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

Mutation Research 654 (2008) 114–132

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Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens III. Appropriate follow-up testing in vivo ¨ David Kirkland a,∗ , Gunter Speit b a b

Covance Laboratories Ltd., Otley Road, Harrogate, HG3 1PY England, United Kingdom Universit¨ at Ulm, Institut f¨ ur Humangenetik, D-89069 Ulm, Germany

a r t i c l e

i n f o

Article history: Received 28 January 2008 Received in revised form 3 May 2008 Accepted 9 May 2008 Available online 16 May 2008 Keywords: Genotoxicity Carcinogens In vivo testing Micronucleus UDS Transgenic mutation Comet assay

a b s t r a c t There has been much discussion in recent years regarding the most appropriate follow-up testing in vivo when positive results are obtained in vitro but the in vivo micronucleus (MN) test (traditionally the most widely-used test) is negative. Not all rodent carcinogens give positive results in the micronucleus test, and so it has been common practice to include a second in vivo assay such as the unscheduled DNA synthesis (UDS) test. This has proved useful but is usually limited to analysis of rodent (usually rat) liver. With the increased evaluation and use of other in vivo assays, e.g. for transgenic mutations (TG) and DNA damage (Comet assay) it was important to investigate their usefulness. We therefore examined the published in vivo UDS, TG and Comet-assay results for 67 carcinogens that were negative or equivocal in the micronucleus test. Between 30 and 41 chemicals were evaluated in each of the three in vivo tests, with some overlap. In general, the UDS test was disappointing and gave positive results with <20% of these carcinogens, some of which induced tumours in rat liver and produced DNA adducts in vivo. The TG assay gave positive responses with >50% of the carcinogens, but the Comet assay detected almost 90% of the micronucleus-negative or equivocal carcinogens. This pattern of results was virtually unchanged when the in vitro profile (gene mutagen or clastogen) was taken into account. High sensitivity (ability to detect carcinogens as positive) is only really useful when the specificity (ability to give negative results with non-carcinogens) is also high. Based on small numbers of publications with non-carcinogens, the TG and Comet assays gave negative results with non-carcinogens on 69 and 78% of occasions, respectively. Although further evaluation of the Comet and TG assays, particularly with non-carcinogens, is needed, these data suggest that they both should play a more prominent role in regulatory testing strategies than the UDS test. © 2008 Elsevier B.V. All rights reserved.

1. Introduction There has been much discussion in recent years over what is the most appropriate follow-up testing in vivo when positive results are obtained in vitro. Traditionally the micronucleus (MN) test in rodent bone marrow or peripheral blood has been the most widely used in vivo test, but not all rodent carcinogens give positive results in this assay. In fact, an analysis of the published literature indicated that out of 218 rodent carcinogens, 120 (i.e. >50%) were either negative or equivocal in the bone-marrow MN test. For these reasons it has been common practice to include a second in vivo test in those cases where positive results are obtained in vitro but the micronucleus test is negative. The unscheduled DNA synthesis (UDS) test has been

∗ Corresponding author. Tel.: +44 1423 848401. E-mail address: [email protected] (D. Kirkland). 1383-5718/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2008.05.002

recommended in several regulatory guidelines as an appropriate complement to the MN test in these circumstances, but is usually limited to the analysis of rodent (usually rat) liver. However, the popularity of the Comet (single-cell gel electrophoresis) and transgenic mutation (TG) assays has increased recently because of their ability to detect genotoxic responses in a wide variety of tissues. It is therefore timely to re-evaluate the strategy for follow-up in vivo testing. The database of in vitro genotoxicity results with rodent carcinogens recently published by Kirkland et al. [1] was used. In vivo MN data were obtained by searching the published literature. We found 120 rodent carcinogens where the MN result was negative or equivocal (i.e. an inconclusive result in a single report or conflicting results across several published papers). These data are summarised in Table 1 together with references. Please note the free base and sodium salt of nitrilotriacetic acid have been combined into one entry. The in vitro genotoxicity data

Table 1 DNA adduct and in vivo genotoxicity results with rodent carcinogens negative or equivocal in the micronucleus test

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Table 1 (Continued )

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Key to abbreviations: Inc. = inconclusive, ip = intraperitoneal, E = equivocal, W = weak, V: IARC monograph on the evaluation of carcinogenic risk to humans. a In vivo, unless mentioned otherwise. b Comets induced at doses causing necrosis considered to be “false” positive by authors.

