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Additional information

Bacterial mutagenicty

Genetic toxicity in bacteria was analyzed in a bacterial reverse mutation assay performed according to GLP and OECD guideline 471, EU method B.13/14 and EPA OPPTS 870.5265 (Thompson, 1999). Bacteria strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with doses of 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the pretest, and doses of 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation in the main test, respectively. Acetone was used as vehicle and the treatments were with and without metabolic activation by cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with phenobarbitone/b-naphthoflavone. No cytotoxicity was noted, but precipitation of the substance occurred at and above of 1500 µg/plate. However, no increase in the number of revertants was observed, so that NPG di-2-ethylhexanoate (2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate) was found to be not genotoxic in bacteria.

In addition, negative results were observed in another bacterial reverse mutation assay, where bacteria strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2P and WP2P uvrA were tested with and without metabolic activation according to OECD guideline 471 (Callander, 1995). 

Mammalian cytogenicity

The potential of NPG di-2-ethylhexanoate (2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate) to induce chromosomal damage in mammalian cells was evaluated in an in vitro mammalian chromosome aberration test performed according to GLP and OECD guideline 473, and EU method B.10 (Buskens, 2010). In a dose range finding study, properly maintained cultured peripheral human lymphocytes were treated with doses of 3, 10, 33, 100 and 333 µg/mL with and without metabolic activation for 3 h and with doses of 3, 10, 33, 100, 333, 1000 and 3333 µg/mL with and without metabolic activation for 24 and 48 h, respectively. Since the substance precipitated at 333 µg/plate and higher, concentrations of 3, 10, 33, 100 and 333 µg/mL were used for a 3 h treatment in the first cytogenetic assay, with and without metabolic activation by a cofactor supplemented post-mitochondrial fraction (S9 mix). A second cytogenetic assay was performed, where the cells were treated for 3 h with 50, 100 and 350 µg/mL with metabolic activation and for 24 and 48 h with doses of 5, 10, 50, 150 and 200 µg/mL only without metabolic activation, respectively. Additionally, a repeat of the 48 h treatment was performed with doses of 10, 30, 50, 60, 70, 80, 90 and 100 µg/mL without metabolic activation. Ethanol was the vehicle in all experiments. After the treatment, the cells were treated with colchicine and stained for analysis, where 1000 cells per dose were analyzed. Cytotoxicity was observed starting at the dose of 100 µg/mL at the 48 h exposure without metabolic activation. However, no increase in number of aberrant cells with or without gaps was found for any concentration and any exposure time used. Thus, NPG di-2-ethylhexanoate (2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate) did not induce chromosome aberration in mammalian cells.

Another in vitro mammalian chromosome aberration test is available were (2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate) was tested in human lymphocytes which were treated with 25, 75 and 150 µg/mL with and without metabolic activation by a S9-mix (Fox, 1995). Reductions in mean mitotic activity compared to control values were noted at 150 mg/mL with (29%) and without metabolic activation (33%), respectively. A reduction of 44% was noted at a dose of 75 µg/mL in the absence of the S9 mix. However, no chromosomal aberrations were noted.

Mammalian mutagenicity

Mutagenicity in mammalian cells induced by NPG di-2-ethylhexanoate (2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate) was assessed in an in vitro mammalian cell gene mutation assay, where mouse lymphoma L5178Y cells were analyzed in accordance with GLP and OECD guideline 476 and EU method B.17 (Verbaan, 2010). The cells were exposed for 3 h in the presence and absence of metabolic activation and for 24 h in the absence of metabolic activation, respectively. In the first experiment, the cells were treated with concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/mL in ethanol with and without metabolic activation containing 8% rat liver S9-mix. In a second experiment, concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/mL with metabolic activation containing 12% rat liver S9-mix, as well as concentrations of 0.3, 1, 3, 10, 33, 66, 85, 100, 125 and 150 µg/mL without metabolic activation were used. As in the absence of metabolic activation fluctuations in toxicity were observed which were not dose dependent, this part of the experiment was repeated with doses of 0.3, 1, 3, 10, 33, 100, 200, 300, 400, 500 and 600 µg/mL without metabolic activation. The cells were cultured for 48 h after the treatment period. Afterwards, the cells were plated for the determination of the cloning efficiency and mutation frequency. For the determination of the mutation frequency cells were incubated for 11-12 d. In all experiments, precipitation was noted at and above 33 µg/mL, but no cytotoxicity was observed. A 12- to 16-fold increase in mutation frequency was observed after treatment of cells with the positive control substance cyclophosphamide. In contrast, no significant increase in mutation frequency was found after treatment with NPG di-2-ethylhexanoate (2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate) in the two experiments at any dose tested. Thus, NPG di-2-ethylhexanoate (2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate) did not induce gene mutation in mammalian cells.

In conclusion, negative results obtained in bacterial and mammalian mutagenicity tests as well as in mammalian cytogenicity tests after treatment with 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate indicate no genotoxic potential of the test substance.


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

Short description of key information:
In vitro:
Gene mutation (Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102: negative with and without metabolic activation (according to OECD TG 471)
Mammalian cytogenicity (chromosome aberration): peripheral human lymphocytes: negative (OECD TG 473)
Mammalian mutagenicity (MLA): negative with and without metabolic activation (OECD TG 476)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.