Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-13 to 2011-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
α-methylcyclohexanepropanol
EC Number:
234-091-5
EC Name:
α-methylcyclohexanepropanol
Cas Number:
10528-67-3
Molecular formula:
C10H20O
IUPAC Name:
4-cyclohexylbutan-2-ol

Method

Target gene:
Salmonella typhimurium strains had mutations in the histidine locus (histidine deficiency), rfa-minus (deficient lipopolysaccharide envelope), uvrB-minus (deficient excision repair) and TA 98 and TA 100 carried R-factor plasmid pKM 101 (ampicillin resistance marker).
The E. coli strain was deficient for tryptophan biosynthesis and had a mutation of uvrA leading to a deficient excision repair.
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:
Phenobarbital/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
0.3; 1; 3; 10; 33; 100; 333; 1000 and 2500 µg/plate
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. Since toxic effects were observed nine concentrations were tested in experiment II and 2500 µg/plate was chosen as maximal concentration.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA 1535, TA 100 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine,
Remarks:
for TA 1537, TA 98 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
for WP2 uvrA without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
for TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: individual values from the plates for each concentration

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants
Evaluation criteria:
A test item was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control was observed.
A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data was not mandatory.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA1537, TA100, TA1535, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
starting from 333 µg/plate
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:
cytotoxicity
Remarks:
starting from 333 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the overlay agar in the test tubes from 1000 to 5000 ug/plate, in the overlay agar on the incubated agar plates from 1000 to 5000 µg/plate in experiment I with and without S9 mix, in the overlay agar on the incubated agar plates from 333 to 2500 µg/plate in the absence of metabolic activation and from 1000 to 2500 µg/plate in the presence of metabolic activation in experiment II. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: yes, with all strains and 8 concentrations (3 - 5000 µg/plate)

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Any other information on results incl. tables

Experiment 1 (range-finding test, plate incorporation)

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
TA1535 TA1537 TA98 TA100 WP2 uvrA
Results without S9
DMSO 18± 4 14 ± 2 33 ± 2 131 ± 18 57 ± 18
Untreated 14 ± 3 9 ± 4 32 ± 4 130 ± 22 50 ± 5
3 17 ± 2 13 ± 3 30 ± 4 124 ± 12 61 ± 9
10 17 ± 1 17 ± 6 26 ± 5 126 ± 9 57 ± 6
33 19 ± 5 15 ± 4 22 ± 6 124 ± 1 53 ± 8
100 19 ± 6 15 ± 0 25 ± 5 111 ± 6 57 ± 8
333 11 ± 3 8 ± 2 13 ± 3 83 ± 18 34 ± 6
1000 6 ± 2 3 ± 2 11 ± 5 34 ± 9 27 ± 3
2500 1 ± 1 0 ± 0 0 ± 0 0 ± 0 13 ± 3
5000 0 ± 0 0 ± 0 0 ± 0 0 ± 0 4 ± 2
NaN3 (10) 1906 ± 38 2085 ± 70
4-NOPD (10) 306 ± 7
4-NOPD (50) 66 ± 12
MMS (3.0 µL) 1125 ± 66
Results with S9
DMSO 23 ± 1 15 ± 3 43 ± 6 149 ± 10 69 ± 7
Untreated 24 ± 5 24 ± 3 44 ± 2 167 ± 28 76 ± 4
3 24 ± 5 18 ± 2 38 ± 10 156 ± 12 74 ± 9
10 22 ± 3 22 ± 3 36 ± 8 147 ± 3 68 ± 13
33 21 ± 7 21 ± 3 46 ± 7 161 ± 16 62 ± 7
100 22 ± 2 20 ± 5 43 ± 4 156 ± 14 60 ± 4
333 22 ± 2 19 ± 3 35 ± 7 101 ± 15 61 ± 4
1000 1 ± 1 1 ± 1 2 ± 3 2 ± 1 12 ± 4
2500 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
5000 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
2-AA (2.5) 349 ± 30 309 ± 24 1592 ± 281 1631 ± 73
2-AA (10.0) 398 ± 21

Experiment 2 (pre-incubation)

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
TA1535 TA1537 TA98 TA100 WP2 uvrA
Results without S9
DMSO 17 ± 4 15 ± 4 29 ± 6 120 ± 20 49 ± 1
Untreated 14 ± 1 21 ± 2 32 ± 9 130 ± 13 49 ± 7
0.3 17 ± 4 123 ± 4 33 ± 3 112 ± 12 47 ± 11
1 15 ± 1 15 ± 1 31 ± 6 108 ± 11 44 ± 10
3 21 ± 6 13 ± 3 31 ± 9 103 ± 9 56 ± 8
10 17 ± 3 16 ± 3 28 ± 12 101 ± 5 40 ± 6
33 20 ± 1 15 ± 4 29 ± 3 113 ± 11 46 ± 4
100 16 ± 2 9 ± 3 28 ± 6 109 ± 5 48 ± 7
333 8 ± 2 3 ± 1 12 ± 0 74 ± 4 20 ± 2
1000 4 ± 1 1 ± 1 1 ± 1 16 ± 2 12 ± 3
2500 0 ± 0 0 ± 0 0 ± 1 0 ± 0 4 ± 1
NaN3 (10) 1612 ± 101 1557 ± 128
4-NOPD (10) 340 ± 14
4-NOPD (50) 94 ± 4
MMS (2.0 µL) 505 ± 62
Results with S9
DMSO 23 ± 4 19 ± 6 42 ± 7 127 ± 6 51 ± 5
Untreated 29 ± 5 17 ± 4 35 ± 2 129 ± 9 59 ± 12
0.3 21 ± 6 16 ± 3 40 ± 5 130 ± 20 50 ± 2
1 20 ± 6 20 ± 4 38 ± 4 125 ± 7 57 ± 8
3 23 ± 7 17 ± 5 38 ± 15 124 ± 14 55 ± 11
10 23 ± 5 20 ± 2 42 ± 7 118 ± 10 50 ± 11
33 20 ± 7 16 ± 4 38 ± 4 132 ± 10 52 ± 5
100 23 ± 4 15 ± 2 37 ± 5 130 ± 19 42 ± 3
333 7 ± 2 4 ± 1 7 ± 1 24 ± 6 39 ± 3
1000 3 ± 1 0 ± 1 1 ± 1 0 ± 1 15 ± 4
2500 0 ± 0 0 ± 0 0 ± 0 0 ± 0 3 ± 1
2-AA (2.5) 191 ± 5 145 ± 11 1062 ± 43 1381 ± 37
2-AA (10.0) 212 ± 13

Applicant's summary and conclusion

Conclusions:
The test substance was determined to be non mutagenic but cytotoxic to the bacteria in high concentrations.
Executive summary:

A bacterial reverse mutation assay (Ames test, OECD 471) was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test substance was tested at several concentrations (Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000µg/plate; Experiment II: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500µg/plate). The plates incubated with the test substance showed reduced background growth in all strains. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains at high concentrations. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used. Therefore the test substance is considered to be non-mutagenic.