Sodium ascorbate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Mutagenic Evaluation of Compound. FDA 75-64 Sodium Ascorbate USP, FCC

Author:

Litton Bionetics, Inc, Kensington, Md

Year:

1976

Bibliographic source:

PB-266 896

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

only three dose levels and only two plates per dose level were tested, no induction of the liver homogenate

GLP compliance:

not specified

Type of assay:

bacterial reverse 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

Molecular formula:

C6H8O6

IUPAC Name:

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

Method

Target gene:

various genes in the histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate prepared from mouse, rat and monkey

Test concentrations with justification for top dose:

0.075, 0.15 and 0. 3 % (w/v)
Based on cytotoxicity data.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water or saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylnitrosoguanidine

Remarks:

(MNNG) without metabolic actication, TA1535, TA100, 2 µg/plate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

(NF), without metabolic activation, TA1538, TA98, 100 µg/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water or saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: quinacrine mustard

Remarks:

(QM), without metabolic activation, TA1537, 20 µg/plate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(ANTH), with metabolic activation, TA1535, TA100, 100 µg/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 8-aminoquinoline

Remarks:

(AMQ), with metabolic activation, TA1537, 100 µg/plate

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

(AAF), with metabolic activation, TA1538, TA98, 100 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: 10E8 cells

DURATION
- Exposure duration: 48 - 72 hours in the dark

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- The test chemical was tested for toxicity against specific indicator strains over a range of doses to determine the 50% survival dose. Bacteria were tested in phosphate buffer, pH 7.4, for one hour at 37°C on a shaker. The 50% survival concentrations and the 1/4 and 1/2 50% doses were calculated. If no toxicity was obtained for the chemical with a given strain, then a maximum dose of S% (w/v) was used.

Evaluation criteria:

1. Strains TA1535, TA1537, and TA1538
If the solvent control value is within the normal range, a chemical which produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic.

2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a chemical which produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control value for TA100 and two to three times the solvent control value for strains TA98 is considered to be mutagenic. For these strains, the dose response increase should start at approximately the solvent control value.

3. Pattern
Because TA1535 and TA100 were both derived from the same parental strain (G-46) and because TA1538 and TA98 were both derived from the same parental strain (D3052), there is a built-in redundancy in the microbial assay. In general the two strains of a set respond to the same mutagen and such a pattern is sought. It is also anticipated that if a given strain, e.g. TA1537, responds to a mutagen in nonactivation tests it will generally do so in activation tests. (The converse of this relationship is not expected.) While similar response patterns are not required for all mutagens, they can be used to enhance tha reliability of an evaluation decision.

4. Reproducibility
If a chemical produces a response in a single test which cannot be reproduced in one or more additional runs, the initial positive test data loses significance. The preceding criteria are not absolute and other extenuating factors may enter into a final evaluation decision. However, these criteria are applied to the majority of situations and are presented to aid those individuals not familiär with this procedure. As the data base is increased, the criteria for evaluation can be more firmly established.

Statistics:

none

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table: Summary of test results - plate incorporation tests

Test	Species	Tissue	Revertants per plate
			TA-1535		TA-1537		TA-1538		TA-98		TA-100
			Plate 1	Plate 2	Plate 1	Plate 2	Plate 1	Plate 2	Plate 1	Plate 2	Plate 1	Plate 2
1. Non-activation
Solvent control	--	--	31	23	31	19	19	18	22	21	201	248
Positive control	--	--	>1000	>1000	895	461	>1000	>1000	>1000	>1000	>1000	>1000
Test 0.30000%	--	--	34	22	26	26	9	26	40	22	291	196
0.15000 %	--	--	23	21	40	30	16	12	30	33	266	212
0.07500 %	--	--	28	36	16	16	19	21	36	29	247	245
2. Activation
Solvent control	Mouse	Liver	25	40	20	12	22	23	24	19	111	123
	Rat	Liver	20	20	14	11	32	28	40	59	89	77
	Monkey	Liver	16	41	12	6	22	36	51	60	57	71
Positive control	Mouse	Liver	202	154	303	516	>1000	>1000	167	129	123	100
	Rat	Liver	94	91	>1000	127	462	500	239	173	154	181
	Monkey	Liver	513	375	80	119	>1000	>1000	142	183	167	285
Test 0.30000%	Mouse	Liver	21	26	25	15	21	20	34	30	150	160
0.15000 %	Mouse	Liver	27	35	26	17	19	21	33	36	141	151
0.07500 %	Mouse	Liver	39	54	15	20	20	17	31	42	152	156
Test 0.30000%	Rat	Liver	15	19	12	14	17	10	65	65	94	101
0.15000 %	Rat	Liver	18	18	6	19	13	18	64	65	84	86
0.07500 %	Rat	Liver	16	23	13	8	30	11	58	52	66	78
Test 0.30000%	Monkey	Liver	37	30	9	13	24	18	55	34	57	57
0.15000 %	Monkey	Liver	31	40	13	10	21	18	62	33	58	78
0.07500 %	Monkey	Liver	25	36	14	8	21	13	61	43	57	67

Applicant's summary and conclusion

Conclusions:

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the substance is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The genetic toxicity of ascorbic acid was tested in-vitro using bacteria (S. typhimurium, strains TA 98; TA100, TA1535, TA1537 and TA1538) with metabolic activation (S-9 from mouse, rat and monkey) using the plate incorporation method. The test substance (maximum dose 0.3 % (w/v)/plate) was incubated at 37°C for 48 to 72 hours. The number of his-independent colonies were counted thereafter. Ascorbic acid was negative in this bacterial reverse mutation test (Litton Bionics, 1976).