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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-28 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 471 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 12-14 March 2014 / signed on 12 May 2014)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Colorless liquid
- Storage condition of test material: Stored at 2-8 °C temperature and protected by Nitrogen.
Specific details on test material used for the study:
- Purity test date: 04 November 2014
- Storage condition of test material: Refrigerated at 4°C in the dark under nitrogen

Method

Target gene:
Histidine and tryptophan
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% S9: S9-mix from the livers of rats treated with phenobarbitone/β-naphthoflavone
Test concentrations with justification for top dose:
Mutation Test – Experiment 1 – Range-finding test (Pre-incubation method):
All Salmonella strains (without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Justification: The maximum concentration was 5000 μg/plate (the maximum recommended dose level).

Mutation Test – Experiment 2 - Main Test (Pre-Incubation Method):
Salmonella strain TA98 and E.coli strain WP2uvrA (without S9-mix) and all tester strains (with S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate.
Salmonella strains TA100, TA1535 and TA1537 (without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate.
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 DMSO at the same concentration in solubility checks performed in-house. DMSO was therefore selected as the vehicle.
- Preparation of test item formulation: The test item was accurately weighed and approximate half-log dilutions prepared in DMSO by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (2.1%) 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 aluminosilicate pellets with a nominal pore diameter of 4 x 10^-4 microns.
Controlsopen allclose all
Untreated negative controls:
yes
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
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:
TEST SYSTEM: The strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

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

DURATION
- Preincubation period: 37 ± 3 °C for 20 minutes (with shaking)
- Exposure duration: Plates were incubated at 37 ± 3 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

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

OTHERS:
- The concurrent negative controls were dosed using the standard plate incorporation method
- After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system.
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 analysis of data as determined by UKEMS (Mahon et al., 1989).

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item was immiscible in sterile distilled water at 50 mg/mL
- 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.

RANGE-FINDING/SCREENING STUDIES
The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix, initially from 150 μg/plate (TA100, TA1535 and TA1537) and 500 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix, weakened lawns were noted to all of the tester strains from 500 μg/plate. Consequently, the toxic limit of the test item was employed in the second mutation test.e appropriate:

HISTORICAL CONTROL DATA
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2012 and 2013 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Main test results once again showed a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix, initially from 150 μg/plate (TA100, TA1535, TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence of S9-mix, weakened lawns were noted to all of the tester strains from 500 μg/plate.

MUTAGENICITY RESULTS
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 in both experiments. Small, statistically significant increases in TA100 revertant colony frequency were observed (presence of S9-mix) at 15 and 50 μg/plate in the main test. 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 colony counts at 15 and 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.2 times the concurrent vehicle control.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:
Under the test condition, test item is not mutagenic with and without metabolic activation in S.typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS
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 using the Ames pre-incubation method (under anaerobic conditions) at up to 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 the range-finding test (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 the range-finding test and ranged between 0.15 and 500 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item. Negative, vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

 

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. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix, initially from 150 μg/plate (TA100, TA1535 and TA1537) and 500 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix, weakened lawns were noted to all of the tester strains from 500 μg/plate. Consequently, the toxic limit of the test item was employed in the second mutation test. Main test results once again showed a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix, initially from 150 μg/plate (TA100, TA1535, TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence of S9-mix, weakened lawns were noted to all of the tester strains from 500 μg/plate. 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 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 in both experiments. Small, statistically significant increases in TA100 revertant colony frequency were observed (presence of S9-mix) at 15 and 50 μg/plate in the main test. 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 colony counts at 15 and 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.2 times the concurrent vehicle control.

 

Under the test condition, test item is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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