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for these carcinogens can be found in Kirkland et al. [1] or at http://www.lhasalimited.org/cgx. Published data from one or more of UDS, TG and Comet assays were found for 67 of this subset of 120 carcinogens, and the results are summarised in Table 2. We have not attempted to define all the carcinogens in the list as acting either via a genotoxic or non-genotoxic mode of action because many are not clearly defined or are contentious. However, we have added DNA-adduct data where they have been published, and this may help the reader decide whether a substance is more or less likely to be genotoxic and therefore whether or not we should expect the genotoxicity studies to give positive responses. As can be seen from the subset of the MN-negative carcinogens in Table 2, many were multi-site, trans-species carcinogens, and therefore we might have expected the bone marrow to be a suitable target for genotoxicity in vivo. The data have been analysed for how frequently each of these three additional in vivo tests produced positive responses with carcinogens that were not clearly detected in the MN test. The tissues in which tumours, TG mutations or Comet-assay responses occurred were also recorded so that they could be evaluated in terms of their relevance to the respective positive and negative findings.

2. Rat liver UDS data A total of 41 rodent carcinogens, negative or equivocal for MN, have been identified for which published UDS data exist. The details are shown in Table 2 and the responses can be summarised as follows: • Only seven of the 41 carcinogens (17.1%) produced positive liver UDS responses. Interestingly, two carcinogens producing positive responses (3,3 -dichlorobenzidine and 2-naphthylamine) did not produce tumours in rat liver. • Six of the 41 carcinogens (14.6%) produced equivocal responses for rat liver UDS, but only three of these (MeIQx, IQ and nitrosodiethanolamine) produced liver tumours in rats. The other three compounds (acrylamide, 3-methylcholanthrene and 2-nitro-pphenylenediamine) may therefore be expected not to produce genotoxic effects in rat liver. • Twenty-eight of the 41 carcinogens (68.3%) produced negative responses in the rat liver UDS test. Epichlorohydrin did produce a positive UDS response in the stomach, which is the target site for tumours. Of these 28: o Five (di[2-ethylhexyl]phthalate, 1,4-dioxane, methyl clofenapate, phenobarbital and poly-brominated biphenyl mixture) are considered to induce tumours via a non-genotoxic mechanism and 1 (pyridine) is a strain-specific carcinogen. Therefore positive liver UDS responses would not be expected for these compounds. o Of the remaining 23 compounds, four (11-aminoundecanoic acid, carbon tetrachloride, coumarin and 1,3-dichloropropene) were found to induce liver tumours in rats and may have been expected to induce UDS. In particular 1,3-dichloropropene has been shown to produce DNA adducts. o Of the remaining 19 compounds, several have been shown to produce DNA adducts, and to induce liver tumours in mice. It is debatable whether the rat liver UDS test should be expected to give positive results with such compounds. Of the six carcinogens that were equivocal for UDS, five were tested for TG mutations and three were tested in the Comet assay, and all were positive (see Table 2). Of the 28 carcinogens that were negative for UDS:

Table 2 Results in other in vivo tests for rodent carcinogens negative or equivocal in bone-marrow micronucleus tests

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Table 2 (continued )

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Table 2 (continued )

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D. Kirkland, G. Speit / Mutation Research 654 (2008) 114–132 Tissue codes: adr = adrenal gland, bmw = bone marrow, brn = brain, cli = clitoral gland, col = colon, eso = esophagus, ezy = ear/Zymbal’s gland, hmo = haematopoietic system, kid = kidney, lgi = large intestine, liv = liver, lun = lung, mgl = mammary gland, nas = nasal cavity, nrv = nervous system, orc = oral cavity, ova = ovary, pan = pancreas, per = peritoneal cavity, pit = pituitary gland, pre = preputial gland, ski = skin, smi = small intestine, spl = spleen, sto = stomach, sub = subcutaneous tissue, tes = testes, thy = thyroid gland, ubl = urinary bladder, ute = uterus, vsc = vascular system, N/A = no data available. a Data from animals treated with o-toluidine hydrochloride, because no tissue data from carcinogenicity studies with the free base. Highlighted tissues (yellow in online version and grey shading in printed version) indicate those in which positive mutation or Comet responses were seen, but tumours were not induced. For simplicity, genotoxic responses in the colon (col) were considered as indicative of tumours in the large intestine (lgi), and genotoxic responses in bone marrow (bmw) were considered as indicative of tumours of the haematopoietic system (hmo).

