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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:
Bacterial Reverse Mutation Assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 January 2011 - 26 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Expiration date of the lot/batch: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, stored protected from light without desiccant
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: not specified

OTHER SPECIFICS:
- Purity: 100%
- Molecular weight : 178.2
- Appearance: clear colorless liquid

Method

Target gene:
Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Initial Toxicity-Mutation Assay (B1): 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate with and without S9-mix;
Confirmatory Mutagenicity Assay (B2): 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate with tester strain TA100 in the absence of S9 activation and 15, 50, 150, 500, 1500 and 5000 µg per plate with and without S9-mix;

The top dose of 5000 µg/plate in the Initial toxicity-mutation assay was selected based on the maximum recommended dose according to the Guideline. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 µg/plate.
Vehicle / solvent:
- Vehicle used: - DMSO
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a soluble and clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9; 1.0 µg/plate for TA98, TA1535 and TA1537; 2.0 µg/plate for TA100; 15.0 µg/plate for WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9; 1.0 µg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9; 1.0 µg/plate for TA100 and TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9; 75 µg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9; 1000 µg/plate for WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water. Bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).
Each plate was labeled with a code system that identified the test substance, test phase, dose level, tester strain and activation, as described in detail inthe laboratory's Standard Operating Procedures.
One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50 μL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

DURATION
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium); Tryptophan (E. coli tryptophan-dependent strains)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants; reductino in the bacterial background lawn

Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 500, 1500 or 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 500, 1500 or 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not specified
- Precipitation: no precipitation has been observed
- Other: sterility results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

RANGE-FINDING/SCREENING STUDIES:
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. Vehicle control, positive controls and a minimum of eight dose levels of the test substance were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 500 or 1500 μg per plate with most test conditions. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate..

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: the positive control chemicals induced clear positive responses
- Negative (solvent/vehicle) historical control data: vehicle control data were with the historical control data range


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Initial Toxicity-Mutation Assay (B1): Toxicity was observed beginning at 500 or 1500 μg per plate with most test conditions.
Confirmatory Mutagenicity Assay (B2): Toxicity was observed beginning at 500, 1500 or at 5000 µg per plate with most test conditions.

Applicant's summary and conclusion

Conclusions:
Interpretation of the results: negative with and without metabolic activation
The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test item did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.