Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens

Mutation Research 584 (2005) 1–256

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夽 David Kirkland a,∗ , Marilyn Aardema b , Leigh Henderson c,1 , Lutz M¨uller d b

a Covance Laboratories Limited, Otley Road, Harrogate HG3 1PY, UK The Procter and Gamble Company, Miami Valley Laboratories, PO Box 538707, Cincinnati, OH 45253-8707, USA c Henderson Scientific Consultancy, Northwich CW8 2UT, UK d Non-Clinical Drug Safety, F Hoffmann-La Roche Ltd., PRBN-S, Bldg 73311b, CH-4070 Basel, Switzerland

Received 3 November 2004; received in revised form 7 February 2005; accepted 25 February 2005

Abstract The performance of a battery of three of the most commonly used in vitro genotoxicity tests—Ames + mouse lymphoma assay (MLA) + in vitro micronucleus (MN) or chromosomal aberrations (CA) test—has been evaluated for its ability to discriminate rodent carcinogens and non-carcinogens, from a large database of over 700 chemicals compiled from the CPDB (“Gold”), NTP, IARC and other publications. We re-evaluated many (113 MLA and 30 CA) previously published genotoxicity results in order to categorise the performance of these assays using the response categories we established. The sensitivity of the three-test battery was high. Of the 553 carcinogens for which there were valid genotoxicity data, 93% of the rodent carcinogens evaluated in at least one assay gave positive results in at least one of the three tests. Combinations of two and three test systems had greater sensitivity than individual tests resulting in sensitivities of around 90% or more, depending on test combination. Only 19 carcinogens (out of 206 tested in all three tests, considering CA and MN as alternatives) gave consistently negative results in a full three-test battery. Most were either carcinogenic via a non-genotoxic mechanism (liver enzyme inducers, peroxisome proliferators, hormonal carcinogens) considered not necessarily relevant for humans, or were extremely weak (presumed) genotoxic carcinogens (e.g. N-nitrosodiphenylamine). Two carcinogens (5-chloro-o-toluidine, 1,1,2,2-tetrachloroethane) may have a genotoxic element to their carcinogenicity and may have been expected to produce positive results somewhere in the battery. We identified 183 chemicals that were non-carcinogenic after testing in both male and female rats and mice. There were genotoxicity data on 177 of these. The specificity of the Ames test was reasonable (73.9%), but all mammalian cell tests had very low specificity (i.e. below 45%), and this declined to extremely low levels in combinations of two and three test systems. When all three tests were performed, 75–95% of non-carcinogens gave positive (i.e. false positive) results in at least one test in the battery. The extremely 夽 The appendices have been posted on the following website www.lhasalimited.org.uk/cgx where any reader can go to make their own analy-

ses. These appendices will be updated on a regular basis. ∗ Corresponding author. Tel.: +44 1423 848401; fax: +44 1423 525152. E-mail address: [email protected] (D. Kirkland). 1 Present address: FSANZ, PO Box 10559, The Terrace, Wellington 6036, New Zealand. 1383-5718/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2005.02.004

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low specificity highlights the importance of understanding the mechanism by which genotoxicity may be induced (whether it is relevant for the whole animal or human) and using weight of evidence approaches to assess the carcinogenic risk from a positive genotoxicity signal. It also highlights deficiencies in the current prediction from and understanding of such in vitro results for the in vivo situation. It may even signal the need for either a reassessment of the conditions and criteria for positive results (cytotoxicity, solubility, etc.) or the development and use of a completely new set of in vitro tests (e.g. mutation in transgenic cell lines, systems with inherent metabolic activity avoiding the use of S9, measurement of genetic changes in more cancer-relevant genes or hotspots of genes, etc.). It was very difficult to assess the performance of the in vitro MN test, particularly in combination with other assays, because the published database for this assay is relatively small at this time. The specificity values for the in vitro MN assay may improve if data from a larger proportion of the known non-carcinogens becomes available, and a larger published database of results with the MN assay is urgently needed if this test is to be appreciated for regulatory use. However, specificity levels of <50% will still be unacceptable. Despite these issues, by adopting a relative predictivity (RP) measure (ratio of real:false results), it was possible to establish that positive results in all three tests indicate the chemical is greater than three times more likely to be a rodent carcinogen than a non-carcinogen. Likewise, negative results in all three tests indicate the chemical is greater than two times more likely to be a rodent non-carcinogen than a carcinogen. This RP measure is considered a useful tool for industry to assess the likelihood of a chemical possessing carcinogenic potential from batteries of positive or negative results. © 2005 Elsevier B.V. All rights reserved. Keywords: Genotoxicity; In vitro tests; Carcinogenicity; Prediction

1. Introduction The National Toxicology Program (NTP) evaluation of four short-term in vitro genotoxicity tests to predict rodent carcinogenicity [1] was the first systematic assessment of the performance of commonly used in vitro tests and demonstrated some of the limitations of these assays used individually and in a battery. For the 73 chemicals that were tested (including 44 carcinogens and 29 non-carcinogens) the sister-chromatid exchange (SCE) test demonstrated the highest sensitivity (ability to produce a positive response with a rodent carcinogen) at 73% and the Ames test was the least sensitive at 45%. A subsequent analysis of 114 chemicals [2,3] confirmed this initial analysis. A more recent analysis [4] of 363 chemicals tested by the NTP in three of the four assays (SCE was not included) revealed that the mouse lymphoma tk gene mutation assay (MLA) was the most sensitive (74%) but was the least specific (ability to give negative results with known rodent noncarcinogens). Although the Ames test had low sensitivity (54%) it demonstrated the best overall concordance (positive results for carcinogens + negative results for non-carcinogens), which was not improved by combining it with chromosomal aberration (CA) or MLA tests in a battery, because adding more tests to the battery increased the numbers of both carcinogens and non-carcinogens giving positive responses. Also of relevance is a recent analysis by Brambilla and Martelli

[5], who reported that the current battery of tests as proposed by ICH for pharmaceuticals [6] appears to fail to detect some crucial rodent and human genotoxic carcinogens. Since the NTP program was initiated some tests have lost favour (e.g. SCE) and new tests have gained favour (e.g. in vitro micronucleus (MN) test, see Kirsch-Volders et al. [7]). In addition, there are now results on many more rodent carcinogens and noncarcinogens than ever before. For some of the newer publications the protocols for conducting these genotoxicity tests will have been refined according to more recent recommendations (e.g. OECD [8]). In this paper we have evaluated how the currently popular in vitro genotoxicity tests – Ames test, MLA and a test for clastogenicity (in vitro micronucleus or chromosomal aberration test) – performed in their ability to discriminate rodent carcinogens and noncarcinogens. This paper deals separately with the performance indicators (sensitivity first, then specificity) of the individual tests and various combinations of two or three tests, and proposes reasons why some rodent carcinogens are not detected in this battery. It also proposes a new method to look at the balance of rising sensitivity accompanied by falling specificity to help choose the best individual or combinations of tests. It is noteworthy to mention that the rodent carcinogenicity database, when dissociated by likely mechanism of action, obviously contains so-called genotoxic and

D. Kirkland et al. / Mutation Research 584 (2005) 1–256

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non-genotoxic carcinogens. However, there are only a few rodent carcinogens for which the non-genotoxic mechanism of action is sufficiently defined to discern whether an absence of response in the in vitro battery is an accurate reflection of mechanism or a true lack of predictivity.

evidence of carcinogenicity based on local sarcomas after subcutaneous injections. Gold [9] does not evaluate studies using this route. • Titanium dioxide—included as a carcinogen (Appendix A) because IARC concluded it is a carcinogen only at high doses by the inhalation route.

2. Selection of chemicals

Chemicals excluded from our analysis due to questions over purity or test substance identity are:

Appendix A contains 756 chemicals that are considered rodent carcinogens based on tumour findings in at least one sex, rats or mice (both sexes and both species did not have to be tested). Appendix B contains 183 chemicals that are non-carcinogenic after thorough testing in both male and female rats and mice. We began the selection of chemicals for these appendices with the carcinogen potency database (CPDB) of Gold [9]. Because this database only includes chemicals tested for carcinogenicity via oral and parenteral routes, we have added some chemicals that are carcinogenic via topical application (e.g. to the skin) and considered to be carcinogenic by reputable organisations such as the International Agency for Research on Cancer (IARC) or the National Toxicology Program. Additional carcinogens identified by Nesnow et al. [10] and Haseman and Clark [11] were also included. We have, however, excluded from both appendices some chemicals where the purity or identity of the substance tested was not clear, and where the carcinogenicity outcome was inconclusive. The following amendments and comments to the database of Gold [9] are of note: • Cyclosporin—recorded as a non-carcinogen by Gold [9] but included as a carcinogen (Appendix A) because with adequate dosing it does produce tumours in animals. • Glycidaldehyde—included as a carcinogen (Appendix A) because IARC concluded it shows evidence of carcinogenicity based on local site tumours only after subcutaneous administration and skin painting, where chronic irritation may be the mechanism of action. Gold [9] does not evaluate studies using these routes. • Pyrimethamine—included as a carcinogen (Appendix A) on the basis of IARC evaluation. • Succinic anhydride—included as a carcinogen (Appendix A) because IARC concluded it shows

• 9,10-Anthraquinone—omitted from both Appendices A and B. The NTP report [15] describes it as carcinogenic but IARC considers the result inconclusive due to the low purity of the test chemical. We are unable to determine whether the same level of purity would have been used in the genotoxicity assay, and therefore comparisons are not possible. • 2,3,4,5,6-Pentachlorophenol (PCP)—three forms of PCP have been tested for carcinogenicity, a pure form, Dowicide EC-7 and technical grade, and the tumour profiles are different. Pure PCP was only tested in female mice but gave no tumours. Dowicide EC-7 produced adrenal and liver tumours in male and female mice but no tumours in rats. Technical grade PCP also produced liver tumours in female rats. It is not clear from the genotoxicity studies what material was used. Therefore, PCP was neither included in Appendix A or Appendix B. • Xylene mixture—two forms of xylene mixture have been tested for carcinogenicity. The mixture referred to as m-xylene, o-xylene, p-xylene produces tumours of the oral cavity at high doses in rats, but was not tested in mice. The mixture described as 60% mxylene, 9% o-xylene, 14% p-xylene, 17% ethylbenzene does not produce tumours either in rats or mice. It is not clear from the genotoxicity data which mixture was used. Xylene mixture has therefore not been included either in Appendix A or Appendix B. The following were also excluded from both appendices because Gold [9] gave an assessment of “inconclusive” for their carcinogenic potency. It is therefore not appropriate to try to correlate results in genotoxicity tests with such inconclusive carcinogenicity results: • allyl chloride; • chloropicrin;

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• • • •

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emetine·2HCl; l-epinephrine·HCl; phosphamidon; proflavine·HCl hemihydrate.

Due to the large number of chemicals in this exercise, the detailed results from the carcinogenicity assays were not reviewed. We hope to analyse the genotoxicity results in terms of tumour profile for a later publication. In the meantime we invite others to add contributions to the analyses described in this report.

