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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Guideline recommends the use of 4 strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537) and either E. coli WP2 uvrA, E. coli WP2 uvrA (pKM 101) or Salmonella strain TA 102.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Guideline recommends the use of 4 strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537) and either E. coli WP2 uvrA, E. coli WP2 uvrA (pKM 101) or Salmonella strain TA 102.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Heptan-2-one
EC Number:
203-767-1
EC Name:
Heptan-2-one
Cas Number:
110-43-0
Molecular formula:
C7H14O
IUPAC Name:
heptan-2-one
Constituent 2
Reference substance name:
606-024-00-3
IUPAC Name:
606-024-00-3
Constituent 3
Reference substance name:
methyl amyl ketone; methyl pentyl ketone; MAK; 1-methylhexanal; butylacetone
IUPAC Name:
methyl amyl ketone; methyl pentyl ketone; MAK; 1-methylhexanal; butylacetone
Details on test material:
Test substance:
-Test substance name (as cited in report): Methyl n-Amyl Ketone
-Lot number: LR 21995-193
-Purity: 99%
-Test substance description: clear, colorless liquid
-Storage conditions: Room temperature, protected from exposure to light
-Test substance receipt date: 09/28/94
-Test substance expiration date: 10/31/95


Study Dates:
-Study initiation: October 14, 1994

Method

Target gene:
His (-)
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 Salmonella typhimurium tester strains were received from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley.
Additional strain / cell type characteristics:
other: TA 98 and TA 100: uvrB, rfa, pKM 101; TA 1535 and TA 1537: uvrB, rfa
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
The Salmonella typhimurium tester strain was received from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley.
Additional strain / cell type characteristics:
other: TA 1538: uvrB, rfa
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate was prepared from the livers of male Sprague-Dawley rats that had been injected (i.p.) with Aroclor 1254 (200 mg/mL in corn oil) at 500 mg/kg.
Test concentrations with justification for top dose:
-Dose-Range Finding Study:
With and without metabolic activation: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, and 5000 µg/plate

-Mutagenicity Assay
With and without metabolic activation: 100, 333, 1000, 3333, 5000 µg/plate
Vehicle / solvent:
Dimethyl sulphoxide (DMSO) (Fisher Scientific Company)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
2-Aminoanthracene (Sigma Chemical Company, practical grade) was used with tester strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 at a concentration of 1.0 µg/plate with metabolic activation.
Positive control substance:
2-nitrofluorene
Remarks:
2-Nitrofluorene (Aldrich Chemical Company, purity 98%) was used with tester strains TA 98 and TA 1538 at a concentration of 1.0 µg/plate without metabolic activation.
Positive control substance:
sodium azide
Remarks:
Sodium azide (Sigma Chemical Company, practical grade) was used with tester strains TA 100 and TA 1535 at a concentration of 1.0 µg/plate without metabolic activation.
Positive control substance:
9-aminoacridine
Remarks:
9-Aminoacridine (Sigma Chemical Company, purity 98%) was used with tester strain TA 1537 at a concentration of 75.0 µg/plate without metabolic activation.
Remarks:
Sterility Controls: The most concentrated test substance dilution was checked for sterility by plating an aliquot volume equal to that used in the assay. The S9 mix and Sham S9 mixes were checked for sterility by plating 0.5 mL of each on selective agar.
Details on test system and experimental conditions:
- S9 Mix:
Each bulk preparation of S9 was assayed for its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA 100. The microsomal enzyme mixture (S9 mix) was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM β-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2, and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture contained 100 mM phophate buffer at pH 7.4.

-Dose Rangefinding study:
A dose rangefinding study using the plate incorporation method was used to establish the dose-range over which the test substance would be assayed. Ten dose levels of the test substance (up to 5000 µg/plate) were plated, one plate per dose, with an overnight culture of TA 100 on selective minimal agar in the presence and absence of metabolic activation.

- Mutagenicity Assay:
The tester strains were exposed to five concentrations of the test substance along with appropriate vehicle and positive controls via the plate incorporation methodology as described by Ames et al. (1975) and Maron and Ames (1983). All dose levels of test substance, vehicle control and positive controls were plated in triplicate.

