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The genetic toxicology of 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB) and Benzyl Chloride (BC) as assessed by the UKEMS genotoxicity trial (1981)
Mutation Research, 100 (1982) 411-416
411
Elsevier Biomedical Press
THE GENETIC TOXICOLOGY OF 4-CHLOROMETHYLBIPHENYL (4CMB), 4-HYDROXYMETHYLBIPHENYL (4HMB) AND BENZYL CHLORIDE (BC) AS ASSESSED BY THE UKEMS GENOTOXICITY TRIAL (1981)
JAMES M. PARRY
Department of Genetics, University College of Swansea, Swansea SA2 8PP (Great Britain) (Received 13 September 1981) (Accepted 17 September 1981)
An overall summary of the results of the UKEMS collaborative exercise to determine the genetic toxicology of 4CMB, 4HMB and BC is shown in Table 1. The response of each chemical in the various assay systems is outlined below.
4-Chloromethylbiphenyl (4CMB) In bacteria, 4CMB was shown to be a potent, broad spectrum, direct-acting mutagen capable of inducing both frameshift mutation at the hisD3052 and hisC3076 loci of Salmonella typhimurium and base-substitution mutation at the trpE ochre locus of Escherichia coli and the hisG46 locus of Salmonella typhimurium. Mutagenesis in Salmonella strains was increased 2-fold at the hisD3052 locus and greater than 10-fold at the hisG46 locus in the presence of the plasmid PKM101. In the presence of a liver microsome $9 mix the potency of 4CMB as a frameshift mutagen was reduced, whereas no such reduction was observed for base-pair substitution mutagenesis. Such observations suggest that the different mutational events induced by 4CMB are the result of different mechanisms (Venitt, 1982). The fungal data on 4CMB produced by the collaborators in the trial was of a more heterogeneous nature which did not allow for the sophisticated quantitative analysis performed on the bacterial data, however, the data obtained did allow for the assessment of the effects of 4CMB upon a wide range of genetic endpoints. 4CMB was shown to be capable of inducing point mutation in Neurospora, mitotic gene conversion in yeast, primary D N A damage in yeast and mitotic crossing-over in both Aspergillus and yeast. There was no evidence in either Aspergillus or yeast that 4CMB was capable of inducing changes in chromosome number. Where tested, the presence of liver microsome $9 mix reduced the overall toxicity of 4CMB to yeast cells a l t h o u g h there was no evidence of any change in the proportion of genetically altered to viable cells in such experiments. The most sensitive assay of genetic activity in the fungi reported in this trial was the induction of prototrophs by 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
412 TABLE 1 SUMMARY OF THE GENETIC TOXICOLOGY OF 4-CHLOROMETHYLBIPHENYL (4CMB), 4-HYDROXYMETHYLBIPHENYL (4HMB) AND BENZYL CHLORIDE (BC) AS REPORTED BY THE COLLABORATORS IN THE UKEMS GENOTOXICITY TRIAL 1981 Assay
Chemicals studied
system
4CMB
4HMB
BC
Bacteria
Potent frameshift and base-pair substitution mutagen
-ve
Base-pair substitution mutagen
Fungi
(a) Potent inducer of point mutation, mitotic gene conversion and crossing-over
(a) - v e for nuclear effect (b) mitochondrial activity
(a) Potent inducer of point mutation, mitotic gene conversion and crossing-over (b) - v e for induction of changes in chromosome number
(b) - v e for induction of changes in chromosome number DNA damage in eukaryotic cells
Inducer of repairable DNA damage
-ve
Inducer of repairable DNA damage
Point mutation in cultured mammalian cells
Inducer of point mutation
-ve
Inducer of point mutation
Cell transformation
Positive in most studies
Positive in one study
Weak positive
Cytogenetics in vitro
(a) Potent inducer of chromosome aberrations and sisterchromatid exchanges (b) - v e for induction of changes in chromosome number
-ve
Inducer of chromosome aberrations
In vivo mammalian assays
(a) - v e in micronucleus, dominant lethal and sperm morphology (b) Weak positive for chromosome aberrations and sisterchromatid exchanges in bone marrow
-ve
-ve
Tumour induction (skin painting)
Weak positive
-ve
-ve
413
