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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a key Ames test with registered substance C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite no increase in mutations were observed in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA with and without metabolic activation up to 5000 µg/plate. In a supporting Ames test with 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 (CAS 68784-08-7) no increase in mutations were observed in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 with and without metabolic activation up to 5000 µg/plate. In a key mammalian gene mutation test in HPRT cells, the read-across test item CAS 68784-08-7did not induce mutations in the absence and presence of metabolic activation when tested up to cytotoxic concentrations of 125 µg and 1000 µg/mL, respectively. Finally, in a key in vitro Micronucleus study in human peripheral lymphocytes with CAS 68784-08-7, no chromosome damage was observed in the absence and presence of metabolic activation when tested up to cytotoxic concentrations of 125 µg/mL (4 and 20h exposure time) and up to 500 µg/mL (4h exposure), respectively.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) number 440/2008 of 30 May 2008
Qualifier:
according to guideline
Guideline:
other: USA, EPA OCSPP harmonized guideline 870.5100- Bacterial Reverse Mutation Test.
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Manufacturer: Evonik Nutrition & Care GmbH, Manufacture date: March 2016 ; Batch: S016318441
- Expiration date of the lot/batch: March 2017
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:
Target gene:
histidine, tryptophan
Species / strain / cell type:
other: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver; Phenobarbital / beta-Naphta flavone induced @ 8 / 10 mg/kg) Lot No. PB/βNF S9 04 March 2016
Test concentrations with justification for top dose:
Experiment1 (plate incorporation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Experiment 2 (pre-incubation): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Experiment 1 (plate incorporation): The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 (pre-incubation): The dose range used for Experiment 2 was determined by the results of Experiment 1. Seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: The test item was fully miscible in sterile distilled water at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
True negative controls:
yes
Remarks:
untreated
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2 μg/plate for WP2uvrA, 3 μg/plate for TA100, 5 μg/plate for TA1535
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 μg/plate for TA1537
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide (4NQO)
Remarks:
0.2 μg/plate for TA98
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, 10 μg/plate for WP2uvrA
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 μg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation)
Experiment 2: preincubation

DURATION
Experiment 1
- Exposure duration: approximately 48 hours
- Selection time (if incubation with a selection agent): approximately 48 hours
Experiment 2:
- Preincubation period: 20 minutes
- Exposure duration: approximately 48 hours
- Selection time (if incubation with a selection agent): approximately 48 hours

SELECTION AGENT (mutation assays): histidine (Salmonella strains), tryptophan (E.coli)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: microscopically examination for evidence of thinning (toxicity): visible reduction in the growth of the bacterial background lawn
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: The sensitivity of the bacterial tester strains to the toxicity of the test item varied between exposures with and without S9-mix and experimental methodology.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: The sensitivity of the bacterial tester strains to the toxicity of the test item varied between exposures with and without S9-mix and experimental methodology.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: The sensitivity of the bacterial tester strains to the toxicity of the test item varied between exposures with and without S9-mix and experimental methodology.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: The sensitivity of the bacterial tester strains to the toxicity of the test item varied between exposures with and without S9-mix and experimental methodology.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: The sensitivity of the bacterial tester strains to the toxicity of the test item varied between exposures with and without S9-mix and experimental methodology.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
POSITIVE CONTROL VALUES 2014
TA100 -S9: min 236, max 2698, mean 728, SD 336.3
TA100 +S9: min 211, max 4564, mean 1199, SD 659.5
TA1535 -S9: min 98, max 3537, mean 828, SD 714.9
TA1535 +S9: min 105, max 1457, mean 240, SD 97.7
TA98 -S9: min 95, max 713, mean211, SD 70.3
TA98 +S9: min 90, max 1325, mean 187, SD 112.7
TA1537 -S9: min 99, max 3014, mean 709, SD 362.6
TA1537 +S9: min 110, max 1033, mean 329, SD 151.5

POSITIVE CONTROL VALUES 2015
TA100 -S9: min 222, max 2266, mean 614, SD 260.6
TA100 +S9: min 250, max 2402, mean 927, SD 452.5
TA1535 -S9: min 79, max 2779, mean 472, SD 434.8
TA1535 +S9: min 118, max 457, mean 246, SD 55.7
TA98 -S9: : min 100, max 502, mean 222, SD 70.2
TA98 +S9: : min 78, max 705, mean 218, SD 107.6
TA1537 -S9: : min 164, max 2318, mean 911, SD 412.4
TA1537 +S9: : min 97, max 823, mean 336, SD 135.7

