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The C2-alkylated acrolein derivatives 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein are mutagenic in Salmonella typhimurium TA100. They are direct mutagens, their mutagenic potency being inversely proportional to the size of the alkylating substituent in the C2 position. In the presence of S9 mix, the mutagenicity of all these substances is considerably reduced; the reduction in mutagenicity is inversely proportional to the direct mutagenic potential of the substance. As shown for 2-methylacrolein, the reduction in mutagenicity is dependent on the concentration of S9 in the S9 mix and is not significantly influenced by heat inactivation of the S9 mix or by addition of TCPO, an inhibitor of epoxide hydrolase, to the testing system. There are no indications of enzymatic activation by the metabolizing microsomal system.

Hexachloro(1,3)butadiene (HCBD) is a well known environmental contaminant. The nephrocarcinogenic potential of HCBD has been shown in long-term studies with rats. Experiments were performed to assist in determining whether this effect is mediated by epigenetic or genotoxic mechanisms and to compare the mutagenic properties of HCBD with those of its monooxidation products, perchloro-3-butenoic acid (PCBA) and perchloro-3-butenoic acid chloride (PCBAC), which are conceivable metabolites of HCBD. All 3 compounds are mutagenic to the Salmonella typhimurium tester strain TA100. The mutagenic effect is dose-dependent and parallels the chemical reactivity of the compounds. HCBD is only mutagenic in the presence of drug-metabolizing enzymes (S9 mix) with an increased protein content. The mutagenic response after incubation with PCBAC and PCBA is 2-3-fold that of HCBD. Additionally, both PCBAC and PCBA exert a mutagenic response in the absence of S9 mix. The experiments support the assumption of a genotoxic potential of HCBD.

Thirty-nine allylic and non-allylic compounds have been tested in the standard 4-(p-nitrobenzyl)pyridine (NBP) alkylating procedure and the Salmonella typhimurium mutagenicity assay. Fourteen of these were found directly mutagenic (without addition of S-9 mix activating enzyme system). With twelve of these compounds, a good correlation of alkylating and mutagenic potencies was established; the remaining two do not meet the chemical conditions of the NBP procedure on account of HCl elimination with these two compounds. The other 25 substances were inactive in both systems. The quantitative correlation proved to be almost linear in the lower activity ranges (E approximately 2; revertants/muml approximately 600). The reasons for some deviations from the linear relationship have been analyzed and discussed on the basis of structural features. In addition to the standard alkylation test, a modified NBP-test was performed in order to obtain kinetic data and activation energy values. The results with 6 representative allylic compounds show that the overall correlation is not substantially improved above that of the standard procedure: nonetheless, additional information on reaction characteristics is obtained with some substances.

Dichloroacetylene (DCA) is a highly reactive compound that decomposes rapidly in contact with air into a series of chlorinated aliphatic hydrocarbons (e.g., phosgene, trichloroacetyl chloride, trichloroacryloyl chloride and hexachlorobutadiene). Experiments were performed to compare the mutagenic properties of DCA and its degradation products on the histidine-dependent tester strains TA98 and TA100 of Salmonella typhimurium. In these experiments, DCA vapour was streamed under analytical control through the bacterial suspensions. DCA is soluble in aqueous solution and was stable under the experimental steady-state conditions of the bacterial exposure. There is a linear correlation between the supply of DCA vapour and solubilized DCA in the range of 1000 and 16 000 ppm. Mutagenic response was observed with strain TA100 if the bacteria were suspended in Oxoid medium. No mutagenicity could be detected with strain TA98. DCA mixtures with acetylene, as used as stabilizer for animal experiments, were not mutagenic in either bacterial strain, irrespective of the presence or absence of S9 mix in the cell suspension. One of the degradation products of DCA, trichloroacryloyl chloride, showed pronounced mutagenic properties with and without drug-metabolizing enzymes. Other degradation products of DCA, such as trichloroacetyl chloride and hexachlorobutadiene, were not mutagenic, either in the presence or absence of liver homogenate.

In the presence of S9 mix all allylic chloropropenes tested exert considerable indirect mutagenic activity which is most pronounced for 1,2,3-trichloropropene. Lower as well as higher chlorinated derivatives are clearly less mutagenic. Longer than standard incubation time (120 min instead of 20 min) at 37 degrees C always leads to an increase in mutagenic activity. An increase in concentration of rat-liver homogenate fraction (S9) in the metabolising system (S9 mix) enhances mutagenicity only for 1,3-dichloropropene, 2,3-dichloro-1-propene and for the cis isomer of 1,1,2,3-tetrachloro-2-propene. According to the effects of the enzyme inhibitors SKF525 1,1,1-trichloropropene-2,3-oxide and cyanamide the allylic chloropropenes fall into 3 groups distinguished by their mode of metabolic activation by S9 mix: (a) allyl chloride and 1,3-dichloropropene are hydrolysed to the corresponding allylic alcohols which can be oxidised to the respective acroleins (hydrolytic-oxidative pathway); (b) 2,3-dichloro-1-propene, 1,1,2,3-tetrachloro-2-propene and hexachloropropene are epoxidised in the C=C double bond, giving rise to reactive epoxides (epoxidative pathway); (c) only 1,2,3-trichloropropene is obviously activated by both these alternative metabolic pathways. Structural parameters like chloro-substitution of the central C atom of the C=C-C sequence and substituent-induced polarisation of the C=C double bond as well as cis/trans isomerism might be responsible for different substrate properties for the enzymes involved in allylic chloropropene metabolism, thus determining different degrees of activation by either one or both pathways.

Allyl isothiocyanate, a naturally occurring compound, component of oil of mustard and human food plants such as cabbage, cauliflower and horseradish, has up to now been regarded as nonmutagenic in bacterial mutagenicity testing systems. Recently, however, it was found to cause transitional-cell papillomas in the urinary bladder of male F344 rats. Contrary to earlier reports, in this study allyl isothiocyanate showed clear mutagenicity for Salmonella typhimurium TA100 in the preincubation assay after longer, non-standard preincubation times (greater than 20 min). The mutagenicity is expressed only in the presence of a rat-liver homogenate metabolising system, i.e. it is indirect. However, high concentrations of rat-liver homogenate suppress the mutagenicity of allyl isothiocyanate. SKF525, inhibitor of microsomal oxygenase, reduces the mutagenic potential which on the other hand is increased in the presence of 1,1,1-trichloropropene-2-oxide, inhibitor of epoxide hydrolase. This indicates the occurrence of an epoxide intermediate in allyl isothiocyanate metabolism. Another metabolic pathway, namely hydrolysis to allyl alcohol and oxidation to acrolein, a known mutagen, also seems possible as cyanamide, inhibitor of aldehyde dehydrogenase, can slightly increase the mutagenic potential. The reason(s) for allyl isothiocyanate's requirement for long preincubation times to express mutagenicity still requires elucidation, and the question arises: is allyl isothiocyanate a single, exceptional case or not?

Contrary to findings published up to now, allyl chloride, a well known directly acting mutagen for Salmonella typhimurium, is efficiently activated by rat-liver homogenate (S9 mix) under non-standard mutagenicity testing conditions. Its indirect, S9-mediated mutagenic activity is greatly enhanced when longer than standard preincubation times are applied. The indirect mutagenicity of allyl chloride, thus revealed, greatly exceeds its direct mutagenic activity. Obviously, standard mutagenicity testing conditions cannot be regarded as reliable tools for the evaluation of the full genotoxic potential of allyl chloride and, possibly, of other related compounds.