OECD SIDS                                                                     L-ASCORBIC ACID

 

 

                  FOREWORD                                           INTRODUCTION

 

 

           L-ASCORBIC ACID

                          CAS N°: 50-81-7

 

 

 

 

SIDS Data Set on L-Ascorbic Acid (Vitamin C)

0. General Information

Name of Sponsor country United Kingdom

Contact point (name, address, telephone and telefax)

Ms M Thomas
Toxic Substances Division
Department of the Environment
Romney House
43 Marsham Street
London
SW1P 3PY

Name of Lead Organisation Roche Products Limited

1. Chemical Identity

1.1 CAS Number: 50-81-7

1.2 Name: L-Ascorbic Acid

1.3 Common Synonyms: Vitamin C

L-threo-2,3,4,5,6-pentahydroxy-1-hexenoic acid-4-lactone

1.4 Empirical Formula: C6H806

1.5 Structural Formula

   OH   OH
    |   |
    C = C
    |   |
0 = C   CH-CHOH-CH2OH
     0

1.6 Purity of Industrial product

1.6.1 Degree of Purity (percentage by weight) > 99.0%

1.6.2 Identity of Major Impurities oxalic acid < 0.2%

1.6.3 Essential Additives (stabilising agents,inhibitors, other additives, if applicable)  None

2. Physical-Chemical Data

2.1 Melting or Decomposition Point

190-192o centigrade with decomposition

Method (e.g. OECD, others): PhEur B.P.

GLP: YES [ ]

NO [v]

Comments: Carried out to B.P./PhEur test requirements in accordance with GMP

Reference: Roche Report No. B 107899 p32 Natural antioxidants exploited commercially

2.2 Boiling Point

Not applicable

2.3 Vapour Pressure

Not applicable

2.4 Partition Coefficient n-Octanol/Water

logP -2.0482

Reference: Non-mutagenic carcinogens are primarily hydrophobic McCoy G D, Rosenkranz H S, Klopman G Carcinogenesis, vol 11(7), pp 1111-7 (1990)

2.5 Water Solubility

33 g/100 ml at 20oC - freely soluble

Reference: Roche Report No. B 107899 p32 Natural antioxidants exploited commercially

2.6 Flash Point (liquids)

Not applicable

2.7 Flammability (solid/gases)

See 2.9

2.8 pH in Water

pH 2.4 - 2.8 (2% aqueous)

pKa1 4.17

pKa2 11.57 (37oC)

Method (e.g. OECD, others):

GLP: YES [ ]

NO [v]

Reference: Roche Report N0. B 107899 pp 32, 35 Natural antioxidants exploited commercially

2.9 Other Data (e.g. relative density, surface tension (of aqueous solution), fat solubility, explosivity, oxidising properties and particle size distribution)

GLP: YES [ ]

NO [v]

Test Results: Minimum ignition energy 25mJ (average particle size 50mm)

Ignition temperature 380oC

KSt 122 bar m/s

pmax 8.6 bar

Dust explosion Class St1

Comments: Dust forms explosible mixtures in air with moderate explosion severity

Reference: Explosion Test Laboratory Report 9.8.88 Ref: 88/054 Dr Pellmont, CH-4102, Binningen, Switzerland

3. Source of Exposure

3.1 Production Levels Expressed as Tonnes per Annum

Information on production levels should be provided in ranges (e.g. 100-1000 tonnes, etc.) per responder or country and the date for which those ranges apply should be given.

Europe >20,000 tonnes per annum 1992

World ca 46,000 tonnes per annum 1992

3.2 Processes

Describe sources of potential human or environmental exposure including workplace concentrations and emission data (in % release), if available, for both manufacturing and user areas.

Exposure during manufacture up to 10mg/m³ (time-weighted average, 8-hour working day) has not resulted in any adverse health effects although normal exposure is significantly below this level.

L-Ascorbic Acid is incorporated into many food and pharmaceutical products at a low level and exposure during handling will be considerably less than that experienced during manufacture of the pure product.

3.3 Information Concerning Uses (including categories and types of uses expressed in percentage terms)

Examples of use categories are dyestuffs, intermediates, solvents, adhesives, building material agents, detergents, cleaning agents, fertilisers, plastic agents, surface treatment agents, etc.

Types of uses are divided into three:

Export: over 95% of UK production is exported

Industrial Use: ca 1%

Public Use: nutrient antioxidant and improving agent

-food 73%

-pharmaceuticals (Vitamin C supplement) 22%

-animal feed 4%

3.4 Options for Disposal

Mode of disposal (e.g. incineration, release to sewage system) for each category and type of use, if appropriate; recycling possibility.

