Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 October 2016 - 2 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 31, 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
colorless
Details on test material:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Test item 207812/A, Batch No: EM14089719
- Purity 99.9%
- Expiration date of the lot/batch: 1 August 2017
- Purity test date: 7 July 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No information available


Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Test item 207812/A, Batch No: EM14089719
- - Purity 99.9%
- Expiration date of the lot/batch: 1 August 2017
- Purity test date: 7 July 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No information available

Method

Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Direct plate:
- Dose-range finding test:
TA 100 and WP2uvrA (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate

Based on the results of the dose range finding test, the following dose range was selected for the first mutation experiment:
-Experiment 1:
TA1535, TA1537 and TA98 (with and without S9): 52, 164, 512, 1600 and 5000 μg/plate.

Preincubation:
-Experiment 2:
Based on the results of the first mutation assay, the following dose range was selected for the second mutation experiment:
TA1535, TA1537, TA98, TA100 and WP2uvrA (with and without S9): 492, 878, 1568, 2800, 5000 μg/plate
Vehicle / solvent:
- Vehicle used: Dimethyl sulfoxide (DMSO)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: in agar (plate incorporation) (independent repeat)

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY:
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background
lawn or a microcolony formation
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times
the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or
TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the
concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is
greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester
strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No information

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation:
-Dose range finding test: Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate and at 1600 and 5000 μg/plate at the end of the incubation period. With the exception of tester strain WP2uvrA in the absence of S9-mix where precipitation was only observed at 5000 μg/plate.
-Experiment 1: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 μg/plate. With the exception of the tester strain TA1535 and TA98 in the absence of S9-mix) where no precipitation was observed at the end of the incubation period.
-Experiment 2: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 μg/plate, except in tester strain TA1535, where no precipitation was observed at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES/EXPERIMENT 1: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA1537, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 1600 μg/plate in the presence of S9-mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 78 - 1381 78 - 1058 55 – 1565 55 – 1112 410 – 2057 263 - 1907 549 – 1848 620 - 2651 127 – 1951 85 - 1390
Mean 785 228 653 387 1155 860 892 1404 1263 342
SD 167 105 290 143 370 323 178 327 461 165
n 1684 1662 1448 1536 1646 1686 1650 1677 1370 1410

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 4 - 36 3 - 34 3 – 25 3 - 28 9 - 50 9 - 57 63 - 153 60 - 156 12 – 68 12 - 70
Mean 14 13 7 9 17 25 100 103 26 32
SD 6 5 3 4 5 7 16 18 7 8
n 1662 1677 1548 1547 1662 1703 1659 1691 1421 1424

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between May 31 2014 and May 31 2016

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 2: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test substance was determined to be not mutagenic and does not need to be classified for mutagenicity in accordance with the criteria outline in Annex I of CLP (1272/2008/EC).
Executive summary:

The mutagenic activity of Octan-3-one (ethyl amyl ketone) was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: both in the absence and presence of S9-mix up to and including 5000 μg/plate. The dose levels were selected based on a dose range finding test with strain TA100 and WP2uvrA. In the first experiment, Cytotoxicity, as evidenced by no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA1537, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 1600μg/plate in the presence of S9-mix. In the second experiment no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 -metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that Octan-3-one (ethyl amyl ketone) is not mutagenic and does not need to be classified for mutagenicity in accordance with the criteria outline in Annex I of CLP (1272/2008/EC).