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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: The procedures used followed the description of Maron and Ames modified in accordance with the procedures outlined in OECD 471. Maron DM and Ames BN, 1983. Revised methods for the Salmonella mutagenicity test. Mutat. Res., 113:173-215.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methylpropan-2-ol
EC Number:
200-889-7
EC Name:
2-methylpropan-2-ol
Cas Number:
75-65-0
Molecular formula:
C4H10O
IUPAC Name:
2-methylpropan-2-ol
Constituent 2
Reference substance name:
tertiary butyl alcohol
IUPAC Name:
tertiary butyl alcohol
Details on test material:
Laboratory A:
-Identity (according to report): t-butyl alcohol
-Supplier: Fisher Scientific
-Purity: 99.92%

Laboratory B:
-Identity (according to report): t-butyl alcohol
-Supplier: Sigma-Aldrich
-Purity: 99.7%

Method

Target gene:
His (-)
Species / strain
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Laboratory A: Salmonella typhimurium TA 102 was obtained from Prof. Bruce Ames, University of California, CA, USA

Laboratory B: Salmonella typhimurium TA 102 was obtained from SafePharm Laboratories, Shardlow, UK
Additional strain / cell type characteristics:
other: TA 102 carries ochre mutations of the hisG428 gene, deep rough (rfa) mutations, and the factor resistance plasmid pKM 101. TA 102 carries this hisG428 mutation on a multiple copy plasmid (pAQ1) and is excision repair competent (uvrB+).
Metabolic activation:
with and without
Metabolic activation system:
Laboratory A: used Aroclor 1254-treated male Sprague-Dawley rat liver S9; Laboratory B: used phenobarbital-β-naphthoflavone-treated Sprague-Dawley rat liver S9
Test concentrations with justification for top dose:
Laboratory A:
Trial 1 – 5, 15, 50, 150, 500, 1500, 5000 μg/plate
Trial 2 – 50, 150, 500, 1500, 5000 μg/plate

Laboratory B:
Trial 1 & 2 – 100, 200, 500, 1000, 2500, 5000 μg/plate
Vehicle / solvent:
Laboratory A: tertiary butyl alcohol was dissolved in dimethyl sulfoxide (DMSO), spectrophotometric grade
Laboratory B: tertiary butyl alcohol was dissolved in either deionized water or in dried DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-dihydroxyanthraquinone
Remarks:
Used by Laboratory A; obtained from Sigma Chemicals; used in the presence of S9 at a dose of 30 μg/plate
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
Used by Laboratory A; obtained from Sigma Chemicals; used in the absence of S9 at a dose of 100 μg/plate
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Used by Laboratory B; obtained from Sigma Chemicals; used in the presence of S9 at a dose of 1 μg/plate
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Used by Laboratory B; obtained from Sigma Chemicals; used in the absence of S9 at a dose of 10 μg/plate
Details on test system and experimental conditions:
The test was run in two independent laboratories.

TEST METHOD:
Laboratory A: pre-incubation method
The top (overlay) agar was prepared with 0.6% (w/v) agar and 0.5% (w/v) NaCl and was supplemented with 10 mL of 0.5 mM histidine/0.5 mM biotin solution per 100 mL agar for selection of histidine revertants. A 0.1 mL sample of a 10-hr bacterial culture and 0.5 mL S9 mix or 0.5 mL 0.1 M sodium phosphate buffer (pH 7.4) were placed in gas-tight glass vials. A 0.1 mL sample of the test compound was injected through septa fitted to the vial caps and these mixtures incubated for 30 min at 37 °C. Two mL of molten overlay agar was added and the mixture poured on plates containing 25 mL minimal agar. The plates were incubated for 72 hr at 37 °C, after which the plates were examined for mutants and signs of toxicity.

Laboratory B: plate incorporation method
The top (overlay) agar was prepared with 0.6% (w/v) agar and 0.5% (w/v) NaCl and was supplemented with 10 mL of 0.5 mM histidine/0.5 mM biotin solution per 100 mL agar for selection of histidine revertants. The test material, a 0.1 mL sample of a 10-hr bacterial culture and 0.5 mL S9 mix or 0.5 mL 0.1 M sodium phosphate buffer (pH 7.4) were placed in plastic vials, 2 mL of molten overlay agar was added and the mixture poured on plates containing 25 mL minimal agar. The plates were incubated for 72 hr at 37 °C, after which the plates were examined for mutants and signs of toxicity.

Solvent control:
Laboratory A: DMSO
Laboratory B: DMSO or water

Number of Replications: All assays were done in duplicate and consisted of positive and vehicle controls and at least 5 dose levels of tertiary butyl alcohol. Although not specifically stated in the publication, it is presumed that each trial consisted of triplicate plates since the study was conducted according to OECD Guideline 471 and results for each dose and trial were presented as mean revertants per plate with standard deviation.
Evaluation criteria:
Laboratory A: Treatment was required to produce increases in numbers of mutant colonies of at least 2.0 times the concurrent vehicle control before a response was considered positive.
Laboratory B: Treatment was required to produce increases of at least 2.0 times the concurrent vehicle control at one or more concentrations, or a significant, dose-related response before a response was considered positive.
Statistics:
Appropriate statistical methods were used. Revertants were presented as the mean ± standard error.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Cytotoxicity: Survival of strain TA 102 was unaffected by tertiary butyl alcohol at 5000 μg/plate both in the presence and absence of S9 from male Sprague-Dawley rats treated with either Aroclor 1254 or phenobarbital-β-naphthoflavone.

Results for test chemical: There was no statistically significant mutagenic response in strain TA102 over the entire range of concentrations tested in both trials, in the presence and absence of S9. In the Laboratory B study, all dose levels tested in water in the absence of S9 were associated with mutant numbers greater than was found in the controls, the 5000 μg/plate dose having a 1.54-fold higher number of mutants per plate than the water control. However, the data were inconsistent and in the repeat trial, there was no dose-related response.

Results for controls: A satisfactory response was obtained with the solvent and positive control plates. There was no difference in response between the solvent controls DMSO and water.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative Tested in two laboratories, using two different methods (pre-incubation and plate incorporation) and two different solvents (DMSO or water).

Tertiary butyl alcohol, at concentrations up to 5000 μg/plate, did not induce mutations in Salmonella typhimurium strain TA 102 with or without induced rat liver S9.

Based on an absence of genotoxic/mutagenic effects in this study, tertiary butyl alcohol is not classifiable for Germ Cell Mutagenicity according to GHS.
Executive summary:

In a reverse gene mutation assay in bacteria using the pre-incubation method, strain TA 102 of Salmonella typhimurium was exposed to tertiary butyl alcohol in DMSO at concentrations of 5, 15, 50, 150, 500, 1500, and 5000 μg/plate in the presence and absence of  metabolic activation.  In an independent laboratory, the study was repeated in the plate-incorporation method using water or dried DMSO as the vehicle and tertiary butyl alcohol concentrations of 100, 200, 500, 1000, 2500 and 5000 μg/plate.  There was no evidence of cytotoxicity in either study and the choice of vehicle had no effect on the response.  The positive and vehicle controls induced the appropriate response.  There was no statistical or dose-related increase in number of mutant colonies over background in either study.