Registration Dossier

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:
From September 09 to 29, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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)
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:
UK GLP Compliance Program (inspected on June 17, 2015 / Signed on September 24, 2015)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Colourless liquid
Specific details on test material used for the study:
Storage conditions of test material: 11-25 °C, dry, keep container closed.

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:
other:
Metabolic activation:
with and without
Metabolic activation system:
10% S9-mix: S9 from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route, for 3 days prior to preparation on day 4).
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 strains TA100 and TA98 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate. The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
- Salmonella strains TA1535 and TA1537 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate. The test item was cytotoxic in Salmonella strains TA1535 and TA1537 at and above 1500 μg/plate in Experiment 1.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: 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 on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (8.8%) of the test item. All formulations were used within four hours of preparation and were assumed to be stable for this period. 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 10-4 microns.
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
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

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. Several manual counts were required due to bubbles in the base agar slightly distorting the actual plate count.
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 in Experiment 2 (main test) in order to achieve both four non-toxic dose levels and the toxic limit of the test item.
- Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate. The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
- Salmonella strains TA1535 and TA1537 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate. The test item was cytotoxic in Salmonella strains TA1535 and TA1537 at and above 1500 μg/plate in Experiment 1.
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 analysis of data as determined by UKEMS (Mahon et al., 1989).

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:
Toxicity to strains TA1535 and TA1537 at and above 1500 μg/plate and from 500 μg/plate in Experiments 1 and 2, respectively, with and without S9-mix.
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

RANGE-FINDING/SCREENING STUDIES:
In both experiments 1 and 2, there were no toxicologically 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 2, small, statistically significant increases in revertant colony frequency were observed in the second mutation test at 5000 μg/plate (TA98) and 0.5 μg/plate (TA1537) 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.

COMPARISON WITH HISTORICAL CONTROL DATA:
The individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.9 times the concurrent vehicle controls.

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 test item was cytotoxic in Salmonella strains TA1535 and TA1537 at and above 1500 μg/plate and from 500 μg/plate in Experiments 1 and 2, respectively, in both the presence and absence of metabolic activation (S9-mix). No toxicity was noted to Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA.

OTHERS:
- The test material formulation, amino acid supplemented top agar and S9-mix used in this experiment were shown to be sterile.
Remarks on result:
other:
Remarks:
In both Experiment 1 and 2

Any other information on results incl. tables

 Table 7.6.1/2. Mutagenic and cytotoxic effect of the test material.

Strain

S9-mix

Test concentration range

(µg/plate)

Lowest mutagenic concentration (µg/plate)

Lowest cytotoxic

concentration (µg/plate)

 

TA100

-

1.5 - 5000

None

None

+

1.5 - 5000

None

None

TA1535

-

1.5 - 5000

None

1500

+

1.5 - 5000

None

1500

WP2uvrA

-

1.5 - 5000

None

None

+

1.5 - 5000

None

None

TA98

-

1.5 - 5000

None

None

+

1.5 - 5000

None

None

TA1537

-

1.5 - 5000

None

1500

+

1.5 - 5000

None

1500

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.

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

Experiment 2 (Pre-Incubation Method):

- Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

- Salmonella strains TA1535 and TA1537 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.

 

Vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

 

The test item was cytotoxic in Salmonella strains TA1535 and TA1537 at and above 1500 μg/plate and from 500 μg/plate in Experiments 1 and 2, respectively, in both the presence and absence of metabolic activation (S9-mix). No toxicity was noted to Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA.

 

In both experiments 1 and 2, there were no toxicologically 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 2, small, statistically significant increases in revertant colony frequency were observed in the second mutation test at 5000 μg/plate (TA98) and 0.5 μg/plate (TA1537) 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 each tester strain and the maximum fold increase was only 1.9 times the concurrent vehicle controls.

 

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.

 

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.