Reaction Products of Diphosphorus Pentaoxide with n-Alcohols, C8-10 (even), salted with Amines, C12-14, Tert-alkyl

Endpoint summary

Additional information

Bacterial genotoxicity test

The test item was evaluated under GLP conditions and equivalent to OECD guideline 471 in the Ames/Salmonella-E. coli Liquid Pre-incubation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100), and at the tryptophan locus in one Escherichia coli tester strain (WP2 uvrA), in the presence and absence of an exogenous metabolic activation system (S9) (Stankowski, 1993). Toxicity of the test item was first evaluated in a prescreen by treating duplicate cultures of strains TA1538, TA100 and WP2 uvrA with the test item at doses of 50.0, 167, 500, 1670 and 5000 µg/plate in the absence of S9. Results of the pre-screen indicated the test item was not toxic to strain WP2 uvrA at a dose of 50.0 µg/plate. Inhibited growth (characterized by a reduced background lawn and/or the presence of pindot colonies) or complete toxicity was observed in strains TA1538 and TA100 at all doses evaluated, and in strain WP2uvrA at doses >167 µg/plate. In addition, the test item was incompletely soluble at a dose of 5000 µg/plate.

Based upon these findings, the test item was evaluated in triplicate cultures in all five Salmonella tester strains at doses of 0.500, 1.67, 5.00, 16.7, 50.0 and 167 µg/plate, and in strain WP2 uvrA at doses of 5.00, 16.7, 50.0, 167, 500 and 1000 µg/plate, in the presence and absence of S9. Six dose levels of the test item were evaluated with and without S9 in the event of unacceptable toxicity at the highest dose levels evaluated in the mutation assay. The S9 mixture included 6% (v/v) Aroclor 1254-induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors. The test item was found to be freely soluble at all doses evaluated, but normal growth was observed in tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA at all doses evaluated with or without S9. Revertant frequencies for all doses of the test item in all tester strains with S9, and in strains TA1537, TA1538, TA100 and WP2 uvrA without S9, approximated or were less than those observed in the concurrent negative control cultures. In contrast, statistically significant increases in revertant frequencies, to approximately 1.5- to 2.2-fold control values, were observed in strain TA1535 and TA98 without S9. However, these increases were not dose dependent.

The test item was re-evaluated in the confirmatory assay in all six tester strains at doses of 16.7, 50.0, 167, 500, 1670 and 5000 µg/plate with S9, in all five Salmonella tester strains at doses of 1.67, 5.00, 16.7, 50.0, 167 and 500 µg/plate without S9, and in strain WP2 uvrA at doses of 5.00, 16.7, 50.0, 167, 500 and 1000 µg/plate without S9 (dose levels were adjusted based upon toxicity observations made in the original trial). The test item again was found to be incompletely soluble at a dose of 5000 µg/plate. Inhibited growth was observed in all tester strains at doses of 16.7, 50.0, 167, 500, 1000, 1670 and/or 5000 µg/plate with and/or without S9. Revertant frequencies for all doses of the test item in all tester strains with and without S9 approximated or were less than control values. However, only three acceptable dose levels were available in strain TA1538 with S9, and in strain TA1535 without S9, due to excessive toxicity.

Therefore, the test item was re-evaluated in all six tester strains at doses of 16.7, 50.0, 167, 500, 1670 and 3330 µg/plate with S9, and in all five Salmonella tester strains at doses of 1.67, 5.00, 16.7, 50.0, 100 and 167 µg/plate without S9 (dose levels again were adjusted based upon toxicity observations made in the previous trial). The test item again was found to be incompletely soluble at a dose of 3330 µg/plate, and inhibited growth again was observed in all tester strains at doses of 50.0, 100, 167, 500, 1670 and/or 3330 µg/plate with and/or without S9. Revertant frequencies for all doses of the test item in all tester strains with S9, and in tester strains TA1535, TA1537, TA1538, TA100 and WP2 uvrA without S9, approximated or were less than control values. In contrast, statistically significant increases in revertant frequencies, to approximately 1.6- to 1.9-fold control values, were observed in strain TA98 at doses of 1.67 and 5.00 µg/plate without S9. However, these increases apparently were not dose dependent.

