Condensation products of N-(2-aminoethyl)octadecanamide and N-(2-aminoethyl)hexadecanamide with sebacic acid

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:

12 February to 19 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

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:

Condensation products of N-(2-aminoethyl)octadecanamide and N-(2-aminoethyl)hexadecanamide with sebacic acid

EC Number:

945-989-8

IUPAC Name:

Condensation products of N-(2-aminoethyl)octadecanamide and N-(2-aminoethyl)hexadecanamide with sebacic acid

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test materal: Kyoeisha Chemical Co., Ltd
- Lot/batch No.of test material: 6033175
- Expiration date of the lot/batch: 17 March 2019
- Purity test date: 3 October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (Ambient). Kept in original container as supplied by the Sponsor. Container kept tightly closed and away from heat or sunlight
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: Formed a homogenous suspension in Dimethyl sulphoxide (DMSO) at 50000 µg/mL after sonication. Assumed stable for the duration of the study
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: DMSO added to test item and sonicated to form a homogenous suspension at 50000 µg/mL before addition to the test system
FORM AS APPLIED IN THE TEST (if different from that of starting material) : The test item is a powder and forms a homogenous suspension in DMSO at 50000 µg/mL after sonication.

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Initial Toxicicty-Mutation Assay: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
A normal bacterial background lawn and no increase in the number of revertant colonies was observed up to 5000 µg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA. The OECD recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate (i.e. 5000 ug/Plate).

Based on the results of the initial toxicity-mutation assay, 6 test concentrations were selected for the confirmatory assay using an increase concentration of S9 mix (10% v/v) in accordence with guidline requirments.
Confrmatory Mutation assay: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle:- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in distilled water (stock A, 50000 µg/mL). A homogeneous suspension was however formed in dimethyl sulfoxide (DMSO) (Stock B 50000 µg/mL), acetone (Stock C 50000 µg/mL) and ethanol 95% (Stock D 50000 µg/mL). After sonication all remained homogenous in suspension. DMSO was therefore selected as the vehicle for treatment. A volume of 100 µL of the test item from stock B (50000 µg/mL) was added to 2 mL of top agar, to assess precipitation. Precipitation was not observed at 5000 µg/plate. Hence 5000 µg/plate was selected as the highest concentration to be tested for the initial toxicity-mutation test both in the absence and presence (5 % v/v S9 mix) of metabolic activation.

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2Aa (2-Aminoanthracene)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation method)
- Cell density at seeding (if applicable): Cell densities (OD at 660 nm) of all tester strains were within the required range to produce cultures with approximately 1 - 2 x 109 bacteria/mL, demonstrating appropriate numbers of bacteria were plated.

DURATION
- Preincubation period: not specified
- Exposure duration: Incubated at 37 ± 1 °C for 48 hours
- Expression time (cells in growth medium):

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is detectable as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn. No cytotoxicity was observed as a normal bacterial background lawn was observed up to the tested concentration of 5000 µg/plate in the absence and presence of the metabolic activation system (10% v/v S9 mix) in all tester strains.

Rationale for test conditions:

This assay measures the ability of test item to induce reverse mutations at specific histidine loci in the tester strains of Salmonella typhimurium i.e., TA1537, TA1535, TA98, TA100, and at the tryptophan locus in Escherichia coli WP2uvrA, that are known for their reliability and reproducibility in this short term mutagenicity assay, and are recommended by the OECD and other regulatory guidelines. All procedures were performed ina cccordence with OECD 471 and EC B.13/14, and GLP audited.

Evaluation criteria:

Assay Acceptance Criteria
Before assay data were evaluated, the criteria for a valid assay were confirmed to have been met. The following were used to determine a valid assay:
Tester strain integrity, Characteristic number of spontaneous revertants, Tester strain culture density, Positive control values in the absence and presence of metabolic activation, Cytotoxicity evaluation criteria.

Assay Evaluation Criteria
Once criteria for a valid assay had been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were: there should be a dose related increase in the mean revertants per plate in at least one tester strain over the range tested and/or at one or more doses of the test item either in the absence or presence of the metabolic activation system.
The biological relevance of the results was considered:

Strains TA1535 and TA1537 and Escherichia coli WP2uvrA
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.

Strain TA98, TA100
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.
Statistical analysis was used as an aid in the evaluation of a dose response.
A response that did not meet all three of the above criteria (magnitude, concentration responsiveness, reproducibility) was not evaluated as positive. Negative results obtained in the initial toxicity-mutation assay were confirmed by a confirmatory mutation assay, using the same method as specified above, with an alteration in concentration spacing.

Statistics:

Simple linear regression analysis was performed for tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA, separately, to assess the dose dependent nature of any increase in revertant colonies.

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

From the results of this study, under the specified experimental conditions, N, N’-bis {6-[12-(hydroxy) octadecanoylamino] hexyl} decanediamide is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA.