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EC number: 200-664-3 | CAS number: 67-68-5
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro DNA damage and/or repair study
- Remarks:
- Type of genotoxicity: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
Data source
Referenceopen allclose all
- Reference Type:
- publication
- Title:
- Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in Vitro V: Results with 46 chemicals
- Author:
- Loveday KS, Anderson BE, Resnick MA and Zeiger E
- Year:
- 1 990
- Bibliographic source:
- Environ. Mol. Mutagen. 16: 272-303
- Reference Type:
- publication
- Title:
- Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro : II Results with 20 chemicals
- Author:
- Loveday KS, Lugo MH, Resnick MA, Anderson BE and Zeiger E
- Year:
- 1 989
- Bibliographic source:
- Environ. Mol. Mutagen. 13: 60-94
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
- Deviations:
- not specified
- GLP compliance:
- not specified
- Type of assay:
- sister chromatid exchange assay in mammalian cells
Test material
- Reference substance name:
- Dimethyl sulfoxide
- EC Number:
- 200-664-3
- EC Name:
- Dimethyl sulfoxide
- Cas Number:
- 67-68-5
- Molecular formula:
- C2H6OS
- IUPAC Name:
- dimethyl sulfoxide
- Details on test material:
- Test compound: Dimethylsulfoxide
CAS no.: 67-68-5
Source: Burdick and Jackson Laboratories
Purity: 99.4%
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CHO cells were obtained from Litton Bionetics (Kensington. MD) at their fifth passage level after cloning. and were designated CHO-LB. A large stock of cells was initially prepared, and vials were stored at -80°C. To ensure karyotypic stability, cells were not used beyond the fifteenth passage after cloning. Cells were tested regularly for mycoplasma contamination using 4,6-diamidino-2-phenylindole (DAPI) fluorescence and were found to be free of mycoplasma for all experiments.
Growth and treatment conditions were based on procedures described by Galloway et al. (1985). Cells were grown and exposed to chemicals at 37°C.
- Metabolic activation:
- with and without
- Metabolic activation system:
- The rat liver microsomal fraction was prepared from Aroclor 1254-induced male Sprague-Dawley rats and was combined with cofactors and culture medium to form the metabolic activation system.
- Test concentrations with justification for top dose:
- up to 5000 µg/ml
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- other: 46 subastances were tested
- Positive controls:
- yes
- Positive control substance:
- other: Mitomycin C (MMC; Sigma) was used in the experiments without metabolic activation, and cyclophosphamide (CP; Sigma) was used in the experiments with activation as positive controls.
- Details on test system and experimental conditions:
- Without metabolic activation
Approximately 24 h after culture initiation at 1.25 x 10e6 cells per 75cm² flask, the medium was replaced and cells were exposed to the test or control chemical for 2 hours to allow interaction before bromodeoxyuridine (BrdUrd) addition. BrdUrd was added (final concentration 10e-5 M), and incubation with the chemical continued for an additional 24 hours.
The chemical and BrdUrd were removed 26 hours after the initiation of the treatment and cells were then rinsed twice with phosphate-buffered saline (PBS), pH 7.3. Fresh medium with BrdUrd and colcemid (Sigma, final concentration 10e-6 M) was added, and cells were incubated at 37 °C for an additional 2-2.5 h.
With metabolic activation
Approximately 24 hours after cultures were initiated at 1.25 v 10e6/75cm²flask, the medium was removed and cells were rinsed with PBS (pH 7.3). Serum-free medium containing cofactors and S9 fraction was added, and the cells were treated with the test chemical. Two hours after the initiation of the chemical treatment, the medium with test chemical was removed, the cells were rinsed twice with PBS, and medium with BrdUrd (final concentration 10e-5 M) was added. The cultures were incubated at 37 °C for 24 hours. Colcemid (final concentration 10e-6 M) was then added, and the incubation was continued for another 2-2.5 hours.
