. Thus, although only a few chemicals were tested, it can be seen that the predictive capacity of the BALB/c 3T3 CTA is satisfactory. On the basis of the outcome of this study, an improved protocol, incorporating some changes related to data interpretation, has been developed. It is recommended that this protocol be used in the future to provide more data that may confirm the robustness of this protocol and the performance of the assay itself. During the study it became clear that selecting the most appropriate concentrations for the transformation assay is crucial.     

Enhancement of the morphological transformation of Syrian hamster embryo (SHE) cells by reducing incubation time of the target cells

Syrian hamster embryo (SHE) cell transformation has been used for many years to study chemical carcinogenesis in vitro. It has been shown that this assay is probably the most predictive short-term test system for identifying rodent carcinogens. Although most of the operational difficulties encountered in the early stage of application of this assay have been overcome by culturing the SHE cells under slightly acidic conditions (pH 6.7), a relatively low level of induction of morphological transformation (MT) by known carcinogens still occurs for many cell isolates. In order to improve the response of this assay system to known carcinogens, the effect of incubation time of target SHE cells on the frequency of morphological transformation induced by benzo(a)pyrene (BaP) was investigated. It was shown that the morphological transformation frequency induced by BaP increased significantly (1.4-2.5-fold) when the incubation time of target cells was reduced from the usual 24h to less than 6h prior to seeding onto feeder layers. This improvement in sensitivity was consistent for different cell isolates. In addition, the enhanced response appeared to be a property of carcinogens because treatment with two non-carcinogens, l-ascorbic acid and 4-nitro-o-phenylenediamine, did not induce significant increases in the transformation frequency under the shortened incubation period for target cells. These results suggest that the response of the SHE cell transformation assay may be improved by optimizing the incubation time of the target SHE cells. In addition, the results of the present study provide further evidence to support the idea that morphological transformation of SHE cells results from a block of cellular differentiation of stem or stem-like cells.     

Analysis of chromate-induced DNA-protein crosslinks with the comet assay

Modifications of the comet assay have been introduced to measure crosslinks by determining the reduction of induced DNA migration. Our previous results indicated that the modified protocol of the alkaline comet assay is a sensitive tool for the detection of formaldehyde-induced DNA-protein crosslinks. But results for mitomycin C and cisplatin suggested that the modified protocol is not well suited for the evaluation of DNA-DNA crosslinkers. We now used the comet assay to investigate in V79 cells the effect of potassium chromate (K(2)CrO(4)), another DNA-protein crosslinker, to see whether the results obtained for formaldehyde can be generalized. However, chromate did not reduce spontaneous or radiation-induced DNA migration in the alkaline (pH 13) comet assay but led to a small but significant induction of DNA migration. A crosslinking effect of chromate could also not be detected with the alkaline comet assay after postincubation of cells in normal medium after chromate treatment to enable repair of other (migration-inducing) lesions that might mask the crosslinking effect. Exposure of slides to proteinase K further increased DNA migration of chromate-treated cells, thus indicating the presence of DNA-protein crosslinks. In contrast to the alkaline comet assay, a "neutral" version at pH 9 was suited to demonstrate reduced induction of DNA migration after gamma-irradiation of chromate-treated cells. The crosslinking effect was seen immediately at the end of the chromate treatment as well as after a 3h postincubation period. Using the "neutral" protocol in combination with proteinase K, we were able to demonstrate the presence of DNA-protein crosslinks as the probable cause for the migration-reducing effect. Further investigations will have to show whether this protocol can be recommended as a universal approach for the detection of DNA-protein crosslinks and also of DNA-DNA crosslinks with the comet assay.     

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.     

