Mercury

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented publication.

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:

Journal of Toxicology and Environmental Health, 9:367–376.

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Forward mutation assay at the thymidine kinase locus (TK+/-) in L5178Y mouse lymphoma cells.

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Mercury chloride

EC Number:

231-430-9

EC Name:

Mercury chloride

Cas Number:

7546-30-7

IUPAC Name:

mercury dichloride

Constituent 2

Reference substance name:

Mercury dichloride

EC Number:

231-299-8

EC Name:

Mercury dichloride

Cas Number:

7487-94-7

IUPAC Name:

mercury dichloride

Details on test material:

- Name of test material (as cited in study report): mercury chloride
- Molecular formula (if other than submission substance): HgCl2
- Physical state: solid
No further details are given.

Method

Target gene:

TK+/-

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: all cells were thawed from frozen stock and maintained in Fisher's medium for leukemic cells of mice containing 10% heat-inactivated horse serum, Pluronic F68, sodium pyruvate, penicillin G and streptomycin sulfate.
- Properly maintained: yes.
- Periodically checked for Mycoplasma contamination: no data.
- Periodically checked for karyotype stability: no data.
- Periodically "cleansed" against high spontaneous background: yes, background spontaneous TK-/- mutant frequencies were reduced weekly by 24-hour treatment of the cells with medium containing thymidine, hypoxanthene, methotrexate and glycine.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

2, 4, 6 and 8 µg/mL

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

with metabolic activation

Migrated to IUCLID6: 50 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium and plated on agar
- 0.1 mL of metal solution was added to a 10-mL suspension containing 6 * 10^6 cells from culture recently cleansed of TK-/- cells.
- When testing with metabolic activation, the 10-mL suspension included 4 mL of S9 mix.

DURATION
- Exposure duration: 4 hours at 37°C
- Expression time (cells in growth medium): after exposure, the cells were washed twice, fresh medium was added, and the cultures were carried throgh a 2-day expression period.
- Selection time (if incubation with a selection agent): 12 days after the end of expression time (approximately 2 weeks). On day 2, a sample of each culture was centrifuged and the cells resuspended in Fischer's medium. The concentrated cells were diluted and appropriate dilutions plated in clonig medium with and without TFT.
- Cells were also cloned on nonselective plates for each test and control tube.

SELECTION AGENT (mutation assays):
- 2 µg/mL TFT (trifluorothymidine)

NUMBER OF REPLICATIONS:
- plated in triplicate

COLONIE COUNTS:
- A New Brunswick Scientific automatic colony counter was used to determine the number of colonies per plate.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth: viable cells were determined by trypan blue staining.
- Total survival was determined by a combination of growth in suspension culture and soft cloning efficiency data.

OTHER:
- The mutation frequency (MF) was calculated as the number of mutants per 10^5 colony-forming cells.

Evaluation criteria:

no data

Statistics:

no data

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

weak positive response at 8 and 6 µg/mL

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Total survival at 8 and 6 µg/mL was well above the cutoff level of 10%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Mutation frequencies determined for HgCl2 were 12.5, 16.4, 19.5 and 31.9 for the tested concentrations of 2, 4, 6 and 8 µg/mL, respectively.
- Mutation frequency of the positive control was increased 27.8-fold over the solvent control.
- Mutation frequency of the test substance was increased 1.4, 1.8, 2.1 and 3.5-fold over the solvent control for the tested concentrations of 2, 4, 6 and 8 µg/mL, respectively.

TEST-SPECIFIC CONFOUNDING FACTORS: no data

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA:
- Values for the solvent and positive controls fall within the range established by previous experiments of the testing laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Percent of cell survival at the doses that evoke weak mutagenic potential (8 and 6 µg/mL) was 24 and 56%, respectively, compared to control.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

Under the test conditions reported, the test item mercury chloride showed a weak mutagenic activity in mouse lymphoma L5178Y cells in the presence of a metabolic activation system.

Executive summary:

Forward mutation assay at the thymidine kinase locus (TK+/-) was performed in L5178Y mouse lymphoma cells at concentrations of 2 -8 µg/mL in the presence of metabolic activation. The results showed that the test item mercury chloride was weakly mutagenic in mouse lymphoma L5178Y cells in the presence of a metabolic activation system at the two highest tested concentrations (8 and 6 µg/mL).