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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It was noted by the European Food Safety Authority (EFSA) that ascorbic acid or sodium ascorbate alone did not show any mutagenic potential. In some in vitro test systems including redox active substances, especially redox active metal ions, ascorbic acid and sodium ascorbate may act as pro-oxidants, thereby increasing the mutagenic potential of redox active metals or other compounds. These in vitro effects have not been confirmed in in vivo studies.

It was considered that it is unlikely that ascorbic acid or sodium ascorbate are genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For justification please refer to read across justification in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
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
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
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:
valid
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:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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
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
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
Target gene:
various genes in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1538
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.
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
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
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
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
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
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
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
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
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
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
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

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

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).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For justification please refer to read across justification in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
wea
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
various genes in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
S-9 from Aroclor 1254-induced rat and hamster liver, 10% and 30%
Test concentrations with justification for top dose:
0, 100, 333, 1000, 1666, 3333, 6666. 10000 µ/plate
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation


DURATION
- Preincubation period: 20 minutes
- Exposure duration: 2 days



SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all histidine-independent (his+) colonies



DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The data were evaluated as described previously [Zeiger et al., 1987].
Statistics:
mean +/+ SD was calculated
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Positive result was found by one lab (SRI) only in the absence of S-9. Negative in the other (Microbiological Associates) with or without S-9.

Table L-Ascorbic acid (solvent: H20)

 

Dose

TA 100

TA 1535

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

(-)

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

(-)

µg/Plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0.000

114

13.1

118

5.5

110

5.9

104

0.7

106

15.8

40

4.7

43

3.8

11

1.2

35

3.7

11

1.2

100.000

120

9.2

109

1.2

103

1.2

118

5.4

107

5.9

32

1.0

39

2.5

7

2.3

40

2.3

9

0.7

333.000

120

1.0

129

14.9

100

8.3

102

2.6

100

3.6

38

1.5

41

2.0

9

2.5

40

3.8

14

1.5

1000.000

120

4.2

121

2.6

115

10.7

104

3.8

107

5.2

46

2.6

42

4.0

7

1.2

37

2.6

9

1.9

3333.000

129

10.5

117

2.4

115

7.5

136

3.1

104

9.7

42

2.9

44

3.0

13

2.0

37

3.8

9

2.6

10000.000

122

7.2

138

3.5

122s

1.7

130

4.0

106s

2.5

40

4.4

44

4.0

10s

2.9

37

3.5

7s

1.5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

POS

422

19.5

291

12.9

397

9.3

292

6.7

324

7.1

333

21.3

102

5.4

110

5.4

96

4.1

97

5.0

 

 

Dose

TA 1537

TA 97

 

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

(-)

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

(-)

 

µg/Plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0.000

4

0.7

5

1.0

20

1.5

10

1.9

11

0.9

87

7.0

292

191.9

158

2.6

133

3.5

166

8.9

100.000

5

1.9

5

2.3

24

2.1

7

0.9

10

2.8

77

2.0

101

9.7

187

1.7

116

5.6

181

10.8

333.000

5

0.9

6

1.7

26

2.2

6

1.9

6

1.5

93

6.1

103

5.8

180

6.1

113

5.0

166

3.0

1000.000

9

0.9

7

0.3

21

3.2

9

0.7

10

1.5

79

6.8

118

11.3

165

9.7

122

3.9

148

1.9

3333.000

5

2.2

10

2.7

27

2.1

5

1.5

9

0.9

102

13.9

128

3.5

162

15.0

111

0.9

166

8.1

10000.000

8

1.8

7

1.2

22s

5.2

6

2.1

11

3.5

113

10.4

117

6.2

167s

8.7

117

2.9

162s

9.6

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

POS

1022

132.8

110

3.8

126

11.0

151

3.5

36

3.5

1110

29.0

676

12.8

998

6.5

555

20.6

376

5.5

Dose

TA 98

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

(-)

µg/Plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0.000

19

3.2

32

3.8

23

2.3

27

3.2

16

1.7

100.000

17

2.1

26

2.4

22

2.1

24

1.9

22

1.2

333.000

21

3.1

27

1.5

22

4.0

23

1.8

23

3.0

1000.000

23

1.7

29

1.7

21

2.6

31

4.1

21

0.7

3333.000

21

2.7

28

3.8

23

1.5

29

0.9

24

2.3

10000.000

23

4.0

25

0.0

19s

0.9

16

2.8

18s

2.4

 

 

 

 

 

 

 

 

 

 

 

POS

188

12.1

177

6.7

62

2.9

63

1.5

107

9.1

Conclusions:
The test item did not clearly induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used (based on the results of two testing labs). Therefore, the substance is considered weakly positive in these bacterial reverse mutation assays.
Executive summary:

The genetic toxicity of ascorbic acid was tested in-vitro by two independent laboratories, using bacteria (S. typhimurium, strains TA97, TA 98; TA100, TA1535, and TA1537) with and without metabolic activation (S-9 from Aroclor 1254-induced rat and hamster liver, each at 10% and 30%). Following preincubation with the test substance (doses: 0, 100, 333, 1000, 1666, 3333, 6666, and 10000 g/plate) the plates were incubated at 37°C for two days. The number of his-independent colonies were counted thereafter, and mean values ± SD of three replicates were calculated.

One of the two labs reported a positive result with TA97 in the absence of S-9, and ambiguous results with TA97 and TA100, both in the absence of S-9. Hence, ascorbic acid was weakly positive according to the results of this lab, whereas the substance was not genotoxic according to the results of the other (Zeiger et al., 1988).

The studies were conducted similar to the OECD guideline and are suitable for assessment.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For justification please refer to read across justification in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium, other: TA92
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium, other: TA94
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
test was perfomred only with metabolic activation an with duplicate plates, no positve control were tested
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
various genes in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
BACTERIA USED
- Source of bacteria: Dr B. N. Ames, University of California, Berkeley, USA
Species / strain / cell type:
S. typhimurium, other: TA92 and TA94
Details on mammalian cell type (if applicable):
BACTERIA USED
- Source of bacteria: Dr B. N. Ames, University of California, Berkeley, USA
Metabolic activation:
with
Metabolic activation system:
S-9 from polychlorinated biphenyls (500 mg/kg bw of Kanechlor KC-400 in olive oil, ip) induced Fischer rat liver
Test concentrations with justification for top dose:
maximum dose 5 mg/plate
Vehicle / solvent:
phosphate buffer
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
phosphate buffer
True negative controls:
no
Positive controls:
no
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 hours in the dark


NUMBER OF REPLICATIONS: 2

Evaluation criteria:
The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).
Statistics:
none
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium, other: TA92
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium, other: TA94
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
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, TA92 and TA94) with metabolic activation (S-9 from polychlorinated biohenyls (500 mg/kg bw of Kanechlor KC-400 in olive oil). Following preincubation with the test substance (maximum dose 5 mg/plate) the plates were incubated at 37°C for two days. The number of his-independent colonies were counted thereafter. Ascorbic acid was negative in this bacterial reverse mutation test (Ishidate et al., 1984).

