Evaluation of the sensitivity and specificity of in vivo erythrocyte

Mutation Research 802 (2016) 1–29

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Evaluation of the sensitivity and specificity of in vivo erythrocyte micronucleus and transgenic rodent gene mutation tests to detect rodent carcinogens Takeshi Morita a,∗ , Shuichi Hamada b , Kenichi Masumura c , Akihiro Wakata d , Jiro Maniwa e , Hironao Takasawa b , Katsuaki Yasunaga b , Tsuneo Hashizume f , Masamitsu Honma c a

Division of Risk Assessment, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Nonclinical Research Center, LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan c Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan d Drug Safety Research Laboratories., Astellas Pharma Inc., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan e Clinical Science Division, Research & Development, AstraZeneca KK, 3-1, Ofuka-cho, Kita-ku, Osaka 530-0011 Japan, Japan f Drug Safety Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan b

a r t i c l e

i n f o

Article history: Received 18 November 2015 Received in revised form 14 March 2016 Accepted 16 March 2016 Available online 21 March 2016 Keywords: Genotoxicity in vivo Rodent erythrocyte micronucleus test Transgenic rodent mutation assay Sensitivity Specificity

a b s t r a c t Sensitivity and/or specificity of the in vivo erythrocyte micronucleus (MN) and transgenic rodent mutation (TGR) tests to detect rodent carcinogens and non-carcinogens were investigated. The Carcinogenicity and Genotoxicity eXperience (CGX) dataset created by Kirkland et al. was used for the carcinogenicity and in vitro genotoxicity data, i.e., Ames and chromosome aberration (CA) tests. Broad literature surveys were conducted to gather in vivo MN or TGR test data to add to the CGX dataset. Genotoxicity data in vitro were also updated slightly. Data on 379 chemicals (293 carcinogens and 86 non-carcinogens) were available for the in vivo MN test; sensitivity, specificity or concordances were calculated as 41.0%, 60.5% or 45.4%, respectively. For the TGR test, data on 80 chemicals (76 carcinogens and 4 non-carcinogens) were available; sensitivity was calculated as 72.4%. Based on the recent guidance on genotoxicity testing strategies, performance (sensitivity/specificity) of the following combinations was calculated; Ames + in vivo MN (68.7%/45.3%), Ames + TGR (83.8%/not calculated (nc)), Ames + in vitro CA + in vivo MN (80.8%/21.3%), Ames + in vitro CA + TGR (89.1%/nc), Ames + in vivo MN + TGR (87.5%/nc), Ames + in vitro CA + in vivo MN + TGR (89.3%/nc). Relatively good balance in performance was shown by the Ames + in vivo MN in comparison with Ames + in vitro CA (74.3%/37.5%). Ames + TGR and Ames + in vivo MN + TGR gave even higher sensitivity, but the specificity could not be calculated (too few TGR data on non-carcinogens). This indicates that in vivo MN and TGR tests are both useful as in vivo tests to detect rodent carcinogens. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction Several guidance documents on genotoxicity testing strategies have been issued by advisory bodies and regulatory agencies for pharmaceuticals via the International Conference on Harmonisation (ICH) [1], chemical substances via the UK Committee on Mutagenicity (COM) [2] or the European Chemical Agency (ECHA) [3], or food chemicals via the European Food Safety Authority (EFSA) [4] or the Japanese Food Safety Committee (FSC) [5]. In

∗ Corresponding author. E-mail address: [email protected] (T. Morita).

general, two in vitro tests covering bacterial gene mutation (Ames test) and chromosome damage (in vitro chromosomal aberration (CA) or micronucleus (MN) test) and one or more in vivo tests (e.g., in vivo MN test, the transgenic rodent mutation (TGR) assay, in vivo comet assay) will be required in the tired approach or standard test battery system for identification of the genotoxic substances in the guidance, especially in case of in vitro positive results. In the EU cosmetics, the standard battery generally consists of 2 (Ames and in vitro MN tests) or 3 (Ames, mammalian cell gene mutation and in vitro MN tests) in vitro tests, in which only in vitro tests are allowed to use as in vivo testing is completely prohibited [6,7]. Sensitivity or specificity of the individual in vitro genotoxicity tests or various combinations of the tests for rodent

http://dx.doi.org/10.1016/j.mrgentox.2016.03.008 1383-5718/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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T. Morita et al. / Mutation Research 802 (2016) 1–29

Table 1 Sensitivity and specificity of in vivo micronucleus (MN) and transgenic rodent mutation (TGR) tests by various investigators. Invetigators [Ref.] in vivo MN Morita et al. (1997) [11] Zeiger et al. (1998) [9] Kim and Margolin (1999) [12] Lambert et al. (2005) [15] OECD (2009) [16] Benigni et al. (2010) [13] Benigni et al. (2012) [14] TGR Lambert et al. (2005) [15] OECD (2009) [16]

No. of chemicals (C, NC)

Sensitivity

Specificity

171 (171a , 0) 83 (50, 33) 82 (55, 27) 69 (67, 2) 94 (Not satated clearlyb ) 183 (143, 40) 132 (103, 29)

52% 28% 36% 66% 71% 40% 69%

Not applicable 82% 78% 50% 83% 75% 41%

105 (92, 13) 154 (Not satated clearlyc )

78% 76%

69% 78%

C, Carcinogens; NC, Non-carcinogens. a IARC Groups 1, 2A and 2B carcinogens. b No. of NC will be less than 10. c No. of NC will be less than 40.

carcinogens has been well discussed [8–10]. Though each of four in vitro assays (i.e., Ames test, mouse lymphoma Tk gene mutation assay (MLA), in vitro MN test, and in vitro CA test) showed relatively high sensitivity (58.8–78.4%), their specificity was low (30.8–44.9%) except for the Ames test (73.9%) in the Carcinogenicity and Genotoxicity eXperience (CGX) database [10]. With respect to the in vivo genotoxicity tests, especially for the in vivo MN test, several investigations on the performance of the assay were conducted using relatively small (less than 200) chemical datasets [9,11–16]. The sensitivity (28–71%) or specificity (41–83%) of the in vivo MN obtained from these investigations showed large variations, which might be due to different chemicals in each dataset (Table 1). For the TGR assay, relatively high sensitivity (76–78%) and specificity (69–78%) were indicated by recent analysis; however, specificity was based on an extremely small (maybe less than 40) chemical dataset [15,16]. Although in vivo genotoxicity assays (e.g., in vivo MN) may be less sensitive to detect carcinogens than in vitro assays, they are considered to be more relevant, because they take in to account the absorption, tissue distribution, metabolism and excretion (i.e., ADME) of the test chemicals. Therefore, at least one in vivo genotoxicity assay will often be included in a genotoxicity test battery or used as a follow-up study in case of a positive in vitro result. In vivo genotoxicity assays are also useful to study mode of action when a rodent bioassay showed carcinogenic effects. The recent ICH S2(R1) guideline for human pharmaceuticals offers two options of the standard test battery for genotoxicity, one in which an in vitro mammalian cell assay is included (i.e., option 1), or the other where mammalian cell assays are excluded (i.e., option 2) [1]. Option 1 consists of i) a test for gene mutation in bacteria (i.e., Ames test), ii) an in vitro cytogenetic test for chromosomal damage (CA, MN or MLA test), and iii) an in vivo test for genotoxicity, generally a test for chromosomal damage using rodent hematopoietic cells (predominantly in vivo MN test). Option 2 consists of i) Ames test and ii) an assessment of two in vivo genotoxicity tests with two different tissues, usually an assay for micronuclei using rodent hematopoietic cells and a second in vivo assay. Thus, the in vivo rodent erythrocyte MN assay has a key role under both options. The in vivo MN assay detects chromosome damage, and is therefore the in vivo counterpart of the in vitro CA and MN tests. The in vitro MN test is recently more used and better counterpart of the in vivo MN test preferred over the in vitro CA test because MN test can detect both clastogenicity and aneugenicity. However, total numbers (n = 115) of chemicals evaluated by the in vitro MN test were about a quarter in comparison with those (n = 488) for the in vitro CA test in the CGX database [10]. Therefore, the in vitro CA test was included in this analysis. On the other hand, the Ames and MLA tests detect gene mutations; the in vivo counterpart of the Ames

test or MLA is the TGR assay. Although a DNA strand breakage assay (comet and alkaline elution assays) in liver is also recommended as a second in vivo assay, a TGR assay is recommended especially in the case of a positive response in the MLA with induction of large colonies, but negative results in the in vitro CA test [1]. Genotoxicity testing strategies in other sectors including COM [2], ECHA [3], EFSA [4] and FSC [5] also recommend a TGR assay or in vivo comet assay in some case of positive in vitro results. Thus, the TGR assay also has an important role to play amongst the in vivo genotoxicity assays. Although the single or combined use of in vitro genotoxicity assays showed relatively high sensitivity and low specificity for rodent carcinogens/non-carcinogens [10], a systematic review with a large chemical dataset for the performance of the in vivo genotoxicity assays, either alone or in combination, has not been performed. The CGX is a database of four in vitro genotoxicity tests with large numbers of rodent carcinogens and non-carcinogens, in which specificity and sensitivity of individual and combinations of the tests is calculated [10]. If in vivo genotoxicity data are added to the CGX database, the extended database is useful for a comprehensive understanding of the performance of genotoxicity tests. The performance will reveal implication of the in vivo MN and TGR assays in the genotoxicity testing strategies. Therefore, the sensitivity and specificity of the in vivo MN and TGR assays, alone or in combination, have been analyzed using the CGX chemical dataset. The in vitro–in vivo correlations for the different endpoints (chromosomal damage and gene mutation i.e., in vitro CA vs in vivo MN; Ames vs TGR) have also been investigated.

2. Materials and methods 2.1. Chemical dataset used A chemical dataset, i.e., the Carcinogenicity and Genotoxicity eXperience (CGX) dataset [10,17] was used to investigate the sensitivity and specificity of in vivo genotoxicity tests (i.e., in vivo MN and TGR tests) for the detection of rodent carcinogens and noncarcinogens. The CGX dataset provides results from four in vitro genotoxicity tests (i.e., Ames test, in vitro CA test, in vitro MN test and MLA) and analysis of sensitivity and specificity, both singly and in combination, in comparison with 756 rodent carcinogens and 183 non-carcinogens. The chemicals employed in the CGX dataset included all types of chemicals, such as industrial chemicals, agrochemicals, pesticides, pharmaceuticals, and natural products, among others. The CGX dataset was updated at April 2007 based on the re-examination of some of the MLA results and additional data on certain chemicals [17].

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2.2. Sources of genotoxicity test results 2.2.1. Ames and in vitro CA tests Data for in vitro genotoxicity tests (i.e., Ames and in vitro CA) were taken from the revised CGX dataset [17]. We have made some additional changes from the original CGX [10]. Examples of changes on the Ames and in vitro CA test results are as follows: The Ames test results for carbon tetrachloride (ID C173, 56-23-5) and chloroform (ID C160, 67-66-3) have been re-evaluated as positive (+) from negative (−) due to the positive data using gaseous exposures [18]. The CA result for DDT (ID C221, 50-29-3) has been changed to equivocal (E) from positive (+) due to the presence of one positive test in B14F28 cells and one negative test in V79 cells [19]. Data on the CA test result for o-phenylphenol sodium salt (ID C632, 132-274) have been added according to the review by Brusick [20]. Positive results in the Ames and CA tests have been added for aristolochic acid (ID C66, 313-67-7) due to a recent publication [21]. In addition, some data were identified during in vivo data collection as follows: • Negative results in the CA or Ames test have been added for capsaicin (ID C130, 404-88-4) [22,23] and ICRF 159 (ID C416, 21416-87-5) [24], respectively. • Negative results in the Ames and CA tests have been added for methyl clofenapate (ID C458, 21340-68-1) [25]. • Positive results in the Ames and CA tests have been added for 4-(methylnitrosamino)-1-(3-pyrridyl)-1-(butanone) (NNK, ID C478, 64091-91-4) [26]. • A negative result in the CA test has been added for procarbazine HCl (ID C645, 366-70-1) [27]. The revised Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 473 [28] for in vitro CA test defines that the highest test concentration should correspond to 10 mM, 2 mg/mL or 2 ␮L/mL, whichever is the lowest. Morita et al. [29] investigated the lowest effective concentrations of chemicals in the in vitro CA test cited in the original CGX database. Therefore, with respect to the CA test results, the following 7 carcinogens and 5 non-carcinogens have been re-evaluated as (−) from (+) due to the positive findings only being found at concentrations of higher than both 10 mM and 2 mg/mL [29]: • Carcinogens: hexanamide (ID C392, 628-02-4), nitrite sodium (ID C509, 7632-00-0), nitrobenzene (ID C514, 98-95-3), Nnitrosodiethylamine (ID C551, 55-18-5), saccharin sodium (ID C664, 128-44-9), trimethylphosphate (ID C734, 512-56-1) and urethane (ID C744, 51-79-6); • Non-carcinogens: o-anthranilic acid (ID NC12, 118-92-3), benzyl alcohol (ID NC20, 100-51-6), 4-nitroanthranilic acid (ID NC124, 619-17-0), 1-phenyl-2-thiourea (ID NC144, 103-85-5) and resorcinol (ID NC152, 108-46-3). 2.2.2. In vivo micronucleus test Test results from the in vivo rodent erythrocyte MN test were obtained from international chemical assessment documents (i.e., European Union Risk Assessment Reports (EURAR), http://echa. europa.eu/web/guest/information-on-chemicals/informationfrom-existing-substances-regulation, OECD Screening Information Data Set (SIDS) documents, http://webnet.oecd.org/hpv/UI/Search. aspx, the International Programme on Chemical Safety (IPCS) Concise International Chemical Assessment Documents (CICAD), http://www.who.int/ipcs/publications/cicad/en/ or International Agency for Research on Cancer (IARC) Monographs on the Evaluation of Carcinogenic Risks to Humans, http://monographs.iarc. fr/ENG/Monographs/suppl7/index.php), several review papers on in vivo MN test data [30–33], several reports from large scale

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studies on the in vivo MN test [11,34–37] and the US National Toxicology Program (NTP) Chemical Effects in Biological Systems (CEBS) database (http://tools.niehs.nih.gov/cebs3/ui/). A PubMed literature search was also employed using search words of “CAS number or chemical name”, “micronucle*” and “rodent” (http:// www.ncbi.nlm.nih.gov/pubmed/).

2.2.3. In vivo transgenic rodent mutation assay A detailed review paper on the TGR assay that was provided to OECD to support the development of a test guideline was used [16]. An earlier version of this review paper was also checked [15]. In addition, unpublished reports on the transgenic rodent mutation studies on food additives by the Japanese Ministry of Health Labor and Welfare (MHLW) were used [38–40].

2.3. Categories of the test results Categories of the test results were based on the criteria by Kirkland et al. [10]; positive (+), negative (−), equivocal (E) and technically compromised (TC). Positive indicates 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. Negative indicates a clearly negative response in all publications found. Equivocal indicates the response is weak or not reproduced between experiments or between laboratories. Weak means that a dose-related increase in effect was noted close to the borderline of biological significance, but they were not biologically and/or statistically significant. Non-reproducible means that there were both positive and negative findings across different studies of apparent equal validity, and the weight of evidence did allow a clear positive or negative overall outcome to be concluded. If a published study was considered inconclusive, it was called E for convenience. TC indicates a test result that was questionable due to failure to meet essential standard criteria for an adequate study. An example for a TC classification in the in vivo MN test will be no proof of target cell exposure. Proof of target cell exposure can be shown by reduction in immature/mature erythrocytes ratio in the study or by proof of systemic exposure in other in vivo studies. However, many data sources of in vivo tests were international evaluation documents, published review papers or national databases. All of them do not provide information of target cell exposure in in vivo MN and TGR tests. Therefore, TC classification was not applied to the in vivo test in this analysis; it was employed to the only in vitro tests based on the previous classifications [10]. References for compounds with in vivo negative result for which no target cell exposure were noted in the review paper or database are marked (as “∼”) in the Appendix tables. E results were included in the total numbers of assays/chemicals evaluated, but not employed for calculations of the definitive performance of the assays. It was based on the clear positive or negative results. However, additional calculations of the performance have been made, in which E results were considered positive or negative. TC results were not included in the total numbers.

