Bis(2-ethylhexyl) carbonate

Endpoint summary

Additional information

For the assessment of the mutagenic potential of Bis(2-ethylhexyl) carbonate several studies are available: a reverse gene mutation assay in bacteria (Ames test) with Bis(2-ethylhexyl) carbonate, as well as with the read-across substance 2-Ethylhexanol a mammalian cell gene mutation assay (Mouse lymphoma) with the read-across-substance 2-Ethylhexanol, and an in vitro chromosome aberration test as well as an in vivo mouse bone marrow micronucleus assay with Bis(2-ethylhexyl) carbonate.

In vitro gene mutation in bacteria

In a reverse gene mutation assay in bacteria according to OECD Guideline 471, July 21, 1997, EU Method B.13/14, June 08, 2000 and EPA Guideline OPPTS 870.5100 (EPA 712 -C-98 -247), August 1998, strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 of S. typhimurium were exposed to Bis(2-ethylhexyl) carbonate (a.i. 100 %) at concentrations of 0.0316 to 5.0 µL/plate in the presence and absence of mammalian metabolic activation (plate incorporation).

No cytotoxic effects of the test item were observed in both experiments up to and including the highest dose of 5 µL/plate in all strains used.

No substantial increases in the revertant colony numbers of any of the five test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments.

Positive controls reference mutagens were tested in parallel to the test item. They showed a distinct increase of induced revertant colonies. There was no evidence of induced mutant colonies over background.

 

In a reverse gene mutation assay in bacteria similar to OECD Guideline 471, strains TA 98, TA100, TA1535, TA 1537, TA 1538 of S. typhimurium were exposed to 2-Ethylhexanol at concentrations of 0.01, 0.05, 0.25, 0.5, 1.0 µL/plate in the presence and absence of mammalian metabolic activation (S9 mix; plate incorporation). The test substance was tested up to cytotoxic concentrations.

No substantial increases in the revertant colony numbers of any of the test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments. Positive controls reference mutagens were tested in parallel to the test item. They showed a distinct increase of induced revertant colonies. There was no evidence of induced mutant colonies over background.

In vitro mammalian cell gene mutation

No data are available for Bis(2-ethylhexyl) carbonate. However, a Mouse lymphoma assay isavailable for the read-across substance 2-Ethylhexanol.

2-Ethylhexanol was tested in an in vitro gene mutation assay using mammalian cells cultures both in the absence and presence of metabolic activation (S9 mix), according to a protocol similar to the OECD Guideline 476.

Mouse lymphoma L5178Y cells (clone 3.7.2) cultured in vitro were exposed to 2-Ethylhexanol (99.97%) at 10 concentrations from 0.013 to µL/mL to 0.24 µL/mL in ethanol. Total growth at highest concentration without and with metabolic activation was approximately 10% and 40 %, respectively. Concentration selection was based on an initial toxicity test in which the substance demonstrated complete toxicity at concentrations >/= 1.0 µL/mL. Appropriate positive controls were used. After a 48 rest period, cells were then incubated mutagenicity evaluation with trifluorothymidine during 10-12 days.

None of the cultures treated with 2-Ethylhexanol at any dose-level, exhibited mutation frequencies that were significantly greater (two-fold greater than background) than that of the corresponding ethanol solvent control.

The positive control chemicals, on the other hand, demonstrated significant increases in mutation frequencies for both S9 activated and non-activated cultures.

Under these experimental conditions, 2-Ethylhexanol was not mutagenic in the L5178Y TK+/- mammalian mutagenicity assay.

 

In vitro cytogenicity in mammalian cells

In a mammalian cell cytogenetics assay (chromosome aberration test) according to OECD Guideline 473, July 21, 1997 and EU Method B.10, June 08, 2000, V79 cell cultures were exposed to Bis(2-ethylhexyl) carbonate (99 - 100 % a.i.) at concentrations of 0.008, 0.02, 0.05, 0.13, 0.32, 0.80, 2.0 and 5.0 µL/mL in the presence of mammalian metabolic activation and at concentrations of 0.06, 0.13, 0.25, 0.5,1.0, 2.5 and 5.0 µL/mL in the absence of metabolic activation.

