Dimethyl sulfoxide

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

Reference

Reference 1

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:

1990

Bibliographic source:

Environ. Mol. Mutagen. 16: 272-303

Reference 2

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:

1989

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

Test material information

Constituent 1

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%

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.