mitotic gene conversion in yeast where levels of induction comparable to that seen in bacteria were observed. In cultured mammalian cells, 4CMB was reported to produce repairable DNA damage in human cells. In the cell transformation assays, the results were ambiguous with 3 positive results and 2 in which no clearcut response could be observed. 4CMB was assayed for its ability to induce point mutations in a number of mammalian cell types and using a number of different selective agents. The results obtained indicate that 4CMB was capable of inducing point mutation in mammalian cells. However considerable differences were observed in the kinetics of response in the different selective systems. In those experiments where it was tested, the presence of $9 mix reduced the mutagenic potential of 4CMB in cultured mammalian cells. In such cultured cells, 4CMB was shown to be capable of inducing both chromosome aberrations (CA) and sister-chromatid exchanges (SCE) with the maximum levels of activity being observed in cultured rat-liver cells. In these experiments where it was tested the presence of liver microsome $9 mix gave higher yields of both CAs and SCEs. 4CMB was shown to be capable of inducing CAs and SCEs at concentrations greater than 10 and 2 #g/ml respectively. As with the fungal assays there was no evidence in the cytogenetic data of any changes in chromosome number after treatment with 4CMB. In the in vivo assays utilized in this trial, 4CMB did not show any ability to induce dominant lethal mutations, micronuclei or aberrations of sperm morphology. Such observations indicate that the in vitro activity of 4CMB was not reproduced in whole animals. However, chromosome aberrations (gaps included) and SCEs were reported in bone marrow studies which suggests that 4CMB is a weak in vivo mutagen (Scott, 1982). The 2 Drosophila assays for the induction of sex-linked recessive lethal mutations gave conflicting results which make the assessment of in vivo activity in Drosophila impossible. At the time of the termination of the short-term tests (i.e. 10 months) there was no clearcut evidence that 4CMB was capable of inducing tumours in the skin painting assays of 'normal' mice (Coombs, 1982). Such a result appears to confirm the apparent lack of activity of 4CMB shown by the in vivo assays of genetic activity. However, the skin painting tests are being continued for a full 2 years and at the time of writing 4CMB treated animals are now showing an increased frequency of skin tumours (Coombs, personal communication) which suggests that 4CMB is a weak skin carcinogen.
Benzyl chloride (BC) Less data were reported on BC and thus this review is based upon a more limited data base than that for 4CMB. In bacteria BC was shown to be a direct-acting base-pair substitution mutagen with about one tenth of the activity of 4CMB. The reproducibility of the data for
414
BC was less than that reported for 4CMB with considerable variation in the results obtained even within a single laboratory. Unlike the reduction in activity of 4CMB in the presence of liver microsome $9, those laboratories which used $9 with BC reported an increase in mutagenic activity in bacteria in its presence. The lower activity of BC as compared to 4CMB in bacteria, was in contrast to the similarity of response of the 2 compounds in a number of the higher order assay systems. In fungi, BC was shown to be a potent inducer of point mutation, mitotic gene conversion and crossing-over with a similar pattern of response to 4CMB. There was no evidence of any induction of changes in chromosome number in fungal cultures treated with BC. In cultured m a m m a l i a n cells BC was shown to induce repairable D N A damage in human cells although it was approximately one fifth as active as 4CMB. As with 4CMB the results of the cell transformation with BC were ambiguous with 2 assays producing weak positive results and 2 assays negative results. Point-mutation assays indicated that BC was an inducer of point mutation using a number of selective systems with a similar activity range to that of 4CMB. BC was shown to induce both chromosome aberrations and sister-chromatid exchanges in cultured m a m m a l i a n cells. These events were induced to similar levels and over essentially the same dose range as that observed for 4CMB. BC was tested in the sperm morphology assays, mouse micronucleus assays, the sex-linked recessive lethal and somatic segregation assays in Drosophila. The only positive response observed was in the Drosophila somatic segregation assay. This assay gave a positive response with all 3 compounds and at the present time the results are difficult to evaluate. Unfortunately no data on BC was made available for in vivo cytogenetics so it is not possible to make a comparison of the effect of BC in those in vivo assays which gave a weak positive response with 4CMB. There was no evidence from the skin painting assays at 10 months nor at the time of writing that BC was capable of inducing skin tumours in ' n o r m a l ' mice (Coombs, 1982).