May be disposed to sewer, landfill or by incineration.

4. Environmental Fate and Pathways

Reporting of studies should give the test method, test conditions (laboratory versus field studies) test results (e.g. % degradation in specified time period) and reference. Information on breakdown products (transient and stable) should be provided when available.

4.1 Biodegradability

4.1.1 Aerobic Biodegradability

Test Substance: L-Ascorbic Acid >99.9%

Method: Modified Zahn-Wellens

A mixture of activated sludge was exposed under controlled aerobic conditions to 733mg/l L-Ascorbic Acid (approximately 300mg DOC/l). The degradation of the test substance was monitored during 15 days to determine the DOC values at regular time intervals and expressed as percentage biodegradation at this time.

GLP: YES [v]

NO [ ]

Test Results:

Day No

 

% Degradation

 
 

Test 1

Test 2

Mean

1

48

57

52.5

5

97

97

97

7

98

97

97.5

9

98

99

98.5

12

99

99

99

15

100

100

100

L-Ascorbic Acid shows a biodegradability of 97% after 5 days and 100% after 15 days.

Reference: Inherent Biodegradability of Ascorbic Acid Schlienger C and Gröner J H Roche Report No BS 67889 14.8.91

4.1.2 Sewage Treatment

Information on treatability of the substance.

The above data indicates L-Ascorbic Acid is fully degraded in sewage treatment systems.

4.1.3 Stability in Water (e.g. hydrolysis)

Test Substance: L-Ascorbic Acid >99.9%

Method: Concurrently with the modified Zahn-Wellens test (4.1.1) L-Ascorbic Acid (733mg/l) was dissolved in water without addition of activated sludge and the degradation monitored by determining the DOC values at regular time intervals during 28 days.

GLP: YES [v]

NO [ ]

Test Results: The physico-chemical degradation of L-Ascorbic Acid (without activated sludge) was 11% after 7 days and 50% after 28 days.

An overview of the decomposition pathways (not determined as part of this study) is given in Appendix 1.

Reference: Inherent Biodegradability of Ascorbic Acid Schlienger C. and Gröner J. H. Roche Report No BS 67889 14.8.91

4.1.4 Identification of Main Mode of Degradability in Actual Use

See Section 5.8

4.2 Bioaccumulation

There is no evidence of bioaccumulation in animals or in man. There was no bioaccumulation in guinea pigs fed 86g ascorbic acid/kg diet for 275 days.

Reference: Rivers J. M. Ann. New York Acad. Sci. 1987, 498 pp 445-453

4.3 Transport and Distribution Between Environmental Compartments (including estimated environmental concentrations and distribution pathways)

L-Ascorbic Acid is essential to animal life and is naturally synthesised by most plants. It is therefore present in the majority of living organisms.

4.4 Monitoring Data (environment)

L-Ascorbic Acid levels in plants are typically in the range of 200-500mg/kg and in humans the total body concentration is in the order of 20mg/kg. An adult weighing 70kg will therefore have a body pool of about 1.4g L-Ascorbic Acid.

5. Ecotoxicological Data

5.1 Toxicity to Fish

Test Substance: L-Ascorbic Acid >99.9%

Method: 96 hour acute toxicity in rainbow trout. Rainbow trout, 10per group, were exposed to L-Ascorbic Acid concentration in water of 0, 400, 600, 800, 1,000 and 1,200mg/l for 96 hours at natural pH. In a separate study concentrations of 0, 400, 600, 800 and 1,000mg/l were used and the Ph adjusted to 7.5 ± 0.5 using NaOH.

GLP: YES [v]

NO [ ]

Test Results: In the first experiment at natural pH the LC50 for L-Ascorbic Acid was 1,020mg/l. There were no deaths at 800mg/l and 10/10 at 1,200mg/l. In the second experiment at pH 7.5 ± 0.5 there were no deaths at 1,000mg/l.

The L-Ascorbic Acid may be characterised as very low toxicity to rainbow trout.

Reference: Acute Toxicity of Ascorbic Acid for Fish Schlienger C. and Gröner J. H. Roche Report No BS 67888 14.8.91

5.1.2 Results of Long-Term Tests (e.g. prolonged toxicity, early life-stage)

Although no formal long-term toxicity studies have been performed L-Ascorbic Acid is widely used to supplement the diet of fish reared for food production. The tolerance to L-Ascorbic Acid has been reviewed by the Sub-Committee on Vitamin Tolerance of the US Board on Agriculture National Research Council. The following dietary levels are reported to have no adverse effect in developing hatchlings.

