Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a Key and Supporting Ames test performed according to OECD/ EC guidelines and GLP principles (Klimisch 1), the registered substance was found to be not mutagenic with or without metabolic activation when tested in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 up to and including cytotoxic concentrations .

Key in vitro Micronucleus and gene mutation assays were available for read across substance butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts (CAS 90268 -36 -3) both tested up to and including cytotoxic concentrations. There were no indications of chromosomal damage in the in vitro micronucleus test and the test substance was negative in the HPRT-V79 mammalian cell mutagenicity test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test was conducted according to OECD/EC guidelines and GLP principles. Test substance applied as received.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
including METI, MHLW and MAFF
Deviations:
no
Principles of method if other than guideline:
No info on version of guidelines used (year).
GLP compliance:
yes (incl. QA statement)
Remarks:
issued 2 December 2002
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine or tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (prepared at SPL from livers of rats exposed to phenobarbitone/ beta-naphtoflavone)
Test concentrations with justification for top dose:
Experiment 1:
Salmonella strains: 15, 50, 150, 500, 1500, 5000 µg/ plate;
E.coli strain: 50, 150, 500, 1500, 5000 µg/ plate.
Experiment 2:
TA100, TA1535: 15, 50, 150, 500, 1500, 5000 µg/ plate;
WP2uvrA, TA98, TA1537: 50, 150, 500, 1500, 5000 µg/ plate.
Vehicle / solvent:
- Vehicle used: Water
- Justification for choice of vehicle: The test material was fully miscible in distilled water at 50 mg/ml in solubility checks performed at SPL.
- The test material was accurately weighed and approximate half-log dilutions were prepared in sterile water by mixing on a vortex mixer on the day of each experiment.
- Formulations adjusted for the stated water content (water content = 51% of the test material).
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/ plate (TA100), 5 µg/ plate (TA1535), 2 µg/ plate (WP2uvrA); 9-Aminoacridine: 80 µg/ plate (TA1537); 4-Nitroquinoline-1-oxide: 0.2 µg/ plate (TA98)
Remarks:
without S9-mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
2-Aminoanthracene: 1 µg/ plate (TA100), 2 µg/ plate (TA1535 and TA1537), 10 µg/ plate (WP2uvrA); Benzo(a)pyrene 5 µg/ plate (TA98)
Remarks:
with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 3

Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 5000 µg/ plate, for TA100 and TA1535 (+/- S9-mix).
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST:
The test material was toxic to TA100 at 5000 µg/ plate (sparse bacterial background lawn) with and without S9-mix. The test substance was not toxic to WP2uvrA when tested up to and including 5000 µg/ plate.

No test material precipitate was observed on the plates at any of the doses tested with or without S9 -mix.

Conclusions:
Interpretation of results: negative

In an Ames test performed according to OECD/ EC guidelines and GLP principles, product XSM 2697 (CT-825-05; a 50% solution of disodium 4-isodecyl sulfosuccinate) was found to be not mutagenic with or without metabolic activation when tested up to and including 5000 µg/ plate.
Executive summary:

An Ames test was performed with product XSM 2697 (CT-825-05; a 50% solution of disodium 4-isodecyl sulfosuccinate) according to OECD/ EC guidelines and GLP principles. The concentration of the test material was adjusted for the water (51%) content and tested up to and including 5000 µg/ plate. The test material was toxic to TA100 at 5000 µg/ plate (sparse bacterial background lawn) with and without S9-mix, but did not show cytotoxicity in the other strains. Appropriate positive controls and vehicle controls were included and gave a response within historical range, demonstrating that test system was valid. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Based on these results, it can be concluded that the test substance was not mutagenic with or without metabolic activation when tested in a reverse mutation assay when tested up to and including cytotoxic concentrations.

Endpoint:
in vitro cytogenicity / micronucleus study
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:
See attached read-across justification
Reason / purpose for cross-reference:
read-across source
Species / strain:
lymphocytes: human peripheral blood lymphocytes
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality:
The pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
pH values: using a digital pH meter type WTW pH 525 (series no. 51039051),
Osmolality: with a semi-micro osmometer .
No relevant changes in pH or osmolality of the formulations were noted.
- Water solubility:
The test item was completely dissolved in aqua ad iniectabilia.
- Precipitation:
Any possible test item precipitation was checked before and after each experiment. Evaluation of precipitation was done by light microscopy at the beginning and end of treatment.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary experiment without and with metabolic activation test item concentrations of 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL medium were employed. 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.

COMPARISON WITH HISTORICAL CONTROL DATA:
The micronucleus frequencies of the vehicle controls without and with metabolic activation for the last 8 or 7 studies (most recent background data, not audited by the QAU-department) are given as follows:
Micronucleus frequency per 1000 cells
Without metabolic activation (4-h or 20-h exposure):
Untreated control (n = 8):
Mean: 4.9
Standard deviation: 2.0
Range: 1-9
Vehicle control (n = 8):
Mean: 7.2
Standard deviation: 4.6
Range: 1-18
Positive control: Mitomycin C (n = 7):
Mean: 95.8
Standard deviation: 66.1
Range: 24-286
Positive control: Colchicine (n = 7):
Mean: 25.4
Standard deviation: 10.2
Range: 7-43
With metabolic activation (4-h exposure):
Vehicle control (n = 8):
Mean: 10.8
Standard deviation: 6.2
Range: 2-25
Positive control: Cyclophosphamide (n = 7):
Mean: 60.3
Standard deviation: 37.8
Range: 20-147


Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, the read-across 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 the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively.
Executive summary:

Test sample of read-across substance butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts (CAS 90268 -36 -3) 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.

