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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:
June 3 - September 25, 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The reported data are reliable with restrictions; quality and stability of the tested substance are not mentioned. The study is performed under GLP conditions. The used methods are well documented and seem equivalent to OECD Guideline 473.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report date:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
quality and stability of test substance was not defined
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Santovar A
- Substance type: white powder
- Physical state: solid
- Analytical purity: "production sample"
- Lot/batch No.: T860036
- Stability under test conditions: not mentioned
- Storage condition of test material: room temperature

Method

Target gene:
chromosomal aberrations
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Range-finding: 0, 5 µg/ml - 5 mg/ml
Assay with activation: 0, 5, 10, 15 µg/ml
Assay without activation: 0, 2, 5, 10 , 15 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
other: at all time-points, except at 48 h. fixation
Positive control substance:
methylmethanesulfonate
Remarks:
at 48 hours positive control is lacking Migrated to IUCLID6: cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 18 - 24 hours
- Expression time (cells in growth medium): 6, 12, 24, 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 100 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; chromosomal aberrations
Evaluation criteria:
not mentioned.
Statistics:
Chi-square analysis and Dunnett's t-test were used to analyze the number of cells with structural aberrations and structural aberrations per cell, respectively. Results were considered statistically significant at the probability level of p < 0.05. Linear regression of linear and log dose versus response was used to analyze for dose-response relationship. A statistically significant probability level of p<0.05 was used.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only at highest dose at t=48 hours
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Additional information on results:
RANGE-FINDING
Cytotoxicity was observed at 10 µglml, as indicated by a lengthening of average cell generation time to approximately 24 hrs and 18 hrs for treatment in the absence and presence of activation, respectively.

SCREENING STUDIES:
Without activation:
Santovar A levels of 2, 5, 10 and 15 µg/ml were chosen as the highest four scorable doses. However, after scoring and decoding, the 15 µg/ml treatment group was found not to contain enough scorable cells (2 or less). The results are therefore not reported. A decrease in mitotic index was observed at 5 µg/ml and higher doses for 6 hours and 10 µg/ml for 12 hours, indicating that cytotoxic levels were tested. Statistically significant increases in percentage cells with structural aberrations and in average structural aberrations per cell were observed at the 10 µg/ml treatment level of Santovar A at the 48 hour harvest time. The aberrations were chromatid deletions and interchanges, and 3 chromosome-type exchanges. However, this response was entirely a factor of the high dose point. No increases were observed at any other lower dose levels.

With activation:
The Santovar A levels of 5, 10 and 15 µg/ml were selected as the three highest scorable doses. A decrease in mitotic index was observed at 15 µg/ml for 6 hours. A statistically significant increase in structural aberrations per cell was observed for the 10 µg/ml treatment group at the 6 hour harvest times. Since 92 metaphases were scored for this dose and all of the structural aberrations were found in one cell, this response was evaluated not to be a test-chemical related effect. This evaluation is supported by the negative responses observed at the other harvest times. No statistically significant dose-response relationship was observed at any harvest times.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The results of the chromosomal aberration assay in CHO cells suggest a negative tendency for 2,5-Di-t-pentylhydroquinone for inducing chromosomal aberrations in experiments with and without S9-activation during various incubation periods.
Executive summary:

Santovar A was tested for its potential to induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells. CHO cells were treated for 5 hours with 1, 2, 5, 10 and 15 μg/ml of the test chemical. The dose levels 2, 5, and 10 μg/ml were scored and reported in the absence of exogenous activation and 5, 10 and 15 μg/ml in the presence of exogenous activation (Aroclor 1254 induced rat liver homogenate). The cells were harvested at 6, 12, 24 and 48 hours after initiation of treatment. The harvest times were calculated for each treatment condition to allow the detection of effects of treatment at different cell cycle stages. In the non-activated study, statistically significant increases in percentage cells with structural aberrations and structural aberrations per cell were observed only for 10 μg/ml Sansovar A at the 48 hr harvest time. A statistically significant dose- response relationship was also observed at this harvest time. No statistically significant increases were observed for Santovar A treatment with activation. Under these conditions, Santovar A was concluded to be negative in the presence of metabolic activation. In the absence of activation, Santovar A was concluded to be a possible clastogen. However, because of the single dose level and time point of the response, and that the response was not observed in the presence of activation, the clastogenicity may not be relevant to the in vivo situation.