Cell Harvest and fixation
Approximately 2-2.5 hours after colcemid addition, the cultures were examined with an inverted microscope for signs of toxicity. Toxicity was determined by estimating the percent of confluence of the cell monolayer in treated flask in comparison with control flasks and noting the presence of mitotic cells. Immediately after the toxicity observation, cells were harvested by mitotic shake-off. The harvested cells were treated for 12 min at 37 °C with hypotonic buffer (0.03 M KCl, 0.01 M sodium citrate) and then re-suspended in three volumes of fixatives (3:1, Methanol: glacial acetic acid).
Slides were prepared, air-dried, stained for 10 min in Hoechst 33258 (0.5 µg/ml in phosphate buffer, pH 7.3), rinsed in water, and mounted in the same buffer. Slides were examined with fluorescence microscopy to assess the frequency of metaphase cells that has completed one or two cell cycle in BrdUrd, i.e., frequency of first division (M1) or second division (M2) cells, respectively. If the test chemical caused cell cycle delay, based on having a high proportion of cells still in M1, the cultures were harvested again 4 hours later. In the experiments reported here, the normal time for obtaining M2 cells was 26 hours after addition of BrdUrd (this includes 2 hours of colcemid). When the harvest time was extended because of cell cycle delay, the cells were exposed to BrdUrd for 30 hours (including 6 hours in colcemid). - Evaluation criteria:
- A trend test of the SCEs per chromosome vs. the log of the concentration was used. A positive trend without a 20 % increase over the solvent control was designated “?”. If the response over from one dose was increased by at least 20 % over the control, the response was designated as weak evidence (+W) for the ability of a chemical to induce SCEs. If at least two doses showed increases of a least 20 % over the control, the result was designated as “+”. The determination of “+W” and “+” are indications not of potency but of strength of the evidence for a positive response.
Results and discussion
Test results
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: > 5000 µg/ml
- Additional information on results:
- DMSO was tested in CHO cells to a maximum concentration of 5000 µg/ml.
DMSO did not induce cell toxicity or cell cycle delay and did not induce an increase in the incidence of SCEs. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Study Result: Negative | |||||||||
Activation | Trial | Trial Call | |||||||
No Activation | 1 | Questionable | |||||||
Induced Rat Liver S9 | 1 | Negative | |||||||
No Activation | 2 | Questionable | |||||||
No Activation | 3 | Negative | |||||||
Trial #:1 Activation: No Activation Date: 1986-07-15 00:00:00.0 Trial Call: Questionable | |||||||||
Dose | No. | No. Chromosomes Examined | Total No. SCEs | SCE / | SCE / | Hours in | % Increase | ||
µg/mL | Cells | Chromosome | Cell | BRDU | Over Solvent | ||||
Examined | Control | ||||||||
Vehicle Control: | |||||||||
Medium | 100 | 50 | 1043 | 356 | 0.341 | 7.120 | 27.000 | 0.000 | |
Test Chemical: | 500 | 50 | 1044 | 465.000 | 0.445 | 9.300 | 27.000 | 30.493 | |
1500 | 50 | 1044 | 372.000 | 0.356 | 7.440 | 27.000 | 4.394 | ||
5000 | 50 | 1049 | 377 | 0.359 | 7.540 | 27.000 | 5.293 | ||
Positive Control: | Mitomycin C | 0.002 | 50 | 1043 | 498.000 | 0.477 | 9.960 | 27.000 | 39.888 |
0.01 | 20 | 417 | 264 | 0.633 | 13.200 | 27.000 | 85.482 | ||
Trend: | -0.396 | ||||||||
Probability: | 0.654 | ||||||||
Trial #:1 Activation: Induced Rat Liver S9 Date: 1986-07-15 00:00:00.0 Trial Call: Negative | |||||||||
