2,2,6,6-tetramethyl-4-piperidone

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptable, well documented study report which meets basic scientific principles. The fith strain (E. coli) was also tested. Deficiencies: non-GLP, repeat experiments not performed for all strains but as shown in the supporting study, the result is reducable and no contradictory results occurred.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only 4 concentrations tested)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2,6,6-tetramethyl-4-piperidone

EC Number:

212-554-2

EC Name:

2,2,6,6-tetramethyl-4-piperidone

Cas Number:

826-36-8

Molecular formula:

C9H17NO

IUPAC Name:

2,2,6,6-tetramethylpiperidin-4-one

Details on test material:

- Expiration date of the lot/batch: July 1996

Method

Target gene:

his- / trp-gene

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

- Range in the cytotoxicity test: 20.57 - 5000 µg/plate;
- Range in the mutagenicity test: with/without metabolic activation: 625 - 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: bidistilled water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below: details on test system and conditions

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (Rat liver S9 fraction: 100 µl/ml, NADP: 4 µmol/ml, MgCl2: 8 µmol/ml, KCl: 33 µmol/ml, Na-phosphate-buffer, pH 7.4: 100 µmol/ml, Glucose-6-phosphate 5 µmol/ml; experiments with activation) and 0.1 ml of a solution of the test substance, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 ml of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl. In the experiment with Salmonella the top agar was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water. In the experiment with E. coli it was supplemented with 10% of 0.5 mM L-tryptophan dissolved in water. The plates were inverted and incubated for about 48 hours at 37 ± 1.5°C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn. Colonies were counted electronically with an Artek counter.

Positive controls (without metabolic activation):

- TA 100: sodium azide in bidist. water (5.0 µg/plate);
- TA 1535: sodium azide in bidist. water (5.0 µg/plate);
- E.coli WP2 uvrA: 4-nitroquinoline-N-oxide in DMSO (2.0 µg/plate);
- TA 98: 2-nitrofluorene in DMSO (20.0 µg/plate);
- TA 1537: 9-aminoacridine in DMSO (150.0 µg/plate);

Positive controls (with metabolic activation):

- TA 100: 2-aminoanthracene in DMSO (2.5 µg/plate);
- TA 1535: cyclophosphamide*H2O in bidist. water (400.0 µg/plate);
- E. coli WP2 uvrA: 2-aminoanthracene in DMSO (50.0 µg/plate);
- TA 98: 2-aminoanthracene in DMSO (2.5 µg/plate);
- TA 1537: 2-aminoanthracene in DMSO (2.5 µg/plate);

Evaluation criteria:

Assay acceptance criteria:

A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.

Criteria for a positive response:

The test substance is considered to be mutagenic in this test system if:
A positive effect is observed in one strain and the effect can be reproduced in a confirmatory experiment. A positive effect is observed in two or more strains. A positive effect is defined as an increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 2.0 with strains TA 98, TA 1535, TA 1537 and WP2 uvrA, or by a factor of at least 1.5 with strain TA 100. Generally a concentration-related effect should be demonstrable. If equivocal results are obtained, the final decision has to be based on scientific judgement.

Statistics:

Means and standard deviations for all mutagenicity assays were calculated by a previously validated computer program.

Results and discussion

Test results

Species / strain:

other:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: not observed


RANGE-FINDING/SCREENING STUDIES:
The concentration range of TK 11831 to be tested in the mutagenicity test was determined in a preliminary toxicity test. The toxicity test was carried out with strains S. typhimurium TA 100 and E.coli WP2 uvrA without and with metabolic activation at six concentrations of the test substance and one negative control. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5°C in darkness. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with metabolic activation.


COMPARISON WITH HISTORICAL CONTROL DATA: negative and positive historical control data and acceptable ranges for negative controls were available

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Strain	Metabolic activation system	mean his^+/trp^+revertant colonies (negative control)	maximum revertant factor (conc. (µg/plate))	dose dependency	Assessment	maximum revertant factor (positive control)
TA 98	no	13	1.31 (1250)	no	negative	107.62 (2-nitrofluorene)
	yes	28.5	1.07 (625 / 5000)	no	negative	30.63 (2-aminoanthracene)
TA 100	no	113	0.95 (5000)	no	negative	8.92 (sodium azide)
	yes	129.5	0.96 (5000)	no	negative	5.75 (2-aminoanthracene)
TA 1537	no	8	0.88 (1250)	no	negative	218.50 (9-aminoacridine)
	yes	5.5	1.64 (1250)	no	negative	16 (2-aminoanthracene)
TA 1535	no	15	1.03 (5000)	no	negative	61.47 (sodium azide)
	yes	13	1.31 (5000)	no	negative	31.19 (cyclophosphamide)
E. coli WP2 uvrA	no	21	1.21 (2500)	no	negative	30.4 (4-NQO)
	yes	30	0.83 (1250)	no	negative	45.87 (2-aminoanthracene)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative