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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
test item identity was not properly determined

Data source

Reference Type:
Chemical mutagenesis at the thymidine kinase locus in L5178Y mouse lymphoma cells: results for 31 coded compounds in the National Toxicology Program
Myhr, B.C. & Caspary, W.J.
Bibliographic source:
Environ. Mol. Mutagen. 18, 51-83

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 1984-04-04
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Constituent 1
Chemical structure
Reference substance name:
Tin dichloride
EC Number:
EC Name:
Tin dichloride
Cas Number:
Molecular formula:
Tin dichloride
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Stannous chloride (received from the NTP Chemical Repository (Radian Corporation, Austin, TX)
- Molecular formula: SnCl2
- Molecular weight: 190
- Storage condition of test material: stored at 4°C or -20°C as directed


Target gene:
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium supplemented with pyruvate (220 µg/mL) and gentamycin (50 µg/mL). Treatment medium was always Fischer’s growth medium with the serum content reduced to 5% by volume, even in experiments where RPMI 1640 medium was used for the culture stock and for the expression and cloning phases of the mutation experiment. Because dose-response curves may be sensitive to changes in the medium environment for some test chemicals, Fischer’s medium was retained for consistency across all experiments. Cloning medium consisted of growth medium with the addition of 0.35% to 0.40% Noble agar, for the cloning efficiency (CE) dishes, plus 3 µg/mL of TFT for the mutant selection dishes. Initially, the serum content was increased to 20%, but this component was returned to 10% when no advantages in growth or CE were evident.
- Periodically checked for Mycoplasma contamination: yes, free of mycoplasma as determined every 2-3 months by cultivating techniques (Flow Laboratories, Rockville, MD).

Master cultures of L5178Y mouse lymphoma cells, clone 3.7.2C (from Dr. D. Clive (Burroughs Wellcome Co., Research Triangle Park, NC)) were maintained in liquid nitrogen at a density of 1 X 10^6 cells/mL in growth medium containing 9% dimethyl sulfoxide (DMSO). Laboratory suspension cultures were initiated every 3-4 months and were maintained in T-250 flasks on an orbital platform shaker at 37°C at approximately 90 rpm. Laboratory cultures were maintained in the same culture medium (RPMI 1640) as used for expression and cloning in the mutation experiments.
Cell densities were determined by hemacytometer, and the cultures were routinely diluted to 2 x 10^5 cells/mL each day during the week and to 9,000 cells/mL for the 3-day weekend cultures. Logarithmic growth was normally maintained with population doubling times of 10-11 hours in RPMI 1640 medium. To prepare cell cultures for mutation experiments, subcultures were established in medium containing THMG (THMG consisted of 9 µg/mL thymidine, 15 µg/mL hypoxanthine, 0.3 µg/mL methotrexate, and 22.5 µg/mL glycine added to RPMI 1640 growth medium [Amacher et al., 1979])*. After growth for 1 day, the cells were transferred to THG medium (THMG but with no methotrexate) for the 3-day weekend growth and to normal growth medium thereafter. Experiments normally were started on Tuesdays and Wednesdays, and a new cycle of THMG treatment (to reduce the spontaneous mutant number) was begun on Thursday, with cells from a continuous laboratory culture never exposed to THMG.

- Amacher DE, Paillet S, Ray VA (1979): Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. J. Application to genetic toxicological testing. Mutat Res 64: 391-406.
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared from the livers of Aroclor 1254-induced male Fischer 344 rats
Test concentrations with justification for top dose:
10, 20, 30, 40, 50, 60, and 80 µg/mL (without metabolic activation)
30, 40, 50, 60, 80, and 100 µg/mL (with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Just prior to each mutation experiment, an aliquot was placed into an appropriate solvent, and several dilutions were performed with the solvent. Test chemical stocks were never filtered; if necessary, maximum solubility was obtained by sonicating and/or heating to 56°C. The treatments were initiated by making dilutions of these solutions into treatment medium containing cells in suspension (1:100 for organic solvents).
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
True negative controls:
not specified
Positive controls:
Positive control substance:
Positive control without metabolic activation: methyl methanesulfonate (5 nL/mL; solved in deionised water); Positive control with metabolic activation: 3-methylcholanthrene (2.5 µg/mL; solved in DMSO)
Details on test system and experimental conditions:
Preliminary studies of test chemical solubility and cytotoxicity were conducted. The solvents used were water, ethanol, DMSO, dimethylformamide, and acetone, and suspensions in solvent were avoided if possible. The solubility of the test chemical in treatment medium was examined in clear tubes and without cells. Changes in pH were noted by the colour of the phenol red indicator in the medium. Test chemical toxicity to 24-hr cell suspension growth was determined for 4-hour treatments with a range of doses.

An appropriate range of doses was chosen for the first mutation experiment such that the relative total growth (RTG) values for cloneable cultures would fall in an expected range of approximately 10-100%.