Syrian hamster embryo (SHE) cell transformation assay with conditioned media (without X-ray irradiated feeder layer) using 2,4-diaminotoluene, 2,6-diaminotoluene and chloral hydrate

The Syrian hamster embryo (SHE) cell transformation assay has traditionally been conducted with a feeder layer of X-ray irradiated cells to provide growth support to the target cells seeded in low numbers. The feeder layer of cells consists of X-ray irradiated cells which are still viable but unable to replicate. We have tried seeding the target cells in conditioned media prepared from the stock culture flasks in lieu of plating them on a feeder layer. Three SHE cell isolates were tested to investigate the feasibility of this approach. With freshly prepared conditioned medium (LeBoeuf's Dulbecco's Modified Eagle's Medium with 2 mM L-glutamine and 20% fetal bovine serum), used within 2 weeks of preparation, there was essentially no difference in the number of target cell colonies in the conditioned medium and in the plates with the X-ray irradiated feeder cell layer. The plating efficiencies of the vehicle controls were within the historical range for the standard SHE cell transformation assay. In each experiment, the positive control benzo(a)pyrene [B(a)P] elicited a significant increase in morphological transformation frequency (MTF), with or without feeder cells. Three compounds, 2,4-diaminotoluene (2,4-DAT), 2,6-diaminotoluene (2,6-DAT), and chloral hydrate were tested in the SHE cell transformation assay without an X-ray irradiated feeder layer and using a 7-day exposure regimen. The results were comparable to those reported in the published literature using the standard methodology with feeder cells, with 2,4-DAT and chloral hydrate eliciting a significant increase in MTF, and 2,6-DAT not eliciting any increase in MTF. The results of this study demonstrate the feasibility of conducting the SHE cell transformation assay without the use of an X-ray irradiated feeder layer, thereby simplifying the test procedure and facilitating the scoring of morphologically transformed colonies.     

An interlaboratory comparison of enhanced morphological transformation of Syrian hamster embryo cells cultured under conditions of reduced bicarbonate concentration and pH

Initial studies performed in our laboratory indicated that early passage Syrian hamster embryo (SHE) cells exhibit optimal clonal proliferation when cultured in medium with a sodium bicarbonate concentration of 8.9 mM and pH of 6.70 instead of 44 mM and pH 7.35 as used previously by others. Subsequent studies indicated that morphological transformation frequency induced by benzo[a]pyrene (BP) was also enhanced at pH 6.70 compared to 7.35 and the level of enhancement was affected by cell density and duration of culture. With optimal conditions identified, the carcinogens BP, 3-methylcholanthrene, N-methyl-N'-nitro-N-nitrosoguanidine, 2-acetylaminofluorene and the non-carcinogen anthracene were tested at pH 6.70 and 7.35 in our laboratory and at Microbiological Assoc. Inc. under code. Additionally, the non-carcinogens 4-acetylaminofluorene, and caprolactam were tested in our laboratory. Results from these studies indicate that all carcinogens tested caused a significant increase in morphological transformation frequency compared to controls at pH 6.70. In contrast, only BP caused a significant increase in the morphological transformation frequency at pH 7.35. The non-carcinogens did not significantly increase the morphological transformation frequency compared to controls. Interlaboratory comparisons were in qualitative agreement despite the fact that different lots of serum and hamster cell isolates were used by the two laboratories. However, different dose-response curves for the various chemicals were observed between the two labs. It was also demonstrated that the enhanced morphological transformation frequency is not due to a decrease in culture medium osmolality or Na concentration, a condition which accompanies media with a reduced bicarbonate concentration and pH. These results demonstrate that the chemicals tested, low pH transformation of SHE cells agrees with carcinogenic potential and that assay variability is minimized. The implications of these results regarding use of the SHE cell assay as a short-term test for predicting the carcinogenic potential of chemicals are discussed.     