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• Eight were tested for TG mutations, two were positive and one was equivocal. • Ten were tested in the Comet assay, eight were positive and two (carbon tetrachloride and trichloroethylene) were negative. Some of the carcinogens negative in rat liver UDS tests were positive in TG and Comet assays because mice were used instead of rats, and others were because tissues other than liver were analysed. Details of species studied and tissues giving positive responses are shown in Table 2. 3. Transgenic mutation data A total of 30 rodent carcinogens that were negative or equivocal for MN, have been identified for which published TG data exist. The details are shown in Table 2 and can be summarised as follows: • Seventeen of the 30 carcinogens (56.7%) produced positive responses. • One carcinogen (phenobarbital) produced an equivocal response. • Twelve of the 30 carcinogens (38.7%) produced negative responses. o Of these 12 chemicals, four (di[2-ethylhexy]phthalate, methyl clofenapate, sodium saccharin and TCDD) are considered to induce tumours via a non-genotoxic mechanism and therefore would not be expected to induce mutations in TG models. o Of the remaining eight carcinogens, five (1,2-dibromoethane, 1,2-dichloroethane, hydrazine sulphate, metronidazole and trichloroethylene) have been shown to induce DNA adducts or DNA methylation either in vitro or in vivo, and p-cresidine is considered a genotoxic carcinogen (used as a positive control in p53 tumour studies, but there are no published adduct data), and so the TG assay may have been expected to produce positive results. Of the carcinogens that gave negative TG results, only 1,2dibromoethane produced a positive UDS response. However, 3 of the 12 carcinogens that gave negative TG results (acrylonitrile, 1,2dibromoethane, 1,2-dichloroethane) produced positive results in the Comet assay. 4. Comet assay data A total of 35 carcinogens that were negative or equivocal for MN, have been identified for which published Comet assay data exist. The details are shown in Table 2 and can be summarised as follows: • Thirty-one of the 35 carcinogens (88.6%) produced positive responses. • One carcinogen (gamma-1,2,3,4,5,6-hexachlorocyclohexane) was equivocal, giving positive responses in nasal and colon tissue in one study [2] but negative results in another [3]. • Only three carcinogens (8.6%) produced negative results, although all three (carbon tetrachloride, sodium nitrite and trichloroethylene) might be expected to induce DNA damage if high enough exposures could be achieved. Carbon tetrachloride and trichloroethylene were also negative in UDS and TG assays. Sodium nitrite has not been tested in these other assays. 5. Evaluation of tissues As can be seen from Table 2, in most cases where positive responses in the TG or Comet assays were observed, such

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responses were seen in the same tissues that developed tumours. However, for styrene and styrene oxide Comet-assay responses were only found in non-tumour tissues. In many cases TG and Comet-assay responses were also seen in non-tumour tissues. An explanation for this may be that DNA damage or mutations are induced by high doses of chemical in these tissues but at the lower, repeated doses used in carcinogenicity studies insufficient DNA alterations are induced to lead to development of overt tumours. Many of the carcinogens that were negative in the bone-marrow MN test induced tumours in rodent liver. Because this is inevitably a tissue of interest, and the in vivo UDS test was developed initially to predict liver carcinogens, we separately analysed the liver carcinogens, and these are shown in Table 3. It can be seen that the rat-liver UDS test only gave positive responses for six carcinogens that produced liver tumours (19 were negative). On the other hand, 9/19 liver carcinogens gave positive TG responses in the liver, but 19/23 liver carcinogens gave positive Comet-assay responses in the liver.

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If TG and/or Comet assays are to be used instead of the UDS assay, at a time in the development of new substances when carcinogenicity data are not available, which tissues should be sampled? Clearly because of the number of chemicals that cause liver tumours, the liver is an appropriate organ in which to measure mutations or DNA damage, and the Comet assay may be particularly useful in this respect. The data in Table 3 support this. Some carcinogens do not require liver activation and may induce tumours in the first tissue of contact. It would be logical therefore to also examine mutations or DNA damage in the stomach for an orally dosed substance, in the skin for a topically dosed substance, or in the lungs for an inhaled product. Unfortunately there are insufficient published TG and Comet-assay data with these tissues to know at this time whether this would be an effective strategy. 6. Effect of the in vitro genotoxicity profile By referring to the published in vitro genotoxicity on the 67 carcinogens considered in this paper ([1] or http://www.