4-Hydroxymethylbiphenyl (4HMB) There was no significant evidence in the data presented for this trial that 4HMB was capable of inducing changes in the nuclear genes of any of the organisms tested. However, Patel and Wilkie (1982) reported activity for this compound for both mitochondrial mutagenesis and the inhibition of mitochondrial transcription. In view of the overwhelming evidence of the lack of activity of 4HMB in both the short- and long-term tests used in this trial, the significance of this reported mitochondrial activity of 4HMB is difficult to evaluate. CONCLUSIONS
4CMB showed potent genetic activity in bacteria, fungi and mammalian cells in
415 culture. However, its genetic activity was weak in whole animals. No significant genetic activity was detected with 4 H M B with the exception of its mitochondrial activity in yeast (Patel and Wilkie, 1982). BC showed a similar spectrum of genetic activity to 4CMB in fungi and cultured mammalian cells. However, it was less potent in bacteria and the in vivo insect and m a m m a l i a n system data were sparse. Some important lessons for genetic toxicology testing in general can be derived f r o m the results of the UKEMS Trial. For example, the trial highlights the value of microbial mutagenicity data particularly when related to the chemistry and potential metabolism of a test compound (Venitt, 1982; Parry and Wilcox, 1982). Without such data it is difficult, if not impossible, to design an optimal protocol for the higher order tests. Secondly, the trial illustrates that valid predictions of the potential genetic toxicology of a compound in mammalian cells cannot be made from data derived from the 'standard' bacterial mutagenicity assays. Some of the reasons for the failure of the bacterial mutagenicity assays to provide predictive data on these chemicals are discussed in detail in the following paper (Ashby et al., 1982). The study highlights the very considerable differences that still exist between the results generated by apparently similar assays such as the sex-linked recessive lethal test in Drosophila (see T o p h a m , 1982) and the significant differences in sensitivity of the various cell types used in the in vitro cytogenetic assays (Scott, 1982). Considerable differences were also observable in the responses of the various selective systems used in the cultured m a m m a l i a n cell mutation assays (Arlett et al., 1982). In view of the differences in the qualitative responses observed in the m a m m a l i a n mutation assays it is difficult to see exactly how such data may be used in evaluation of the genetic toxicology of environmental chemicals. The mammalian mutation tests performed in the collaborative exercise were expensive in both time and labour and when adequately performed could not be considered as 'short-term assays'. The overall results of this collaborative exercise clearly indicate the value of a range of assay systems in the assessment of the genetic toxicology of environmental chemicals. However, it is also obvious from the results that the selection of an 'optimal' testing package suitable for all chemicals is a far from easy task. The results of this exercise indicate that a more rational approach would be the selection of a range of assay systems based upon a knowledge of the chemical characteristics of each tested chemical. REFERENCES Arlett, C.F., W.J. Muriel, J. Cole and J. Lowe (1982) The induction of mutants in L51787 mouse lymphoma cells by 4CMB, Mutation Res., 100, 253-256. Ashby, J., P.A. Lefevre, B.M. Elliott and J.A. Styles (1982) An overviewof the chemical and biological reactivity of 4CMB and structurally related compounds: possible relevance to the overall findings of the UKEMS 1981 study, Mutation Res., 100, 417-433. Coombs, M. (1982) The UKEMS GenotoxicityTrial: A summary of the assays for skin tumour induction
416 in mice, the subcutaneous implant test and the sebaceous gland suppression test, Mutation Res., 100, 407-409. Parry, J.M., and P. Wilcox (1982) The genetic toxicology in fungi of 4CMB, 4HMB and BC: Survey of the results of the UKEMS collaborative Genotoxicity Trial (1981), Mutation Res., 100, 185-200. Patel, R., and D. Wilkie (1982) Mitochondrial toxicity in Saccharomyces as a measure of carcinogenicity, Mutation Res., 100, 179-183. Scott, D. (1982) UKEMS Collaborative Genotoxicity Trial: cytogenetic tests of 4CMB, BC and 4HMB: Summary and appraisal, Mutation Res., 100. Topham, J.C. (1982) UKEMS Genotoxicity Trial, In vivo assay systems, Mutation Res., 100, 381-387. Venitt, S. (1982) UKEMS Collaborative Genotoxicity Trial: bacterial mutation tests of 4CMB, 4HMB and BC: analysis of data from 17 laboratories, Mutation Res., 100, 313-331.
