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Key value for chemical safety assessment

Additional information

All in vitro assays were performed with 2-phosphonobutane-1,2,4-tricarboxilic acid ("PBTC"), the corresponding parent acid of tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate ("PBTCNa4").

In aqueous media, PBTCNa4 and PBTC dissociate into the corresponding anion (2-phosphonatobutane-tricarboxylate ion) and the sodium ion and hydrogen ion (proton), respectively. The toxicological properties of PBTC and its tetrasodium salt are thought to be an effect of the phosphonato-carboxylate ion rather than of the sodium ion or the hydrogen ion (proton), which are normal constituents in body fluids and have no relevant toxic properties in low concentrations.

Therefore a read-across between PBTCNa4 and PBTC acid is justified.

An Ames test (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100) was performed according to OECD Guideline 471 with a 45-50% (w/w) solution of PBTC in water, at nominal concentrations of 16; 80; 400; 2500 and 12500 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, PBTC is considered non-mutagenic in this bacterial reverse mutation assay (Herbold B, 1979).

The fact that only four instead of at least five strains of bacteria were used in the Ames assay 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, PBTC 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 C, 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 S, 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 three in-vitro tests, PBTC is to be considered as non-mutagenic.


Justification for selection of genetic toxicity endpoint
The results of the Ames tests, the chromosome aberration test and the HPRT test were considered.
As there is no data available for tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate a read-across approach with the corresponding parent acid 2-phosphonobutane-1,2,4-tricarboxylic acid is proposed.
In aqueous media, tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid dissociate into the corresponding anion (2-phosphonatobutane-tricarboxylate ion) and the sodium ion and hydrogen ion (proton), respectively. The toxicological properties of 2-phosphonobutane-1,2,4-tricarboxylic acid and its tetrasodium salt are thought to be an effect of the phosphonato-carboxylate ion rather than of the sodium ion or the hydrogen ion (proton), which are normal constituents in body fluids and have no relevant toxic properties in low concentrations.
Therefore a read-across between tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid is justified.

Short description of key information:
An Ames test was performed with 2-phosphonobutane-1,2,4-tricarboxilic acid ("PBTC") as a surrogate for tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate ("PBTCNa4"), at nominal concentrations of 16; 80; 400; 2500 and 12500 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.
In two further in-vitro assays, Mammalian Chromosome Aberration and V79-HPRT Forward Mutation assay, both with and without metabolic activation, PBTC 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 three in-vitro tests with 2-phosphonobutane-1,2,4-tricarboxilic acid ("PBTC") as a surrogate for tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate ("PBTCNa4"), which all coincide in the final conclusion that the test substance is not mutagenic, PBTC and PBTCNa4 are 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). A read-across between PBTCNa4 and PBTC acid is justified, because in aqueous media, PBTCNa4 and PBTC dissociate into the corresponding anion (2-phosphonatobutane-tricarboxylate ion) and the sodium ion and hydrogen ion (proton), respectively. The toxicological properties of PBTC and its tetrasodium salt are thought to be an effect of the phosphonato-carboxylate ion rather than of the sodium ion or the hydrogen ion (proton), which are normal constituents in body fluids and have no relevant toxic properties in low concentrations