UKEMS trial: Bacterial mutation tests of 4-chloromethylbiphenyl, 4-hydroxymethylbiphenyl,and benzyl chloride,using E. coli WP2uvrA(pKM101) and S. typhimurium TA98 and TA100

4CMB, 4HMB and BC were assayed in plate tests, using E. coli WP2uvrA(pKM101), and S. typhimurium TA98 and TA100, in the presence or absence of microsomal activation. 4CMB was also assayed in fluctuation tests using E. coli WP2uvrA(pKM101). 4HMB was uniformly negative, and 4CMB was mutagenic to all 3 strains. BC was negative in TA98 and positive in TA100 and WP2uvrA(pKM101). The presence or absence of S9 made no substantial difference to the mutagenicity of 4CMB or BC.     

UKEMS trial compounds: In vitro bacterial mutagenicity

4CMB, 4HMB and BC were examined in the Ames test using Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100. 4CMB was mutagenic for all of the indicator strains, 4HMB was inactive and BC was weakly mutagenic for TA100 only.     

4-chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethylbiphenyl (4HMB): Reverse mutation tests with Salmonella typhimurium

In this study 4CMB was shown to be a strong, direct-acting, mutagen for S. typhimurium strains TA1538, TA1537, TA98 and TA100. However, for strain TA1535 the compound was only weakly mutagenic. No conclusive evidence of mutagenic activity was seen in tests with BC or 4HMB.     

Bacterial mutagenicity tests on 4-chloromethylbiphenyl and 2 structural analogues

4CMB, 4HMB and BC were tested in 5 strains of S. typhimurium and 2 strains of E. coli without S9. 4HMB was negative in all strains. 4CMB was a strong positive mutagen in TA1535, TA1537, TA1538, TA98, TA100 and WP2uvrA⁻(pKM101), and BC was a weak mutagen in TA100 and WP2uvrA⁻(pKM101). Positivity was determined as a dose response over 3 or more points, in repeat experiments, giving a significant correlation coefficient.     

The induction of mitotic gene conversion in the yeast,Saccharomyces cerevisiae JD1 by 4-chloromethylbiphenyl (4CMB), Benzyl Chloride (BC) and 4-hydroxymethylbiphenyl (4HMB)

The induction of mitotic gene conversion by 4CMB, BC and 4HMB was studied in both log-phase and stationary-phase cultures of the yeast, Saccharomyces cerevisiae JD1. Assays were performed both in the presence and in the absence of S9 microsomal fraction obtained from a liver homogenate from rats pretreated with Aroclor 1254.Exposure of both stationary-phase and log-phase cultures to 4CMB and BC resulted in an increase in mitotic gene conversion, both in the presence and in the absence of a microsomal activation system; the magnitude of response was greater in stationary-phase cultures. 4HMB did not increase the gene conversion frequency in log-phase or stationary-phase cultures.     

Chromosome analysis of cultured rat-liver epithelial cells (RL4) treated with 4-chloromethylbiphenyl, 4-hydroxymethylbiphenyl and benzyl chloride

Cultured rat-liver epithelial cells, RL₄, were exposed to maximum concentrations of 24 μg/ml 4CMB, 100 μg/ml 4HMB and 30 μg/ml BC. 4CMB and BC induced highly significant numbers of aberrations; 4HMB did not cause an increase in aberration levels.     

The activity of 4CMB, 4HMB and BC in Saccharomyces cerevisiae JD1

3 structurally related compounds, 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB), and benzyl chloride (BC) were assayed for their ability to induce mitotic gene conversion in stationary phase cultures of the yeast, Saccharomyces cerevisiae JD1. This strain allows gene conversion to be scored at 2 independent loci, trp 5 and his 4.The results reported in this paper indicate that both 4CMB and BC are genetically active in yeast, producing dose-related increases in mitotic gene conversion at both the loci tested; 4HMB showed no such activity. At high survival levels 4CMB and BC showed comparable activity. However, as toxicity increased BC showed much more potent convertogenic activity, whereas with 4CMB a reduction in induced gene conversion was observed. The presence of a microsomal activation system derived from the livers of Aroclor-induced male rats did not significantly affect the activity of any of the compounds.     

