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Additional information

Bacterial Reverse Mutation assay:

One reverse bacterial gene mutation test (Harlan, 2012) was available and was selected as a key study (OECD 471, reliability 1). In this study, Reaction Mass of "Diisobutyl hydrogen phosphate and Isobutyl phosphate" (IBAP) diluted in Dimethyl sulphoxide (DMSO) was tested in S. typhimurium TA1535, TA1537, TA100 and TA98 and in E. coli WP2 uvr A in the presence and the absence of mammalian metabolic activation (S9) using the direct incorporation or the preincubation method. Six known mutagens (4-nitroquinoline-N-oxide; 9-Aminoacridine; N-ethyl-N-nitro-N-nitrosoguanidine; 2-Aminoanthracene and Benzoapyrene), dissolved in dimethylsulfoxide, were used to check the sensitivity of the test system. 

A preliminary study (one plate/concentration) was performed in order to determine the appropriate concentrations for the two independent main studies. In the first experiment (plate incorporation method; 15, 50, 150, 500, 1500 and 5000 µg/plate) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains at 5000 µg/plate in both the absence and presence of S9-mix. In the second experiment (pre-incubation method with S9 mix, direct plate incorporation without S9 mix; 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) the test item induced a weaker toxic response with weakened bacterial background lawns noted at 5000 µg/plate to TA1537 only in both the absence and presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Under the test conditions, the Reaction Mass of "Diisobutyl hydrogen phosphate and Isobutyl phosphate" (IBAP) did not show any mutagenic activity in the bacterial reverse mutation test using S. typhimurium and E. coli.

- In vitro Mammalian Cell Gene Mutation test:

One fully reliable study is available (Harlan, 2012) conducted according to OECD TG 476 and in compliance with GLP (reliability: 1). In this study, Reaction mass of "Diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate" (purity of 96%) diluted in Dimethylsulphoxide (DMSO) was tested in the L5178Y Tk +/- 3.7.2c mouse lymphoma cell line in the presence and the absence of mammalian metabolic activation (S9).

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 (DMSO) and positive controls (Ethylmethanesulphonate (EMS) or Cyclophosphamide (CP) for the without and with metabolic activation condition respectively) using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). 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 (1% S9) 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 in Experiment 1 was 78.13 to 2000 µg/ml in both the absence and presence of metabolic activation. In Experiment 2 the dose range was 125 to 2500 µg/ml in the absence of metabolic activation, and 250 to 2500 µg/ml in the presence of metabolic activation.

The overall maximum dose level used was limited by a combination of test item-induced toxicity and pH issues. Precipitate of test item was observed at 2500 µg/ml in the mutagenicity test experiment 2. The vehicle controls had acceptable mutant frequency values that were within the normal range 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 the second experiment.

Under the conditions of the test, the test item was considered to be non-mutagenic to L5178Y cells with and without metabolic activation.

 

In vitro Mammalian chromosome aberration test:

One fully reliable study is available (CiToxLAB, 2013) conducted according to OECD TG 473 and GLP (mammalian chromosome aberration test, with and without liver microsomal activation, 3, 20 and 44 hours treatment). The dose-levels used for treatment were 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL for the first experiment, both with and without S9 mix, 156.3, 312.5, 625, 1250, 1875, 2500 and 5000 µg/mL for the second experiment with S9 mix and 39.06, 78.13, 156.3, 312.5, 625, 1250, 1875 and 2500 µg/mL for the second experiment without S9 mix. The cytotoxicity was observed from 625 µg/mL (without S9 mix) and 1875 µg/mL (with S9 mix). No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments after the 3-, 20- as well as 44-hour treatment and in absence or presence of S9 mix. Therefore, Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate was considered to be not clastogenic in this in-vitro mammalian chromosome aberration assay.

In vivo:

No data available. Based on REGULATION (EC) N° 1907/2006 as at July 2011 and the absence of positive results in the three above mentioned in vitro tests no additional testing for genetic toxicity in-vivo is necessary.

General:

For each endpoint (bacterial gene mutation, mammalian gene mutation and mammalian chromosome aberration) reliable, GLP compliant in-vitro studies are available that all gave negative results. Therefore it can be concluded that Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate is neither clastogenic nor mutagenic. Accordingly it can be concluded that Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate is not genotoxic.


Justification for selection of genetic toxicity endpoint
No study was selected, since all in vitro studies were negative.

Short description of key information:
- Ames test (Kr. 1, 2012): negative +/- S9 mix
- Mouse Lymphoma assay (Kr. 1, 2012): negative +/- S9 mix
- An in vitro Mammalian Chromosome Aberration Test: negative +/- S9 mix

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Regarding the overall negative results from the in vitro genotoxicity studies, it is likely that Reaction mass of Diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate doesn’t present genotoxic activity potential, therefore no classification is required according to the CLP Regulation and to the Directive 67/548/EEC.