Magnesium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Comparable to guideline study with acceptable restrictions (not individual results reported...) and well-documented publication report which mees basic scientific principles.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1982

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium chloride

EC Number:

232-094-6

EC Name:

Magnesium chloride

Cas Number:

7786-30-3

Molecular formula:

Cl2Mg

IUPAC Name:

magnesium dichloride

Details on test material:

MgCI2 from Sigma Chemical Co was certified ACS grade.
Examination of the lot analysis of MgCl2 indicated that amounts of contaminants, if present, were insufficient to induce mutagenesis

Method

Target gene:

Thymidine kinase (TK) +/- -3.7.2 heterozygote of L5178 mouse lymphoma cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Media, serum, and reagents have been described by Clive and Spector (1975). All cells were thawed from frozen stock and maintained in Fischer's medium for leukemic cells of mice containing 10% heat-inactivated horse serum, Pluronic F68, sodium pyruvate, penicillin G, and streptomycin sulfate.
Background spontaneous TK -/- mutant frequencies were reduced weekly by 24-h treatment of the cells with medium containing thymidine, hypoxanthene, methotrexate, and glycine.

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

36,000 - 32,000 - 30,000 - 28,000 - 26,000 - 24,000 - 22,000 µg/ml.

Vehicle / solvent:

Sterile glass distilled water.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

with and without S9 metabolic activation. EMS was purchased from Eastman Chemicals (Eastman Kodak, Rochester, N.Y.)

Details on test system and experimental conditions:

The test system was based on the procedure described by Clive et al. (1975, 1979) with modifications to the cloning procedure.
MgCl2 were diluted in sterile glass-distilled water, and 0.1 ml of dilution was added to a 10-ml suspension containing 6 X 10E6 cells from a culture recently cleansed of TK-/- cells. When testing with activation, the 10-ml suspension included 4 ml of an appropriate dilution of S9 with cofactor mix. Cultures containing either MgCl2, positive or solvent controls were incubated for 4 h at 37°C.
After exposure, the cells were washed twice, fresh medium was added, and the cultures were carried through a 2-day expression period. The cultures were counted after day 1 and readjusted to 3 X 10E5 cells per milliliter if necessary.

On day 2 a modified cloning procedure was followed. A sample from each culture was centrifuged and the cells resuspended at 500,000 viable cells per milliliter in Fischer's medium. The concentrated cells were serially diluted and appropriate dilutions plated in triplicate in cloning medium with and without trifluorothymidine (TFT). Approximately 500,000 viable cells (as determined by exclusion of trypan blue) were plated on each of three selective medium plates containing 2 Mg/ml TFT (Sigma), and 100 cells were cloned on each of three nonselective plates for each test and control tube. Cell inocula were added directly into 100-mm tissue culture plates, followed by the addition of about 30 ml cloning medium. The plates were swirled gently to ensure even dispersal of the inocula, allowed to gel, and then incubated at 37°C for approximately 12 d before they were counted. A New Brunswick Scientific automatic colony counter was used to determine the number of colonies per plate.

Evaluation criteria:

Total survival was determined by the method of Clive and Spector (1975) which combines growth in suspension culture and soft cloning efficiency data.
The mutation frequency (MF) was calculated as the number of mutants per 105 colony-forming cells.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See table in "any other information"

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: Thymidine kinase +/-

Any other information on results incl. tables

Mutagenic responses of Mouse lymphoma L5178Y Cells following exposure to MgCl2

Chemical	Dose (µg/ml)	Percent total survival	Mutation frequency	Increase over solvant
Solvent	0	100	17.9	-
EMS	620	20	136.3	7.6
MgCl2	36,000	1	19.0	-
	32,000	15	13.3	-
	30,000	15	16.2	-
	28,000	46	15.6	-
	26,000	75	11.6	-
	24,000	101	13.1	-
	22,000	94	14.6	-

Applicant's summary and conclusion

Conclusions:

Interpretation of results : negative

The in vitro gene mutation test in mouse lymphoma cells (thymidine kinase locus in L5178Y) on MgCl2 shown no treatment related increase in mutation frequency.

Executive summary:

MgCl2 was examined for its potential to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells.

Test doses of MgCI2 evoked little or no enhancement of mutation compared to the solvent control. Only at 36,000 µg/ml was the response to mutation frequency greater than the solvant control, and this was at 1% total survival. These results were not altered by metabolic activation of the test system. While toxicity from exposure to MgCl2 might be related only to abnormal osmotic conditions, these results suggest that low survival levels (<10%) do not necessitate nonspecific enhancement in Mouse Lymphoma.

In conclusion, a negative response was obtained with MgCl2