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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:
25th to 30th July 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Principles of method if other than guideline:
No deviations affected the outcome of the study.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Physical state: Yellow to brown, fused solid
- Analytical purity: 98.5 %
- Lot/batch No.: BM5C01A001
- Expiration date of the lot/batch: 12/05
- Storage condition of test material: Refrigeration (+2 to +8°C)

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12 medium supplemented with L-glutamine, sodium bicarbonate, antibiotics and 5 or 10% of fetal bovine serum (F-12 FBS 5/10).
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes (Karyotype - Hypodiploid with a modal chromosome number 20)
Additional strain / cell type characteristics:
other: K1 strain
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
For the experiment in the presence of metabolic activation, CHO cells were exposed to the test Item in quintuplicate for 3 hours at concentrations of 75, 150 and 300 µg/ml.
For the experiment in the absence of metabolic activation, CHO cells were exposed to the test item at concentrations of 55, 110 and 220 µg/ml of the medium for 3 hours.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Direct acting mutagen
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Indirect acting mutagen
Details on test system and experimental conditions:
METHOD OF APPLICATION: Ham's F-12 medium

DURATION
- Pre-incubation period: Exponentially growing CHO-K1 cells were plated at a density of approximately 10ˆ6 cells in 75 cm2 flasks in quintuplicate with 15 ml of F12 FBS10 and incubated at 36.4°C to 36.5°C for 24 hours.
- Exposure duration: 3 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Each culture from the solvent control, positive control and treatment groups was harvested at 21 hours and 25 minutes after the beginning of the treatment and processed separately for the preparation of chromosomes.

SPINDLE INHIBITOR (cytogenetic assays): Colchicine at 0.2 µg/ml

DETERMINATION OF CYTOTOXICITY
- Method:
Concurrent cytotoxicity for all treated and control cultures was recorded soon after trypsinization, based on cell counts as explained under the preliminary cytotoxicity study. At the time of chromosome preparation, about 0.2 ml of mitotic cells of each group from individual replicates were pooled into respective test tubes, mixed well and the cell counts were determined separately using a haemocytometer.
Mitotic Arrest:
At 19 hours and 30 minutes after the start of the treatment, 300 µl of Colchicine (10 µg/mi) was transferred into tubes containing 15 ml of F12 FBS10, mixed well and poured into respective flasks after removal of the existing medium.

OTHER EXAMINATIONS:
The chromosome and chromatid aberrations observed have been grouped into three categories - gaps, breaks (includes deletions and displacements) and exchanges. Endoreduplication and ring chromosomes were also observed.

Evaluation criteria:
Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results, but statistical significance should not be the only determining factor for a positive response. However, there are certain reservations based on the differences in ranking the types of aberrations. Gaps are ranked lowest and exchange configurations are ranked highest.
A positive result is strengthened by the demonstration of a dose-related increase of the effect. This is particularly crucial if only the frequency of gaps is increased.
Exchanges are such rare events (<1 in 1000 cells) that they are seldom observed in control samples. Thus, the observation of exchanges in experimental groups, even without a dose-related increase, is a strong indication for a positive response.
Statistics:
The data analysed were the proportions of aberrant metaphases in each sample, both including and excluding gaps as aberrations. The pooled data from each test concentration and the positive control data was compared with the solvent control using one-tailed Fisher exact test.
All analysis and comparisons was evaluated at 5% (P<0.05) level.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the test medium at the end of the treatment period ranged from 6.93 to 7.40 with 7.29 in the DMSO control.
- Water solubility: soluble in DMSO
- Precipitation: No precipitation was observed even at the highest tested concentration of 5000 µg/ml.

RANGE-FINDING/SCREENING STUDIES:
There was no monolayer attachment at 1200 µg/ml and above, when CHO cells were exposed to the test item for 3 hours in the presence of and also continuously for 21 hours in the absence of metabolic activation. Similarly, there was no monolayer attachment at 600 µg/ml and above, when CHO cells were exposed to the test item for 3 hours in the absence of metabolic activation.
There was evidence of significant growth inhibition (> 50% inhibition) at 300 µg/ml and above in the presence of metabolic activation, when compared with the DMSO control. There was significant growth inhibition at 300 µg/ml compared to the DMSO control, when CHO cells were exposed to the test item in the absence of metabolic activation. Similarly, when CHO cells were exposed continuously to the test item for 21 hours, there was a significant reduction in the growth of the cells compared to the DMSO control at 150 µg/ml. However, at 300 µg/ml, the cells were dead and disfigured.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
At the highest concentration tested, the reduction in the cell growth was 53.94 and 56.39 % over the DMSO control, in the presence and absence of S-9 activation, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Presence of metabolic activation:

The incidence of aberrant metaphases both including and excluding gaps was statistically comparable to the DMSO control at the lowest tested concentration of 75 µg/ml. There was a biological, as well as a statistically significant, increase in the incidence of aberrant metaphases both including and excluding gaps at and above 150 µg/ml test concentration over the DMSO control. Exchange figures were observed in all the three tested concentrations. Ring chromosome was observed at the highest tested concentration of 300 µg/ml. The positive control, Cyclophosphamide, caused a statistically significant increase in the aberrant metaphases both including and excluding gaps.

Absence of metabolic activation:

There was a biological as well as a statistically significant increase in the incidence of aberrant metaphases both including and excluding gaps at all the three tested concentrations over the DMSO control. Exchange figures were observed in all the three tested concentrations. Ring chromosome was observed at 55 and 220 µg/ml test concentrations.

Ethylmethanesulphonate caused a statistically significant increase in aberrant metaphases both including and excluding gaps.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

From the results of the study, it is concluded that the test item 2-sec butyl-4,6 dinitrophenol (CAS RN: 88-85-7) is clastogenic in CHO cells at the tested concentrations and under the conditions of testing employed.
Executive summary:

The genotoxic potential of the test item 2-sec butyl-4,6 dinitrophenol (CAS RN: 88-85-7) to induce chromosome aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO) cells. One trial each, in the presence and absence of metabolic activation was conducted.

For the experiment in the presence of metabolic activation, CHO cells were exposed to the test item in quintuplicate for 3 hours at concentrations of 75, 150 and 300 µg/ml of the medium in the presence of exogenous metabolic activation system, containing post-mitochondrial supernatant (S-9 homogenate) of the liver of rats treated with Aroclor 1254.

For the experiment in the absence of metabolic activation, CHO cells were exposed to the test item at concentrations of 55, 110 and 220 µg/ml of the medium for 3 hours. In a similar way, concurrent solvent control (DMSO) and appropriate positive controls viz., Cyclohosphamide in the presence of metabolic activation and Ethylmethanesulphonate in the absence of metabolic activation were also tested in quintuplicate. In each case, the cells in C-metaphase were harvested at 21 hours and 25 minutes after the start of the treatment from the DMSO control, all three concentrations of the test item and the positive controls and the slides were prepared for chromosome analysis. A total of 200 metaphases from quintuplicate cultures from the DMSO control, each treatment group and the positive control were evaluated for chromosome aberrations. The data from the treatment groups and the positive control were statistically compared with the DMSO control.

There was evidence of induction of chromosome aberrations with experiments in the presence and absence of metabolic activation, both including and excluding gaps. In each of these experiments, the respective positive control items produced a large and statistically significant increase in aberrant metaphases, under identical conditions. At the highest concentration tested, the reduction in the cell growth was 53.94 and 56.39 % over the DMSO control, both in the presence and absence of metabolic activation, respectively.

The study indicated that the test item has the potential to cause chromosome aberrations at the concentrations tested and under the conditions of testing.