- Negative (solvent/vehicle) historical control data:
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2014
TA100 -S9: min 63, max 172, mean 91, SD 14.1
TA100 +S9: min 65, max 137, mean 92, SD 13.1
TA1535 -S9: min 8, max 37, mean 15, SD 3.9
TA1535 +S9: min 8, max 27, mean 13, SD 3.2
TA98 -S9: min 9, max 37, mean20, SD 4.3
TA98 +S9: min 9, max 38, mean 23, SD 4.2
TA1537 -S9: min 4, max 27, mean 12, SD 3.4
TA1537 +S9: min 5, max 23, mean 13, SD 3.1
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2015
TA100 -S9: min 60, max 166, mean 91, SD 19.3
TA100 +S9: min 61, max 175, mean 95, SD 19.1
TA1535 -S9: min 7, max 31, mean 16, SD 4.5
TA1535 +S9: min 7, max 29, mean 14, SD 4.0
TA98 -S9: : min 11, max 45, mean 21, SD 6.2
TA98 +S9: : min 10, max 46, mean 24, SD 6.1
TA1537 -S9: : min 4, max 27, mean 12, SD 3.8
TA1537 +S9: : min 6, max 27, mean 13, SD 3.4

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The sensitivity of the bacterial tester strains to the toxicity of the test item varied between exposures with and without S9-mix and experimental methodology:
TA1535:
Exp. 1: Reductions in revertant colony frequency (no significant weakening of the bacterial background lawns) from 1500 µg/plate in absence of S9
Exp. 2: Weakened bacterial background lawns from 1500 µg/plate in the absence of S9
TA1537:
Exp.1: Reductions in revertant colony frequency (no significant weakening of the bacterial background lawns) from 1500 µg/plate in absence of S9
Exp. 2: Reductions in revertant colony frequency (no significant weakening of the bacterial background lawns) at 5000 µg/plate in presence of S9
TA98:
Exp. 1: no cytotoxicity
Exp. 2: Weakened bacterial background lawns noted at 5000 µg/plate in the absence of S9-mix
TA100:
Exp. 1: Weakened bacterial background lawns at 5000µg/plate in the absence of S9; Reductions in revertant colony frequency (without a significant weakening of the bacterial background lawns) at 5000 µg/plate in presence of S9.
Exp. 2: Weakened bacterial background lawns from 1500 µg/plate in absence of S9; weakened lawns at and above 1500 µg/plate in presence of S9.
Conclusions:
C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate.

Seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawn of Salmonella strain TA100 at 5000 µg/plate in the absence of metabolic activation (S9-mix). Reductions in revertant colony frequency (without a significant weakening of the bacterial background lawns) were also noted from 1500 µg/plate to TA1537 and TA1535 dosed in the absence of S9-mix and to TA1537 and TA100 at 5000 µg/plate dosed in the presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate in the second mutation test. The test item induced a stronger toxic response after employing the pre-incubation method in Experiment 2 with weakened bacterial background lawns noted in the absence of S9-mix from 1500 µg/plate (TA100 and TA1535) and at 5000 µg/plate (TA98 and TA1537). In the presence of S9-mix, weakened lawns were noted to TA100 at and above 1500 µg/plate with substantial reductions in TA1537 revertant colony frequency noted at 5000 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test item varied both between strain type, exposures with and without S9-mix and experimental methodology. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite was considered to be non-mutagenic under the conditions of this test.

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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2
Reason / purpose for cross-reference:
read-across source
Key result
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:
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 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 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 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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2
Reason / purpose for cross-reference:
read-across source
Key result
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:
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 10-6clonable 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 106clonable 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 10-6clonable 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 106clonable 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 10-6clonable cells in the case of EMS and ranging from 319.22 to 909.47 x 10-6clonable 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 10-6clonable 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 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)

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore, the results of the in vitro data are regarded as relevant for humans.

Additional information

Additional information from genetic toxicity in vitro:  

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 (CAS 68784-08-7) was examined for bacterial and mammalian gene mutation as well as for chromosomal aberration by means of a liquid test item containing 41.5% active ingredient.

 

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, 2013a). 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.

 

Mammalian mutagenicity

A key study was conducted in cultured mammalian cells (V79, genetic marker HPRT) both in the presence (4 hours) and absence (4 and 24 hours) of metabolic activation (Flügge, 2013b). In the 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 1000µg/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.

 

Chromosome aberration

A key in vitro micronucleus test (Flügge, 2013c) was conducted using human peripheral lymphocytes both in the presence and absence of metabolic activation, employing 2 exposure times (4 hours and 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. The test item was completely dissolved in aqua ad iniectabilia, which also served as the vehicle control. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only.

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.

Both in the tests without metabolic activation (4- and 20-hour exposure) and with metabolic activation (4 -h exposure), there was no increase in micronuclei up to the cytotoxic concentrations 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.

 

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