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Key value for chemical safety assessment

Additional information

Ames study:

On study of reliability 1 according to Klimisch cotation critera is available (Sokolowski A., 2007) and was selected as a key study. This study was performed to investigate the potential of Dimethyl 2-methyl glutarate to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 Lg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 Lg/plate

 

In experiment I, reduced background growth was observed from 333 up to 5000 Lg/plate without metabolic activation in all strains used. No reduction of the background growth was observed in the presence of metabolic activation, and with and without metabolic activation in experiment II.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with dimethyl 2 -methyl glutarate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, Dimethyl 2-methyl glutarate is considered not to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

Chromosomal aberration:

On study of reliability 1 according to Klimisch cotation critera is available (Bohnenberger S., 2008) and was selected as a key study. In this study,Dimethyl 2-methyl glutarate, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in human lymphocytes in vitro.

The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation (S9 mix). In each experiment, two cell cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.The cytotoxicity was determined by the % of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate.

The highest applied concentration in this study (1950μg/mL, approx.10 mM) was chosen with respect to the current OECD Guideline 473.

Exposure periods were 4 hours, 22 hours and 46 hours in the absence of S9 mix and 4 hours in the presence of S9 mix.

 

In this study, at both preparation intervals (22 and 46 hours), in the absence as well as in the presence of S9 mix, no cytotoxicity indicated by reduced mitotic indices could be observed. In both experiments, no visible precipitation of the test item in the culture medium was observed.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls and induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, Dimethyl 2-methyl glutarate is considered not to be clastogenic in thisin vitromammalian chromosome aberration assay.

 

Micronucleus in vivo:

Three studies, of reliability 1 according to Klimisch cotation critera, are available (Pössnecker A. et al., 2007 and Honarvar N. 2008). One of the 2008 studies (RCC-CCR report no. 1060302, 2008) was selected as it is the reference method endorsed by REACH. The 2 other studies performed to clarify the results obtained in the key study were selected as supporting studies.

In one mouse study (RCC-CCR report no. 1060302, 2008) a dose-dependent increase in micronucleated bone marrow cells was observed, with a statistically significant difference at 24 hours compared with the control group only at the highest dose (1500 mg/kg i.p., which corresponds to the Maximum tolerated Dose (MTD)). In that treatment group, the overall elevated numbers of micronucleated cells were attributable to particularly high numbers in two animals, rather than an effect occuring across all or most animals in the treatment group.

However, no signs of increased micronuclei were observed in a second mouse study (RCC-CCR report no. 1114100, 2008). In this study, measurement of rectal temperature was added to the protocol to investigate the hypothesis of a hypothermia-induced micronuclei increase based on the growing evidence that hypothermia effect can induce micronucleus formation in bone marrow cells as a secondary, non-genotoxic effect. Althought treatment at 1500 mg/kg i.p. induced a marked hypothermia within 1 hour and lasting up to 8 hours post-administration, no significant increase in micronuclei was observed in this study in the treatment group as a whole or among any individual mice.

In addition, micronucleus formation was also assessed in male rats treated with the test substance by oral gavage for 28 days in conjunction with an OECD 422 study (RCC report no. B05670, 2007). Although the dose in rats was lower than in the micronucleus study in mice treated by the intraperitoneal route, the substance was administered for 28 days. This is therefore compliant with the OECD 474 guideline which recommends a limit dose of 1000 mg/kg/day for treatment longer than 14 days. No increase in micronuclei was observed in this study.However, as no cytotoxic effect was observed at the maximum dose tested, there is no evidence that the test substance reached the target tissue.

In summary, based on the following considerations:

- the effects observed in mice at the MTD only, which are attributable to effects in only two mice that made the effect statistically significant in the entire group, were not reproducible,

- a marked hypothermia was observed at the MTD in the second study though no increase in micronuclei was observed,

- no increase in micronuclei was observed in a second species (rat) repeatedly exposed to the substance by oral gavage for 28 days,

it is considered that the statistically significant effect observed in the initial micronucleus study was artefactual and not biologically significant.

In conclusion, Dimethyl 2-methyl glutarate is considered not to be clastogenic based on the availablein vivomicronucleus assays.

 

No data are available for mammalian cell mutagenicity testing. However, the hydrolysis products of Dimethyl 2 -methylglutarate, namely methanol, 2 -methylglutaric acid and/or monoester of dimethylglutaric acid, are not considered to be clastogenic or genotoxic. Indeed, methanol is considered not genotoxic to bacteria or mammalian cells based on negative results obtained in the majority of the numerous in vitro and in vivo assays performed (OECD SIDS Initial Asessement profile for methanol, 2004). Although no specific data on the toxicological properties of 2 -methylglutaric acid and monoester of dimethylglutaric acid could be found in the literature, data on the analogous substance Glutaric acid indicate that these structures areinactive for mutagenic and clastogenic effects (US EPA-HPV, Dicarboxylic acids, 2002). Moreover, 2-Methylglutaric acid is an organic acid normally found in human urine of different age groupsand therefore highly unlikely to be genotoxic at relevant exposure levels to humans (Human Metabolome Database reference no.HMDB00422, 2009).

In addition, in the 90 -day inhalation study, there was no evidence of pre-neoplastic lesions at any dose levels that could suggest genotoxic/carcinogenic potential. Therefore, there is sufficient data available to conclude that this substance is not mutagenic to mammalian cells.


Justification for selection of genetic toxicity endpoint
No specific study was selected since all available studies were used to assess the genotoxic potential of the registered substance.

Short description of key information:
Negative in in vitro bacterial reverse mutation assay and in in vitro chromosomal aberration test. Ambigous in one in vivo mouse micronucleus test but negative in one other mouse and in one rat in vivo micronucleus assays.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Overall, the genetic toxicity of Dimethyl 2 -methyl glutarate is considered to be negative and therefore no classification is warranted according to the classification criteria of Annex VI Directive 67/548/EEC or EU Regulation 1272/2008 (CLP).