3. Results Appendices A and B show each genotoxicity test result from the chemical dataset of the 756 carcinogens and 183 non-carcinogens, respectively.

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Table 2 Summary performance of individual in vitro assays in detecting rodent carcinogens or non-carcinogens. Carcinogenicity

Ames

in vitro CA

+

E

−

Total

+

E

−

Total

+ − Total

321 40 361

8 6 14

215 130 345

544 176 720

225 56 281

15 14 29

118 66 184

358 136 494

Sensitivitya Specificitya Concordancea

59.0% (321/544) 73.9% (130/176) 62.6% (451/720)

62.8% (225/358) 48.5% (66/136) 58.9% (291/494)

+, Positive; −, Negative; E, Equivocal. a Equivocal (E) results were not counted either as positive or negative, but they were included in the total number. If E results are considered positive, the performanceis as follows: Ames: sensitivity, 60.5% (329/544); specificity, 73.9% (130/176), concordance, 63.8% (459/720). In vitro CA: sensitivity, 67.0% (240/358); specificity, 48.5% (66/136), concordance, 61.9% (306/494). If E results are considered negative, the performance is as follows: Ames: sensitivity, 59.0% (321/544); specificity, 77.3% (136/176), concordance, 63.5% (457/720). In vitro CA: sensitivity, 62.8% (225/358); specificity, 58.8% (80/136), concordance, 61.7% (305/494). Table 3 Summary performance of individual in vivo assays in detecting rodent carcinogens or non-carcinogens. Carcinogenicity

in vivo MN

TGR

+

E

−

Total

+

E

−

Total

+ − Total

120 24 144

11 10 21

162 52 214

293 86 379

55 0 55

0 0 0

21 4 25

76 4 80

Sensitivitya Specificitya Concordancea

41.0% (120/293) 60.5% (52/86) 45.4% (172/379)

72.4% (55/76) Not calculated due to small numbers of non-carcinogens Not calculated

+, Positive; −, Negative; E, Equivocal. a Equivocal (E) results were not counted either as positive or negative, but they were included in the total number. If E results are considered positive, the performance is as follows: In vivo MN: sensitivity, 44.7% (131/293); specificity, 60.5% (52/86), concordance, 48.3% (183/379). If E results are considered negative, the performance is as follows: In vivo MN: sensitivity, 41.0% (120/293); specificity, 72.1% (62/86), concordance, 48.0% (182/379).

3.1. Performance of in vitro assays The summary performances (sensitivity, specificity and concordance) of the Ames and in vitro CA tests in discriminating between rodent carcinogens and non-carcinogens are shown in Table 2. TC results were not included in the table. 3.1.1. Ames test Ames test results were available for 544 carcinogens (excluding 1 TC result) and 176 non-carcinogens from the dataset. Of the 544 carcinogens, 321 clearly gave positive results (59.0%, sensitivity), 8 gave equivocal results, and 215 clearly gave negative results. Of the 176 non-carcinogens, 130 clearly gave negative results (73.9%, specificity), 6 gave equivocal results, and 40 gave positive results. Concordance was calculated as 62.6% (451/720) (Table 2). These performance indicators are consistent with those reported by Kirkland et al. (sensitivity, specificity and concordance were 58.8%, 73.9% and 62.5%, respectively) with the original CGX dataset [10]. If equivocal results are considered positive or negative, the sensitivity or specificity rises to 60.5% (329/544) or 77.3% (136/176), respectively.

positive or negative, the sensitivity or specificity rises to 67.0% (240/358) or 58.8% (80/136), respectively. 3.2. Analysis of in vivo genotoxicity results with carcinogens 3.2.1. In vivo MN test MN results were available for 293 of the 756 carcinogens. Specific comments and evaluations assigned for the current analysis on the following 16 chemicals are as follows: • ID C17, Acrylonitrile, 107-13-1 (negative) Almost all studies showed negative results [11,41]. Positive results were obtained in rat bone marrow cells treated by intravenous (i.v.) injection, but not in the peripheral blood [11,35]. In mice treated by i.v. injection, negative results were obtained in both bone marrow cells and peripheral blood [11]. Acrylonitrile was regarded as negative based on the results with a relevant route of exposure. • ID C179, Chlorpromazine hydrochloride, 69-09-0 (positive)

3.1.2. In vitro CA test CA results were available for 358 carcinogens (excluding 11 TC results) and 136 non-carcinogens (excluding 9 TC results) from the dataset. Of the 358 carcinogens, 225 clearly gave positive results (62.8%, sensitivity), 15 gave equivocal results, and 120 clearly gave negative results. Of the 136 non-carcinogens, 69 clearly gave negative results (48.5%, specificity), 14 gave equivocal results, and 52 gave positive results. Concordance was calculated as 58.9% (291/494) (Table 2). These performance indicators are also very similar to those reported by Kirkland et al. (sensitivity, specificity and concordance were 65.6%, 44.9% and 59.8%, respectively) with the original CGX dataset [10]. If equivocal results are considered

The reported positive finding is considered to be due to hypothermia [42]. The MN induction by hypothermia is due to an indirect genotoxic mode of action (i.e., secondary effect) and threshold related, which indicates a genotoxic hazard. If core body temperature changes do not occur under conditions of human exposure, there is unlikely to be a genotoxic risk [43]. Therefore, a positive call was assigned. • ID C197, C.I. Solvent yellow 3 (o-aminoazotoluene), 97-56-3 (positive)

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Differences in species were found (positive in mice [11], but negative in rats [35]).

Differences in species were found (positive in mice [30,32,50], but negative in rats [32]).

• ID C246, 1,2-Dibromoethane, 106-93-4 (equivocal)

• ID C711, o-Toluidine, 95-53-4 (positive)

Differences in route of exposure were found; negative by oral gavage [44,45] or by intraperitoneal (i.p.) injection [11], but positive by inhalation [35].

Differences in species were found (positive in rats [49,50], but negative in mice [11,50]).

No in vivo MN data were available on this chemical. However, a negative result was obtained with the free base (3,3 dimethoxybenzidine, 119-90-4) [11].

3.2.2. TGR assay TGR results were available for 76 of the 756 carcinogens. Specific comments on 6 chemicals (indicated as ‘+ˆ’ in Appendix A) which showed positive results in the non-target organ(s)/tissue(s) of carcinogenicity in rodents (mouse, rat and/or hamster) are as follows:

• ID C378, Haloperidol, 52-86-8 (positive)

• ID C244, 1,2-Dibromo-3-chloropropane, 96-12-8

The positive finding is considered to be due to hypothermia in mice [46]. On the other hand, rats showed a negative result [46].

1,2-Dibromo-3-chloropropane showed a positive gene mutation result in the testis, but was negative in the liver. The target organs of carcinogenicity are the nasal cavity, oral cavity, stomach, adrenal gland and mammary gland in rats, and the lung, nasal cavity and stomach in mice [15].

• ID C285, 3,3 -Dimethoxybenzidine 2HCl, 20325-40-0 (negative)

• ID C439, Mercuric chloride, 7487-94-7 (positive) Differences in species were found (positive in mice [47,48], but negative in rats [48]). • ID C466, 4,4 -Methylenedianiline 2HCl, 13552-44-8 (positive) This compound showed a positive result [34], however, there were negative results with the free base (4,4 -Methylenedianiline, 101-77-9) [11,49]. • ID C478, 4-(Methylnitrosamino)-1-(3-pyrridyl)-1-(butanone) (NNK), 64091-91-4 (equivocal) There was one positive [26] and one negative [50] result. In addition a positive result was obtained in mice treated by subcutaneous injection for up to 52 weeks [51]. • ID C509, Nitrite, sodium, 7632-00-0 (equivocal) There was one positive [30] and one negative [50] result. The OECD SIDS document evaluated this compound as positive [52]. • ID C631, Phenylhydrazine HCl, 59-88-1 (positive) No in vivo MN data were available on this chemical. However, a positive result was obtained with the free base (phenylhydrazine, 100-63-0) [30]. • ID C657, Pyrimethamine, 58-14-0 (positive) Differences in species were found (positive in rats [45,53], but negative in mice [51]). • ID C660, Reserpine, 50-55-5 (positive) The positive finding in mice might be due to hypothermia, whilst a negative result was obtained in rats [43]. • ID C672, Sodium dichromate, 10588-01-9 (positive)

• 1D C246, 1,2-Dibromoethane, 106-93-4 1,2-Dibromoethane showed a positive gene mutation result in the nasal mucosa and testes, but was negative in the lung and liver. The target organs of carcinogenicity are the nasal cavity, peritoneal cavity, pituitary gland, stomach, liver, lung and mammary gland [15]. • ID C340, Ethyl methanesulphonate, 62-50-0 Ethyl methanesulphonate showed positive gene mutation results in the bone marrow, epididymal sperm and liver, but was negative in the brain and small intestine. The target organs of carcinogenicity are the kidney, lung and thymus [15]. • ID C457, 3-Methylcholanthrene, 56-49-5 3-Methylcholanthrene showed a positive gene mutation result in the liver. The target organs of carcinogenicity are the lung, skin and mammary gland [15]. • ID C492, Mitomycin C, 50-07-7 Mitomycin C showed positive gene mutation results in the bone marrow and liver, but was negative in the small intestine and testis. The target organs of carcinogenicity are the intestine, mammary gland and peritoneal cavity [15]. • ID C702, Thio-tepa, 52-24-4 Thio-tepa showed a positive gene mutation result in splenic lymphocytes. The target organs of carcinogenicity are the ear/Zymbal’s gland, haematopoietic system, skin, mammary gland and preputial gland [15]. Furthermore, specific comments on the 4 chemicals (indicated as ‘-ˆ’ in Appendix A) which showed negative results in the nontarget organ/tissue(s) of carcinogenicity are as follows: • ID C17, Acrylonitrile, 107-13-1

A positive result was obtained by i.p. injection, but a negative result was obtained by oral administration (gavage) [32]. • ID C676, Styrene, 100-42-5 (positive)

Acrylonitrile showed negative gene mutation results in the bone marrow, brain, lung, splenic lymphocytes and testicular germ cells [16]. The target organs of carcinogenicity are the ear/Zymbal’s

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gland, nervous system, oral cavity, small intestine, mammary gland and nasal cavity [15]. • ID C257, 1,2-Dichloroethane, 107-06-2 1,2-Dichloroethane showed negative gene mutation results in the liver and testis. The target organs of carcinogenicity are the stomach, subcutaneous tissue, vascular system, mammary gland, lung and uterus [15]. • ID C489, Metronidazole, 443-48-1 Metronidazole showed a negative gene mutation result in the stomach. The target organs of carcinogenicity are the pituitary gland, testes, liver, mammary gland, lung and haematopoietic system [15]. • ID C683, SX Purple, 2611-82-7 SX Purple showed negative gene mutation results in the liver and stomach [40]. No clear target organ of carcinogenicity was identified (“all tumor bearing animals”) in the Carcinogenic Potency Database [54].

3.4. Performance of in vivo assays The summary performances of the in vivo MN and TGR assays in discriminating between rodent carcinogens and non-carcinogens are shown in Table 3. 3.4.1. Performance of the in vivo MN test In vivo MN results were available for 293 of the 756 carcinogens and for 86 of the 183 non-carcinogens. Of the 293 carcinogens, 120 clearly gave positive results (41.0%, sensitivity), 11 gave equivocal results, and 162 clearly gave negative results. Of the 86 noncarcinogens, 52 clearly gave negative results (60.5%, specificity), 10 gave equivocal results, and 24 gave positive results. Concordance was calculated as 45.4% (172/379) (Table 3). If equivocal results are considered positive or negative, the sensitivity or specificity rises to 44.7% (131/293) or 72.1% (62/86), respectively. 3.4.2. Performance of the TGR assay TGR results were available for 76 of the 756 carcinogens and for 4 of the 183 non-carcinogens. Of the 76 carcinogens, 55 clearly gave positive results (72.4%, sensitivity) and 21 clearly gave negative results (Table 3). All 4 non-carcinogens showed negative results. Specificity and concordance could not be calculated due to the very limited TGR data for non-carcinogens. 3.5. Performance of the combination of two, three or four tests

3.3. Analysis of in vivo genotoxicity results with non-carcinogens 3.3.1. In vivo MN test In vivo MN results were available for 86 of the 183 noncarcinogens. Specific comments and evaluations assigned for the current analysis on the following 4 chemicals are as follows: • ID NC8, dl-Amphetamine sulfate, 60-13-9 (positive) The positive result assigned for the current analysis was based on the data obtained with the free base (dl-amphetamine, 300-629) [30]. • ID NC73, EDTA, trisodium salt trihydrate, 150-38-9 (negative) The negative result assigned for the current analysis was from data obtained with the disodium salt (6381-92-6) [55]. Note that a positive result in mice after oral administration of the disodium salt was reported [56]. However, EURAR commented that the positive result seems to be of low reliability, and that EDTA does not induce micronuclei in bone marrow cells [55]. • ID NC91, Fluoride, sodium, 7681-49-4 (equivocal) Kirkland et al. assigned this as positive based on a single positive result [33]. However, 3 negative studies were identified [50,57,58]. • ID NC138, Phenol, 108-95-2 (positive) The positive finding is considered to be due to hypothermia [43,49]. 3.3.2. TGR assay TGR results were available for only 4 of the 183 non-carcinogens. Such limited data meant that specificity and concordance values could not be calculated.

Based on the recent recommendations for genotoxicity test batteries or testing approaches as defined by advisory bodies and regulatory agencies (e.g. ICH, COM. ECHA, EFSA, FSC), the performances of the following combinations of tests were analyzed: Ames + in vitro CA, Ames + in vivo MN, Ames + TGR, Ames + in vitro CA + in vivo MN, Ames + in vitro CA + TGR, Ames + in vivo MN + TGR, Ames + in vitro CA + in vivo MN + TGR. The Ames test was included in all combinations employed due to it being the most basic genotoxicity test. The sensitivity calculations were based on at least one positive result in all of the genotoxicity tests in the combination among the carcinogens tested. The specificity calculations were based on there being negative results in all of the genotoxicity tests in the combination among the non-carcinogens tested. Performance of the combinations (i.e., sensitivity and specificity) is described below. The specificity values for the combination of tests including the TGR assay were not calculated due to the very low number of non-carcinogens tested by TGR. 3.5.1. Performance of Ames + in vitro CA Results were available from both Ames and in vitro CA tests for 350 carcinogens and 136 non-carcinogens. For the carcinogens, 149 or 111 chemicals were clearly positive (i.e., equivocal results considered negative) in both tests or in one of the two tests when both were performed, respectively (Table 4). Therefore, the sensitivity value that was calculated from the results where both tests were performed was 74.3% (260/350). If equivocal results are considered positive, the sensitivity rises to 76.9% (269/350). For the non-carcinogens, clearly negative results were obtained in both tests for 51 of the 136 non-carcinogens (Table 5). Therefore, the specificity value that was calculated from the results where both tests were performed was 37.5% (51/136). If equivocal results are considered negative, the specificity rises to 47.8% (65/136). 3.5.2. Performance of Ames + in vivo MN Results were available from both Ames and in vivo MN tests for 284 carcinogens and 86 non-carcinogens. For the carcinogens, 79 or 116 chemicals gave clearly positive in both tests or in one of the two tests when both were performed, respectively (Table 6). Therefore, the sensitivity value that was calculated from the results

T. Morita et al. / Mutation Research 802 (2016) 1–29 Table 4 Performance of combinations of Ames and in vitro CA tests in detecting rodent carcinogens when both performed. Ames

in vitro CA +

Table 7 Performance of combinations of Ames and in vivo MN tests in detecting rodent noncarcinogens when both performed. Ames

E

−

+ 149 8 35 5 0 2 E 63 7 81 − Total 217 15 118 No. of carcinogens tested in both tests (A): 350 No. (%) of clear positive results in both tests (B): 149 (42.6%) No. (%) of clear positive results in only 1 of the two tests (C): 111 (31.7%) Sensitivity (i.e., clearly positive in at least 1 test when both conducted ([B + C]/A)a : 74.3%

Total 192 7 151 350

7

in vivo MN +

E

+ 6 2 E 2 0 − 16 8 24 10 Total No. of non-carcinogens tested in both tests (A): 86 No. of clear negative results in both tests (B): 39 Specificity (B/A)a : 45.3%

−

Total

11 2 39 52

19 4 63 86

+, Positive; −, Negative; E, Equivocal a: If E results are considered negative, the specificity is 60.0% (49/86)

+, Positive; −, Negative; E, Equivocal a: If E results are considered positive, the sensitivity is 76.9% (269/350) Table 5 Performance of combinations of Ames and in vitro CA tests in detecting rodent noncarcinogens when both performed. Ames

Ames

in vitro CA +

−

Total

15 2 51 66

34 5 97 136

+, Positive; −, Negative; E, Equivocal a: If E results are considered negative, the specificity is 47.8% (65/136)

in vivo MN E

−

+ 79 7 73 E 2 0 2 − 34 4 83 Total 115 11 158 No. of carcinogens tested in both tests (A): 284 No. (%) of clear positive results in both tests (B): 79 (27.8%) No. (%) of clear positive results in only 1 of the two tests (C): 116 (40.8%) Sensitivity (i.e., clearly positive in at least 1 test when both conducted ([B + C]/A)a : 68.7%

E

−

Total

+ 48 0 8 E 1 0 0 5 0 12 − Total 54 0 20 No. of carcinogens tested in both tests (A): 74 No. (%) of clear positive results in both tests (B): 48 (64.9%) No. (%) of clear positive results in only 1 of the two tests (C): 14 (18.9%) Sensitivity (i.e., clearly positive in at least 1 test when both conducted ([B + C]/A)a : 83.8%

56 1 17 74

+, Positive; −, Negative; E, Equivocal a: If E results are considered positive, the sensitivity is 83.8% (62/74)

Table 6 Performance of combinations of Ames and in vivo MN tests in detecting rodent carcinogens when both performed.

