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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read across was performed for the genotoxicity endpoints with category members Docusate sodium (CAS 577-11-7) and Sodium di (1,3-dimetylbutyl) sulfosuccinate (CAS 2373-38-8) based on the read across justification for the Diester Sulfosuccinate Category group. Based on the experimental data, there was no evidence for bacterial and mammalian gene mutation potential, nor was there any indication of chromosome aberration potential. In conclusion, based on the read across with experimental data, there is no genotoxic potential for the test substance.

Link to relevant study records

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read across argumentation is provided in Section 13.
Reason / purpose:
read-across source
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect
- Effects of osmolality:no effect

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the read-across test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells ( Chinese hamster cell line) in vitro.
Therefore, Butanedioic acid, sulfo, 1,4-bis(1,3-dimethylbutyl) ester, sodium salt (80% active ingredient) is considered to be non clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.
Executive summary:

The read-across test item Butanedioic acid, sulfo, 1,4-bis(1,3-dimethylbutyl) ester, sodium salt (80% active ingredient), dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments up to 5000 µg/mL(approx. 10 mM of the active ingredient). In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations and 500 cells were scored for polyploidy. Toxic effects indicated by reduced cell numbers and/or mitotic indices of about and below 50% of control were observed in all experimental parts. However, in experiment I in the absence and the presence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. The observed statistical significance's and dose-dependency are regarded as being biologically irrelevant. No 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. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be non-clastagenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read across argumentation is provided in Section 13.
Reason / purpose:
read-across source
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The highest concentration used in the pre-test was chosen with regard to the purity (80 % active substance) and the molecular weight of the test item (400 g/mol). Test item concentrations between 39.1 and 5000 μg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Following 4 hour treatment distinct toxic effects leading to RSG (relative suspension growth) values below 50 % were observed at 156.3 μg/mL and above in the absence and at 312.5 μg/mL and above in the presence of metabolic activation. Following continuous treatment (24 hours) a reduced relative suspension growth was determined at 312.5 μg/mL and above. The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was noted at 2500 μg/mL and above at the end of the 4 h treatment. Following 24 hours treatment, precipitation was observed at the maximum concentration of 5000 μg/mL.

The dose range of the first main experiment was selected according to the data generated in the pre-experiment. However, the onset of toxicity shifted and the analysable toxic range was not covered in the first experiment. Therefore, this experiment was repeated at higher concentrations without metabolic activation and at more narrowly spaced concentrations in the presence of metabolic activation. To overcome problems with possible deviations in toxicity both main experiments were started with more than four concentrations.

Relevant toxic effects indicated by a relative cloning efficiency 1 (survival) and/or a relative total growth (RTG) of less than 50 % in both parallel cultures were observed at 150 μg/mL and above in experiment IA without metabolic activation. The recommended toxic range of approximately 10 – 20 % of survival or RTG was covered. In the presence of metabolic activation toxic effects as described above were noted at 300 μg/mL. Although the recommended 10 – 20 % range of toxicity was not covered severe toxic effects occurred. The cell growth compared to the corresponding solvent control was severely reduced 24 h after treatment (20.8 - 21.3%). The surviving cells recovered and grew at a quite normal rate resulting in higher values of survival and RTG. Still, a reduction of the cell population down to about 20 % of the initial value is indicating severe toxicity, any further reduction may lead to undesired selection processes resulting in irreproducible artefacts.

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. The threshold of 126 plus each solvent control count was not reached or exceeded at any test point even at toxicity levels below 10 % of survival or RTG.

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion it can be stated that under experimental conditions reported the read-across test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the abscence and presence of metabolic activation.
Executive summary:

The study was performed to investigate the potential of the read-across test item, Butanedioic acid, sulfo-, 1,4 -bis(1,3 -dimethylbutyl) ester, sodium salt (ca. 80% act. ingr.) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The assay was performed in two independent experiments, using two parallel cultures each. Both main experiments were performed with and without liver microsomal activation and a treatment period of 4h. The second experiment (experiment IA) was required to verify the results obtained in experiment I and to cover highly toxic concentrations using an adjusted concentration range.
The highest applied concentration in the pre-test on toxicity (5000 µg/mL) was chosen with regard to the molecular weight of the test item. The dose range of the main experiments was limited by toxicity of the test item.
No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was limited by toxicity of the test item.
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.
Under experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read across argumentation is provided in Section 13.
Reason / purpose:
read-across source
Species / strain:
other: TA98,TA100, TA1535, TA1537, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not provided
- Effects of osmolality: not provided
- Evaporation from medium: not provided
- Water solubility: not provided
- Precipitation: some thinning of the background lawn at 1000 µg/plate
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:
An initial toxicity range-finder experiment was carried out in
TA100 only, using final concentrations of sodium dioctyl sulphosuccinate at 8, 40, 200, 1000 and
5000 µg/plate plus a solvent and positive control. In the absence S-9, complete killing of the test
bacteria was observed at the highest concentration of 5000µg/plate. In addition, a thinning of the
background lawn at the second highest concentration (1000µg/plate) also indicated toxicity. In the
presence of S-9, toxicity was only observed at the highest dose. In view of these results , maximum
test concentrations of 1000 and 2500µg/plate were chosen for Experiment 1 treatments, in absence
and presence of S-9 respectively.