The biological activity of 4-chloromethylbiphenyl, benzyl chloride and 4-hydroxymethylbiphenyl in 4 short-term tests for carcinogenicity. A report of an individual study in the UKEMS genotoxicity trial 1981

4-Chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethyl-biphenyl (4HMB) were tested for biological activity in the following assays: (i) the Salmonella/microsome assay; (ii) a bacterial 'fluctuation' assays; (iii) a DNA repair assay in Hela cells, and (iv) a mouse lymphoma mutation assay. 4CMB was active in assays (i), (ii) and (iii) but not in (iv); BC was active in assays (i), (ii), (iii) but not in (iv) while 4HMB was inactive in all assays. Where biological activity was seen this did not require addition of a liver S9 preparation. 4CMB was more active than BC in all the test systems in which a positive response was obtained. The implication of these results for a test battery approach to in vitro testing is discussed.     

The biological activity of 4-chloromethylbiphenyl,benzyl chloride and 4-hydroxymethylbiphenyl in 4 short-term tests for carcinogenicity: A report of an individual study in the UKEMS genotoxity trial 1981

4-Chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethyl-biphenyl (4HMB) were tested for biological activity in the following assays: (i) the Salmonella/microsome assay; (ii) a bacterial ‘fluctuation’ assays; (iii) a DNA repair assay in Hela cells, and (iv) a mouse lymphoma mutation assay. 4CMB was active in assays (i), (ii) and (iii) but not in (iv); BC was active in assays (i), (ii), (iii) but not in (iv) while 4HMB was inactive in all assays. Where biological activity was seen this did not require addition of a liver S9 preparation. 4CMB was more active than BC in all the test systems in which a positive response was obtained. The implication of these results for a test battery approach to in vitro testing is discussed.     

A comparison of the response to 4CMB, 4HMB and BC of 5 yeast strains differing in their radiosensitivities

The 3 test compounds 4CMB, 4HMB and BC were assayed for their genotoxicity using stationary phase cultures of 5 yeast strains which differ in their mutagen sensitivity. It was found that 4HMB produced no differences in survival between the 5 strains whereas 4CMB and BC caused more lethality in the triple rad strain than the other 4 strains. The results indicate that both BC and 4CMB are capable of inducing DNA damage which results in cell lethality in the repair-deficient triple mutant.     

The effects of 4CMB, 4HMB and BC on SCE,chromosome aberration and point mutation in cultured Chinese hamster ovary cells

Cultured Chinese hamster ovary cells (CHO-KI-BH₄) were treated for 2 h with 4CMB, 4HMB and BC, in the absence of any exogenous metabolic activation system. The cells were subjected to tests for survival, sister-chromatid exchange, chromosome aberration and mutation to thioguanine resistance.4HMB had no effect in any test at concentrations up to 100 μg/ml. 4CMB was slightly more toxic than BC. Both 4CMB and BC induced SCE and chromosome aberrations, but the effects were more marked with BC. With 4CMB, SCE increased with dose only up to about 7 μg/ml and then levelled off. A weak mutagenic effect was observed with both BC and 4CMB, but in each case, the response reached a peak and was not evident at higher doses.     

An assay for mutagenic activity of 4CMB, 4HMB and BC,using the “microtitre” fluctuation test

4CMB, 4HMB and BC were assayed for mutagenic activity using the ‘microtitre’ bacterial fluctuation test without metabolic activation. 4CMB was positive in strains of Salmonella typhimurium detecting both base-substitution and frameshift mutation. BC was weakly positive only in the strain which detects base-substitution mutation. 4HMB was negative in both strains. 4CMB and 4HMB were equally toxic to the strains, whilst BC was comparatively less toxic.     

The mutagenic activity of 4-chloromethylbiphenyl (4CMB) and Benzyl Chloride (BC) in the bacterial/microsome assay

The mutagenic activity of 4CMB was investigated in agar layer cultures of Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100, and Escherichia coli WP2 and WP2 uvrA. The mutagenic activity of BC was investigated in the Salmonella strains only. Assays were performed both in the absence and in the presence of S9 microsomal fraction obtained from a liver homogenate from rats pretreated with Aroclor 1254.     

Attempts to initiate skin tumours in mice in the 2-stage system using 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB), and benzyl chloride (BC). Report of the experiment at 10 months

Groups of T.O. mice (Theiler's Original, derived from Swiss albino mice) were treated topically, with a single dose of 1.0 mg of 4CMB, 4HMB, or BC, or with 0.4 mg benzo[a]pyrene (B[a]P). Twice-weekly promotion of the treated skin area with croton oil was begun 1 week later. At 10 months the skin-tumour incidence in the positive control (B[a]P) was 8/19, with a mean latent period of 20 weeks. Both 4CMB and 4HMB have so far produced 1 papilloma each (1/36 at 40 weeks, and 1/39 at 34 weeks, respectively), while BC has produced none. Further time is required in order to ascertain whether these single papillomas will develop into carcinomas and thereby herald weak initiating activity for 4CMB and 4HMB.     