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For justification please refer to read across justification in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: results for source substance; adopted for the target substance
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
- Lot no: 310418
- Purity: not stated
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Ciba, Summit, NJ
- Doubling time: 14-16 hrs
- Modal number of chromosomes: 19


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F-10 medium with L-glutamine, supplemented with 10% foetal calf serum and 1% penicillin (10000 IU/mL) and streptomycin (10000 µg/mL).
Metabolic activation:
with and without
Metabolic activation system:
in medium 1% of S-9 fraction from rat livers, phenobarbital / ß-naphthoflavone induced
Test concentrations with justification for top dose:
without metabolicactivation: 0, 50, 100, 200, 300, 400, 500, 600 µg/mL. With meatbolic activation: 0, 400, 700, 1000, 1300 µg/mL
Vehicle / solvent:
culture medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: bleomycin
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension
- Cell density at seeding: 9000/mL

DURATION

- Exposure duration: 48 hrs (without S-9); 3 hrs (with S-9)
- Expression time (cells in growth medium): 48 /45 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hrs

STAIN: Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: washing with PBS, treatment withg RNAse, washing with water, air-drying, staining for 15 minutes

NUMBER OF CELLS EVALUATED: 2000 / dose level (2 replicates, 1000 cells each)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- MN clearly surrounded by a nuclear membrane
- area less than one-third of the area of the main nucleus
- nonrefractility
- not linked to the main nucleus via nucleoplasmic bridges
- location within the cytoplasma of the cell
- only mononucleated cells with well-preserved cytoplasm containing five or fewer MN were scored to exciude apoptosis and nuclear fragmentation.


DETERMINATION OF CYTOTOXICITY
- Method: reduction of cell density by at last 25% compared to teh control
Rationale for test conditions:
state of the art
Evaluation criteria:
Taking into account variability of the MN rates, as a positive result a dose-related effect was defined as showing an increase of the MN frequency over the control by about threefold or higher in at least one dose tested.
Statistics:
not needed
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
no cytotoxicity up to 1730 µg/mL; whereas cytotoxicity was seen at 500 /mL without S-9

Micronuclei:

Without metabolic activation: the percentage of MN increased with dose and reached 7 to 8%, a factor of about 3 over the control.

Cytotoxicity:

With S-9 no cytotoxicity up to 1730 µg/mL (10 mM) ascorbic acid.

Without S-9, cytotoxicity was seen at above 400 /mL.

Conclusions:
Ascorbic acid was weakly positive without metabolic activation.
Executive summary:

Ascorbic acid was weakly positive in a mammalian cell (CHO cells) micronucleus test. Without metabolic activation, the percentage of MN reached 8.1% and 6.9% at 400 µg/mL, which corresponds to a factor of 3 over the control, thus exactly meeting the evaluation criterion. Cytotoxicity was seen at doses above 400 µg/mL in the first experiment, other than in the second where MN reached 6.9%.

In the experiments with metabolic activation, no cytotoxicity or increased number of MN was seen up to 1730 µg/mL, the highest tested dose (Miller et al., 1995).

It is concluded that ascorbic acid was weakly positive in this test in the absence of S-9, but not with S-9. It cannot be excluded that this is influenced by a low pH value; at slightly higher doses (500 and 600 µg/mL; wo S-9) marked cytotoxicity was noted in some of the experiments.

Overall, ascorbic acid gave positive results in an in-vitro mammalian cell micronucleus test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For justification please refer to read across justification in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
mammalian cell line, other: Chinese Hamster Fibroblasts (CHL)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
yes
Remarks:
only long time treatment (24 and 48 hrs) without metabolic activation, only 100 metaphases evaluated
GLP compliance:
not specified
Type of assay:
other: Chromosome Aberration Test
Species / strain / cell type:
other:
Remarks:
Chinese Hamster Fibroblast (CHL) cell line
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970)
Cytokinesis block (if used):
Colchemid
Metabolic activation:
without
Test concentrations with justification for top dose:
three doses, maximum dose of 0.3 mg/mL
The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer.
Vehicle / solvent:
physiological saline
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
physiological saline
True negative controls:
no
Positive controls:
no
Details on test system and experimental conditions:
DURATION
- Exposure duration: 24 and 48 hrs

SPINDLE INHIBITOR: Colchemid (final concentration 0.2 µg/mL was added to the culture 2 hrs before cell harvesting

STAIN: Giemsa Solution (1.5%, at pH 6.8; E. Merck)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: cells were then trypsinized and suspended in a hypotonic KCl Solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa Solution for 12-15 min.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100

DETERMINATION OF CYTOTOXICITY
- Method: other: 50% cell-growth inhibition using a cell densitometer


Evaluation criteria:
Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were
considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.
Statistics:
none
Key result
Species / strain:
mammalian cell line, other: Chinese Hamster Fibroblasts (CHL)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined

Observations: 1 % polyploid cells, 2 % structurel aberration (48 hrs treatment time)

Conclusions:
The test item did not induce chromsome aberrations in the Chinese Hamster Fibroblast (CHL) cell line. Therefore, the substance is considered non-mutagenic in this chromsome aberration test in vitro.
Executive summary:

The genetic toxicity of ascorbic acid was tested in vitro using Chinese Hamster Fibroblast cells without metabolic activation, treated for 24 and 48 hrs. Following tretament with the test substance (maximum dose 0.3 mg/mL) 100 cells/dose were examined for chromsome aberrations. Ascorbic acid was negative in this chromsome aberration test in vitro (Ishidate et al., 1984).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For justification please refer to read across justification in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not specified
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2015
Deviations:
yes
Remarks:
only short term treatment
GLP compliance:
not specified
Type of assay:
other: Mutation assay at the thymidine kinase locus in L5178Y mouse lymphoma cells
Target gene:
The L5178Y mouse lymphoma cell mutation assay measures the increases in the frequency of 5-trifluorothymidine (TFT)-resistant cells in cell populations exposed to chemicals in vitro, and such increases are related to mutational events occurring at the thymidine kinase (TK) locus.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. D. Clive (Burroughs Wellcome Co., Research Triangle Park, NC)