Species

No in

Study

Approximate Weight in g

Concentration in Diet

Duration

Adverse Effect

Reference

Trout

300

1

10g/kg

16 weeks

None

Lann et al 1985

Catfish

60

25

5g/kg

150 days

None

Mayer et al 1978

These dietary concentrations are approximately 100 times the normal requirements for healthy growth and wound healing in trout and catfish which are respectively 100 mg/kg and 60 mg/kg.

Reference: Vitamin Tolerance of Animals

Sub-Committee of Vitamin Tolerance Committee on Animal Nutrition of the US Board on Agriculture National Research Council

National Academy Press 1987

5.2 Toxicity to Daphnids

5.2.1 Results of Acute Tests

Not available

5.2.2 Results of Long-Term Tests (e.g. reproduction)

Dietary intake of L-Ascorbic Acid is essential for normal growth and development and there are no reported adverse effects on reproduction at supplemented dietary levels.

5.3 Toxicity to Algae

L-Ascorbic Acid is naturally present in all aquatic systems.

5.4 Toxicity to Other Aquatic Organisms

L-Ascorbic Acid is naturally present in all aquatic systems.

5.5 Toxicity to Bacteria

L-Ascorbic Acid is naturally present in all biotic systems.

5.6 Toxicity to Terrestrial Organisms

5.6.1 Toxicity to Soil Dwelling Organisms

L-Ascorbic Acid is naturally present in the soil environment.

5.6.2 Toxicity to Plants

L-Ascorbic Acid is naturally synthesised by most plant species. The concentration found in some natural foodstuffs is listed below:

  Approximate Concentration (mg/kg)
Acerola cherries 20,000
Parsley 1,700
Blackcurrants 1,400
Green pepper 1,300
Green kale 1,100
Lemons 450
Oranges 360
Tomatoes 230
Potatoes 160
Mushrooms 50
Apples 40

5.6.3 Toxicity to Birds

No formal toxicity data are available but the following dietary supplements in chickens have been found to have no adverse effect.

Species

No in study

Age

Concentration in Diet

Duration

Adverse Effect

Reference

Chicken

24

30wks

3.3g/kg

>28 days

None

Chen & Nockels 1973

Chicken

200

1 day

3.3g/kg

32 weeks

None

Chen & Nockels 1971

This supplement is in addition to the normal endogenous production of L-Ascorbic Acid in the chicken.

Reference: Vitamin Tolerance of Animals Sub-Committee of Vitamin Tolerance Committee on Animal Nutrition of the US Board on Agriculture National Research Council National Academy Press 1987

5.7 Biological Effects Monitoring (including biomagnification)

Studies on variation of predominant species in certain ecosystems and monitoring of biological effects (e.g. thinning of eggshell etc.) are included.

L-Ascorbic Acid is essential for life for all animals.

5.8 Biotransformation and Kinetics in Environmental Species

Under this item, studies on absorption, distribution, metabolism and excretion etc. should be given.

Metabolism and Excretion

In animals such as guinea pigs and rats there is a considerable conversion of carboxy-group labelled L-Ascorbic Acid to carbon dioxide. In guinea pigs the major product of catabolism of (L-14C) L-Ascorbic Acid is (14C) carbon dioxide which is excreted via the lungs. Urinary and faecal excretion contribute only to a minor part. Of the approximately 8-10% of label recovered in urine, unchanged L-Ascorbic Acid, dehydro-ascorbic acid and 2,3-diketogulonic acid accounted for about 2-3% of the given dose. Most of the excreted label was incorporated in oxalate (7% of the given dose). Other metabolites were isolated and identified in rat and/or guinea pig urine: 2-0-methyl ascorbate, L-Ascorbic Acid 2-sulfate, and saccharo-ascorbic acid, which are excreted in small amounts.

The metabolism of L-Ascorbic Acid in the monkey and man follows a similar pattern. The size of the body pool varies with the level of dietary intake. Half-lives are substantially prolonged when lower amount of L-Ascorbic Acid are ingested. The percentage of unmetabolised L-Ascorbic Acid excreted in urine increases with the dietary intake. Following ingestion of (L-14C) L-Ascorbic Acid two main fractions of the urinary radioactivity were identified as unmetabolised (L-14C) ascorbate and (14C) oxalate. With low ascorbate intake, unmetabolised ascorbate accounted for 10-20% and oxalate for 25-48% of the urinary radioactivity. But with increasing intakes of ascorbate (larger than 10 mg/kg bodyweight) unmetabolised ascorbate (about 75%) was identified as the major fraction; oxalate contributed only 7% suggesting the metabolic conversion of ascorbate to oxalate to be limited. The conversion of ascorbate to 5-keto metabolites suggests that there may be other metabolites of L-Ascorbic Acid of functional importance. As in the monkey, L-Ascorbic Acid is not primarily converted to carbon dioxide in man. Less than 5% of (L-14C) L-Ascorbic Acid was exhaled during a 10-day period following intravenous injection.

