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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
November 10, 2004 - March 21, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
RCC Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
866625-93-6
Cas Number:
866625-93-6
IUPAC Name:
866625-93-6
Test material form:
liquid: viscous
Details on test material:
- Name: Benzotriazole, 1(or 2)-[1-(cyclohexyloxy)heptyl]methyl-
- Physical state: light yellow liquid
- Purity: 99.7%
- storage: at room temperature

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Minimal Essential Medium) supplemented with 10 % fetal calf serum (FCS)
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/p-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I, 4h, without S9 mix: (1.56), 3.13, 6.25, 12.50, (25.0), (50.0) µg/ml
Experiment I, 4h, with S9 mix: (3.13), 6.25, 12.50, 25.0, 50.0, (100.0) µg/ml
Experiment II, 18h, without S9 mix: (0.39), (0.78), (1.56), 3.13, 6.25, 12.50 µg/ml
Experiment II, 28h, without S9 mix: (1.56), 3.13, 6.25, (12.50) µg/ml
Experiment II, 4h, with S9 mix: 3.13, (6.25), 12.50, 25.0, (50.0), (100.0) µg/ml
Concentrations in parentheses were not analyzed
Vehicle / solvent:
ethanol 0.5% (v/v)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: with metabolic activation: 4h, without metabolic activation: 4, 18 and 28h
- Expression time (cells in growth medium): 18 or 28 h
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.2 µg/mL culture medium)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control group in Experiment I after 4 hrs treatment without metabolic activation, in Experiment II after 28 hrs treatment without metabolic activation, and in Experiment II after 4 hrs treatment with metabolic activation, where only 50 metaphase plates were scored due to strong genotoxicity.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, cell number
For evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs and 28 hrs, respectively.

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells in 500 metaphase cells per culture was determined
- Determination of endoreplication: yes

OTHER:
A pre-test on cell growth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item.
Evaluation criteria:
A test item is classified as non-dastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: In the absence of S9 mix, precipitation of the test item in culture medium was observed in Experiment II at preparation interval 18 hrs at 6.25 µg/mL and above. In the presence of S9 mix, test item precipitates in culture medium were observed in Experiment I at preparation interval 18 hrs at 12.5 µg/mL and above and in Experiment II at preparation interval 28 hrs at 100 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 hrs treatment with 25.8 µg/mL and above in the absence of S9 mix. In addition, 4 hrs treatment with 51.6 µg/mL and above in the presence of S9 mix induced strong toxic effects. Considering the toxicity data and the occurrence of test item precipitation of-the pre-test, 50 µg/mL (without S9 mix) and 100 µg/mL (with S9 mix) were chosen as top concentrations in Experiment I. Dose selection of Experiment II was also influenced by test item toxicity and the occurrence of precipitation. In the range finding experiment clearly reduced cell numbers were observed after 24 hrs exposure with 25.8 µg/mL and above. Therefore, 12.5 µg/mL were chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix 100 µg/mL were chosen as top treatment concentration with respect to the results obtained in Experiment I.
In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 51.6pg/mL and above in the absence and the presence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The aberration rates of the cells after treatment with the test item (0.5-4.0% aberrant cells, exclusive gaps) were close to the range of the solvent control values (1.5 - 2.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In this study, toxic effects indicated by reduced cell numbers and/or mitotic indices of about or below 50% of control were observed. However, in Experiment I at 18 hrs preparation interval in the presence of S9 mix and in Experiment II at 28 hrs preparation interval in the absence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In detail, in the absence of S9 mix, toxic effects indicated by reduced cell numbers of about and below 50 % of control were observed after 4 hrs treatment with 12.5 µg/mL (46 % of control) in Experiment I and after 18 hrs continuous treatment with 12.5 µg/mL (51 % of control) in Experiment II. In the presence of S9 mix, cell numbers were clearly reduced after 4 hrs treatment with 25 µg/mL (15 % of control) in Experiment II.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results

Aberrant cells
Exp Exposure Period Preparation Interval concentration (µg/ml) S9 Mix Polyploid cells (%) cell numbers in % of control Mitotic indices in % of control incl. gaps* excl. gaps* with exchanges
I 4 h 18 h neg. control - 1.4 n.t. 100 1.5 1.0 0.0
4 h 18 h solvent control1 - 1.8 100 100 2.0 2.0 0.5
4 h 18 h pos. control2# - 1.0 n.t. 87 36.0 34.0S 12.0
4 h 18 h 3.13 - 1.7 71 83 3.5 2.0 1.0
4 h 18 h 6.25 - 1.8 102 104 5.0 0.5 0.0
4 h 18 h 12.5 - 1.7 46 76 5.5 3.5 0.0
II 18 h 18 h neg. control - 1.9 n.t. 100 1.0 0.5 0.0
18 h 18 h solvent control1 - 2.1 100 100 3.5 2.5 0.5
18 h 18 h pos. control2 - 1.4 n.t. 104 11.5 10.5S 2.0
18 h 18 h 3.13 - 2.3 79 114 1.0 0.5 0.0
18 h 18 h 6.25P - 1.3 69 105 6.0 4.0 0.0
18 h 18 h 12.5P - 1.8 51 136 1.0 1.0 0.0
II 28 h 28 h neg. control - 3.1 n.t. 100 2.5 2.0 1.0
28 h 28 h solvent control1 - 2.6 100 100 2.5 1.5 0.0
28 h 28 h pos. control2# - 1.2 n.t. 84 49.0 49.0S 24.0
28 h 28 h 3.13 - 2.5 88 90 1.5 1.0 0.5
28 h 28 h 6.25 - 1.5 56 89 0.5 0.5 0.0
I 4 h 18 h neg. control + 1.9 n.t. 100 4.5 2.5 1.0
4 h 18 h solvent control1 + 2.4 100 100 2.5 1.5 0.0
4 h 18 h pos. control3 + 2.1 n.t. 96 8.5 8.0S 4.0
4 h 18 h 6.25 + 2.0 104 102 5.0 2.0 0.5
4 h 18 h 12.5P + 2.8 111 85 1.5 1.0 0.5
4 h 18 h 25P + 1.2 96 103 2.0 1.5 0.5
4 h 18 h 50P + 1.6 86 88 3.0 2.0 0.5
II 4 h 28 h neg. control + 3.1 n.t. 100 1.0 1.0 0.5
4 h 28 h solvent control1 + 1.3 100 100 2.5 2.5 0.0
4 h 28 h pos. control4# + 1.1 n.t. 92 46.0 45.0S 15.0
4 h 28 h 3.13 + 1.9 78 87 2.5 2.0 0.0
4 h 28 h 12.5 + 2.7 53 101 1.0 0.5 0.0
4 h 28 h 25 + 1.9 15 131 1.0 0.5 0.0

* inclusive cells carrying exchanges

# evaluation of 50 metaphase plates per culture

P precipitation occurred

n.t. not tested

S aberration frequency statistically significant higher than corresponding control values

1 ethanol 0.5 % (v/v)

2 EMS 300.0 µg/ml

3 CPA 1.0 µg/ml

4 CPA 1.4 µg/ml

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to cytotoxic test item concentrations.