Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 11 to August 15, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 471 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer
EC Number:
204-102-8
EC Name:
5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer
Cas Number:
115-71-9
Molecular formula:
C15H24O
IUPAC Name:
5-(2,3-dimethyltricyclo[2.2.1.0~2,6~]hept-3-yl)-2-methylpent-2-en-1-ol
Constituent 2
Chemical structure
Reference substance name:
[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol
EC Number:
201-027-2
EC Name:
[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol
Cas Number:
77-42-9
Molecular formula:
C15H24O
IUPAC Name:
2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)pent-2-en-1-ol
Test material form:
liquid
Details on test material:
-Storage conditions: Ambient temperature, protected from light

Method

Target gene:
Histidine and tryptophan for S. typhimurium and E. coli, respectively.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: purchased from Molecular Toxicology, Inc. (Lot No. 3234, containing 37.5 mg/mL protein). The homogenate was prepared from male Sprague-Dawley rats that had been injected (intraperitoneally) with Aroclor 1254 (200 mg/mL in corn oil) at 500 mg/kg, 5 days before sacrifice.
- method of preparation of S9 mix: S9 mix was prepared on the day of use, maintained on ice, and contained (in 1 mL):
H2O 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
Liver Homogenate (S9) 0.10 mL
- volume of S9 mix in the final culture medium: 500 µL added to 2 mL of undiluted molten selective top agar
- quality controls of S9: The most concentrated test article dilution and the S9 mix (100 and 500 µL, respectively; the same volumes used in the assay) were checked for sterility by plating on selective agar.
Test concentrations with justification for top dose:
Initial assay: 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 µg/plate with and without S9.
Confirmatory assay: 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 µg/plate with S9 and 1.60, 5.00, 16.0, 50.0, 160, 500, and 1600 µg/plate without S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on solubility assessment.
- Justification for percentage of solvent in the final culture medium: Not reported.
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
benzo(a)pyrene
other:
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate)
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation)

TREATMENT SCHEDULE:
- Exposure duration/duration of treatment: 52 +/- 4 hours at 37 +/- 2°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
Evaluation criteria:
- Criteria for a Negative Response:
A test article is considered to have produced a negative response if no dose-dependent, ≥ 2.0-fold or ≥3.0-fold increases are observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively
- Criteria for an Equivocal Response
Even after repeated trials, a test article may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are reproducible). In those rare instances, the test article may be considered to have produced an equivocal response.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98 & TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Possibility of evaporation from medium: low volatility based on vapor pressure of constituents
- Precipitation and time of the determination:
Initial assay: Precipitate was observed at ≥1600 μg/plate in all tester strains with and without S9 except WP2uvrA, where precipitate was observed at 5000 μg/plate with S9.
Confirmatory assay: Precipitate was observed at 5000 μg/plate in all tester strains with and without S9

STUDY RESULTS
- Sign of toxicity
Initial assay: Toxicity, as evident by the absence or reduction in the mean number of revertant colonies and/or reduction in the background bacterial lawn, was observed at ≥1600 μg/plate in all tester strains with S9 except TA98 and WP2uvrA, where toxicity was observed at 5000 μg/plate. Toxicity, as evident by the absence or reduction in the mean number of revertant colonies and/or absence or reduction in the background bacterial lawn, was observed at ≥160 μg/plate in all tester strains without S9 except WP2uvrA, where toxicity was observed at ≥1600 μg/plate.
Confirmatory assay: Toxicity, as evident by the absence or reduction in the mean number of revertant colonies and/or reduction in the background bacterial lawn, was observed at ≥1600 μg/plate in all tester strains with S9 except TA98 and WP2uvrA, where toxicity was observed at 500 and 5000 μg/plate, respectively. Toxicity, as evident by the absence or reduction in the mean number of revertant colonies and/or absence or reduction in the background bacterial lawn, was observed at ≥160 μg/plate in all tester strains without S9 except TA1537 and WP2uvrA, where toxicity was observed at ≥50.0 μg/plate in TA1537 and no toxicity was observed in WP2uvrA.
- Individual plate counts & Mean number of revertant colonies per plate and standard deviation are in the attached document.

HISTORICAL CONTROL DATA
- Positive historical control data: inside (cf attached document)
- Negative (solvent/vehicle) historical control data: inside (cf attached document)

Applicant's summary and conclusion

Conclusions:
Under the test condition, test material was not mutagenic to S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E.coli WP2 uvrA- in the presence and absence of metabolic activation.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E. coli strain WP2 uvrA- were exposed to the test material both in the presence and absence of metabolic activation system (liver S9 in standard co-factors) using the plate incorporation method.

In the first experiment the dose levels ranged from 5 to 5000 µg/plate with and without S9. Precipitate was observed at ≥1600 μg/plate in all tester strains with and without S9 except WP2uvrA with S9, where precipitate was observed at 5000 μg/plate. Toxicity was observed in all tester strains at ≥1600 μg/plate with S9 except TA98 and WP2uvrA, where toxicity was observed at 5000 μg/plate. Toxicity was observed at ≥160 μg/plate in all tester strains without S9 except WP2uvrA, where toxicity was observed at ≥1600 μg/plate.

Based on the results of the first, a second confirmatory mutagenicity assay was conducted in all five tester strains at dose levels of 5 to 5000 µg/plate with S9 and from 1.60 to 1600 µg/plate without S9. Precipitate was observed at 5000 μg/plate in all tester strains with and without S9. Toxicity was observed in all tester strains at ≥1600 μg/plate with S9 except TA98 and WP2uvrA, where toxicity was observed at ≥500 and 5000 μg/plate, respectively. Without S9, toxicity was observed at ≥160 μg/plate in all tester strains except TA1537 and WP2uvrA, where toxicity was observed at ≥50.0 μg/plate in TA1537 and no toxicity was observed in WP2uvrA.

No increase in the mean number of revertant colonies was observed at any tested dose level in any tester strain with and without S9 in the initial or the confirmatory assay. All positive and vehicle control values were within the acceptable ranges, and all criteria for a valid study were met.  

Under the test condition, test material was not mutagenic to S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E.coli WP2 uvrA- in the presence and absence of metabolic activation.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.