Reduction of misleading ("false") positive results in mammalian cell genotoxicity assays. II. Importance of accurate toxicity measurement

In a previous publication, Fowler et al. [4] demonstrated that the seemingly high rate of false or misleading positive results obtained in in vitro cytogenesis assays for genotoxicity - when compared with in vivo genotoxicity or rodent carcinogenicity data - was greater when rodent cell lines were used that were also reported to have mutant or non-functional p53. As part of a larger project for improvement of in vitro mammalian cell assays, we have investigated the impact of different toxicity measures, commonly used in in vitro cytogenetic assays, on the occurrence of misleading positive results. From a list of 19 chemicals that produce "false" positive results in in vitro mammalian cell assays [10], six substances that had given positive responses in CHO, CHL and TK6 cells [4], were evaluated for micronucleus induction in vitro, with different measures of toxicity for selection of the top concentration. The data show that estimating toxicity by relative cell count (RCC) or replication index (RI) consistently underestimates the toxicity observed by other measures (Relative Population Doubling, RPD, or Relative Increase in Cell Count, RICC). RCC and RI are more likely to lead to selection of concentrations for micronucleus scoring that are highly cytotoxic and thus could potentially lead to artefacts of toxicity being scored (elevated levels of apoptosis and necrosis), generating misleading positive results. These results suggest that a further reduction in the frequency of misleading positive results in in vitro cytogenetic assays can be achieved with this set of chemicals, by avoiding the use of toxicity measures that underestimate the level of toxicity induced.     

Reduction of misleading ("false") positive results in mammalian cell genotoxicity assays. I. Choice of cell type

Current in vitro mammalian cell genotoxicity assays show a high rate of positive results, many of which are misleading when compared with in vivo genotoxicity or rodent carcinogenicity data. P53-deficiency in many of the rodent cell lines may be a key factor in this poor predictivity. As part of an European Cosmetics Industry Association initiative for improvement of in vitro mammalian cell assays, we have compared several rodent cell lines (V79, CHL, CHO) with p53-competent human peripheral blood lymphocytes (HuLy), TK6 human lymphoblastoid cells, and the human liver cell line, HepG2. We have compared in vitro micronucleus (MN) induction following treatment with 19 compounds that were accepted as producing misleading or "false" positive results in in vitro mammalian cell assays [6]. Of these, six chemicals (2-ethyl-1,3-hexandiol, benzyl alcohol, urea, sodium saccharin, sulfisoxazole and isobutyraldehyde) were not toxic and did not induce any MN at concentrations up to 10mM. d,l-Menthol and ethionamide induced cytotoxicity, but did not induce MN. o-Anthranilic acid was not toxic and did not induce MN in V79, CHL, CHO, HuLy and HepG2 cells up to 10mM. Toxicity was induced in TK6 cells, although there were no increases in MN frequency up to and above the 55% toxicity level. The other 10 chemicals (1,3-dihydroxybenzene, curcumin, propyl gallate, p-nitrophenol, ethyl acrylate, eugenol, tert-butylhydroquinone, 2,4-dichlorophenol, sodium xylene sulfonate and phthalic anhydride) produced cytotoxicity in at least one cell type, and were evaluated further for MN induction in most or all of the cell types listed above. All these chemicals induced MN at concentrations <10mM, with levels of cytotoxicity below 60% (measured as the replication index) in at least one cell type. The rodent cell lines (V79, CHO and CHL) were consistently more susceptible to cytotoxicity and MN induction than p53-competent cells, and are therefore more susceptible to giving misleading positive results. These data suggest that a reduction in the frequency of misleading positive results can be achieved by careful selection of the mammalian cell type for genotoxicity testing.     

Comparison of different methods for an accurate assessment of cytotoxicity in the in vitro micronucleus test. II: Practical aspects with toxic agents

Appropriate measures of cytotoxicity need to be used when selecting test concentrations in in vitro genotoxicity assays. Underestimation of toxicity may lead to inappropriately toxic concentrations being selected for analysis, with the potential for generation of irrelevant positive results. As guidance for the in vitro micronucleus test is being developed, it is clearly important to compare the different measures of cytotoxicity that can be used both with and without cytokinesis blocking. Therefore, relative cell counts (RCC), relative increase in cell counts (RICC) and relative population doubling (RPD) for treatments without cytokinesis block were compared with replication index (RI) for treatments with cytokinesis block, and the corresponding induction of micronucleated cells was evaluated. A wide range of chemicals and gamma irradiation were used, and in almost all cases, RCC underestimated cytotoxicity when compared with all other measures such that RCC would have resulted in the selection of inappropriately high concentrations for micronuclei analysis. In the absence of cytokinesis block, RICC or RPD is more comparable with RI with cytokinesis block, and therefore considered more appropriate measure of survival. Furthermore, using these estimations of cytotoxicity and the limit of 50% survival, all the mutagens and aneugens tested were appropriately identified as positive in the in vitro micronucleus assay. Accordingly, it was clear that testing beyond 50% survival was not necessary to identify the potential of these agents to induce micronuclei.     