411
Elsevier Biomedical Press
THE GENETIC TOXICOLOGY OF 4-CHLOROMETHYLBIPHENYL (4CMB), 4-HYDROXYMETHYLBIPHENYL (4HMB) AND BENZYL CHLORIDE (BC) AS ASSESSED BY THE UKEMS GENOTOXICITY TRIAL (1981)
JAMES M. PARRY
Department of Genetics, University College of Swansea, Swansea SA2 8PP (Great Britain) (Received 13 September 1981) (Accepted 17 September 1981)
An overall summary of the results of the UKEMS collaborative exercise to determine the genetic toxicology of 4CMB, 4HMB and BC is shown in Table 1. The response of each chemical in the various assay systems is outlined below.
4-Chloromethylbiphenyl (4CMB) In bacteria, 4CMB was shown to be a potent, broad spectrum, direct-acting mutagen capable of inducing both frameshift mutation at the hisD3052 and hisC3076 loci of Salmonella typhimurium and base-substitution mutation at the trpE ochre locus of Escherichia coli and the hisG46 locus of Salmonella typhimurium. Mutagenesis in Salmonella strains was increased 2-fold at the hisD3052 locus and greater than 10-fold at the hisG46 locus in the presence of the plasmid PKM101. In the presence of a liver microsome $9 mix the potency of 4CMB as a frameshift mutagen was reduced, whereas no such reduction was observed for base-pair substitution mutagenesis. Such observations suggest that the different mutational events induced by 4CMB are the result of different mechanisms (Venitt, 1982). The fungal data on 4CMB produced by the collaborators in the trial was of a more heterogeneous nature which did not allow for the sophisticated quantitative analysis performed on the bacterial data, however, the data obtained did allow for the assessment of the effects of 4CMB upon a wide range of genetic endpoints. 4CMB was shown to be capable of inducing point mutation in Neurospora, mitotic gene conversion in yeast, primary D N A damage in yeast and mitotic crossing-over in both Aspergillus and yeast. There was no evidence in either Aspergillus or yeast that 4CMB was capable of inducing changes in chromosome number. Where tested, the presence of liver microsome $9 mix reduced the overall toxicity of 4CMB to yeast cells a l t h o u g h there was no evidence of any change in the proportion of genetically altered to viable cells in such experiments. The most sensitive assay of genetic activity in the fungi reported in this trial was the induction of prototrophs by 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
412 TABLE 1 SUMMARY OF THE GENETIC TOXICOLOGY OF 4-CHLOROMETHYLBIPHENYL (4CMB), 4-HYDROXYMETHYLBIPHENYL (4HMB) AND BENZYL CHLORIDE (BC) AS REPORTED BY THE COLLABORATORS IN THE UKEMS GENOTOXICITY TRIAL 1981 Assay
Chemicals studied
system
4CMB
4HMB
BC
Bacteria
Potent frameshift and base-pair substitution mutagen
-ve
Base-pair substitution mutagen
Fungi
(a) Potent inducer of point mutation, mitotic gene conversion and crossing-over
(a) - v e for nuclear effect (b) mitochondrial activity
(a) Potent inducer of point mutation, mitotic gene conversion and crossing-over (b) - v e for induction of changes in chromosome number
(b) - v e for induction of changes in chromosome number DNA damage in eukaryotic cells
Inducer of repairable DNA damage
-ve
Inducer of repairable DNA damage
Point mutation in cultured mammalian cells
Inducer of point mutation
-ve
Inducer of point mutation
Cell transformation
Positive in most studies
Positive in one study
Weak positive
Cytogenetics in vitro
(a) Potent inducer of chromosome aberrations and sisterchromatid exchanges (b) - v e for induction of changes in chromosome number
-ve
Inducer of chromosome aberrations
In vivo mammalian assays
(a) - v e in micronucleus, dominant lethal and sperm morphology (b) Weak positive for chromosome aberrations and sisterchromatid exchanges in bone marrow
-ve
-ve
Tumour induction (skin painting)
Weak positive
-ve
-ve
413
