1,4-Dihydroxy-2,2,6,6-tetramethylpiperidine

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

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:

-

EC Number:

434-880-6

EC Name:

-

Cas Number:

3637-10-3

Molecular formula:

C9H19NO2

IUPAC Name:

2,2,6,6-tetramethylpiperidine-1,4-diol

Test material form:

solid: crystalline

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

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/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test (with and without S9 mix): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 1 and Experiment 2 (with and without S9 mix): 50, 150, 500 and 1500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of bacterial strains

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

The positive controls 2AA and BP were used in the series of plates with S9-mix.

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of bacterial strains

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

The positive controls ENNG, 9AA and 4NQO were used in the series of plates without S9-mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar, direct plate incorporation for Experiment 1 and Experiment 2.

DURATION
- 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 background lawn.

Evaluation criteria:

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
-All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks
-All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
-All tester strain cultures should be in the range of 1 to 9.9 x 10E9 bacteria per ml.
-Each mean positive control value should be at least two times the respective control value for each strain, thus demonstrating both the intrinsic sensitivity ofthe tester strains to mutagenic exposure and the integrity of the S9-mix.
-There should be a minimum of four non-toxic test item dose levels.
-There should be no evidence of excessive contamination.

Evaluation Criteria:
The test material should have induced a reproducible, dose related and statistically significant increase in revertant count in at least one strain of bacteria..

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

Remarks:

Tested up to the maximum recommended dose level of 5000 µg/plate

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

Remarks:

Tested up to the maximum recommended dose level of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST:
The test material was non-toxic to the strains of bacteria (TA100 and WPuvrA-). The test material formulation and the S9-mix used in this experiment were both shown to be sterile.

The number of revertant colonies for the toxicity are shown in the attached background information.

MUTATION TEST:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in the attached background information.and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in the attached background information.).

A history profile of vehicle and positive control values is presented in the attached background information..

No toxicity was exhibited to any of the strains of bacteria used. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B14 of Commission Directive 92/69/EECand the USA, EPA (TSCA) OPPTS harmonized guidelines.

Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia colistrainWP2uvrA were treated with the test item 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 was determined in a preliminary toxicity assay and ranged between 50 and 5000µg/plate, in the first experiment. The experiment was repeated on a separate day using the same dose range as in experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.

 

Results

The vehicle (dimethyl sulphoxide) 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 item caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was therefore tested upto the maximum recommended dose of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial starins, with any dose of the test material, either with or without metabolic activation.

Conclusion

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