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 May 12 to July 01, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD test Guideline No. 471 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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:
inspected on September 15, 2009 / Signed on November 26, 2009
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Target gene:
Histidine and tryptophan.
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain 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:
Preliminary Toxicity Test (Plate Incorporation Method): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Experiment 1 – Range-finding test (Plate Incorporation Method): 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix
Experiment 2 - Main Test (Pre-Incubation Method):
- All tester strains in the absence of S9-mix: 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate.
- All strains in the presence of S9-mix: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test material was not fully miscible 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 material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at approximately 40 °C on the day of each experiment. Prior to use, the solvent was dried to remove water using molecular sieves ie 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.
Controlsopen allclose all
Negative controls:
yes
Solvent 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
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and conditions:
TEST SYSTEM: The four strains of Salmonella used in the test were obtained either from the University of California, Berkeley, on culture discs, on 04 August 1995 or from Syngenta CTL, Alderley Edge, as frozen vials, on 20 March 2007. E. coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.

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

DURATION
- Exposure duration: Plates were incubated at 37 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was based on the growth of the bacterial background lawn.

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using a Domino colony counter.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (Kirkland, 1989) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Statistical analysis of data as determined by UKEMS (Kirkland, 1989).

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test material was not fully miscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.
- Precipitation: A particulate precipitate was observed at 5000 μg/plate, this did not prevent the scoring of revertant colonies.

PRELIMINARY TOXICITY TEST
- The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

RANGE-FINDING/SCREENING STUDIES: In the range-finding test, the test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

COMPARISON WITH 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 2008 and 2009 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the main test, the test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains initially at and above 1500 and 150 μg/plate, with and without S9-mix, respectively. The test material was, therefore, either tested up to the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on exposures to S9 and exposure method. A particulate precipitate was observed at 5000 μg/plate, this did not prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. A small but statistically significant increase in revertant colony frequency was observed in TA100, in the presence of S9, at and above 1500 μg/plate in the main test only. The increases observed were considered to be of no biological or toxicological relevance because in both cases, the bacterial background lawns were considered to be toxic.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
- The test material formulation, amino acid supplemented top agar and S9-mix used in this experiment were shown to be sterile.

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

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- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and of the Directive 67/548/EEC.
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 material diluted in dimethyl sulphoxide both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).The dose range for the range-finding test (direct plate incorporation method) was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The main mutation test was performed on a separate day by pre-incubation method and using an amended dose range to the range-finding test because of test material induced toxicity. The dose range for this main test ranged between 0.5 and 5000 μg/plate. Fresh cultures of the bacterial strains and fresh test material formulations were prepared on the day of test. Negative, vehicle (dimethyl sulphoxide) 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.

 

In the range-finding test, the test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. However, in the main test, the test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains initially at and above 1500 and 150 μg/plate, with and without S9-mix, respectively. A particulate precipitate was observed at 5000 μg/plate, this did not prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. A small but statistically significant increase in revertant colony frequency was observed in TA100, in the presence of S9, at and above 1500 μg/plate in the main test only. The increases observed were considered to be of no biological or toxicological relevance because in both cases, the bacterial background lawns were considered to be toxic.

 

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- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and of the Directive 67/548/EEC.

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