Magnesium diniobate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity of metal salt in the L5178Y mouse lymphoma assay

Author:

Oberly, T.J. et al.

Year:

1982

Bibliographic source:

Journal of Toxicology and Environmental 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)

Version / remarks:

1984-04-04

Deviations:

yes

Remarks:

purity and stability were missing; individual results not reported; rationale for selection of concentrations missing

GLP compliance:

not specified

Type of assay:

other: in vitro 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

Test material form:

not specified

Details on test material:

Supplier: Sigma Chemical Co.

Specific details on test material used for the study:

not specified

Method

Target gene:

TK

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.2 heterozygote

Details on mammalian cell type (if applicable):

CELLS USED
- Methods for maintenance in cell culture: 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.

MEDIA USED
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix: S9 fraction added in appropriate dilution with cofactor mix containg NADP (8 mg/mL) and isocitric acid (15 mg/mL) in Fischer's medium. A 10 % dilution of S9 in medium was utilized.

Test concentrations with justification for top dose:

22000, 24000, 26000, 28000, 30000, 32000, and 36000 µg/mL (without metabolic activation; assumed that these concentrations were also tested with metabolic activation (data not presented))

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile glass-distilled water

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

sterile glass-distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

ethylmethanesulphonate

Details on test system and experimental conditions:

FORWARD MUTATION ASSAY
- test system was based on the procedure described by Clive et al. (1975, 1979)* with modifications to the cloning procedure.
- test item was diluted in the vehicle
- 0.1 mL of each test item dilution was added to a 10-mL suspension containing 6 X 10^6 cells.
- when testing with activation, the 10-mL suspension included 4 mL of an appropriate dilution of S9 with cofactor mix.
- cultures containing either test chemical, positive or negative controls were incubated for 4 hours 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 10^5 cells per mL if necessary.

- on day 2 a modified cloning procedure was followed.
- a sample from each culture was centrifuged and the cells resuspended at 500000 viable cells/mL in Fischer's medium.
- the concentrated cells were serially diluted and appropriate dilutions plated in triplicate in cloning medium with and without trifluorothymidine (TFT).
- approx. 500000 viable cells (as determined by exclusion of trypan blue) were plated on each of three selective medium plates containing 2 μg/mL TFT, 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 to ensure even dispersal of the inocula, allowed to gel, and then incubated at,37°C for approx. 12 days before they were counted.
- a New Brunswick Scientific automatic colony counter was used to determine the number of colonies per plate.
- 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 10^5 colony-forming cells.

*Refernces:
- Clive, D. and Spector, J. F. S. 1975. Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutat. Res. 31 :17 - 29.
- Clive, D., Johnson, K.O., Spector, J. F.S., Batson, A.B., and Brown, M.M.M. 1979. Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system. Mutat. Res. 59: 61 - 108.

Rationale for test conditions:

not specified

Evaluation criteria:

Generally, a test agent will be considered positive in the L5178Y mouse lymphoma assay if a dose-related response is obtained in which two or more concentrations elicit a greater than 2-fold increase in mutation frequency over the solvent control with a minimum of 10% survival (Clive et al., 1979)*.

*Reference:
- Clive, D., Johnson, K.O., Spector, J, F.S., Batson, A.B., and Brown, M.M.M. 1979. Validation and characterization of the L5178Y /TK+/- mouse lymphoma mutagen assay system. Mutat. Res. 59: 61- 108.

Statistics:

not specified

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.2 heterozygote

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1 % survival at the 36000 µg/mL concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

FORWAR MUTATION ASSAY
- without metabolic activation: test doses of magnesium chloride evoked little or no enhancement of mutation compared to the solvent control. Only at 36000 μg/mL was the response to mutation frequency greater than the negative control (not significant finding), and this was at 1 % total survival.
- with metabolic activation: results were not altered by metabolic activation of the test system.
- toxicity from exposure to the chemical might be related only to abnormal osmotic conditions.
Please also refer to the field "Any other information on results incl. tables" below

Values for the solvent and positive controls fall within the range established by previous experiments in our laboratory.

Any other information on results incl. tables

Table 1. Muatgenic Response of Mouse Lymphoma L5178Y Cells following Exposure to the test item

Chemical	Dose (µg/ml)	Percent Total survival	Mutation frequency	Increase over solvet (-fold)
Solvent	0	100	17.9	-
EMS	620	20	136.3	7.6
MgCl2	36,000	1	19.0	1.1
	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:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.