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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern GLP compliant guideline study, no restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TAEE, tert-amyl ethyl ether, CAS No. 919-94-8
- Lot/batch No.: 00931302
- Description: colourless liquid
- Purity: 99.4%
- Date received: 22 October 2008

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced S9 fraction from male SD rats (Lot 2308 from Molecular Toxicology Incorporated, Boone, NC, USA)
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced S9 fraction from male SD rats (Lot 2308 from Molecular Toxicology Incorporated, Boone, NC, USA)
Test concentrations with justification for top dose:
Following test concentrations used (based on preliminary toxicity test with TA100):
Experiment 1: 1.6, 8, 40, 200, 1000 and 5000 µg/plate
Experiments 2 and 3: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: based on preliminary solubility experiments
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
other: strain-specific positive control substances used in the absence and presence of S9: see below for full details
Details on test system and conditions:
METHOD OF APPLICATION
- Experiment 1: plate incorporation
- Experiments 2 and 3: liquid pre-incubation
Comment: only incubations containing TA98 (-S9), TA102 (-S9) and TA1537 (+S9) were repeated in experiment 3.
- triplicate plates prepared per exposure concentration for each experiment

DURATION
- Exposure duration: 48 hr at 37 degrees C

NUMBER OF EXPERIMENTS:
- 2 independent repeats

DETERMINATION OF CYTOTOXICITY
- Method: TA100 exposed to 0.15-5000 ug/plate (10 concentrations) by plate incorporation, +/- S9, for 48 hr at 37 degrees C. An oily precipitate was observed at > 1500 μg/plate, but this was not toxic to the bacteria.

ANALYSIS OF ACHIEVED CONCENTRATION:
Duplicate samples of all test substance preparations were analysed by GC-FID; results +/-10% of nominal.
Evaluation criteria:
Test article considered mutagenic if (i) Dunnett's test gave a significant concentration-related response; and (ii) the effect was reproducible.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
CYTOTOXICITY: The test substance was practically non-toxic to TA100.

STUDY RESULTS: Following statistically significant but not clearly dose-related increases in revertant number for TA98 (-S9), TA102 (-S9) and TA1537 (+S9) in experiment 2, this portion of the study was repeated using the same tester strain/S9/test concentration conditions. No mutagenic was response was seen in the repeat trial. Overall it was concluded that the test substance was not mutagenic to Salmonella tester strains either in the absence or presence of Aroclor 1254-induce rat S9 fraction.

POSITIVE CONTROLS: a satisfactory response was obtained with all of the positive control substances.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Not mutagenic in 5 standard Salmonella typhimurium tester strains (TA100, TA1535, TA1537, TA102, or TA98) in the absence or presence Aroclor 1254-induced rat S9 fraction.
Executive summary:

Mutagenic potential assessed in a GLP-compliant guideline bacterial mutation assay (reverse mutation assay, method B13/14 of directive 2004/73/EC) using Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA102 exposed via plate incorporation (one experiment) and liquid pre-incubation (one experiment with partial repeat). Based on results obtained from an initial toxicity screen, test concentrations up to 5000 ug/plate were used in the absence and in the presence of Aroclor 1254 -induced rat S9 fraction. The test substance was not cytotoxic nor was there any consistent increase in the frequency of revertant colonies in any strain both with and without metabolic activation. The results indicate that TAEE (tert-amyl ether) is not mutagenic in this bacterial assay (Ames test) when tested up to the maximum concentration required in current test guidelines.