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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: GLP guideline study conducted according to OECD Guideline 471.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000
Reference Type:
publication
Title:
Unnamed
Year:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
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
Type:
Constituent
Details on test material:
Identity (as provided in report): Tertiary Butyl Acetate
-Appearance: Clear colorless liquid
-Storage conditions: Room temperature
-Lot number: 1031267
-Purity: ≥95%
-Date received: 30 March 1998
-Expiry date: > 1 year from date of receipt; assumed stable for duration of study

Method

Target gene:
His (-), Trp (-)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rats (S9 mix)
Species / strain:
S. typhimurium TA 102
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rats (S9 mix)
Species / strain:
E. coli WP2 uvr A pKM 101
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rats (S9 mix)
Test concentrations with justification for top dose:
5-5000 µg/plate
Vehicle:
Dimethyl sulphoxide (DMSO)
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
Dimethyl sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
0.5 µg/plate for strains TA 1535 and TA 100 in the absence of S9
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
30 µg/plate for strain TA 1537 in the absence of S9
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
1 µg/plate for strain TA 98 in the absence of S9
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide; AF-2
Remarks:
0.05 µg/plate for strain WP2 uvr A pKM 101 (CM891) in the absence of S9
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
100 µg/plate for strain TA 102 in the absence of S9
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
2 µg/plate for strain TA 1535 and 10 µg/plate for WP2 uvr A pKM 101 (CM891) in the presence of S9
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for strains TA 1537, TA 98, and TA 100 in the presence of S9
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxyanthraquinone; Danthron
Remarks:
30 µg/plate for strain TA 102 in the presence of S9
Details on test system and conditions:
Strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium were obtained from the National Collection of Type Cultures, London, England. Strain TA 102 was obtained from Professor B. N. Ames, University of California Berkeley, California, USA. The strain of E.coli was obtained from the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland.

The test was conducted twice. In the first test (range-finding), the test or reference substance was added to cultures of the six tester strains at seven concentrations (0, 5, 15, 50, 150, 500, 1500, 5000 μg/plate) separated by half-log10 intervals. The highest concentration of tertiary butyl acetate tested was 50 mg/mL in the chosen solvent, which provided a final concentration of 5000 µg/plate.

An aliquot of 0.1 mL of a 10-hour bacterial culture and 0.5 mL S9 mix or 0.5 mL 0.1M sodium phosphate buffer (pH 7.4) were placed in gas–tight glass vials. An aliquot of 0.1 mL of the test dilution was added via septa fitted in the vial caps and the mixtures pre-incubated at 37 °C with shaking at 120 rpm for 30 minutes. Following the pre-incubation period, 2 mL of molten agar containing 0.5mM histidine/biotin/tryptophan was added. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 mL minimal agar. Three petri dishes were used for each concentration. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix, and sodium phosphate buffer. All plates were incubated at 37 °C for 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies were counted using a Domino automated colony counter. The second test was an exact repeat of the first test, except only five concentrations were used (0, 50, 150, 500, 1500, 5000 μg/plate). Concurrent positive and solvent controls were run with each test.
Evaluation criteria:
For a test to be considered valid, the mean of the solvent/vehicle revertant colony numbers of each strain should lie within the 99% confidence limits of the current historical control of the laboratory. The historical range is maintained as a rolling record over a minimum of two and a maximum of five years. The positive control compounds must cause at least a doubling of mean revertant colony numbers over the negative control. The mutagenic activity of a test substance was assessed by applying the following criteria:
-If treatment with the test substance produced an increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, the test substance was considered to show evidence of mutagenic activity in this test system. No statistical analysis will be performed.
- If treatment with the test substance did not produce reproducible increases of at least 1.5 times the concurrent solvent/vehicle controls in either mutation test, the test substance was considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed.
- If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response given in the previous two examples, even after the additional testing outlined in the mutation test procedure, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al., (1989) and will usually be analysis of variance followed by Dunnett’s test (Dunnett, 1964).
See references below in "Any other information on materials and methods".
Statistics:
No statistical analyses were necessary in this study since the results clearly showed no evidence of mutagenicity.

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:
negative
Cytotoxicity:
yes
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'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
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'.
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
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:
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to tertiary butyl acetate at any concentration in either the presence or absence of S9 mix.

Toxicity towards the tester strains (thinning of the background lawn and/or reduction in revertant colony counts) was observed in both mutation tests following exposure to tertiary butyl acetate at a concentration of 5000 μg/plate.

Applicant's summary and conclusion

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

At a maximum concentration of 5000 μg/plate, tertiary butyl acetate did not induce mutations in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 or TA 1537, or in E. coli strain WP2 uvr A pKM 101 when tested in the Salmonella /microsome bacterial mutagenicity assay (Ames assay) in the presence and absence of a metabolic activation system.

Based on an absence of genotoxic/mutagenic effects in this study, tertiary butyl acetate is not classifiable for Germ Cell Mutagenicity according to Directive 67/548/EEC, the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 of S. typhimurium and strain WP2 uvr A pKM 101 of E. coli were exposed to tertiary butyl acetate in DMSO at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method. Tertiary butyl acetate was tested up to the limit concentration of 5000 μg/plate. There was a reduction in the number of mutant colonies and a thinning of the background lawn of micro-colonies, indicative of toxicity, at 5000 μg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.