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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:
From July 16 to August 03, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 471 Guideline without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on May 13, 1998/ signed on November 05, 1998
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical state: White solid (but some samples were received as liquid, due to stability in supercooled state)
- Storage condition of test material: At room temperature, protected from light and under nitrogen atmosphere


Method

Target gene:
Salmonella typhimurium strains: Histidine gene
Escherichia coli strain WP2uvrA: Tryptophan gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9 (v/v); S9 from liver of rats injected with Aroclor 1254
Test concentrations with justification for top dose:
Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 µg/plate in TA 98, TA 100 and WP2 uvrA strains, with and without S9 mix, using direct plate incorporation method

Mutagenicity tests without S9 mix (direct plate incorporation method):
- 93.75, 187.5, 375, 750 and 1500 µg/plate: for any tester strains in the first experiment as well as for the WP2 uvrA strain in the second experiment,
- 46.875, 93.75, 187.5, 375 and 750 µg/plate: for the TA 1535 and TA 100 strains in the second experiment,
- 23.438, 46.875, 93.75, 187.5 and 375 µg/plate: for the TA 1537 and TA 98 strains in the second experiment.

Mutagenicity tests with S9 mix:
- 93.75, 187.5, 375, 750 and 1500 µg/plate: for all tester strains in the first experiment (direct plate incorporation method) as well as for the WP2 uvrA strain in the second experiment (pre-incubation method),
- 46.875, 93.75, 187.5, 375 and 750 µg/plate: for Salmonella typhimurium strains in the second experiment (pre-incubation method).
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Test substance was freely soluble in DMSO at 50 mg/mL.

Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Anthramine
Remarks:
With S9-mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from B.N. Ames Laboratory (University of California, Berkeley, U.S.A.) whilst
Escherichia coli strain WP2uvrA- was obtained from S. Venitt's Laboratory (lCR, Sutton, England).

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 minutes, 37 °C

- Exposure duration: 48-72 hours at 37 °C

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Mutation study: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: 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.

OTHER:
- After 48 to 72 hours of incubation at 37 °C, revertants were scored with an automatic counter (Artek counter, model 880, O.S.l., 75015 Paris, France).
- The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
Evaluation criteria:
A reproducible two-fold increase 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.
Statistics:
not applicable

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additional information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: Not expected
- Water solubility: None
- Precipitation: None
- Other confounding effects: None

P^$^$^$^$^=RELIMINARY TOXICITY TEST
No precipitate was observed in the Petri plates when scoring the revertants at any dose level.
The test substance was highly toxic at dose levels ≥ 2500 µg/plate, in the three tester strains, both with and without S9 mix.
At dose-levels of 500 and 1000 µg/plate, the test substance was slightly to moderately toxic in the TA 100 strain, both with and without S9 mix.

MUTAGENICITY EXPERIMENTS
1) Experiment without S9 mix:
A slight to strong toxicity was induced in Salmonella typhimurium strains mainly at dose-levels ≥ 750 µg/plate. In the WP2 uvrA strain, a slight to moderate toxicity was noted at 1500 µg/plate.
The test substance did not induce any noteworthy increase in the number of revertants in any of the five strains.

2) Experiment with S9 mix:
In the first experiment, a slight to marked toxicity was generally noted at 1500 µg/plate (direct plate incorporation method).
In the second experiment (preincubation method), a slight to marked toxicity was generally induced at dose-levels ≥ 375 µg/plate.
The test substance did not induce any noteworthy increase in the number of revertants in any of the five strains.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the untreated controls.

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

Conclusions:
The test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471/EU Method B.13/14 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 uvr A were exposed to the test material diluted in DMSO at the following concentrations:

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 µg/plate in TA 98, TA 100 and WP2 uvrA strains, with and without S9 mix, using direct plate incorporation method

Mutagenicity tests without S9 mix (direct plate incorporation method):

- 93.75, 187.5, 375, 750 and 1500 µg/plate: for any tester strains in the first experiment as well as for the WP2 uvrA strain in the second experiment,

- 46.875, 93.75, 187.5, 375 and 750 µg/plate: for the TA 1535 and TA 100 strains in the second experiment,

- 23.438, 46.875, 93.75, 187.5 and 375 µg/plate: for the TA 1537 and TA 98 strains in the second experiment.

Mutagenicity tests with S9 mix:

- 93.75, 187.5, 375, 750 and 1500 µg/plate: for all tester strains in the first experiment (direct plate incorporation method) as well as for the WP2 uvrA strain in the second experiment (pre-incubation method),

- 46.875, 93.75, 187.5, 375 and 750 µg/plate: for Salmonella typhimurium strains in the second experiment (pre-incubation method).

Vehicle and positive control groups were also included in mutagenicity tests.

 

 

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. Experiment without S9 mix: A slight to strong toxicity was induced in Salmonella typhimurium strains mainly at dose-levels ≥ 750 µg/plate. In the WP2 uvrA strain, a slight to moderate toxicity was noted at 1500 µg/plate. The test substance did not induce any noteworthy increase in the number of revertants in any of the five strains.

Experiment with S9 mix: In the first experiment, a slight to marked toxicity was generally noted at 1500 µg/plate (direct plate incorporation method). In the second experiment (preincubation method), a slight to marked toxicity was generally induced at dose-levels ≥ 375 µg/plate. The test substance did not induce any noteworthy increase in the number of revertants in any of the five strains.

 

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

 

Under the test condition, the test material is not mutagenic with and without metabolic activation to S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.