+

TGR +

E

+ 19 2 3 0 E 34 12 − 56 14 Total No. of non-carcinogens tested in both tests (A): 136 No. of clear negative results in both tests (B): 51 Specificity (B/A)a : 37.5%

Ames

Table 8 Performance of combinations of Ames and TGR tests in detecting rodent carcinogens when both performed.

Total 159 4 121 284

+, Positive; −, Negative; E, Equivocal a: If E results are considered positive, the sensitivity is 70.8% (201/284)

where both tests were performed was 68.7% (195/284). If equivocal results are considered positive, the sensitivity rises to 70.8% (201/284). For the non-carcinogens, clearly negative results were obtained in both tests for 39 of the 86 non-carcinogens (Table 7). Therefore, the specificity value that was calculated from the results

where both tests were performed was 45.3% (39/86). If equivocal results are considered negative, the specificity rises to 60.0% (49/86). 3.5.3. Performance of Ames + TGR Results were available from both Ames and TGR tests for 74 carcinogens. For the carcinogens, 48 or 14 chemicals gave clearly positive in both tests or in one of the two tests when both were performed, respectively (Table 8). Therefore, the sensitivity value that was calculated from the results where both tests were performed was 83.8% (62/74). Even if equivocal results are considered positive, the sensitivity is not changed. The sensitivities are not changed in other combinations including the TGR test. 3.5.4. Performance of Ames + in vitro CA + in vivo MN Results in all three tests were available for 224 carcinogens and 75 non-carcinogens. For the carcinogens, 51 or 130 chemicals gave clear positive results in all three tests or in one or two of the tests when all three were performed (Table 9). Therefore, the sensitivity value that was calculated from the results where all three tests were performed was 80.8% (181/224). If equivocal results are con-

Table 9 Summary performance of three or four genotoxicity assays in detecting rodent carcinogens when all tests performed. Test combination

No. of carcinogens tested in all three or four test systems No. (%) of clear positive results in all three or four test systems No. (%) of clear positive results in one or two of the three assays No. (%) of clear positive results in one, two or three of the four assays Sensitivity (i.e., clearly positive in at least one assay when all three or four conducted)a

Ames + in vitro CA + in vivo MN

Ames + in vitro CA + TGR

Ames + in vivo MN + TGR

Ames + in vitro CA + in vivo MN + TGR

224 51/224 (22.8%) 130/224 (58.0%) Not applicable 181/224 (80.8%)

64 36/64 (56.3%) 21/64 (32.8%) Not applicable 57/64 (89.1%)

64 31/64 (48.4%) 25/64 (39.1%) Not applicable 56/64 (87.5%)

56 25/56 (44.6%) Not applicable 25/56 (44.6%) 50/56 (89.3%)

a If equivocal results are considered positive, the sensitivity of the combination of Ames + in vitro CA + in vivo MN is 83.0% (186/224). The sensitivities of other test combinations are not changed.

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T. Morita et al. / Mutation Research 802 (2016) 1–29

Table 10 Summary performance of three or four genotoxicity assays in detecting rodent non-carcinogens when all three or four tests performed. Test combination

No. of non-carcinogens tested in all three or four test systems (A) No. (%) of clear negative results in all three or four test systems (B) Specificity (B/A)a a

Ames + in vitro CA + in vivo MN

Ames + in vitro CA + TGR

Ames + in vivo MN + TGR

Ames + in vitro CA + in vivo MN + TGR

75

4

3

3

16

0

1

0

16/75 (21.3%)

Not calculated

Not calculated

Not calculated

If equivocal results are considered negative, the specificity is 29.3% (22/75).

Table 11 Concordance of Ames and TGR tests in detecting rodent carcinogens and noncarcinogens. Ames

TGR

+ E − Total Concordancea

+ (C, NC)

E (C, NC)

− (C, NC)

Total

48 (48, 0) 1 (1, 0) 5 (5, 0) 54 (54, 0) 79.5% (62/78)

0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0)

10 (8, 2) 0 (0, 0) 14 (12, 2) 24 (12, 2)

58 1 19 78

+, Positive; −, Negative; E, Equivocal; C, Carcinogens; NC, Non-carcinogens. a Equivocal (E) results were not counted either as positive or negative, but they were included in the total number. If E results are considered positive, the concrdance is 80.8% (63/78). If E results are considered negative, the concrdance is 79.5% (62/78).

sidered positive, the sensitivity rises to 83.0% (186/224). For the non-carcinogens, clearly negative results were obtained in all three tests for 16 of the 75 non-carcinogens (Table 10). Therefore, the specificity value that was calculated from the results where all three tests were performed was 21.3% (16/75). If equivocal results are considered negative, the specificity rises to 29.3% (22/75). 3.5.5. Performance of Ames + in vitro CA + TGR Results in all three tests were available for 64 carcinogens and 4 non-carcinogens. For the carcinogens, 36 or 21 chemicals gave clear positive results in all three tests or in one or two of the tests when all three were performed, respectively (Table 9). Therefore, the sensitivity value that was calculated from the results where all three tests were performed was 89.1% (57/64). For the non-carcinogens, none of the four chemicals were clearly negative in all three tests (Table 10). 3.5.6. Performance of Ames + in vivo MN + TGR Results in all three tests were available for 64 carcinogens and 4 non-carcinogens. For the carcinogens, 31 or 25 chemicals gave clear

positive results in all three tests or in one or two of the tests when all three were performed, respectively (Table 9). Therefore, the sensitivity value that was calculated from the results where all three tests were performed was 87.5% (56/64). For the non-carcinogens, none of the four chemicals were clearly negative in all three tests (Table 10).

3.5.7. Performance of Ames + in vitro CA + in vivo MN + TGR Results in all four tests were available for 56 carcinogens and 3 non-carcinogens. For the carcinogens, 25 or another 25 chemicals gave clear positive results in all four tests or in one, two or three of the tests when all four tests were performed, respectively (Table 9). Therefore, the sensitivity value that was calculated from the results where all four tests were performed was 89.3% (50/56). For the noncarcinogens, none of the 3 chemicals were clearly negative in all four tests (Table 10).

3.6. Comparison analysis between in vitro and in vivo tests with a similar endpoint Extrapolation of test results from in vitro to in vivo is one of the major issues in chemical risk assessment. The Ames and TGR tests can detect gene mutations, whereas the in vitro CA and in vivo MN tests can detect chromosome damage; the MN test can detect aneugenicity in addition to clastogencity. Therefore, concordance of these in vitro and in vivo tests in detecting rodent carcinogens and/or non-carcinogens was investigated. Though the results of in vitro MN tests were presented in the CGX database, the number (n = 89) of carcinogens with in vitro MN data in the dataset was quite small in comparison with that (n = 352) for the in vitro CA test [8]. Thus, for this evaluation the in vitro CA test was selected as the most appropriate in vitro test system that detects chromosome damage for comparison with the in vivo MN test.

Table 12 Incosistent results between Ames and TGR tests. ID

Chemical

Chemical grouping

CAS

Carcinogenicity

Ames

TGR

C84 C384 C605 C645 C742 C17 C137 C160 C257 C395 C489 C509 C622 NC52 NC126

Benzene Hexachlorobutadiene Oxazepam Procarbazine HCl Uracil Acrylonitrile Carbon tetrachloride Chloroform 1,2-Dichloroethane Hydrazine sulphate Metronidazole Nitrite, sodium Phenobarbital 2,6-Diaminotoluene 2HCl 1-Nitronaphthalene

Benzene Halogenated alkene Aromatic amine or amide Mono- or di-alkylhydrazine Substituted pyrimidine or purine Alpha-, beta-unsaturated nitrile Halogenated methane Halogenated methane vic-Dihalide Hydrazine or monoacyl- or monosulphonyl-hydrazine Aromatic nitro compound Alkyl nitrite, nitrous acid or nitrite salt (Thio)urea Aromatic amine or amide Aromatic nitro compound

71-43-2 608-73-1 604-75-1 366-70-1 66-22-8 107-13-1 56-23-5 67-66-3 107-06-2 10034-93-2 443-48-1 7632-00-0 50-06-6 15481-70-6 86-59-7

+ + + + + + + + + + + + + − −

− − − − − + + + + + + + + + +

+ + + + + − − − − − – − − − −

T. Morita et al. / Mutation Research 802 (2016) 1–29 Table 13 Concordance of in vitro CA and in vivo MN tests in detecting rodent carcinogens and non-carcinogens. in vitro CA

in vivo MN

+ E − Total Concordancea

+ (C, NC)

E (C, NC)

− (C, NC)

Total

82 (72, 10) 3 (1, 2) 27 (17, 10) 112 (90, 22) 50.3% (151/300)

12 (6, 6) 0 (0, 0) 4 (3, 1) 16 (9, 7)

91 (70, 21) 12 (7, 5) 69 (49, 20) 172 (126, 46)

185 15 100 300

+, Positive; −, Negative; E, Equivocal; C, Carcinogens; NC, Non-carcinogens. a Equivocal (E) results not counted either as positive or negative, but they were included in the total number. If E results are considered positive, the concrdance is 55.3% (166/300). If E results are considered negative, the concrdance is 55.7% (167/300).

3.6.1. Ames test and TGR tests Concordance of the Ames and TGR tests in detecting rodent carcinogens and non-carcinogens is shown in Table 11. Results were available from both Ames and TGR tests for 74 carcinogens and 4 non-carcinogens. Forty eight or 14 chemicals gave clearly positive or negative results in both tests, respectively. The concordance value calculated was 79.5% (62/78). Fifteen chemicals which showed inconsistent results are listed in Table 12. They consisted of 5 carcinogens with negative results in the Ames test but positive in the TGR test, 8 carcinogens with positive results in the Ames test but negative in the TGR test, and 2 non-carcinogens with positive results in the Ames test but negative in the TGR test. 3.6.2. In vitro CA and in vivo MN tests Concordance of the in vitro CA and in vivo MN tests in detecting rodent carcinogens and non-carcinogens is shown in Table 13. Results were available from both in vitro CA and in vivo MN tests for 225 carcinogens and 75 non-carcinogens. Eighty two or 69 chemicals gave clearly positive or negative results in both tests, respectively. The concordance value calculated was 50.3%

9

(151/300). There were many chemicals (70 carcinogens and 21 non-carcinogens) which showed positive results in the in vitro CA, but gave negative results in the in vivo MN. On the other hand, 27 chemicals (17 carcinogens and 10 non-carcinogens) showed negative results in the in vitro CA test, but were positive in the in vivo MN test. They are listed in Table 14. 4. Discussion The in vivo MN and TGR tests are the most important in vivo genotoxicity tests for identification of chemical genotoxic hazard for regulatory bodies [1–5]. Therefore, performances (sensitivity and specificity) of these tests, alone and in combination with other assay(s), including in vitro tests, were investigated. The performance of the in vivo MN test was 41.0% for sensitivity or 60.5% for specificity. The sensitivity is lower than in both the Ames test (59.0%) and the in vitro CA test (62.8%), but the specificity fell between that for the two tests considered singly, i.e., 73.9% and 48.5% (Table 15 and Fig. 1). The performance of the in vivo MN test previously reported by several investigators was wide ranging, namely 28–71% for sensitivity or 41–83% for specificity (Table 1). These variations might be due to different chemical sets, including IARC carcinogens by Morita et al. [11], NTP testing chemicals by Zeiger et al. [9] or Kim and Margolin [12], chemicals tested by TGR assay by Lambert et al. [15] or OECD [16] and ISSMIC chemicals by Benigni et al. [13,14]. Some of these references do not provide chemical names [9,13,14] or original data sources [15,16] in their texts. Therefore, it will be difficult to analyze the variations. Our investigation with a large number of chemicals gave sensitivity and specificity values that fell in the middle of the ranges given in those previous investigations. The in vivo MN test is recognized as demonstrating a relatively low sensitivity for the detection of carcinogens. Although the assay is sensitive to many different chemical classes, based on a study of IARC carcinogens (groups 1, 2A and 2B) [11], it is less sensitive to dialkyl type N-nitroso compounds, silica and metal compounds, aromatic amines excluding

Table 14 Incosistent results between in vitro CA (negative) and in vivo MN (positive) tests. ID

Chemical

Chemical grouping

CAS

Carcinogenicity

in vitroCA

in vivoMN

C179 C185 C198 C217 C226 C240 C277 C305 C425 C645 C660 C691 C705 C706 C734 C738 C744 NC8 NC13 NC49 NC59 NC119 NC120 NC133 NC152 NC173 NC178

Chlorpromazine hydrochloride C.I. Direct black 38 C.I. Solvent yellow 14 D&C Red 9 Decabromodiphenyl oxide Diazepam 3,4-Dihydrocoumarin Dimethylvinyl chloride Isoprene Procarbazine HCl Reserpine 1,1,2,2-Tetrachloroethane Titanium dioxide Toluene Trimethylphosphate Tris(2-chloroethyl)phosphate Urethane dl-Amphetamine sulfate l-Ascorbic acid Deltamethrin 1,1-Dichloroethane Methyl parathion Monochloroacetic acid Oxytetracycline HCl Resorcinol Tolbutamide Triphenyltin hydroxide

Aromatic amine or amide Aromatic azo compound Aromatic azo compound Aromatic azo compound Polyhalogenated aromatic Aromatic amine or amide Glycidyl ether, amine, ester or amide Halogenated alkene Alkene Mono- or di-alkylhydrazine Phenol or precursor Gem-dihalide Alkali, alkali earth, metal salt Benzene Alkyl ester of phosphoric or phosphonic acid Alkylating agent Alkyl carbamate Amine Carboxylic acid Halogenated alkene Gem-dihalide Alkyl ester of phosphoric or phosphonic acid Alkylating agent Substituted vinyl ketone Resorcinol or precursor Aryl sulphonamide Alkali, alkali earth, metal salt

69-09-0 1937-37-7 842-07-9 5160-02-1 1163-19-5 439-14-5 119-84-6 513-37-1 78-79-5 366-70-1 50-55-5 79-34-5 13463-67-7 108-88-3 512-56-1 115-96-8 51-79-6 60-13-9 50-81-7 52918-63-5 75-34-3 298-00-0 79-11-8 2058-46-0 108-46-3 64-77-7 76-87-9

+ + + + + + + + + + + + + + + + + − − − − − − − − − −

− − − − − − − − − − − − − − −* − −* − − − − − − − −* − −

+a + + + + + + + + + +b + + + + + + + + + + + + + + + +

*: positive response at both >10 mM and 2 mg/mL. a: due to hypothermia. b: mice, due to hypotheramia; negative in rat.

