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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:
August 2013 - November 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 37060B
- Expiration date of the lot/batch:
- Purity test date: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark


Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Plate Incorporation Method (Experiment 1, range finding test):
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
The maximum concentration was 5000 µg/plate (the maximum recommended dose level).

Pre-Incubation Method (Experiment 2, main test)):
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 0.5 to 500 µg/plate for the Salmonella strains and 5 to 5000 µg/plate for Escherichia coli strain WP2uvrA.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The test item was fully soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Plate Incorporation Method (experiment , range-finding test): in agar (plate incorporation);
- Pre-Incubation Method (experiment 2, main test): preincubatio

Plate Incorporation Method (experiment , range-finding test):
The test was performed with and without activation (S9-mix).
Each concentration of the test item, appropriate positive control, and each bacterial strain, was assayed using triplicate plates.
All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were performed to confirm the automated scores.

Pre-Incubation Method (experiment 2, main test):
As Experiment 1 (the range-finding test) was deemed negative, Experiment 2 (main test) was performed using the pre-incubation method in the presence and absence of metabolic activation.
The test was performed with and without activation (S9-mix).
All testing for this experiment was performed in triplicate.
All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Some manual counts were required due to the colonies spreading slightly.



Evaluation criteria:
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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
Additional information on results:
The maximum dose level of the test item in Experiment 1 (plate incorporation method) was selected as the maximum recommended dose level of 5000 (µg/plate. There was a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 |ng/plate in the absence of metabolic activation (TA100) and at and above 500 µg/plate in the presence of metabolic activation (all Salmonella strains). Consequently in the second mutation test (pre-incubation method), the same maximum dose level was used for Escherichia colt strain WP2uvrA and the toxic limit (500 µg/plate) for the Salmonella strains. Once again the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 µg/plate in the absence of metabolic activation (TA1537) and at and above 150 µg/plate in the presence of metabolic activation (TA1537 again). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation.

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 in the range-finding test (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 in the main test (pre-incubation method).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. 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:
Interpretation of results:
negative without metabolic activation
negative with metabolic activation

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 in the range-finding test (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 in the main test (pre-incubation method).
Therefore, the test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, 40 CFR 799.9510 TSCA bacterial reverse mutation test and the USA, EPA (TSCA) OCSPP harmonized guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre­incubation methods 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 Experiment 1 (plate incorporation method) 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 (plate incorporation method) and was 0.5 to 500 µg/plate for theSalmonellastrains and 5 to 5000 µg/plate for Escherichia coli strain WP2uvrA.

Additional dose levels and an expanded dose range were selected in Experiment 2 (pre-incubation method) in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

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 Experiment 1 (plate incorporation method) was selected as the maximum recommended dose level of 5000 µg/plate. There was a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 |ug/plate in the absence of metabolic activation (TA100) and at and above 500 µg/plate in the presence of metabolic activation (all Salmonella strains). Consequently in the second mutation test (pre-incubation method), the same maximum dose level was used for Escherichia coli strain WP2uvrA and the toxic limit (500 µg/plate) for the Salmonella strains. Once again the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 µg/plate in the absence of metabolic activation (TA1537) and at and above 150 µg/plate in the presence of metabolic activation (TA1537 again). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation.

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 in the range-finding test (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 in the main test (pre-incubation method).

The Reaction mass of AminoPhosphonium salt and Bisphenol AF (XA 31) was considered to be non-mutagenic under the conditions of this test.