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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:
29 November 2013 to 14 January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 10 July 2012 / signed on 30 November 2012)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Clear colorless liquid
- Storage Conditions: ca. 4 °C in the dark under nitrogen
Specific details on test material used for the study:
- Purity test date: 14 January 2014

Method

Target gene:
Histidine and tryptophan for S. typhimurium and E. coli, respectively.
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 male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).
Test concentrations with justification for top dose:
Experiment 1 – Range-finding test (Pre-incubation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix
Justification: Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - Main Test (Pre-Incubation Method):
Salmonella strain TA100 (without S9-mix) and Salmonella strains TA98, TA1535 and TA1537 and E.coli strain WP2uvrA (with S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate.
Salmonella strains TA98, TA1535 and TA1537 and E.coli strain WP2uvrA (without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate.
Salmonella strain TA100 (with S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Justification: Seven 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.
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 substance 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. 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 μm.
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 bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and 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: 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: Plates were assessed microscopically for evidence of thinning (toxicity).

OTHERS:
After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system. Several manual counts were required, predominantly due to revertant colonies spreading slightly, thus distorting the actual plate count.

*concurrent negative controls were dosed using the standard plate incorporation method
Rationale for test conditions:
Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - Seven 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.
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:
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: None

RANGE-FINDING/SCREENING STUDIES:
- Experiment 1 (Range finding test): 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 (TA1537), 500 μg/plate (TA1535, TA98 and WP2uvrA) and 1500 μg/plate (TA100). In the presence of S9-mix, weakened lawns were noted from 1500 μg/plate (TA1535, TA98, WP2uvrA and TA1537) and at 5000 μg/plate (TA100). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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 2011 and 2012 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 2 (main test): Main test results once again showed a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 μg/plate (TA1537) and 500 μg/plate (TA100, TA1535, TA98 and WP2uvrA) in the absence of S9-mix. In the presence of S9-mix, weakened lawns were noted from 500 μg/plate (TA1535) and 1500 μg/plate (TA100, TA98, WP2uvrA and TA1537). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

MUTAGENICITY:
- 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. A small, statistically significant increase in TA1537 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the range-finding test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the response was accompanied by weakened bacterial background lawns and was, therefore considered spurious.

OTHERS:
- 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.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test condition, test substance 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 TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test substance using the Ames pre-incubation method 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 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day 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 0.5 and 5000 μg/plate, depending on bacterial strain type and presence or absence of metabolic activation (S9-mix). Seven 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. Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

 

The vehicle 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 (TA1537), 500 μg/plate (TA1535, TA98 and WP2uvrA) and 1500 μg/plate (TA100). In the presence of S9-mix, weakened lawns were noted from 1500 μg/plate (TA1535, TA98, WP2uvrAand TA1537) and at 5000 μg/plate (TA100). Consequently, depending on bacterial strain type, the same maximum dose level and the toxic limit 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, initially from 150 μg/plate (TA1537) and 500 μg/plate (TA100, TA1535, TA98 and WP2uvrA) in the absence of S9-mix. In the presence of S9-mix, weakened lawns were noted from 500 μg/plate (TA1535) and 1500 μg/plate (TA100, TA98, WP2uvrA and TA1537). 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. A small, statistically significant increase in TA1537 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the range-finding test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the response was accompanied by weakened bacterial background lawns and was, therefore considered spurious.

 

Under the test condition, test substance 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.