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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: reliable without restrictions; study conducted according to established guidelines and performed according to GLPs.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
-Test Substance: EC 98-0268, MIAK
-Purity: ≥98%
-Lot Number: 12-98
-Date received: 12/21/1998
-Physical Description: Transparent, colorless liquid

Method

Target gene:
His (-), Trp (-)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The tester strains were received directly from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley.
Additional strain / cell type characteristics:
other: The tester strains contain 2 additional mutations: rfa wall mutation and a deletion of the uvrB gene. Strains TA 98 and TA 100 also contain the R-factor plasmid, pKM101, which increases the sensitivity of these strains to mutagens.
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
The tester strain, WP2uvrA(pKM101) was received from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).
Additional strain / cell type characteristics:
other: The tester strain contains a uvrA DNA repair deficiency which enhances the strain's sensitivity to some mutagenic compounds. The strain also contains the R-factor plasmid, pKM101, which increases the sensitivity of these strains to mutagens.
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomal enzymes (S9 homogenate) were purchased from Molecular Toxicology, Inc (Batch 0883).
Test concentrations with justification for top dose:
Dose Range-Finding Study:
6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330, and 5000 µg/plate

Mutagenicity Assay:
100, 333, 1000, 3330, 5000 µg/plate
Vehicle / solvent:
Dimethyl sulphoxide (DMSO)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoananthracene
Remarks:
2-aminoananthracene (Sigma Chemical Co., purity ≥97%) was used with tester strains TA 98, TA 100, TA 1535, TA 1537 at a concentration of 2.5 µg/plate and with tester strain WPuvrA(pKM101) at a concentration of 5.0 µg/plate with metabolic activity.
Positive control substance:
2-nitrofluorene
Remarks:
2-nitrofluorene (Aldrich Chemical Co., purity ≥98%) was used in tester strain TA 98 at a concentration of 1.0 µg/plate without metabolic activity.
Positive control substance:
sodium azide
Remarks:
Sodium azide (Sigma Chemical Co., purity ≥98%) used with tester strains TA 100 and TA 1535 at a concentration of 2.0 µg/plate without metabolic activity.
Positive control substance:
other: ICR-191
Remarks:
ICR-191 (Sigma Chemical Co., purity ≥98%) used with tester strain TA 1537 at a concentration of 2.0 µg/plate without metabolic activity.
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
4-nitroquinoline-N-oxide (Sigma Chemical Co., purity ≥99%) used with tester strain WPuvrA(pKM101) at a concentration of 2.0 µg/plate without metabolic activity.
Remarks:
Sterility Controls: The test substance at the greatest concentration tested was checked for sterility by plating a 50 μl aliquot on selective agar. Likewise, the S9 mix was also checked for sterility by plating 0.5 ml on selective agar.
Details on test system and experimental conditions:
Dose Range-Finding Study:
This study was performed using tester strains TA 100 and WP2uvrA(pKM101) with and without metabolic activation at concentrations up to 5 mg/plate.

Mutagenicity Assay:
The tester strains were exposed to the test substance via the plate incorporation methodology as described by Ames et al. (1975) and Maron and Ames (1983).

The plating procedure was the following: When S9 was not required, 100 µl of the tester strain and 50 µl of the vehicle or test substance dose was added to 2.5 ml of molten top agar. When S9 was required, 500 µl of S9 mix, 100 µl of tester strain, and 50 µl of vehicle were added to 2.0 ml of molten top agar. The mixture was vortexed and overlaid onto the surface of 25 ml of minimal bottom agar and after the mixture solidified the plates were inverted and incubated for 48 ± 8 hours at 37 ± 2°C. Positive control materials were plated using a 50 µl plating aliquot.

The condition of the bacterial background lawn was evaluated for evidence of cytotoxicity and test substance precipitate and was scored relative to the vehicle control plate. The number of revertant colonies per plate for the vehicle controls and all plates containing test substance were counted manually while the positive controls were counted by an automated colony counter with few exceptions.

Metabolic Activation System:
The homogenate was prepared from male Sprague-Dawley rats that had been injected (i.p.) with Aroclor 1254 (200 mg/ml in corn oil) at 500 mg/kg. The S9 mix was prepared immediately prior to use. Components of the S9 mix included: Water (0.70 ml), 1M NaH2PO4/Na2HPO4, pH 7.4 ( 0.10 ml), 0.25M Glucose-6-phosphate (0.02 ml), 0.10M NADP (0.04 ml), 0.825M KCL/0.2M MgCl2 (0.04 ml), and S9 homogenate (0.10 ml).
Evaluation criteria:
Dose Range-Finding Study:
Cytotoxicity, defined as a detectable decrease in the number of revertant colonies/plate and/or by a thinning or disappearance of the bacterial background lawn, was the main criterion evaluated in the dose range-finding study. The maximum dose for the mutagenicity assay was the dose at which no cytotoxicity was observed.

Mutagenicity Assay:
-Criteria for a Valid Assay:
Tester strain integrity was demonstrated by rfa wall mutation sensitivity towards crystal violet for Salmonella and pKM101 plasmid sensitivity towards ampicillin for both Salmonella and E coli. The characteristic number of spontaneous revertants was determined by the tester strain cultures exhibiting a characteristic number of spontaneous revertants per plate when plated along with the vehicle. The acceptable ranges are: TA 98: 8-60, TA 100: 60-240, TA 1535: 4-45, TA 1537: 2-25, and WP2uvrA(pKM101) 80-350.

-Positive Controls:
To demonstrate that the tester strains in the absence or presence of S9 mix were capable of identifying a mutagen, the mean value of a positive control needed to exhibit at least a 3-fold increase over the vehicle control.

-Cytotoxicity:
A minimum of 3 non-toxic doses were required to evaluate the assay data.

-Criteria for a Positive Response:
For a test substance to be considered positive, it had to produce at least a 2-fold (TA 98, TA 100, and WP2uvrA(pKM101) or 3-fold (TA 1535 and TA 1537) increase in the mean revertants per plate of at least one of the tester strains over the mean revertants per plate of the appropriate vehicle control. The increase had to be accompanied by a dose response to increasing concentrations of the test substance.

The results of the initial mutagenicity assay were confirmed in an independent experiment.
Statistics:
For all replicate platings, the mean revertants per plates and the standard deviation were calculated. Statistical analyses were not needed due to the absence of an increase in the number of revertant colonies at any dose level beyond the positive control.

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 / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
-Test Substance:
The test substance formed a solution in DMSO up to concentrations of 499 mg/ml. At 100 mg/ml, the most concentrated stock solution used in this study, the test substance formed a transparent colorless solution and remained dissolved in all succeeding solutions prepared for the study.

Dose Range-Finding Study:
No cytotoxicity was observed with either strain with or without metabolic activity as evidenced by a normal background lawn and no decrease in the number of revertants per plate. No test substance precipitate was observed on the plates at any of the doses tested.

Mutagenicity Assay:
All criteria for a valid assay were met, all data was acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of metabolic activity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

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

It is concluded that, under the conditions of this test, methyl isoamyl ketone showed no evidence of mutagenic activity in this bacterial system with and without metabolic activation. Based on an absence of genotoxic/mutagenic effects in this study, methyl isoamyl ketone is not classified for “Germ Cell Mutagenicity” according to GHS.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium and strain WP2 uvr A pKM 101 of E. coli were exposed to methyl isoamyl ketone in DMSO at concentrations of 100, 333, 1000, 3330, 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method. The positive controls induced the appropriate responses in the corresponding strains and there was no evidence of induced mutant colonies over background. The results of the initial mutagenicity assay were confirmed in an independent second experiment.