3. Sources of genotoxicity test results For a number of pharmaceuticals that are listed in Appendices A and B, there are summaries of genotoxicity findings presented in the Physicians Desk Reference [12]. However, the information in most cases is very sparse, and insufficient to be able to judge whether the study designs met current criteria. Only the PDR data on omeprazole (Appendix B) were considered sufficiently detailed to be included in our analysis. The sources for the majority genotoxicity data are described below. 3.1. Ames assay results Ames test results for many chemicals were provided by Zeiger [13] in his compilation of the NTP genotoxicity database. Additional data were obtained from Gold et al. [14] in their paper on target organs for carcinogenicity. Other Ames data came directly from the NTP website [15], when entries could not be found in Zeiger [13], and from the evaluations of the US EPA Gene-Tox Program [16]. We knew that many of the early NTP Ames tests did not include strains such as TA102 or E. coli WP2uvrA that could have detected oxidising and cross-linking mutagens. However, if all other criteria for a valid assay were met (i.e. testing in at least four strains in the absence and presence of S9, up to 5000 ␮g/plate or toxicity or solubility limit) we accepted negative results as definitive. Additional publications were obtained by searching the databases of the Environmental Mutagen Information Center (EMIC—accessed via http://toxnet.nlm.nih.gov/cgibin/sis/search), Toxline and PubMed (to be found at http://toxnet.nlm.nih.gov).

3.2. Mouse lymphoma assay (MLA) results Evaluation and interpretation of the mouse lymphoma assay has changed over the years. Some of the early studies used very high concentrations or reached very high levels of toxicity, and the consequences of high osmolality, high ionic strength, high cytotoxicity and shifts in pH were not understood at that time. Such possible indirect influences on genotoxic responses, particularly, became apparent in the late 1980s [17–19]. However, the re-evaluation of MLA results by the panel of experts involved in the US EPA Gene-Tox Program [20] concluded many studies were inadequate, inconclusive or equivocal. We therefore checked the original data and made our own judgements according to current criteria [8] for a valid assay. MLA results for 67 carcinogens and 46 non-carcinogens were checked, and only four results remained unchanged (see Appendices A and B for details). The main reason for change was related to insolubility, and we did not have a category for “inconclusive results due to insolubility” in our analysis (see Section 4 below). Specifically we considered tests conducted up to the solubility limit as valid, whereas Mitchell et al. [20] considered these as inconclusive. Thus, a large number of the Mitchell calls had to be changed. As current guidelines only require one precipitating concentration to be evaluated in mouse lymphoma cells, because of the difficulty in removing the precipitate when the cells are centrifuged, we made such “calls” negative. As before, additional publications were obtained through searches of EMIC, Toxline and PubMed. 3.3. In vitro micronucleus assay The in vitro micronucleus test has not been used as long or as widely as the Ames, MLA and chromosomal aberration tests. However, there are some major reviews [21,22] and key papers [23,24] which provided the bulk of data used in this assessment. However, many chemicals had been tested for induction of chromosomal aberrations in vitro that had not been tested for MN. As the above publications (and others) clearly show a very high correlation between induction of structural aberrations and induction of MN, we decided to include in vitro chromosomal aberration data (see below) to supplement the in vitro MN data. It should be noted that, as with the chromosomal aberration test (see

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below) the methods for measuring toxicity and the levels of toxicity required at the top concentration for a valid micronucleus study, have changed over the years. Wherever possible we attempted to judge the response in the MN test according to current criteria [7]. As before, additional publications were obtained through searches of EMIC, Toxline and PubMed.

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Although not as rigorous by today’s standards, if all other criteria for a valid assay were met, we accepted negative results in an NTP study as definitive. As before, if data were not available from these sources, if studies were incomplete (e.g. negative but only tested in the absence of S9), or if the results were inconclusive we obtained additional publications through searches of EMIC, Toxline and PubMed.

3.4. In vitro chromosomal aberration assay Data for the in vitro chromosomal aberration test were taken from compilations such as that of Ishidate et al. [25] and the reports of NTP studies published by Galloway et al. [26], Loveday et al. [27,28] and Anderson et al. [29]. All cell types (CHO, CHL, human lymphocytes, etc.) were considered. Some of these studies were difficult to evaluate because toxicity data were not provided, and, similarly to the MLA, the extent of toxicity required at the highest concentration has changed over the years. For example in the 1983 OECD guideline it was recommended that up to 75% toxicity should be induced whereas the 1997 revision recommends at least 50% toxicity. It is now accepted [6,30–32] that positive results at high levels of cytotoxicity, particularly in CA tests, may not be biologically relevant. Judgement of which published positive CA results might have been irrelevant because they only occurred at high levels of cytotoxicity was therefore very difficult, and in many cases, where no toxicity data were provided, impossible. The methods of measuring toxicity have also changed; early studies tended to measure colony formation or confluency whereas cell counts or mitotic index measures have been more common in recent times. Wherever possible we attempted to judge the response in the CA test according to current criteria [8]. However, we knew that it was common in the NTP studies to use early sampling times (e.g. 10–15 h) whereas Ishidate and colleagues often used 24 or 48 h continuous treatments. We carefully reviewed the “calls” of Ishidate et al. [25] where a number of negative findings were reported from incomplete tests (e.g. lacking metabolic activation) and we reclassified some of these as “technically compromised”. We also assessed carefully those chemicals where conflicting calls came from different sources. In total we reevaluated the CA calls from 12 carcinogens and 18 non-carcinogens. Four calls remained unchanged (see Section 4 below, and Appendices A and B for details).

4. Response categories Where possible we have taken the consensus conclusions of experts such as Zeiger, Ishidate, etc. and those assembled in EPA Gene-Tox panels. For most clear positive and negative results we have accepted their conclusions. However, for negative results in old publications it was necessary to check whether an adequate protocol had been followed (see TC below). Also in cases where results were inconclusive, inconsistent, equivocal or extreme conditions pertained, we examined the original data and searched the literature for more recent papers. In the case of NTP studies, we first viewed the testing status on the “ntp-server” site (http://ntp-server.niehs.nih.gov/cgi), and then checked the results as far as possible (not all were available) on the “ntp-apps” site (http://ntpapps.niehs.nih.gov/ntp tox). Where the key publications listed above did not have data from one of the test systems, we searched the literature, primarily using EMIC at Oak Ridge National Laboratory and (particularly for publications since 2000) Toxline and PubMed. In many cases, genotoxicity data were located from different publications on the same chemical. In some cases the different publications all gave the same outcome, so deciding the response category was easy. In other cases the results in different publications were contradictory and a panel decision was made. We developed the following four categories for summarising the genotoxicity results of the chemicals evaluated in this assessment. While others will certainly evaluate individual responses differently from our classification in this exercise, the benefit of dealing with such a large dataset is that changes in “calls” for a small number of chemicals will not significantly impact the overall findings. We do, however, encourage other scientists to check the details of chemicals of particular

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interest to them, and update the database as appropriate: ‘+’ is a definitive positive response, either in a single publication or across the majority of publications with the chemical in question. Any negative results could be outweighed by overwhelming dominance of positive publications, or by viewing the data in detail and deciding that the negative test was not adequate. ‘−’ is a clearly negative response in all publications found. ‘E’ is equivocal, indicating that the results with a given chemical were not consistent (both positive and negative results obtained) either within an experiment, in repeated experiments in the same laboratory, or between laboratories (and therefore between publications). Weak responses, where there was some evidence of a chemically-induced effect but no clear dose–response or not reaching biologically significant levels, were also categorised E. Because equivocal results may be considered as either negative or positive, the sensitivity and specificity of the different assays and combinations of assays were evaluated considering E as negative and also considering E as positive. ‘TC’ is technically compromised, indicating a test result that was questionable due to failure to meet essential standard criteria for an adequate study. Some examples would be if a test compound was negative but only tested in the absence of S9, or if the test compound did not reach adequate levels of toxicity, or was not tested according to accepted criteria for upper concentrations for non-toxic or insoluble compounds. In such cases where negative results could not be completely judged as conclusive, we called these TC. As none of the studies categorised as TC contributed valid results from an acceptable study they were excluded from the comparison with rodent carcinogenicity. There is clearly a need for new studies to be conducted in these cases. In addition there was one chemical (dimethyl methylphosphonate) where the authors concluded a positive clastogenicity result was probably due to high osmolality, and another chemical (trichloracetic acid) where the authors concluded a positive clastogenicity result was probably due to low pH. In both these cases we considered that the control of pH and osmolality currently exercised in modern protocols would possibly have avoided the positive responses. We therefore concluded these re-

sults should also be excluded from the comparison with rodent carcinogenesis, and for convenience we also categorised these results as TC. Where we have checked and reclassified a call, the author’s original call is given in the reference column for those publications in Appendices A and B. For example: • 5-Chloro-o-toluidine was “I” (inconclusive) in the MLA according to Mitchell et al. [20] but the chemical was negative at concentrations giving >80% reduction in relative total growth (RTG) and only one concentration that induced >90% reduction in RTG was positive. We reclassified this as negative. • Succinic anhydride was I in the MLA according to Mitchell et al. [20] but was only tested in the absence of S9. We reclassified this as TC. • Thioacetamide was I in the MLA according to Mitchell et al. [20] but produced a weak (two to three-fold) increase in mutant frequency at <90% reduction in RTG although it was clearly positive at concentrations inducing >90% toxicity. We reclassified this weak response as E. • Decabromodiphenyl oxide was “#” (testing limited by solubility) in the MLA according to Mitchell et al. [20] but was clearly negative up to concentrations producing precipitation. As a sufficient test in the MLA (according to OECD guidelines [8]) only requires one precipitating concentration to be evaluated (because the precipitate cannot be separated from the cells when centrifuged at the end of treatment) we reclassified this as negative. • Calciferol was negative in the CA test according to Ishidate et al. [25] but was only tested in the absence of S9. We reclassified this as TC. If, during the course of collecting these data, any results were found which could not be categorised according to these rules, we discussed them as a panel and arrived at a decision based on scientific judgement.

