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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:
01 - 11 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation.

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
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)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
solid - liquid: aqueous solution
Details on test material:
- Physical state: Clear colourless liquid (determined at NOTOX)
- Expiration date of the lot/batch: 18 December 2010 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- Other:
Density: 1.189 kg/L at 21°C
pH: 3.0 ± 0.5% (20°C)

Method

Target gene:
S. typhimurium: Histidine gene
E. coli: Tryptophan gene
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:
5 and 10% (v/v) S9-fraction; Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1:
Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate, without and with S9 in TA100 and WP2uvrA
Main test:
Experiment 1: 100, 333, 1000, 3330 and 5000 μg/plate, without and with 5% (v/v) S9-mix in TA1535, TA1537 and TA98
Experiment 2: 33, 100, 333, 1000, 3330 and 5000 μg/plate without and with 10% (v/v) S9-mix in TA1535, TA1537, TA98, TA100 and WP2uvrA
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water.
- Justification for choice of solvent/vehicle: Test compound was soluble in water and water has been accepted and approved by authorities and international guidelines.
Test substance preparation: The test substance was dissolved in Milli-Q water (Millipore Corp., Bedford, MA., USA). The stock solution was filter (0.22 μm)-sterilized. Test substance concentrations were used within 3 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 metabolic activation
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 metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames); Escherichia coli strain was obtained from Trinova Biochem GmbH, Germany (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK).

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Treated plates were inverted and incubated in the dark at 37 ± 1 °C 48 ± 4 hours

NUMBER OF REPLICATIONS:
Triplicate plates per dose level for test substance, vehicle and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

OTHERS
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.
Rationale for test conditions:
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix.
Evaluation criteria:
No formal hypothesis testing was done.
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) in the test 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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, 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:
not applicable
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 μg/plate

RANGE-FINDING/SCREENING STUDIES:
- No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed at up to 5000 μg/plate in TA100 and WP2uvrA strains.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative 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:
Experiment 1: In the absence of S9-mix, cytotoxicity, as evidenced by a decrease or total absence in the number of revertants, was observed in all three tester strains at test substance concentrations of 3330 and 5000 μg/plate. In the presence of S9-mix, cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 at test substance concentrations of 3330 and 5000 μg/plate.
Experiment 2: In the absence of S9-mix,cytotoxicity, as evidenced by a total absence of revertant colonies, was observed in the tester strains TA1537 and TA98 at test substance concentrations of 3330 and 5000 μg/plate. A decrease in the number of revertants less than the minimal value of the historical control data range was observed in tester strain TA1535 at the test substance concentration of 5000 μg/plate. In the presence of S9-mix, cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 at the test substance concentrations of 3330 and 5000 μg/plate.
In tester strain TA100 in the presence of S9-mix, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose relationship was observed, the reductions are not considered to be caused by toxicity of the test substance.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test conditions, test substance is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvr A strains.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli WP2 uvr A strains were exposed to test substance at the following concentrations using plate incorporation method: 

Experiment 1:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate, without and with S9 in TA100 and WP2uvrA

Main test:

Experiment 1: 100, 333, 1000, 3330 and 5000 μg/plate, without and with 5% (v/v) S9-mix in TA1535, TA1537 and TA98

Experiment 2: 33, 100, 333, 1000, 3330 and 5000 μg/plate without and with 10% (v/v) S9-mix in TA1535, TA1537, TA98, TA100 and WP2uvrA

 

Metabolic activation system used in this test 5 and 10 % S9 mix; S9 fraction prepared from liver homogenates of male Wistar rats induced with phenobarbital and ß-naphthoflavone. Vehicle and positive control groups were also included in mutagenicity tests.

 

In the dose range finding test, no precipitation was observed up to the top dose of 5000 μg/plate in the strains TA100 and WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first experiment of the mutation assay. In main tests, cytotoxicity, as evidenced by a decrease in the number of revertants or total absence of revertants, was observed in TA1535, TA1537 and TA98 strains in the absence of S9-mix and in tester strain TA1537 in the presence of S9-mix at test substance concentrations of 3330 and 5000 μg/plate.

 

Test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative 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. 

 

Under the test conditions, test substance is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvr A strains.

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