Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 August - 10 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Change of supplier of tester strain TA 100: this deviation did not influence the quality or integrity of the study
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
4-Cyclohexan-1-ol, dodecyl-, branched
EC Number:
938-702-2
Molecular formula:
not applicable UVCB
IUPAC Name:
4-Cyclohexan-1-ol, dodecyl-, branched
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Cyclohexanol, 4-C11-12-alkyl, branched
- Substance type: pure active substance
- Physical state: liquid
- Lot/batch No.: 04152/MA
- Expiration date of the lot/batch: May 2014
- Storage condition of test material: room temperature

Method

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:
S9 mix preparation according to Ames et al. containing S9 liver microsomal fraction of male Wistar rats, phenobarbital (80 mg/kg bw) and ß-Naphthoflavone (100 mg/kg bw) induced
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
A. dest., treated in the same way as all dose groups
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, treated in the same way as all dose groups
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
A. dest., treated in the same way as all dose groups
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, treated in the same way as all dose groups
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) in the first experiment, preincubation in the repeat experiment

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

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approx. ≤ 0.5 in relation to the solvent control

OTHER:
The properties of the S. typhimurium and E. coli strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al.
Evaluation criteria:
Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least ont of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA1537 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Criteria of validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100)
- the mean values of the spontaneous reversion frequencies of the control plates with and without S9 mix are within the historical control data range
- corresponding background growth on both negative control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate.
Statistics:
A statistical evaluation of the results was not regarded as necessary as the biological relevance of the results is the criterion for the interpretation of results (according to guideline).

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation of the test item was observed in all tester strains used in experiment I at a concentration of 5000 µg/plate and in experiment II at concentrations of 2500 µg/plate and higher (with and without metabolic activation)

RANGE-FINDING/SCREENING STUDIES: no toxicity was observed in the pre-experiment (strains TA98 and TA100 were tested in the presence and absence of S9 mix in the following concentrations: solvent control, 3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate)

COMPARISON WITH HISTORICAL CONTROL DATA: The control plates with and without S9 mix were within the historical control data range.

