R.D. Callander

Chloracetamide-N-metholol: an example of an in vitro and in vivo clastogen which is non-mutagenic to Salmonella

The industrial biocide chloracetamide-N-metholol (CAM) has been shown to be non-mutagenic to 6 strains of Salmonella using both the plate-incorporation and a pre-incubation test protocol. Its biocidal activity is unlikely to have influenced these results since Kathon 886, a more potent biocide, was concomitantly detected as mutagenic to strain TA100. In contrast, CAM was weakly clastogenic to human lymphocytes cultured in vitro and elicited a positive response in the mouse bone marrow micronucleus test when assayed using the intraperitoneal, but not the oral route of administration. A positive response was concomitantly observed for the rodent carcinogen and formaldehyde-releasing agent hexamethylphosphoramide (HMPA) in these 2 clastogenicity assays. Data are presented showing the slow hydrolysis of CAM to formaldehyde in vitro, and both [carbonyl-14C]CAM and [metholol-14C]CAM have been shown to interact covalently with calf-thymus DNA in vitro. It is concluded that CAM may be a direct-acting carcinogen to rodents, but that both the qualitative and quantitative outcome of its bioassay for carcinogenicity will be influenced critically by the bioassay protocol adopted; in particular, by the route of administration selected. These findings emphasize the need to complement the Salmonella gene-mutation assay with an in vitro assay for the induction of chromosomal aberrations if in vivo genotoxins are to be detected efficiently in vitro.

Studies on the genotoxicity of beryllium sulphate in vitro and in vivo

There is limited evidence that beryllium is a lung carcinogen to man, and several compounds of beryllium are carcinogenic to the lungs of the rat, rabbit and monkey. One such compound is beryllium sulphate (BeSO4.4H2O). This soluble salt has been evaluated in a range of genotoxicity tests. It was non-mutagenic to Salmonella typhimurium (strains TA1535, 1537, 1538, 98 and 100) when evaluated in the plate-incorporation assay at dose levels up to 5 mg/plate (+/- induced rat-liver S9 mix). It was also non-clastogenic to Chinese hamster lung (CHL) cells cultured in vitro. When dosed to male CBA mice via oral gavage at dose levels of 80% and 50% of the medium lethal dose (2.3 and 1.4 g/kg, respectively) it failed to increase the incidence of micronucleated polychromatic erythrocytes in the bone marrow (sampled at 24, 48 and 72 h post-dosing). However, a marked depression of erythropoiesis was evident 24 h after dosing suggestive of beryllium-mediated bone-marrow toxicity. When tested in the strain A mouse lung tumour bioassay, BeSO4 induced a significant increase in the number of tumour-bearing animals but not in the number of lung tumours per animal. These findings are discussed within the contexts of other genotoxicity data published for BeSO4, and of current strategies for the detection of possible human carcinogens.

Weak mutagenicity to Salmonella of the formaldehyde-releasing anti-tumour agent hexamethylmelamine

Hexamethylmelamine (HEMLA) is a metabolism-dependent formaldehyde-releasing agent related in structure to hexamethylphosphoramide (HMPA). Both compounds are known to be mutagenic to Drosophila. HMPA, in common with the other formaldehyde-releasing agents studied, is non-mutagenic to Salmonella. The present paper describes the mutagenicity of HEMLA to strain TA100 of Salmonella typhimurium. Activity is dependent upon both the use of a pre-incubation assay protocol and on high concentrations of Aroclor-induced rat liver in the S9 mix. HMPA was inactive under similar conditions of test.

N-Chloropiperidine and calcium hypochlorite: Possible examples of toxicity-dependent clastogenicity in vitro

N-Chloropiperidine (NCP) has been reported to be both toxic and mutagenic in a wide range of in vitro and in vivo genotoxicity assays, however, few experimental details or numerical data have been presented to support these claims. The latter facts, together with the lack of any clear structural precedent for the mutagenicity of this agent, led us to re-evaluate it using the Salmonella mutation assay and the mouse bone marrow micronucleus test. The absence of mutagenic activity observed in both of these systems indicates that the genotoxicity of NCP and related chloramines remains to be unequivocally established. In particular, the potent clastogenicity to CHO cells reported for NCP may be related solely to oxidative denaturation of cellular proteins induced by hypochlorous acid, a hydrolysis product of NCP. Separate reports indicate that calcium hypochlorite (bleaching powder) is also clastogenic in vitro but not in vivo. We therefore suggest that N-chloropiperidine may prove of value as a model compound for studies designed to distinguish genotoxic clastogenicity (specifically DNA-reactive) from general toxicity-mediated clastogenicity.