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T. Morita et al. / Mutation Research 802 (2016) 1–29

Table 15 Summary of performance for individual assays or their combinations. Measure

Ames

in vitro CA

in vivo MN

TGR

Ames + in vitro Ames + in vivo CA MN

Ames + TGR

Ames + in vitro Ames + in vitro Ames + CA + in vivo CA + TGR in vivo MN + MN TGR

Ames + in vitro CA + in vivo MN + TGR

Sensitivity (%) Specificity (%) Concordance (%)

59.0 73.9 62.6

62.8 48.5 58.9

41.0 60.5 45.4

72.4 NC NC

74.3 37.5 NA

83.8 NC NC

80.8 21.3 NA

89.3 NC NA

68.7 45.3 NA

89.1 NC NA

87.5 NC NA

NC: Not calculated. NA: Not applicable.

90 80

Sensitivity (%) Specificity (%)

70 60 50 40 30 20 10 0

Fig. 1. Sensitivity and specificity for individual assays of Ames, in vitro CA, in vivo MN or TGR and their selected combinations.

aminobiphenyl and benzidine derivatives or heterocyclic amines, halogenated compounds or steroids and other hormones. As the CGX dataset includes many N-nitroso compounds, aromatic amines and halogenated compounds, it was to be expected that the in vivo MN test would have relatively low sensitivity when compared to previous investigations. Aromatic amines/halogenated or N-nitroso compounds would most likely produce effects in the liver or at a site of contact, respectively. Therefore, by knowledge of the chemical class it should be possible to determine whether a bone marrow MN test, a test for genotoxicity in liver or a site-of-contact assay would be most appropriate. Another important in vivo genotoxicity test, i.e., TGR test, also showed good sensitivity (72.4%). However, its specificity, either alone or in combination with other test(s), could not be calculated due to the limited number of non-carcinogens in the CGX dataset that have been tested in the TGR assay. The sensitivity and specificity of the TGR test were previously reported to be 78% and 69% [15] or 76% and 78% [16], respectively. Similar sensitivity was obtained in our investigation. Sensitivity and specificity of the combination of Ames + in vivo MN was 68.7% and 45.3%, respectively. These values are similar to those obtained with the combination of Ames + in vitro CA (sensitivity 74.3%, specificity 37.5%) (Table 15 and Fig. 1). Similar sensitivity

(62%) was also seen for the combination of Ames + in vivo MN, but the specificity was much higher (67%) [12]. Higher sensitivity (83.8%) was seen for the combination of Ames + TGR; the specificity could not be calculated as discussed above. Similar sensitivity (88%) was also seen for the combination of Ames + TGR [15,16]. When one considers option 2 of ICH where two different in vivo tests are required in addition to the Ames test, then the combination of Ames + in vivo MN + TGR showed an even higher sensitivity (87.5%). This is similar to the sensitivity achieved by the combination of Ames + in vitro CA + in vivo MN (80.8%) as would often be used in option 1 of ICH. The sensitivity of the combination of Ames + in vitro CA + TGR was also high (89.1%). Apparently, option 1 and option 2 from ICH gave comparable sensitivities. These data indicate that, if the second in vivo test is selected adequately, option 2 of ICH can work equally as well as option 1 in terms of detecting carcinogens with a high sensitivity. In those cases where 4 tests will be performed (e.g., Ames + in vitro CA + in vivo MN + TGR), usually where follow-up to an in vitro positive result is required, the sensitivity (89.3%) remained equally high. Good sensitivities were seen in the combinations of in vitro and in vivo tests, suggesting the usefulness of both in vivo MN and TGR tests as in vivo genotoxicity tests. The performances of Ames + in vitro CA and Ames + in vivo MN were also comparable, which is an important finding for the testing cosmetic ingredients in Europe [6,7]. Comparative analyses of in vitro and in vivo test results for the same or similar genotoxic endpoints were also investigated (i.e., Ames and TGR test, or in vitro CA and in vivo MN test). Concordance for the Ames and TGR tests was good (79.5%, Table 11). Fifteen chemicals were identified as exhibiting inconsistent results (Table 12), but no clear explanations can be given for these inconsistencies. With respect to oxazepam (ID C605), chronic administration was required to detect a mutagenic response in the TGR test, which might be due to oxidative damage as a possible primary mechanism for this chemical [16]. For carbon tetrachloride (ID C137) and chloroform (ID C160), positive results in the Ames test were obtained from gaseous exposure [18]. On the other hand, carbon tetrachloride was administered by gavage and chloroform was administered by inhalation in the TGR test [16]. Some factors may differently affect chemical accessibility, metabolism and toxicity in bacteria and intact mammals. They include DNA structure, xenobiotic metabolism, antioxidant activity, and DNA damage repair. High levels of reductive enzymes of bacteria, compared to mammalian cells, can activate efficiently nitro and azo compounds to electrophilic metabolites [60]. Therefore, differences in metabolism or exposure levels between bacteria and mammalian tissues in whole animals might be explain the different responses. Seven out of the above 15 chemicals were halogenated alkenes or aromatic compounds (Table 12). For these chemicals, consideration of the route of exposure and the impact on metabolism may be important. Concordance of the in vitro CA and in vivo MN tests was not so high (50.3%) based on a 300 chemical dataset (Table 13). Ninety one chemicals were positive in the in vitro CA, but negative in the in vivo MN test. Factors of the inconsistency will include detoxification/elimination, different metabolism or poor absorption in

T. Morita et al. / Mutation Research 802 (2016) 1–29

in vivo, or extreme high concentration in in vitro [61,62]. Noncarcinogens within these chemicals are called misleading or false positives. Several recommendations including use of p53 competent cells or accurate cytotoxicity measurement have been made to avoid generation of false positives [61,63]. Twenty seven chemicals (17 carcinogens and 10 non-carcinogens) showed the opposite responses, i.e., negative in the in vitro CA test, but positive in the in vivo MN test (Table 14). Benigni et al. reported that the concordance for these 2 tests was 60.2% (68/113) based on a 113 chemical dataset, and that the quite high number (n = 22) of in vivo MN positives that are negative in vitro CA may be due to aneugenic mechanisms of action that are not detected by the in vitro CA test [14]. However, this does not seem to be a logical explanation for the 27 inconsistent chemicals in Table 14, which do not seem to include many aneugens. The differences in concordance between the current analysis and Benigni et al. [14] may be due to the different chemical datasets used. The induction of MN in vivo by chlorpromazine (ID C179), reserpine (ID C660) and phenol (ID NC138) might be due to hypothermia [42,43,59]. For 3 of the chemicals, including trimethylphosphate (ID C734), urethane (ID C744) and resorcinol (ID NC152), the original CGX database expressed positive results in the in vitro CA test. However, as the positive responses were only seen at concentrations greater than both 10 mM and 2 mg/mL, they were assigned as negative in this analysis [28,29]. No specific chemical classes were identified amongst these inconsistent chemicals (Table 14), and, therefore, no clear explanations can be given to the other inconsistent in vitro CA and in vivo MN results. Some of the chemicals might induce MN via an indirect genotoxic mode of action (i.e., secondary effect) such as body temperature change (hypothermia or hyperthermia) or erythropoiesis

11

[52], or differences in metabolism including detoxification might be involved. 5. Conclusions The combination of Ames and the in vivo MN test showed similar values to Ames + in vitro CA test in the performance of the different batteries (sensitivity and specificity to rodent carcinogenicity). This indicates that, as long as the second in vivo test is appropriately selected, option 2 in the ICH S2(R1) recommendations can work equally as well as option 1 in detection of carcinogens. Usefulness of the strategy of only in vitro test battery by the EU cosmetics is also indicated. Higher sensitivities with the combined use of the TGR test indicate the usefulness of this test in the genotoxicity testing strategies by COM, ECHA, EFSA, FSC, or ICH in case of in vitro positive results. An assay which can detect genotoxic effects in the liver or at a site of contact will be important as a second in vivo test. The standard test battery (Ames + in vitro CA + in vivo MN) is effective to detect rodent carcinogens, but it should be noted that this battery has low specificity. Conflict of interest There are no conflicts of interests. Acknowledgements This work was supported by the Health and Labor Sciences Research Grants (H24-Food-General-011 and H27-ChemicalDesignation-005).

12

T. Morita et al. / Mutation Research 802 (2016) 1–29

Appendix A. : Genotoxicity test results with rodent carcinogens. ID

Chemical

CAS No.

Ames

in vitro CA

in vivo MN

in vivo MN Ref.

C1 C2 C3 C4 C5

Acetaldehyde Acetaldehyde methylformylhydrazone Acetamide Acetaminophen Acetone[4-(5-nitro-2-furyl)-2thiazolyl]hydrazone Acetoxime N-Acetoxy-2-acetylaminofluorene 1 -Acetoxysafrole 4-Acetylaminobiphenyl 2-Acetylaminofluorene N -Acetyl-4-(hydroxymethyl)phenylhydrazine 1-Acetyl-2-isonicotinoylhydrazine 1-Acetyl-2-phenylhydrazine Acifluorfen Acronycine Acrylamide Acrylonitrile Actinomycin D Aflatoxicol Aflatoxin B1 Aflatoxin, crude Aldrin Allyl glycidyl ether Allyl isothiocyanate Allyl isovalerate 1-Allyl-1-nitrosourea Allylhydrazine HCl 2-Aminoanthracene 2-Aminoanthraquinone 4-Aminoazobenzene 4-Aminobiphenyl 4-Aminobiphenyl HCl 1-Amino-2,4-dibromoanthraquinone 2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) 2-Amino-3,8-dimethylimidazo[4,5f]quinoxaline (MeIQx) 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole acetate (Trp-P-1 acetate) 2-Aminodiphenylene oxide 2-Aminodipyrido[1,2-a:3 ,2 -d]imidazole (Glu-P-2) 3-Amino-4-ethoxyacetanilide 3-Amino-9-ethylcarbazole HCl 3-Amino-9-ethylcarbazole mixture 2-Aminofluorene 2-Amino-6-methyldipyridol[1,2-a:3 ,2 d]imidazole (Glu-P-1) 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) 2-Amino-3-methylimidazo[4,5-f]quinoline HCl (IQ.HCl) 2-Amino-1-methyl-6-phenylimidazo-[4,5b]pyridine hydrochloride (PhiP.HCl) 3-Amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2 acetate) 2-Amino-5-(5-nitro-2-furyl)-1,3,4-oxadiazole 2-Amino-5-(5-nitro-2-furyl)-1,3,4-thiadiazole 2-Amino-4-(5-nitro-2-furyl)thiazole trans-5-Amino-3[2-(5-nitro-2-furyl)vinyl]1,2,4-oxadiazole 2-Amino-4-nitrophenol 2-Amino-5-nitrophenol 4-Amino-2-nitrophenol 2-Amino-4-(p-nitrophenyl)thiazole 2-Amino-5-nitrothiazole 2-Amino-9H-pyrido(2,3-b)indole (A-alpha-C)

75-07-0 16568-02-8 60-35-5 103-90-2 18523-69-8

− − − −

+

+

[11]

+

− −

127-06-0 6098-44-8 34627-78-6 4075-79-0 53-96-3 65734-38-5 1078-38-2 114-83-0 50594-66-6 7008-42-6 79-06-1 107-13-1 50-76-0 29611-03-8 1162-65-8 – 309-00-2 106-92-3 57-06-7 2835-39-4 760-56-5 52207-83-7 613-13-8 117-79-3 60-09-3 92-67-1 2113-61-3 81-49-2 77094-11-2

− + +

+

+

+

E + − + +

77500-04-0

+

68808-54-8

+

+

3693-22-9 67730-10-3

+

+

17026-81-2 6109-97-3 Mixture 153-78-6 67730-11-4

+ + + + +

− −

76180-96-6

+

–

+

105650-23-5

+

+

72254-58-1

+

+

C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33 C34 C35

C36

C37 C38 C39 C40 C41 C42 C43

C44 C45 C46

C47

C48 C49 C50 C51 C52 C53 C54 C55 C56 C57

3775-55-1 712-68-5 38514-71-5 28754-68-9 99-57-0 121-88-0 119-34-6 2104-09-8 121-66-4 26148-68-5

TGR

TGR Ref.

[11]∼ [64]

−

[16]∼

+

[30]

+

[16]

+ + +

+ −a +

[65,66] [11,41] [50]

+ −ˆ

[16,67] [16]∼

+

+

[30]

+

[16]

− + E −

+ + + +

− + −

[68] [69] [34]

+ + + + + + +

E + + +

[11] [11] [70]

+

[16]

+

[16]

+

+ − +

+

[71]

+

[16]

−

[72]

+

[16]

+

[16]

+

[16]

+

[16]

+

+

[73]

−

[50]

+

+ + +

+ + +

+ +

+ +

T. Morita et al. / Mutation Research 802 (2016) 1–29 C58 C59 C60 C61 C62 C63 C64 C65 C66 C67 C68 C69 C70 C71 C72 C73 C74 C75 C76 C77 C78 C79 C80 C81 C82 C83 C84 C85 C86 C87 C88 C89 C90 C91 C92 C93 C94 C95 C96 C97 C98 C99 C100 C101 C102 C103 C104 C105 C106 C107 C108 C109 C110 C111 C112 C113 C114 C115 C116 C117 C118 C119 C120 C121 C122 C123 C124 C125 C126 C127 C128 C129 C130

3-Amino-1,2,4-triazole (Amitrole) 11-Aminoundecanoic acid 1-Amyl-1-nitrosourea Amylopectin sulphate Aniline HCl o-Anisidine HCl Aramite Arecoline HCl Aristolochic acid Aroclor 1254 Aroclor 1260 Asbestos Atrazine Auramine O 5-Azacytidine Azaserine Azathioprine Azobenzene Azoxymethane 1-Azoxypropane 2-Azoxypropane Barbital, sodium Bemitradine Benomyl Benzaldehyde Benz[a]anthracene Benzene Benzidine Benzidine 2HCl Benzofuran Benzo[a]pyrene 1,4-Benzoquinone Benzotrichloride Benzoyl hydrazine Benzyl acetate Benzyl chloride o-Benzyl-p-chlorophenol Benzylhydrazine 2HCl 2-Biphenylamine HCl 2,2-Bis(bromomethyl)-1,3-propanediol, technical grade Bis(2-chloro-1-methylethyl)ether, technical grade Bis-2-chloroethylether Bis-1,2-(chloromethoxy)ethane Bis-1,4-(chloromethoxy)-p-xylene Bis-(chloromethyl)ether Bis(2,3-dibromopropyl)phosphate, magnesium salt Bis(dimethylamino)benzophenone (Michler’s ketone) 4-Bis(2-hydroxyethyl)amino-2-(5-nitro-2Thienyl)quinazoline Bis-2-hydroxyethyldithiocarbamic acid, potassium Bromate, potassium Bromocriptine mesylate Bromodichloromethane Bromoethane 2-Bromoethanol 7-Bromomethyl-12-methylbenz[a]anthracene Budesonide 1,3-Butadiene tert-Butyl alcohol Butylated hydroxyanisole Butylated hydroxytoluene Butylbenzyl phthalate N-n-Butyl-N-formylhydrazine N-Butylhydrazine HCl N-Butyl-N-(4-hydroxybutyl)nitrosamine N-n-Butyl-N-nitrosourea beta-Butyrolactone Cadmium chloride Cadmium sulphate Caffeic acid Calciferol Calcium chromate Calcium valproate Capsaicin