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 experiment with S9 mix was carried out twice. The harvesting time was 20 hours after the end of exposure. The study was conducted in duplicate.

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.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item concentrations of 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL medium were employed. Cytotoxicity was noted starting at a concentration of 156.3 µg test item/mL. Hence, 156.3 µg/mL was 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.

Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide in the presence of metabolic activation.

Tests without metabolic activation (4- and 20-hour exposure)

The micronucleus frequencies of cultures treated with the test item at concentrations of 9.77, 19.53, 39.1, 78.1 or 156.3 µg/mL medium (4 h and 20-h exposure) in the absence of metabolic activation ranged from 5.5 to 15.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487; (in this test: vehicle control: 6.0 or 12.0 micronuclei per 1000 binucleated cells, untreated controls: 4.0 or 9.0 micronuclei per 1000 binucleated cells (4-hour and 20-hour exposure, respectively). Vehicle and untreated control values fell within acceptation ranges.

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the test item at concentrations of 9.77, 19.53, 39.1, 78.1 or 156.3 µg/mL medium (4-h exposure) in the presence of metabolic activation ranged from 6.0 to 18.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487; (in this test: vehicle control: 3.5 or 13.0 micronuclei per 1000 binucleated cells, untreated controls: 4.5 or 8.5 micronuclei per 1000 binucleated cells). Vehicle and untreated control values fell within acceptation ranges.

Under the present test conditions, the Registered substance, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) is also considered devoid of chromosomal damage in the in vitro micronucleus test.

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:
See attached read-across justification
Reason / purpose for cross-reference:
read-across source
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
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.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No changes in the pH values in the medium were noted.
- Effects of osmolality: No relevant changes in osmolality of the formulations were noted.

RANGE-FINDING/SCREENING STUDIES:
- In the preliminary study cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL in the experiment without and with metabolic activation, respectively. Hence, 39.06 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 156.3 µg/mL in the presence of metabolic activation.
- The next higher concentrations resulted in complete cytotoxicity in the preliminary experiment. In addition, the number of mutants was also already considerable decreased in the main experiments at the highest employed concentrations pointing to general pronounced cytotoxicity. Finding a higher concentration with viable and evaluable cells/mutants was therefore not considered realistic.
- 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.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 106 survivors in non-activation solvent controls and 6 to 46 per 106 survivors in S9 activation solvent controls.
The mutation frequency of the cultures treated with concentrations of 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL culture medium without metabolic activation ranged from 3.78 to 15.74 x 10-6 clonable cells. These results are within the normal range of the vehicle controls.
The mutation frequency of the cultures treated with concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL culture mediumwith metabolic activation ranged from 4.44 to 13.04 x 10-6 clonable cells. These results are within the normal range of the vehicle controls.
The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 475.00 to 780.00 x 10-6 clonable cells in the case of EMS and ranging from 530.71 to 855.00 x 10-6 clonable cells in the case of DMBA, indicating the validity of this test system.
The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6 clonable cells for EMS and 130.0 to 2693.3 x 106 clonable cells for DMBA.



Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, the read-across 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.

Executive summary:

The read-across substance butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts (CAS 90268 -36 -3) 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.

Main study

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.

Cytotoxicity

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.

Experiments without metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 14.35 and 16.67 x 10-6clonable cells. Hence, the vehicle controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL culture medium ranged from 4.44 to 13.04 x 10‑6clonable cells. These results are within the normal range of the vehicle controls.

Experiments with metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 17.87 and 16.05 x 10-6clonable cells. Hence, the vehicle controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL culture medium ranged from 3.78 to 15.74 x 10‑6clonable cells. These results are within the normal range of the vehicle controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 475.00 to 780.00 x 10-6clonable cells in the case of EMS and ranging from 530.71 to 855.00 x 10-6clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6clonable cells for EMS and 130.0 to 2693.3 x 10-6clonable cells for DMBA.

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

A key Ames test was performed with registered substance according to OECD/ EC guidelines and GLP principles (Bowles, 2005). The concentration of the test material was adjusted for the water (51%) content and tested up to and including 5000 µg/ plate. The test material was toxic to TA100 at 5000 µg/ plate (sparse bacterial background lawn) with and without S9-mix, but did not show cytotoxicity in the other strains. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

A supporting AMES study was performed with same formulation according to OECD guideline 471, but no E.coli-strain was included (Mecchi, 2005). All bacterial strains showed negative responses up to and including 5000 µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity and/or precipitation of the test substance was observed. Based on information in other reports on the test substance, the concentration of 50% allow to conclude that disodium isodecyl sulfosuccinate is not mutagenic in the Ames test with or without metabolic activation when tested up to and including 2500 µg/plate.

 

A key in vitro Micronucleus assay was available for read across substance butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts (CAS 90268 -36 -3) using human peripheral lymphocytes both in the presence and absence of metabolic activation, employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours (Flügge, 2013). 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.

 

A key in vitro gene mutation assay was available for read across (source) substance butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts (CAS 90268 -36 -3) using cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation (Flügge, 2013). 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. 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 without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test. 


Justification for classification or non-classification

The available data do not indicate that disodium isodecyl sulfosuccinate is genotoxic. The substance is thus not classified for genotoxic properties according to CLP Regulation (EC) No. 1272/2008.