Table 3 Analyis of in vivo test results for rodent-liver carcinogens Chemical carcinogen (CAS no.)

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (77500-04-0) 2-Amino-3-methylimidazo[4,5-f]quinolone (IQ) (76180-96-6) 11-Aminoundecanoic acid (2432-99-7) Benzyl acetate (140-11-4) Carbon tetrachloride (56-23-5) Chlorodibromomethane (124-48-1) Coumarin (91-64-5) p-Cresidine (120-71-8) 2,4-Diaminotoluene (95-80-7) 1,2-Dibromoethane (106-93-4) 1,4-Dichlorobenzene (106-46-7) Dichloromethane (75-09-2) 1,3-Dichloropropene (542-75-6) Diethanolamine (111-42-2) Di(2-ethylhexyl)phthalate (117-81-7) N,N-dimethyl-4-aminoazobenzene (60-11-7) 3,3 -Dimethylbenzidine (119-93-7) 2,4-Dinitrotoluene (121-14-2) 1,4-Dioxane (123-91-1) N,N -ethylene thiourea (96-45-7) HC Blue 1 (impure and purified) (2784-9493) Gamma-1,2,3,4,5,6-Hexachlorocyclohexane (58-89-9) Hydrazine sulphate (10034-93-2) Methyl tert-butyl ether (1634-04-4) Methyl clofenapate (21340-68-1) 4,4 -Methylenebis(2-chloroaniline) (101-14-4) 4-(Methylnitrosamino)-1-(3-pyrridyl)-1-(butanone) (64091-91-4) Metronidazole (443-48-1) 2-Naphthylamine (91-59-8) Nitrite sodium (7632-00-0) 2-Nitro-p-phenylenediamine (5307-14-2) Ninitrosodiethanolamine (1116-54-7) N-nitrosodiethylamine (55-18-5) N-nitrosodipropylamine (621-64-7) N-nitrosopyrrolidine (930-55-2) Oxazepam (604-75-1) Phenobarbital (50-06-6) Phenobarbital sodium (57-30-7) Polybrominated biphenyl mixture (67774-32-7) Pyridine (110-86-1) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (1746-01-6) o-Toluidine (95-53-4) Trichloroacetic acid (76-03-9) Trichloroethylene (with and without epichlorohydrin) (79-01-6) Tris(2,3-dibromopropyl)phosphate (126-72-7) Vinyl bromide (593-60-2) E = equivocal; N/A = not available.

Liver tumours in

Genotoxic responses in livers for

Rats

Mice

UDS

Transgenic mutations (species)

Comets (species)

+ + + (Male) − + − + + + + − − + − + + (Female) + + + − − − + − + + + (Male) + − + − + + + + − E E + E + − − (Males) − − +

+ + − + + + (Female) − − + − + + + + − N/A N/A − + + + + − + + N/A N/A − + − + N/A N/A N/A − (Males) + + + + + + + + + + N/A

E E − − − − − N/A + + − − − N/A − + N/A + − − − N/A N/A − − N/A N/A N/A + N/A E E + N/A N/A N/A − N/A − − N/A N/A − − N/A N/A

+ (M) + (R&M) N/A N/A − (M) N/A N/A N/A + (M) − (M) N/A − (M) N/A N/A − (M) N/A N/A N/A N/A N/A N/A N/A − (M) N/A − (M) N/A + (M) N/A N/A N/A + (M) N/A + (M) + (M) + (R) + (M) E (R) − (M) N/A N/A N/A − (R) N/A N/A − (M) − (M) N/A

+ (R) + (R) N/A + (R) − (M) + (R&M) N/A − (M) + (M) + (M) + (M) + (M) + (M) N/A N/A + (M) + (M) N/A N/A + (M) N/A E (R) N/A N/A N/A + (R) + (R) N/A + (M) − (M) N/A N/A + (R&M) N/A N/A N/A N/A + (R) N/A N/A N/A + (R) N/A N/A N/A + (R)

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Table 4 Influence of gene mutation or clastogen profile in vitro on in vivo results In vitro results