61-82-5 2432-99-7 10589-74-9 9047-13-6 142-04-1 134-29-2 140-57-8 61-94-9 313-67-7 27323-18-8 11096-82-5 12001-29-5 1912-24-9 2465-27-2 320-67-2 115-02-6 446-86-6 103-33-3 25843-45-2 17697-55-1 17697-53-9 144-02-5 88133-11-3 17804-35-2 100-52-7 56-55-3 71-43-2 92-87-5 531-85-1 271-89-6 50-32-8 106-51-4 98-07-7 613-94-5 140-11-4 100-44-7 120-32-1 20570-96-1 2185-92-4 3296-90-0

13

− −

− − +

− −

[74]∼ [34]

− +

+

+ −

[75] [50]

+** −

+**

+ −

[30] [76]

− − + + + + + +

+ + + +

− +

[11]∼ [77]

+ + + +

[50] [11] [30] [26]

+

[78]

+ + + +

[11] [30] [30] [50]

+ +

[30] [79]

+ E

+

[16]

+

[16]

+

[16]

+

[16]

+ − − − + − + + − + − +

+ + + + − +

− + −

− + −

− −

[34] [30]∼

+ +

+ +

− E

[34] [50]

108-60-1

+

+

111-44-4 13483-18-6 56894-91-8 542-88-1 36711-31-6

+

+

+

90-94-8

+

E

33372-39-3

TC

+ − −

[30] [80] [50,81]

+

[16]

+ − − − −

[30] [36,50] [50] [50] [50]

+

[16]

+ +

[11] [82]

−

[30]∼

−

[83]

−

[22,23]

23746-34-1 7758-01-2 22260-51-1 75-27-4 74-96-4 540-51-2 16238-56-5 51333-22-3 106-99-0 75-65-0 25013-16-5 128-37-0 85-68-7 16120-70-0 56795-65-4 3817-11-6 869-01-2 3068-88-0 10108-64-2 10124-36-4 331-39-5 50-14-6 13765-19-0 33433-82-8 404-86-4

+

+

− + + +

+ −

+ − − − −

+ + + + − −

− + − −

+

+

+ + + TC +

+

−**

14 C131 C132 C133 C134 C135 C136 C137 C138 C139 C140 C141 C142 C143 C144 C145 C146 C147 C148 C149 C150 C151 C152 C153 C154 C155 C156

C157 C158 C159 C160 C161 C162 C163 C164 C165 C166 C167 C168 C169 C170 C171 C172 C173 C174 C175 C176

C177 C178 C179 C180 C181 C182 C183 C184 C185 C186 C187 C188 C189 C190 C191 C192 C193 C194 C195 C196 C197 C198 C199

T. Morita et al. / Mutation Research 802 (2016) 1–29 Captafol Captan Carbamyl hydrazine HCl 1-Carbamyl-2-phenylhydrazine Carbaryl Carbazole Carbon tetrachloride Carboxymethylnitrosourea Carrageenan, acid-degraded Catechol Chloral hydrate Chloramben Chlorambucil Chlordane, technical grade Chlordane, analytical grade Chlorendic acid Chlorinated paraffins: C12 Chlorinated paraffins: C23 Chlornaphazine Chloroacetaldehyde 4-Chloro-4 -aminodiphenylether p-Chloroaniline HCl Chlorobenzene Chlorobenzilate Chlorodibromomethane 2-Chloro-5-(3,5dimethylpiperidinosulphonyl)benzoic acid Chloroethane 1-Chloroethylnitroso-3-(2hydroxypropyl)urea Chlorofluoromethane Chloroform Chloromethyl methyl ether 3-Chloro-2-methylpropene, technical grade 3-(Chloromethyl)pyridine HCl 1-Chloro-2-nitrobenzene 1-Chloro-4-nitrobenzene 3-(p-Chlorophenyl)-1,1-dimethylurea (AKA monuron) 4-Chloro-m-phenylenediamine 4-Chloro-o-phenylenediamine 1-(4-Chlorophenyl)-1-phenyl-2-propynyl carbamate 2-Chloropropanal 1-Chloropropene Chlorothalonil 5-Chloro-o-toluidine 4-Chloro-o-toluidine HCl 2-Chloro-1,1,1-trifluoroethane [4-Chloro-6-(2,3-xylidino)-2pyrimidinylthio]acetic acid (AKA Wyeth 14,643) 4-Chloro-6-(2,3-xylidino)-2-pyrimidinylthio (N-beta-hydroxyethyl)acetamide Chlorozotocin Chlorpromazine hydrochloride Chrysazin C.I. Acid orange 3 C.I. Acid red 26 (AKA D&C Red 5 and Ponceau MX) C.I. Acid red 114 C.I. Basic red 9 (pararosaniline HCl) C.I. Direct black 38 C.I. Direct blue 6 C.I. Direct blue 14 (Trypan blue) C.I. Direct blue 15 C.I. Direct blue 218 C.I. Direct brown 95 C.I. Disperse blue 1 C.I. Disperse orange 2 (1-amino-2-methyl-anthrquinone) C.I. Disperse yellow 3 Cinnamyl anthranilate C.I. Pigment red 3 Ciprofibrate C.I. Solvent yellow 3 (o-Aminoazotoluene) C.I. Solvent yellow 14 Citrinin

2425-06-1 133-06-2 563-41-7 103-03-7 63-25-2 86-74-8 56-23-5 60391-92-6 9000-07-1 120-80-9 302-17-0 133-90-4 305-03-3 12789-03-6 57-74-9 115-28-6 108171-26-2 63449-39-8 494-03-1 107-20-0 101-79-1 20265-96-7 108-90-7 510-15-6 124-48-1 37087-94-8

+ + −

+ +

+ − +** − − + + + + − − − − + +

75-00-3 –

+

593-70-4 67-66-3 107-30-2 563-47-3 6959-48-4 88-73-3 100-00-5 150-68-5 5131-60-2 95-83-0 10473-70-8 683-50-1 590-21-6 1897-45-6 95-79-4 3165-93-3 75-88-7 50892-23-4

+ − − +

+ −

[30] [84]

+

+

[68]

− +

−

[11]

+ + + +

+ +

[85] [50]

+

[11]

+

[86]

+ −

[50] [34]

−

[50]

−

[87]

E E − −

[88] [11] [34] [89]

+ +

[90] [34]

+

[11]

−

[91]

−

[16]∼

+

[16]

−

[16]∼

+

[16]

+

[16]

+

[16]

− + +

+ − +

+** − − + + + −

−

+ +

+ +

− − − −

+ − E

+ + E + +

65089-17-0 54749-90-5 69-09-0 81-55-0 6373-74-6 3761-53-3

+ − + + E

6459-94-5 569-61-9 1937-37-7 2602-46-2 72-57-1 2429-74-5 28407-37-6 16071-86-6 2475-45-8 82-28-0

+ + + + + + − + + +

− − − −

2832-40-8 87-29-6 2425-85-6 52214-84-3 97-56-3 842-07-9 518-75-2

+ − +

− − − +

+ + −

− + +

− − − + +

−

+b

[42]

− +

[50] [92]

−

[11]∼

−

[50]

− −

[34] [34]

+c +

[11] [35]

T. Morita et al. / Mutation Research 802 (2016) 1–29 C200 C201 C202 C203 C204 C205 C206 C207 C208 C209 C210 C211 C212 C213 C214 C215 C216 C217 C218 C219 C220 C221 C222 C223 C224 C225 C226 C227 C228 C229 C230 C231 C232 C233 C234 C235 C236 C237 C238 C239 C240 C241 C242 C243 C244 C245 C246 C247 C248 C249 C250 C251 C252 C253 C254 C255 C256 C257 C258 C259 C260 C261 C262 C263 C264 C265 C266 C267 C268 C269 C270 C271 C272 C273 C274 C275 C276

C.I. Vat yellow 4 Clivorine Clofibrate Clophen A 30 Cobalt Sulfate heptahydrate Compound LY171883 Coumarin m-Cresidine p-Cresidine Crotonaldehyde Cupferron Cyclamate, sodium Cyclochlorotine Cyclopenta[c,d]pyrene Cyclophosphamide monophydrate Cyclosporin A Cytembena D&C Red 9 D&C Yellow 11 (AKA C.I. Solvent Yellow 33) p,p -DDD p,p -DDE DDT Dacarbazine Daminozide Danthron Dapsone Decabromodiphenyl oxide Dehydroepiandrosterone Dehydroepiandrosterone acetate Dextran sulphate sodium N-1-Diacetamidofluorene Diallate 1,1-Diallylhydrazine 1,2-Diallylhydrazine 2HCl Diallylnitrosamine 2,4-Diaminoanisole sulphate 4,6-Diamino-2-(5-nitro-2-furyl)-s-triazine 2,4-Diaminophenol 2HCl 2,4-Diaminotoluene 2,4-Diaminotoluene 2HCl Diazepam 3-Diazotyramine HCl Dibenz[a,h]anthracene 3-Dibenzofuranamine 1,2-Dibromo-3-chloropropane Dibromodulcitol 1,2-Dibromoethane Dibromomannitol 1,1-Di-N-butylhydrazine 1,2-Di-N-butylhydrazine HCl 1,3-Dibutyl-1-nitrosourea Dichloroacetic acid Dichloroacetylene 1,4-Dichlorobenzene 3,3 -Dichlorobenzidine trans-1,4-Dichlorobutene-2 3,5-Dichloro(N-1,1-dimethyl-2propynyl)benzamide 1,2-Dichloroethane Dichloromethane 2,6-Dichloro-p-phenylenediamine 1,2-Dichloropropane 1,3-Dichloropropene (AKA Telone II) Dichlorvos Dicofol Dieldrin Diethanolamine Diethylacetamide Diethylene glycol Di(2-ethylhexyl)adipate Di(2-ethylhexyl)phthalate N,N-diethyl-4-(4 -[pyridyl-1 oxide]azo)aniline Diethylstilbestrol Diethylstilbestrol dipropanate N,N -Diethyl-2-thiourea 1,2-Diformylhydrazine Diftalone Diglycidyl resorcinol ether, technical grade

128-66-5 33979-15-6 637-07-0 55600-34-5 10026-24-1 88107-10-2 91-64-5 102-50-1 120-71-8 123-73-9 135-20-6 139-05-9 12663-46-6 27208-37-3 6055-19-2 59865-13-3 21739-91-3 5160-02-1 8003-22-3 72-54-8 72-55-9 50-29-3 4342-03-4 1596-84-5 117-10-2 80-08-0 1163-19-5 53-43-0 853-23-6 9011-18-1 63019-65-8 2303-16-4 5164-11-4 26072-78-6 16338-97-9 39156-41-7 720-69-4 137-09-7 95-80-7 636-23-7 439-14-5 – 53-70-3 4106-66-5 96-12-8 10318-26-0 106-93-4 488-41-5 7422-80-2 78776-28-0 56654-52-5 79-43-6 7572-29-4 106-46-7 91-94−1 110-57-6 23950-58-5

15

−

−

−

+

−

[45]

+ + E

−

[50]

−

−

[16]∼

[11]∼

+

[16]

+ − − + −

[37] [30]∼ [34] [93] [36]

+

[16]

+ E −

[11] [50] [50]

+

[50]

− +

−

[11]∼

+

[16]

−

+

[94]

+

[11]

+ + + + + +

+ + − + + + − − − + − + − − −

+

+ + − + + E** − + E −

+

+ + + + + + −

+

+ + + + +

+

+

[11]

+ˆ

[16]

+ +

E +

+[36];-[44,45] [50]

+ˆ

[16]

+ +

+ +

−

[50]

+

[16]

− + +

−

− E

[11,36,50] [11]

107-06-2 75-09-2 609-20-1 78-87-5 542-75-6 62-73-7 115-32-2 60-57-1 111-42-2 685-91-6 111-46-6 103-23-1 117-81-7 7347-49-1

+ + + + + + − − −

+ + + + − + − + −

− − − − − −

[11,36] [11]∼ [34] [95] [11,34] [50]

-ˆ

[16]∼

+ −

[96] [36]

− − −

E −

− −

[34] [97]

−

[16]∼

56-53-1 130-80-3 105-55-5 628-36-4 21626-89-1 101-90-6

− − −

+

E −

[11,50] [98]

+

+

−

[34]

−

16 C277 C278 C279 C280 C281 C282 C283 C284 C285 C286 C287 C288 C289 C290 C291 C292 C293 C294 C295 C296 C297 C298 C299 C300 C301 C302 C303 C304 C305 C306 C307 C308 C309 C310 C311 C312 C313 C314 C315 C316 C317 C318 C319 C320 C321 C322 C323 C324 C325 C326 C327 C328 C329 C330 C331 C332 C333 C334 C335 C336 C337 C338 C339 C340 C341 C342 C343 C344 C345 C346 C347 C348 C349 C350

T. Morita et al. / Mutation Research 802 (2016) 1–29 3,4-Dihydrocoumarin 1,2-Dihydro-2-(5-nitro-2-Thienyl)quinazolin4(3H)-one 3,6-Dihydro-2-nitroso-2H-1,2-oxazine Dihydrosafrole 1,2-Dihydro-2,2,4-trimethylquinoline Dimethoxane 2,5-Dimethoxy-4 -aminostilbene 3,3 -Dimethoxybenzidine-4,4 -diisocyante 3,3 -Dimethoxybenzidine 2HCl 5,6-Dimethoxysterigmatocystin N,N-Dimethyl-4-aminoazobenzene 6-Dimethylamino-4,4-diphenyl-3-heptanol acetate HCl Dimethylaminoethylnitrosoethylurea, nitrite salt trans-2-[(Dimethylamino)methylimino]-5[2-(5-nitro-2-furyl)vinyl]-1,3,4-oxadiazole N,N-Dimethylaniline 7,12-Dimethylbenz[a]anthracene 3,3 -Dimethylbenzidine 3,3 -Dimethylbenzidine 2HCl Dimethylcarbamoyl chloride 1,1-Dimethylhydrazine 1,2-Dimethylhydrazine 2HCl 2-(2,2-Dimethylhydrazino)-4-(5-nitro-2furyl)thiazole Dimethyl hydrogen phosphite Dimethyl methylphosphonate Dimethyl morpholinophosporamidate Dimethylnitramine 4,6-Dimethyl-2-(5-nitro-2-furyl)pyrimidine 1,2-Dimethyl-5-nitroimidazole Dimethylvinyl chloride Dinitrosohomopiperazine Di-(N-nitroso)-perhydropyrimidine Dinitrosopiperazine 2,4-Dinitrotoluene 2,6-Dinitrotoluene Dinitrotoluene, technical grade 1,4-Dioxane Dipentylnitrosamine 5,5-Diphenylhydantoin Dipyrone Doxylamine succinate alpha-Ecdysone Enovid Epichlorhydrin 1,2-Epoxybutane 17-B-Estradiol Estradiol mustard Estragole Ethinyl estradiol Ethionamide Ethionine DL-Ethionine o-Ethoxybenzamide Ethyl acrylate Ethyl alcohol Z-Ethyl-O,N,N-azoxyethane Z-Ethyl-O,N,N-azoxymethane Ethylbenzene Ethylene glycol monobutyl ether Ethylene imine (AKA Aziridine) Ethylene oxide N,N -Ethylenethiourea N-Ethyl-N-formylhydrazine Ethylhydrazine HCl Ethyl methanesulphonate N-Ethyl-N -nitro-N-nitrosoguanidine Ethylnitrosocyanamide 1-Ethylnitroso-3-(2-hydroxyethyl)-urea 1-Ethylnitroso-3-(2-oxopropyl)-urea 1-Ethyl-1-nitrosourea 4-Ethylsulphonylnaphthalene-1-sulfonamide FD&C Green 1 FD&C Green 2 FD&C Red 1 (Ponceau 3R) FD&C Red 2

