Reaction mass of N, N'-hexane-1,6-diylbis [12-hydroxyoctadecanamide] and 12-hydroxy-N-[6-[1-oxoalkyl)amino] hexyl ] octadecanamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February - 20 February 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): AD-1000
- Substance type: white powder
- Physical state: powder
- Storage condition of test material: at room temperature in the dark

Method

Target gene:

Histidine gene in S. typhimurium
Tryptophan gene in E. coli

Species / strain

Species / strain / cell type:

bacteria, other: Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and Escherichia coli (WP2uvrA).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix
Concentration range in the main test (with metabolic activation): 3, 10, 33, 100, 333, 1000 µg/plate
Concentration range in the main test (without metabolic activation): 3, 10, 33, 100, 333, 1000 µg/plate

Vehicle / solvent:

Solvent: Dimethyl sulfoxide

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: sodium azide (SA) 5µg in Saline for TA1535

Remarks:

Without metabolic activation

Controls 2

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Migrated to IUCLID6: 60 µg in Milli-Q water for TA1537

Controls 3

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Migrated to IUCLID6: 10 µg in DMSO for TA98

Controls 4

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Migrated to IUCLID6: 650 µg in DMSO for TA100

Controls 5

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Migrated to IUCLID6: 10 µg in DMSO for WP2uvrA

Controls 6

Positive control substance:

other: 2-aminoanthracene (5 and/or 10%) for all strains

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 100 µg/plate

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: doses of the test substance were tested in triplicate in each strain. Two independent experiments were
conducted.


OTHER EXAMINATIONS:
precipitation of test substance

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not biologically relevant.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Test results 1

Species / strain:

other: Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and Escherichia coli (WP2uvrA).

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

>=1000 µg/plate

Test results 2

Species / strain:

other: Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and Escherichia coli (WP2uvrA).

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

>= 100 µg/plate

Additional information on results:

Observations: The test substance did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each
of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9- metabolic activation. These results were confirmed in an independently repeated experiment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: preliminary test

Any other information on results incl. tables

In the first and second experiment 5% (v/v) and 10% (v/v) S9 -mix was used, resepectively.

In this study, the negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that AD-1000 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.