5. Analysis of genotoxicity results with carcinogens From the carcinogens database (Appendix A), for 203 of the 756 carcinogens there were either no published genotoxicity data in any of the four in vitro tests

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Table 1 Summary performance of individual in vitro assays in detecting rodent carcinogens Test system

No. of carcinogens tested in each test system No. of clear positivesa Sensitivity (%) No. of equivocal results Sensitivity (%) if equivocal results counted positive a

Ames

MLA

MN

CA

541 318 318/541 (58.8%) 8 326/541 (60.3%)

245 179 179/245 (73.1%) 19 198/245 (80.8%)

89 70 70/89 (78.7%) 2 72/89 (80.9%)

352 231 231/352 (65.6%) 14 245/352 (69.6%)

All conditions; see text for details.

of interest or, as discussed above, studies were considered invalid. Therefore, the following analyses were made on a total database of 553 carcinogens. 5.1. Individual assays The performances of the four in vitro tests in individually detecting carcinogens are summarised in Table 1. 5.1.1. Ames test Ames test results were available for 541 chemicals (97.8% of the analysable database). Of these 541 chemicals, 318 (58.8%) clearly gave positive results (i.e. equivocal results considered negative), including: • Nalidixic acid, a gyrase poison, which was only positive in TA102 (mechanistically correct). • Four chemicals (azathioprine, C.I. Direct blue 6, C.I. Direct blue 15 and C.I. Direct brown 95) that needed reductive or anaerobic conditions. Some reports indicate some of the dyes are positive using standard (aerobic) S9 conditions. There were eight equivocal results. If these are considered positive the sensitivity (326/541) rises to 60.3%. 5.1.2. Mouse lymphoma assay MLA results were available for 245 chemicals (44.3% of the analysable database). Of these 245 chemicals, 179 (73.1%) gave clearly positive results (i.e. equivocal results considered negative). There were 19 equivocal results. If these are considered positive the sensitivity (198/245) rises to 80.8%. 5.1.3. In vitro micronucleus test Being a more recent test there were many fewer results in the database of the MN, only 89 chemicals

(16.1% of the analysable database). Of these 89 chemicals, 70 (78.7%) gave clearly positive results (i.e. equivocal results considered negative), which was a very credible performance, and the best of the individual assays. However, because most of the published results were from validation studies, there is expected to be a bias towards positive chemicals because of the attempts to compare MN and CA results. There were two equivocal results. If these are considered positive the sensitivity (72/89) rises to 80.9%. 5.1.4. In vitro chromosomal aberration test CA results were available for 352 chemicals (63.7% of the analysable database). Of these 352 chemicals, 231 (65.6%) gave clearly positive results (i.e. equivocal results considered negative), including: • Vanadium pentoxide which only induced hyperdiploidy in human lymphocytes. • Four chemicals (chlorobromomethane, 4,4 -methylenebis[2-chloroaniline], pentachloroethane and 1,1,1,2-tetrachloroethane) which only induced numerical aberrations such as increases in polyploidy. There were 14 equivocal results. If these are considered positive the sensitivity (245/352) rises to 69.6%. 5.1.5. Discussion of individual tests The sensitivity of the Ames and CA tests were somewhat better in our analyses than found either by Tennant et al. [1] (45.5 and 54.4%, respectively) or Zeiger [4] (54.1 and 52.2%, respectively). The sensitivity of the MLA in our analysis was very similar to that found by Tennant et al. [1] (70.5%) and by Zeiger [4] (74.3%). This is interesting because, although we also took most results from the NTP databases, we needed to re-evaluate many of the calls

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in the EPA GeneTox reassessment of the MLA [20] to fit only the four categories we selected as appropriate. Although the individual calls are important for individual chemicals, these data suggest that re-evaluation of “difficult” and unclear sets of data has not made a significant difference to the overall performance of the assay. This is probably because the re-evaluated calls fell into all categories (+, −, E and TC) and must have done so in proportions largely similar to the remainder of the database. The MN test had the highest sensitivity in our analysis, but on a small database of chemicals that consisted mainly of carefully selected established clastogens and aneugens, against which the test was validated. It has been observed in the past with the Ames test, for example, that the sensitivity is higher when fewer, select chemicals are tested, and drops as more diverse chemicals (whose outcome cannot be anticipated) are studied. Thus, it is expected that the sensitivity of the in vitro MN test will decrease as a wider spectrum of chemicals is tested. 5.2. Combinations of two tests The performances of the various combinations of two in vitro tests for detecting rodent carcinogens are

summarised in Table 2B. Table 2A is a “working table” showing the different combinations of results (in this case for carcinogens and the Ames + MLA) that may be obtained. For several carcinogens in each combination there was a negative result in one assay but no data from the other. As we could not know whether a positive result would have been obtained had the other test been conducted, these tests were eliminated from the calculations. In our first evaluation of sensitivity (Table 2B, Part i), carcinogens were included for which both genotoxicity tests were conducted, and positive results obtained in one or both tests. There were many situations where only one of the tests had been performed but gave a positive or equivocal response (the other test was blank). We included these positive (or equivocal) results where only one of the two assays had been performed (Table 2B, Part ii) because the outcome for the pair of tests would have been positive whatever the result in the other assay. This gave an “upper limit” of the level of sensitivity (Table 2B, Part iii). It should be noted that where the dataset for a pair of tests is quite small (e.g. MLA + MN), the number of positive chemicals increases quite significantly with the addition of positives from an incomplete pair of tests (in this example, mainly from the MLA). This may have been expected to produce an unusually high upper

Table 2A Working table demonstrating how figures in Table 2B were derived Ames

MLA

No. of chemicals

Totals

+ + Equivocal + − Equivocal Equivocal − −

+ Equivocal + − + Equivocal − Equivocal −

108  8  4 11 65  2 1 9 34

Total +ve in both tests = 108 (B) Total +ve in one of two tests when both performed = 88 (C)

242

242 (A)

Total both tests + Blank Equivocal Blank − Blank Total single test

Blank + Blank Equivocal Blank −



191  2 1  0 107 1 302

Total equivocal in one or both tests when both performed = 12

Total −ve in both tests = 34

Total +ve in one test when only one of two performed = 193 (D) Total equivocal in one test when only one of two performed = 1 (E) Total −ve in one test when only one of two performed = 108 302

The totals for A, B, C, D and E (in parentheses) are the same as in Table 2B, and illustrate how the sensitivity and upper limit of sensitivity values have been calculated.

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Table 2B Summary performance of combinations of two in vitro assays in detecting rodent carcinogens Combinations of two tests

Part (i): when both tests performed No. of carcinogens tested in both tests (A) No. (%) of clear positive results in both tests (B) No. (%) of clear positives (C) in only 1 of the two assaysa Sensitivity (i.e. clearly positive in at least 1 assay when both conducted) B + C/A

Ames + MLA

Ames + MN

Ames + CA

MLA + MN

MLA + CA

242

85

344

54

203

108 (44.6%)

37 (43.5%)

151 (43.9%)

38 (70.4%)

113 (55.7%)

88 (36.4%)

36 (42.4%)

108 (31.4%)

9 (16.7%)

52 (25.6%)

196/242 (81.0%)

73/85 (85.9%)

259/344 (75.3%)

47/54 (87.0%)

165/203 (81.3%)

278

139

164

133

9

1

17

10

398/484 (82.2%)

211/235 (89.8%)

298/346 (86.1%)

9

19

23c

407/484 (84.1%)

230/235 (97.9%)

321/346 (92.8%)

Part (ii): when only one of the two tests is performed No. of carcinogens (D) tested in 193 only one of the two tests and producing +ve resultd No. of carcinogens (E) tested in 1 only one of the two tests and producing an equivocal result

Part (iii): overall performance whether one or both tests performed Upper limit of sensitivity 389/436 (89.2%) 351/372 (94.4%) B + C + D/A + D + E (%) 13b 10 No. of carcinogens with equivocal result in one assay but negative or no test in the othera Upper limit of sensitivity (%) if 402/436 (92.2%) 361/372 (97.0%) equivocal results counted positive a b c d

All conditions; see text for details. Includes two chemicals equivocal in both assays. Includes one chemical equivocal in both assays. Chemicals tested in only one test but giving a −ve result were excluded from the analysis.

limit of sensitivity but, as can be seen from Table 2B and the discussion below, the difference between sensitivity calculated on the basis of two completed tests and the upper limit is no greater than for those combinations where more pairs of tests are complete than incomplete (e.g. Ames + CA). This is partly due to the contribution of the proportion of equivocal results in this data set and when equivocal results are considered negative, this contributes to lowering the sensitivity.

(i.e. equivocal results considered negative) were obtained in one or both assays:

5.2.1. Ames + MLA Results were available from both Ames and MLA tests for 242 carcinogens, and positive or equivocal results from a single test were available for a further 194 carcinogens (total 436). The following positive results

Therefore, the number of carcinogens that were clearly detected as positive in at least one of the two assays was 389, giving an upper limit of sensitivity of 89.2%. The sensitivity value calculated from the results where both tests were performed was 81.0%.

• 108/242 (44.6%) chemicals were clearly positive in both assays (including those positive in the Ames test under reductive or anaerobic conditions). • 88/242 (36.4%) chemicals were clearly positive in one of the two assays when both were performed. • 193 chemicals were positive in one assay when only one was performed.

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In addition, 11 chemicals were equivocal in one assay but negative in the other, and two chemicals were equivocal in both assays. If these are considered positive the upper limit of sensitivity (402/436) rises to 92.2%. 5.2.2. Ames + MN Results were available from both Ames and in vitro MN tests for 85 carcinogens, and positive or equivocal results from a single test were available for a further 287 carcinogens (total 372). The following positive results (i.e. equivocal results considered negative) were obtained in one or both assays: • 37/85 (43.5%) chemicals were clearly positive in both assays. • 36/85 (42.4%) chemicals were clearly positive in one of the two assays when both were performed. • 278 chemicals were clearly positive in one assay when only one was performed. Therefore, the number of carcinogens that were clearly detected as positive in at least one of the two assays was 351 giving an upper limit of sensitivity of 94.4%. The sensitivity value calculated from the results where both tests were performed was 85.9%. In addition, 10 chemicals were equivocal in one assay but negative in the other. If these are considered positive the upper limit of sensitivity (361/372) rises to 97.0%. 5.2.3. Ames + CA Results were available from both Ames and in vitro CA tests for 344 carcinogens, and positive or equivocal results from a single test were available for a further 140 carcinogens (total 484). The following positive results (i.e. equivocal results considered negative) were obtained in one or both assays: • 151/344 (43.9%) chemicals were clearly positive in both assays (including those positive in the Ames test under reductive or anaerobic conditions and those inducing numerical aberrations in the CA test). • 108/344 (31.4%) chemicals were clearly positive in one of the two assays when both were performed. • 139 chemicals were clearly positive in one assay when only one was performed. Therefore, the number of carcinogens that were clearly detected as positive in at least one of the two

assays was 398, giving an upper limit of sensitivity of 82.2%. The sensitivity value calculated from the results where both tests were performed was 75.3%. In addition, nine chemicals were equivocal in one assay but negative in the other. If these are considered positive the upper limit of sensitivity (407/484) rises to 84.1%. 5.2.4. MLA + MN Results were available from both MLA and in vitro MN tests for 54 carcinogens, and positive or equivocal results from a single test were available for a further 182 carcinogens (total 236). The following positive results (i.e. equivocal results considered negative) were obtained in one or both assays: • 38/54 (70.4%) chemicals were clearly positive in both assays. • 9/54 (16.7%) chemicals were clearly positive in one of the two assays when both were performed. • 164 chemicals were clearly positive in one assay when only one was performed. Therefore, the number of carcinogens that were clearly detected as positive in at least one of the two assays was 211 giving an upper limit of sensitivity of 89.8%. The sensitivity value calculated from the results where both tests were performed was 87.0%. In addition, 19 chemicals were equivocal in one assay but negative in the other. If these are considered positive the upper limit of sensitivity (231/236) rises to 97.9%. 5.2.5. MLA + CA Results were available from both MLA and in vitro CA assays for 203 carcinogens, and positive or equivocal results from a single test were available for a further 143 carcinogens (total 346). The following positive results (i.e. equivocal results considered negative) were obtained in one or both assays: • 113/203 (55.7%) chemicals were clearly positive in both assays (including those inducing numerical aberrations). • 52/203 (25.6%) chemicals were clearly positive in one of the two assays when both were performed. • 133 chemicals were clearly positive in one assay when only one was performed.

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Therefore, the number of carcinogens that were clearly detected as positive in at least one of the two assays was 306, giving an upper limit of sensitivity of 86.1%. The sensitivity value calculated from the results where both tests were performed was 81.3%. In addition, one chemical was equivocal in both assays and 22 chemicals were equivocal in one assay but negative in the other. If these are considered positive the upper limit of sensitivity (321/346) rises to 92.8%.