MEDIA USED
Fischer's and RPMI 1640 media, donor horse serum, L-glutamine, and antibiotics were purchased from Quality Biological (Gaithersburg, MD). Pluronic F68 was obtained from BASF (Wyandotte, MI), sodiumpyruvate from Gibco,
(Grand Island, NY), and Noble agar from Difco Laboratories (Detroit, MI). Growth medium consisted of Fischer's medium supplemented with heat-treated horse serum (10% v/v), 110 µg/mlsodium pyruvate, 2 mM L-glutamine, 0.05% Pluronic F68 (nonionic Surfactant), and pen-strep (95 U/mL, 95 pg/mL). The horse serum was freshiy thawed and then brought to 56°C and held at this temperature for 30 min. Later in the study, RPMI 1640 medium was routinely used because of consistently better colony growth in soft agar; with this change, the pyruvate was increased to 220 fxg/ml and
pen-strep was replaced with gentamycm at 50 pg/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 (to obtain a soft consistency), for the cloning efficiency (CE) dishes, plus 3 pg/mL of TFT for the
mutant selection dishes, Initially, the serum content was increased to 20%, but this component was retumed to 10%
when no advantages in growth or CE were evident.
Metabolic activation:
with and without
Metabolic activation system:
prepared from Aroclor 1254-induced male Fischer 344 rats livers
Test concentrations with justification for top dose:
A dose range of 200 - 2000 µg/mL was tested with and without metabolic activation.
Vehicle / solvent:
not specified
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
methylmethanesulfonate
other:
Remarks:
without metabolic activation: 5 nL/mlL MMS or 250 nL/mL EMS with metabolic activation: 2.5 pg/mL MCA
Details on test system and experimental conditions:
Cells from THMG treated stock cultures were seeded at 6 X 10E6 cells into 50-mL centirifuge tubes and were resuspended in Fischer's treatment medium. The final volumes were 10 mL after adding S9 mix in activation experiments (or additional medium in nonactivation experiments) and the test chemical solution. The tubes were closed tightly and placed on a rollet drum for 4 hr at 10-15 rpm at 37°C for the treatment period. After treatment, the cells were centrifuged at low speed (approximately 150g for 8-10 min), 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 20 mL of Fischer's or RPMI 1640 growth medium to obtain densities of 3 x 10E5 cells/ml, and the cultures were retumed 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 10E5 cells/mL after Day 1, the culture was diluted to 3 x 10E5 cells/mL with fresh growth medium (maintaining the 20 mL volume). On Day 2, the cultures showing an increase in cell density were diluted to 3 x 10E5 cells/mL, and samples were cloned in soft agar medium. A 10 mL sample containing 3 X 10E5 cells was added to a flask containing 90 mL cloning medium and 3 pg/mL TFF. The contents were mixed by swirling and were poured into three 100-mm dishes such that each dish contained approximately 1 x 10E6 cells in 33 mL medium. The CE was determined by serially diluting a 1 mL sample from each culture and seeding 600 cells into a flask containing 100 mL of cloning medium. This flask was emptied into three 100-mm dishes to obtain approximately 200 cells/dish in 33 mL of medium. The dishes were incubated for 11-12 days at 37°C with 5% CO2/humidified air to allow colony development.
Rationale for test conditions:
An appropriate range of doses was chosen for the first mutation experiment such that the relative total growth (RTG) values for clonable cultures would fall in an expected range of approximately 10-100%. Doses for subsequent mutation experiments were often adjusted to allow better coverage of the toxicity range.
Evaluation criteria:
See - Any information on materials and methods
Statistics:
Statistical analyses were performed for both the MF trend and comparisons between each dose level and the solvent controls in each experiment. 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 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 cortesponded 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. The colony distribution plots did not directly represent colony size groupings, because the counter gave only accumulative numbers of large and small colonies as the size discriminator was progressively set toward zero. Bimodal distributions were not always resolved due to large proportions of either small or large colonies, or to too few colonies in the solvent controls, or to substandard colony growth conditions.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
Ascorbic acid was evaluated as mutagenic to mouse lymphoma cells under the acidic conditions caused by doses in the range of 750-1,500 µg/mL although no single experiment showed even a twofold increase in MF or a clear dose-related response. The addition of S9 mix seemed to have no particular effect. Variable toxicity was obtained at 1,500 µg/mL (88-51% RTG), and 2,000 µg/mL was lethal in four of five trials. At this point, however, the critical influence of pH on the toxicity was revealed in one nonactivation experiment that was performed at approximately pH 7 (Trial 4). The chemical stock Solution was neutralized with NaOH prior to dosing the cells, and it was subsequently observed that heatment with 200 pg/ml was toxic (25% RTG) and that 300 µg/mL was lethal. No mutagenic activity was observed, despite the dramatic increase in toxicity.

The compound was decoded, and further studies on pH effects were performed. Amacher and Paillet (1981) had reported that sodium ascorbate was nonmutagenic and became highly toxic to L5178Y cells at concentrations above 1 mM. These results were in close agreement with our pH 7 experiment (Nonactivation Trial 4), in which the 200 µg/mL treatment corresponded to 1.1 mM sodium ascorbate. Ascorbic acid was reported as less toxic than ascorbate and nonmutagenic up to the highest tested dose of 3.5 mM (-5% RTG). Ascorbic acid lowers the pH of Fischer's medium, and the lethal concentration of 2,000 µg/ml (11.4 mM) lowered the pH to ~5.0 in the current study. In contrast, the same amount of ascorbic acid added to RPMI 1640 medium reduced the pH only to 6.5. Amacher and Paillet (1981] stated that the pH of their test medium (RPMI 1640) remained above 7.0, so much of the added ascorbic acid was actually converted to (the more toxic) ascorbate by the buffering ability of the medium.

To reexamine the mutagenicity of ascorbic acid, one nonactivation experiment and one S9 activation experiment were repeated, but the pH was held constant in the 6.0-6.5 range by adding NaOH to the highest doses and HCl to the lower ones. The pH range was chosen to keep the ratio of ascorbic acid to ascorbate ion nearly constant as high as possible without exposing the cells to unduly acid conditions. Without S9, high toxicity was induced (at 1,500 µg/mL) without any increase in mutant frequency (Nonactivation Trial 5). With S9 (see Trial 3), a 4.3-fold increase in MF was obtained for a moderately toxic treatment with 1,000 µg/mL (28% RTG). However, a smaller increase in MF occurred for the less toxic treatment with 1,500 µg/mL, and the higher concentrations of 1,800 and 2,000 µg/mL were both non toxic and nonmutagenic. The experiment was evaluated as positive because the significant increases in MF could not be ignored, but the dose relationship was not interpretable. The increase in MF at 1,000 µg/mL was primarily in the small mutant colony fraction (Table 3).