Therefore, the test item was re-evaluated in strains TA1535 and TA98 at doses of 0.167, 0.500, 1.67, 5.00, 16.7, 50.0, 100 and 167µg/plate without S9. The test item again was found to be freely soluble at all doses evaluated, and inhibited growth again was observed in both tester strains at doses of 16.7, 50.0, 100 and/or 167 µg/plate. Revertant frequencies for all doses of the test item in both tester strains again approximated or were less than control values. Thus, the slight increases observed in strains TA1535 and TA98 are considered to be statistical aberrations due to random fluctuation of the spontaneous revertant frequencies. All positive and negative control values in all assays were within acceptable limits.

Therefore, the results for the test item were negative in the Ames/Salmonella-E. coli Liquid Pre-incubation Assay under the conditions and according to the criteria of the test protocol.

In vitro Mammalian Chromosome Aberration Test

In a mammalian cell chromosome aberration assay, Chinese hamster lung (CHL) cells cultured in vitro were exposed to the test item at concentrations up to 160 µg/mL in the main tests in the presence and absence of mammalian metabolic activation (S9) (Wright, 1995).

Duplicate cultures of CHL cells were treated with the test material at a minimum of four dose levels, in each treatment case, together with vehicle and positive controls in each of two experiments. In Experiment 1 a single cell-harvest time-point at 12 hours, both with and without metabolic activation, was used. In Experiment 2 six treatment regimes were used: 6 hours exposure both with and without the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 18 hours treatment-free incubation); 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 8 hours treatment-free incubation); continuous exposures of 12, 24 and 48 hours.

The dose range for metaphase analysis was selected from a series of at least four dose levels chosen on the basis of the results of a preliminary toxicity test. The test material was evaluated at doubling dose levels between 1.25 and 120 µg/ml depending on the particular treatment regime used, the continuous exposures were more toxic than the pulse exposures.

The vehicle (solvent) controls gave frequencies of aberrations within the range expected for the CHL cell line. The positive control treatments gave significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. A typical poor response was seen in the 12-hour treatment case which was probably due to toxicity-induced cell cycle delay, such that any clastogenic responses induced by the positive control materials were not apparent at the 12-hour harvest time.

The test material demonstrated no statistically significant dose-related increases in the frequency of cells with aberrations in Experiment 1, or in Experiment 2. The test material was shown to be toxic to CHL cells in vitro in all six treatment cases, with a very steep dose response curve. The test material was shown to be non-clastogenic to CHL cells in vitro.

This study is classified as acceptable and study satisfies the requirements for EU Method B.10 and the requirements of the Japanese New Chemical Substance Law (MITI).

In vivo Mammalian Erythrocyte Micronucleus Test

A study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered via the intraperitoneal route to male and female albino CD-1 mice (Durward, 1995). The method followed has been designed to comply with Method B12 of the EEC Commission Directive 92/69/EEC which constitutes Annex V of Council Directive 57/548/EEC

Following a preliminary range-finding study to confirm the toxicity of the test material, the micronucleus study was conducted using the test material at the maximum tolerated dose level of 150 mg/kg with 37.5 and 75 mg/kg as the lower two dose levels.

In the micronucleus study, groups of ten mice (five males and five females) were given a single intraperitoneal dose of the test material at 37.5, 75 or 150 mg/kg. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

Further groups of mice were dosed via the intraperitoneal route with arachis oil or orally with 50 mg/kg bw cyclophosphamide, to serve as vehicle and positive controls respectively.

There was no evidence of a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the concurrent vehicle control groups. A statistically significant change in the PCE/NCE ratio was observed after dosing with the test material at 150 mg/kg in the 24-hour group indicative of a cytotoxic response in the bone marrow and thus confirming systemic absorption and exposure of the target tissue.

The positive control material produced a significant increase in the frequency of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the concurrent vehicle control groups.

The test material was considered to be non-genotoxic under the conditions of the test.

The study is classified as acceptable and satisfies the requirement for EU method B.12 for in vivo cytogenetic mutagenicity data.

Justification for selection of genetic toxicity endpoint
The in vivo study was selected because the results are consistent with those from the in vitro studies and because the in vivo studies assess a significant endpoint in whole animals where all organ systems and homeostatic process are present.

Short description of key information:
The substance was clearly negative in the vitro studies and in the in vivo study in mouse.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No evidence of genotoxicity was observed in two in vitro tests and in one in vivo test. Bacterial gene mutation test, in vitro chromosome aberration study in CHL cells, and in vivo Mammalian Erythrocyte Micronucleus Test conducted with the test substance were all negative.

In accordance with EU CLP (Regulation (EC) No. 1272/2008) classification is not required for genotoxicity.