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Toxicological information

Genetic toxicity: in vitro

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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 March 11 to 22, 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
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: granules
Details on test material:
- Name of test material (as cited in study report): 202030/A
- Physical state: White granules
- Storage condition of test material: Stored at room temperature in the dark.
- Stability under storage conditions: Stable

Method

Target gene:
Histidine and tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
Experiment-1
Dose range finding test (without and with 5 % (v/v) S9-mix): 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in TA 100 and WP2uvrA strains.
Main test (without and with 5 % (v/v) S9-mix): 100, 333, 1000, 3330 and 5000 µg/plate in TA 1535, TA 1537 and TA 98 strains.

Experiment-2 (without and with 10 % (v/v) S9-mix): 33, 100, 333, 1000, 3330 and 5000 µg/plate in TA 1535, TA 1537, TA 98 and TA 100 strains; 100, 333, 1000, 3330 and 5000 µg/plate in WP2uvrA strain.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: Test substance was dissolved in Milli-Q water.
- The stock solution was filter (0.22 μm)-sterilized. Test substance concentrations were used within 2.5 hours after preparation.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 h at 37.0 ± 1.0 °C

NUMBER OF REPLICATIONS: 3 plates/dose for Experiment 1 and 2

DETERMINATION OF CYTOTOXICITY
- Method: Test material toxicity was determined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

OTHER:
Colony counting: - The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present. If more than 40 colonies were present, these could be counted automatically with a Biocount 4000 Pro-S-colony counter. Plates with abundant test article precipitate which interfered with automated colony counting were counted manually and the evidence of test substance precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.
Evaluation criteria:
- A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
- A test substance is considered positive (mutagenic) if:
(a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
(b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
- The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
- No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium 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:
not applicable
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 applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: Soluble in water.
- Precipitation: No test material precipitation was observed on the plates at the start or at the end of the incubation period.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: The bacterial background lawn was not reduced at any of the concentrations tested. The number of revertants of TA100 was moderately decreased at 5000 µg/plate in the absence of S9-mix. In the presence of S9-mix, no biologically relevant decrease in the number of revertants was observed in tester strain WP2uvrA.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 3330 µg/plate and above and with S9: 3330 µg/plate and above
TA1537: without S9: 1000 µg/plate and above and with S9: 3330 µg/plate and above
TA98: without S9: 333 µg/plate and above and with S9: 5000 µg/plate
TA100: without S9: 3330 µg/plate and above and with S9: 5000 µg/plate

See the “Attached background material” section for further details
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

None

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 WP2uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli strain WP2 uvrA were exposed to test material at the following concentrations both in the presence and absence of metabolic activation system (rat liver S9-mix) using the plate incorporation method in two independent experiments.

Experiment-1: 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in TA 100 and WP2uvrA strains; 100, 333, 1000, 3330 and 5000 µg/plate in TA 1535, TA 1537 and TA 98 strains, without and with 5 % (v/v) S9-mix).

Experiment-2: 33, 100, 333, 1000, 3330 and 5000 µg/plate in TA 1535, TA 1537, TA 98 and TA 100 strains; 100, 333, 1000, 3330 and 5000 µg/plate in WP2uvrA strain, without and with 10 % (v/v) S9-mix)

Vehicle and positive control groups were also included in mutagenicity tests.

The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. Toxicity was observed in tester strains TA1535, TA1537, TA98 and TA100 in the absence and presence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation in two independently repeated experiments.

 

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 WP2uvrA.