References:
Ames et al., 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Research 31:347-364.

Maron DM and Ames BN, 1983. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research 113:173-215.



-Plating Procedure:
Each plate was labeled with a code system. Test substance dilutions were prepared immediately before use. One-half (0.5) milliliter of S9 mix or Sham S9 mix, 100 µL of tester strain and 50 µL of vehicle or test substance were added to 2.0 mL of molten selective top agar (45 ± 2 °C). Positive control substances were plated using a 50 µL plating aliquot. After the required components had been added, the mixture was vortexed, overlaid onto the surface of 25 mL bottom agar, and allowed to solidify. Plates were inverted and incubated for 48-72 hours at 37± 2 °C.

-Plate Scoring and Counting:
Plates that were not evaluated immediately following the incubation period were held at 4 ± 2 °C. The numbers of revertant colonies per plate were counted by an automated colony counter.

-Bacterial Background Lawn Evaluation:
The condition of the bacterial background lawn was evaluated both macroscopically and microscopically for indications of cytotoxicity and test substance precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate.
Evaluation criteria:
Dose Rangefinding study:
Cytotoxicity was detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn.

Mutagenicity Assay:
The following criteria had to be met for the assay to be considered valid:
-All tester strain cultures must have demonstrated the presence of the deep rough mutation (rfa); the deletion in the uvrB gene; and the characteristic mean number of spontaneous revertants in the vehicle control (TA 98: 10-50; TA 100: 80-240; TA 1535: 5-45; TA 1537: 3-21; and TA 1538: 5-35).
-Cultures of tester strains TA 98 and TA 100 must demonstrate the presence of the pKM 101 plasmid R-factor.
-Tester strain culture titers must be >/= 0.3 x 10^9 cells/mL.
-The mean of each positive control must exhibit at least a 3-fold increase in the number of revertants over the mean value of the vehicle control.
-A minimum of 3 non-toxic dose levels are required.

A dose level is considered toxic if one or both of the following criteria are met:
-A >50% reduction in the mean number of revertants/plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count.
-A reduction in the background lawn.

Assay Evaluation Criteria:
Tester Strains TA 98 and TA 100:
For a test substance to be considered positive, it must have produced at least a 2-fold increase in the mean revertants per plate relative to the respective vehicle control and must be accompanied by a dose response to increasing concentrations of the test substance.

Tester Strains TA 1535, TA 1537 and TA 1538:
For a test substance to be considered positive, it must have produced at least a 3-fold increase in the mean revertants per plate relative to the respective vehicle control and must be accompanied by a dose response to increasing concentrations of the test substance.
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 all dose levels. Results for the positive control materials were in line with historical data on those substances.

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:
S. typhimurium TA 1538
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:
Dose Range-Finding Assay:
The results indicate that neither precipitate nor appreciable toxicity was observed. The test material was not cytotoxic over the dose range used in this study.

Mutagenicity Assay:
The results indicate that neither precipitate nor appreciable toxicity was observed. No positive responses were observed in the presence or absence of metabolic (S9) activation. All criteria for a valid assay were met.
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

At a maximum concentration of 5000 µg/plate, methyl n-amyl ketone did not induce mutations in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or TA 1538 when tested in the Salmonella/microsome bacterial mutagenicity assay in the presence and absence of a metabolic activation system.

Methyl n-amyl ketone is not classified for genotoxic or mutagenic effects in Annex I of Directive 76/548/EEC. Based on the absence of genotoxic or mutagenic effects in this study with and without metabolic activation, methyl n-amyl ketone is not classified for Germ Cell Mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) 1272/2008 or the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to methyl n-amyl ketone in DMSO at concentrations of 100, 333, 1000, 3333, and 5000 µg/plate in the presence and absence of metabolic activation using the plate incorporation method. There was no evidence of cytotoxicity or an increase in the number of mutant colonies over background level at concentrations up to the limit dose of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains.