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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct.
Qualifier:
according to
Guideline:
other: EC Commission Directive 2000/32/EC, Annex 4E No. L136
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: >99.9%

Method

Target gene:
histidine (S. typhimurium), tryptophan (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Toxicity-mutation test: 1.5, 5.0, 15, 50, 150, 500, 1500, and 5000 μg/plate

Confirmatory mutagenicity assay: 15, 50, 150, 500, 1500, and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: Based on the solubility of the test substance, compatibility with the target cells, and in order to maintain the test substance dilutions on ice during all dilution and plating procedures.
Controls
Untreated negative controls:
yes
Remarks:
distilled water
Positive controls:
yes
Remarks:
2-aminoanthracene for all strains +S9; 2-nitrofluorene for TA98 -S9; sodium azide for TA100 and TA1535 -S9; 9-aminoacridine for TA1537 -S9; methyl methanesulfonate for WP2 urvA -S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: In the non-activated assays, 0.5 mL of sham mix, 100 μL of tester strain, and 100 μL of vehicle or test substance dilution were added to glass culture tubes pre-heated to 37±2°C. All tubes receiving test substance were capped during the preincubation period with a Teflon-lined screw cap. After vortexing, these mixtures were incubated with shaking for 60±2 minutes at 37±2°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and 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 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 counted.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: approximately 48-72 hours at 37±2°C
- Expression time (cells in growth medium): approximately 48-72 hours

NUMBER OF REPLICATIONS: initial toxicity-mutation tests were plated in duplicate; confirmatory mutagenicity tests plated in triplicate

NUMBER OF CELLS EVALUATED: To ensure that appropriate numbers of bacteria were plated, tester strain culture titers were greater than or equal to 0.3E9 cells/mL. Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity.


DETERMINATION OF CYTOTOXICITY
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes below:

Code 1 (Normal): Distinguished by a healthy microcolony lawn
Code 2 (Slightly Reduced): Distinguished by a noticeable thinning of the microcolony lawn and possibly a slight increase in the size of the microcolonies compared to the vehicle control plate.
Code 3 (Moderately Reduced): Distinguished by a marked thinning of the microcolony lawn resulting in a pronounced increase in the size of the microcolonies compared to the vehicle control plate.
Code 4 (Extremely Reduced): Distinguished by an extreme thinning of the microcolony lawn resulting in an increase in the size of the microcolonies compared to the vehicle control plate such that the microcolony lawn is visible to the unaided eye as isolated colonies.
Code 5 (Absent): Distinguished by a complete lack of any microcolony lawn over more than or equal to 90% of the plate.
Code 6 (Obscured by Particulate): The background bacterial lawn cannot be accurately evaluated due to microscopic test substance particulate.
Code NP (Non-Interfering Precipitate): Distinguished by precipitate on the plate that is visible to the naked eye but any precipitate particles detected by the automated colony counter total less than or equal to 10% of the revertant colony count (e.g., less than or equal to 3 particles on a plate with 30 revertants).
Code IP (Interfering Precipitate): Distinguished by a precipitate on the plate that is visible to the naked eye and any precipitate particles detected by the automated colony counter exceed 10% of the revertant colony count (e.g., more than 3 particles on a plate with 30 revertants). These plates were counted manually.

A dose level is considered toxic if one or both of the following criteria are met:
- A 50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count.
- At least a moderate reduction in the background lawn (background lawn code 3, 4, or 5)
Evaluation criteria:
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 equal to or greater than 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 equal to or greater than 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 was evaluated as negative, if it was neither positive nor equivocal.

The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of 3 non-toxic dose levels was required to evaluate assay data.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated.

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
Remarks:
Toxicity was observed beginning at 1500 µg/plate in the absence of S9 activation
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was observed beginning at 1500 µg/plate in the absence of S9 activation
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
STERILITY RESULTS: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions, and the S9 and Sham mixes.

RANGE-FINDING/SCREENING STUDIES: In the toxicity-mutation assay, the maximum dose tested was 5000 µg/plate; this dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500, and 5000 µg/plate. The test substance formed soluble but cloudy solutions in water from 15 to 50 mg/mL and soluble and clear solutions from 0.015 to 5.0 mg/mL. No precipitate was observed. Toxicity was observed beginning at 1500 µg/plate in the absence of S9 activation. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In the confirmatory mutagenicity assay, no precipitate was observed. Toxicity was observed beginning at 1500 µg/plate in the absence of S9 activation. In the initial confirmatory mutagenicity assay, no positive mutagenic responses were observed with tester strains TA98, TA1535, TA1537, and WP2 uvrA in the presence of S9 activation and with any of the tester strains in the absence of S9 activation. Due to an unacceptable positive control value, tester strain TA100 in the presence of S9 activation was not evaluated but was retested. Due to the lack of bacterial growth on all assay plates, the retest of the confirmatory mutagenicity assay with tester strain TA100 in the presence of S9 activation was not evaluated but was retested in a second test. In the second retest of the confirmatory mutagenicity assay, no positive mutagenic response was observed with tester strain TA100 in the presence of S9 activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

All criteria for a valid study were met. Under the conditions of this study, the test substance did not cause a positive response in either the presence or absence of Aroclor-induced rat liver S9.

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

The test substance was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the preincubation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. Water was selected as the solvent of choice based on the solubility of the test substance, compatibility with the target cells and in order to maintain the test substance dilutions on ice during all dilution and plating procedures. The test substance formed a workable suspension in water at approximately 50 mg/mL, the highest concentration tested, when diluted in a tube with a Teflon-lined screw cap.

In the toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. The test substance formed soluble but cloudy solutions in water from 15 to 50 mg/mL and soluble and clear solutions from 0.015 to 5.0 mg/mL. No precipitate was observed. Toxicity was observed beginning at 1500 μg per plate in the absence of S9 activation. Based on the findings of the initial toxicity mutation assay, the maximum dose tested in the mutagenicity assay was 5000 μg per plate. In the confirmatory mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 1500 μg per plate in the absence of S9 activation. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, test substance did not exhibit any mutagenic responses in either the presence or absence of Aroclor-induced rat liver S9.