5.2.6. Discussion of two-test combinations In previous analyses, it has been commented that by combining assays “sensitivity has improved somewhat” [1]. Clearly one would expect to detect more carcinogens by adding test systems with different endpoints, so the key question becomes “is the improvement significant?” In our analysis, the sensitivity of Ames, MLA and CA all improved significantly on combination with a second test. The sensitivity of the Ames test (excluding equivocal responses) improved from 58.8% to an upper limit of 89.2% (with MLA), 94.4% (with MN) and 82.2% (with CA) based on the analysis of a positive result in one or both tests. These are considered meaningful improvements in sensitivity for the addition of a second test. Even the sensitivity of the MN test (which was highest of the individual tests) was significantly improved when combined with a second test. However, it should be noted that the performance of the MN may well be distorted by the narrow selection of clastogens and aneugens against which it has been validated. Its true performance as an individual test, and in combination, can only be assessed when it has been performed with a wider spectrum of chemicals. The improvements in sensitivity must, however, be balanced against deterioration in specificity (picking up more false positives) as is discussed later. Perhaps more important than the sensitivity figures is consideration of which carcinogens that were negative in a single test, were positive when a second test was introduced. The Ames test is the quickest and cheapest of the in vitro tests to perform and is conducted by most laboratories. However, it has the lowest sensitivity of the four individual test systems (Table 1). Its ability to detect rodent carcinogens is therefore significantly enhanced by combining with a mammalian cell test. For example, 215 carcinogens were negative in the Ames test, and of these:

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• 108 were tested in MLA and 65 (60.2%) were positive. • 43 were tested in MN and 31 (72.1%) were positive. • 150 were tested in CA and 67 (44.7%) were positive. By contrast, there is much less improvement in sensitivity by adding an Ames test when a mammalian cell test has already been performed and given a negative result, for example: • 47 carcinogens were negative in MLA, 46 of these were also tested in Ames but only 11 (23.9%) were positive. • 17 carcinogens were negative in MN, 16 of these were also tested in Ames but only 5 (31.25%) were positive. • 107 carcinogens were negative in CA, all of these were also tested in Ames but only 28 (26.2%) were positive. These data suggest that, if resources and time are available, a much better sensitivity in detection of rodent carcinogens can be obtained by first performing one of the mammalian cell tests. If this is negative, then a small improvement in prediction can be obtained by then conducting an Ames test. However, it would be wrong to make firm recommendations for testing based solely on sensitivity, and, as is discussed later, the specificity of these various tests and combinations of tests does not necessarily support this approach to testing. 5.3. Combinations of three completed and valid tests The performance of the combination of three in vitro tests (as recommended in the UK COM guidelines [33], but considering the chromosomal aberration test and the micronucleus test as alternative assays) for detecting rodent carcinogens is summarised in Table 3. 5.3.1. Ames + MLA + MN Results in all three assays were available for 54 carcinogens. Of these 54 chemicals, 24 (44.4%) gave clear positive results (i.e. equivocal results considered negative) in all three assays. A further 25 chemicals were clearly positive in one or two of the assays when all three were performed. Therefore, when all three tests were performed, 49 (90.7%) of the 54 carcinogens in this dataset gave clear positive results in at least one of

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Table 3 Summary performance of combinations of three in vitro assays in detecting rodent carcinogens when all three tests performed Test combination

No. of carcinogens tested in all three test systems No. (%) of clear positive results in all three test systems No. (%) of clear positive results in one or two of the three assays Sensitivity (i.e. clearly positive in at least one assay when all three conducted) No. of carcinogens giving equivocal but no positive results Sensitivity if equivocal results counted as positive a

Ames + MLA + MN

Ames + MLA + CA

54 24/54 (44.4%) 25/54 (46.3%) 49/54 (90.7%) 1 50/54 (92.6%)

202 73/202 (36.1%) 98/202 (48.5%) 171/202 (84.7%) 10a 181/202 (89.6%)

Includes two chemicals giving equivocal results in two assays but negative in the third.

the assays. This is not significantly improved over any two-test combination discussed above. One chemical (tetrachloroethylene) was negative in Ames and MN but equivocal in the MLA. If this chemical is considered positive the sensitivity increases to 92.6%. Four chemicals (di[2-ethylhexyl]adipate, di[2ethylhexyl]phthalate, N-nitrosodiphenylamine and titanium dioxide) were negative in all three tests. The latter three of these were also negative in the CA test. Di(2-ethylhexyl)adipate was equivocal in the CA test. 5.3.2. Ames + MLA + CA Results in all three assays were available for 202 carcinogens. Of these 202 chemicals, 73 (36.1%) gave clear positive results (i.e. equivocal results considered negative) in all three assays. A further 98 chemicals were clearly positive in one or two of the assays when all three were performed. Therefore, when all three tests were performed, 171 of the 202 carcinogens in this dataset gave clear positive results in at least one of the assays. Therefore, the sensitivity of this combination, when all three tests were completed, was 84.7%. Again, the combination of three tests was not significantly improved over any two-test combination. A further eight chemicals gave equivocal results in a single test (whilst the other two tests were negative or inconclusive) and two chemicals gave equivocal results in two of the tests whilst being negative in the third. If these equivocal results are considered positive the sensitivity (181/202) rises to 89.6%. Three chemicals (17-␤-estradiol, nitrilotriacetic acid and oxazepam) were negative in Ames + MLA + CA but were positive in the MN test. If positive responses in either MN or CA are considered as predictive, then the sensitivity, counting equivocal responses

as negative, increased to 86.1%, and counting equivocal responses as positive, the sensitivity increased to 91.1%. 5.3.3. Discussion of three-test combinations The combination of three tests (Ames + MLA + either MN or CA) was clearly very sensitive. Only 19 carcinogens were negative in the Ames + MLA + CA/MN combination (these are discussed later). Sensitivity compared with the individual assays (Table 1) was greatly improved. If all tests were conducted, sensitivity did increase above the levels seen when only two tests were conducted, but not dramatically (Tables 2B and 3). The addition of a third test did detect some important chemicals found negative by the two-test combinations, for example: • Four chemicals (17-␤-estradiol, nitrilotriacetic acid, oxazepam and DDT) that were negative in the combination Ames + MLA were positive in the MN test. However, nitrilotriacetic acid is highly acidic and pH effects cannot be ruled out. DDT was also positive in the CA test. By adding MN to Ames + MLA, and completing all three tests, the sensitivity improved markedly from 81.0 to 90.7%. This is considered a meaningful improvement for the additional effort. • A further four chemicals (naphthalene, pentachloronitrobenzene, sodium saccharin and zearalenone) that were negative in the combination Ames + MLA were positive in the CA test. However, the sensitivity improvement from two tests to three was only from 81.0 to 84.7%. This is partly because of the larger dataset than when MN was in-

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cluded, but fewer chemicals (202) were evaluated in the three-test combination than in the two-test combination (242). • By contrast, the MLA detected 21 carcinogens that were negative in the combination of Ames + CA. This increased the sensitivity, when all tests were performed, from 75.4 to 84.7%, which, on a reduced dataset (202 instead of 345 chemicals), is again considered a meaningful increase in sensitivity for the additional effort. • However, Ames + MLA + MN only detected one carcinogen (urethane) that was negative in the combination MLA + MN, and so the sensitivity, on a dataset of identical size, increased slightly. This does not appear to be a meaningful reward for effort. However, this is a small dataset restricted by the particular chemicals that have been selected for validation of the MN. Considering the comments made in Section 5.2.6 above, these data would suggest that the most effective sequence of testing for sensitivity would be to first perform a mammalian cell test (e.g. the MN test), then conduct the Ames test, and, if both are negative, finally perform the MLA. This way the greatest sensitivity is achieved with the first test (MN sensitivity 78.7%), then increases meaningfully with the addition of the second test (Ames + MN sensitivity 85.9%), and can be improved to a sensitivity of 90.7% by the addition of the MLA. However, it would be wrong to make firm recommendations for testing based solely on sensitivity, and, as is discussed later, the specificity of these various tests and combinations of tests does not necessarily support this approach to testing.

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5.4. Results from one, two or three tests For some chemicals technically acceptable data from a full battery of three tests was not always available. In such cases it is important also to ask “How many carcinogens were detected as positive in at least one test even if the battery of three in vitro tests was not completed?” A summary of this analysis is given in Table 4. As mentioned earlier, there are published genotoxicity data that are technically acceptable for 553 of the carcinogens in the database in Appendix A. For 82 chemicals there were negative results, but from an incomplete battery (i.e. only one or two tests conducted). Therefore, we could not know whether a positive result would have been obtained if the battery had been completed. There were therefore either complete sets, or positive/equivocal data from an incomplete set, for 471 chemicals. Of these 471 chemicals there were positive results (i.e. equivocal results considered negative) in at least one of the three assays (considering MN and CA as alternatives) for 438 chemicals, giving a sensitivity value of 93.0%. For a further 14 chemicals there were equivocal results in at least one assay. If these are considered positive, the upper limit of sensitivity rises to 96.0%. As above, one would expect the sensitivity to improve with the addition of one or more tests with different endpoints. As before it is important to question whether the improvement in sensitivity is sufficient to be worth the effort. When we compare the occurrence of positive results in one, two or three tests with the occurrence in one or two tests, the improvement in both sensitivity (based on completed batteries) and upper limit of sensitivity (based on positive results in both

Table 4 Summary performance of one, two or three in vitro tests in detecting rodent carcinogens No. of carcinogens tested in one, two or three tests (A) No. of chemicals giving negative results with an incomplete battery (i.e. only one or two tests) (B) Chemicals evaluated (A − B) i.e. positive in at least one assay or negative/inconclusive across all three tests Upper limit of sensitivity i.e. no. (%) of chemicals giving clear positive results in at least one assaya No. of chemicals giving an equivocal (rather than positive) result in at least one assayb Upper limit of sensitivity if equivocal results counted as positive

553 82 471 438/471 (93.0%) 14 452/471 (96.0%)

a Includes three chemicals (17-B-estradiol, nitrilotriacetic acid and oxazepam) which were negative in Ames, MLA and CA but positive in MN. b Includes (di[2-ethylhexyl]adipate] which was negative in Ames, MLA and MN but equivocal in CA and tetrachloroethylene which was negative in Ames, MN and CA but equivocal in MLA.

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incomplete and complete batteries) in some cases is quite small and in others declines (e.g. 94.4% upper limit of sensitivity for Ames + MN falls to 93.0% when MLA is added). These are fluctuations based on different datasets (471 chemicals with data on one, two or three tests versus 372 chemicals with data from one or two tests) rather than true reductions in sensitivity. However, the sensitivity values do suggest that perhaps there is little improvement to be gained by adding a third test. Another way of looking at the data in Tables 1–4 is as follows. If the only test chosen is the Ames test then there is a 58.8% probability of clearly detecting a rodent carcinogen (Table 1). If both Ames and MLA are available and either or both are performed, the probability that a positive result will predict carcinogenic potential is in the range 81.0–89.2% (Table 2). With Ames + MLA + MN available, whether we proceed with testing negative calls, or stop testing at the first positive call, the probability of detecting a rodent carcinogen is >90% (Tables 3 and 4).

