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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:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to recent guieline and under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
AN-2
IUPAC Name:
AN-2
Constituent 2
Reference substance name:
4,4'-methylene bis(2,6-di-tert-butylphenol)
IUPAC Name:
4,4'-methylene bis(2,6-di-tert-butylphenol)
Constituent 3
Chemical structure
Reference substance name:
2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol
EC Number:
204-279-1
EC Name:
2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol
Cas Number:
118-82-1
Molecular formula:
C29H44O2
IUPAC Name:
2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): AN-2
- Analytical purity: 99/45%

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
other: K-1 cell line
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 mix from male rats
Test concentrations with justification for top dose:
Experiment 1 with and without S9 mix:
0 (DMSO solvent), 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.2, 0.1 micro-g/ml
Experiment 2 with S9 mix:
0 (DMSO solvent), 300, 200, 150, 100, 75, 50, 30, 10, 5 micro-g/ml
Experiment 2 without S9 mix:
0 (DMSO solvent), 300, 200, 150, 100, 75, 50, 30, 10, 5, 2.5, 1.25 micro-g/ml
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: based on a solubility test wih solvents suitable for the test system. The test substance coudl be dissolved up to 5mg/ml in DMSO after sonification for 15 min at 40 deg. C
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
cyclophosphamide in the presence of S9, mitomycin c in the absence of S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in tissue culture flasks with culture medium

DURATION
- Exposure duration:
Experiment 1 with and without S9: 4 h
Experiment 2:
with S9: 4 h
without S9: 18 h
- Expression time (cells in growth medium): 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): 16 h (2 h before the end of the total incubation period)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 micro-g/ml)

NUMBER OF REPLICATIONS: 2 independent experiments, 2 cultures per concentration

NUMBER OF CELLS EVALUATED: 100 metaphases per culturr, 200 per concentration
Three concentrations of the test substance were selected for the evaluation based on the cytotoxicity (highest should reduce mitotiic index at least 50% but not more than 70%).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
Positive response criteria: concentration relaed increase or reproducible increase in number of cells with structural chromosomal aberrations.

Equivocal response: percentage of cells with structural chromosomal aberrations is statistically significantly higher than that of the negative controls (0.05
A test substance is considered clasogenic if a concentration related increase of the percentage of cells with structural chromosomal aberrations over the concurrent control frequency is observed, or if a single positive test point is observed in both tests at approximately the same dose level.

A test substance is considered negative if it produces neither a dose rleated increase in the numberstructural chromosomal aberrations, nor a reproducible positive response at any of the test points.
Both statistical significance and biologicall relevance are consideed together in the evaluation.
Statistics:
Fisher's exact probability test (two-sided)

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 300 and 200 micro-l/ml precipitation of the TS occurred in cultures with and without S9 mix. Turbidity of the medium was observed down to concentrations of 50 micro-g/ml
Test concentrations selected for CA scoring:
Exp. 1 with S9 mix:
DMSO control, cyclophosphamide control 5 micro-g/ml, TS: 200, 100, 50 micro-l/ml
without S 9 mix:
DMSO control, mitomycin c control 0.1 micro-g/ml, TS: 200, 100, 50 micro-l/ml
Experiment 2:
with S9 mix:
DMSO control, cyclophosphamide control 5 micro-g/m, TS: 300, 200, 100 micro-l/ml
without S 9 mix:
DMSO control, mitomycin c control 0.1 micro-g/ml, TS: 10, 5, 2.5 micro-l/ml
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1 with S9 mitotic index reduced: 71% of controls at 200 micro-g/ml, without S9: 54% at 200 and 59 at 100 micro-g/ml
Experiment 2: with S9 mitotic index reduced: 40% of controls at 300 micro-g/ml, 49% ot 200 micro-g/ml, 80% at 100 micro-g/ml, without S9: 47% at 10 and87% at 5 micro-g/ml, 91% at 2.5 micro-g/ml.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of this study the test substance was not clastogenic to Chinese Hamster Ovary cells
Executive summary:

The test substance AN-2 was examined for its potential to induce structural chromosomal aberrations in Chinese Hamster Ovary (CHO) cells, in both the absence and presence of a metabolic activation system (S9-mix). Dimethylsulfoxide (DMSO) was used as vehicle for the test substance. Two chromosomal aberration tests were conducted. In all instances, duplicate cultures were used. Dose levels, ranging from 1.25 to 300 micro-g/ml, were tested. Both solubility and cytotoxicity were used for dose level selection. Mitotic index reduction was used as indication for cell toxicity.

In the first chromosomal aberration test, in both the absence and presence of a metabolic activation system (S9-mix). treatment/harvesting time was 4/18 hours (pulse treatment) and three concentrations of the test substance (50, 100 and 200 micro-g/ml) were selected for chromosomal aberration analysis. In the pulse treatment group with metabolic activation (S9-mix), the highest concentration was weakly cytotoxic to the cells. In the pulse treatment group without metabolic activation (S9- mix), the two highest concentrations were clearly cytotoxic to the cells.

In the second chromosomal aberration test, in the presence of S9-mix. treatment/harvesting time was 4/18 hours (pulse treatment) and three concentrations of the test substance (100, 200 and 300 micro-g/ml) were selected for chromosomal aberration analysis. Dose-related cytotoxicity was induced by the test substance. In the absence of S9-mix, treatment/harvest time was 18/18 hours (continuous treatment) and three concentrations of the test substance (2.5, 5 and 10 micro-g/ml) were selected for chromosomal aberration analysis. The test substance was weakly cytotoxic to the cells at the two lowest concentrations and clearly cytotoxic to the cells at the highest concentration. At the next higher concentration (30 micro-g/ml), the test substance was too cytotoxic and therefore not selected for chromosomal aberration analysis.

In both the first and second chromosomal aberration lest, the test substance AN-2 did not induce a statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment periods analysed. when compared to the number of aberrant cells found in the vehicle (DMSO) control cultures. In both chromosomal aberration tests, the numbers of aberrant cells. found in the vehicle (DMSO) control cultures. were within the historical range and the positive control substances mitomycin C (in the absence of the S9·mix) and cyclophosphamide (in the presence of the S9-mix) induced the expected statistically significant increases in the incidence of structural chromosomal aberrations. This demonstrates the validity of the assay. Under the conditions used in this study. the test substance AN-2 was not clastogenic to Chinese Hamster Ovary (CHO) cells.