Any other information on results incl. tables

Table #1: Salmonella typhimurium reverse mutation assay without metabolic activation, Experiment I (plate incorporation test)
Concentration [µg/plate] Revertant colonies / plate
TA 98 TA 100 TA 1535 TA 1537 E. coli WP2 uvrA
mean±SD MF mean±SD MF mean±SD MF mean±SD MF mean±SD MF
Negative control (A. dest.) 20±4.6 0.8 110±5.1 1.1 6 ± 2.0 0.8 7 ± 1.5 0.7 49 ± 13.0 1.2
Solvent control (DMSO) 25±6.4 1.0 101 ± 6.2 1.0 8 ± 2.0 1.0 9 ± 2.6 1.0 42 ± 1.7 1.0
31.6 12±3.1 0.5 87 ± 15.7 0.9 9 ± 4.0 1.2 9 ± 4.2 1.0 41 ± 6.1 1.0
100 19±6.1 0.8 84 ± 6.4 0.8 6 ± 2.1 0.7 7 ± 4.6 0.8 37 ± 9.1 0.9
316 23±4.5 0.9 90 ± 4.7 0.9 7 ± 1.5 0.9 7 ± 1.5 0.8 43 ± 5.9 1.0
1000 18±7.5 0.7 116 ± 12.6 1.1 5 ± 1.5 0.7 9 ± 4.0 1.0 48 ± 7.1 1.2
2500 22±6.7 0.9 102 ± 19,1 1.0 7 ± 1.0 0.9 6 ± 2.0 0.7 41 ± 7.1 1.0
5000 17±5.6 P 0.7 86 ± 18.0 0.9 10 ± 1.5 P 1.3 5 ± 1.0 P 0.6 43 ± 9.6 P 1.0
Positive control 319 ± 34.5 12.9 709 ± 64.0 7.0 631 ± 155.9 78.9 80 ± 13.6 8.9 436 ± 23.7 10.4
SD = Standard deviation    P = Precipitiation
Table #2: Salmonella typhimurium reverse mutation assay with metabolic activation, Experiment I (plate incorporation test)
Concentration [µg/plate] Revertant colonies / plate
TA 98 TA 100 TA 1535 TA 1537 E. coli WP2 uvrA
mean±SD MF mean±SD MF mean±SD MF mean±SD MF mean±SD MF
Negative control (A. dest.) 26±4.4 0.9 89 ± 1.5 0.8 5 ± 1.5 1.1 12 ± 2.5 1.6 61 ± 16.9 1.0
Solvent control (DMSO) 28±2.5 1.0 105 ± 20.1 1.0 5 ± 0.6 1.0 7 ± 2.1 1.0 62 ± 9.5 1.0
31.6 25±4.0 0.9 116 ± 3.1 1.1 8 ± 2.9 1.8 16 ± 2.5 2.2 45 ± 7.5 0.7
100 23±4.7 0.8 120 ± 2.9 1.1 7 ± 1.7 1.5 18 ± 6.9 2.5 50 ± 10.5 0.8
316 24±1.0 0.9 110 ±14.6 1.1 6 ± 1.2 1.4 8 ± 4.9 1.0 47 ± 3.1 0.8
1000 29±7.0 1.0 119 ± 17.3 1.1 6 ± 4.6 1.3 11 ± 3.0 1.5 56 ± 4.2 0.9
2500 22±4.9 0.8 104 ± 17.5 1.0 6 ± 3.2 1.4 12 ± 2.5 1.6 51 ± 1.5 0.8
5000 22±6.0 P 0.8 79 ± 5.7 P 0.8 6 ± 2.0 P 1.3 15 ± 1.5 P 2.1 47 ± 5.5 P 0.8
Positive control 2014 ± 336.5 72.8 1489 ± 147.4 14.2 94 ± 26.1 20.2 187 ± 7.5 25.5 170 ± 21.5 2.8
SD = Standard deviation    P = Precipitiation
Table #3: Salmonella typhimurium reverse mutation assay without metabolic activation, Experiment II (pre-incubation test)
Concentration [µg/plate] Revertant colonies / plate
TA 98 TA 100 TA 1535 TA 1537 E. coli WP2 uvrA
mean±SD MF mean±SD MF mean±SD MF mean±SD MF mean±SD MF
Negative control (A. dest.) 25 ± 8.5 1.0 96 ± 7.0 1.3 8 ± 3.5 1.0 7 ± 2.5 1.1 49 ± 3.5 1.0
Solvent control (DMSO) 24 ± 2.9 1.0 74 ± 9.6 1.0 8 ± 1.2 1.0 6 ± 3.2 1.0 48 ± 8.7 1.0
31.6 21 ± 3.0 0.9 69 ± 3.2 0.9 4 ± 1.5 0.6 6 ± 1.5 1.0 43 ± 11.3 0.9
100 21 ± 4.0 0.9 61 ± 4.7 0.8 7 ± 1.2 1.0 6 ± 3.5 0.9 40 ± 5.0 0.8
316 23 ± 6.7 1.0 60 ± 3.2 0.8 5 ± 1.0 0.7 5 ± 1.2 0.7 36 ± 2.5 0.7
1000 25 ± 4.5 1.1 67 ± 8.0 0.9 6 ± 0.6 0.7 6 ± 3.1 0.9 35 ± 6.5 0.7
2500 21 ± 8.4 P 0.9 64 ± 8.6 P 0.9 2 ± 1.2 P 0.2 4 ± 2.0 0.6 43 ± 9.8 P 0.9
5000 35 ± 4.6 P 1.5 66 ± 8.5 P 0.9 4 ± 2.3 P 0.6 5 ± 1.2 0.7 45 ± 5.1 P 0.9
Positive control 311 ± 26.6 13.1 1251 ± 425.0 16.8 1302 ± 108.9 169.8 89 ± 4.0 14.1 584 ± 61.6 12.2
SD = Standard deviation    P = Precipitiation
Table #4: Salmonella typhimurium reverse mutation assay with metabolic activation, Experiment II (pre-incubation test)
Concentration [µg/plate] Revertant colonies / plate
TA 98 TA 100 TA 1535 TA 1537 E. coli WP2 uvrA
mean±SD MF mean±SD MF mean±SD MF mean±SD MF mean±SD MF
Negative control (A. dest.) 35 ± 4.9 1.1 101 ± 18.2 1.2 8 ± 2.5 1.2 5 ± 2.0 0.7 63 ± 17.9 1.2
Solvent control (DMSO) 31 ± 4.6 1.0 86 ± 17.2 1.0 6 ± 2.3 1.0 7 ± 0.0 1.0 51 ± 8.7 1.0
31.6 29 ± 5.5 0.9 78 ± 18.8 0.9 5 ± 2.0 0.8 8 ± 1.5 1.1 39 ± 7.5 0.8
100 28 ± 4.7 0.9 72 ± 13.5 0.8 9 ± 3.0 1.4 6 ± 2.3 0.9 46 ± 7.5 0.9
316 25 ± 8.1 0.8 70 ± 10.0 0.8 6 ± 6.4 0.9 5 ± 3.1 0.8 54 ± 5.2 1.1
1000 23 ± 2.5 0.7 68 ± 19.0 0.8 4 ± 2.6 0.6 4 ± 1.7 0.6 50 ± 5.9 1.0
2500 24 ± 4.6 P 0.8 78 ± 16.6 P 0.9 9 ± 1.5 P 1.5 6 ± 3.0 0.9 56 ± 2.6 P 1.1
5000 31 ± 7.8 P 1.0 65 ± 12.3 P 0.8 8 ± 0.0 P 1.3 5 ± 2.0 0.7 39 ± 6.4 P 0.8
Positive control 2336 ± 744.3 74.6 2196 ± 234.4 25.6 50 ± 13.9 7.9 155 ± 6.6 22.1 164 ± 24.4 3.2
SD = Standard deviation    P = Precipitiation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the chosen experimental conditions Cyclohexanol, 4-C11-12-alkyl, branched did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore Cyclohexanol, 4-C11-12-alkyl, branched is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The test item Cyclohexanol, 4 -C11 -12 -alkyl, branched was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.

Precipitation of the test item was observed in all tester strains used in experiment I at a concentration of 5000 µg/plate and in experiment II at concentrations of 2500 µg/plate and higher (with and without metabolic activation).

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II. The reduction in the number of revertants down to a mutation factor of 0.5 was found in experiment I in tester strain TA 98 at a concentration of 31.6 µg/plate and down to a mutation factor of 0.2 found in experiment II in tester strain TA 1535 at a concentration of 2500 µg/plate (both without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Cylcyclohexanol, 4 -C11 -12 -alkyl, branched at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Cyclohexanol, 4 -C11 -12 -alkyl, branched did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore Cyclohexanol, 4 -C11 -12 -alkyl, branched is considered to be non-mutagenic in this bacterial reverse mutation assay.