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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data were used from read across substance 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts.' In a key Ames test no increase in mutations were observed in different Salmonella typhimureum strains with and without metabolic activation up to 5000 µg/plate. In a key Mouse mammalian gene mutation test in HPRT cells, the test item did not induce mutations in the absence and presence of metabolic activation when tested up to cytotoxic concentrations of 125 and 1000 µg/mL, respectively. In a key in vitro Micronucleus study in human peripheral lymphocytes, no chromosome aberrations were observed with and without metabolic activation when tested up to cytotoxic concentrations of 125 µg/mL.

Link to relevant study records

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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:
see attached read-across justification
Reason / purpose:
read-across source
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
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 read-across test item Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]¬esters, disodium salts tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains 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.

Executive summary:

Read-across test item Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts was examined in the 5 Salmonella typhimurium strains 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 substance was completely dissolved in aqua ad iniectabilia. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only. The vehicle served as the negative control.

Preliminary test

The read-across test substance was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Ten concentrations ranging from 0.316 to 5000 µg act.ingr./plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate.

Hence, 5000 µg act.ingr./plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 3.16 to 5000 µg act.ingr./plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg act.ingr./plate in all test strains.

No increase in revertant colony numbers as compared with control counts was observed up to a concentration of 5000 µg act.ingr./plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies 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 read-across test substance tested up to a concentration of 5000 µg act.ingr./plate, caused no mutagenic effect in the Salmonella typhimurium strains 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.

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:
read-across source
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
125 µg/mL were employed as the top concentration for the first and second experiment, 500 µg/mL were employed in the third experiment. It was thought that a concentration of 125 µg/mL had not resulted in sufficient clear-cut 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:
The pH of the negative control and the test item formulations in the medium were determined employing a digital pH meter type WTW pH 525 (series no. 51039051). No changes in the pH values were noted (pH range: 7.66 – 7.73).

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

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 absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of any chromosomal damage in the in vitro micronucleus test.
In the same test, Mitomycin C and cyclophosphamide induced significant damage.


Executive summary:

Test samples of the read-across test item were 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: two experiments with an exposure time of 4 hours and two different concentration ranges and one experiment with an exposure time of 20 hours. The experiment with S9 mix was carried out threefold with one exposure time of 4 hours employing two different concentration ranges. The harvesting time was 24 hours after the end of exposure. Each treatment was conducted in duplicate.

The read-across test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only. The vehicle aqua ad iniectabilia served as the vehicle control.

Preliminary experiment

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 10, 25, 100, 250, 1000, 2500 and 4358 µg active ingredient/mL medium were employed. Cytotoxicity was noted starting at a concentration of 100 µg test item/mL in the experiment without and with metabolic activation.

Hence, 125 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation in two independent experiments, each (4-hour and 20-hour exposure). In a third experiment without and with metabolic activation (4-hour exposure) 500 µg/mL were employed as the top concentration for the mutagenicity tests. A third experiment with two higher concentrations was added as it was thought that a concentration of 125 µg/mL had not resulted in sufficient clear-cut cytotoxicity.

Main study

In the main study cytotoxicity was noted starting at a concentration of 125 µg active ingredient/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. Positive controls induced significant increases in micronuclei in both experiments with/without metabolic activation.

 

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

The micronucleus frequencies of cultures treated with the read-across test item at concentrations of 7.81, 15.63, 31.3, 62.5 or 125 µg active ingredient/mL medium in the first and second experiment (4 h and 20-h exposure) or 31.3, 62.5, 125, 250 or 500 µg active ingredient/mL in the third experiment (4-hour exposure) in the absence of metabolic activation ranged from 3.0 to 8.0 micronuclei per 1000 binucleated cells. There was no increase in micronuclei up to the cytotoxic concentration when compared to control (in this test: vehicle control: 8.0, 6.0 or 6.5 micronuclei per 1000 binucleated cells, untreated controls: 5.5, 4.5 or 5.5 micronuclei per 1000 binucleated cells (4-hour and 20-hour exposure, respectively)).