Reference: Application for Renewal of the Product Authorizations for REDOXON 'C' Tablets 250mg and 500mg Orange Flavour March 1989, pp 10-11

6. Toxicological Data (oral, dermal and inhalation, as appropriate)

6.1 Acute Toxicity

A summary of acute toxicity test results is given below.

L-Ascorbic Acid, acute toxicity, LD50

All doses in mg/kg bodyweight

Species Mode of Administration
  Orally s.c. i.v. i.p.
Mouse 8,021 5,000 1,058 2,000
Rat >5,000 5,000 1,000  
Guinea-pig >5,000 >1,000 500 2,000
Rabbit >2,000 >1,000 >1,000 >1,000
Cat >1,000 > 500 > 500  
Dog >5,000 > 200 > 200  

Reference: Tolerance and Effects of High Doses of L-Ascorbic Acid Hanck A. Lecture at 3rd International Symposium on Vitamin C San Paulo 1980 (GCR B95327)

6.2 Corrosiveness/Irritation

6.2.1 Skin Irritation

Test Substance: L-Ascorbic Acid Technical Grade >99.0%

Test Species/Strain: Rabbit: New Zealand White

Method (e.g. OECD, others): OECD Guidelines 404 12 May 1981

GLP: YES [ ]

NO [v]

Test Results: give maximum scores after 4 hours. When applied to the intact and abraded skin under semi-occlusive conditions for 4 hours the primary irritation score was 0.00.

Comments: L-Ascorbic Acid (Technical Grade) was found to be non-irritating to the skin

Reference: Roche Research Report B-157452 31.8.90

6.2.2 Eye Irritation

Test Substance: L-Ascorbic Acid Technical Grade >99.0%

Test Species/Strain: Rabbit: New Zealand White

Method (e.g. OECD, others): OECD Guidelines 405 February 1987

GLP: YES [ ]

NO [v]

Test Results: give maximum scores after 72 hours (not rinsed)

0.1 ml of a saturated solution (0.3 g/ml) instilled into the conjunctival sac gave a primary irritation score of 0.33 after 24 hours. A 1:3 dilution of saturated solution in distilled water gave an irritation score of 0.00.

Comment: L-Ascorbic Acid is classified as non-irritating for the rabbit eye.

Reference: Roche Research Report B-157442 16.8.90

6.3 Skin Sensitisation

Not available

6.4 Repeated Dose Toxicity

Test Substance: L-Ascorbic Acid

Test Species/Strain: Rat, Sprague-Dawley, Female

Method (e.g. OECD, others):

Dose Levels: 0, 500, 20,000 mg/kg

No per Group: 20

Duration: 10 months

GLP: YES [ ]

NO [v]

Test Results: L-Ascorbic Acid (20 g/kg) increased urinary oxalate, calcium and iron but had no significant effect on calcification in the kidneys nor did it effect kidney calcium concentration. The rats remained generally healthy and vigorous.

Reference: Effects of High L-Ascorbic Acid and Iron Intake on the Renal Excretion of Oxalate, Calcium and Iron and on the Kidney of Rats Keith, M. O. et al Nutr. Rep. International 1974 Vol.10 p357-369

6.5 Genetic Toxicity

6.5.1 Bacterial Tests

Test Substance: L-Ascorbic Acid

Test Species/Strain: S.typhimurium TA100

Method (e.g. OECD, others): Modified Ames bacterial system using a 15 minute liquid pre-incubation of the organism with the test compound

GLP: YES [ ]

NO [v]

Test Results: L-Ascorbic Acid (3mM) was a weak mutagen when incubation medium was prepared with sterilised tap water.

L-Ascorbic Acid (68mM) was not mutagenic when deionised water was used to prepare the incubation medium.

