[No public or meaningful name is available]

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:

Experimental phase: 23 August 2017 to 20 October 2017: Report issue: 13 February 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Molecular formula:

not applicable as UVCB

IUPAC Name:

Fatty acids, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Test material form:

liquid

Method

Target gene:

Histidine for Salmonella typhimuriun
Tryptophan for E.coli

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

E. coli strain = WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone / beta-naphthoflavone induced rat liver S9.

Test concentrations with justification for top dose:

Experiment one (plate incorporation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Experiment two (pre-incubation method):

1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate: WP2uvrA and TA98 with and without S9.
0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate: TA100 with and without S9 and TA1537 without S9 only.
0.5, 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate; TA1537 with S9 only.
5, 15, 50, 100, 150, 250 and 500 µg/plate: TA1535 with S9.
0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate: TA1535 without S9.

No correction for purity was made due to the UVCB nature of the test substance.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Choosen following solubility checks performed in-house.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Withuot S9

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of E. Coli WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of E. coli WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: iin agar with plate incorporation for experiment 1 and preincubation for experiment 2.

DURATION
- Preincubation period for bacterial strains: 10 hrs
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: triplicate plating

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial lawn.

Evaluation criteria:

Any one or all of the following criteria could 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.

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle 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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test substance was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The mutagenicity of the test substance in bacteria was assessed using a test method was designed to be compatible with OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test".

Methods

Salmonella typhimuriums strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with solutions of the test substance 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 metabolising system (10% liver S9 in standard co-factors) over four separate experiments (two experiments with two further confirmatory experiments).

The dose range for Experiment 1 (plate incorporation) was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using a dose range that ranged between 0.15 and 5000 µg/plate, depending on the strain type and presence or absence metabolic activation (S9). An expanded dose range was selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

Due to differences in the results obtained in Experiments 1 and 2, a confirmatory test (Confirmatory Test 1) was dosed at 5 to 500 µg/plate using TA1535 (in the presence of S9-mix only) under pre-incubation conditions only. A second confirmatory experiment (Confirmatory Test 2) was dosed, again using TA1535, with and without S9-mix and using both plate incorporation and pre-incubation methods to confirm the difference in results obtained in Experiment 2 and Confirmatory Test 1. The dose range was the same as Confirmatory Test 1.

Results

The vehicle (dimethyl sulphoxide (DMSO)) 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.

Following analysis of the results of all the experiments, it was concluded that no biologically relevant increases in the frequency of revertant colonies were observed at any dose of the test item, either with or without metabolic activation (S9-mix).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix in any of the experiments detailed above.

Conclusion

The test substance was considered to be non-mutagenic under the conditions of this test.