6. Rodent carcinogens not detected by the in vitro battery From the above it will be clear that, for the 206 completed sets of three in vitro tests (using MN and CA as alternatives in the battery as appropriate), there were 19 chemicals which were clearly negative when Ames + MLA + either MN or CA were performed. It is important that the possible reasons for the failure of the three-test in vitro battery to detect these carcinogens are discussed. Comments are as follows: 6.1. 3-Amino-1,2,4-triazole (amitrole) Amitrole produced thyroid and liver tumours in both rats and mice. The mechanism is thought to be due to hormonal effects and prolactin secretion [34], which cannot be adequately represented in in vitro genotoxicity tests. Rodents are considered more sensitive than humans to thyroid tumour induction due to hormonal imbalance causing raised TSH levels (IARC Scientific Publication 147). However, Krauss and Eling [35] have also speculated that reactive metabolites of amitrole may be generated by the action of thyroid peroxidase and/or prostaglandin H synthase, which again

may not be represented in the bacterial and cell cultures conventionally used for genotoxicity testing. In addition, amitrole interferes with histidine biosynthesis in Salmonella and would not be expected to be mutagenic in a system using histidine reversion. 6.2. tert-Butyl alcohol tert-Butyl alcohol induced mainly kidney and bladder tumours in rats and mice. Significant increases in kidney weight were seen in the latter stages of a 2year study, accompanied by increased mineralisation and chronic inflammation of both kidneys and bladder [36]. The tumours therefore most likely result from a non-genotoxic mechanism based on excessive mineralisation and inflammation. 6.3. 5-Chloro-o-toluidine 5-Chloro-o-toluidine induced mouse liver tumours and haemangiosarcomas (mainly in adipose tissue), typical of aromatic amines. It was non-carcinogenic in the rat. There are no DNA binding data although 5-chloro-o-toluidine does bind to haemoglobin. Analogy to 4-chloro-o-toluidine suggests it is a genotoxic carcinogen [37]. The initial metabolism of 5-chloro-otoluidine, probably via N-oxidation, is said to produce a nitrosoarene that can bind covalently to haemoglobin (IARC Monographs, volume 77, 2000). 5-Chloro-otoluidine induced replicative DNA synthesis in mouse hepatocytes in vitro, which has been linked with nongenotoxic mechanisms of tumour induction [38]. However, it may be considered that this chemical is a carcinogen for which a genotoxic mechanism of action plays a role in the aetiology of tumours, and the failure of the in vitro test battery to give any positive results is an anomaly. 6.4. Decabromodiphenyl oxide (DCBDO) Decabromodiphenyl oxide induced mainly hepatocellular adenomas in the livers of rats (and possibly also male mice) at high doses. There is some evidence these tumours are related to stimulation of thyroid function, and would therefore not be relevant for humans [34]. In addition these were considered benign neoplasms by Huff et al. [39]. DCBDO also induced acinarcell adenomas of the pancreas and mononuclear-cell

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leukaemia in male rats (IARC Monographs, volume 48, 1990). The data are insufficient to clearly classify a non-genotoxic mechanism of action. 6.5. Diethanolamine Diethanolamine induced tumours in the mouse liver and renal tubules. The mechanism is probably via choline deficiency [40,41]. Chronic disruption of choline metabolism in the liver can lead to hepatic foci and subsequently tumours. This is a nongenotoxic mechanism that is not represented in the cultures used for in vitro genotoxicity testing (see also dl-ethionine). 6.6. Di(2-ethylhexyl)phthalate Di(2-ethylhexyl)phthalate induced only liver tumours in rats and mice, and is considered a peroxisome proliferator [34]. Peroxisome proliferation probably leads to an excess generation of reactive oxygen species which can contribute to the tumorigenic process, thus an indirect genotoxic mechanism of action. Such carcinogens are considered not to indicate carcinogenic risk for humans because of their low level of peroxisome proliferation. It is considered a nongenotoxic carcinogen by Jackson et al. [42]. 6.7. 1,4-Dioxane The primary target organs for tumours induced by 1,4-dioxane were the liver (rats and mice) and nasal cavity (rats), however the relevance of nasal cavity tumours for humans has been questioned. The nasal tumours probably resulted from severe irritation. The liver tumours were accompanied by degenerative changes and appear to occur only at high doses, accompanied by saturation of clearance mechanisms and liver toxicity. The tumours probably resulted from a cytotoxic-epigenetic mechanism. 1,4-Dioxane also induced hepatocyte proliferation and can act as a tumour promoter in rat liver and mouse skin carcinogenicity assays [43] although it only gave an equivocal response for the induction of replicative DNA synthesis in rat hepatocytes in vitro [44]. Hence, this chemical appears to be carcinogenic in rodents by a predominantly nongenotoxic mechanism.

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6.8. dl-Ethionine dl-Ethionine produced liver tumours in rats. One postulated mechanism is alteration of histone methylation. However, it also affects choline metabolism [45] and the chronic disruption can result in tumours via a non-genotoxic mechanism. 6.9. Melamine Melamine produced urinary bladder and ureteral carcinomas in male rats but only urinary bladder hyperplasia in male mice. The occurrence of urinary bladder tumours in male rats correlated strictly with calculus formation and exposure to high doses. This is considered a non-DNA-reactive mechanism involving epithelial hyperplasia resulting from the presence of bladder stones (IARC Monographs volume 73, 1999). 6.10. Methyl carbamate Methyl carbamate induced liver tumours in male and female rats but was not carcinogenic for mice. Metabolism and clearance of methyl carbamate is much greater in mice, whereas bioaccumulation is found in rats, and this may explain the species differences in carcinogenicity [46]. Methyl carbamate only gave an equivocal response for induction of replicative DNA synthesis in rat hepatocytes in vitro [45]. It is probably unable to form a reactive epoxide in the way that ethyl carbamate (urethane) and vinyl carbamate can [47–49]. There is therefore insufficient evidence to clearly classify a non-genotoxic mechanism of action. 6.11. Nitrilotriacetic acid, trisodium salt, monohydrate (NaNTA) NaNTA induced tumours of the urinary system in male and female rats but was non-carcinogenic in mice. It was nephrotoxic and induced urinary microcrystals and urothelial cytotoxicity in rats, but not mice, at doses higher than those inducing nephrotoxicity. The carcinogenicity of NaNTA is probably the result of severe irritation (IARC Monograph, volume 73, 1999). It should be noted that although NaNTA was negative in the three in vitro tests this did not include the MN test. Nitrilotriacetic acid did give a positive result in the MN, and it is difficult to envisage that nitrilotriacetic acid and

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its sodium salt would have different mechanisms of action (certainly the tumour profiles are very similar [9]). Therefore, NaNTA may be genotoxic if tested in MN. 6.12. N-Nitrosodiphenylamine N-Nitrosodiphenylamine induced transitional-cell carcinomas of the urinary bladder in rats at high doses but was not carcinogenic for mice. It is about 100× weaker as a carcinogen than the alkylnitrosamines [50] and Purchase et al. [51] actually concluded it was noncarcinogenic. Although there is no proven relationship between mutagenic activity in vitro and carcinogenic potency in vivo we may expect that such a weak or doubtful carcinogen may not give rise to significant genotoxicity in vitro. 6.13. Progesterone Progesterone induced ovarian, uterine and mammary tumours in mice after subcutaneous and intramuscular injection (IARC Monographs, volume supplement 7, 1987). As these are all hormonally dependent tumours, they probably result from the chronic disruption of the hormonal system in mice and are not due to a DNA-reactive mechanism. 6.14. Pyridine Pyridine is a strain-specific renal tubule carcinogen in rats (F344 but not Wistar) and induced hepatocellular carcinomas and hepatoblastomas in male and female mice (see NTP website [15]). The effects in both species can be argued as strain-specific as the effects in B6C3F1 mouse liver were related to a high spontaneous background incidence and the effects in Fischer rats were not reproduced in comparably exposed Wistar rats. As pyridine was also negative in a p53+/− short-term tumour assay [52] it can be argued that it is a non-genotoxic carcinogen as well as being strainspecific. 6.15. Reserpine Reserpine induced testicular and mammary gland tumours in male and female mice, respectively, and adrenal gland tumours in male rats. It is a dopamine modulator affecting prolactin secretion [53]. It is con-

sidered a non-genotoxic carcinogen by Jackson et al. [42]. 6.16. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) TCDD induced a variety of tumours in rodents including tumours of the lung, oral and nasal cavities, thyroid and adrenal glands, livers, subcutaneous tissues, skin, integumentary system and as lymphomas. These effects have been induced with doses as low as 0.001 ␮g/kg. It is widely accepted that the mechanism is a non-genotoxic one and involves consequences of its binding to the Ah receptor. Activation of the Ah receptor leads downstream to the activation of various oncogenic pathways (e.g. SRC) and “crosstalk” with various elementary cell cycle signalling pathways [54]. Another effect of TCDD is induction of CYP 1A1 and 1A2 enzymes [55] and hence an increased risk from exposure to ultimate carcinogens that are activated by these routes (e.g. benzo[a]pyrene). This latter mechanism is not a determining factor for its own carcinogenesis. 6.17. 1,1,2,2-Tetrachloroethane (TTCE) TTCE induced liver tumours in male and female mice but was not carcinogenic in rats. There is some evidence it bound covalently to DNA in mice in vivo, and binding to DNA in vitro was enhanced by induced microsomal and cytosolic activating systems [56]. TTCE induced replicative DNA synthesis (indicative of a non-genotoxic mechanism) in mouse hepatocytes [38]. However, it may be argued that TTCE should be detected as genotoxic in vitro, and the lack of effects in the battery of tests under discussion here is an anomaly. 6.18. Titanium dioxide Inhalation of titanium dioxide increased incidences of lung adenomas in rats of both sexes and of cystic keratinizing lesions diagnosed as squamous-cell carcinomas in female rats that had inhaled the high but not the low doses. Oral, subcutaneous, intratracheal and intraperitoneal administration did not produce a significant increase in the frequency of any type of tumour in any species (IARC Monographs, volume 47, 1989). The particles do not produce lung fibrosis, which is

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considered a precursor of lung cancer in humans, but persistent lung inflammation [57], probably through persistent coating of the alveoli preventing oxygen uptake. This can therefore be considered a non-genotoxic mechanism at the dose levels employed. 6.19. Tris(2-ethylhexyl)phosphate Tris(2-ethylhexyl)phosphate induced liver tumours only in female mice at a high dose. It was not carcinogenic for rats. The mechanism probably involves peroxisome proliferation, which is not considered genotoxic. 6.20. Discussion of carcinogens reported to give negative genotoxicity results From the above discussion it can be seen that most of the carcinogens in our database (16/19) not detected by the three-test in vitro battery are suggested to act via non-genotoxic mechanisms, which conventional genotoxicity tests cannot be expected to mimic. Only three chemicals (5-chloro-o-toluidine, Nnitrosodiophenylamine and 1,1,2,2-tetrachloroethane) may have a genotoxic component involved in their carcinogenicity. The Syrian hamster embryo (SHE) cell transformation assay has been suggested as a valuable assay for detecting non-genotoxic carcinogens [200]. It is interesting then that 14 of these 19 carcinogens that were negative in conventional in vitro genotoxicity tests have been tested in the SHE assay; 7/14 (50%) gave positive results and 7/14 (50%) gave negative results. As expected, some mechanisms of non-genotoxic carcinogens are unique to the in vivo situation and would not be manifest in SHE cells or any other in vitro system. This will be discussed further in future publications. The above list of carcinogens not detected by the three-test battery is almost completely different from the list published by Brambilla and Martelli [5] as not being detected by the normal battery of two in vitro and one in vivo test for pharmaceuticals. The only carcinogen giving negative genotoxicity results that we both identify is trisodium nitrilotriacetic acid. The differences between our evaluations may be attributable as much to the specific chemicals studied as to the test battery used. For the other chemicals listed by Bram-