High frequency of constitutive alkali-labile sites in mouse major satellite DNA, detected by DNA breakage detection-fluorescence in situ hybridization

Electromagnetic fields (EMFs) have been associated with increased incidence of cancer suggested by epidemiological studies. To test the carcinogenic potency of EMF, the in vitro micronucleus assay with SHE cells has been used as a screening method for genotoxicity. A 50Hz magnetic field (MF) of 1mT field strength was applied either alone or with the tumour initiator benzo(a)pyrene (BP) or the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). All three treatments were applied in single, double or triple treatment regimes. MF or TPA (1nM) alone did not affect the number of micronuclei (MN) in initiated and non-initiated SHE cells. Changing the schedule of the typical initiation protocol, namely applying the initiator (BP) during exposure to MF, results in an 1.8-fold increased MN formation compared to BP treatment alone. Combined experiment with BP, TPA and MF did not cause further MN formation. Since initiation during MF exposure caused a significant increased MN formation, our findings suggest that MFs enhance the initiation process of BP. We think that this MF-enhanced co-carcinogenic effect is caused by an indirect "cell activation" process. The resulting genomic instability is proposed to be due to free radicals and/or to the unscheduled "switching-on" of signal transduction pathways.     

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: III. Cell transformation in C3H/10T1/2 mouse embryo cell, BALB/c 3T3 mouse fibroblast and Syrian hamster embryo cell cultures

A standardized protocol and guidelines for the performance of cell transformation testing in mouse embryo (C3H/10T1/2), mouse fibroblast (BALB/c 3T3) and Syrian hamster embryo (SHE) cells have been developed. The protocol is based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North American and European governmental, university and contract laboratories involved with cell transformation experimentation. This report identifies those modifications to previously described methodologies which are being used on a regular basis and also serves to clarify confusing or inconsistent practices.     

Insect virus: assays for toxic effects and transformation potential in mammalian cells

The nuclear polyhedrosis virus of Autographa californica (AcNPV) was evaluated by using in vitro test systems for toxicity and transforming potential in mammalian cells. Mass cell cultures of CV-1 and WI38 cells appeared unaffected by AcNPV at a multiplicity of infection of 5. Human foreskin cells grew more slowly after inoculation but eventually produced healthy monolayers. The sensitivities of the inhibition of reproductive survivability assays were greater and demonstrated slight AcNPV toxicity to CV-1, WI38, and human foreskin cells. Toxicity was not ameliorated when gradient-purified or psoralen-inactivated virus was used, suggesting that the toxic component of the preparation is part of the virion or copurifies with it. AcNPV was not toxic to and did not transform BALB/c 3T3 cells or primary cell cultures derived from Syrian hamster embryo cells (SHE). Unlike the BALB/c 3T3 transformation assay, the SHE assay detected no spontaneous transformants. The SHE transformation assay can employ simian adenovirus 7 as a positive control. SHE are transformed by numerous viruses and so are useful in assessment protocols. This study suggests that in vitro assessment of viral pesticide toxicity should employ the inhibition of reproductive survivability assay and that transformation assessment is best done with the SHE-simian adenovirus 7 procedure.     

Insect virus: assays for toxic effects and transformation potential in mammalian cells.

The nuclear polyhedrosis virus of Autographa californica (AcNPV) was evaluated by using in vitro test systems for toxicity and transforming potential in mammalian cells. Mass cell cultures of CV-1 and WI38 cells appeared unaffected by AcNPV at a multiplicity of infection of 5. Human foreskin cells grew more slowly after inoculation but eventually produced healthy monolayers. The sensitivities of the inhibition of reproductive survivability assays were greater and demonstrated slight AcNPV toxicity to CV-1, WI38, and human foreskin cells. Toxicity was not ameliorated when gradient-purified or psoralen-inactivated virus was used, suggesting that the toxic component of the preparation is part of the virion or copurifies with it. AcNPV was not toxic to and did not transform BALB/c 3T3 cells or primary cell cultures derived from Syrian hamster embryo cells (SHE). Unlike the BALB/c 3T3 transformation assay, the SHE assay detected no spontaneous transformants. The SHE transformation assay can employ simian adenovirus 7 as a positive control. SHE are transformed by numerous viruses and so are useful in assessment protocols. This study suggests that in vitro assessment of viral pesticide toxicity should employ the inhibition of reproductive survivability assay and that transformation assessment is best done with the SHE-simian adenovirus 7 procedure.