L-Ascorbic acid, 2-(dihydrogen phosphate), sodium salt (1:3)

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 from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not guideline conformant, GLP: no data

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not applicable

Principles of method if other than guideline:

Method: other: according to Amacher, D.E. et al.: Mutat. Res. 64, 391-406

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ascorbic acid

EC Number:

200-066-2

EC Name:

Ascorbic acid

Cas Number:

50-81-7

IUPAC Name:

5-(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one (non-preferred name)

Details on test material:

- Name of test material : Ascorbic acid (in parallel sodium ascorbate was applied)
- Analytical purity: 99.9%

Method

Target gene:

Thymidine Kinase (TK)

Species / strain

Details on mammalian cell type (if applicable):

L5178Y mouse lymphoma cells used in these experiments were the 3.7.2C line and were negative when screened for mycoplasma via growth on agar and Hoechst staining methods. Weekly treatment of these cells to reduce the TK-/- background mutant frequencies were performed. Culture medium was RPM1-1640 (Microbiological Associates) containing 50 units/ml penicillin-50 µg/mL streptomycin, 0.05 % Pluranic F-68 (BASF Wyandotte), and either 3 % horse serum (R3 or test medium) or 5 % horse serum (R5 or growth medium).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

up to ca. 704 µg/mL (4 mM; ascorbic acid); up to ca. 475 µg/mL (2.4 mM; sodium ascorbate)

Vehicle / solvent:

saline (1%)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: 1 h
- Exposure duration: 3 h
- Expression time (cells in growth medium): 48 h

The dosing of cells, removal of test chemicals after 3-h exposure, and maintenance of cells in lag phase growth for the 48-h expression period were previously described [Armacher, D.E. et al., 1979, Mutat. res., 64, 391-406]. Individual test cultures contained 6 x E06 cells in R3 medium, ascorbate or ascorbic acid initially dissolved in saline, and in same cases, 0.1 mg/mL freshly prepared catalase. Final solvent (saline) concentration was always 1%. Negative controls received saline only; concurrent
positive controls were dosed with 2.5 X 10-3 M ethylmethanesulfonate (EMS) dissolved in saline and filter-sterilized. In one set of experiments, ascorbate in R3 medium with or without catalase was preincubated for 1 h at 37°C prior to the addition of cells for 3-h exposure. Same of these preincubated samples were heated to 56°C for 15 min then coaled before cells were added. Where indicated, pH was measured before the 3-h incubation period with a Copenhagen Radiometer an duplicate cultures prepared for that purpose (sterility cannot be maintained after pH measurements). Test chemicals were removed by repeated washing and cells maintained at 37°C at cell densities permitting log phase growth in a New Brunswick Roller-drum. Cloning in soft-agar medium containing 0.37 % Difco Noble agar, 10 % serum and no antibiotics. Viable colony counts and counts of large trifluarathymidine-resistant (TFTr) colonies were made after 7 days incubation af plates at 37 °C in a humid 5 % CO2 atmosphere via a calibrated Artek model 870. Cell survival was determined as the product of both daily growth and cloning efficiency values relative to saline controls. This method differs from the colony forming method used by Rosin et al. and is sensitive to minor day-to day fluctuations.

Evaluation criteria:

Viable colony counts and counts of large trifluarathymidine-resistant (TFTr) colonies were made after 7 days incubation of plates at 37 °C in a humid 5 % CO2 atmosphere via a calibrated Artek model 870.

Statistics:

mean and standard deviation

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

ca. 264 µg/mL (1.5 mM; ascorbic acid); ca. 99 µg/mL (0.5 mM; sodium ascorbate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Ascorbic acid as well as sodium ascorbate was tested. Ascorbic acid was tested at concentrations up to 4.0 mM (ca. 704 µg/mL). It was cytotoxic at concentrations above 1.5 mM (ca. 265 µg/mL) but not mutagenic up to the highest concentration used. Sodium ascorbate was tested at concentrations up to 2.4 mM (ca. 475 µg/ml). It was cytotoxic at concentrations above 0.5 mM (ca. 99 µg/ml) but not mutagenic up to the highest concentration used.
Mutagenicity for the concurrent positive control (2.5 X 10-3 M EMS) appears to vary considerably; however, the results represent pooled data from independent trials spanning a 6 months.
Very high levels of ascorbic acid exceed the buffering capacity of the tissue culture medium and cause a precipitous drop in pH. To determine what effect increased hydrogen ion concentration might have an observed mutagenicity and cytotoxicity, R3 medium was titrated with 1 N HCl, filtered, measured for pH, then added to cultures of L5178Y cells. A pH of less than 6 results in sharply increased cytotoxicity, but no dose-dependent increase in mutant frequency. Since none of the ascorbic acid concentrations produced an initial pH lower than 7, reduced pH was not responsible for ascorbic acid toxicity. Representative pH measurements of sodium ascorbate dissolved in R3 medium showed no initial change (pH 7.6-7.8) compared to untreated medium.

Remarks on result:

other: strain/cell type: L5178Y

Remarks:

Migrated from field 'Test system'.