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Bacterial reverse gene mutation assay

In a reverse gene mutation assay in bacteria equivalent to OECD guideline 471 (1983), strains of S. typhimurium (TA 1535, TA 1537, TA 98, T 100) were exposed to Stearic acid, esters with methyl α-D-glucoside (a.i. 100 % according to producer information) at concentrations of 8, 40, 200, 1000 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (plate incorporation method). Stearic acid, esters with methyl α-D-glucoside did not induce a significant dose-related increase in the number of revertant colonies in all four tested strains, both with and without mammalian metabolic activation. These results were confirmed in an independently repeated experiment. No cytotoxic effects of the test substance were observed. Precipitation was observed at concentrations of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. There was no evidence of induced mutant colonies over background. The study was performed equivalent to the OECD guideline 471 of 1983. Therefore, the additional strain S. typhimurium TA 102 or E. coli WP2 was not tested as required by the current guideline.

However, similar effects were observed in a reverse gene mutation assay in bacteria (plate incorporation method) according to OECD guideline 471, in S. typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 and E. coli (WP2 uvrA) with the read-across substance Isostearic acid, esters with methyl α-D-glucoside. At concentrations of 33, 100, 333, 1000, and 3330 µg/plate Isostearic acid, esters with methyl α-D-glucoside did not induce a significant dose-related increase in the number of revertant colonies in each of the five tester strains, both in the absence and presence of mammalian metabolic activation. These results were confirmed in an independently repeated experiment. No cytotoxic effects of the test substance were observed. Precipitation was observed at concentrations of 1000 and 3330 µg/plate. Based on the results of this study it is concluded that Isostearic acid, esters with methyl α-D-glucoside is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.  

 

Mammalian cell cytogenetic assay

Reliable data are available from a mammalian cell cytogenetic assay with the read across substance Isostearic acid, esters with methyl α-D-glucoside.

Human peripheral blood lymphocyte cultures were exposed to Isostearic acid, esters with methyl α-D-glucoside

in dimethyl sulfoxide at concentrations of 0, 33, 100, and 333 µg/ml culture medium (3 h exposure time, 24 h fixation time with and without metabolic activation in the first cytogenetic assay). In the second cytogenetic assay concentrations of 0, 100, 300, 400, 500, 600, 700, and 800 µg/ml (24 h and 48 h exposure time and fixation time) without S9-mix and 0, 33, 100, and 300 µg/ml (3 h exposure time, 48 h fixation time) with S9-mix were tested, respectively.

Isostearic acid, esters with methyl α-D-glucoside was tested up to cytotoxic and precipitating concentrations.

In the first cytogenetic assay, Isostearic acid, esters with methyl α-D-glucoside was tested up to 333 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Isostearic acid, esters with methyl α-D-glucoside precipitated in the culture medium at this dose level. In the second cytogenetic assay, Isostearic acid, esters with methyl α-D-glucoside was tested up to 800 µg/ml for 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at 600 µg/ml. In the presence of S9-mix Isostearic acid, esters with methyl α-D-glucoside was tested up to 300 µg/ml for a 3 h exposure time with a 48 h fixation time. Isostearic acid, esters with methyl α-D-glucoside precipitated in the culture medium at this dose level.

Isostearic acid, esters with methyl α-D-glucoside did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No biologically relevant effects of Isostearic acid, esters with methyl α-D-glucoside on the number of polyploid cells and cells with endoreduplicated chromosomes were observed, both in the absence and presence of S9-mix.

Therefore it can be concluded that Isostearic acid, esters with methyl α-D-glucoside does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

It is concluded that this test is valid and that Isostearic acid, esters with methyl α-D-glucoside is not clastogenic in human Iymphocytes under the experimental conditions described in this report.

Positive controls induced the appropriate response. There was no evidence of chromosome aberrations induced over background. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.

 

Mammalian cell gene mutation assay

In the first experiment Mouse lymphoma L5178Y cells cultured in vitro were exposed to Isostearic acid, esters with methyl α-D-glucoside in DMSO up to concentrations of 300 µg/ml in the absence of S9-mix and up to concentrations of 500 µg/ml in presence of 8% (v/v) S9-mix with an incubation time of 3 hours.

Since an enhanced cytotoxicity was observed in the first experiment, concentrations up to 240 µg/mL and 350 µg/mL were selected for evaluation of mutation frequencies without and with S9- mix (8 %), respectively. Dose depending cytotoxicity with RTG (relative total grow) values of 12 % and 14 % at the highest concentrations were observed without and with S9-mix, respectively.

