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Toxicological information

Genetic toxicity: in vitro

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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:
30th May 2012 - 5th July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with the appropriate guideline and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
other: USA, EPA (TSCA) OPPTS harmonised guidelines
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas

Method

Target gene:
Histidine for Salmonella strains.
Tryptophan for E.coli strains.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fraction prepared from livers of male rats induced with phenobarbitone/ β-naphthoflavone given orally for 3 consecutive days.
Test concentrations with justification for top dose:
Mutation Test –Experiment 1 (plate incorporation):
50, 150, 500, 1500 and 5000 μg/plate were assayed in triplicate, with and without metabolic activation.

Mutation Test- Experiment 2 (pre-incubation):
1000, 1500, 2000, 3000, 4000 and 5000 μg/plate were assayed in triplicate, with and without metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Substance appeared immiscible in sterile distilled water at 50 mg/ml but was fully miscible with DMSO.
Controls
Untreated negative controls:
yes
Remarks:
2-Aminoanthracene, Benzo(a)pyrene (BP)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1) and pre-incubation (Experiment 2)
DURATION
- Pre-incubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C for both application methods.
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells):N/A

NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
A preliminary toxicity test was performed at 11 test concentrations in the range 0 - 5000µg/plate using bacterial strains TA 100 and WP2uvrA. The number of revertant colonies and effects on the growth of the bacterial background lawn were used to assess the toxicity of the test substance to the test system.
Evaluation criteria:
There are several criteria for determining a positive result, such as 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
metabolic activation. Biological relevance of the results will be considered first, statistical methods, can also be used as an aid to
evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a
definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
In particular, strains TA100 and TA98 at the upper dose levels.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not determined
- Effects of osmolality: Not determined
- Evaporation from medium: Not determined
- Water solubility: Not applicable - vehicle DMSO
- Precipitation: None detected

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed on 11 concentrations in the range 0 - 5000µg/plate using bacterial strains TA100 and WP2uvrA.
In addition the sterility of the test item was assessed.
After 48 hours incubation at 37°C, the plates were assessed for number of revertant colonies and effects on the growth of the bacterial lawn.
The test item was found to be non-toxic to the strains of bacteria used. The test item and the s9 mix used were found to be sterile.
COMPARISON WITH HISTORICAL CONTROL DATA:
At the upper end of the statistically significant dose levels the number of revertant colonies was either at or above the upper limit of the vehicle/negative control range. A large data set was available for the historical control data as a high number of Ames tests are performed. It is considered that excursions outside the maxima ranges, particularly when reproducibility is apparent must be considered to be a biological response.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results for concurrent negative controls and experiments 1 and 2 with and without metabolic activation are attached as Tables 1 - 5 below.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

The test item, 4-Oxo Tempo, was considered to be weakly mutagenic under the conditions of this test.
Executive summary:

The genetic toxicity of 4-Oxo Tempo was examined in a bacterial reverse mutation assay (Ames test) performed to GLP and in accordance with OECD 471 and EU method B.13/14. Salmonella Typhimurium strains TA1535, TA 1537, TA98 and TA100, along with Escherichia coli strain WP2uvrA, were tested with and without S9 rat liver fraction metabolic activation.

A preliminary toxicity test proved the test substance to be non-toxic to the TA100 and WP2uvrA strains tested. Two mutation tests were performed, Experiment 1 used the plate incorporation method, test substance concentrations of 50, 150, 500, 1500 and 5000µg/plate were assessed. Experiment 2 was performed using the pre-incubation method at concentrations of 1000, 1500, 2000, 3000, 4000 and 5000 μg/plate. Negative, positive and vehicle controls were also tested.

The results from Experiment 1, showed a small but statistically significant increase in the revertant colony frequencies in the majority of tester strains at the upper dose levels, with and without the S9 mix. This was noted particularly in strains TA100, WP2uvrA and TA98.

The results for Experiment 2 were similar to Experiment 1, again statistically significant increases in the revertant colony frequency were noted for strains TA100 (presence and absence of S9 mix), WP2uvrA and TA98 (absence of S9 mix only) at the majority of the dose levels.

At all statistically significant dose levels, a modest dose-response relationship was observed. The responses, although small were reproducible and in the case of TA100, noted in three separate experiments, the preliminary toxicity test included. 

The results for all control tests were considered acceptable.

The test material, 4-Oxo Tempo, was considered to be weakly mutagenic under the conditions of the test.