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billa and Martelli as false negatives for genotoxicity [5] we can make the following comments (see Appendix A for more details): • Polychlorinated biphenyls—none of the mono-, bi-, tri-, tetra-, penta-, hexa- or heptachlorobiphenyls are listed in the CPDB [9]. We therefore did not search for genotoxicity data and there are no entries for these chemicals either in Appendix A or Appendix B. Aroclor 1254 is a rodent carcinogen [9] but is negative in an Ames test [13]. We could find no data in the four genotoxicity tests we examined for Aroclor 1260, also listed in the CPDB [9] as a rodent carcinogen. • trichloroethylene—is positive in both the MLA [20] and MN tests [58]. • tetrachloroethylene—gave equivocal results in the MLA according to our evaluation. It was reported as “inconclusive” by Mitchell et al. [20]. • o-Toluidine—is positive in Ames [13] and CA tests [25]. • Carbon tetrachloride—is positive for MN in vitro [58]. • Cyproterone acetate—is an interesting chemical that did not appear in the CPDB [9]. Brambilla and Martelli [5] note that the evidence for rodent carcinogenicity is “limited”. • p-Dichlorobenzene—is positive in the MLA [20] and induces CA [27]. • Hexachlorobenzene—induces MN in vitro [59]. • Hexachloroethane—is negative in Ames [13], MN [58] and CA tests [15] but has not been tested in the MLA. • Ochratoxin A—is negative in Ames [13] and CA tests [15] but the MLA test did not meet current regulatory standards (was technically compromised) according to our evaluation. It was deemed “inconclusive” by Mitchell et al. [20]. Thus, many of the chemicals said to be “missed” in standard genotoxicity tests [5] do, in fact, produce positive results in one of more of the tests we have evaluated. Others may give positive results if the battery were to be completed. Some of the carcinogens above are accepted as exerting their tumourigenicity via a non-genotoxic mechanism and therefore would not be expected to give positive results in in vitro genotoxicity tests. It is nevertheless appreciated that standard in vitro conditions may not adequately resemble

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important metabolic activation pathways for such compounds. With knowledge gained about their mechanisms of action, adequately designed standard in vitro tests should yield positive results.

equivocal results considered positive). The specificity for the Ames test was therefore 73.9%. There were six equivocal results. If these are considered negative, the specificity (136/176) improves to 77.3%.

7. Analysis of genotoxicity results with non-carcinogens

7.1.2. Mouse lymphoma assay MLA results were available for 105 noncarcinogens (59.3% of the database). Of these 105 chemicals only 41 correctly gave negative results (i.e. equivocal results considered positive). The specificity of the MLA was therefore only 39.0%. There were nine equivocal results. If these are considered negative, the specificity (50/105) improves to 47.6%.

To be included in our table of non-carcinogens (Appendix B) chemicals had to be negative both in males and females, rats and mice. The likelihood of chemicals being selected for testing in such a rigid programme to yield clearly negative results is small and therefore this was a much smaller database (183 chemicals) than for the carcinogens where a single positive tumour response in a single sex was sufficient. There were no published genotoxicity results in any of the four tests for six of the non-carcinogens. Therefore, the database available for analysis consisted of 177 noncarcinogens. As before, equivocal (E) results could be taken as indicative either of negative or positive results. Therefore, in the following analysis of non-carcinogens, E results are considered separately as indicating positive or negative responses. 7.1. Individual assays The performances of the four in vitro tests in individually detecting non-carcinogens are summarised in Table 5. 7.1.1. Ames test Ames test results were available for 176 noncarcinogens (99.4% of the database). Of these 176 chemicals, 130 correctly gave negative results (i.e.

7.1.3. In vitro micronucleus test MN results were available for only 26 noncarcinogens (14.7% of the database). Of these 26 chemicals only 8 correctly gave negative results (i.e. equivocal results considered positive). The specificity of the MN was therefore only 30.8%. There were six equivocal results. If these are considered negative, the specificity (14/26) improves to 53.8% 7.1.4. In vitro chromosomal aberration test CA results were available for 136 non-carcinogens (76.8% of the database). Of these 136 chemicals only 61 correctly gave negative results (i.e. equivocal results considered positive). The specificity of the CA was therefore only 44.9%. There were 14 equivocal results. If these are considered negative, the specificity (75/136) improves to 55.1% In summary, the specificity of the Ames test was acceptably high at >70% but a little lower than that

Table 5 Summary performance of individual in vitro assays in detecting rodent non-carcinogens Test system

No. of non-carcinogens tested in each test system No. of clear negatives Specificity (%) No. of equivocal results Specificity (%) if equivocal results counted negative

Ames

MLA

MN

CA

176 130 130/176 (73.9%) 6 136/176 (77.3%)

105 41 41/105 (39.0%) 9 50/105 (47.6%)

26 8 8/26 (30.8%) 6 14/26 (53.8%)

136 61 61/136 (44.9%) 4 75/136 (55.1%)

D. Kirkland et al. / Mutation Research 584 (2005) 1–256

seen previously [1,2,4]. However, the specificity of all three of the mammalian cell tests was very low, even when equivocal results were considered negative—a non-carcinogen was just as likely to give a positive result as a carcinogen. For the MLA it was similar and for the CA it was lower than in the previous published analyses. 7.2. Combinations of two tests If only one of the two tests was performed but gave a negative result, we could not know if a positive response would have been obtained had the second test been conducted. Such results could therefore not be taken as a definitive indication of lack of genotoxicity. Our first prediction of non-carcinogenic status (specificity) was taken when both tests were clearly negative, i.e. equivocal results considered positive (Table 6B, Part i). An upper limit of specificity was calculated by considering equivocal results as negative. Thus, if both tests gave equivocal results, or one gave an equivocal result and the other was negative, then the combination could be considered overall negative. Chemicals giving a positive result in a single assay but not tested in the other would have been positive for the battery (whatever the outcome of the second assay). If chemicals

19

tested in a single assay and giving equivocal results are also considered positive, then we can calculate a “lower limit” of specificity by including these in the denominator (Table 6B, Part iii). Table 6A is a working table showing the different combinations of results for two tests (in this case Ames + MLA) with non-carcinogens, and demonstrates how some of the figures in Table 6B were derived. The performances of the five different pairs of tests in detecting non-carcinogens are summarised in Table 6B. The specificity obtained for each individual assay (Table 5) was reduced to even worse levels by combination with a second test (Table 6B). The pair combination with the lowest specificity (i.e. negative results in both tests) was MLA + MN with a value of only 10%, although this improved to 30% if equivocal results were considered negative. However, it must be noted that the database for these two tests was very small, consisting of only 20 non-carcinogenic chemicals evaluated in both test systems. The lower limits of specificity, calculated because a positive (or equivocal when counted as positive) result when only one of the tests had been performed meant that, whatever the result of the second test, the overall outcome for the pair of tests would be positive, were even lower.

Table 6A Working table demonstrating how figures in Table 6B were derived Ames

MLA

No. of chemicals

Totals

+ + Equivocal + − Equivocal Equivocal − −

+ Equivocal + − + Equivocal − Equivocal −

16  2  2  5 37  0 2 7 34

Total +ve in both tests = 16 Total +ve in 1/2 tests when both performed = 46

105 (A)

Blank + Blank Equivocal Blank −

105  17 0  2 0  52 0

Total both tests + Blank Equivocal Blank − Blank Total single test

71

Total equivocal in one or both tests when both performed = 9 (E)

Total −ve in both tests = 34 (B) Total +ve in 1 test when only ½ performed = 17 (C) Total equivocal in 1 test when only ½ performed = 2 (D) Total −ve in 1 test when only ½ performed = 52 71

The totals for A, B, C and D (in parentheses) are the same as in Table 6B, and illustrate how the specificity and lower limit of specificity values have been calculated.

20

D. Kirkland et al. / Mutation Research 584 (2005) 1–256

Table 6B Summary performance of combinations of two in vitro assays in detecting rodent non-carcinogens Combinations of two tests

Part (i): when both tests performed No. of non-carcinogens tested in both tests (A) No. of clear negative results in both tests (B) Specificity (%) B/A No. of non-carcinogens equivocal in both tests or equivocal in one test and negative in the other (E) Upper limit of specificity (%) if equivocal counted as positive B + E/A Part (ii): when only one of the two tests performed No. of non-carcinogens tested in only one of the two tests and producing a +ve result (C) No. of non-carcinogens tested in only one of the two tests and producing an equivocal result (D)

Ames + MLA

Ames + MN

Ames + CA

MLA + MN

MLA + CA

105 34 34/105 (32.4%) 9

25 3 3/25 (12.0%) 5

136 47 47/136 (34.6%) 14

20 2 2/20 (10.0%) 4

96 26 26/96 (27.1%) 6

43/105 (41.0%)

8/25 (32.0%)

61/136 (44.9%)

6/20 (30.0%)

32/96 (33.3%)

17

31

6

45

21

2

5

1

7

5

3/61 (4.9%)

47/142 (33.1%)

2/72 (2.8%)

26/122 (21.3%)

Part (iii): overall performance whether one or both tests performed Lower limit of specificity if equivocal results 34/124 (27.4%) counted as positive B/A + C + D (%)

With the Ames test being the best individual assay for specificity, it was interesting to see how many non-carcinogens that were Ames-negative gave positive results (i.e. false positives) when different mammalian cell tests were added. For example, 130 noncarcinogens were negative in Ames, and of these: • 78 were also tested in MLA and 37 (47.4%) gave positive results. • 15 were also tested in MN and 7 (46.7%) gave positive results. • 97 were also tested in CA and 38 (39.2%) gave positive results. Thus, the specificity is unacceptably low and false positive rates are unacceptably high. 7.3. Combinations of three tests As above for pairs of tests, if only one or two of the three tests was performed but gave a negative result(s), we could not know if a positive response would have been obtained had the second (or third) test been conducted. Such results could therefore not be taken as a definitive indication of lack of genotoxicity. Our first prediction of non-carcinogenic status (specificity) was taken when all three tests were clearly negative, i.e.

equivocal results considered positive (Table 7B, Part i). An upper limit of specificity was calculated by considering equivocal results as negative. Thus, if one or two tests gave equivocal results but the other test(s) in the battery was negative, then the combination could be considered overall negative. These values are denoted by (E) in Table 7B, Part i. Chemicals giving a positive result in a one or two assay battery but not tested in the (second or) third would have been positive for the battery (whatever the outcome of the second or third assays). If equivocal results from similar situations were considered positive then we could calculate a “lower limit” of specificity by including these in the denominator (Table 7B, Part ii). Table 7A is a working table to show how the values for incomplete batteries (as used in Table 7B) were derived (in this case for Ames + MLA + CA) and therefore how the lower limit of specificity was obtained. The performances of the two combinations of the three-test battery in detecting non-carcinogens are therefore summarised in Table 7B. There were 96 noncarcinogens that had been tested in the complete battery of Ames + MLA + CA, and 19 of these were also tested for MN. One compound, omeprazole, was tested in Ames + MLA + MN but was not tested for CA. The specificity for the combination Ames + MLA + CA was

D. Kirkland et al. / Mutation Research 584 (2005) 1–256 Table 7A Working table demonstrating how some of the figures in Table 7B were derived Categories of results

Total no. of non-carcinogens

+ve in two tests but no result (blank) in third +ve in one test, equivocal in second test, but blank in third +ve in one test, −ve in second test but blank in third +ve in one test but blank in other two tests Total non-carcinogens +ve in one or two tests from incomplete battery Equivocal in one test, −ve in second test but blank in third Equivocal in two tests but blank in third Equivocal in one test but blank in other two tests Total non-carcinogens giving only equivocal results from incomplete battery

9 1 19 3 32 (C) 7 0 0 7 (D)

The totals for C and D are the same as in Table 7B.

again lower (at 22.9%) than any of the respective pairs, and remained so even when equivocal results were counted as negative (upper limit of specificity only 29.2%). The database for completed Ames + MLA + MN was small and consisted of the same 20 non-carcinogens as in the pair MLA + MN. It is interesting therefore that by adding the Ames test to MLA + MN for the same 20 non-carcinogens the specificity fell from 10 to a mere 5%. In detail, whereas dimethyl terephthalate and N-(1-naphthyl)ethylenediamine·2HCl were

21

the only 2 (of 20) non-carcinogens to give negative results in both MLA and MN, only dimethyl terephthalate was also negative in Ames. Not only was N(1-naphthyl)ethylenediamine·2HCl positive in Ames it was also positive in CA. Even by considering equivocal results as negative, the upper limit of specificity only improved to 15%. The very low specificity values for this combination of three tests (Ames + MLA + MN) is probably quite unreliable due to the small number of non-carcinogens evaluated in this combination. The lower limits of specificity obtained by adding to the denominator those incomplete batteries where positive results (or equivocal results considered as positive) had been obtained (because that battery would have been overall positive whatever the outcome of the tests to be completed) were extremely low. Again, because there were so few completed batteries for Ames + MLA + MN the lower limit of specificity fell to 1%! If only a few of the incomplete batteries (where two tests were already negative) were to be completed, and negative results were obtained in the third test, the specificity would change dramatically. There were 31 chemicals (listed in Table 8) which were negative for Ames + MLA but there were no test results in the MN. However, 20 out of the 31 had given negative results in the CA. Therefore, if MN tests were conducted on (say) a further 10 chemicals, the specificity could quickly rise from 5 to >30%. Thus, an accurate determination of the specificity of the Ames + MLA + MN battery (in particular) is dependent on obtaining additional test data.

Table 7B Summary performance of combinations of three in vitro assays in detecting rodent non-carcinogens (all three tests performed) Test combination Ames + MLA + MN

Ames + MLA + CA

Part (i): when all three tests performed No. of non-carcinogens tested in all three test systems (A) No. of clear negative results in all three test systems (B) Specificity B/A (%) No. of non-carcinogens equivocal in one or two tests and negative in the other(s) (E) Upper limit of specificity (%) if equivocal counted as negative B + E/A

20 1 1/20 (5.0%) 2 3/20 (15.0%)

96 22 22/96 (22.9%) 6a 28/96 (29.2%)

Part (ii): when only one or two tests performed No. of non-carcinogens +ve in one or two tests (C) No. of non-carcinogens Equivocal in one or two tests and −ve or blank in the other(s) (D) Lower limit of specificity B/A + C + D (%)

69 10 1/99 (1.0%)

32 7 22/135 (16.3%)

a

Not including ascorbic acid which was negative in MLA and CA, and equivocal in Ames, but was positive in MN.

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D. Kirkland et al. / Mutation Research 584 (2005) 1–256

Table 8 Non-carcinogens negative in Ames + MLA but not tested in MN Chemical

CAS Number

Result in CA

Acetohexamide Acetonitrile [AKA ethyl nitrile] Ampicillin trihydrate Benzoin n-Butyl chloride Caprolactam (2-Chloroethyl)trimethylammonium chloride C.I. pigment yellow 12 Cyclohexanone N,N-Dicyclohexylthiourea Dimethylformamide Diphenhydramine·HCl EDTA, trisodium salt trihydrate Endrin Ephedrine sulphate Erythromycin stearate Etodolac FD and C green no. 3 [AKA C.I. Food green 3] FD and C red no. 3 [AKA fluorescein, 2 , 4 , 5 , 7 -tetraiodo, disodium salt] Fluometuron [AKA urea, 1,1-dimethyl 3- (alpha, alpha, alpha-trifluoro-m-tolyl)-] 4,4 -Isopropylidenediphenol d-Mannitol Phenformin·HCl Phthalamide 3-Sulfolene Tetracycline·HCl Tin (II) chloride Tolbutamide 1,1,1-Trichloroethane, technical grade Tricresyl phosphate l-Tryptophan

968-81-0 75-05-8 7177-48-2 119-53-9 109-69-3 105-60-2 999-81-5

+ E − + − E −

6358-85-6 108-94-1 1212-29-9 68-12-2 147-24-0 150-38-9 72-20-8 134-72-5 643-22-1 41340-25-4 2353-45-9

− − − − + − − − − − TC

16423-68-0

+

2164-17-2

−

80-05-7 69-65-8 834-28-6 88-96-0 77-79-2 64-75-5 7772-99-8 64-77-7 71-55-6

+ − − − − + + − E

1330-78-5 73-22-3

− −

It is interesting to see how many chemicals became “false positives” because the third test was positive when the first two tests had been negative: • 34 chemicals were negative in Ames + MLA. - Three were tested in MN and none (0%) gave positive results, although two were equivocal. - 32 were tested in CA and seven (21.9%) gave positive results.

• Three chemicals were negative in Ames + MN, only one was also tested in MLA and was negative (0%). • 47 chemicals were negative in Ames + CA, 36 were also tested in MLA and 12 (33.3%) were positive. This trend has already been recognised when the ICH genotoxicity guidelines for pharmaceuticals were discussed [6]. Hence, these guidelines describe two in vitro tests (plus one in vivo test) as sufficient for most test compounds. For three Ames-negative chemicals, positive results were obtained in all three mammalian cell tests (MLA, MN and CA). These were: • o-anthranilic acid; • phenol; • resorcinol. Such reproducibility of positive findings in mammalian cell systems suggests these are genotoxic noncarcinogens, and that the Ames test is insensitive to them. A further 20 Ames-negative non-carcinogens were positive in two of the mammalian cell tests. They were: • positive in MLA and CA - benzyl alcohol - C.I. acid orange 10 - diallyl phthalate - diazinon - ethyl tellurac - eugenol - methotrexate - nickel(II)sulfate hexahydrate - penicillin VK - 1-phenyl-2-thiourea - phthalic anhydride - propyl gallate - tetraethylthiuram disulfide - tetrakis(hydroxymethyl)phosphonium chloride* - tetrakis(hydroxymethyl)phosphonium sulfate* (*) means that the chemicals may be considered as one in that they become the same chemical in solution or when absorbed by an animal. • positive in MLA and MN - rotenone - triphenyltin hydroxide • positive in MN and CA - fenvalerate - malathion.

D. Kirkland et al. / Mutation Research 584 (2005) 1–256

Again, positive results in two mammalian cell assays would tend to suggest these 20 chemicals are probably genotoxic non-carcinogens. As the criteria for an acceptable top dose in the mammalian cell tests (in particular the MLA and CA) have changed in the last two decades, it was interesting to see if many of the non-carcinogens that were positive in the MLA gave positive responses only at high levels of toxicity. A preliminary analysis of the MLA results for about half of the Ames-negative non-carcinogens that gave positive results in two mammalian cell tests indicated most chemicals were clearly positive (i.e. greater than two-fold increases in mutant frequency at RTG values >20%). For a further 16 Ames-negative noncarcinogens that were positive only in the MLA (i.e. negative, equivocal or no test data in MN and CA tests), a preliminary analysis indicated also that the positive responses were similarly quite clear (i.e. greater than two-fold at >20% RTG). Thus there is no indication from preliminary analysis that many (if any) of the MLA results with these non-carcinogens were “borderline” or limited to high levels of cytotoxicity. We need to investigate further to see whether there are differences in attained fold increases in mutant frequency over controls for carcinogens and non-carcinogens. Analysis of the positive CA results with these Amesnegative non-carcinogens was more difficult because many of the publications and data sources (e.g. NTP website) contained no toxicity data. However, preliminary analysis of the Ames-negative non-carcinogens that were positive in two mammalian cell tests showed many gave quite strong CA responses (e.g. 20–30% cells with CA), sometimes at low or middle concentrations where high cytotoxicity would not likely be responsible for an indirect effect. Thus, these results can also not be easily dismissed as “borderline” or “due to cytotoxicity”. However, the responses seemed more borderline for a further 18 Ames-negative non-carcinogens that were positive only in the CA test. Although toxicity measurements were again not reported for most of these chemicals, a preliminary analysis indicated that generally weak CA responses (e.g. <10% cells with CA) were seen at concentrations where all cells could be scored. It appeared that higher CA responses were seen where it had not been possible to score a full complement of cells, and therefore toxicity may have been severe. Thus, these isolated positive CA responses with

23

Ames-negative non-carcinogens are not as convincing and may be due indirectly to high levels of cytotoxicity. We hope to conduct a more detailed examination of these results, and to look at possible mechanisms of action, for a future publication.

8. Relative predictivity With such high sensitivity (positive genotoxicity results with carcinogens) but poor specificity values (negative genotoxicity results with non-carcinogens), particularly with combinations of two or three in vitro tests, it becomes difficult to evaluate the overall performance of these assays. Companies developing new products clearly desire sensitive tests to predict whether they have a safe chemical. However, a high falsepositive rate can mean that important substances (possibly life-saving drugs) may be dropped from development. An appropriate balance between sensitivity and specificity therefore needs to be found, as a basis for judging whether individual tests, or combinations of tests, provide the best information from which to make decisions on product development. In the past, previous authors [1,2,4] have placed much emphasis on concordance. For comparison, concordance data for the individual assays is given in Table 9. This is the fraction of both positive and negative results that are correct. Consider Example 1 (see Table 10) where 30/40 carcinogens give positive results and 20/30 non-carcinogens give negative results in a particular assay (or combination of assays) then the concordance is 50/70 = 0.71 (or 71%). The same concordance, however, could be obtained in Example 2 where 39/40 carcinogens gave positive results but only 11/30 non-carcinogens gave negative results. In other words, concordance does not help us see the respective false positive and false negative attributes of the test(s) we are evaluating. Table 9 also shows that for the individual assays, similar concordance values are obtained with quite variable sensitivity and specificity values. We therefore decided to investigate a measure we have called “relative predictivity” or RP. This is analogous to relative risk in the study of health effects in respect of various human exposures. Relative predictivity is the ratio of real results to false results. If we use the example above, and (for simplicity) we assume that the carcinogens failing to give positive results in fact

24

D. Kirkland et al. / Mutation Research 584 (2005) 1–256

Table 9 Summary of sensitivity, specificity and concordance values for individual assays Measurea

Carc.

