Cyclic hydrazides are mutagenic for Salmonella typhimurium

At the International Workshop on the Standardisation of Genotoxicity Test Procedures, in Melbourne (27–28 February 1993), the current international guidelines for the correct conduct of bacterial mutation assays were considered, and the major differences between them were examined. An attempt was made to construct a scientifically based, internationally harmonised protocol. The main points of agreement were as follows. The consensus opinion was that there are currently insufficient data to justify a preference for either the preincubation or plate-incorporation methodologies as the initial test. Whichever method is used there was consensus agreement that the bacterial test battery should consist of S. typhimurium TA1537, TA1535, TA98 and TA100. There was also consensus that the 3 strains TA97a, TA97 and TA1537 could be used interchangeably. Although it was not possible to achieve a consensus, the majority of the working group members agreed that strains for the detection of mutagens acting specifically on AT base pairs should be routinely included within the test battery. These strains may be S. typhimurium TA102 or E. coli WP2 strains (WP2 pKM101 and WP2 uvrA or WP2 uvrA pkM101). With regard to study design it was universally agreed that 5 doses of test compound should be used in each experiment, and a majority agreement was obtained for 3 plates per dose. The use of 2 plates per dose is acceptable ONLY if the experiment is repeated. It is recommended that the negative controls may consist of solvent control alone provided that historical data are available to demonstrate lack of effect of the solvent in question. Positive control compounds should be included in all experiments, although the nature of these control compounds need not be specified in the guidelines. There was consensus agreement that for non-toxic freely soluble test agents, an upper limit of 5 mg/plate should be tested (5 μl per plate for liquids). For insoluble or toxic compounds, the recommendations were the same as those for other in vitro tests (see appropriate paper). A consensus agreement was reached on the need to carry our further tests if equivocal results are obtained in the initial test, although it was generally agreed that the design of the repeat study should be left flexible. As there are little or no data to support the use of an exact repeat assay, a majority of the group recommended that negative results in the first test should be further investigated by either conducting a modified repeat (e.g. S9 titration) or by conducting the alternative methodology. If a preincubation assay is carried out, an incubation time within the range 20–60 min is recommended, usually at a temperature of 37°C.

Lastly, a consensus agreement on the acceptable criteria for a positive or negative result could not be reached. There was majority agreement that a reproducible dose-response was necessary for a chemical to be classified as positive.

A number of limitations of the proposed test methodology were recognised and included the adequate testing of specific chemical classes or groups, (i.e. azo-dyes and diazo compounds, gases, volatile chemicals and glycosides). Such ‘special cases’ should be evaluated using alternative published procedures.

The first evidence for the reductive metabolism of a noncyclic nitrosamine to the corresponding hydrazine derivative in vivo and in vitro is provided. Under anaerobic conditions, N-nitrosodiphenylamine was reduced by guinea pig liver 9000g supernatant to 1,1-diphenylhydrazine in the presence of 2-hydroxypyrimidine or to acetaldehyde diphenylhydrazone in the presence of acetaldehyde. These metabolites were identified unequivocally by comparative study with authentic samples. In addition, the study shows that such reductive reactions of the nitrosamine can be catalyzed by guinea pig and rabbit liver aldehyde oxidase in the presence of its electron donors. When the nitrosamine was given orally to acetaldehyde-treated guinea pigs, a metabolite was detected from plasma and identified as acetaldehyde diphenylhydrazone by comparison with the authentic sample.

Five newly synthesized hydrazides: l-aspartic acid dihydrazide, dl-ureidosuccinic acid dihydrazide, dl-dihydroorotic acid hydrazide, orotic acid hydrazide, and 2,2′-anhydro-1-(β-d-arabinofuranosyl)-orotic acid hydrazide were tested for mutagenicity in the Ames test. All these hydrazides except dl-ureidosuccinic acid dihydrazide were found to be non-mutagenic. The mutagenic activities of the latter compound which causes base-pair substitution was decreased in the presence of metabolic activator.