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An Ames test (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100) was performed according to OECD Guideline 471 with BAYHIBIT AM containing 45-50% (w/w) 2-phosphonobutane-1,2,4-tricarboxylic acid in water, at nominal concentrations of 16; 80; 400; 2500 and 12,500 µg /plate. Strain-specific bacteriotoxicity was observed at doses >500 µg/plate and higher. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution strains used. Hence, BAYHIBIT AM is considered non-mutagenic in this bacterial reverse mutation assay (Herbold, 1979). This result is supported by a further Ames test (same strains) performed with Phosphonosuccinic acid (PBS, CAS No. 5768-48-9), a structural analogue substance and very similar to 2-phosphonobutane-1,2,4-tricarboxylic acid. The test was performed at nominal concentrations of 16; 80; 400; 2000 and 10,000 µg /plate. Strain-specific bacteriotoxicity was observed at 400 µg/plate and higher doses. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, PBS is considered non-mutagenic (Herbold, 1992) in this bacterial reverse mutation assay. The fact that only four instead of at least five strains of bacteria were used in both AMES assays is compensated by two other in-vitro assays (Mammalian Chromosome Aberration and V79 -HPRT Forward Mutation assay). In an in-vitro Mammalian Chromosome Aberration Assay according to OECD Guideline 473 with Chinese hamster lung fibroblasts (V79) cultures that were treated with the test substance with and without metabolic activation showed a biologically relevant increase in the number of gaps. As the only observed increase was in gaps, a cytotoxic effect rather than a genotoxicity effect of the test substance is favourable. Hence, under the test conditions, 1,2,4 -Butanetricarboxylic acid, 2 -phosphono does not induce structural chromosomal aberrations in cultured mammalian somatic cells (V79) tested with and without an exogenous metabolic system. Following a cytotoxicity assay, test substance concentrations were selected for the chromosomal aberration assay, with and without metabolic activation (S9 -mix). Results of cultures with and without S9 -mix showed no biologically significant increase in the number of breaks, exchanges or other aberrations in cultures from two independent experiments (May, 1996). In a Chinese hamster lung cells (V79) forward mutation assay, no cytotoxic effects were observed up to a concentration of 3000 µg/mL (Brendler; Schwaab, 1997), no significant dose-related or increase in mutant frequency above that of the negative controls was observed; on the contrary to positive controls which induced clear mutagenic effect. Thus, based on the available data derived from four in-vitro tests, BAYHIBIT AM is to be considered as non-mutagenic.


Short description of key information:
The Ames test was performed with BAYHIBIT AM, at nominal concentrations of 16; 80; 400; 2500 and 12,500 µg /plate. Under the experimental conditions, the test item did not induce gene mutations by frameshift or base-pair substitution strains used and is therefore to be considered as non-mutagenic in this bacterial reverse mutation assay. Strain-specific bacteriotoxicity was observed at doses >500 µg/ plate and higher. These results are supported by an Ames test performed with Phosphonosuccinic acid, a structural analogue substance, at nominal concentrations of 16; 80; 400; 2000 and 10,000 µg /plate. No mutations by frameshift or base-pair substitution were induced in the strains used. Strain-specific bacteriotoxicity was observed at 400 µg/ plate and higher doses.
Both assays were performed in four strains (TA1535, TA 100, TA 1537, TA98).

In two further in-vitro assays, Mammalian Chromosome Aberration and V79 -HPRT Forward Mutation assay, both with and without metabolic activation, BAYHIBIT AM was found to be non-mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data derived from four in-vitro tests, that all coincide in the final conclusion that the test substance is not mutagenic, BAYHIBIT AM is not to be classified as mutagen according to EU Directive 67/548/EC and according to EU regulation No. 1272/2008 (GHS; amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006).

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