Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-14 to 2019-03-22
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cyclohex-2-en-1-ol
Cas Number:
22972-51-6
Molecular formula:
C10H16O
IUPAC Name:
(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cyclohex-2-en-1-ol
Test material form:
liquid
Details on test material:
Test item: (1S,4R)-1-Methyl-4-(prop-1-en-2-yl)cyclohex-2-enol
CAS number: 22972-51-6
Physical state: Liquid
Storage: Room temperature
Specific details on test material used for the study:
Test item: (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cyclohex-2-en-1-ol (PMDOL)
CAS number: 22972-51-6
Physical state: Liquid
Storage: room temperature

Method

Target gene:
Histidine locus (histidine-dependent S. typhimurium strains)
Species / strain
Species / strain / cell type:
other: S. typhimurium: TA97a, TA98, TA100, TA102, TA1535
Details on mammalian cell type (if applicable):
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver enzyme solution (S9 mix)
Test concentrations with justification for top dose:
0.625 µl/plate caused no significant toxicity and was highest dose tested in the study
Dose-rangeused in study: 0.625, 0.3125, 0.15625, 0.078125 and 0.0390625 μL/plate in with and without 5% S9-mix
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (NPD)
Remarks:
Without S9-mix; 10 μg/plate (TA97a and TA98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9-mix; 0.5 μg/plate (TA102)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9-mix; 0.4 μg/plate (TA100 and TA1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Remarks:
4.0 μg/plate (TA97a with S9-mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
4.0 μg/plate (TA98 with S9-mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
4.0 μg/plate (TA100, TA102 and TA1535 with S9-mix)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate

METHOD: Plate incorporation method
The following solutions were added to 2 ml soft agar:
- 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains
- 0.1ml of the test article solution or control solution and
- either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.2 M sodium phosphate buffer (in case of nonactivation assays).
The mixtures was mixed thoroughly on a Vortex and was poured onto a minimal glucose agar plate and incubated at 37.0 °C for 48h. After this period revertant colonies were counted.
Evaluation criteria:
A test article is considered negative (not mutagenic) in the test if:
The total number of revertants in the tester strain TA 97a, TA98, TA100 and TA102 was less than twice or thrice (TA1535) the concurrent negative control (Ethanol).

Results and discussion

Test results
Key result
Species / strain:
other: TA97a, TA98, TA100, TA102, and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Based on the study results, no classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).