Cells from THMG-treated stock cultures were seeded at 6 X 10^6 cells into centrifuge tubes and were resuspended in Fischer's treatment medium. S9 mix in activation experiments (or additional medium in nonactivation experiments) and the test chemical solution were added. The tubes were closed and placed on a roller drum for 4 hour at 37°C for the treatment period. After treatment, the cells were centrifuged at low speed (for 8-10 minutes), and the supernatant treatment medium was removed. Each culture was washed twice by resuspension and centrifugation in fresh growth medium. The cells were then resuspended in RPMI 1640 growth medium to obtain densities of 3 X 10^5 cells/mL, and the cultures were returned to the roller drum for a 2-day expression and growth period.
Viable cell densities were determined by hemacytometer each day using trypan blue dye exclusion. If the density exceeded 4 x 10^5 cells/mL after day 1, the culture was diluted to 3 x 10^5 cells/mL with fresh growth medium.
On day 2, the cultures showing an increase in cell density were diluted to 3 x 10^5 cells/mL, and samples were cloned in soft agar medium. A sample containing 3 x 10^6 cells was added to a flask containing cloning medium and 3 µg/mL 5-trifluorothymidine (TFT). The contents were mixed and were poured into three dishes such that each dish contained approximately 1 x 10^6 cells in medium. The cloning efficiency (CE) was determined by serially diluting a sample from each culture and seeding 600 cells into a flask containing of cloning medium. This flask was emptied into three dishes to obtain approximately 200 cells/dish in medium. The dishes were incubated for 11-12 days at 37°C with 5% CO2/humidified air to allow colony development.

Colonies were counted with an Artek 880 electronic counter modified by the manufacturer and fitted with a ten-turn potentiometer as the colony size discriminator. The modification permitted the size resolution necessary to demonstrate the bimodal distribution of mutant colony sizes, which allowed the calculation of the numbers of colonies in the small (S) and large (L) classes of TFT-resistant mutants.
For some cultures, the mutant colony size distribution was obtained by recording counts at increments of 0.2 on the size discriminator. The colonies from all three dishes corresponding to a single culture were added together at each discriminator setting prior to performing the analysis of S and L mutant populations.

- solvent control: quadruplicate (exception: the second trial with metabolic activation)
- positive controls: triplicate
- test chemical concentrations: triplicate

Three trials were performed without metabolic activation and two trials were performed with metabolic activation.
Evaluation criteria:
Please refer to the field "Any other information on materials and methods incl. tables" below.
The calculations for cloning efficiency (CE), relative total growth (RTG), and mutant frequency (MF) have been described (Mitchell et al., 1988)*. Statistical analyses were performed for both the MF trend and comparisons between each dose level and the solvent controls in each experiment [Lee and Caspary, 1983; Murphy et al., 1988)*.
The analysis for S and L mutant colonies was performed by plotting the difference in counts between successive settings of the size discriminator on the Artek counter as a function of the discriminator setting. Usually, a bimodal distribution was obtained, which defined an intersection position on the size discriminator. The colony count obtained at the bimodal intersection corresponded to the large mutant colony count. The small mutant colony count was obtained
as the difference between the total colony count (zero setting) and the large mutant colony count. The CE of the culture was applied to both the small and large colony counts in calculations of the MF and changes in the MF relative to the concurrent solvent control culture.

- Mitchell AD, Myhr BC, Rudd CJ, Caspary WJ, Dunkel VC (1988): Evaluation of the L5178Y mouse lymphoma cell mutagenesis assay: Methods used and chemicals evaluated. Environ Mol Mutagen 12[Suppl 13]: 1-18.
- Lee YJ, Caspary WJ (1983): Mathematical model of L5178Y mouse lymphoma forward mutation assay. Mutat Res 113:417-430.
- Murphy SA, Caspary WJ, Margolin BH (1988): A statistical analysis for the mouse lymphoma cell forward mutation assay. Mutat Res 203: 145-154.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- Effects of pH:
without metabolic activation: acidic pH shift at 50 µg/mL in one trial and acidic pH shift at 60 µg/mL in another trial
with metabolic activation: acidic pH shift at 80 µg/mL in one trial

- Water solubility: the test chemical was dosed into medium from a solution in DMSO because it was not soluble in water, even at 100 µg/mL, which shifted the pH to 3.5. According to the authors, this observation suggested that the actual material tested was stannous chloride dihydrate, which forms an insoluble basic salt in excess water according to the Merck Index. Furthermore, the authors stated that the reduced toxicity in the presence of S9 mix suggested that stannous chloride, a powerful reducing agent, is capable of interacting (in as yet unknown ways) with metabolic activation systems.

- Precipitation:
without metabolic activation: precipitation at 80 µg/mL in two trials
with metabolic activation: precipitation at 80 µg/mL in two trials

Stannous chloride was not detectably mutagenic to L5178Y cells. Some erratic increases in mutant frequency of 1.5-1.8-fold were observed, but these changes bore no relation to toxicity and were not repeatable among the three nonactivation assays and two S9 activation assays. The lowest average relative total growth values obtained for treatments with soluble concentrations ranged from 30% to 35% without S9 to -60% with S9.

Applicant's summary and conclusion

Interpretation of results: negative

According to the authors, stannous chloride was not detectably mutagenic to L5178Y cells.

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