Effect of reducing the top concentration used in the in vitro chromosomal aberration test in CHL cells on the evaluation of industrial chemical genotoxicity

A current concern with in vitro mammalian cell genotoxicity testing is the high frequency of false or misleading positive results caused in part by the past use of excessively high test concentrations. A dataset of 249 industrial chemicals used in Japan and tested for genotoxicity was analyzed. Of these, 116 (46.6%) were positive in the in vitro chromosomal aberration (CA) test, including 6 that were positive only at test concentrations >10mM. There were 59 CA-positive chemicals at test concentrations ≤ 1mM. At >1mM, 51 chemicals were CA-positive, including 13 Ames-positive chemicals, which were therefore not "missed" by the test battery. Thus, 38 potentially positive chemicals would not have been detected in the test battery if the top test concentration was limited to 1mM in CA test. Analysis of the relevance of CA results on the 38 missed chemicals was conducted based on a weight of evidence approach, including evaluations of effects of extreme culture conditions (low pH, high toxicity, or precipitation), in silico structural alert analysis, in vivo genotoxicity and carcinogenicity test data (where available), mode of action, or information from closely related chemicals. After an exhaustive review, there were four chemicals with some concern for human health risk assessment, nine with minimal concern, and the remaining 25 with negligible concern. We apply different top concentrations to the 38 missed chemicals to identify the most accurate approach for predicting the genotoxicity of industrial chemicals. Of these 2mM or 1mg/mL, whichever is higher, was the most effective in detecting these chemicals, i.e., relatively higher (8/13) or lower (17/25) detection among 13 chemicals with some or minimal concern, or 25 with negligible concern, respectively. Lower top concentration limits, 1mM or 0.5mg/mL, whichever is higher, are not as effective (2/13) for detecting these chemicals with concern. Therefore, we conclude 2mM or 1mg/mL, whichever is higher, would be an appropriate top concentration limit for testing industrial chemicals for chromosome damage.     

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.     

Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test.

The in vitro micronucleus (MN) test was carried out simultaneously with the conventional chromosomal aberration (CA) test on 11 clastogenic chemicals or spindle poisons with different modes of action using a Chinese hamster cell line (CHL). The method of slide preparation for the MN test was the same as that for the conventional metaphase analysis, except that 1% acetic acid in methanol was used as the cell suspension medium for air-drying (to preserve the cytoplasm around the nucleus). All chemicals tested induced micronuclei reproducibly and dose-dependently in good agreement with the results of metaphase analysis (r = 0.99). Since the MN test methodology is simple and the observation of MN is less subjective than that of CA, we conclude that the in vitro MN test would be a good alternative to the conventional CA test for screening the genotoxicity of chemicals.     

Involvement of p53 function in different magnitude of genotoxic and cytotoxic responses in in vitro micronucleus assays

In in vitro micronucleus (MN) assays the sensitivity to MN induction or cytotoxicity can vary depending on the kind of cells employed. This study was conducted to examine the involvement of the p53 function in the different sensitivities between Chinese hamster lung (CHL) cells and human lymphoblastoid TK6 cells in MN assays. MN induction and cytotoxicity were compared using MN-inducing chemicals reported as DNA reactive clastogens, non-DNA reactive clastogens or aneugens. The study revealed that the maximum levels of MN induction in p53-compromised CHL cells were higher than those in p53-competent TK6 cells, but MN were significantly induced in TK6 cells at lower concentrations than in CHL cells. Most of the test chemicals produced a more severe cytotoxicity in TK6 cells, suggesting TK6 cells are more sensitive for cytotoxicity than CHL cells. An additional experiment with 9 MN inducers revealed that the magnitude of MN induction and cytotoxicity were comparable between p53-competent TK6 cells and its p53-null mutant NH32 cells at the same concentrations. Furthermore, the MN frequencies induced by methylmethane sulfonate, aphidicolin and hydroxyurea in NH32 cells were identical to those in TK6 cells at different recovery times. From these results, it is suggested that the p53 abrogation does not explain the difference in sensitivity to MN induction or cytotoxicity between CHL and TK6 cells. In this regard, p53 abrogated NH32 cells can be an option for the in vitro MN assay.     

