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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:
21 March to 15 May 2012
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:
2013
Report Date:
2013

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 19-21 July 2011 / signed on 31 August 2011)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Pale yellow liquid
- Storage condition of test material: Approximately 4 °C in the dark under nitrogen

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 rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/ kg bw/day by oral route).
Test concentrations with justification for top dose:
Preliminary toxicity test (Plate incorporation method): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate in TA100 or WP2uvrA strains with and without S9 mix

Mutation Test – Experiment 1 – Range-finding test (Plate incorporation method):
All Salmonella strains (without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate.
All Salmonella strains (with S9-mix) and E.coli strain WP2uvrA (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate.
Mutation Test – Experiment 2 - Main Test (Pre-Incubation Method):
All Salmonella strains (without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate.
All Salmonella strains (with S9-mix) and E.coli strain WP2uvrA (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 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 50 mg/ml 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 dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated impurity content (1.9%) 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
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, from Syngenta CTL, Alderley Edge, as frozen vials, on 20 March 2007 or from the British Industrial Biological Research Association, on nutrient agar plates, 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 °C for 20 minutes (with shaking) at approximately 130 rpm
- Exposure duration: Plates were incubated at 37 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed for visible reduction in the growth of the bacterial background lawns indicating toxicity.

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.
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).
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 judgement 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: A test item precipitate (light and oily in appearance) was observed at 5000 μg/plate.

PRELIMINARY TOXICITY TEST:
The test item was toxic to TA100 from 150 μg/plate and to WP2uvrA from 500 μg/plate.

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 2010 and 2011 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1 and 2: The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 μg/plate after employing plate incorporation methodology (range-finding test) and from 15 μg/plate following the pre-incubation method (main test). 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. The test item was tested up to the toxic limit. A test item precipitate (light and oily in appearance) was observed at 5000 μg/plate. The test item was tested up to the toxic limit in the range-finding and main tests, therefore, this observation was only noted in the preliminary toxicity test.

MUTAGENICITY:
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 or exposure method.

Any other information on results incl. tables

The test item formulation and S9-mix used in this experiment were both shown to be sterile.

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.

Tables: cf Attached background material.

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-.
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, ST 01 C 11, using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 0.15 and 500 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. The experiment was repeated on a separate day (pre-incubation method) using the same dose range, fresh cultures of the bacterial strains and test item formulations. Additional dose levels and an expanded dose range were selected in each experiment 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 test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 μg/plate after employing plate incorporation methodology (range-finding test) and from 15 μg/plate following the pre-incubation method (main test). 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. The test item was tested up to the toxic limit. A test item precipitate (light and oily in appearance) was observed at 5000 μg/plate. The test item was tested up to the toxic limit in the range-finding and main tests, therefore, this observation was only noted in the preliminary toxicity test.

 

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 or exposure method.

 

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-.

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