119-84-6 33389-33-2 3276-41-3 94-58-6 147-47-7 828-00-2 5803-51-0 91-93-0 20325-40-0 65176-75-2 60-11-7 43033-72-3

− +

− − +

−

+

[36]

− +

−

[36]

−d

[11]∼

−

[11]∼

+ −

[30] [11]∼

− + +

[11] [11] [11]

+ +

+

+

+

142713-77-7 55738-54-0

+

121-69-7 57-97-6 119-93-7 612-82-8 79-44-7 57-14-7 306-37-6 26049-69-4

− + + + + + + +

+ + + + +

868-85-9 756-79-6 597-25-1 4164-28-7 59-35-8 551-92-8 513-37-1 55557-00-1 15973-99-6 140-79-4 121-14-2 606-20-2 25321-14-6 123-91-1 13256-06-9 57-41-0 68-89-3 562-10-7 3604-87-3 8015-30-3 106-89-8 106-88-7 50-28-2 22966-79-6 140-67-0 57-63-6 536-33-4 13073-35-3 67-21-0 938-73-8 140-88-5 64-17-5 16301-26-1 57497-29-7 100-41-4 111-76-2 151-56-4 75-21-8 96-45-7 74920-78-8 18413-14-4 62-50-0 63885-23-4 38434-77-4 – 110559-84-7 759-73-9 842-00-2 4680-78-8 5141-20-8 3564-09-8 915-67-3

+ − − +

+ TC E

+

[34]

−

+

[50]

−

− −

[30]∼ [45,99]

−

−

[11]∼

−

E

−

[50]

−

−

−

[100]

+ + −

+ + −

− − −

[11]∼ [50] [50,101]

− −

[50] [50]

− −

[11] [102]

+ + + + + + + −

− − − − − − − −

− + − + +

+

[16]

−

[16]∼

− − + + E

− −

− −

[36] [36]

+ −

+ −

[30] [11]∼

+

[16]

+ + +

+ +

+

[50]

+ˆ

[16]

+

+

+

[50]

+

[16]

−

[11]

− − −

T. Morita et al. / Mutation Research 802 (2016) 1–29 C351 C352 C353 C354 C355 C356 C357 C358 C359 C360 C361 C362 C363 C364 C365 C366 C367 C368 C369 C370 C371 C372 C373 C374 C375 C376 C377 C378 C379 C380 C381 C382 C383 C384 C385 C386 C387 C388 C389 C390 C391 C392 C393 C394 C395 C396 C397 C398 C399 C400 C401 C402 C403 C404 C405 C406 C407 C408 C409 C410 C411 C412 C413 C414 C415 C416 C417 C418 C419 C420 C421 C422 C423 C424

FD&C Red 4 (AKA C.I. Food Red 1) FD&C Violet 1 (AKA Benzyl Violet 4B) Finasteride Fluconazole N-(2-Fluorenyl)-2,2,2-trifluoroacetamide 4 -Fluoro-4-aminobiphenyl N-4-(4 -Fluorobiphenyl)acetamide 2-Fluoroethyl-nitrosourea 5-Fluorouracil Fluvastatin Formaldehyde Formic acid 2-(4-methyl-2-thiazolyl)hydrazide Formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl]hydrazide Formylhydrazine Fosetyl AI Fumonisin B1 Furan Furfural Furfuryl Alcohol Furosemide Furylfuramide (AF-2) Gallium arsenide Gentian violet (AKA Hexamethyl-p-rosaniline chloride) N2-gamma-Glutamyl-p-hydrazinobenzoic acid Glycidaldehyde Glycidol Griseofulvin Haloperidol HC Blue 1 (impure and purified) HCDD mixture Hematoxylin Heptachlor Hexachlorobenzene Hexachlorobutadiene Hexachlorocyclohexane, technical grade alpha-1,2,3,4,5,6-Hexachlorocyclohexane beta-1,2,3,4,5,6-Hexachlorocyclohexane gamma-1,2,3,4,5,6-Hexachlorocyclohexane (AKA Lindane) Hexachloroethane Hexamethylmelamine Hexanal methylformylhydrazone Hexanamide N-Hexylnitrosourea Hydrazine Hydrazine sulphate 2-Hydrazino-4-(p-aminophenyl)thiazole 2-Hydrazino-4-(5-nitro-2-furyl)thiazole 2-Hydrazino-4-(p-nitrophenyl)thiazole p-Hydrazinobenzoic acid HCl Hydrazobenzene Hydrogen Peroxide Hydroquinone N-Hydroxy-2-acetylaminofluorene 4-Hydroxyaminoquinoline-N-oxide 1-Hydroxyanthraquinone 3-Hydroxy-p-butyrophenetidide 1 -Hydroxyestragole 4-(2-Hydroxyethylamino)-2-(5-nitro-2Thienyl)quinazoline 1-(2-Hydroxyethyl)-3-[(5-nitrofurfurylidene) amino]-2-imidazolidinone 1-(2-Hydroxyethyl)-nitroso-3-ethylurea 1-(2-Hydroxyethyl)-1-nitrosourea 2-Hydroxyethylhydrazine 1-(3-Hydroxypropyl)-1-nitrosourea 1 -Hydroxysafrole ICR 170 ICRF 159 Indolidan Iodinated glycerol Isobutyl nitrite Isomazole Isoniazid Isonicotinic acid vannillylidenehydrazide Isophorone Isophosphamide

4548-53-2 1694-09-3 98319-26-7 86386-73-4 363-17-7 324-93-6 398-32-3 69112-98-7 51-21-8 93957-54-1 50-00-0 32852-21-4 3570-75-0 624-84-0 39148-24-8 116355-83-0 110-00-9 98-01-1 98-00-0 54-31-9 3688-53-7 1303-00-0 548-62-9 – 765-34-4 556-52-5 126-07-8 52-86-8 2784-94-3 Mixture 517-28-2 76-44-8 118-74-1 87-68-3 608-73-1 319-84-6 319-85-7 58-89-9

17

− +

− − +

+ − +

+ − −

[30] [103] [11]∼

+ + + E + +

+ E

[104] [50]

− − + −

[50] [50] [11] [36]

+ − +e −

[50] [11]∼ [46] [105]

+

− − − E − − + − E

+ + −

−

+

+ + + +

− − − −

TC + TC −

− −

−

67-72−1 531-18-0 57590-22-4 628-02-4 18774-85-1 302-01-2 10034-93-2 26049-71-8 26049-68-3 26049-70-7 24589-77-3 122-66-7 7722-84-1 123-31-9 53-95-2 4637-56-3 129-43-1 1083-57-4 51410-44-7 33389-36-5

− −

−

− + + +

−*

+ + − + +

+ + + +

5036-03-3

+

– 13743-07-2 109-84-2 71752-70-0 5208-87-7 146-59-8 21416-87-5 100643-96-7 5634-39-9 542-56-3 86315-52-8 54-85-3 149-17-7 78-59-1 3778-73-2

−

[11]∼

+

[11]

+

− +

[106] [50]

+

[24]

− +

[50] [36]

−

[108]

− +

+ + − + −** − + + − + − +

E + + +

−

[16]∼

+

[16]

−

[16]∼

− +

[107] [16]

18 C425 C426 C427 C428 C429 C430 C431 C432 C433 C434 C435 C436 C437 C438 C439 C440 C441 C442 C443 C444 C445 C446 C447 C448 C449 C450 C451 C452 C453 C454 C455 C456 C457 C458 C459

C460 C461 C462 C463 C464 C465 C466 C467 C468 C469 C470 C471 C472 C473 C474 C475 C476 C477 C478

C479 C480 C481 C482 C483 C484 C485 C486 C487 C488 C489 C490 C491 C492 C493 C494 C495

T. Morita et al. / Mutation Research 802 (2016) 1–29 Isoprene Kepone (AKA Chlordecone) Lasiocarpine Lead Acetate Lead acetate, basic Leupeptin D-limonene Luteoskyrin Malonaldehyde sodium salt Manganese ethylenebisthiocarbamate MeA-alpha-C acetate Melamine Melphalan 2-Mercaptobenzothiazole Mercuric chloride Mercurymethylchloride Metepa Methapyrilene hydrochloride Methidathion Methimazole 3-Methoxy-4-aminoazobenzene 2-Methoxy-3-aminodibenzofuran 3-Methoxycatechol 4-Methoxyphenol (AKA Hydroquinone monomethyl ether) 8-Methoxypsoralen Z-Methyl-O,N,N-azoxyethane Methylazoxymethanol acetate alpha-Methylbenzyl alcohol 3-Methylbutanal methylformylhydrazone Methyl tert-butyl ether Methyl carbamate 4-Methylcatechol 3-Methylcholanthrene Methyl clofenapate 1-Methyl-1,4-dihydro-7-[2-(5nitrofuryl)vinyl]-4oxo-1,8-naphthyridine-3-carboxylate, potassium 3 -Methyl-4-dimethylaminoazobenzene N-Methyl-N,4-dinitrosoaniline 4,4 -Methylenebis(2-chloroaniline) 4,4 -Methylene-bis(2-chloroaniline) 2HCl 4,4 -Methylenebis(N,N-dimethylaniline) 4,4 -Methylene-bis(2-methylaniline) 4,4 -Methylenedianiline 2HCl Methyleugenol N-Methyl-N-formylhydrazine Methylhydrazine Methylhydrazine sulphate Methyl iodide Methyl methanesulphonate Methylnitramine 2-Methyl-1-nitroanthraquinone 4-Methyl-1-[(5-nitrofurfurylidene)amino]-2imidazoline N-Methyl-N’-nitro-N-nitrosoguanidine 4-(Methylnitrosamino)-1-(3-pyrridyl)-1butanol 4-(Methylnitrosamino)-1-(3-pyrridyl)-1(butanone) (NNK) 4-(4-N-Methyl-N-nitrosaminostyryl)quinoline (N-6)-(Methylnitroso)adenine (N-6)-(Methylnitroso)adenosine N-Methyl-N-nitrosobenzamide N-(N-Methyl-N-nitrosocarbamoyl)-L-ornithine Methylnitrosocyanamide R(−)-2-Methyl-N-nitrosopiperidine S(+)-2-Methyl-N-nitrosopiperidine N-Methylolacrylamide Methylphenidate HCl Metronidazole Mirex Mirex, photoMitomycin C Molybdenum trioxide Monoacetyl hydrazine Monocrotaline

78-79-5 143-50-0 303-34-4 301-04-2 1335-32-6 24365-47-7 5989-27-5 21884-44-6 24382-04-5 12427-38-2 – 108-78-1 148-82-3 149-30-4 7487-94-7 115-09-3 57-39-6 135-23-9 950-37-8 60-56-0 3544-23-8 5834-17-3 934-00-9 150-76-5

− − + −

− − + +

− − −

−

+ − + − − −

+

[36]

+ −

[50] [11]

−

[16]∼

+ˆ −

[16] [16]∼

− + − + + + +

− + − +f

[34] [11] [50] [47,48]

+

[109]

− −

[110] [111]

E

[50]

− − − + −

[50] [50] [50] [112] [25]

−

[11,35]∼

+ −

+g −

[34] [36]

+

+

[30]

+

[16]

E − − +

+

−

+

298-81-7 57497-34-4 592-62-1 98-85-1 57590-21-3 1634-04-4 598-55-0 452-86-8 56-49-5 21340-68-1 –

+

+

+ −

+ +

− − − + −**

− −

55-80−1 99-80-9 101-14-4 64049-29-2 101-61-1 838-88-0 13552-44-8 93-15-2 758-17-8 60-34-4 302-15-8 74-88-4 66-27-3 598-57-2 129-15-7 21638-36-8

+

+

+ + + + + −

+

+ +

+

70-25-7 76014-81-8

+

+

+

[30]

+

[16]

64091-91-4

+**

+**

E

+[26,51];−[50]

+

[16]

16699-10-8 21928-82-5 41286-73-1 63412-06-6 63642-17-1 33868-17-6 14026-03-0 36702-44-0 924-42-5 298-59-9 443-48-1 2385-85-5 39801-14-4 50-07-7 1313-27-5 1068-57-1 315-22-0

+

+ −**

−

− + +

+ +

+

− − + −

+ + + −

− − −

[113] [50] [11]∼

−ˆ

[16]∼

+ −

+ −

+

[30]

+ˆ

[16]

−

+

+ +

[114] [11]

T. Morita et al. / Mutation Research 802 (2016) 1–29 C496

C497 C498 C499 C500 C501 C502 C503 C504 C505 C506 C507 C508 C509 C510 C511 C512 C513 C514 C515 C516 C517 C518 C519 C520 C521 C522 C523 C524 C525 C526 C527 C528 C529 C530 C531 C532 C533 C534 C535 C536 C537 C538 C539 C540 C541 C542 C543 C544 C545 C546 C547 C548 C549 C550 C551 C552 C553 C554 C555 C556 C557 C558 C559 C560

L-5-Morpholinomethyl-3-[(5nitrofurfurylidene) amino]-2-oxazolidinone HCl 4-Morpholino-2-(5-nitro-2Thienyl)quinazoline Nafenopin Nalidixic acid Naphthalene 1,5-Naphthalenediamine 2-Naphthylamine Nickel sulfate heptahydrate Nicotinic acid hydrazide Nithiazide Nitrilotriacetic acid Nitrilotriacetic acid, trisodium salt Nitrilotriacetic acid, trisodium salt, monohydrate Nitrite, sodium 5-Nitroacenaphthene 3-Nitro-p-acetophenetide 5-Nitro-o-anisidine o-Nitroanisole Nitrobenzene 6-Nitrobenzimidazole p-Nitrobenzoic acid 4-Nitrobiphenyl Nitrofen 5-Nitro-2-furaldehyde semicarbazone (AKA Nitrofurazone) 1-[(5-Nitrofurfurylidene)amino]hydantoin (AKA Nitrofurantoin) 1-[(5-Nitrofurfurylidene)amino]-2imidazolidinone 3-(5-Nitro-2-furyl)-imidazo(1,2alpha)pyridine 5-(5-Nitro-2-furyl)-1,3,4-oxadiazole-2-ol N-{[3-(5-Nitro-2-furyl)-1,2,4-oxadiazole5-yl]-methyl}acetamide N-[5-(5-Nitro-2-furyl)-1,3,4-thiadazol-2yl]acetamide 4-(5-Nitro-2-furyl)thiazole N-[4-(5-Nitro-2-furyl)-2-thiazolyl]acetamide N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide N,N -[6-(5-Nitro-2-furyl)-s-triazine-2,4-diyl] bisacetamide Nitrogen mustard Nitrogen mustard N-oxide 3-Nitro-3-hexene Nitromethane 2-Nitro-p-phenylenediamine 1-Nitropyrene 8-Nitroquinoline 4-Nitroquinoline-N-oxide N-Nitrosoallylethanolamine N-Nitrosoallyl-2,3-dihydroxypropylamine N-Nitrosoallyl-2-hydroxypropylamine N-Nitrosoallyl-2-oxopropylamine Nitrosoamylurethan Nitrosoanabasine Nitroso-Baygon N-Nitrosobenzthiazuron N-Nitrosobis(2-hydroxypropyl)amine N-Nitrosobis(2-oxopropyl)amine N-Nitroso-bis-(4,4,4-trifluoro-N-butyl)amine Nitrosodibutylamine N-Nitrosodiethanolamine N-Nitrosodiethylamine (diethylnitrosamine) 1-Nitroso-5,6-dihydrouracil Ninitroso-2,3-dihydroxypropylethanolamine N-Nitroso-2,3-dihydroxypropyl-2hydroxypropyl-amine Nitroso-2,3-dihydroxypropyl-2oxopropylamine N-Nitrosodimethylamine (dimethylnitrosamine) 1-Nitroso-3,5-dimethyl-4-benzoylpiperazine N-Nitrosodiphenylamine p-Nitrosodiphenylamine N-Nitrosodipropylamine

19

3031-51-4

58139-48-3

+

3771-19-5 389-08-2 91-20-3 2243-62-1 91-59-8 10101-98-1 553-53-7 139-94-6 139-13-9 5064-31-3 18662-53-8