All the experiments considered together indicate that, while pH is certainly a controlling factor in ascorbic acid toxicity, some other factor in the exposure conditions must be controlled to demonstrate mutagenic activity consistentiy. This is hardly surprising in view of the fact that ascorbic acid has yielded unexplained, contradictory genotoxic effects in many test systems. Ascorbic acid is very unstable to dissolved oxygen, and the production of active intermediates is dependent on pH and metal ions, such as those found in culture medium. Amacher and Paillet (1981) have demonstrated that catalase eliminates the toxicity to L5178Y cells that is associated with ascorbate ion exposures. Thus the curtent results only underscore the need for a precise and systematic study of the exposure conditions in order to understand the multiple effects of ascorbic acid.

Reference
Amacher DE and Paillet SC, 1981. Ascorbate is not detectably mutagenic in the L5178Y TK+/−cell mutation assay. Cancer Letters, 14, 151–158.

Table 1 – Evolution Summary

 

Chemical

Evaluation

Assay

-S9 Test

+S9 Test

1. Ascorbic acid

?

? (-, +, +, -a, -)

? (-, ?, +)

 a     Experiment performed with treatments at neutral pH.

 Evaluation symbols:

+ Positive

- Toxic and negative

? Questionable

Table 2 – Summary of Mutagenic Activities

Chemical

Test

LEC or [HTC]

µg/mL

RTG (%) at LEC or [HTC]

Max. fold-change in MF

Evaluation

1. Ascorbic acid

-S9

+S9

750-1500

600-1000

52-56

28-68

1.7-1.9

1.8-4.3

?

?

Table VIII – Chemical-induced changes in the large and small classes of mutant colonies

Chemical treatment

Trial

Mutant colony count and CE

Mutant frequency

Mutant frequency change

Treatment

Solv. control

Treatment

Solv. Control

Difference

Fold-change

L

S

CE

L

S

CE

L

S

L

S

L

S

L

S

Ascorbic acid

1500 µg/mL

NA 2

61

72

73

26

40

73

28

33

12

18

16

14

2.3

1.8

1500 µg/mL

NA 3

62

100

79

49

42

80

26

42

20

17

6

25

1.3

2.4

1500 µg/mL

A 1

98

86

91

95

42

97

36

32

33

14

3

17

1.1

2.2

2000 µg/mL

A 2

54

80

87

40

65

99

21

31

13

22

7

9

1.6

1.4

1000 µg/mL

A 3

175

384

112

69

41

111

52

114

21

12

31

102

2.5

9.3

A     Induced S9 activation trial keyed to Table VII.

A*    Noninduced S9 activation trial keyed to Table VII.

NA   Nonactivation trial keyed to Table VII.

L      Large mutant colonies, sum of the three dishes.

S      Small mutant colonies, sum of the three dishes.

CE   Cloning efficiency of the culture, as percent.

Mutant frequency and mutant frequency change (often called induced mutant frequency) are exparessed as mutants per 10E6 cells.

Conclusions:
The test item tested both without and with metabolic activation, did induce ambiguous increases in mutant frequency in this in vitro test in L5178Y cells.
Executive summary:

The genetic toxicity of ascorbic acid was tested in vitro at the thymidine kinase locus in L5178Y cells without metabolic activation, treated for 4 hrs. Following treatment with the test substance (maximum dose 2000 µg/mL) ascorbic acid was ambiguous in this mouse lymphoma test in vitro, dependend on  the pH value in the test system. All the experiments considered together indicate that pH is a controlling factor in ascorbic acid toxicity. Thus, it was not possible to conclude the test item as mutagenic or not mutagenic under the conditions of this study. The curtent results underscore the need for a precise and systematic study of the exposure conditions in order to understand the multiple effects of ascorbic acid. (Myhr and Caspary, 1984).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Several in-vivo studies on the induction of micronuclei or SCE (sister chromatid exchange) following the administration of ascorbic acid are known to exist (cf. EFSA ANS PANEL, 2015).

Overall, it is concluded that ascorbic acid gave weak positive results in some of the available in-vitro genotoxicity assays but that is not genotoxic in-vivo (EFSA ANS PANEL, 2015). This is supported by the finding that ascorbic acid was not carcinogenic in male and female rats and mice fed diets containing ascorbic acid at 25,000 and 50,000 ppm for 103 weeks (cf. NTP, 1983).

It could also be mentioned that ascorbic acid is contained in Annex IV of the REACH regulation (EC 1907/2006) as it is considered that sufficient information is available showing that this substance is sufficiently safe and no registration is required (EC 1907/2006, Article 2, No. 7a).

This can be adopted for sodium ascorbic acid because the ascorbate anion is the conjugate base of ascorbic acid; following oral intake, both ascorbic acid and the conjugate base will be present in the protonated form at the low pH in the stomach, i.e. there is no difference which of the substances was orally administered regarding the subsequent absorption from the gut.Based on this it is concluded that sodium ascorbate is also not carcinogenic or genotoxic in-vivo.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For justification please refer to read across justification in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
shorter preparation time of the cells as recommended, highest dose not justified
GLP compliance:
not specified
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: animal production section of this institute for drug research and control, Warzawa, Polland
- Weight at study initiation: 23 - 30 g
- Diet: ad libitum
- Water: ad libitum
Route of administration:
oral: gavage
Vehicle:
water
Frequency of treatment:
First treatment at the start of the study and second treatment at 24 hrs after study start.
Post exposure period:
6 hrs
Dose / conc.:
622 mg/kg bw/day
No. of animals per sex per dose:
3 male and female animals in the ascrobic acid dose group
Control animals:
yes, concurrent vehicle
Tissues and cell types examined:
polychromatic erythrocytes
Details of tissue and slide preparation:
The bone-marrow smears were prepared and stained according to the method of Schmid except that Sorensen buffer (pH 6.8) was used instead of distilled water.

Reference
Schmid W.: The micronucleus test. Mutation Res., 1975, 31, 9 - 15.
Evaluation criteria:
Only polychromatic erythrocytes with clearly identifiable micronuclei were scored. The frequency, of micronuclei was calculated for each animal (2000 cells per mouse) and used to determine the mean value for the group. The results were evaluated according to Kastenbaum and Bowman. Smears from all groups of animals were also used to determine the ratio of erythrocytes to nucleated cells by counting about 500 cells for each mouse.

Reference
Kastenbaum M.A., Bowman K.O.: Tables for determining the statistical significance of mutation frequencies. Mutation Res., 1970, 9, 527-549.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid

Compound Dose
mg/kg bw
Polychromatic erythrocytes
scored with MN
n n
Control   16000 19 1.2
Ascorbic acid 2 x 622 12000 26 2.17
Conclusions:
The test item did not induce a significant increase in the frequency of micronuclei in polychromatic erythrocytes. Therefore, the substance is considered non-mutagenic in this micronucleus test in vivo.
Executive summary:

The genetic toxicity of ascorbic acid was tested in vivo in a micronucelus test in mice. Ascorbic acid was tested as a satellite group in this study. Following oral treatment with the test substance (dose 2 x 622 mg/kg bw) ascorbic acid induced no significant increase in the frequency of micronuclei and was therefore non-mutagenic in the test system (Pienkowska, 1985).