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the read-across test item at concentrations of 7.81, 15.63, 31.3, 62.5 or 125 µg active ingredient/mL medium in the first and second experiment or 31.3, 62.5, 125, 250 or 500 µg active ingredient/mL in the third experiment in the presence of metabolic activation ranged from 3.0 to 9.5 micronuclei per 1000 binucleated cells. There was no increase in micronuclei up to the cytotoxic concentration when compared to control (in this test: vehicle control: 4.5 or 9.0 micronuclei per 1000 binucleated cells, untreated controls: 5.0, 6.0 or 4.5 micronuclei per 1000 binucleated cells).

Under the present test conditions, the read-across test item tested up to cytotoxic concentrations, 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 any chromosomal damage in the in vitro micronucleus test.

In the same test, Mitomycin C and cyclophosphamide induced significant damage.

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:
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 125 or 1000 µg/mL in the absence and presence of metabolic activation, respectively.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
Along with toxicity, changes in the pH of the test solutions was assessed. The pH was measured at the highest test item treatment level.
The pH of the vehicle control and the test item formulations in the medium were determined employing a digital pH meter type WTW pH 525 (series no. 51039051). No changes in the pH values in the medium were noted.
- Effects of osmolality:
Along with toxicity, the Osmolality of the test solutions was assessed. Osmolality determination were carried out in test solutions without target cells both in the presence and absence of metabolic activation. The Osmolality of the highest test item treatment condition, lowest precipitating test item level and the highest soluble test item level in test solution was measured.
- Precipitation:
Along with toxicity, the ability of the test item to cause precipitation in the test solution was assessed.


RANGE-FINDING/SCREENING STUDIES:
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 10, 25, 100, 250, 1000, 2500 and 4150 µg/mL medium were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 125 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 10 6 survivors in non-activation solvent controls and 6 to 46 per 10 6 survivors in S9 activation solvent controls (BRADLEY, M. O., B. BHUYAN, M. C. FRANCIS, R. LANGENBACH, A. PETER¬SON and E. HUBERMANN. Mutagenesis by chemical agents in V79 Chinese hamster cells: a report and analysis of the literature. A report of the Gene-Tox Program. Mutation Research 87, 81 - 142 (1981)).
The background data obtained at LPT are given at the end of chapter 'Results and Discussion'. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
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 test item 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 read-across test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only. 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 10, 25, 100, 250, 1000, 2500 and 4150 µg/mL medium were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 125 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation.

Main study 

Five concentrations 7.81,15.63, 31.3, 62.5 or 125 and 62.5, 125, 250, 500 or 1000 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 125 or 1000 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 15.47 and 17.27 x 10-6 clonable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 7.81, 15.63, 31.3, 62.5 or 125µg test item/mL culture medium ranged from 3.20 to 12.62x 106 clonable 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 14.29 and 14.32 x 10-6 clonable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 62.5, 125, 250, 500 or 1000 µg test item/mL culture medium ranged from 7.33 to 13.13 x 106 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 307.73 to 830.00 x 10-6 clonable cells in the case of EMS and ranging from 319.22 to 909.47 x 10-6 clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPTranges 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 10-6 clonable cells for DMBA.

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.

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

Additional information

Additional information from genetic toxicity in vitro:  

No test data were available for current substance, however read across data were available from 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts'. Justification for read across within the subgroup of N-containing sulphosuccinates (N2) is documented in a separate document attached in Section 13. A liquid test item containing 41.5% active ingredient was examined for bacterial and mammalian gene mutation as well as for chromosomal aberration.