+ − Sensitivity (%) Specificity (%) Concordance (%) a

Ames

MLA

MN

CA

+

E

−

+

E

−

+

E

−

+

E

−

318 40 58.8 73.9 62.5

8 6

215 130

179 55 73.1 39.0 62.9

9 9

57 41

70 12 78.7 30.8 67.8

2 6

17 8

231 61 65.6 44.9 59.8

14 14

107 61

Equivocal (E) results not counted either as positive or negative.

gave (false) negative results, and the non-carcinogens failing to give negative results in fact gave (false) positive results, then the data in Table 10 are obtained. Clearly with Example 2, the low false negative rate with the carcinogens (39/40 carcinogens were positive in this short-term test) means that a negative result in this test is nearly 15× more likely to indicate a non-carcinogen than a carcinogen. However, the higher false positive rate with the non-carcinogens (19/30 noncarcinogens gave positive results in this short-term test) means that a positive result is less than 2× more likely to indicate a carcinogen than a non-carcinogen. Thus, although these two examples gave the same concordance, the predictions of rodent carcinogenicity from positive results, or non-carcinogenicity from negative results, are quite different. We believe that relative predictivity could be a useful tool in decision making. In studies of human health effects, relative risk values of 1.4 or 1.6 are often considered important (e.g. a 40% or 60% greater chance of getting skin cancer if sun-tanning). However, many publications indicate that a relative risk of less than two should not be consid-

ered significant (see http://www.numberwatch.co.uk/ RR.htm). In the data discussed below we therefore accepted that a RP of less than two was not significant. The relative predictivity values are calculated and presented for the individual assays in Table 11, for pairs of assays in Table 12 and for the three-test combinations in Table 13. Examination of Table 11 shows that none of the individual assays had RP values of >2.0 for prediction of both carcinogenic potential from positive results and non-carcinogenic potential from negative results. Examination of Table 12 shows that only the combination of Ames + MLA gave RP values of >2.0 for prediction of both carcinogenic potential from positive results and non-carcinogenic potential from negative results. Examination of Table 13 shows that Ames + MLA + CA gives RP values of >2.0 for both calculations, and has the highest prediction of carcinogenic potential from positive results of any of the choices of individual of combinations of tests. Thus positive results in each of Ames, MLA and CA tests indicate that the chemical is 3.47× more likely to be a rodent

Table 10 Example to illustrate how relative predictivity can vary between sets of results with the same concordance % chemicals giving +ve genotoxicity results (actual numbers)

Example 1; concordance 71% Example 2; concordance 71%

Carcinogens (A)

Non-carcinogens (B)

75.0 (30/40)

33.3 (10/30)

97.5 (39/40)

63.3 (19/30)

Relative predictivity of +ve result for carcinogenicity (A/B)

% chemicals giving −ve genotoxicity results (actual numbers)

Relative predictivity of −ve result for non-carcinogenicity (C/D)

Non-carcinogens (C)

Carcinogens (D)

2.25

66.7 (20/30)

25.0 (10/40)

2.67

1.54

36.7 (11/30)

2.5 (1/40)

14.68

D. Kirkland et al. / Mutation Research 584 (2005) 1–256

25

Table 11 Prediction of carcinogen/non-carcinogen status from results in a single in vitro assay Test system

Ames MLA MN CA

% of chemicals positive in a single assay Carcinogens (A)

Non-carcinogens (B)

58.8 73.1 78.7 65.6

22.7 52.4 46.2 44.9

Relative predictability that a single positive result indicates a carcinogen A/B 2.59 1.40 1.70 1.46

% of chemicals negative in a single assay Non-carcinogens (C)

Carcinogens (D)

73.9 39.0 30.8 44.9

39.7 19.2 19.1 30.4

Relative predictability that a single negative result indicates a non-carcinogen C/D 1.86 2.03 1.61 1.48

Table 12 Prediction of carcinogen/non-carcinogen status from combinations of two in vitro tests Two-test combination

Ames + MLA Ames + MN Ames + CA MLA + MN MLA + CA

% of chemicals positive in both tests Carcinogen (A)

Non-carcinogen (B)

44.6 43.5 43.9 70.4 55.7

15.2 16.0 14.7 40.0 32.3

Relative predictability that two positive results indicate a carcinogen (A/B) 2.93 2.72 2.99 1.76 1.72

carcinogen than a non-carcinogen. Also, negative results in each of Ames, MLA and CA tests indicate that the chemical is 2.1× more likely to be a rodent noncarcinogen than a carcinogen. Whilst the positive RP value for Ames + MLA + MN is very acceptable (2.96) the negative RP value is poor, and is <1.0. However, as mentioned earlier the database of chemicals for which results in all three of these tests are available is very small, and the predictivity could change dramatically with the publication of a few more results. It was mentioned earlier that, in terms of sensitivity measures alone, the most effective sequence for testing

% of chemicals negative in both tests Non-carcinogen (C)

Carcinogen (D)

32.4 12.0 34.6 10.0 27.1

14.0 12.9 22.4 9.3 12.8

Relative predictability that two negative results indicate a non-carcinogen (C/D) 2.31 0.93 1.54 1.08 2.12

would be to perform MN first, followed by Ames and finally MLA. The RP values, however, suggest that the most effective sequence for testing would be Ames first, followed by MLA and finally CA, which is, in fact, the practice in most laboratories. We have not provided RP values for those situations where, in a pair of tests one was positive and the other negative or, with combinations of three tests, there was a mixture of positive and negative results. As might be expected, all RP values for these situations were not significant (<2) and in some cases were even <1, indicating a better prediction of carcinogenic potential

Table 13 Prediction of carcinogen/non-carcinogen status from combinations of three in vitro tests Combination of three % of chemicals positive in all tests three tests

Ames + MLA + MN Ames + MLA + CA

Carcinogens (A)

Non-carcinogens (B)

44.4 36.1

15.0 10.4

Relative predictability that three positive results indicate a carcinogen (A/B)

% of chemicals negative in all three tests Non-carcinogens (C)

Carcinogens (D)

2.96 3.47

5.0 22.9

7.4 10.9

Relative predictability that three negative results indicate a non-carcinogen (C/D) 0.68 2.10

26

D. Kirkland et al. / Mutation Research 584 (2005) 1–256

might be obtained by flipping a coin! Such findings tend to emphasise the need to understand the mechanisms by which chemicals might be positive only in one out of three tests in the battery, leading to an erroneous prediction of rodent carcinogenicity. In these cases evaluation of carcinogenic potential must involve mechanism of action and weight of evidence approaches.

9. Implications for chemical safety screening and regulatory assessment Genetic toxicology testing has a long-standing tradition in chemical safety screening and regulatory guidances as an approach to test for potential carcinogenesis of chemicals. While earlier evaluations have already cast doubt on the usefulness to do this basically with an in vitro battery only, the present evaluation underlines the problems encountered on the specificity side using as large a database as currently possible. Further to what we have discussed in previous sections of this paper, we realise that there are potentially very serious implications of this analysis on the generally accepted chemical safety screening approaches. In this paper, we will not discuss these in detail. Nevertheless, we would like to pose as follows a series of questions that arise from this analysis and which obviously call for an answer in due course. a) What is the appropriate follow-up procedure on an Ames test negative compound? b) What is the appropriate follow-up procedure on an Ames test positive compound? c) Can an Ames test positive be “de-risked” with negative results from mammalian cell testing in vitro? d) Is there a correlation with “potency” of carcinogenesis (trans-sex, trans-species, multi-organ versus single-sex, single-species, single organ) and either number of positives in a genotoxicity battery in vitro or fold increases in single assays over spontaneous events or lowest observed effect concentrations? e) Is a radical change in assessing criteria for positivity in mammalian cell assays in vitro a possible way forward towards a higher specificity whilst at the same time retaining the desired sensitivity? f) Do we need to completely reassess the conditions for in vitro testing (solubility, cytotoxicity, etc.) in

g)

h)

i)

j)

order to improve the specificity of the existing tests without sacrificing (much) sensitivity? Do we need to devise a completely new set of in vitro tests that can provide both acceptable specificity and sensitivity (e.g. mutation in transgenic cell lines, systems with inherent metabolic activity avoiding the need for S9, measurement of mutation in more cancer-relevant genes or hotspots of genes)? If mammalian cell tests in vitro do react towards chemical characteristics that do not lead to cancer, are there other serious toxicological concerns that are addressed with them (e.g. relevant cell cycle influences, mitochondrial toxicity, reactivity with macromolecules similar to protein adduct screening etc.)? If the appropriate follow-up testing for a single positive result consists of one or two mammalian in vivo genotoxicity tests, and if in vitro mammalian cell tests do provide only a very low specificity, are the arguments for reducing use of animals still valid enough to proceed with the current strategy for an (extensive) in vitro test battery? Would it be better to proceed with two in vivo endpoints in different organs (e.g. bone marrow and liver, possibly in the same animals) together with the Ames test? Is the SHE cell transformation assay an appropriate follow-up test for chemicals producing negative results in the standard in vitro tests?

10. Summary and conclusions We have assessed the largest database of chemicals for comparison of rodent carcinogenicity with results in a battery of in vitro genotoxicity tests. Whilst the dataset is large it may be considered a rather selected set of chemicals, as those that are chosen for cancer bioassays may contain a higher proportion of chemicals that are biologically active and are therefore likely to show some activity in genotoxicity tests. In comparison with previous evaluations (Tennant et al. [1]; Zeiger et al. [2]; Zeiger [4]) the sensitivity of Ames and CA tests were better, the MLA was similar but the MN test had the highest sensitivity of the individual assay systems. As expected, combinations of two and three tests showed improved sensitivity in detecting carcinogens, although the improvement from two to three tests

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was not that dramatic. Specificity for the Ames test alone was reasonable, but mammalian cell tests and combinations of two and three tests had unacceptably low specificity. It was very difficult to assess the performance of the MN test, particularly in combination with other tests, because of the small database of chemicals. Its specificity may improve if data from a larger proportion of known non-carcinogens were to become available, and a larger published database of results with the MN assay is urgently needed if this test is to be assessed for regulatory use. In order to balance the conflicting sensitivity and specificity values we adopted a measure called relative predictivity which is the ratio of real:false results. The combination of Ames + MLA gave significant (RP > 2) predictions of both carcinogenicity from positive results in both assays and noncarcinogenicity from negative results in both assays. A slightly higher positive RP was obtained if a three-test battery of Ames + MLA + CA was adopted. This measure is considered a useful tool for industry to predict the likelihood of a chemical possessing carcinogenic or non-carcinogenic potential from positive or negative

Appendix A. Genotoxicity results with rodent carcinogens

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results across a battery of three tests. However, mixed positive and negative results across a battery give no meaningful prediction of rodent carcinogenicity. The disappointing findings from these analyses (particularly the extremely low specificity) suggest it may be time for a complete rethink on in vitro genotoxicity testing. In the short term this may come from a reassessment of the conditions (cytotoxicity, solubility, etc.) for in vitro testing, but in the longer term, new, more robust assays may be needed. It may be time to acknowledge that “if nature is tortured to its limits, in its desperation it will give an answer”. This may not always be the answer we want or expect!

Acknowledgements The authors are extremely grateful to Dr. Lois Swirsky Gold for providing and maintaining the CPDB, and for her helpful criticism in the preparation of this manuscript, and to Joan Fisher of Procter & Gamble (Cincinnati) for providing the chemical structures in the appendices. The authors wish to state that they did not receive any funding for the work involved in this exercise and do not have any conflicts of interest.

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