Activity of bromochlorodifluoromethane (BCF) in three mutation tests

The halocarbon BCF was tested in 3 assays to assess its mutagenicity and clastogenicity. It produced a positive response in Salmonella typhimurium strain TA1535 but was negative in TA1537, TA1538, TA98 and TA100. In an L5178Y mouse lymphoma microwell assay (TK locus), BCF was negative. BCF was administered at 5000 and 50 000 ppm in air for 6 h to groups of C57B1/6J mice of both sexes. Animals were killed at 24, 48 and 72 h after cessation of exposure and the incidence of bone marrow micronuclei per 1000 PCEs determined. There was no significant difference in the incidences of micronuclei between untreated animals and those exposed to either concentration of BCF at any of the sampling times. These results suggest that BCF is mutagenic in vitro in only one strain of Salmonella; in mammalian cells the compound induced no gene mutation in vitro nor clastogenic activity in vivo at doses that also produced clear evidence of toxicity.

Lack of mutagenicity to S. typhimurium of neopentyl bromide and pentaerythrityl tetrachloride: relation to chemical structure.

Neopentyl bromide and pentaerythrityl tetrachloride were shown here to be non-mutagenic to 7 strains of Salmonella typhimurium. These inactivities are reflected in the inability of either compound to produce a colour reaction in the chemical alkylation test of Epstein (4-nitrobenzylpyridine, NBP). It is suggested that the lack of biological reactivity of these two alkyl halides is due to steric crowding of the halogen group. These findings are significant within the context of the potent mutagenicity of mono-haloalkanes in general. The similarity between the odour of pentaerythrityl tetrachloride and camphor is discussed.

Mutagenicity to Salmonella of the mono methylamino and N-cyanoethyl analogues of 4-dimethylaminoazobenzene (DAB) and 6-dimethylaminophenylazobenzthiazole (6BT)

Replacement of one of the methyl groups of the carcinogens 4-dimethylaminoazobenzene (DAB) and 6-dimethylaminophenylazobenzthiazole (6BT) with a cyanoethyl (-CH2CH2CN) substituent dramatically increases their mutagenic potency to Salmonella (strain TA98). The corresponding monomethylamino derivatives (-NHCH3) are more mutagenic than the parent dimethylamino [-N(CH3)2] compounds, but substantially less mutagenic than the cyanoethyl derivatives. All of these mutagenic activities are liver-S-9-dependent. The very similar dose response curves observed for the two cyanoethyl compounds argues for the formation of a common electrophilic intermediate from each.

Non-mutagenicity of 3-dimethylamino-ψ-saccharin to Salmonella typhimurium

3-Dimethylamino-psi-saccharin has been confirmed as non-mutagenic to Salmonella typhimurium when evaluated in vitro in the presence of induced rat liver S9 mix.

The Weak Mutagenicity of CDA to Strain TA98 of Salmonella typhimurium

4-Cyanodimethylaniline (CDA) was purified by thick layer chromatography followed by preparative HPLC isolation. It was found to be weakly but reproducibly mutagenic to strain TA98 of S. typhimurium but only when evaluated using a preincubation test protocol. Similar activity was observed for the main study sample of CDA (Sample B). This test protocol and test strain have previously been established as optimum for the detection of DAB as a bacterial mutagen.

Enhanced Mutagenicity of DAT to Salmonella (-S9) when Evaluated under Conditions of Reduced Light

Preliminary experiments conducted in separate laboratories indicate that the S9-independent mutagenicity of DAT to Salmonella is enhanced by testing under conditions of reduced light.