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

Endpoint summary

Additional information

Bacterial reverse mutation assay (Ames-Test):

In a reverse gene mutation assay in bacteria (BASF AG, 1997), strains TA 1535, TA 1537, TA 98 and TA 100 of Salmonella typhimurium and Escherichia coli WP2 uvr A were exposed to Sodium ascorbyl phosphate (85.4%) at concentrations of 0; 24; 120; 600; 3,000 and 6,000 µg/plate in the presence and absence of mammalian metabolic activation (plate co-incubation and pre-incubation). 

Sodium ascorbyl phosphate was tested up to cytotoxic concentrations (6,000 µg/plate). The positive controls induced appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study satisfies the requirement for Test Guideline EU B1.13/14 and OECD 471/472 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Chromosome aberation test:

In a mammalian cell cytogenetics assay (Chromosome aberration) (BASF AG, 1998), V79 cell cultures were exposed to Sodium ascorbyl phosphate (85.4 %) dissolved in water at concentrations of 0, 500, 2000, 3800 µg/ml, with and/or without metabolic activation (S9 mix).

Sodium ascorbyl phosphate was tested (up to cytotoxic and limit concentration). No test article precipitation and no influence of the test article on the pH value or osmolarity was observed. In both independent experiments (plate incorporation (4 h) and pre-incubation test (18 h, 28 h)), neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test article. In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Positive controls induced the appropriate response. There was no evidence or a concentration related positive response of Chromosome aberration induced over background.

This study satisfies the requirement for Test Guidelines (EU-method B.10 and OECD 473) for in vitro cytogenetic mutagenicity data. 

Mouse Lymphoma assay (read across from ascorbic acid):

In a mammalian cell gene mutation assay (TK locus, Amacher 1991), mouse lymphoma L5178Y cells cultured in vitro were exposed to ascorbic acid (99.9 %, dissolved in saline 1%) up to a concentration of 4 mM (704 µg/mL) in the absence of mammalian metabolic activation.

Ascorbic acid was tested up to cytotoxic concentrations. Replicate experiments with pure ascorbic acid in medium resulted in cytotoxicity at ascorbic concentrations greater than 1.5 mM ca. 265 µg/mL), but no reproducible dose-dependent increase in mutant frequencies over background levels were seen. The positive controls induce the appropriate response.There was no evidence of induced mutant colonies over background.

In vivo bone marrow micronucleous assay (read across from ascorbic acid):

In a CD-1 mouse bone marrow micronucleous assay (Tsuchimoto, 1981), (2 animals/sex/dose) were treated i.p. with ascorbic acid at doses of 0, 80, 160, 320 mg/kg bw. Bone marrow cells were harvested at 6 hrs post-treatment. The vehicle was DMSO. The substance was administered twice, 24 hrs apart.

There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. This study was not performed according to actual OECD or EU guideline but is well documented and considered scientificaly valid to evaluate the mutagenic potential of the test item in vivo.

Short description of key information:
One bacterial reverse mutation assay (Ames test) and a Chromosome aberration test with the test substance Sodium ascorbyl phosphate showed no mutagenic effects up to limit concentrations. No mouse lymhoma or HPRT test is available for Sodium ascorbyl phosphate.
No further studies with Sodium ascorbyl phosphate are available, but the parent compound ascobic acid was tested in a mouse lymphoma assay without metabolic activation and an in vivo bone marrow micronucleous assay demonstrating no mutagenic effects of ascorbic acid and are confirming the findings obtained with Sodium ascorbyl phosphate directly.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results (negative Ames test, negative chromosome aberration test, negative mouse lymphoma assay - read across, negative in vivo bone marrow micronucleous assay - read across) Sodium ascorbyl phosphate is not classified mutagenic according to EU Directive 67/548/EC amended and repealed by EU Regulation No.1272/2008 and CLP.