C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: USA, EPA OCSPP harmonized guideline 870.5100- Bacterial Reverse Mutation Test.

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-[1-methyl-2-[(1-oxooctadec-9-enyl)amino]ethyl] 2-sulphonatosuccinate

EC Number:

267-199-6

EC Name:

Disodium 4-[1-methyl-2-[(1-oxooctadec-9-enyl)amino]ethyl] 2-sulphonatosuccinate

Cas Number:

67815-88-7

Molecular formula:

C25H45NO8S.2Na

IUPAC Name:

disodium 4-[1-methyl-2-(oleoylamino)ethoxy]-4-oxo-2-sulfonatobutanoate

Test material form:

liquid

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:

Method

Target gene:

histidine, tryptophan

Species / strain

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.

Controls

Controls 1

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

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 μg/plate for TA1537

Controls 3

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline-1-oxide (4NQO)

Remarks:

0.2 μg/plate for TA98

Controls 4

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

Controls 5

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Applicant's summary and conclusion

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.