Butanedioic acid, sulfo-, C-C16-18-alkyl esters, disodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial mutagenicity - Ames test: negative (with/without S9)

- Mammalian mutagenicity - HPRT test: negative (with/without S9)

- Chromosome aberration - MN test: negative (with/without S9)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial mutagenicity

- Key: The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium (TA 1535, 100, 1537, 98) and Escherichia coli WP2 uvrA, in a reverse mutation assay. Standard plate test (SPT) and preincubation test (PIT) were performed with and without metabolic activation. Precipitation was found from 100 µg/plate onward with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 10 µg/plate onward. Under the experimental  conditions of the study, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and presence of metabolic activation (BASF, 2011).

- Supporting:

In a supporting study, the test item (C12 -18 sulfosuccinate) containing ≥ 90 % a.i. was tested for mutagenic activity in the bacterial tester strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 1538 and TA 98 with and without metabolic activation (BASF SE, 1986). Concentrations tested were 8, 40, 200, 1000 and 5000 µg per plate in a first and second test. The test substance did not induce reverse mutations in the presence and absence of metabolic activation in all tested strains.

The read-across substance (C12 -18 sulfosuccinate) was further examined in the 5 Salmonella typhimuriumstrains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

The test item was completely dissolved inaqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item.Aqua ad iniectabiliawas used as vehicle control.

The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were tested. Cytotoxicity (scarce background lawn and reduction of the number of revertants)was noted at concentrations of 316 µg/plate and higher. Hence, 316 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Six concentrations of 1.0, 3.16, 10.0, 31.6, 100 and 316 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
Inthe plate incorporation test and in the preincubation test, each carried out without and with metabolic activationcytotoxicity (scarce background lawn and reduction of the number of revertants)was noted at the top concentration of 316 µgtest item/plate in all test strains.
No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to a cytotoxic concentration of 316 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).
The results for the vehicle controls were within the range of historical control data of the laboratory. The positive control items showed a significant increase in the number of revertant colonies compared to the vehicle controls of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.
In conclusion, under the present test conditions the test item tested up to a cytotoxic concentration of 316 µg/plate, caused no mutagenic effect in theSalmonella typhimuriumstrains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

- Supporting: The read-across substance (16 -18, C18-unsatd. sulfosuccinate) was tested in the Salmonella typhimurium Reverse Mutation Assay for the induction of reverse mutations in a bacterial test system. The assay was performed with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in two independent experiments, both with and without metabolic activation by S9 -mix. Solutions of the test substance were prepared in bidest. water and diluted with the same solvent just before use. Toxic effects were noted at concentrations of approx. 200 µg/plate or higher. No enhanced revertant rates compared to concurrent negative controls, induced by the test substance, were observed in all tested strains, neither in the presence nor in the absence of metabolic activation. The direct plate incorporation test was used. The test substance did not induce reverse mutations in the tested strains in this bacterial mutagenicity test, neither with nor without metabolic activation by S9-mix.

Thus, the test substance is considered not to be muatgenic in this bacterial mutagenicity test in vitro.

In conclusion, negative results were obtained for bacterial mutagenicity in a key study with the registered substance and in supporting studies with read-across substances.

Mammalian mutagenicity

No information is available for the registered substance on mammalian mutagenicity, the substance is evaluated based on information on a read-across substance.

The read-across substance (C12 -18 sulfosuccinate) was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix. The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item.  The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary study test item concentrations of 19.53, 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/mL medium were employed . Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 39.06 µg test item/mL was employed as the top concentration for the mutagenicity tests in the absence and 156.3 µg/mL in the presence of metabolic activation. Five concentrations 2.44, 4.88, 9.77, 19.53 or 39.06 and 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL were selected for the experiments without and with metabolic activation, respectively. In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2)was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.

Under the present test conditions, the test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

In conclusion, negative results were obtained for mammalian mutagenicity in a key study with a read-across substance tested in V79 cells with genetic marker HPRT.

Chromosome aberration

No information is available on the registered substance, the substance is evaluated based on information on a read-across substance.

The test item (C12 -18 sulfosuccinate) containing >95% active ingredient was assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals (key study; Flügge, 2013e). The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. Aqua ad iniectabilia served as the vehicle control. In the preliminary experiment , cytotoxicity was noted starting at a concentration of 156.3 µg test item/mL. Hence, 156.3 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation. In the main study cytotoxicity was noted at the top concentration of 156.3 µg/mL in the experiments without and with metabolic activation. Both in the experiments with and without metabolic activation, the mutation frequencies of treated cell cultures were within the normal range of the vehicle controls. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Under the present test conditions, the test item tested up to a cytotoxic concentration of 156.3 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test. In conclusion,negative results were obtained for chromosome aberration in a key study with registered substance tested in an in vitro Micronucleus test in human peripheral lymphocytes.

Conclusion

Standard information requirements according to REACH Guidance Part 3 R7a were fulfilled for genotoxicity testing, including bacterial and mammalian mutagenicity and chromosomal aberration. Based on the available results,there were no indications of mutagenicity or genotoxicity, and no further testing is needed. The substance can be considered to have no mutagenic or genotoxic potential.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

As there was no indication for genotoxic potential, classification for genotoxicity is not warranted according to the EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008).