Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
Version / remarks:
November 24, 2000.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-cyclohexylphenol
EC Number:
214-465-4
EC Name:
4-cyclohexylphenol
Cas Number:
1131-60-8
Molecular formula:
C12H16O
IUPAC Name:
4-cyclohexylphenol

Method

Target gene:
histidine / tryptophan
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 homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (in all experiments)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, 1st experiment); preincubation (2nd experiment)

DURATION
- Preincubation period: 20 Min
- Exposure duration: 48h

SELECTION AGENT (mutation assays):
His-negative plates

NUMBER OF REPLICATIONS: Per strain and dose, three plates with and three plates without S9 mix were used.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

- OTHER
- Study controls: The solvent (vehicle) control used was dimethyl sulphoxide. The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.
The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.
- Concentrations of positive controls:
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) - 2 μg/plate for WP2uvrA, 3 μg/plate for TA100, 5 μg/plate for TA1535
9-Aminoacridine (9AA) - 80 μg/plate for TA1537
4-Nitroquinoline-1-oxide (4NQO) - 0.2 μg/plate for TA98
2-Aminoanthracene (2AA) - 1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, 10 μg/plate for WP2uvrA
Benzo(a)pyrene (BP) - 5 μg/plate for TA98
- The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results
Key result
Species / strain:
bacteria, other: S.typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
plate incorporation: from 150 μg/plate (Salmonella strains) & 500 μg/plate (E.coli WP2uvrA); pre-incubation: without S9 from 50 μg/plate (TA100, TA1535 & TA1537) & 150 μg/plate WP2uvrA & TA98), with S9 from 150 μg/plate (all tester strains)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The study was conducted under GLP according to OECD guideline 471 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of the test item to induce reverse mutations in bacteria. The test substance was non-mutagenic in the Salmonella typhimurium test strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA in the absence and presence of metabolic activation under the experimental conditions in the present study.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471, GLP), Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (Experiment 2, pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range for Experiment 2 was amended, following the results of Experiment 1, and was 0.15 to 500 μg/plate. Eight test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

Formulation analysis was carried out in Experiment 1 to determine the concentration of the test item concentration (maximum dose).

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation method), the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, in both the presence and absence of metabolic activation (S9-mix), initially from 150 μg/plate (Salmonella strains) and 500 μg/plate (E.coli strain WP2uvrA). Consequently the toxic limit of the test item was employed as the maximum dose in the second mutation test. The test item induced a stronger toxic response in Experiment 2 after incorporating the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9-mix from 50 μg/plate (TA100, TA1535 and TA1537) and 150 μg/plate WP2uvrA and TA98). In the presence of S9-mix, weakened bacterial background lawns were noted to all of the tester strains from 150 μg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy in appearance) was noted in both experiments at and above 500 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA1535 revertant colony frequency were observed in the first mutation test at 5 and 15 μg/plate in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the mean maximum fold increase was only 1.5 times the concurrent vehicle control. Hence, the test substance was considered to be non-mutagenic under the conditions of this test.