Genetic activities of 4-chloromethylbiphenyl,the 4-hydroxy derivative and benzyl chloride in the soma and germ line of Drosophila melanogaster

The genetic activities of 4CMB, 4HMB and BC were assayed as regards the induction of somatic alterations in gene expression on an unstable w⁺ locus with an intragenic TE and all the simultaneously induced germinal mutations on the X-chromosome carrying this locus. The compounds were applied topically in solution at equimolar doses on late embryos and newly hatched larvae. The somatic events were scored as aberrantly pigmented eye sectors in the emerging adult males and the germinal mutations in their F₂ progeny, according to the Muller-5 technique.The somatic events were expressed as red or white mosaic eye sectors; the former could be an outcome of the repression or deletion of the zeste-regulatory proximal subunits of w⁺ locus, and the latter generally attributable to deletions (w⁻) within its structural part. All 3 compounds were effective in the induction of red sectors at the higher tested doses (0.5–2.0 mM) and the level of this activity was virtually the same for 4CMB and 4HMB, but was 2-fold higher for BC. In contrast, the frequencies of the simultaneously scored white sectors were not raised significantly above the controls with 4CMB, but showed decisive increases above this level with both 4HMB and BC.The germinal X-chromosome mutations (recessive lethals and visibles) were only induced at the highest tested dose (2.0 mM), and their frequencies were virtually the same for all 3 compounds reaching a common level of about 0.6%, which is some 3-fold the normal control level for the test system. Specific-locus mutability at the TE w⁺ was suggestively positive only with BC.     

Assay of the induction of mitotic crossing-over and aneuploidy in yeast by BC, 4CMB and 4HMB

Both BC and 4CMB but not 4HMB were shown to be capable of inducing mitotic crossing-over in exponential phase cells of the D6 strain of the yeast Saccharomyces cerevisiae. In contrast, none of the 3 test compounds were capable of inducing mitotic chromosome aneuploidy.     

Mutagenicity testing with TA97 and TA102 of 30 DNA-damaging compounds, negative with other Salmonella strains

In a comparative study on 135 compounds of various chemical classes, 30 agents inducing direct nonreparable DNA damage in repair-deficient E. coli failed in reverting strains TA1535, TA1537, TA1538, TA98 and TA100 of S. typhimurium (De Flora et al., 1984b). These compounds were re-assayed in the Ames test using strains TA97 and TA102. A dose-dependent mutagenic response was detected with aminoantipyrine and p-rosaniline in TA97 and with streptomycin and formaldehyde in TA102. p-Rosaniline was the only mutagen requiring metabolic activation. 5 compounds, i.e. o-aminophenol in TA97 and methanol, ethanol, cadmium chloride and cadmium sulfate in TA102, induced a reproducible increase in revertants over controls, but this was less than 2-fold. The remaining 21 chemicals--including amino compounds, aliphatics, aromatics, heterocycles, hydrazine derivatives and inorganics--confirmed their inactivity in the Ames test. Overall data for 135 compounds, comparing the Ames test (7 strains) and the DNA-repair test (3 strains), are re-assessed on the basis of these findings.     

UKEMS trial compounds: Induction of unscheduled DNA synthesis in HeLa S3 cells

4CMB, 4HMB and BC were assayed for their ability to induce unscheduled DNA synthesis (UDS) in HeLa S3 cells. 4CMB elicited a positive response in the presence and absence of S9; 4HMB and BC produced no significant increase in UDS.     

Transformation of BHK 21 C13 cells by BC, 4CMB and 4HMB using the method of styles

4CMB, 4HMB and BC were examined for their cell transforming potential, as judged by baby hamster kidney (BHK) cells growing without attachment in soft agar.     

Carcinogenicity bioassay of 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB) and benzyl chloride (BC) on mouse skin: interim (7 month) report

4CMB, 4HMB, BC of benzo[a]pyrene (BP, positive control) dissolved in toluene have been applied twice weekly to the shaved dorsal region of groups of 20 Alderley Park Swiss albino mice. After 7 months of treatment, 6/20 of the BP test group were confirmed as having developed squamous cell carcinoma of the skin. However, none of the animals in the test groups appeared to have been affected by the test chemicals. This interim observation was supplemented by intercurrent sacrifice of 1 animal from each of the test groups, followed by histopathological examination of sections of the subcutaneous tissue. No neoplastic changes were discerned.