Reaction products of amines, dicoco alkyl and glycollic acid

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:

2005/10/28 to 2005/11/10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study done according to OECD 471 guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: dark amber slightly viscous liquid
- Analytical purity: >99%
- Storage condition of test material: room temprature in the dark

Method

Target gene:

Histidine and tryptophan

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:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta-naphthoflavone induced S9

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500 and 5000 ug/plate

Vehicle / solvent:

Acetone

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: 2 ug/plate for WP2 uvr A, 3 ug/plate fro TA100 and 5 ug/plate for TA 1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 80ug/plate for TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: 0.2 ug/plate for TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1 ug/plate fro TA100, 2 ug/plate for TA1535 and TA1537, 10 ug/plate for WP2 uvr A

Remarks:

2-aminoanthracene (2AA)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 5 ug/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate plates per dose level, experiment repeated on seperate occasion.

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of growth of background lawn of bacteria and numbers of revertant colonies

Evaluation criteria:

A dose-related increase in revertant frequency over the dose-range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolica activation. Biological relevance of the results will be considered first, statistical methods also used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

Statistics:

Dunnett's t-test, as recommended by the UKEMS (1989).

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Not soluble in water
- Precipitation: Observed at upper three dose levels but did not interfere with the test

RANGE-FINDING/SCREENING STUDIES: A prelimiary test was performed using a dose range of 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 ug/plate

COMPARISON WITH HISTORICAL CONTROL DATA: Control data were within the range of the historical dataset.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1 – Mean colony counts of triplicate plates

	TA100		TA1535		WP2uvrA		TA98		TA1537
S9	-	+	-	+	-	+	-	+	-	+
0	111	104	29	16	19	26	24	21	13	19
50	104	97	33	12	18	27	23	20	10	20
150	111	97	30	11	19	19	20	19	16	18
500	112	104	34	12	19	20	20	24	9	24
1500	106	85	33	11	17	24	21	21	13	24
5000	107	86	25	13	20	29	22	19	10	18
Positive	657	517	413	194	977	673	234	162	905	413

 

Experiment 2 – Mean colony counts of triplicate plates

	TA100		TA1535		WP2uvrA		TA98		TA1537
S9	-	+	-	+	-	+	-	+	-	+
0	111	99	26	10	19	21	18	23	14	13
50	110	90	21	8	18	29	17	19	17	11
150	110	106	23	12	19	25	16	21	19	9
500	115	105	18	12	18	26	18	13	15	7
1500	117	99	25	12	17	22	18	19	10	8
5000	105	89	21	19	24	28	20	13	10	10
Positive	421	1417	245	251	616	712	152	233	481	503

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test, at up to the maximum recommended dose level and beyond the precipitating limit.

Executive summary:

Introduction. The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B 13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TAl535, TA1537, TA98 and TAl00 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-fmding test was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Results. The vehicle (acetone) 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 test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 pg/plate. A precipitate (oily in appearance) was observed at and above 500 ug/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

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