Number of carcinogens In vivo UDS result

Positive in Ames Positive in MLA Positive in MN Positive in CA

In vivo transgenic mutation result

In vivo Comet-assay result

+

−

E

+

−

E

+

−

E

7 5 1 5

13 14 3 11

5 3 3 3

13 8 5 8

5 4 3 5

1 1 0 1

21 13 7 17

1 1 2 1

0 0 0 0

lhasalimited.org/cgx) we were able to determine whether a predominantly gene-mutation profile (i.e. positive in Ames or mouse lymphoma assays), or a predominantly clastogenic profile (i.e. positive in in vitro micronucleus or chromosomal aberration assays) had any bearing on the ability of each of the three in vivo tests to detect the carcinogenic potential. A breakdown of these findings is given in Table 4. Of course the mouse lymphoma assay (MLA) can detect both clastogens and gene mutagens. For most of the published data reviewed here, small and large colony mutant frequencies were not evaluated. Thus, we cannot know whether the mode of action of the chemical was predominantly clastogenic or mutagenic. Knowing that there will be some errors we have assumed all the MLA-positive results reflect a mutagenic mode of action. The UDS test seems to be equally poor at producing positive results with carcinogens that have either a gene mutation or clastogen profile in vitro, giving negative responses much more often (around 2–3-fold) than positive responses. The TG assay does produce a higher frequency of positive results (2–3 times more likely than a negative result) with carcinogens showing a gene-mutation profile. Positive TG results are <2-fold more likely than negative results for clastogens. This analysis is consistent with that published by Lambert et al. [4]. The Comet assay seems equally effective at detecting carcinogens that are gene mutagens or clastogens, and only declines slightly in sensitivity with compounds positive for MN in vitro. As can be seen from Table 4, the two compounds positive for MN in vitro that were negative in the Comet assay in vivo were carbon tetrachloride and trichloroethylene, both of which may exhibit aneuploidy potential.

Comet assay detects DNA strand breakage, which may be repaired or lead to cell death and not necessarily lead to tumours. We examined how the TG and comet assays responded to non-carcinogens. From the non-carcinogens in the CGX database (Appendix B of [1]) we found only three non-carcinogens that had been tested for TG mutations – eugenol, phthalic anhydride and l-tryptophan – and all were negative. However, Lambert et al. [4] reported only 9/13 non-carcinogens gave expected negative results (specificity 69%), however the positive conclusions with acetic acid and sucrose may be argued to be overstated (certainly there are conflicting positive and negative results in the published literature) and conclusions of equivocal may be more appropriate. For the non-carcinogens in the CGX database (Appendix B of [1]) we found in vivo Comet-assay data for 13 non-carcinogens, of which 11 were negative. However, Sasaki et al. [7] summarised in vivo Comet-assay data on a much larger number and reported 24/30 non-carcinogens were negative. Closer inspection revealed that some of these chemicals were not tested to a maximum tolerated dose, or the group sizes of rats and mice used were small, or the duration of exposure was considerably shorter than the usual minima of 104 weeks for rats and 80 weeks for mice. Between these two databases we arrived at a total of 27 chemicals with in vivo Comet results for which there was convincing evidence of a lack of carcinogenic potential. These chemicals are listed in Table 5. Only five chemicals (benzyl alcohol, p-chloroaniline, 2,4-dichlorophenol, dimethoate and phenol) gave positive Comet-assay responses and one (2,4-dimethoxyaniline) was considered equivocal (negative in mice but positive in rats). For these five or six chemicals there may be valid explanations as to why a positive Comet-assay result was obtained even though they were non-carcinogenic, for example:

7. Discussion From the above analysis it is clear that the rat-liver UDS test is not very sensitive (<20%) at detecting genotoxic responses with rodent carcinogens that have been “missed” in the conventional in vivo micronucleus test. The TG and Comet assays are much more likely to give positive genotoxic responses with rodent carcinogens that are “missed” in the micronucleus test. In particular the Comet assay has a high sensitivity for detecting carcinogens acting via both mutagenic and clastogenic mechanisms. A full analysis of the performance of these assays across the wider database of rodent carcinogens (not just those that may be negative of equivocal in the micronucleus test) is on-going in order to see if these trends are maintained. In the meantime, the current analysis suggests that the TG and Comet assays should be given a higher priority in selection of follow-up in vivo tests for genotoxins that are positive in vitro but where negative results are obtained in the in vivo micronucleus test. Whilst the Comet and TG assays may be appropriately sensitive to detect carcinogens that are negative in the bone-marrow MN assay, are they equally reliable at giving negative responses with non-carcinogens? In particular it should be noted that gene mutations are only partly responsible for tumourigenesis and that the

