Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic 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:

11/07/2017 - 12/11/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid

IUPAC Name:

Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid

Test material form:

solid: bulk

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0001230562
- Expiration date of the lot/batch: 08/06/2019
- Purity test date: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark.
- Stability under test conditions: Assumed to be stable.
- Solubility and stability of the test substance in the solvent/vehicle: Assumed to be stable and solubility measured (best solvent was tetrahydrofuran).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None assumed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: Disolved in solution up to a maximum concentration of 200 mg/mL.
- Final preparation of a solid: Disolved in tetrahydrofuran.

FORM AS APPLIED IN THE TEST: Dissovled in tetrahydrofuran.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: frame shift mutations

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: base-pair substitutions

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: base-pair substitution

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The maximum concentration was 5000 µg/plate (the maximum recommended dose level).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran.
- Justification: test item had highest solubility in this solvent.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: The plate incorporation method and the pre-incubation method.

DURATION
- Preincubation period: 48 hours.
- Exposure duration: 48 hours .
- Expression time (cells in growth medium): 48 hours .
- Selection time (if incubation with a selection agent): Automated (except at 5000 ug/plate where it was performed manually) at approximately 48 hours.
- Fixation time (start of exposure up to fixation or harvest of cells): N/A.

SELECTION AGENT (mutation assays): reverse mutation.

NUMBER OF REPLICATIONS: 3.

NUMBER OF CELLS EVALUATED: cells growth per plate per bacterial strain per treatment type.

Rationale for test conditions:

The study was based on the in vitro technique described by Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item.

Evaluation criteria:

The following criteria were used to determine a positive interaction:

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)).

It is stated that a test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Dunnetts Regression Analysis (* = p < 0.05) to indicate statistical significance.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The study examines the potential for Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid to cause gene mutations in a reverse mutation assay 'Ames Test' using stains of Salmonella typhimurium and Escherichia coli. The study is GLP compliant and is performed according to OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test" and the EU method B13/14 of Commission Regulation. The organisms used in the study are as follows: Salmonella typhimurium (Strains: TA1537; TA98; TA1535; TA100) and Escherichia coli. (Strains:WP2uvrA). The study includes a valid solvent control, negative control and positive control. Controls and test item experiments are performed in triplicate. The results from the experiment show that Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid does not cause gene mutation in any of the bacterial strains used in this experiment.