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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:
05 January 2012 - 23 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 1,3-Diphenyl-2-thiourea
- Physical state: white powder
- Lot/batch No.: 1012001047
- Analytical purity: 99.4%
- Expiry date: 12 January 2013
- Storage conditions: protected from moisture and heat, at room temperature.

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Experiments without S9 mix
The selected treatment-levels were 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the first and second experiments.

Experiments with S9 mix
.           78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the first and second experiments,
.           312.5, 625, 937.5, 1250, 1875 and 2500 µg/plate for the third and fourth experiments.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide
- Justification for choice: test item is soluble at 100 mg/mL in the vehicle and using a treatment volume of 50 µL/plate, the highest recommended dose-level of 5000 µg/plate is achievable.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

The first experiment (with and without S9 mix) and the second experiment without S9 mix were performed according to the direct plate incorporation method, contrary to the second, third and fourth experiments with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C).
Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to six dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.
The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
DURATION
- Preincubation period: 60 minutes at 37°C.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.
Statistics:
no

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid.
Since the test item was found poorly soluble in the preliminary test, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines.

Experiments without S9 mix
The selected treatment-levels were 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the first and second experiments.
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.
In the first experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at dose‑levels of 1250 µg/plate in the strains TA 1535 and TA 1537, and at 2500 µg/plate in the TA 100 strain.
In the second experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at 2500 µg/plate in the TA 98 strain.
No noteworthy toxicity was noted towards the strain TA 102 in either experiment.
The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.
 
Experiments with S9 mix:
In the first experiment performed using the direct plate incorporation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.
Using the pre-incubation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >=1250 µg/plate, except for the strains TA 100 and TA 1537 in the second experiment where a moderate precipitate was observed at 1250 µg/plate only. In the fourth experiment, a strong toxicity (decrease in the number of revertants) was noted at 2500 µg/plate towards the strain TA 1537.
No noteworthy toxicity was noted towards the other strains used.
 
Noteworthy increases in the number of revertants were noted at 2500 µg/plate in the strains TA 1535, TA 1537 and TA 100 in the second experiment performed with S9 mix. These increases exceeded the threshold of 2-fold the vehicle control (2.6-fold the vehicle control for the strain TA 100) and of 3-fold the vehicle control (up to 30.3-fold the vehicle control for the strains TA 1535 and TA 1537). Two additional experiments were performed using the same experimental conditions (pre-incubation method) and a closer range of dose-levels to check the reproducibility and therefore the reliability of these increases. In these experiments, no increases in the number of revertants were noted at any of the dose-levels tested. Thus, the increases noted in the second experiment were not reproducible in two additional independent experiments. Consequently, they were not considered as biologically relevant.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, in the presence or in the absence of a rat metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD No. 471 and Council Regulation (EC) No. 440/2008 of 30 May 2008, Part B13/14 p. 248) and in compliance with the principles of Good Laboratory Practice.

 

Methods

The first experiment (with and without S9 mix) and the second experiment without S9 mix were performed according to the direct plate incorporation method, contrary to the second, third and fourth experiments with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to six dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Results

The test item was dissolved in dimethylsulfoxide (DMSO).

The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid.

Since the test item was found poorly soluble in the preliminary test, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines.


Experiments without S9 mix

The selected treatment-levels were 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the first and second experiments.

A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.

In the first experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at dose‑levels of 1250 µg/plate in the strains TA 1535 and TA 1537, and at 2500 µg/plate in the TA 100 strain.

In the second experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at 2500 µg/plate in the TA 98 strain.

No noteworthy toxicity was noted towards the strain TA 102 in either experiment.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

 

Experiments with S9 mix

In the first experiment performed using the direct plate incorporation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.

Using the pre-incubation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >=1250 µg/plate, except for the strains TA 100 and TA 1537 in the second experiment where a moderate precipitate was observed at 1250 µg/plate only. In the fourth experiment, a strong toxicity (decrease in the number of revertants) was noted at 2500 µg/plate towards the strain TA 1537.

No noteworthy toxicity was noted towards the other strains used.

 

Noteworthy increases in the number of revertants were noted at 2500 µg/plate in the strains TA 1535, TA 1537 and TA 100 in the second experiment performed with S9 mix. These increases exceeded the threshold of 2-fold the vehicle control (2.6-fold the vehicle control for the strain TA 100) and of 3-fold the vehicle control (up to 30.3-fold the vehicle control for the strains TA 1535 and TA 1537). Two additional experiments were performed using the same experimental conditions (pre-incubation method) and a closer range of dose-levels to check the reproducibility and therefore the reliability of these increases. In these experiments, no increases in the number of revertants were noted at any of the dose-levels tested. Thus, the increases noted in the second experiment were not reproducible in two additional independent experiments. Consequently, they were not considered as biologically relevant.

 

Conclusion

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, in the presence or in the absence of a rat metabolizing system.