An evaluation of the in vitro cytotoxicities of 50 chemicals by using an electrical current exclusion method versus the neutral red uptake and MTT assays

According to the 2001 National Institutes of Health guidance document on using in vitro data to estimate in vivo starting doses for acute toxicity, the performance of the electrical current exclusion method (ECE) was studied for its suitability as an in vitro cytotoxicity test. In a comparative study, two established in vitro assays based on the quantification of metabolic processes necessary for cell proliferation or organelle integrity (the MTT/WST-8 [WST-8] assay and the neutral red uptake [NRU] assay), and two cytoplasm membrane integrity assays (the trypan blue exclusion [TB] and ECE methods), were performed. IC50 values were evaluated for 50 chemicals ranging from low to high toxicity, 46 of which are listed in Halles Registry of Cytotoxicity (RC). A high correlation was found between the IC50 values obtained in this study and the IC50 data published in the RC. The assay sensitivity was highest for the ECE method, and decreased from the WST-8 assay to the NRU assay to the TB assay. The consistent results of the ECE method are based on technical standardisation, high counting rate, and the ability to combine cell viability and cell volume analysis for detection of the first signs of cell necrosis and damage of the cytoplasmic membrane caused by cytotoxic agents.     

Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish

In ecotoxicology, in vitro assays with fish cells are currently applied for mechanistic studies, bioanalytical purposes and toxicity screening. This paper discusses the potential of cytotoxicity assays with fish cells to reduce, refine or replace acute lethality tests using fish. Basal cytotoxicity data obtained with fish cell lines or fish primary cell cultures show a reasonable to good correlation with lethality data from acute toxicity tests, with the exception of compounds that exert a specific mode of toxic action. Basal cytotoxicity data from fish cell lines also correlate well with cytotoxicity data from mammalian cell lines. However, both the piscine and mammalian in vitro assays are clearly less sensitive than the fish test. Therefore, in vivo LC50 values (concentrations of the test compounds that are lethal to 50% of the fish in the experiment within 96 hours) currently cannot be predicted from in vitro values. This in vitro-in vivo difference in sensitivity appears to be true for both fish cell lines and mammalian cell lines. Given the good in vitro-in vivo correlation in toxicity ranking, together with the clear-cut difference in sensitivity, the role of cytotoxicity assays in a tiered alternative testing strategy could be in priority setting in relation to toxic hazard and in the toxicity classification of chemicals and environmental samples.     

Genotoxicity evaluation of selenium sulfide in in vivo and in vivo/in vitro micronucleus and chromosome aberration assays

Selenium monosulfide (Ses) was reported to be carcinogenic to livers of male and female rats and livers and lungs of female mice. However, its genotoxicity profile in short-term assays is somewhat equivocal. A multiple endpoint/multiple tissue approach to short-term genetic toxicity testing has been developed in our laboratory. In the present paper, the effect of SeS in in vivo and in vivo/ in vitro micronucleus and chromosome aberration assays in rat bone marrow and spleen are reported. In the in vivo assay, small but statistically significant increases in bone marrow micronucleated polychromatic erythrocytes (MNPCEs) were observed 24 h after treatment of rats with 50 mg/kg SeS and 48 h after treatment with 12.5 mg/kg. A significant decrease in the PCE/total erythrocyte (TE) ratio, indicative of cytotoxicity, was observed at the 50 mg/kg dose at the 24-h timepoint. In spleen, no increases in MNPCEs or decreases in the PCE/TE ratios were observed. No evidence of a significant increase in aberrations was observed in bone marrow or spleen. In the in vivo/in vitro assay, no increase in micronucleated binucleated cells or cells with aberrations was observed in SeS-treated rats. The small but statistically significant increases in MN observed in the in vivo study are considered likely not to be biologically significant since no dose-response was observed and all the values obtained were within historical control range in our laboratory. Given the overall genetic toxicity profile of SeS, it appears that SeS may be a weak mutagen and that differences between testing protocols may be very important in determining whether or not it is found to be negative or positive. Histological evidence was obtained in this study that suggests that the liver is the acute target organ of SeS in rats. Given the fact that SeS is selectively hepatocarcinogenic, we are currently testing the hypothesis that the genotoxicity of SeS in rats may be more readily detectable in liver than in bone marrow or spleen.     