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For justification please refer to read across justification in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Negative controls validity:
valid
Positive controls validity:
not examined
Remarks on result:
other: results for source substance
Additional information on results:
results for source substance can be adopted for sodium ascorbate
Endpoint:
genetic toxicity in vivo, other
Remarks:
mammalian cells; cytogenicity; bone marrow SCE
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Principle of test:
- Short description of test conditions:
The SCE test in vivo was carried out according to Allen et al, (1978). Chinese hamsters 6—12 months old were used. To implant a 50 mg tablet of pure BrdU, an incision was made in the lower abdominal region of an etherized animal. After Insertion of the tablet, the wound was closed with 2 surgical clips. 1 h afterwards, aqueous solutions of ascorbic acid were given by intraperitoneal injections or the animals were fed using a syringe fitted with a sound which was inserted in the Oesophagus. After 24 h, colchicine (50 mg/kg body weight) was injected, and, after another 2 h, metaphases were prepared firom the bone marrow by routine methods.

- Parameters analysed / observed:
The method of Epplen et al. (1975) was used for differential staining. In each experiment, the SCEs were counted in 50 metaphases from 2 animals, each of which received the same dose of ascorbic acid. The significance. of the effects was determined by the t test.
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay
Species:
hamster, Chinese
Sex:
not specified
Details on test animals or test system and environmental conditions:
number of animals: 2 per dose and route
Route of administration:
other: oral and i.p.
Vehicle:
- Vehicle used: water
Details on exposure:
Dosing: single dosing
Oral routeg: a solution containing the test material was given by oral gavage
i.p route: injection of a solution a solution containing the test material

Doses: 200 to 10000 mg/kg bw
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Post exposure period:
26 hours
Dose / conc.:
0 mg/kg bw/day
Remarks:
control
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
oral and i.p.
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
oral and i.p.
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
oral and i.p.
Dose / conc.:
10 000 mg/kg bw/day (actual dose received)
Remarks:
oral and i.p.
No. of animals per sex per dose:
not specified
Control animals:
yes, concurrent vehicle
Positive control(s):
none
Tissues and cell types examined:
bone marrow metaphases
Details of tissue and slide preparation:
24 hours after dosing, colchicine (50 mg/kg body weight) was injected, and, after another 2 h, metaphases were prepared from the bone marrow by routine methods. The method of Epplen et al. (1975) was used for differential staining.
Evaluation criteria:
Allen et al. (1978)
Statistics:
t test
Key result
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
not examined
Remarks on result:
other: SCE incidence not increased after oral or i.p. dosing

Incidence of SCEs by ascorbic acid in the bone marrow of Chinese hamsters in vivo (n=2):

 Dose (mg/kg bw) oral dosing  i.p. injection
 0  3.0 ± 0.2
 100  3..4 ± 0.3   n.d.
 1000  3.3 ± 0.2   3.3  ± 0.3 
 2000  3.7  ± 0.3   2.7  ± 0.2
 10000  3.2  ± 0.2  2.8   ±  0.2

Induction of SCEs by ascorbic acid in V79 Chinese hamster cells in vitro:

 Ascorbic acid

(mM)

 number of SCEs per cell

(mean ± SD)

 0  6.5 ± 0.4
 0.001  6.7 ± 0.4
 0.01  9.1 ± 0.5
 0.1 10.9 ± 0.5
 1  13.8 ± 0.6
Conclusions:
The test item did not induce a significant increase in the frequency of sister chromatid exchanges (SCEs). Therefore, the substance is considered non-mutagenic in this sister chromatid exchange assay in vivo.
Executive summary:

Ascorbic acid did not induce sister-chromatid exchanges (SCEs) in the bone marrow of Chinese hamsters (n=2) in the SCE test in vivo. The test dose ranged from 200 to 10 000 mg/kg body weight and was administered orally as well as by intraperitoneal injection. Only one tested concentration led to a statistically significant (t≤1%) increase in the incidence of SCEs, but this was neglected because all other results were homogeneous and the significant increase was not observed at the highest tested dose, i.e. there was no dose relationship.

In contrast, the same study reported a dose-dependent increase of SCEs in V79 cells in vitro which could be decreased by the simultaneous exposure to either cysteine or reduced glutathione which themselves did not increase the number of SCEs. Oxidised glutathione did not reduce the number of SCEs, i.e. the sulfhydryl group had a protective effect (data not shown).

Overall, ascorbic acid was genotoxic in vitro but this was not confirmed in vivo even with high doses administered per os or i.p. injection to Chinese hamsters. This observation is in line with many other in-vitro and in-vivo genotoxicity studies on ascorbic acid (Speit et al., 1980).

Despite some shortcomings the study is considered to be valid and suitable for assessment.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ascorbic acid or sodium ascorbate were intensively evaluated by the European Food Safety Authority (EFSA) in 2015 ( EFSA Journal 2015;13(5):4087). The evaluation of the in vitro and in vivo gentox data is summarized below.

In vitro gentox data

Gene mutation assay in bacteria

CIR (2005), in a review, reported 11 studies performed in Salmonella typhimurium strains (Litton Bionetics, 1975, as referred to by CIR (2005); Litton Bionetics, 1976, as referred to by CIR (2005); Stich et al., 1976; Omura et al., 1978; Bruce and Heddle, 1979; Weitzman and Stossel, 1982; Ishidate et al., 1984, 1988; Zeiger et al., 1988; Anderson et al., 1995) or Staphylococcus aureus strains (Amabile-Cuevas, 1988).

It was considered that, based on the data available, there is no evidence for a mutagenic effect of ascorbic acid in bacteria.

Gene mutation assay in yeast

CIR (2005) reported, in a review, an unpublished report from Litton Bionetics (1975, as referred to by CIR (2005)) in which ascorbic acid was tested in Saccharomyces cerevisiae strain D4 without metabolic activation and with metabolic activation using the liver, lung and testes of mice, rats and primates (Macaca mulatta) S9. No mutagenic activity was demonstrated. In another unpublished report from Litton Bionetics (1976, as referred to by CIR (2005)), sodium ascorbate was tested in the same strain using with the same metabolic activation systems at 0.075, 0.150 and 0.1 %; no mutagenic activity was observed.

