Registration Dossier

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 2010-03-31 to 2010-05-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline 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)
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
Details on test material:
- Expiration date of the lot/batch: February 2011
- Storage conditions of test material: at room temperature, protected from light and under nitrogen gas in a dry and well-ventilated room.

Method

Target gene:
Histidine operon.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix
The selected treatment-levels were:
- 0.39, 0.78, 1.56, 3.13, 6.25 and 12.5 µg/plate for the first experiment,
- 0.63, 1.25, 2.5, 5, 10 and 20 µg/plate for the second experiment.

Experiments with S9 mix
The selected treatment-levels were:
- 3.13, 6.25, 12.5, 25, 50 and 100 µg/plate for the first experiment,
- 10, 20, 40, 80, 160 and 320 µg/plate for the second experiment.
Vehicle / solvent:
- Vehicle used: ethanol.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar.
The test item was tested in a preliminary test and two mutagenicity experiments.
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate
incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 or 72 hours.
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 in the evaluation of the data obtained.

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:
S. typhimurium TA 102
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:
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid.
Experiments without S9 mix:
In the first experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at 12.5 μg/plate towards all the tested strains.
In the second experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels ≥ 5 μg/plate in the TA 1537 strain, ≥ 10 μg/plate in the TA 98 and TA 102 strains and at 20 μg/plate in the TA 1535 and TA 100 strains.
Experiments with S9 mix:
In the first experiment performed using the direct plate incorporation method, no noteworthy toxicity was noted towards any of the five strains tested up to 100 μg/plate.
In the second experiment performed using the preincubation method, a moderate to strong toxicity was noted at dose-levels ≥ 80 μg/plate in the TA 100 strain, ≥ 160 μg/plate in the TA 1537 and TA 98 strains and at 320 μg/plate in the TA 1535 and TA 102 strains.
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, either in the presence or 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 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.

   

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

  Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes,).

  Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at, 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. The test item was diluted in ethanol.

  The dose-levels of the positive controls were as follows:

  without S9 mix

.           1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

.           50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

.           0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

.           0.5 µg/plate of Mitomycin C (MMC): TA 102 strain.

  with S9 mix

.           2 µg/plate of 2-Anthramine (2AM): TA 1535, TA 1537 and TA 98 strains,

.           5 µg/plate of Benzo(a)pyrene (BAP): TA 100 strain,

.           10 µg/plate of 2-Anthramine (2AM): TA 102 strain.

 

Results:

 

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

  Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines

Experiments without S9 mix

The selected treatment-levels were:

.           0.39, 0.78, 1.56, 3.13, 6.25 and 12.5 µg/plate for the first experiment,

.           0.63, 1.25, 2.5, 5, 10 and 20 µg/plate for the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

 

In the first experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at 12.5 µg/plate towards all the tested strains.

In the second experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels >= 5 µg/plate in the TA 1537 strain, >= 10 µg/plate in the TA 98 and TA 102 strains and at 20 µg/plate in the TA 1535 and TA 100 strains.

 

An increase in the number of revertants was noted in the TA 102 strain in the second experiment. This increase reached the threshold of 2‑fold the vehicle control value, however it was neither dose-related nor reproducible (not observed in the first experiment performed under the same experimental conditions). Therefore, this increase was considered not to be biologically significant.

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

Experiments with S9 mix

The selected treatment-levels were:

.           3.13, 6.25, 12.5, 25, 50 and 100 µg/plate for the first experiment,

.           10, 20, 40, 80, 160 and 320 µg/plate for the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

 

In the first experiment performed using the direct plate incorporation method, no noteworthy toxicity was noted towards any of the five strains tested up to 100 µg/plate.

In the second experiment performed using the preincubation method, a moderate to strong toxicity was noted at dose-levels >= 80 µg/plate in the TA 100 strain, >= 160 µg/plate in the TA 1537 and TA 98 strains and at 320 µg/plate in the TA 1535 and TA 102 strains.

 

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

 

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