COMPARISON WITH HISTORICAL CONTROL DATA:
Individual plate counts from both experiments were recorded separately and the mean and
standard deviation of the plate counts for each treatment were determined.




Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
It is concluded that read-across substance docusate sodium failed to induce mutation in 5 strains of Salmonella thyphimurium, when tested up to concentrations close to or within the toxic range, in the absence and presence of a rat liver metabolic activation system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

No test data were available for current substance, however read acros data were available from Docusate sodium (CAS No. 577-11-7) and Sodium di (1,3-dimetylbutyl) sulfosuccinate (CAS No. 2373-38-8) from the same subgroup. Justification for read across within the category of Di-ester sulfosuccinates is documented in a separate document attached in Section 13.

Bacterial mutagenicity
- A key study for bacterial mutagenicity was available for Docusate sodium (CAS 577 -11 -7) with 5 Salmonella thyphimurium strains (Clare, 1993); the study was conducted according to OECD 471 and GLP guidelines, and was considered to be reliable, adequate and relevant. After a range-finder experiment showing cytotoxicity at the highest concentration of 5000µg/plate, maximum test concentrations of 1000 and 2500 µg/plate were chosen for the main experiment 1. In experiment 1, concentrations were close to the limit of toxicity, therefore for experiment 2, concentrations for all strains were maximally 2000 µg/plate without S9 and 2500 µg/plate with S9. In both experiments, Docusate sodium did not result in statistically significant increases in revertant number of colonies, both with and without S9.
- Further a disregarded screening Ames test study was available for the registered substance, where the test item (>90% pure) was tested to be negative for mutagenicity without S-9 activation (Culbreth, 1977). The highest dose, however, was only 1000µg/mL and there were no positive controls tested.

Mammalian mutagenicity
A key study was available for the mutation assay at the thymidine kinase locus (TK+/-) in Mouse lymphoma L5178Y cells for read across substance Sodium di (1,3-dimetylbutyl) sulfosuccinate (CAS 2373 -38 -8) according to OECD 476 and GLP guidelines, and was considered to be reliable, adequate and relevant (Wollny, 2006). The assay was performed in two independent experiments, using two parallel cultures each. Both main experiments were performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment (experiment IA) was required to verify the results obtained in experiment I and to cover highly toxic concentrations using an adjusted concentration range. The highest applied concentration in the pre-test on toxicity (5000μg/mL) was chosen with regard to the molecular weight of the test item. The dose range of the main experiments was limited by toxicity of the test item. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item. In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Chromosomal aberration
A key study for chromosome aberration potential in Chinese Hamster V79 cells was available for a read across substance Sodium di (1,3-dimetylbutyl) sulfosuccinate (CAS 2373-38-8) from the same category (Schulz, 2003).The study was conducted according to OECD 473 and GLP guidelines, and was considered to be reliable, adequate and relevant. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction. A pre-test for cytotoxicity testing was performed up to 5000 µg/mL, resulting in reduced cell numbers after 4h treatment with 625 µg/mL and above in the absence and the presence of S-9 mix. Considering the toxicity data of the pre-test, 800 µg/mL (without S9) and 1000 µg/mL (with S-9) were chosen as top concentrations in the main experiment I. Dose selection of experiment II was also influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24h exposure with 312,5 µg/mL and above. Therefore 600 µg/mL was chosen as top treatment concentration for continuous exposure in the absence of S-9 and 800 µg/mL in the presence of S-9.In both experiments, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed after treatment with the test item. No increase in frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate positive controls induced statistically significant increases in cells with structural chromosome aberrations. In conclusion, the test material was considered to be non clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.

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.

Justification for classification or non-classification

Based on the negative findings with read across substances, the test item does not need to be classified and has no obligatory labelling requirement for genotoxicity according to the EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008).