Gene mutation assay in mammalian cells

Rosin et al. (1980) exposed Chinese hamster ovary (CHO) cells to ascorbate (unknown salt) (2 to 1 × 10E–3 M) for three hours without metabolic activation only.This treatment resulted in the induction of mutations at the HPRT locus. The authors noted that the concentration at which ascorbate was positive in inducing the mutants was very narrow: the peak of mutation induction occurred with 5 × 10E−4 M ascorbate (two assays) or 2 × 10E−4 M (two assays) and a concentration of 1 × 10E−4 M ascorbate resulted in a weak decrease in cell survival but induced no increase in mutation frequency. They also demonstrated that the addition of catalase prevented both mutagenesis and toxicity, suggesting that mutagenic metabolites of ascorbate may involve oxygen radicals. The Panel noted the absence of any consistent dose–response relationship (only one concentration in each of the four trials was reported as mutagenic) and the variability between assays without a clear indication of cell toxicity level in each assay. The absence of this information makes results difficult to interpret. The consideration of the positive data reported was impaired by the absence of a dose response relationship and information on cytotoxicity.

Amacher and Paillet (1981) performed a mouse lymphoma assay (L5178Y cells) at the TK locus. Cells were treated for two hours without metabolic activation only. No mutagenic activity was observed up to the maximum toxicity level that appears for concentrations above 1.5 mM with ascorbic acid and 0.5 mM with sodium ascorbate. The concentrations of 10 % survival were about 3.5 mM with ascorbic acid and 1 mM with sodium ascorbate.

In an additional mouse lymphoma assay at the TK locus in L5178Y cells, Myhr and Caspary (1991) evaluated ascorbic acid as a mutagenic compound, in both the absence and the presence of rat S9 metabolism in the range 750–1 500 μg/mL when acidic conditions caused by treatments were not controlled. However, when stock solutions of ascorbic acid were neutralised with NaOH to keep pH at values close to 7, no mutagenicity was observed, although toxicity was markedly enhanced (25 % RTG at 200 μg/mL and lethal effects at 300 μg/mL).

Gene mutation assay in Drosophila melanogaster

Tripathy et al. (1990) in a wing spot test in Drosophila melanogaster (three days of age) at concentrations of 0, 10, and 300 mM ascorbic acid in food for 48 hours, found that no gene mutation was induced.

Khan and Sinha (2008) performed a Muller-5 test (X-chromosome-linked recessive lethal mutations assay) in Drosophila melanogaster. First-instar larvae (age: 24 ± 2 hours) collected from culture stock were transferred to food vials containing supplementation with vitamin C (10 mg/L of food). Virgin females of Muller-5 stock were mated to treated males in vials containing normal food to obtain M1 flies. Brother–sister crosses were arranged for these M1 flies by keeping one female with one male in each vial to increase the M2 generation. A statistically significant increase (about three times) in mutation frequency was observed with respect to controls when the food was mixed with vitamin C.

Chromosomal aberration assay

Stich et al. (1980) observed clastogenic activity of neutralised culture medium extract of three different ascorbic acid pills (one pill containing 100 mg in 20 mL culture medium) in CHO cells at concentrations given for 3 hours followed by a 16-hour recovery period with extracts diluted from 1/100 to 1/1 000 (theoretically from 1 to 0.1 mg/mL ascorbic acid). Authors observed that 24–42 % of cells treated with the extract dilutions were binding chromosomal aberrations. However, CIR (2005), in a review, pointed out that the authors of this study noted that the study demonstrated chromosome damaging capacity of vitamin C in one in vitro test system and that they did not provide information on the possible mutagenic or clastogenic action of ascorbic acid in vitro in mammals, including in humans. It was noted that this assay did not study ascorbic acid itself but three extracts of three different pills, and that the clastogenic and cytotoxic effects are very different between extracts; that no control of the ascorbic acid extracted was performed and that excipients of the pills could interfere in the assay; that no information on the use of metabolic activation was given; and that no result of negative control was reported. It was considered that this study had some shortcomings, e.g. poor reporting of the methodology used for the preparation of the test substance.

Ishidate et al. (1984), in an in vitro chromosomal aberration test using a Chinese hamster fibroblast cell line, found no clastogenic activity of ascorbic acid when tested at up to 0.3 mg/mL in saline solution. The maximum dose was selected by a preliminary test which identified the dose needed for 50 % cell-growth inhibition. The cells were exposed to three different concentrations for 24 and 48 hours. No information about the use of metabolic activation was provided.

CHO cells were treated with L-ascorbic acid (purity 99.3 %) for 24 hours without S9 and 2 hours followed by a 24-hour recovery period (Gulati et al., 1989). L-Ascorbic acid was negative for the induction of chromosomal aberrations in either the presence or the absence of S9, although in the first trial without S9 a positive response was observed at the highest dose tested (300 μg/mL); the highest non-lethal dose achievable in three subsequent trials without S9 was 1 600 μg/mL and no increase in aberrations occurred at this level. The authors noted that a noticeable decrease in pH of the culture medium occurred at doses of 500 μg/mL and above. Greggi et al. (1999) investigated the clastogenic effect of ascorbic acid (at concentrations of 100, 200, 500 and 1 000 mg/mL) without metabolic activation for 48 hours on human peripheral blood lymphocytes in vitro, in which 100 metaphases/concentrations were observed. Ascorbic acid did not show any clastogenic effect, except at 1 000 mg/mL that demonstrated an excessive cytotoxic level (60 % decrease of mitotic index). It was noted that no neutralisation of the acidification of the culture medium was performed.

Robichová et al. (2004), found no induction of chromosomal aberrations in human Hep G2 cells in vitro with ascorbic acid at 0.5 mM for 1 hour without metabolic activation.

Micronucleus assay

Miller et al. (1995) tested ascorbic acid in CHO cells without S9 with a 48-hour continuous treatment and with S9 (from livers of phenobarbital/β naphtoflavone induced-rats) with a 3-hour treatment followed by a 45-hour recovery period in only one culture/dose. Only 1 000 cells/culture were observed. There was a dose-related increase in two independent assays without metabolic activation, statistically significant at 400 μg/mL in the first assay and at 500 and 600 μg/mL in the second assay. No induction of micronuclei was observed with metabolic activation. A shorter treatment (25 to 46 hours) at concentrations of 1300 and 1730 μg/mL (10mM) in the absence of metabolic activation did not induce a significant increase in micronuclei. The authors explained this effect by the production of oxidative radicals produced at high doses only. It was noted that no adjustment of pH was performed and that an acidification of the medium could be at the origin of the clastogenic activity.

Comet assay

Frenzilli et al. (2000) investigated the genotoxic potential of ascorbic acid in the alkaline single-cell gel assay (Comet assay) in human leucocytes in vitro at concentrations up to 1 500 μM without metabolic activation for four hours and no DNA strand breaks were noted.

The genotoxic potential of vitamin C was assessed on human lymphocytes, isolated by centrifugation in a density gradient, using single-cell gel electrophoresis (Comet assay) (Blasiak et al., 2000). Fifty cells were randomly selected from each sample and the comet tail moment (a product of DNA fraction in tail and tail length) was measured. Vitamin C at 20 and 100 μmol/L, for one hour at 37 °C, significantly increased the tail moment of lymphocytes. It was noted that no adjustment of pH was performed and that an acidification of the medium could be the origin of the clastogenic activity.

