Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

The Derek software prediction for the mutagencity of the registered substance is performed (Cehtra, 2013). No alert was found.

One in vitro study (in vitro micronucleus) was available on the registered substance, it was recent (Institut Pasteur de Lille, 2013) and reliable (Kr. 1).

Two in vitro studies were available on an analog substance of the registered substance: an Ames test and a mouse lymphoma assay. Both studies were recent and reliable (Kr.2).

In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 guideline, the reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (96% purity) 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. Formulated concentrations were adjusted (correction factor of 1.04) to allow for the stated water/impurity content (4%) of the test item.

A preliminary study (one plate/concentration) was performed in order to determine the appropriate concentrations for the two independent main studies (3 plates/concentration). In the preliminary study, the bacterial strains TA100 and WP2 uvrA were exposed to the test substance at the following concentrations: 0,0.15,0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test item was freely soluble up to the highest tested concentration. Cytotoxicity, assessed by the decrease in the number of revertants and/or the thinning of the bacterial lawn, was observed in all strains. The test item exhibited weakened background lawns to both strains (TA100 and WP2uvrA) at 5000 µg/plate in the absence and presence of

S9-mix. Therefore, 5000 µg/plate was chosen as the highest tested concentration for the main study.

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.

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.

Under the test conditions,the reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate did not show any mutagenic activity in the bacterial reverse mutation test using S. typhimurium and E. coli. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 471.

In an in vitro mammalian mutation assay, performed according to the OECD No.476 and in compliance with the GLP,  the 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 an in vitro mammalian cell micronucleus test , performed according to the OECD No.487 and in compliance with the GLP,  the reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate (purity of 96%) diluted in Dimethylsulphoxide (DMSO) was tested in a human lymphocyte culture in the presence and the absence of mammalian metabolic activation (S9).

In the short treatment either with or without metabolic activation followed by a 24 -hour recovery period (assay S9 - 4h/+ 24h), the substance induced neither statistically nor biologically significant increase in the number of micronucleated cells at all the concentrations analyzed.

In the continuous treatment without metabolic activation without recovery period (assays S9- 24h/+0h), the substance induced neither statistically nor biologically significant increase in the number of micronucleated cells at all the concentrations analyzed from 545 and 940μg/mL. The highest dose tested of 940μg/mL induced an increase in the number of micronuclei in mononucleated cells over the doubling when compared to the negative control. Nevertheless, no dose effect relationship was observed, the mean value was close to the historical data and a slight difference between the slides was observed. This increase was thus considered as devoid of mutagenic hazard. It should be noted that a statistically significant decrease in the number of micronucleated binucleated cells was observed at the intermediary concentration analyzed of 817μg/mL, but this effect had no meaning for genotoxicity assessment.

In conclusion, under the conditions of the test, The genotoxic activity of the substance was assessed by means of the in vitro micronucleus test in human lymphocytes treated in presence and in absence of metabolic activation, either with a short or with a continuous treatment.

The acceptance criteria for the assay were fulfilled. The study is thus considered as valid. Under these experimental conditions, no genotoxic activity was observed.

Overall conclusion: Based on the results for the registered substance (in vitro micronucleus) and based on results for an analog substance and on the DEREK software prediction, the registered substance is considered as not mutagenic (weight of evidence strategy)

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. 2, 2012): negative +/- S9 mix (study available on an analog of the registered substance). Negative (registered substance, Derek prediction (Kr. 2, 2013))
Mouse Lymphoma assay (Kr. 2, 2012): negative +/- S9 mix (study on an analog of the registered substance)
Mammalian mutagenicity (Kr. 2, 2013): negative (registered substance, Derek predicition)
In vitro micronucleus test (Institut Pasteur, Lille, France) (Kr. 1, 2013): negative +/- S9 mix (registered substance).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Regarding the overall negative results from the in vitro genotoxicity studies and QSAR predictions, it is likely that Phosphoric acid, mono- and bis(branched and linear pentyl) esters doesn't present genotoxic activity potential, therefore no classification is required according to the Regulation (EC) No 1272/2008 and the Directive 67/548/EEC criteria.