Bis(2,4,4-trimethylpentyl)phosphinic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative with and without metabolic activation in bacterial reverse mutation assay (OECD 471)

Negative with and without metabolic activation in the vitro gene mutation study in mammalian cells (OECD 476)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Does not induce micronucleus in the mouse bone marrow cells (OECD 474)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

The registered substance was assessed for its genotoxic potential in in vitro (Ames and Mouse Lymphoma assay) and in vivo (Micronucleus test in the Mouse) tests.

In a reverse gene mutation assay in bacteria, performed according to the OECD guideline No.471, and in compliance with the GLP, strains TA 98, TA 100, TA1535, TA1537, TA1538 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to the test substance diluted in ethanol in the presence or absence of mammalian metabolic activation (fraction of S9 from the liver of rats treated with Aroclor 1254). The method of direct incorporation was tested.

Based on results of cytotoxicity obtained in a preliminary assay, the maximum dose that was plated in the main mutagenicity assay was 10000 µg/plate. Bis(2,4,4-trimethylpentyl)phosphinic acid was therefore tested up to the highest recommended limit concentration (5000 µg/plate) and cytotoxicity was observed at concentrations equal and higher than 1000 or 3000 µg/plate depending on the bacteria strain and the metabolic activation condition (with or without).

Under the test conditions, the test item did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium TA 98, TA 100, TA1535, TA1537 and TA1538 and with Escherichia coli WP2 uvrA as there was no evidence of induced mutant colonies over background.

In an in vitro mammalian mutation assay, performed according to the OECD No.476 and in compliance with the GLP, the test substance diluted in DMSO was tested in the L5178Y TK+/-mouse lymphoma cell line in the presence and the absence of mammalian metabolic activation. Previously to the mutagenicity experiments, a preliminary assay was conducted in order to assess the cytotoxicity of the test substance. L5178Y TK +/- mouse lymphoma cells were treated with the substance at eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls (Methylmethane sulphonate (MMS) or Cyclophosphamide (CPA) for the without and with metabolic activation condition respectively) using 3-hour and 24-hour exposure groups both in the absence of metabolic activation. The cells were also treated with the substance at eight dose levels using a 3‑hour exposure group in the presence of metabolic activation. The test item is cytotoxic in both metabolic conditions. Without metabolic activation, no noteworthy increase in the mutation frequency was observed even at cytotoxic concentration when the exposure period to the test item was 3h. Following a 24h exposure period without S9 mix, a slight increase in the mutation frequency was noted at the dose-level of 0.25 mM, which showed a severe decrease in Adj. RTG of 100%. However, at the lower dose-level of 0.13 mM, which showed the recommended level of toxicity (Adj. RTG comprised between 10 and 20%), no increase in the mutation frequency was induced. Therefore, it can be considered that the test item is negative in terms of mutagenicity without metabolic activation.

With metabolic activation, following the first experiment, no noteworthy increase in the mutation frequency was noted up to the dose-level of 0.25 mM inducing 91 % decrease in the Adj. RTG. Following the second experiment, a slight increase in the mutation frequency was noted at 0.25 mM. This increase (IMF of 92 x 10-6) did not reach the GEF but a dose-response relationship was demonstrated (p < 0.001) by statistical analysis. Since a dose-related increase in the mutation frequency was observed in this experiment where the recommended level of toxicity (Adj. RTG between 10 and 20%) was not reached, an additional experiment was performed using the same experimental conditions but a closer range of dose-levels. In this third experiment, no noteworthy increase in the mutation frequency was noted up to the dose-level of 0.38 mM inducing 95% decrease in the Adj. RTG. Therefore, the test item is considered as non mutagenic in the presence of metabolic activation.

Under the experimental conditions of this study, the test item, did not show any mutagenic activity in the mouse lymphoma assay, in the presence and in the absence of a rat metabolizing system.

Genetic toxicity in vivo

In an in vivo micronucleus assay, performed according to the OECD No.474 and in compliance with the GLP, the test material diluted in aqueous solution of 1% methylcellulose was administered by gavage to CD1 mice (males and females). A preliminary test was performed on mice to determine the suitable dose levels for the main micronucleus test. Eight doses from 191 to 5000 mg/kg bw were tested; as death occurred at dose levels higher than 1080 mg/kg bw the maximum tolerated dose was estimated to be approximately 900 mg/kg bw.

Thus, in the initial main test, male and female mice were dosed with 900 mg/kg bw of the test material in aqueous solution of 1% methylcellulose. Mice were also treated in the same experimental conditions with the vehicle only (negative control) and other mice were dosed with Mitomycin C at 12mg/kg bw (positive control). 5 mice per sex were then sacrificed at 24, 48 and 72 h after dosing in order to analyse the number of micronucleated polychromatic erythrocytes (in bone marrow). Hunched posture, lethargy, piloerection, ptosis, twitching were observed in the first day after the oral administration of the test substance. Furthermore, five male and ten females died after the treatment. The reasons of this high mortality incidence were not clearly understood. The test substance did not cause any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes (mnp) at any time point compared with the concurrent control. However slides from the 48h sampling time showed an unusually high incidence of mnp for animals treated with the test substance. Since this result did not meet the criteria for a positive or a negative response, it was regarded as equivocal and a confirmatory micronucleus test was performed only in male rats (10 animals for each time point (36 and 48h)) at the dose level of 1000 mg/kg bw. In this second assay, the results were negative at either sampling time. In both experiments, no decreases in the ratio of polychromatic to normochromatic erythrocytes (ratio PCE/NCE) were obtained for animals treated with the test substance at any sampling time.

In conclusion, under the test conditions, the test substance did not induce micronucleus in the mouse bone marrow cells. As no decrease of the ratio PCE/NCE was observed in animals treated with the test substance, it can not be assumed with certitude that the test substance reach the target tissue (i.e. bone marrow). However as signs of systemic toxicity were obtained in the oral repeated dose toxicity study performed on the registered substance (OECD 422), it can be considered that the substance is absorbed by the oral route and reached the systemic circulation. Therefore it can be assumed that the target organ in the micronucleus assay was reached by the test item.

Justification for classification or non-classification

Based on all available data obtained in the two in vitro studies (Bacterial reverse mutation assay and in vitro gene mutation assay) and in the in vivo study (mammalian erythrocyte micronucleus test) the test substance is not classified as mutagenic according to the CLP Regulation (EC) No. 1272/2008