 

Bacterial mutagenicity

In a key test for bacterial mutation, 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments were tested without and with metabolic activation with test item dissolved in aqua ad iniectabilia based on a correction factor of 2.41 for active ingredient (Flügge, 2013c). In a preliminary test, ten concentrations ranging from 0.316 to 5000 µg act.ingr./plate were tested without metabolic activation in strain TA 100. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. In the main study, six concentrations ranging from 3.16 to 5000 µg act.ingr./plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. No signs of cytotoxicity were noted without and with metabolic activation up to the top concentration of 5000 µg act.ingr./plate in all test strains. Under the present test conditions the test item tested up to a concentration of 5000 µg act.ingr./plate, caused no mutagenic effect in the Salmonella typhimurium strains 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.

In conclusion, no mutagenic effect was exerted in bacterial strains without and with metabolic activation, therefore no mutagenic potential is expected for registered substance.

 

Mammalian mutagenicity

A key study was performed in cultured mammalian cells (V79, genetic marker HPRT) both in the presence (4 hours) and absence (4 and 24 hours) of metabolic activation (Flügge, 2013d). In this preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 4150 µg/mL medium were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 125 µg test item/mL was employed as the top concentration for the mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation. Five concentrations 7.81,15.63, 31.3, 62.5 or 125 and 62.5, 125, 250, 500 or 1000 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 125 or 1000 µg/mL in the absence and presence of metabolic activation, respectively. 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. The positive controls caused a pronounced increase in the mutation frequencies, indicating the validity of this test system. 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, no mutagenic effect was exerted in mammalian V79 cells without and with metabolic activation, therefore no mutagenic potential is expected for registered substance.

 

Chromosome aberration

A key in vitro micronucleus test was conducted 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 (Flügge, 2013e). The test was carried out employing 2 exposure times without S9 mix: two experiments with an exposure time of 4 hours and two different concentration ranges and one experiment with an exposure time of 20 hours. The experiment with S9 mix was carried out threefold with one exposure time of 4 hours employing two different concentration ranges. The harvesting time was 24 hours after the end of exposure. Each treatment was conducted in duplicate. The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only. The vehicle aqua ad iniectabilia served as the vehicle control.

Based on a preliminary experiment, cytotoxicity was noted starting at a concentration of 100 µg test item/mL in the experiment without and with metabolic activation. Hence, 125 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation in two independent experiments, each (4-hour and 20-hour exposure). In a third experiment without and with metabolic activation (4-hour exposure) 500 µg/mL were employed as the top concentration for the mutagenicity tests. A third experiment with two higher concentrations was added as it was thought that a concentration of 125 µg/mL had not resulted in sufficient clear-cut cytotoxicity.

In the main study cytotoxicity was noted starting at a concentration of 125 µg active ingredient/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. Positive controls induced significant increases in micronuclei in both experiments with/without metabolic activation.

In the tests without metabolic activation (4- and 20-hour exposure), the micronucleus frequencies of cultures treated ranged from 3.0 to 8.0 micronuclei per 1000 binucleated cells. There was no increase in micronuclei up to the cytotoxic concentration when compared to control. In the test with metabolic activation (4-hour exposure), the micronucleus frequencies of cultures treated ranged from 3.0 to 9.5 micronuclei per 1000 binucleated cells. There was no increase in micronuclei up to the cytotoxic concentration when compared to control.

Under the present test conditions, the test item tested up to cytotoxic concentrations, 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 any chromosomal damage in the in vitro micronucleus test. In the same test, Mitomycin C and cyclophosphamide induced significant damage.

In conclusion, no chromosomal aberration was exerted in human peripheral lymphocytes without and with metabolic activation, therefore no clastogenic potential is expected for registered substance.

 

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 selection of genetic toxicity endpoint Although the bacterial gene mutation study was selected, the mammalian gene mutation and chromosomal aberration tests are equivalent endpoints.

Justification for classification or non-classification

Based on these results and CLP (No. 1272/2008 of 16 December 2008), the test item does not have to be classified and has no obligatory labelling requirement for genetic toxicity.