mitotic gene conversion in yeast where levels of induction comparable to that seen in bacteria were observed. In cultured mammalian cells, 4CMB was reported to produce repairable DNA damage in human cells. In the cell transformation assays, the results were ambiguous with 3 positive results and 2 in which no clearcut response could be observed. 4CMB was assayed for its ability to induce point mutations in a number of mammalian cell types and using a number of different selective agents. The results obtained indicate that 4CMB was capable of inducing point mutation in mammalian cells. However considerable differences were observed in the kinetics of response in the different selective systems. In those experiments where it was tested, the presence of $9 mix reduced the mutagenic potential of 4CMB in cultured mammalian cells. In such cultured cells, 4CMB was shown to be capable of inducing both chromosome aberrations (CA) and sister-chromatid exchanges (SCE) with the maximum levels of activity being observed in cultured rat-liver cells. In these experiments where it was tested the presence of liver microsome $9 mix gave higher yields of both CAs and SCEs. 4CMB was shown to be capable of inducing CAs and SCEs at concentrations greater than 10 and 2 #g/ml respectively. As with the fungal assays there was no evidence in the cytogenetic data of any changes in chromosome number after treatment with 4CMB. In the in vivo assays utilized in this trial, 4CMB did not show any ability to induce dominant lethal mutations, micronuclei or aberrations of sperm morphology. Such observations indicate that the in vitro activity of 4CMB was not reproduced in whole animals. However, chromosome aberrations (gaps included) and SCEs were reported in bone marrow studies which suggests that 4CMB is a weak in vivo mutagen (Scott, 1982). The 2 Drosophila assays for the induction of sex-linked recessive lethal mutations gave conflicting results which make the assessment of in vivo activity in Drosophila impossible. At the time of the termination of the short-term tests (i.e. 10 months) there was no clearcut evidence that 4CMB was capable of inducing tumours in the skin painting assays of 'normal' mice (Coombs, 1982). Such a result appears to confirm the apparent lack of activity of 4CMB shown by the in vivo assays of genetic activity. However, the skin painting tests are being continued for a full 2 years and at the time of writing 4CMB treated animals are now showing an increased frequency of skin tumours (Coombs, personal communication) which suggests that 4CMB is a weak skin carcinogen.
Benzyl chloride (BC) Less data were reported on BC and thus this review is based upon a more limited data base than that for 4CMB. In bacteria BC was shown to be a direct-acting base-pair substitution mutagen with about one tenth of the activity of 4CMB. The reproducibility of the data for
414
BC was less than that reported for 4CMB with considerable variation in the results obtained even within a single laboratory. Unlike the reduction in activity of 4CMB in the presence of liver microsome $9, those laboratories which used $9 with BC reported an increase in mutagenic activity in bacteria in its presence. The lower activity of BC as compared to 4CMB in bacteria, was in contrast to the similarity of response of the 2 compounds in a number of the higher order assay systems. In fungi, BC was shown to be a potent inducer of point mutation, mitotic gene conversion and crossing-over with a similar pattern of response to 4CMB. There was no evidence of any induction of changes in chromosome number in fungal cultures treated with BC. In cultured m a m m a l i a n cells BC was shown to induce repairable D N A damage in human cells although it was approximately one fifth as active as 4CMB. As with 4CMB the results of the cell transformation with BC were ambiguous with 2 assays producing weak positive results and 2 assays negative results. Point-mutation assays indicated that BC was an inducer of point mutation using a number of selective systems with a similar activity range to that of 4CMB. BC was shown to induce both chromosome aberrations and sister-chromatid exchanges in cultured m a m m a l i a n cells. These events were induced to similar levels and over essentially the same dose range as that observed for 4CMB. BC was tested in the sperm morphology assays, mouse micronucleus assays, the sex-linked recessive lethal and somatic segregation assays in Drosophila. The only positive response observed was in the Drosophila somatic segregation assay. This assay gave a positive response with all 3 compounds and at the present time the results are difficult to evaluate. Unfortunately no data on BC was made available for in vivo cytogenetics so it is not possible to make a comparison of the effect of BC in those in vivo assays which gave a weak positive response with 4CMB. There was no evidence from the skin painting assays at 10 months nor at the time of writing that BC was capable of inducing skin tumours in ' n o r m a l ' mice (Coombs, 1982).