Cytotoxic effects of the test item were observed with and without metabolic activation. Without metabolic activation no biologically relevant increase of the aberration rates was noted after treatment with the test item. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In the main experiment with metabolic activation no biologically relevant increase of aberrant cells was observed. All dose groups evaluated were within the historical control data or within the range of the corresponding solvent control. No dose-response relationship was indicated with and without metabolic activation. No biologically relevant increase in the frequencies of polyploid cells was observed after treatment with the test item.

As positive controls reference mutagens EMS and CPA were tested in parallel to the test item. They induced distinct and biologically relevant increases in cells with structural chromosomal aberration.

There was no evidence of chromosome aberration induced over background.

 

In vivo cytogenicity in mice

In a NMRI mouse bone marrow micronucleus assay according to OECD Guideline No. 474, July 21, 1997, EU Method B12, June 08, 2000 and US EPA Guideline OPPTS 870.5395, EPA 712-C-98-226, August 1998, 5 male and 5 female animals/harvest time were treated i.p. with Bis(2-ethylhexyl) carbonate (99- 100% a.i.) at the limit dose of 2000 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hours post-treatment. The vehicle was cotton seed oil.

There were no signs of toxicity during the study. Bis(2-ethylhexyl) carbonate was tested at an adequate dose, as the tested dose is the limit dose recommended by the guidelines. The positive control induced the appropriate response.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

Although no signs of toxicity were observed in this study, it can be expected from the route of administration (intraperitoneal injection) that the test item was systemically available. Moreover, the in vitro cytogenicity study was also negative rising no concern for cytogenicity.

 

Although no in vitro gene mutation data in mammalian cells are available for Bis(2-ethylhexyl) carbonate, the substance is considered to have no mutagenic potential. An in vitro gene mutation study in mammalian cells is available for the metabolite of Bis(2-ethylhexyl) carbonate, 2-Ethylhexanol. This also showed no evidence of mutagenicity, neither in bacteria nor in mammalian cells.

The read-across approach is justified based on metabolism. As Bis(2-ethylhexyl) carbonate does not penetrate the skin and has a very low vapour pressure and a use pattern which will not generate inhalative exposure, the only route of exposure relevant for risk assessment is oral ingestion. Bis(2-ethylhexyl) carbonate is expected to be acid labile and thus will break down to its carbonate and alcohol moieties at contact with the acidic gastric juice. This metabolic pathway has been verified in an in vitro hydrolysis study.

The resulting carbonic acid, respectively the carbonate anion and carbon dioxide are uncritical from a toxicological view due to natural regulation mechanisms. The released alcohol moiety 2-Ethylhexanol might be systemically available and thus might have a systemic effect and is therefore the relevant substance for read across.

Based on the available reliable, relevant and adequate data, there was no evidence of genotoxicity for Bis(2-ethylhexyl) carbonate. There are no data gaps for the endpoint genotoxicity. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

Justification for selection of genetic toxicity endpoint
No single key study has been selected since all available studies were negative.

Short description of key information:
Data on the genotoxic potential of Bis(2-ethylhexyl) carbonate are available from an in vitro gene mutation study in bacteria , an in vitro chromosome aberration test in Chinese Hamster V79 cells and an in vivo mammalian erythrocyte micronucleus test in mice. Further tests (in vitro gene mutation study in bacteria and in mammalian cells) are available for the metabolite 2-Ethylhexanol. All test were considered negative. There is no evidence for a genotoxic potential of Bis(2-ethylexyl) carbonate.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Bis(2-ethylhexyl) carbonate is considered to have no genotoxic properties. Data on the genotoxic potential of Bis(2-ethylhexyl) carbonate are available from an in vitro gene mutation study in bacteria, an in vitro chromosome aberration test in Chinese Hamster V79 cells and an in vivo mammalian erythrocyte micronucleus test in mice. An in vitro mammalian cell gene mutation test is available for the metabolite 2-Ethylhexanol. All these tests were clearly negative and gave no indication on a genotoxic potential of Bis(2-ethylhexyl) carbonate.

Therefore, Bis(2-ethylhexyl) carbonate does not comply with the requirements regarding germ cell mutagenicity outlined in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC.