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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (not individual results reported), acceptable, well-documented publication/study report which meets basic scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Mutagenicity of metal salts in the L5178Y mouse lymphoma assay
Author:
Oberly, T.J. et al.
Year:
1982
Bibliographic source:
J. Tox. Environ. Health 9, 367-376

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Magnesium chloride
EC Number:
232-094-6
EC Name:
Magnesium chloride
Cas Number:
7786-30-3
IUPAC Name:
magnesium dichloride
Test material form:
solid: compact
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):
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: TK +/-
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:
Migrated to IUCLID6: 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.
Statistics:
no data

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:
Cytotoxicity observed at concentration of 28 mg/mL and above due to changes in osmotic pressure. See table in "any other information"
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: Thymidine kinase +/-
Remarks:
Migrated from field 'Test system'.

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 

 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 (migrated information):
negative

The in vitro gene mutation test in mouse lymphoma cells (thymidine kinase locus in L5178Y) on the test substance 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 (