Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 April, 2016 - 16 June, 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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
Reaction mass of 2-[3-(3,3-dimethylcyclohex-1-en-1-yl)propyl]-2-ethyltetrahydrofuran and rel-(3aR,9aR,9bR)-3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan and rel-(3aR,9aS,9bS)-3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan and rel-2-ethyl-2-{2-[(1R,6R)-2,2,6-trimethylcyclohexyl]ethyl}tetrahydrofuran
Molecular formula:
C17H30O + C17H32O
IUPAC Name:
Reaction mass of 2-[3-(3,3-dimethylcyclohex-1-en-1-yl)propyl]-2-ethyltetrahydrofuran and rel-(3aR,9aR,9bR)-3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan and rel-(3aR,9aS,9bS)-3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan and rel-2-ethyl-2-{2-[(1R,6R)-2,2,6-trimethylcyclohexyl]ethyl}tetrahydrofuran
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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 experiment 1, the following dose levels were used:
- Experiment 1:
TA 1535, TA 1537 and TA 98 (without and with S9): 17, 52, 164, 512, 1600 and 5000 μg/plate
Preincubation:
- Experiment 2:
Based on the results of experiment 1, the following dose levels were used:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 17, 52, 164, 512 and 1600 μg/plate
- Experiment 3:
Since in the second mutation test not enough non-toxic dose levels were present in tester strains TA1535 and TA100 in the absence of S9-mix, an additional experiment was performed:
TA 1535 (without S9): 1.7, 5.4, 17, 52, 164 and 512 μg/plate
TA 100 (without S9): 0.056, 0.18, 0.55, 1.7, 5.4, 17 and 52 μg/plate
- Experiment 4:
Since in the third mutation test again not enough non-toxic dose levels were present in tester strain TA1535, an additional experiment was performed.
TA 1535 (without S9): 0.056, 0.18, 0.55, 1.7, 5.4, 17 and 52 μg/plate
Vehicle / solvent:
- Vehicle used: Ethanol
- Justification for choice of vehicle: A solubility test was performed. The test item was dissolved in ethanol.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
100 μL/plate Ethanol
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: (independent repeat): preincubation

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 tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent 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 tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat 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:
Not performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: direct plate: no cytotoxicity up to and including 5000 μg/plate; pre-incubation assay: with S9 at 164, 512 and 1600 μg/plate, and without S9 at 5.4, 17 and 52 μg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: direct plate: no cytotoxicity up to and including 5000 μg/plate; pre-incubation assay: with S9 at 1600 μg/plate, and without S9 at 5.4, 17, 52, 164 and 512 μg/plate.
Vehicle 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:
other: direct plate: no cytotoxicity up to and including 5000 μg/plate; pre-incubation assay: with S9 no cytotoxicity up to and including 1600 μg/plate, and without S9 at 1600 μg/plate.
Vehicle 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
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of GRISALVA on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 and 5000 μg/plate.

RANGE-FINDING/SCREENING STUDIES: In the dose range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 78 - 1932 81 - 1332 62 – 1565 55 – 1112 347 – 1967 261 - 1885
Mean 791 234 662 409 976 821
SD 261 98 206 126 251 298
n 1732 1737 1409 1428 1721 1737

TA100 WP2uvrA
S9-mix - + - +
Range 549 – 1798 640 - 2760 123 – 1958 85 - 1390
Mean 914 1387 1367 261
SD 150 324 276 276
n 1734 1752 1373 1404

SD = Standard deviation
n = Number of observations

Historical control data from experiments performed between November 2013 and January 2016.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 5 - 36 3 - 34 3 – 25 3 - 28 9 - 50 9 - 57 63 - 153 60 - 156 13 – 68 12 - 70
Mean 177 14 7 9 18 26 104 105 28 34
SD 6 5 3 3 6 7 17 17 7 7
n 1644 1716 1425 1443 1707 1730 1725 1739 1368 1404

SD = Standard deviation
n = Number of observations

Historical control data from experiments performed between November 2013 and January 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY (see also Results and discussion):
Experiment 1: No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed. In the tester strains TA1535 (absence of S9-mix) and TA1537 (absence and presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.
Experiment 2: Cytotoxicity, as evidenced by a decrease in the bacterial background lawn, was observed in several tester strains, TA1535 (absence and presence of S9-mix), TA1537 (absence of S9-mix), and TA100 (absence and presence of S9-mix).
In the tester strain TA1537 (presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.
Experiment 3: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or bacterial background lawn, was observed in both tester strains.
Experiment 4: Cytotoxicity was observed, as evidenced by a decrease in the number of revertants and/or bacterial background lawn.

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 (1997) and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct plate assay was performed in the absence and presence of S9-mix up to and including the precipitating concentration of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Secondly a pre-incubation assay was performed in the absence and presence of S9-mix up to and including the precipitating concentration of 1600 μg/plate. Cytotoxicity, as evidenced by a decrease in the bacterial background lawn and/or decrease in the number of revertants was observed in several tester strains, TA1535 (absence and presence of S9-mix), TA1537 (absence of S9-mix), and TA100 (absence and presence of S9-mix). Since in the second mutation experiment, only dose levels with cytotoxicity were present in the tester strains TA1535 and TA100 in the absence of S9-mix, an additional pre-incubation experiment was performed. In this third mutation experiment, the test item was tested at a concentration range of 1.7 to 512 μg/plate in tester strain TA1535 and 0.056 to 52 μg/plate in tester strain TA100. The test item did not precipitate on the plates at these dose levels. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or bacterial background lawn, was observed in both tester strains. Since in the third mutation experiment, only dose levels with cytotoxicity were present in tester strain TA1535, an additional pre-incubation experiment was performed. In this fourth mutation experiment, the test item was tested at a concentration range of 0.056 to 52 μg/plate. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.