− + − + +

+ − + + +

+ −

− −

−

−

7632-00-0 602-87-9 1777-84-0 99-59-2 91-23-6 98-95-3 94-52-0 62-23-7 92-93-3 1836-75-5 59-87-0

+ + + + + − + + + + +

67-20-9

+

−

[32]∼

−

[11]∼

−

[115]

−* E

E −

+ [30,52];− [50] [11]∼

− + −* + +

−

[116]

−

[36]

− +

− −

[11]∼ [50]

+

−

[50]

−

[38]

+

[16]

555-84-0 75198-31-1 2122-86-3 36133-88-7

+

2578-75-8

+

53757-28-1 531-82-8 24554-26-5 51325-35-0

+ + + +

51-75-2 126-85-2 4812-22-0 75-52-5 5307-14-2 5522-43-0 607-35-2 56-57-5 91308-69-9 88208-16-6 91308-70-2 91308-71-3 64005-62-5 1133-64-8 38777-13-8 51542-33-7 53609-64-6 60599-38-4 83335-32-4 924-16-3 1116-54-7 55-18-5 16813-36-8 89911-78-4 89911-79-5

+

+

+

[11]

− + + + +

− + +

− −

[36] [117]

+

[16]

+

+

[30]

+

[16]

−*

− − −

[11]∼ [11] [11]∼

+

[16]

+

[30]

+

[16]

−

[11]∼

+

[16]

+

+ + + + +

92177-50-9 62-75-9

+

+

61034-40-0 86-30-6 156-10-5 621-64-7

− − + +

− +

20 C561 C562 C563 C564 C565 C566 C567 C568 C569 C570 C571 C572 C573 C574 C575 C576 C577 C578 C579 C580 C581 C582 C583 C584 C585 C586 C587 C588 C589 C590 C591 C592 C593 C594 C595 C596 C597 C598 C599 C600

C601 C602 C603 C604 C605 C606 C607 C608 C609

C610 C611 C612 C613 C614 C615 C616 C617 C618 C619 C620 C621 C622 C623 C624 C625 C626 C627 C628 C629 C630 C631 C632 C633

T. Morita et al. / Mutation Research 802 (2016) 1–29 Nitrosododecamethyleneimine N-Nitrosoephedrine Nitrosoethylmethylamine Nitrosoethylurethan Nitrosoheptamethyleneimine N-Nitrosohexamethyleneimine 1-Nitrosohydantoin 1-Nitroso-1-hydroxyethyl-3-chloroethylurea 1-Nitroso-1-(2-hydroxypropyl)-3chloroethylurea N-Nitroso-(2-hydroxypropyl)-(2hydroxyethyl)amine N-Nitroso-3-hydroxypyrrolidine N-Nitroso-N-isobutylurea 2-Nitrosomethylaminopyridine Nitrosomethylaniline N-Nitroso-N-methyldecylamine N-Nitrosomethyl-2,3-dihydroxypropylamine N-Nitroso-N-methyl-N-dodecylamine N-Nitroso-N-methyl-4-fluoroaniline N-Nitrosomethyl-(2-hydroxyethyl)amine N-Nitrosomethyl-2-hydroxypropylamine N-Nitrosomethyl-(3-hydroxypropyl)amine N-Nitrosomethyl(2-oxopropyl)amine Nitroso-N-methyl-N-(2-phenyl)ethylamine N-Nitroso-N-methyl-N-tetradecylamine N-Nitrosomethyl-(2-tosyloxyethyl)amine Nitrosomethylundecylamine N-Nitroso-N-methylurea N-Nitrosomorpholine N -Nitrosonornicotine-1-N-oxide 3-Nitroso-2-oxazolidinone Nitroso-2-oxopropylethanolamine N-Nitrosopiperazine N-Nitrosopiperidine N-Nitrosopyrrolidine Nitroso-1,2,3,6-tetrahydropyridine N-Nitrosothialdine N-Nitrosothiomorpholine o-Nitrosotoluene N-Nitroso(2,2,2-trifluoroethyl)ethylamine N-Nitroso-2,2,4-trimethyl-1,2dihydroquinoline polymer 1-Nitroso-3,4,5-trimethylpiperazine 5-Nitro-o-toluidine Norlestrin Ochratoxin A Oxazepam N-(9-Oxo-2-fluorenyl)acetamide Oxolinic acid 4,4 -Oxydianiline N-Oxydiethylene thiocarbamyl-N-oxydiethylene sulphenamide Oxymetholone Ozone Pentachloroanisole Pentachloroethane Pentachloronitrobenzene Pentanal methylformylhydrazone n-Pentylhydrazine HCl Petasitenine Phenacetin Phenazone Phenazopyridine HCl Phenesterin Phenobarbital Phenobarbital, sodium Phenolphthalein Phenoxybenzamine HCl Phenylbutazone 1-Phenyl-3,3-dimethyltriazene o-Phenylenediamine 2HCl Phenylethylhydrazine sulphate Phenylglycidyl ether Phenylhydrazine HCl o-Phenylphenol, sodium o-Phenylphenol

40580-89-0 17608-59-2 10595-95-6 614-95-9 20917-49-1 932-83-2 42579-28-2 96806-34-7 96806-35-8

+ + + + + +

−

[11]∼

+ +

[50] [11]

− −

75896-33-2 56222-35-6 760-60-1 16219-98-0 614-00-6 75881-22-0 86451-37-8 55090-44-3 937-25-7 26921-68-6 75411-83-5 70415-59-7 55984-51-5 13256-11-6 75881-20-8 – 68107-26-6 684-93-5 59-89-2 78246-24-9 38347-74-9 92177-49-6 5632-47-3 100-75-4 930-55-2 55556-92-8 81795-07-5 26541-51-5 611-23-4 82018-90-4 29929-77-9

75881-18-4 99-55-8 8015-12-1 303-47-9 604-75-1 3096-50-2 14698-29-4 101-80-4 13752-51-7

434-07-1 10028-15-6 1825-21-4 76-01-7 82-68-8 57590-20-2 1119-68-2 60102-37-6 62-44-2 60-80-0 136-40-3 3546-10-9 50-06-6 57-30-7 77-09-8 63-92-3 50-33-9 7227-91-0 615-28-1 156-51-4 122-60-1 59-88−1 132-27-4 90-43-7

+ +

+

+ +

+

+

[16]

[11]∼ [11]∼

+

[16]

−

[36]

+

[16]

+ +

+

[34]

− + − + +

−

[50,118]

−

[50]

+ +

+

[30,35]

+

+

[11]

+

+ − + − − +

[30] [11]∼ [36,50] [11] [50] [119]

−

[16]∼

− +h

[120] [30]

−

[121]

+ + + + +

+

+

− −

− −

+ +

− + + − −

+ − E − + − − + − + + + + + − +

+ + +

TC −** +

T. Morita et al. / Mutation Research 802 (2016) 1–29 C634 C635 C636 C637 C638 C639 C640 C641 C642 C643 C644 C645 C646 C647 C648 C649 C650 C651 C652 C653 C654 C655 C656 C657 C658 C659 C660 C661 C662 C663 C664 C665 C666 C667 C668 C669 C670 C671 C672 C673 C674 C675 C676 C677 C678 C679 C680 C681 C682 C683 C684 C685 C686 C687 C688 C689 C690 C691 C692 C693 C694 C695 C696 C697 C698 C699 C700 C701 C702 C703 C704 C705 C706 C707 C708 C709 C710 C711

Phorbol Piperonyl butoxide Piperonyl sulphoxide Pivalolactone Polybrominated biphenyl mixture Potassium bicarbonate Prednimustine Prednisolone Primaclone (primidone) Probenecid Procarbazine Procarbazine HCl (Natulan) Progesterone Propane sultone beta-Propiolactone 1,2-Propylene oxide N-N’-Propyl-N-formylhydrazine Propylhydrazine HCl N-Propyl-N’-nitro-N-nitrosoguanidine N-Propyl-N-nitrosourea Propylthiouracil Pyridine Pyrilamine maleate Pyrimethamine Quercetin p-Quinone dioxime Reserpine Retinol acetate Rifampicin Ripazepam Saccharin, sodium Safrole Salbutamol SDZ 200-110 Selenium diethyldithiocarbamate Selenium sulphide Senkirkine Sesamol Sodium dichromate Sterigmatocystin Streptozotocin Strobane Styrene Styrene oxide Succinic anhydride Sulfallate Sulfamethazine Sulfamethoxazole 4,4 -Sulfonylbisacetanilide SX Purple Symphytine Tamoxifen citrate Terbutaline Testosterone 3,3 ,4,4 -Tetraaminobiphenyl 4HCl 2,3,7,8-Tetrachlorodibenzo-p-dioxin 1,1,1,2-Tetrachloroethane 1,1,2,2-Tetrachloroethane Tetrachloroethylene Tetrachlorvinphos 12-O-Tetradecanoylphorbol 13-acetate Tetrafluoro-m-phenylenediamine 2HCl Tetrahydrofuran Tetrahydro-2-nitroso-2H-1,2-oxazine Tertanitromethane Thioacetamide 4,4 -Thiodianiline beta-Thioguanine deoxyriboside Thio-tepa Thiouracil Thiourea Titanium dioxide Toluene 2,4-Toluene diisocyanate Toluene diisocyanate, commercial grade (2,4 and 2,6) o-Toluenesulfonamide m-Toluidine HCl o-Toluidine

17673-25-5 51-03-6 120-62-7 1955-45-9 67774-32-7 298-14-6 29069-24-7 50-24-8 125-33-7 57-66-9 671-16-9 366-70-1 57-83-0 1120-71-4 57-57-8 75-56-9 77337-54-3 56795-66-5 13010-07-6 816-57-9 51-52-5 110-86-1 59-33-6 58-14-0 117-39-5 105-11-3 50-55-5 127-47-9 13292-46-1 26308-28-1 128-44-9 94-59-7 18559-94-9 – 5456-28-0 7446-34-6 2318-18-5 533-31-3 10588-01-9 10048-13-2 18883-66-4 8001-50-1 100-42-5 96-09-3 108-30-5 95-06-7 57-68-1 723-46-6 77-46-3 2611-82-7 22571-95-5 54965-24-1 23031-25-6 58-22-0 7411-49-6 1746-01-6 630-20-6 79-34-5 127-18-4 961-11-5 16561-29-8 63886-77-1 109-99-9 40548-68-3 509-14-8 62-55-5 139-65-1 64039-27-6 52-24-4 141-90-2 62-56-6 13463-67-7 108-88-3 584-84-9 26471-62-5 88-19-7 638-03-9 95-53-4

− − + −

− + − − − − + + +

21

TC − − −

[34]

− −

−

[50]

−** − + + +

+

[30,122]

+

[16]

+ − +

[11] [11] [11]

+

[16]

+

[123]

+

[16]

− − +i − − +e

[50] [50] [45,53] [32]∼ [124] [83]

−

[16]∼

+

[16]

−ˆ

[39]

−

[16]∼

−

[16]∼

+ˆ

[16]

+ + − −

+ + TC −

− + + −

+ + + − +

− −

−* +

−

[125]

+ + − + + +

+ TC

−

[50]

+

+j

[126]

+

[127,128]

− + − + − −

+ + −

+k −

[30,32,50] [11,32]

−

[81]

+

[129,130]

−

[131] [132] [50] [50] [50]

−

− − − − − −

− + − − − +

− + + −

−

−

−

[36]

+ − +

+ TC +

+

[30]

+

+

+

[119]

− − − + +

− − − −

− + +

[133] [34] [30]

−

[134]

−

[135]

+l

[49,50]

− − +

+

22

T. Morita et al. / Mutation Research 802 (2016) 1–29

C712 C713 C714 C715 C716 C717 C718 C719 C720 C721 C722 C723 C724 C725 C726 C727 C728

C729 C730 C731 C732 C733 C734 C735 C736 C737 C738 C739 C740 C741 C742 C743 C744 C745 C746 C747 C748 C749 C750 C751 C752 C753 C754 C755 C756

o-Toluidine HCl p-Toluidine HCl p-Tolylurea Toxaphene Trenimon Triamcinolone acetonide Triamterene Tribromomethane Trichloracetic acid 2,4,6-Trichloroaniline 1,1,2-Trichloroethane Trichloroethylene (with and without epichlorohydrin) N-(Trichloromethylthio)phthalimide 2,4,6-Trichlorophenol 1,2,3-Trichloropropane Triethanolamine 2,2,2-Trifluoro-N-[4-(5-nitro-2-furyl)-2thiazolyl acetamide Trifluralin, technical grade 2,4,5-Trimethylaniline 2,4,5-Trimethylaniline HCl 2,4,6-Trimethylaniline HCl 1,2,4-Trimethylbenzene Trimethylphosphate Trimethylthiourea 2,4,6-Trinitro-1,3-dimethyl-5-tertbutylbenzene Trinitroglycerin Tris(2-chloroethyl)phosphate Tris-1,2,3-(chloromethoxy)propane Tris(2,3-dibromopropyl)phosphate Tris(2-ethylhexyl)phosphate Uracil Uracil (uracil mustard) Urethane Vanadium Pentoxide Vinyl acetate Vinyl bromide Vinyl carbamate Vinyl chloride 4-Vinylcyclohexene Vinylidene chloride (1,1-Dichloroethylene) 2,4-Xylidine HCl 2,5-Xylidine HCl Zearalenone Zinc dimethyldithiocarbamate (Ziram) Zinc ethylenebisthiocarbamate (Zineb)

636-21-5 540-23-8 622-51-5 8001-35-2 68-76-8 76-25-5 396-01-0 75-25-2 76-03-9 634-93-5 79-00-5 79-01-6

+ + − + +

E

−

[89]

+

+

[136]

− + − − − −

+ + TC

− −

[50] [32]

+ −

− −

[137] [34]

133-07-3 88-06-2 96-18-4 102-71-6 42011-48-3

+ − + − +

+ − + −

−

[36]

1582-09-8 137-17-7 21436-97-5 6334-11-8 95-63-6 512-56-1 2489-77-2 81-15-2

+ + +

− +

− + −

−* −

− +

[138] [139]

55-63-0 115-96-8 38571-73-2 126-72-7 78-42-2 66-22-8 66-75−1 51-79-6 1314-62-1 108-05-4 593-60-2 15805-73-9 75-01-4 100-40-3 75-35-4 21436-96-4 51786-53-9 17924-92-4 137-30-4 12122-67-7

+ −

−

+

[30]

+ −

+ −

[11] [50]

+ − − + + − − + + + − − + + − + −

−* +

− TC

+ +

+ − + − + + − −

[30,122] [50] [30] [11]∼ [140] [30] [141] [30]∼

+ − −

[142] [143] [144]

−

[16]∼

+

[16]

+

[16]

+

[16]

+

[16]

Ames, Ames test; CA, chromosome aberration test; MN, rodent erythrocytes micronucleus test; TGR, transgenic rodent mutation assay. Results of genotoxicity data are given as follows: +, positive;−, negative. E, equivocal result, when response is weak or not reproduced between experiments or between laboratories. TC, technically compromised. *, positive response at both >10 mM and >2 mg/mL [29]. **, new data or alteration of the results from the original CGX [10]. ˆ, not in the target tissue(s) of carcinogenicity. ∼, no information on target cell exposure in the review paper or database for negative result in vivo. a, positive in rat treated by intravenous injection [11,35]; b, maybe due to hypothermia; c, negative in rat [35]; d, as free base (119-90-4). e, maybe due to hypothermia in mouse, but negative in rat; f, negative in rat [48]. g, negative as free base (101-77-9) [11,49]; h, as free base (100-63-0); i, positive in rat, but negative in mouse. j, positive by intraperitoneal injection, but negative by oral gavage; k, negative in rat [32]. l, positive in rat, but negative in mouse [11,50].

T. Morita et al. / Mutation Research 802 (2016) 1–29

23

Appendix B. : Genotoxicigty test results with rodent non-carcinogens. ID

Chemical

CAS No

Ames

in vitro CA

in vivo MN

in vivo MN Ref.