In the second experiment an extended 24 hours exposure with concentrations up to 260 µg/mL was used without S9-mix. The experiment with mammalian metabolic activation was performed at concentrations up to 375 µg/mL with 12 % S9-mix with 3 hours exposure time. As well an extended cytotoxicity was observed with RTG values of 19 % and 41 % at concentrations of 210 µg/mL without S9 mix and 375 µg/mL with S9-mix (12 %), respectively.

The test substance was tested beyond the limit of solubility; however the precipitating dose levels did not interfere with the scoring. Precipitation in exposure medium was observed at concentrations of 130 mg/mL and above.

The numbers of small and large colonies in the treated cultures of both experiments were comparable to the numbers of small and large colonies of the solvent controls.

Isostearic acid, esters with methyl α-D-glucoside did not induce a significant increase in the mutation frequency in the presence or absence of mammalian metabolic activation. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9-mix and exposure period.

Isostearic acid, esters with methyl α-D-glucoside is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report. The positive controls induced the appropriate response. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

Conclusion:

Finally, it can be concluded that there is no evidence for a genotoxic intrinsic property of Stearic acid, esters with methyl α-D-glucoside. Results from a full set of genotoxic studies required by REACH regulation are negative, including data from the read-across substance Isostearic acid, esters with methyl α-D-glucoside.

 

Justification for read-across:

Read-across is considered acceptable, based on the structural analogue approach from Isostearic acid, esters with methyl α-D-glucoside to Stearic acid, esters with methyl α-D-glucoside.

Stearic acid, esters with methyl α-D-glucoside is a reaction product of Methyl glucoside with Stearic acid, a linear saturated mainly C18 fatty acid whereas for Isostearic acid, esters with methyl α-D- glucoside the branched Isostearic acid is used. Composition of chain length is very similar; more than 90 % of the fatty acids have a carbon number of 18, with some chain length distribution between C16 and C20.

The main components of Isostearic acid are mono- and poly branched C18 fatty acids, in which the branching occurs mainly medium-chained, mostly methylenic, which accounts for its good biodegradability. However, a defined structure of isostearic acid does not exist.

The reaction leads to a fatty acid ester in which the four OH-groups of glucose are partially esterified.

Due to its esterification with the branched Isostearic acid, isostearate is more hydrophobic than the sesquistearate.

Stearic acid, esters with methyl α-D-glucoside is composed of Methylglucoside (2.4 %), Methyl glucoside ester (approx. 78 % mono-, di-, tri-, and tetraester (mainly di, and triester)) and of approx. 19 % free fatty acids. Whereas Isostearic acid, esters with methyl α-D-glucoside is composed of Methylglucoside (4 %), Methyl glucoside ester (approx. 83 % mono-, di-, and triester (mainly diester)) and of approx. 13 % free fatty acids.

Likewise intrinsic toxicological properties of Stearic acid, esters with methyl α-D-glucoside and Isostearic acid, esters with methyl α-D-glucoside are proven for acute oral toxicity, skin and eye irritation, skin sensitization and genmutation in bacteria. The read-across substance Isostearic acid, esters with methyl α-D-glucoside, containing branched chains, is considered to be more critical in its toxicological impacts and can be understood as a worst case in this analogue approach. However, no intrinsic toxicological property leading to an intrinsic health hazard could be identified for the Isostearic acid, esters with methyl α-D-glucoside.

In conclusion this structural analogue approach is scientifically justified by close similarities of structural aspects and physico-chemical properties and finally the comparable harmless toxicological profile of both substances.


Justification for selection of genetic toxicity endpoint
Results from a full set of genotoxic studies required by REACH regulation are negative, including data from the read-across substance Isostearic acid, esters with methyl α-D-glucoside.

Short description of key information:
Gene mutagenicity in bacteria (Reverse Mutation Assay OECD TG 471) is negative with Stearic acid, esters with methyl α-D-glucoside in Salmonella typh. strains TA98, TA100, TA1535, TA1537. Additional negative results are observed in a full set of in-vitro genotoxicity studies with the read across substance Isostearic acid, esters with methyl α-D-glucoside (Ames test with Salmonella typh. strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA; Chromosome aberrations in Peripheral Human Lymphocyte Cultures; Mammalian Cell Gene Mutation Test (Mouse Lymphoma L5178Y TK+/- cells).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

There were no indications of mutagenic activity in the Bacterial Reverse Mutation Assay, of clastogenicity in-vitro in cultured Peripheral Human Lymphocyte Cultures and of mutagenicity in the mammalian cell culture (Mouse Lymphoma) test system. Therefore according to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008 no classification and labelling for mutagenic toxicity is necessary.