Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 30 to February 23, 2017
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)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on 05 July 2016 / signed on 28 October 2016
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Appearance: clear, colorless to pale yellow, liquid, mobile
Specific details on test material used for the study:
Storage conditions: Room temperature in the dark until 19 January 2017 when stored at approximately 4°C in the dark

Method

Target gene:
Histidine and tryptophan gene for Salmonella typhimurium and Escherichia coli, respectively.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fraction was pre-prepared using standardized in-house procedures (outside the confines of this study). The 10% S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
Test concentrations with justification for top dose:
Experiment 1 (Plate Incorporation Method):
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix. The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 (Pre-Incubation Method):
- Salmonella strain TA100 (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
- Salmonella strains TA1535 and TA1537 (absence of S9-mix) and TA100 (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
- E.coli strain WP2uvrA and Salmonella strain TA98 (absence and presence of S9-mix) and
- Salmonella strains TA1535 and TA1537 (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
Eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: 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. Dimethyl sulphoxide was therefore selected as the vehicle.
- Preparation of test materials: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment. No correction for purity was required. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10E-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated: spontaneous mutation rates
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
Remarks:
Without S9-mix
Untreated negative controls:
yes
Remarks:
untreated: spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from the University of California, Berkeley, and from the British Industrial Biological Research Association.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes with shaking
- Exposure duration: approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level in experiment 1 and experiment 2.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning.

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Rationale for test conditions:
Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - test item dose levels were selected based on Experiment 1 results in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.
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:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases 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 results
Key result
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:
cytotoxicity
Remarks:
-S9: from 50 μg/plate (TA100), 150 μg/plate (TA1537), 500 μg/plate (TA1535 and WP2uvrA), 1500 μg/plate (TA98). (+S9) from 500 μg/plate (TA100), 1500 μg/plate (TA1535, TA98 and TA1537) and 5000 μg/plate (WP2uvrA).
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA:
Not needed (no statistical significant increase were noted)

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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
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, the test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains in the absence of S9-mix and to TA100, TA1535 and TA1537 in the presence of S9-mix. Reductions in the bacterial background lawn growth were initially noted in the absence of S9-mix from 150 μg/plate (TA100), 500 μg/plate (TA1535) and 1500 μg/plate (TA98 and TA1537). In the presence S9-mix reduced bacterial background lawns were noted from 500 μg/plate (TA100), 1500 μg/plate (TA1535) and 5000 μg/plate (TA1537). No toxicity was noted for Escherichia coli strain WP2uvrA (absence and presence of S9-mix) or the Salmonella strain TA98 dosed in the presence of S9-mix. Consequently for the second mutation test, the same maximum dose level (5000 μg/plate) or toxic limit was used, depending on bacterial strain type and presence or absence of S9-mix. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with reductions in bacterial background lawn growth in the absence of S9-mix initially noted from 50 μg/plate (TA100), 150 μg/plate (TA1537), 500 μg/plate (TA1535 and WP2uvrA) and 1500 μg/plate (TA98). In the presence S9-mix, reductions in bacterial lawn growth were initially noted from 500 μg/plate (TA100), 1500 μg/plate (TA1535, TA98 and TA1537) and 5000 μg/plate (WP2uvrA). 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. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

OTHERS:
There were no toxicological 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 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 TA98 revertant colony frequency was observed in the absence of S9-mix at 5000 μg/plate in the first mutation test. This increase was considered to have no biological relevance because weakened bacterial background lawns were noted alongside the dose concentration. Therefore the responses noted would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and present as non-revertant colonies.

Any other information on results incl. tables

Cf Tables of results in attached background material

Applicant's summary and conclusion

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the Ames plate incorporation and pre‑incubation methods in Experiment 1 and Experiment 2, respectively.

The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.15 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix.

Eight test item dose levels per bacterial strain were again selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

 

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 or 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, the test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains in the absence of S9-mix and to TA100, TA1535 and TA1537 in the presence of S9-mix. Reductions in the bacterial background lawn growth were initially noted in the absence of S9-mix from 150 μg/plate (TA100), 500 μg/plate (TA1535) and 1500 μg/plate (TA98 and TA1537). In the presence S9-mix reduced bacterial background lawns were noted from 500 μg/plate (TA100), 1500 μg/plate (TA1535) and 5000 μg/plate (TA1537). No toxicity was noted for Escherichia coli strain WP2uvrA (absence and presence of S9-mix) or the Salmonella strain TA98 dosed in the presence of S9-mix. Consequently for the second mutation test, the same maximum dose level (5000 μg/plate) or toxic limit was used, depending on bacterial strain type and presence or absence of S9-mix. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with reductions in bacterial background lawn growth in the absence of S9-mix initially noted from 50 μg/plate (TA100), 150 μg/plate (TA1537), 500 μg/plate (TA1535 and WP2uvrA) and 1500 μg/plate (TA98). In the presence S9-mix, reductions in bacterial lawn growth were initially noted from 500 μg/plate (TA100), 1500 μg/plate (TA1535, TA98 and TA1537) and 5000 μg/plate (WP2uvrA). 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. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no toxicological 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 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 TA98 revertant colony frequency was observed in the absence of S9-mix at 5000 μg/plate in the first mutation test. This increase was considered to have no biological relevance because weakened bacterial background lawns were noted alongside the dose concentration. Therefore the responses noted would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and present as non-revertant colonies.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

 

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.