Robichová et al. (2004) found no induction of DNA strand breaks using the Comet assay in human Hep G2 cells in vitro with ascorbic acid at 0.5 mM for one hour.

Duarte et al. (2007), studying the clastogenic potential of ascorbic acid, demonstrated that high concentrations of ascorbic acid (100 to 300 μM) induced DNA strand breakage in a dose-dependent manner in skin human diploid fibroblasts. The genotoxic effect of ascorbic acid was transient, required the formation of extracellular H2O2 and the presence of intracellular iron, but not of extracellular transition metal ions. It was noted that no adjustment of pH was performed and that an acidification of the medium could be the origin of the clastogenic activity.

In a study by Sharma et al. (2011), blood samples were treated with various concentrations (90, 180 and 360 μM) of antioxidants for one hour at 37 °C and analysed with the Comet assay. The results showed that there was a significant decrease in DNA damage in the samples treated with ascorbic acid at the lower concentration (90 μM), whereas there was a significant increase in DNA damage at higher concentrations > 180 μM. Glutathione treatment resulted in decreasing DNA damage at only the highest dose (360 μM). The Panel noted that this study was briefly presented.

Other genotoxicity assays

Lo and Stich (1978) demonstrated that sodium ascorbate at concentration below 1 × 10E–2 M had no detectable inhibitory effect on the ultraviolet-elicited DNA repair synthesis of primary human skin fibroblasts in culture.

Galloway and Painter (1979) found that sodium ascorbate caused, at concentrations from 1 × 10E–4 to 1 × 10E–2 M for 27–29 hours, a dose-dependent increase in sister chromatid exchanges (SCEs) in CHO cells and from 5.4 × 10E–4 to 5.4 × 10E–3 M for 73 hours in human lymphocytes. In the DNA synthesis inhibition test with HeLa cells, ascorbate gave results typical of DNA-damaging chemicals at concentrations of 2 to 20 mM. No effects were seen below 1 mM. Catalase-reduced SCE induction by ascorbate prevented its cytotoxicity in CHO cells and DNA synthesis inhibition in HeLa cells, a result which is in favour of the role of radical oxygen species from H2O2 produced in the oxidation of ascorbate in the culture medium.

Macrae and Stich (1979) demonstrated genotoxic activity with sodium ascorbate in a SCE assay in Chinese hamster cells treated with 1 × 10E−4 to 1 × 10E−2 M sodium ascorbate for two to three hours. They also showed an increase of this effect when using a 24-hour continuous treatment and by the addition of copper(II) ions in the culture medium.

Speit et al. (1980) found that sodium ascorbate in concentrations from 1 × 10E–6 to 1 × 10E–3 M induced SCEs in V79 Chinese hamster cells in vitro. As it was not possible to confirm this effect in vivo with doses up to 10 000 mg/kg bw/day, the authors concluded that the actual mutagenic potential in humans did not appear to be as great as expected from the in vitro study findings. In addition, in this study, it was suggested that the positive effects in vitro were caused by the production of hydrogen peroxide. This mechanism has been confirmed in experiments where the genotoxic effect of ascorbic acid or ascorbates was inhibited by the addition of cysteine or reduced glutathione but not by oxidised glutathione.

Novicki et al. (1985), in an inhibition of DNA synthesis assay in rat hepatocytes, demonstrated no primary DNA damage with 1 × 10E–4 to 1 × 10E–6 mM ascorbic acid with a 48-hour incubation period.

In a study by Weitberg (1987), CHO cells, when exposed to sodium ascorbate at 1 × 10E–3 and 1 × 10E–4M for 10 minutes without metabolic activation, developed increased numbers of SCEs without a dose–response relationship. Superoxide dismutase and catalase caused a significant reduction in the number of SCEs induced by vitamin C, which suggests that free radicals may play a role in the genotoxic effect of high doses (≥ 0.1 M).

Gulati et al. (1989) performed an SCE test in CHO cells at concentrations of 0–1 600 μg L-ascorbic acid/mL with and without metabolic activation ((S9) rat liver fractions). The authors noted that there was a noticeable decrease in pH at doses ≥ 500 μg/mL. A dose-related increase in SCE frequencies was observed following exposure of the cells to 500–1 000 μg/mL. No increase in SCEs was observed with metabolic activation for all tested doses.

Littlefield and Hass (1995) performed a DNA damage assay determining the in vitro formation of DNA double-strand breaks in a cultured human B lymphoblastoid cell line (AHH-1) using a fluorescent dye that interacts with only double-strand DNA in alkaline conditions, the rate of unwinding being directly related to the presence or amount of alkali labile breaks in the DNA. Ascorbic acid at 500 μM did not produce primary DNA damage.

References

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Amacher DE and Paillet SC, 1981. Ascorbate is not detectably mutagenic in the L5178Y TK+/cell mutation assay. Cancer Letters, 14, 151–158.

Anderson D, Basaran N, Blowers SD and Edwards AJ, 1995. The effect of antioxidants on bleomycin treatment inin vitroandin vivogenotoxicity assays.Mutation Research, 329, 37–47.

Bruce R and Heddle JA, 1979. The mutagenic activity of 61 agents as determined by the micronucleus,Salmonella, and sperm abnormality assays.Canadian Journal of Genetics and Cytology, 21, 319–334.

CIR (Cosmetic Ingredient Review), 2005. Final report of the safety assessment of L-ascorbic acid, calcium ascorbate, magnesium ascorbate, magnesium ascorbyl phosphate, sodium ascorbate, and sodium ascorbyl phosphate as used in cosmetics.Report prepared by Elmore AR. International Journal of Toxicology, 24, 51–111.

Duarte TL, Almeida GM and Jones GDD, 2007. Investigation of the role of extracellular H2O2and transition metal ions in the genotoxic action of ascorbic acid in cell culture models.Toxicology Letters, 170, 57–65.

Frenzilli G, Bosco E and Barale R, 2000. Validation of single cell gel assay in human leucocytes with 18 reference compounds.Mutation Research, 468, 93–108.

Galloway SM and Painter RB, 1979. Vitamin C is positive in the DNA synthesis inhibition and sister-chromatic exchange test.Mutation Research, 60, 321–327.

Greggi Antunes LM and Takahashi CS, 1999. Protection and induction of chromosomal damage by vitamin C in human lymphocyte cultures.Teratogenesis, Carcinogenesis and Mutagenesis, 19, 53–59.

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Littlefield NA and Hass BS, 1995. Damage to DNA by cadmium or nickel in the presence of ascorbate.Annals of Clinical & Laboratory Science, 25, 485–492.