Table 5 Non-carcinogens tested in the in vivo Comet assay Chemical

Result

Chemical

Result

l-Ascorbic acid [286] Benzoin [7] Benzyl alcohol [7]

− − +

− − −

Caprolactam [7]

−

p-Chloroaniline [7] 3-Chloro-p-toluidine [7] 2,5-Diaminotoluene [91] 2,6-Diaminotoluene [7]

+ − − −

1,2-Dichlorobenzene [7]

−

2,4-Dichlorophenol [7] Dimethoate [7] 2,4-Dimethoxyaniline [91]

+ + E

EDTA trisodium [7] 8-Hydroxyquinoline [7]

− −

Lithocholic acid [7] dl-Menthol [7] 4-Nitroanthranilic acid [91] 4-Nitro-ophenylenediamine [91] Phenol [7] p-Phenylenediamine [7] Phenylephrine [7] N-phenyl-pphenylenediamine [7] Sodium diethyldithiocarbamate [287] Sodium fluoride [288] Sodium hypochlorite [289] Tetramethylthiuram disulfide [290] l-Tryptophan [7]

−

+ − − −

−

− − − −

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• p-chloroaniline may be considered a genotoxic non-carcinogen, i.e. a substance that demonstrates widespread positive genotoxic responses in vitro and in vivo but where tissue levels are not sufficient to lead to tumour induction in a long-term bioassay, • although phenol is considered a non-carcinogen, there are increased frequencies of lymphomas and leukaemias in the treated groups in the cancer studies. If all six chemicals are considered to have given positive responses in the Comet assay, out of a total of 27 non-carcinogens this would give a specificity for the Comet assay of 77.8%. 8. Conclusions For rodent carcinogens that are negative in the conventional bone-marrow micronucleus assay, the Comet assay is the best follow-up with which to find them positive. The transgenic mutation assay is also a useful follow-up, particularly for those substances producing a predominantly “gene-mutation signature” in vitro. The rat-liver UDS test was very poor at detecting these “missed” carcinogens (<20%) and was not even sensitive to the subset of carcinogens giving a gene-mutation signature in vitro. Although the Comet and TG assays show high sensitivity to these particular rodent carcinogens, they show generally acceptable specificity (69 and 78% negative results with non-carcinogens) when evaluated against all published results with non-carcinogens. It is recommended that the Comet and TG assays should be preferred to the rat-liver UDS assay for regulatory testing. Although the TG assay measures “fixed” mutations, whereas the Comet-assay measures DNA strand breakage, and each endpoint has some relevance to carcinogenicity, it is not possible at this time to state a preference for one assay over the other for follow-up in vivo testing. A recent recommendation has been made for a simple and straightforward approach to genotoxicity testing for those cases where in vivo testing is indicated, and that is to perform a single study combining the bone-marrow MN test with the Comet assay in appropriately selected tissues [5]. Although TG assays were suggested as a potential alternative to the Comet assay, some arguments seem to speak in favour of the Comet assay, e.g. its ease of use, and ability to detect a broader spectrum of primary DNA alterations (relevant for the formation of chromosome and gene mutations). On the other hand, there is still a need for standardisation and validation of the Comet assay, and such activities are already initiated [6]. There is also a need to check the accuracy and reproducibility of the results so far obtained, as many of the published in vivo Comet-assay results have all been generated by one research group (Sasaki et al. [3,7,31,40,41,53,79,91,210]). In any case, more data are required before a general recommendation can be made. Conflict of interest statement None. References ¨ [1] D. Kirkland, M. Aardema, L. Henderson, L. Muller, Evaluation of the ability of a battery of 3 in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1–256. [2] B.L. Pool-Zobel, C. Guigas, R. Klein, Ch. Neudecker, H.W. Renner, P. Schmezer, Assessment of genotoxic effects by lindane, Food Chem. Toxicol. 31 (1993) 271–283. [3] Y.F. Sasaki, F. Izumiyama, E. Nishidate, N. Matsusaka, S. Tsuda, Detection of rodent liver carcinogen genotoxicity by the alkaline single-cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow), Mutat. Res. 391 (1997) 201–214. [4] I.B. Lambert, T.M. Singer, S.E. Boucher, G.R. Douglas, Detailed review of transgenic rodent mutation assays, Mutat. Res. 590 (2005) 1–280.

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