Bis(D-gluconato-O1,O2)zinc

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:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

Difference and principle of test are detailed in the section "principles of method if other than guidline"

Deviations:

yes

Principles of method if other than guideline:

- Principle of test:
The experiment is designed to test and compare some known or suspected carcinogenic metal ions, some noncarcinogenic, essential metal ions, and a platinum compound with no reported antitumor activity for mutagenic activity in the L5178Y/TK somatic cell point mutation assay.

- Short description of test conditions:
Cells deficient in thymidine kinase (TK) due to the mutation TK+/- to TK-/- are resistant to the cytotoxic effects of trifluorothymidine (TFT). Thymidine kinase proficient cells (TK+/-) are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not able to proliferate.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

1

Reference substance name:

Zinc chloride

EC Number:

231-592-0

EC Name:

Zinc chloride

Cas Number:

7646-85-7

Molecular formula:

Cl2Zn

IUPAC Name:

Zinc chloride

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name of test material (as cited in study report): Zinc chloride

Method

Target gene:

Thymidine kinase locus/TK +/-

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: TK+/- 3.7.2C (Clive, 1973) cells originally obtained from D. Clive had been thawed from a frozen ampule within 1 month of the completion of these experiments.
- Suitability of cells:
These cells were determined free of mycoplasma via 3 methods including fluorescent antibody to M. hyorhinus by an independent laboratory and were routinely suspended in R5 so as to remain in exponential growth. Routine growth conditions and the weekly suppression of background TK-/- mutants have been described by the same author elsewhere (Amacher et al., 1979). A known TK-/- line, GT2, had been growing in Rs (non-selective) medium for >190 generations when used in these studies.

MEDIA USED
- Type and identity of media: RPMI 1640 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes


Reference :
Clive, D, (1973) Recent developments with the L5178Y TK-heterozygote mutagen assay system, Environ. Health Perspect., 6,119--125.
Amacher, D.E., S. Paillet and V.A. Ray (1979) Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, I. Application to genetic toxicology testing, Mutation Res., 64, 391--406.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

1.21-12.13 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Normal saline (1 %)
- Justification for choice of solvent/vehicle: Not reported

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION:
- Preincubation period: Not reported
- Exposure duration: 3 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 7 d (at 37 °C in 5 % C02-95 % humidified air)

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT) - 4 μg/mL

NUMBER OF REPLICATIONS: Duplicate cell culture for test material treatment while triplicate plates were prepared for both survival and mutation frequency determinations for each of the 2 replicate cultures

NUMBER OF CELLS EVALUATED: Cells densities were 30000/mL (1 x 100,000/plate) for mutant selection and 15/mL (500/plate) for viability detrmination

DETERMINATION OF CYTOTOXICITY
- Method: Cell survival for each culture was the product of growth in suspension culture and cloning efficiency in soft-agar medium, each relative to solvent controls

OTHER: Cell culture contained 6 x 100,000 cells each in 10 mL test medium
Light exposure was minimal during treatment of cells.
Test conducted at 37 °C
For the recovery and mutant expression, all cells were maintained at 37 °C for 48 h in log phase growth after treatment with test material

Evaluation criteria:

TFTres colonies were counted to identify the mutants. The average mutant counted is compared to a solvent control, in order to determine the causality of the tested compound (zinc chloride).

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Graph showing Average trifluorothymidine resistance (TFTRes) mutant counts versus test material has been attached as 'attached

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
The test material was found to be non-mutagenic under the test conditions.

Executive summary:

A study was conducted to assess the potential mutagenicity of test material in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The mouse lymphoma cells (TK+/-) were treated with test material at 1.21 - 12.13 µg/mL for 3 h. 48 h after treatment, cells were treated with 4 µg/mL trifluorothymidine (TFT) for 7 d. Colonies growing in the presence of triflurothymidine (TFT resistant) were counted. TFT resistant colonies were scored as mutants.

The test material was found to be non-mutagenic under the test conditions.