NC1 NC2 NC3 NC4 NC5 NC6 NC7 NC8 NC9 NC10 NC11 NC12 NC13 NC14 NC15 NC16 NC17 NC18 NC19 NC20 NC21 NC22 NC23 NC24 NC25 NC26 NC27 NC28 NC29 NC30 NC31 NC32 NC33

Acetohexamide Acetonitrile [AKA ethyl nitrile] Acrolein Adipamide Agar Aldicarb Aluminum potassium sulfate dl-Amphetamine sulfate Ampicillin trihydrate Anilazine p-Anisidine HCl o-Anthranilic acid l-Ascorbic acid Aspirin, phenacetin, and caffeine Azinphosmethyl [AKA gusathion] Barium chloride dihydrate Benzoate, sodium Benzoin 1H-Benzotriazole Benzyl alcohol Beryllium sulfate Black PN [AKA Food Black 1] Bromomethane n-Butyl chloride N-Butylurea gamma-Butyrolactone Caffeine Caprolactam Carbromal 2-Chloroacetophenone 4-(Chloroacetyl)-acetanilide p-Chloroaniline o-Chlorobenzalmalonitrile [AKA malonitrile, o-chlorobenzylidene] Chlorodifluoromethane [AKA fluorocarbon 22] (2-Chloroethyl)trimethylammonium chloride 2-(Chloromethyl)pyridine HCl 3-Chloro-p-toluidine [AKA 4-methyl-5-chloro-1-aniline] Chlorpheniramine maleate Chlorpropamide C.I. acid orange 10 C.I. food red 3 [AKA Acid red 14] C.I. pigment red 23 [AKA pigment red 23] C.I. pigment yellow 12 Codeine Coumaphos Cyanamide, calcium Cyclohexanone Cyclohexylamine HCl Deltamethrin Diallyl phthalate 4,4-Diamino-2,2-stilbenedisulfonic acid, disodium salt 2,6-Diaminotoluene 2HCl 2,5-Diaminotoluene sulfate Diazinon Dibenzo-p-dioxin 1,2-Dichlorobenzene 2,7-Dichlorodibenzo-p-dioxin Dichlorodifluoromethane 1,1-Dichloroethane 2,4-Dichlorophenol N,N-Dicyclohexylthiourea Dieldrin, photoDimethoate Dimethoxane, commercial grade [AKA acetic acid ester with 2,6-dimethyl-m-dioxan-4-ol] 2,4-Dimethoxyaniline HCl 6-Dimethylamino-4,4-diphenyl-3-heptanone HCl Dimethylformamide Dimethyl terephthalate

968-81-0 75-05-8 107-02-8 628-94-4 9002-18-0 116-06-3 10043-67-1 60-13-9 7177-48-2 101-05-3 20265-97-8 118-92-3 50-81-7 8003-03-0 86-50-0 10326-27-9 532-32-1 119-53-9 95-14-7 100-51-6 13510-49-1 2519-30-4 74-83-9 109-69-3 592-31-4 96-48-0 58-08-2 105-60-2 77-65-6 532-27-4 140-49-8 106-47-8 2698-41-1

− − + −

+ E −

+ −

[33] [50]

− − E − − + − E − + − − − + − −

− TC − − −

E

[33]

+a −

[30] [31]∼

−* −

− +

[89] [34]

− + + + −*

−

[34,145]

− −

[81] [146]

+ − − − − − − − + + E

E − TC

+

[36]

− E −

[147] [33] [148]

E −

[33] [149]

75-45-6

+

999-81-5 6959-47-3 95-74-9

− + −

− + E

−

[50]

113-92-8 94-20-2 1936-15-8 3567-69-9 6471-49-4 6358-85-6 76-57-3 56-72-4 156-62-7 108-94-1 4998-76-9 52918-63-5 131-17-9 7336-20-1

− − − + + − − − + – − − − −

+ + + − − −

− E − −

[50] [33] [34] [34]

− − −

[150] [151] [152]

− +

+ −

[33] [33,34]

15481-70-6 6369-59-1 333-41-5 262-12-4 95-50-1 33857-26-0 75-71-8 75-34-3 120-83-2 1212-29-9 13366-73-9 60-51-5 828-00-2

+ + − − − − − − E − + + +

+ + +

E − +

[33] [33]∼ [153]

−

−

[34,50]

− + − − + +

+ −

[154] [155]

+

[33]

54150-69-5 1095-90-5

+

+

−

[50]

68-12-2 120-61-6

− −

− −

− −

[156] [34]

NC34 NC35 NC36 NC37 NC38 NC39 NC40 NC41 NC42 NC43 NC44 NC45 NC46 NC47 NC48 NC49 NC50 NC51 NC52 NC53 NC54 NC55 NC56 NC57 NC58 NC59 NC60 NC61 NC62 NC63 NC64 NC65 NC66 NC67 NC68

+ E + E + + +

− − −

TGR

TGR Ref.

−

[16]∼

24 NC69

NC70 NC71 NC72 NC73 NC74 NC75 NC76 NC77 NC78 NC79 NC80 NC81 NC82 NC83 NC84 NC85 NC86 NC87

NC88 NC89

NC90 NC91 NC92 NC93 NC94 NC95 NC96 NC97 NC98 NC99 NC100 NC101 NC102 NC103 NC104 NC105 NC106 NC107 NC108 NC109 NC110 NC111 NC112 NC113 NC114 NC115 NC116 NC117 NC118 NC119 NC120 NC121 NC122 NC123 NC124 NC125 NC126 NC127 NC128 NC129 NC130 NC131 NC132 NC133

T. Morita et al. / Mutation Research 802 (2016) 1–29 Dioxathion [AKA phosphorodithioic acid, S,S -p-dioxane-2,3-diyl-O,O,O ,O -tetraethyl2,3-diyl-O,O,O ,O -tetraethyl ester] Diphenhydramine HCl Diphenyl-p-phenylenediamine 2,5-Dithiobiurea EDTA, trisodium salt trihydrate Endrin Ephedrine sulphate Erythorbate, sodium Erythromycin stearate Estazolam p,p-Ethyl-DDD [AKA perthane] Ethyl tellurac Etodolac Eugenol FD & C green no. 3 [AKA C.I. Food green 3] FD & C red no. 3 [AKA fluorescein, 2 , 4 , 5 , 7 -tetraiodo, disodium salt] FD & C yellow no. 5 [AKA tartrazine] FD & C yellow no. 6 [AKA Food yellow 3] Fenaminosulf, formulated [AKA p-dimethylaminobenzenediazo sulphonic acid, sodium salt] Fenthion Fenvalerate [AKA cyano-3-phenoxyphenylmethyl-4-chloroalpha-1-methylethylbenzene acetate] Fluometuron [AKA urea, 1,1-dimethyl 3(alpha, alpha, alpha-trifluoro-m-tolyl)-] Fluoride, sodium Gemfibrozil Guar gum Gum arabic HC blue no. 2 [AKA ethanol, 2,2 ((4-(2hydroxyethylamino)-3-nitrophenyl)imino)di-] HC yellow 4 Hexachlorocyclopentadiene Hexachlorophene Hexamethylenetetramine 4-Hexylresorcinol Hydrochlorothiazide 8-Hydroxyquinoline [AKA 8-quinolinol] Iodoform [AKA methane, triiodo-] Isopropyl-N-(3-chlorophenyl)carbamate 4,4 -isopropylidenediphenol Lead dimethyldithiocarbamate Levobunolol HCl Lithocholic acid Locust bean gum Malaoxon Malathion Maleic hydrazide Manganese (II) sulfate monohydrate d-Mannitol Methotrexate Methoxychlor alpha-Methyldopa sesquihydrate Methyl methacrylate Methyl parathion [AKA phosphorothioic acid, O, O-dimethyl o-(p-nitrophenyl)ester] Monochloroacetic acid N-(1-Naphthyl)ethylenediamine 2HCl [AKA PL-89779] Nickel (II) sulfate hexahydrate p-Nitroaniline 4-Nitroanthranilic acid 3-Nitro-4-hydroxyphenylarsonic acid (AKA roxarsone) 1-Nitronaphthalene 4-Nitro-o-phenylenediamine 3-Nitropropionic acid Omeprazole gamma-Oryzanol Oxamyl Oxprenolol HCl Oxytetracycline HCl

78-34-2

+

−

147-24-0 74-31-7 142-46-1 150-38-9 72-20-8 134-72-5 6381-77-7 643-22-1 29975-16-4 72-56-0 20941-65-5 41340-25-4 97-53-0 2353-45-9 16423-68-0

− + − − − − − −

+ + − − − − − −

+ − − − − −

− + − + TC +

1934-21-0 2783-94-0 140-56-7

− − +

+ − −

55-38-9 51630-58-1

E −

2164-17-2

−b

[55]

− E E

[34] [33] [33]

−

[34]

+ +

+

[33]

−

−

−

[157]

7681-49-4 25812-30-0 9000-30-0 9000-01-5 33229-34-4

−

+

E

+[33];−[50,57,58]

− − +

−

−

[105]

59820-43-8 77-47-4 70-30-4 100-97-0 136-77-6 58-93-5 148-24-3 75-47-8 101-21-3 80-05-7 19010-66-3 27912-14-7 434-13-9 9000-40-2 1634-78-2 121-75-5 123-33-1 10034-96-5 69-65-8 59-05-2 72-43-5 41372-08-1 80-62-6 298-00-0

+ − − + − − + + − − +

− +

−

[36]

−

[50]

−

[34]

− −

[147] [158]

− − − − − + − − − − − +

+ E + E + − + −

+ − + − +

[33] [159] [33] [34] [33,122]

+ −

– +

[160] [33]

79-11-8 1465-25-4

− +

− +

+

[161]

10101-97-0 100-01-6 619-17-0 121-19-7

− + + −

+ + −* −

−

[11]

86-57-7 99-56-9 504-88-1 73590-58-6 11042–64-1 23135-22-0 6452-73-9 2058-46-0

+ + + − − − − −

+ + E

−

[162]

E

[33]

E +

[33] [163]

− − + − E + +

−

−

[16]

−

[16]∼

T. Morita et al. / Mutation Research 802 (2016) 1–29 NC134 NC135 NC136 NC137 NC138 NC139 NC140 NC141 NC142 NC143 NC144 NC145 NC146 NC147 NC148 NC149 NC150 NC151 NC152 NC153 NC154 NC155 NC156 NC157 NC158 NC159 NC160 NC161 NC162 NC163 NC164 NC165 NC166 NC167 NC168 NC169 NC170 NC171 NC172 NC173 NC174 NC175 NC176 NC177 NC178 NC179 NC180 NC181 NC182 NC183

Parathion Penicillin VK Pentaerythritol tetranitrate with 80% d-lactose monohydrate Phenformin HCl Phenol p-Phenylenediamine 2HCl Phenylephrine HCl 1-Phenyl-3-methyl-5-pyrazolone Phenyl-beta-naphthylamine [AKA N-phenyl-2-naphthylamine] N-Phenyl-p-phenylenediamine HCl [AKA C.I. Oxidation base 2A] 1-Phenyl-2-thiourea Phthalamide Phthalic anhydride Picloram, technical grade Polysorbate 80 Promethazine HCl Propylene [AKA propene] Propyl gallate Resorcinol Rhodamine 6 G [AKA basic red 1] Rotenone Sodium chlorite Sodium diethyldithiocarbamate trihydrate [AKA carbamic acid, diethyldithio, sodium salt] Sodium hypochlorite Sorbic acid Sotalol HCl Sulfisoxazole 3-Sulfolene Tara gum 2,3,5,6-Tetrachloro-4-nitroanisole Tetracycline HCl Tetraethylthiuram disulfide [AKA disulfide, bis(diethylthiocarbamoyl)] 1-trans-delta-9-Tetrahydrocannabinol Tetrakis(hydroxymethyl)phosphonium chloride Tetrakis(hydroxymethyl)phosphonium sulfate Tetramethylthiuram disulfide 4,4-Thiobis(6-tert-butyl-m-cresol) [AKA santonox-R] Tin (II) chloride Tolazamide Tolbutamide 1,1,1-Trichloroethane, technical grade Trichlorofluoromethane 2,4,5-Trichlorophenoxyacetic acid Tricresyl phosphate Triphenyltin hydroxide Triprolidine HCl monohydrate l-Tryptophan Turmeric oleoresin (79%-85% curcumin) Urea Vinyl toluene (65-71% m- and 32-35% p-) [AKA benzene,ethenylmethyl-]

56-38-2 132-98-9 78-11-5

− − −

− + −

834-28-6 108-95-2 624-18-0 61-76-7 89-25-8 135-88-6

− − + − − −

− + + − − E

2198-59-6

−

103-85-5 88-96-0 85-44-9 1918-02-1 9005-65-6 58-33-3 115-07-1 121-79-9 108-46-3 989-38-8 83-79-4 7758-19-2 148-18-5

− − − − − − + − − − − + −

−* − + TC TC −

7681-52-9 110-44-1 959-24-0 127-69-5 77-79-2 39300-88-4 2438-88-2 64-75-5 97-77-8

− − − − − − − −

1972-08-3 124-64-1

− −

55566-30-8 137-26-8 96-69-5

− + −

+

7772-99-8 1156-19-0 64-77-7 71-55-6 75-69-4 93-76-5 1330-78-5 76-87-9 6138-79-0 73-22-3 8024-37-1 57-13-6 25013-15-4

− E − − − − − − − − − − −

+ TC − E

25 − −

[110] [164]

+c − −

[43,59] [162] [50]

− + +

[165] [33] [50]

− +

[166] [33]

E TC

− −

[81] [167]

− −

−

[34]

+ −

[33] [50]

− +

[168] [33]

−

[33,34]

+ −

[33] [50]

+ −* TC − + −

[1] The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, Step 4 in November 2011. Available at http://www.ich.org/ fileadmin/Public Web Site/ICH Products/Guidelines/Safety/S2 R1/Step4/ S2R1 Step4.pdf (accessed 26.09.15). [2] UK Committee on Mutagenicity (COM), Guidance on a strategy for genotoxicity testing of chemical substances, 1 December, 2011, Available at https://www.gov.uk/government/uploads/system/uploads/attachment data/file/315793/testing chemicals for genotixicity.pdf (accessed 05.11.15). [3] European Chemical Agency (ECHA), Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a, Version 4.1, October, 2015, Available at http://echa.europa.eu/documents/10162/13632/information requirements r7a en.pdf (accessed 05.11.15).

[40]

E + +

+

−

−

[33]∼

− −

+

[169]

− E TC +

+

[33]

For explanation of the in vitro results, in vivo MN and TGR results and other symbols see footnotes to Appendix A. a, as free base (300-62-9). b, as disodium salt. c, maybe due to hypothermia. References

−

[4] HEuropean Food Safety Authority (EFSA), Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment, 3 October 2012, EFSA Journal 9 (9) (2011) 2379, Available at http://www.efsa.europa. eu/sites/default/files/scientific output/files/main documents/2379.pdf (accessed 05.11.15). [5] M. Hayashi1, M. Honma, M. Takahashi, A. Horibe, J. Tanaka1, M. Tsuchiya, T. Morita, Identification and evaluation of potentially genotoxic agricultural and food-related chemicals, Food Saf. 1 (2013) 32–42, Available at https:// www.jstage.jst.go.jp/article/foodsafetyfscj/1/1/1 2013003/ pdf (accessed 05.11.15). [6] EU, Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003 amending Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products, Off. J. Eur. Union L66 (2003) 10. [7] EU, Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products, Off. J. Eur. Union L342 (2009) 151. [8] R.W. Tennant, B.H. Margolin, M.D. Shelby, E. Zeiger, J.K. Haseman, J. Spalding, W. Caspary, M. Resnick, S. Stasiewicz, B. Anderson, R. Minor,

26

T. Morita et al. / Mutation Research 802 (2016) 1–29

[9]

[10]

[11]

[12]

[13] [14]

[15] [16]

[17]

[18]

[19]

[20] [21]

[22]

[23]

[24] [25]

[26]

[27] [28]

[29]

[30]

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