4-Hydroxymethylbiphenyl (4HMB) There was no significant evidence in the data presented for this trial that 4HMB was capable of inducing changes in the nuclear genes of any of the organisms tested. However, Patel and Wilkie (1982) reported activity for this compound for both mitochondrial mutagenesis and the inhibition of mitochondrial transcription. In view of the overwhelming evidence of the lack of activity of 4HMB in both the short- and long-term tests used in this trial, the significance of this reported mitochondrial activity of 4HMB is difficult to evaluate. CONCLUSIONS
4CMB showed potent genetic activity in bacteria, fungi and mammalian cells in
415 culture. However, its genetic activity was weak in whole animals. No significant genetic activity was detected with 4 H M B with the exception of its mitochondrial activity in yeast (Patel and Wilkie, 1982). BC showed a similar spectrum of genetic activity to 4CMB in fungi and cultured mammalian cells. However, it was less potent in bacteria and the in vivo insect and m a m m a l i a n system data were sparse. Some important lessons for genetic toxicology testing in general can be derived f r o m the results of the UKEMS Trial. For example, the trial highlights the value of microbial mutagenicity data particularly when related to the chemistry and potential metabolism of a test compound (Venitt, 1982; Parry and Wilcox, 1982). Without such data it is difficult, if not impossible, to design an optimal protocol for the higher order tests. Secondly, the trial illustrates that valid predictions of the potential genetic toxicology of a compound in mammalian cells cannot be made from data derived from the 'standard' bacterial mutagenicity assays. Some of the reasons for the failure of the bacterial mutagenicity assays to provide predictive data on these chemicals are discussed in detail in the following paper (Ashby et al., 1982). The study highlights the very considerable differences that still exist between the results generated by apparently similar assays such as the sex-linked recessive lethal test in Drosophila (see T o p h a m , 1982) and the significant differences in sensitivity of the various cell types used in the in vitro cytogenetic assays (Scott, 1982). Considerable differences were also observable in the responses of the various selective systems used in the cultured m a m m a l i a n cell mutation assays (Arlett et al., 1982). In view of the differences in the qualitative responses observed in the m a m m a l i a n mutation assays it is difficult to see exactly how such data may be used in evaluation of the genetic toxicology of environmental chemicals. The mammalian mutation tests performed in the collaborative exercise were expensive in both time and labour and when adequately performed could not be considered as 'short-term assays'. The overall results of this collaborative exercise clearly indicate the value of a range of assay systems in the assessment of the genetic toxicology of environmental chemicals. However, it is also obvious from the results that the selection of an 'optimal' testing package suitable for all chemicals is a far from easy task. The results of this exercise indicate that a more rational approach would be the selection of a range of assay systems based upon a knowledge of the chemical characteristics of each tested chemical. REFERENCES Arlett, C.F., W.J. Muriel, J. Cole and J. Lowe (1982) The induction of mutants in L51787 mouse lymphoma cells by 4CMB, Mutation Res., 100, 253-256. Ashby, J., P.A. Lefevre, B.M. Elliott and J.A. Styles (1982) An overviewof the chemical and biological reactivity of 4CMB and structurally related compounds: possible relevance to the overall findings of the UKEMS 1981 study, Mutation Res., 100, 417-433. Coombs, M. (1982) The UKEMS GenotoxicityTrial: A summary of the assays for skin tumour induction
416 in mice, the subcutaneous implant test and the sebaceous gland suppression test, Mutation Res., 100, 407-409. Parry, J.M., and P. Wilcox (1982) The genetic toxicology in fungi of 4CMB, 4HMB and BC: Survey of the results of the UKEMS collaborative Genotoxicity Trial (1981), Mutation Res., 100, 185-200. Patel, R., and D. Wilkie (1982) Mitochondrial toxicity in Saccharomyces as a measure of carcinogenicity, Mutation Res., 100, 179-183. Scott, D. (1982) UKEMS Collaborative Genotoxicity Trial: cytogenetic tests of 4CMB, BC and 4HMB: Summary and appraisal, Mutation Res., 100. Topham, J.C. (1982) UKEMS Genotoxicity Trial, In vivo assay systems, Mutation Res., 100, 381-387. Venitt, S. (1982) UKEMS Collaborative Genotoxicity Trial: bacterial mutation tests of 4CMB, 4HMB and BC: analysis of data from 17 laboratories, Mutation Res., 100, 313-331.