Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 November 2016 - 24 November 2016
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
other: USA, EPA OCSPP harmonized guideline 870.5100 - Bacterial Reverse Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
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:
Rat liver S9-mix
Test concentrations with justification for top dose:
Experiment 1: Direct plate incorporation method:
- The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA: doses 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (with and without S9 mix).

Experiment 2: Preincubation method:
Based on the results of experiment 1, the following dose levels were used:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA: doses 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate (with and without S9 mix).
Vehicle / solvent:
- Solvent used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent: a solubility test was performed. The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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 (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: (independent repeat) preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation

ACCEPTABILITY OF THE ASSAY:
The reverse mutation assay may be considered valid if the following criteria are met: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000). All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows: TA1535: 7 to 40, TA100: 60 to 200, TA1537: 2 to 30, TA98: 8 to 60, WP2uvrA: 10 to 60.

All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL. Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, which are at least the minimum positive control value over the previous two years, both with or without metabolic activation. There should be a minimum of four non-toxic test item dose levels. There should be no evidence of excessive contamination.
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.
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 resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment 1 from 500 μg/plate (without S9) and from 1500 μg/plate (with S9). In experiment 2 from 150 μg/plate (with and without S9).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment 1 from 500 μg/plate (without S9) and from 1500 μg/plate (with S9). In experiment 2 from 150 μg/plate (with and without S9).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment 1 from 500 μg/plate (without S9) and from 1500 μg/plate (with S9). In experiment 2 from 150 μg/plate (with and without S9).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment 1 from 500 μg/plate (without S9) and from 1500 μg/plate (with S9). In experiment 2 from 150 μg/plate (with and without S9).
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:
In experiment 1 from 1500 μg/plate (with and without S9). In experiment 2 from 150 μg/plate (with and without S9).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
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 in excess of the minimum positive control values over the previous two years, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Results for the negative controls (spontaneous mutation rates) were generally considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. Two counts for TA1535 (second mutation test) were just below the minimum level. These counts were still considered acceptable as the other vehicle and untreated control counts were within expected range and the tester strain responded very well with the respective positive controls in both the presence and absence of S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. Toxicity was noted to Escherichia coli strain WP2uvrA from 1500 μg/plate in both the presence and absence of S9-mix.
Experiment 2: weakened bacterial background lawns noted to all of the tester strains in both the absence and presence of S9-mix from 150 μg/plate.
The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between exposures with or without S9-mix and experimental methodology.

VALIDITY CRITERIA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

ADDITIONAL INFORMATION ON RESULTS:
There were no significant 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 toxicologically significant 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). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the second mutation test at 15 µg/plate in the presence of S9-mix only. This increase was 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 level were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.9 times the concurrent vehicle control.


Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test substance was determined to be not negative in the Ames test.
Executive summary:

The mutagenic activity of Dimeth Cyclormol was evaluated in accordance with OECD TG 471 and according to GLP principles (Klimisch 1). The test was performed in two independent experiments: the first using the direct plate incorporation method, and the second using the pre-incubation method, both in the absence and presence of S9-mix, with strains TA1535, TA1537, TA 98, TA100 and WP2uvrA. The dose range for experiment 1 was predetermined and was 1.5 to 5000 μg/plate, in all strains, with or without S9-mix. The dose range for experiment 2 was amended slightly, following the results of Experiment 1, and was 0.5 to 1500 µg/plate, in all strains, with and without S9. In experiment 1 the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 500 µg/plate in the absence of S9-mix and 1500 µg/plate in the presence of S9-mix. Toxicity was noted to Escherichia coli strain WP2uvrA from 1500 µg/plate in both the presence and absence of S9-mix. In experiment 2, cytotoxicity as evidenced by weakened bacterial background lawns was noted to all of the tester strains in both the absence and presence of S9-mix from 150 µg/plate. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 -metabolic activation, in experiment 1 and 2. Based on the results of this study it is concluded that Dimeth Cyclormol is negative in the Ames test.