Dose | No. | No. Chromosomes Examined | Total No. SCEs | SCE / | SCE / | Hours in | % Increase | ||
µg/mL | Cells | Chromosome | Cell | BRDU | Over Solvent | ||||
Examined | Control | ||||||||
Vehicle Control: | |||||||||
Medium | 100 | 50 | 1040.000 | 450 | 0.433 | 9.000 | 25.500 | 0.000 | |
Test Chemical: | 500 | 50 | 1049 | 494.000 | 0.471 | 9.880 | 25.500 | 8.836 | |
1500 | 50 | 1045 | 506.000 | 0.484 | 10.120 | 25.500 | 11.906 | ||
5000 | 50 | 1050.000 | 479 | 0.456 | 9.580 | 25.500 | 5.431 | ||
Positive Control: | Cyclophosphamide | 0.4 | 50 | 1034 | 706.000 | 0.683 | 14.120 | 25.500 | 57.799 |
2.5 | 10 | 208.000 | 371 | 1.784 | 37.100 | 25.500 | 312.222 | ||
Trend: | 0.868 | ||||||||
Probability: | 0.193 | ||||||||
Trial #:2 Activation: No Activation Date: 1986-09-03 00:00:00.0 Trial Call: Questionable | |||||||||
Dose | No. | No. Chromosomes Examined | Total No. SCEs | SCE / | SCE / | Hours in | % Increase | ||
µg/mL | Cells | Chromosome | Cell | BRDU | Over Solvent | ||||
Examined | Control | ||||||||
Vehicle Control: | |||||||||
Medium | 100 | 50 | 1033.000 | 437 | 0.423 | 8.740 | 26.500 | 0.000 | |
Test Chemical: | 1530 | 50 | 1023 | 537.000 | 0.525 | 10.740 | 26.500 | 24.085 | |
3050 | 50 | 1036 | 480.000 | 0.463 | 9.600 | 26.500 | 9.522 | ||
5090 | 50 | 1027.000 | 455 | 0.443 | 9.100 | 26.500 | 4.727 | ||
Positive Control: | Mitomycin C | 0.0003 | 50 | 1028 | 879.000 | 0.855 | 17.580 | 26.500 | 102.122 |
0.01 | 10 | 212.000 | 763 | 3.599 | 76.300 | 26.500 | 750.761 | ||
Trend: | -0.007 | ||||||||
Probability: | 0.503 | ||||||||
Trial #:3 Activation: No Activation Date: 1988-03-23 00:00:00.0 Trial Call: Negative | |||||||||
Dose | No. | No. Chromosomes Examined | Total No. SCEs | SCE / | SCE / | Hours in | % Increase | ||
µg/mL | Cells | Chromosome | Cell | BRDU | Over Solvent | ||||
Examined | Control | ||||||||
Vehicle Control: | |||||||||
Medium | 100 | 50 | 1035.000 | 371 | 0.358 | 7.420 | 26.000 | 0.000 | |
Test Chemical: | 500 | 50 | 1037 | 435.000 | 0.419 | 8.700 | 26.000 | 17.025 | |
1000 | 50 | 1036 | 375.000 | 0.362 | 7.500 | 26.000 | 0.981 | ||
1500 | 50 | 1042 | 361.000 | 0.346 | 7.220 | 26.000 | -3.349 | ||
3000 | 50 | 1047.000 | 403 | 0.385 | 8.060 | 26.000 | 7.380 | ||
Positive Control: | Mitomycin C | 0.002 | 50 | 1028 | 722.000 | 0.702 | 14.440 | 26.000 | 95.934 |
0.01 | 10 | 208.000 | 420 | 2.019 | 42.000 | 26.000 | 463.316 | ||
Trend: | -0.296 | ||||||||
Probability: | 0.617 |
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
Under these experimental conditions, DMSO did not induce an increase of the incidence of SCE. - Executive summary:
The potential of Dimethylsulfoxide (DMSO) to induce Sister Chromatic Exchanges in CHO cells was evaluated according to a protocol similar to the OECD guidelines 479. CHO cells were treated with and without metabolic activation at concentrations up to 5000 µg/ml. In the absence of limitations on solubility or toxicity, the maximum test chemical concentration was 5 mg/ml. In tests without metabolic activation, cell cultures were exposed to DMSO for 24 hr. In tests with metabolic activation, cultures were exposed to DMSO and rat liver S-9 for 2 hr. Cell toxicity was determined by comparing cell monolayer in treated flasks with control cultures. Mitotic cells were harvested, treated with hypotonic buffer, and re-suspended in fixative. Slides were stained and 50 second-division M2 cells from each of the top three concentrations were scored for SCEs.
DMSO did not induce cell toxicity or cell cycle delay, and did not induce an increase in the incidence of SCEs.
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