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Additional information

There is a mutagenicity study in mammalian cells available for the target substance (Mouse Lymphoma Test). No further genetic toxicity studies are available for the target substance. Therefore, the available studies for the read-across substance (Phosphoric acid, mono- and di-(C8 -C10) ester, compound with C12 -14 amine) were used to evaluate the potential of genetic toxicity of the target substance

Mouse Lymphoma Test

The study was conducted with the target substance (Reaction product of diphosphorus pentaoxide and alcohol C7-9-iso, C8 rich, salted with 2-ethylhexylamine) according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (OECD 476, Flanders, 2013, Project No. 41204422). Two independent experiments were performed. In Experiment 1, L5178Y TK. +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9 final concentration). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (2 % S9 final concentration) and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item was selected following the results of a preliminary toxicity test and for Experiment 1 were 5 to 160 µg/mL in the absence of metabolic activation, and 2.5 to 120 µg/mL in the presence of metabolic activation. In Experiment 2 the dose range was 5 to 70 µg/mL in the absence of metabolic activation, and 10 to 80 µg/mL in the presence of metabolic activation. The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. In conclusion, the test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Genotoxicity in bacterial cells

In a key study, the read-across substance (Phosphoric acid, mono- and di-(C8 -C10) ester, compound with C12 -14 amine) was tested according to Japanese Regulatory Authorities (MITI, MHW, MOL and MAFF), OECD Guideline 471, EU Method B.13/14 and the USA, EPA (TSCA) OPPTS harmonised guidelines in order to determine its genetic toxicity in bacterial strains (Thompson, 1998, Project No. 525/131). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate depending on bacterial tester strain type and presence or absence of S9-mix. The experiment was repeated on a separate day using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Extra dose levels were included in both experiments to allow for the toxicity of the test material and to ensure there were a minimum of four non-toxic doses plated out.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial lawn of all the tester strains, initially at 500 and 1500 µg/plate without and with S9-mix respectively. The test material was, therefore, tested up to either the maximum recommended dose of 5000 µg/plate or its toxic limit depending on tester strain type and presence or absence of S9-mix. An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

Chromosome Aberration Test

An in vitro test for the detection of structural chromosomal aberrations in cultured mammalian cells according to OECD 473 and EU Method B.10 was performed with the read across substance (Phosphoric acid, mono- and di-(C8 -C10) ester, compound with C12 -14 amine) (Wright and Durward, 1999, Project No. 525/133). Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. The dose-levels for scoring of chromosomal aberrations were selected on the basis of the qualitative slide assessment and mitotic index (MI) inhibition. Four treatment conditions were used for the study, i.e. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system (S9) with cell harvest after a 16-hour expression period and a 4-hour exposure in the absence of activation with a 16-hour expression period, this was Experiment 1. In Experiment 2, the 4-hour exposure with addition of S9 was repeated, whilst in the absence of activation the exposure time was increased to 20 hours. In the first experiment, the final concentrations of the test material were 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL with and without metabolic activation. In the second experiment, the final concentrations of the test material were 5, 10, 20, 40, 80, 160, 320 and 480 µg/mL without S9-mix and 10, 20, 40, 80, 160, 320, 480 and 640 µg/mL, for the with S9 treatments.

Precipitate observations during pH and osmolality determinations showed that there was precipitate of test material at 1250 and 2500 µg/mL. In the first experiment, the qualitative assessment determined that there were few scorable metaphases at and above 312.5 µg/mL both with and without metabolic activation (S9). A steep dose-related toxicity curve was observed. In the second experiment, the test material was more toxic. There were no scorable metaphases at and above 320 µg/mL in the absence of metabolic activation and only a few scorable metaphases were present at and above 480 µg/mL in the presence of metabolic activation. The dose-related inhibition of mitotic index confirmed the qualitative observations in both experiments. The dose-levels both with and without S9 mix selected for metaphase analysis were 78.13, 156.25, and 312.5 µg/mL for the first experiment and 40, 80 and 160 µg/mL (without S9 mix) and 80, 160 and 320 µg/mL (with S9 mix) for the second experiment.
In both experiments, all of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method itself was operating as expected. The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the presence or absence of metabolic activation in two independent experiments. The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the treatment cases.

In conclusion, the test material was shown to be non-clastogenic to human lymphocytes in vitro.

In vivo Micronucleus Test

In an in vivo study, the read-across substance Phosphoric acid, mono- and di-(C8 -C10) ester, compound with C12 -14 amine was tested for the potential to produce damage to chromosomes or aneuploidy when administered to mice (OECD 474, Durward, 1998, Project No. 525/132) . A range-finding study was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes; therefore the main study was performed using only male mice. The micronucleus study was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) of 125 mg/kg with 31.25 and 62.5 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow was extracted and smears preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls, respectively.
There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. However, the presence of premature deaths and clinical signs was taken to indicate that systemic absorption had occurred. It was considered that the premature loss of three animals in the 48-hour 125 mg/kg test material dose group did not affect the integrity of the study. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes. The test material was considered to be non-genotoxic under the conditions of the test.


Justification for selection of genetic toxicity endpoint
No study is selected since all available tests are negative.

Short description of key information:
1. Mouse Lymphoma Test (OECD 476): L5178Y cells, target substance, tested up to cytotoxic dose levels; negative (with and without metabolic activation);
2. Ames test (OECD 471): Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- , read-across substance, tested up to 5000 µg/plate; negative (with and without metabolic activation);
3. Chromosome Aberration (OECD 473): human lymphocytes, read-across substance, tested up to cytotoxic dose levels, not clastogenic (with and without metabolic activation);
4. In vivo Micronucleus Test (OECD 474): CD-1 mice, i.p. administration, bone marrow/erythrocytes, tested at 31.25, 62.5 and 125 mg/kg bw (MTD); clinical signs, deaths, PCE/NCE similar to controls, non-genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to the classification criteria outlined in section 3.5.2.2 (Guidance on the Application of CLP criteria, 2012), the test item needs to be classified if the substance induces heritable mutations in the germ cells of humans (positive evidence) or the substance causes concern for humans owing to the possibility that they may induce such heritable mutations in humans. The target substance 8Reaction products of diphosphorus pentaoxide and alcohol C7-9-iso, C8 rich, salted with 2-ethylhexylamine) was negative in the Mouse Lymphoma Test. The read-across substance (Phosphoric acid, mono- and di-(C8 -C10) ester, compound with C12 -14 amine) was negative in all available genetic toxicity tests in vitro and in vivo. Therefore, the target substance does not meet the requirement under EU CLP (Regulation (EC) No. 1272/2008) for classification as a mutagen.