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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
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: OECD Guidance study with minor deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
test article treatment is 4 hours instead of 5; whole fetal bovine serum is used instead of dialyzed calf serum
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: granules
Details on test material:
- Name of test material (as cited in study report): Benzotriazol Granulat
- Substance type: yellow granulat
- Physical state: solid
- Analytical purity: 99.83 %
- Lot/batch No.: 909

Method

Target gene:
HGPRT (Hypoxanthine guanine phophoribosyl transferase
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Clone: CHO-K1BH4
- Type and identity of media: Ham's Nutrient Mixture F10 supplemented with L-glutamine, penicillin G, strptomycin sulfate, fungizone fetal bovine serum
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver homogenate
Test concentrations with justification for top dose:
50 to 1000 µg/ml see tables
Vehicle / solvent:
DMSO
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
other: 5-Bromo-2'-deoxyuridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 6 to 7 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN: Giemsa

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED:

Evaluation criteria:
Relative survival
Relative Population Growth
Absolute cloning efficiency
Mutant Frequency
Statistics:
mean values and standard deviation; significance calculated

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
concentrations ranging from 2 to 1000µg/ml were tested for cytotoxic effects.
Without activation, only in the highest concentration a decrease in the relative survival to 80 % was obtained.
With activation, a concentration of 125 µg/ml lead to a survival of 70 % and at the highest concentration the rate was 0%.

MUTAGENICITY STUDY:
Non activation
Three trials were performed under nonactivation conditions, but the first trial was not used for evaluation, because the cloning efficiency of the vehicle controls was too low. In the second trial the assayed cultrues showed a low toxicity up to 1000 µg/ml. The lowest two concentrations were not assyed, because enough concentrations were left for cloning. None of the ten treated cultrues showed increases in mutant frequency that were statistically significantly elevated over the concurrent vehicle controls. Therefore, this nonactivation assay was evaluated as negative. In the independent repeat test concentrations from 400 to 1000 µg/ml were cloned, inducing low toxicities. The mutatnt frequencies varied for concentrations up to the maximum applied concentration of 1000µg/ml. The test material was therefore evaluated as nonmutagenic in this trial.

Activation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In this study the toxicity and mutagenicity of Benzotriazole was assayed.
Under non-activating conditions only low toxicity could be induced up to the maximum applied concentration (1000 µg/ml).
With S9 metabolic activation the substance is more toxic to the used cells as seen by decreases in relative survival and relative population growth.
In all assays the mutant frequencies were within or near the range that is typical of vehiclecontrol variation between trials (1-15*10^-6), while no dose-respons relationships were evident.
Based on these conclusions, the substance 1H-benzotriazole is considered to be nonmutagenic