Litton Bionetics, 1975. Mutagenicity evaluation of compound FDA71–65 ascorbic acid. Report No 223-74-2104. Submitted by FDA in response to an FOI request in 1999, 46 pp.(as referred to by CIR, 2005).

Litton Bionetics, 1976. Mutagenicity evaluation of sodium ascorbate USP, FCC, FDA 75–64 final report. Report No 223-76-2101. Submitted by FDA in response to an FOI request in 1999.32 pp. (as referred to by CIR, 2005).

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Macrae WD and Stich HF, 1979. Induction of sister chromatid exchanges in Chinese hamster cells by the reducing agents bisulfite and ascorbic acid.Toxicology, 13, 167–174.

Miller B, Pujadas E and Gocke E, 1995.Evaluation of the micronucleus test in vivo using Chinese hamster cells.Environmental and Molecular Mutagenesis, 26, 240–247.

Myhr BC and Caspary WJ, 1991. Chemical mutagenesis at the thymidine kinase locus in L5178Y mouse lymphoma cells: results for 31 coded compounds in the National Toxicology Program.Environmental and Molecular Mutagenesis, 18, 51–83.

Novicki DL, Rosenberg MR and Michalopoulos G, 1985. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes. Cancer Research, 45, 337–344.

Robichová S, Slamenová D, Chalupa I and Lvia Šebová L, 2004. DNA lesions and cytogenetic changes induced byN-nitrosomorpholine in HepG2, V79 and VH10 cells: the protective effects of Vitamins A, C and E. Mutation Research, 560, 91–99.

Rosin MP, San RHC and Stich HF. 1980. Mutagenic activity of ascorbate in mammalian cell cultures.Cancer Letters, 8, 299–305.

Sharma S, Naravaneni R, Bhaumick D and Mehta RD, 2011. Effect of four antioxidant on DNA damage measurement by the Comet assay. Environmental Mutagen Society 42 Annual meeting, Abstract.

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Speit G, Wolf M and Vogel W, 1980. The SCE-inducing capacity of vitamin C: investigationsin vitroandin vivo.Mutation Research,78, 273–278.

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Stich HG, Wei L and Whitting RF, 1980. Chromosome aberrations in mammalian cells exposed to vitamin C and multiple vitamin pills.Food and Cosmetics Toxicology, 18, 497–501.

Tripathy NK, Wurgler FE and Frei H, 1990. Genetic toxicity of 6 carcinogens and 6 noncarcinogens in the drosophila wing spot-test.Mutation Research, 242, 169–180.

Weitberg AB, 1987. Antioxidants inhibit the effect of vitamin C on oxygen radical-induced sister chromatid exchange.Mutation Research, 191, 53–56.

Weitzman SA and Stossel TP, 1982.Effects of oxygen radical scavengers and antioxidants on phagocyte-induced mutagenesis. Journal of Immunology, 128, 2770–2772.

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In vivo gentox data

Micronucleus assay

In the study by Bruce and Heddle (1979), ascorbic acid was investigated for its capability to induce micronuclei in the bone-marrow reticulocytes of C57BL/6xC3H/He female mice. Three animals per group were treated daily by intraperitoneal injection at concentrations close to 0.125, 0.25, 0.5 and 1 of the LD50 (actual doses not mentioned in the report) for five consecutive days and were sacrificed four hours after the last injection. A vehicle control animal group (tap water) was included and negative results were reported. However, the Panel noted that the study bears major shortcomings related to the limited number of animals employed and the number of cells scored (approximately 330 reticulocytes/animal). Furthermore, no positive control was employed. On this basis, this study was considered of limited validity.

A study in mice (six animals/group) examined bone marrow micronuclei after oral administration of ascorbic acid at doses up to 622 mg/kg bw/day for two days (Pienkowska et al., 1985). The animals were sacrificed four hours after the last administration and 2 000 reticulocytes were scored per animal. No statistically significant increase in the number of micronuclei was reported. This study was considered to be mainly consistent with the current OECD Guideline 474 (OECD, 2014), except that the choice for the maximum dose was not justified by the authors.

In the study by Shelby et al. (1993), which aimed to evaluate 49 chemicals (25 carcinogens and 24 non-carcinogens) in a mouse bone marrow micronucleus test, ascorbic acid was assessed for its capability to induce micronuclei in the bone marrow erythrocytes of male B6C3F1 mice. Groups of five mice were administered with the test compound by intraperitoneal injection on three consecutive days and bone marrow smear slides were prepared 24 hours after the last administration. Dose levels of 500, 1 000 and 1 500 mg/kg bw/day selected in a preliminary dose-range finding experiment were used. Groups of solvent and positive control-treated animals were also included. For induction of micronuclei, a minimum number of 2 000 polychromatic erythrocytes (PCEs) per animal were scored in bone marrow smears stained with acridine orange. The results obtained indicate that ascorbic acid induced dose-related increases in the incidence of micronucleated PCEs, which achieved statistical significance at the highest dose-level (1 500 mg/kg bw/day). However, it was noted that acceptability of results based on positive and negative control values and biological relevance of the results obtained, according to historical control range values, were not evaluated by the authors. In addition, it should be noted that the intraperitoneal route of administration employed is not recommended, as it is not a typical relevant route of human exposure, and should be used with specific justification only (OECD Guideline 474; OECD, 2014). On this basis, it was concluded that the positive outcome of this study should be critically considered.

Sister chromatid exchanges assay

Speit et al. (1980) tested SCEs in the bone marrow of Chinese hamsters. Ascorbic acid was administered either orally or by intraperitoneal injection at doses ranging from 200 to 10 000 mg/kg bw/day. Animals were sacrificed 16 hours after the treatment. The SCEs were counted in 50 metaphasesfrom two animals. No induction of SCEs was noted.

Krishna et al. (1986) reported that no SCEs were induced in bone marrow and spleen cells of mice seven hours after a single intraperitoneal administration of ascorbic acid at doses up to 6 680 mg/kg bw/day.

References

Bruce R and Heddle JA, 1979. The mutagenic activity of 61 agents as determined by the micronucleus,Salmonella, and sperm abnormality assays.Canadian Journal of Genetics and Cytology, 21, 319–334.

Krishna G, Nath J and Ong T, 1986. Inhibition of cyclophosphamide and mitocycin C-induced sister chromatid exchanges in mice.Cancer Research, 46, 2670–2674.

Pienkowsha K, Gajcy H and Koziorowska J, 1985. Protective effect of ascorbic acid against mutagenicity of aminopyrine plus nitrite.Polish Journal of Pharmacology and Pharmacy, 37, 601–607.

Speit G, Wolf M and Vogel W, 1980. The SCE-inducing capacity of vitamin C: investigationsin vitroandin vivo.Mutation Research,78, 273–278.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified as mutagenic or clastogenic according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.