Use of an established cell line in the evaluation of the cytotoxic effects of various chemicals.

The HTC hepatoma cell line was used as an "in vitro" model to detect the cytotoxicity of eighteen chemicals, chosen on the basis of different biological activities and physicochemical characteristics. Two different cytotoxicity assays measuring cell lethality (CS) or inhibition of cell growth (CF) were applicated to confluent cell monolayers or to colony-forming cells, respectively. Cells were exposed to the chemicals at doses ranging from 10(-6) M to 10(-2) M for 24 h. The results indicated a wide range of IC 50 (the concentration resulting in 50% inhibition of toxicity parameters) from as low as 1 microM (Potassium dichromate) to as high as 407.5 mM (Ethanol), the sensitivity of the CF test being greater than that of the CS test. A battery of cytotoxicity tests could be established in order to offer simple, rapid and economic methods which can be complementary and, in part, alternative to the use of laboratory animals.     

Relevance and follow-up of positive results in in vitro genetic toxicity assays: an ILSI-HESI initiative

In vitro genotoxicity assays are often used to screen and predict whether chemicals might represent mutagenic and carcinogenic risks for humans. Recent discussions have focused on the high rate of positive results in in vitro tests, especially in those assays performed in mammalian cells that are not confirmed in vivo. Currently, there is no general consensus in the scientific community on the interpretation of the significance of positive results from the in vitro genotoxicity assays. To address this issue, the Health and Environmental Sciences Institute (HESI), held an international workshop in June 2006 to discuss the relevance and follow-up of positive results in in vitro genetic toxicity assays. The goals of the meeting were to examine ways to advance the scientific basis for the interpretation of positive findings in in vitro assays, to facilitate the development of follow-up testing strategies and to define criteria for determining the relevance to human health. The workshop identified specific needs in two general categories, i.e., improved testing and improved data interpretation and risk assessment. Recommendations to improve testing included: (1) re-examine the maximum level of cytotoxicity currently required for in vitro tests; (2) re-examine the upper limit concentration for in vitro mammalian studies; (3) develop improved testing strategies using current in vitro assays; (4) define criteria to guide selection of the appropriate follow-up in vivo studies; (5) develop new and more predictive in vitro and in vivo tests. Recommendations for improving interpretation and assessment included: (1) examine the suitability of applying the threshold of toxicological concern concepts to genotoxicity data; (2) develop a structured weight of evidence approach for assessing genotoxic/carcinogenic hazard; and (3) re-examine in vitro and in vivo correlations qualitatively and quantitatively. Conclusions from the workshop highlighted a willingness of scientists from various sectors to change and improve the current paradigm and move from a hazard identification approach to a "realistic" risk-based approach that incorporates information on mechanism of action, kinetics, and human exposure..     

Guidance for understanding solubility as a limiting factor for selecting the upper test concentration in the OECD in vitro micronucleus assay test guideline no. 487

The OECD guideline for the in vitro mammalian cell micronucleus test (OECD 487) was recently adopted in July 22, 2010. Since its publication, it has become apparent that the guidance for testing chemicals where solubility is a limiting factor can be interpreted in a variety of ways. In this communication, we provide clarification for testing insoluble chemicals. The intent of the OECD 487 guideline is for the high dose to be the lowest precipitating concentration even if toxicity occurs above the solubility limit in tissue culture medium. Examination of precipitation can be done by the unaided eye or microscopically. Precipitation is examined at the onset or end of treatment, with the intent to identify precipitate present during treatment.     

Assessing the data quality in predictive toxicology using a panel of cell lines and cytotoxicity assays

In vitro cell viability assays have a central role in predictive toxicology, both in assessing acute toxicity of chemicals and as a source of experimental data for in silico methods. However, the quality of in vitro toxicity databanks fluctuates dramatically because information they contain is obtained under varying conditions and in different laboratories. The aim of this study was to identify the factors responsible for these deviations and thus the quality of the data extracted for predictive toxicology. Three cell viability assays measuring LDH leakage, WST-1 reduction, and intracellular ATP were compared in an automated environment using four mammalian cell lines: Caco-2, Calu-3, Huh-7, and BHK. Using four standard compounds--polymyxin B, gramicidin, 5-fluorouracil, and camptothecin--a significant lack of sensitivity in LDH assay compared with the other assays was observed. Because the viability IC(50) values for the standards were similar among the cell lines, the biochemical characteristics of different cell lines seem to play only a minor role, with an exception being the hepatocellular Huh-7 cell line. Toxicity assessment of new 1,2,4-triazoles revealed significant differences in their toxic potential, and the results indicate the same sensitivity profile among the assays as observed with the standard compounds. Overall, it can be argued that the assay selection is the most important factor governing the uniform quality of the data obtained from in vitro cell viability assays.     

In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances

A collaborative study with 10 participating laboratories was conducted to evaluate a test protocol for the performance of the in vitro micronucleus (MN) test using the V79 cell line with one treatment and one sampling time only. A total of 26 coded substances were tested in this study for MN-inducing properties. Three substances were tested by all 10 laboratories and 23 substances were tested by three or four laboratories in parallel. Six aneugenic, 7 clastogenic and 6 non-genotoxic chemicals were uniformly recognised as such by all laboratories. Three chemicals were tested uniformly negative by three laboratories although also clastogenic properties have been reported for these substances. Another set of three clastogenic substances showed inconsistent results and one non-clastogenic substance was found to be positive by one out of three laboratories. Within the study, the applicability of the determination of a proliferation index (PI) as an internal cytotoxicity parameter in comparison with the determination of the mitotic index (MI) was also evaluated. Both parameters were found to be useful for the interpretation of the MN test result with regard to the control of cell cycle kinetics and the mode of action for MN induction. The MN test in vitro was found to be easy to perform and its results were mainly in accordance with results from chromosomal aberration tests in vitro.     

The in vitro genotoxicity of benzoic acid in human peripheral blood lymphocytes

The chromosomal aberration (CA), sister chromatid exchange (SCE) and micronucleus test (MN) were employed to investigate the in vitro effect of antimicrobial food additive benzoic acid on human chromosomes. Lymphocytes were incubated with various concentrations (50, 100, 200 and 500 μg/mL) of benzoic acid. The results of used assays showed that benzoic acid significantly increased the chromosomal aberration, sister chromatid exchange and micronucleus frequency (200 and 500 μg/mL) without changing the pH of the medium in a dose-dependent manner. Also this additive significantly decreased the mitotic index (MI) at the highest concentration for 24 h and 100, 200 and 500 μg/mL for 48 h. This decrease was dose-dependent as well. However, it did not effect the replication (RI) and nuclear division (NDI) indices.     

Species differences in the genotoxicity of cyclophosphamide and styrene in three in vivo assays.

Species differences in dispositional factors such as distribution, metabolism and excretion may often account for species differences in the toxic responses to foreign chemicals. In this study we compared the genotoxic responses of cyclophosphamide (CP) and styrene (ST) between Porton rats and LACA Swiss mice in three in vivo assays (bone marrow micronucleus (MN), sperm morphology (SM) and sister-chromatid exchange (SCE) assays). The sensitivities of the three assays were compared by the doses of the compounds required to elicit a significant genotoxic response. The baseline levels for the MN, SCE and SM assays were 1.1-1.4 and 1.2-1.3 MNPCEs/1000 PCEs, 0.23-0.24 and 0.20-0.21 SCEs/chromosome, 3.5-5.7% and 1.6-1.9% abnormal sperm in mice and rats, respectively. CP was a potent genotoxin in the MN and SCE assays but weakly genotoxic in the SM assay. At comparable doses, the rat was approximately 3-, 2.5- and 1.8-fold more sensitive to CP than mice in the MN, SM and SCE assays, respectively. ST produced weak genotoxic responses in all assays in mice and only in the SM and SCE assays in rats. The mice were more sensitive to ST in the MN and SM assays, while it was difficult to compare the species in the SCE assay. For both compounds the sensitivity of the three assays, in decreasing order, were SCE greater than MN much greater than SM. For CP the relative responses in the Porton rats and LACA Swiss mice were qualitatively similar to previous reports. Although the use of different strains may explain differences between the studies in the magnitude of the responses observed. The results for ST in the rat shows that the choice of genotoxic endpoint can determine whether a response is detectable. Moreover, the discrepancies between the results for ST in this study and others, suggest that as well as using a battery of in vivo tests, it may be prudent to select more that one strain or species to fully assess a compound's ability to produce DNA damage.