Minimum concentration of test substance at which toxicity to bacteria was observed:

Tap Water Deionised Water

with metabolic activation - -

without metabolic activation 3mM 68mM

(71% cell survival) (96.8% cell survival)

Concentration of the Test Compound Resulting in Precipitation

Not applicable

Genotoxic Effects

+ ? -
with metabolic activation X
without metabolic activation X

Comments: If tap water is used to prepare the incubation medium L-Ascorbic Acid gives a weak positive response in the Ames test. If the incubation medium is prepared in deionised water the results are negative. The reason for the weak positive result is believed to be the generation of hydrogen peroxide by the reaction of L-Ascorbic Acid with oxygen and metal ions present in the tap water. Hydrogen peroxide is both toxic and mutagenic to the test system.

To show that a similar effect is not produced when L-Ascorbic Acid is given in diet, guinea-pigs were given a feed admix equivalent to 5,000 mg/kg L-Ascorbic Acid and S.typhimurium TA100 was administered by i.v. injection. Bacteria subsequently harvested from the liver showed no mutagenic response.

Reference: Studies on the Mutagenic Activity of L-Ascorbic Acid In-Vitro and In-Vivo Norkus, E.P. et al. Mut. Res. 117 (1983) p183-191

Test Substance: L-Ascorbic Acid

Test Species/Strains: S.typhimurium TA 1535, 1537, 1538, 98 and 100

GLP: YES [ ]

NO [v]

Test Method and Results: L-Ascorbic Acid was one of the reference compounds investigated in a large international collaborative study on short-term tests, the results of which were reported in 1981. Negative results were obtained by 17/20 laboratories, Three/20 laboratories reported positive results. In one case this was against TA100 in the absence of S-9 and consisted only of an increase of 1.7 times backgrounds at the higher dose only, i.e. 5mg/plate, this effect was reproducible. In the second laboratory the positive was reported using TA98 and 500mg/plate in the presence of S-9. In the third case, positive results were obtained using both TA98 and TA100.

The reason for these isolated positive results is not clear but it was the consensus of those collaborating in this validation exercise that the results were not sufficiently consistent nor strong enough to allow L-Ascorbic Acid to be considered a mutagen.

Reference: In Evaluation of Short-Term Tests for Carcinogens Report of the International Collaborative Programme Ed. deSerres, F. J., Ashby, J. Bridges, B. A., Zeiger E., McGregor D. B. Progress in Mut. Research, 1 (1981) p49-67

6.5.2 Non-Bacterial In-Vitro Test

Test Substance: Sodium Ascorbate and L-Ascorbic Acid

Type of Cell Used: Mouse lymphoma L5178Y TK+/-

Method (e.g. OECD, others):

GLP: YES [ ]

NO [v]

Test Results

Lowest Concentration Producing Cell Toxicity:

Sodium Ascorbate L-Ascorbic Acid
with metabolic activation > 2mM -
without metabolic activation 0.5mM 1.5mM

Genotoxic Effects

Sodium Ascorbate

L-Ascorbic Acid

+ ? - + ? -
with metabolic activation X
without metabolic activation X X

Comments: Both sodium ascorbate and L-Ascorbic Acid were tested at millimolar concentrations in the mouse lymphoma L5178Y TK+/- cell for chemically-induced cytotoxicity and the induction of gene mutations at the thymidine kinase locus as detected by increased trifluorothymidine-resistance. Neither chemical caused any dose related increases in trifluorothymidine resistance, even at toxic levels. Increased hydrogen ion concentration was not itself a contributing factor to L-Ascorbic Acid toxicity. Ascorbate toxicity was due to products formed in-vitro in the absence of cells via chemical reactions with medium components. The formation or persistence of these toxic substances could be prevented by co-incubation with catalase prior to the addition of L5178Y cells. These results suggest that L-Ascorbic Acid would not be a mammalian cell mutagen under normal physiological conditions.

Reference: Ascorbate is not detectably mutagenic in the L5178Y TK+/- cell mutation assay Amacher, D.E. et al. Cancer Letters 14 (1981) p151-158

Test Substance: L-Ascorbic Acid

Type of Cell Used: HeLa Cells

Method (e.g. OECD, others): Induction of UDS using liquid scintillation method and hydroxyurea to inhibit S-phase synthesis.

GLP: YES [ ]

NO [v]

Test Results

Compound reported to be positive both in the presence and absence of rat S-9 (phenobarbitone induced). A dose response was noted in both cases.

Reference: Testing of 42 coded compounds for their ability to induce unscheduled DNA repair synthesis in HeLa cells.

In Evaluation of Short-Term Test for Carcinogens

Report of an International Collaborative programme

Martin, C. N., McDermid A. C.

Progress in Mut. Research, 1 (1981) p533-537

Test Substance: L-Ascorbic Acid

Type of Cell Used: CHO

Method (e.g. OECD, others): Induction of SCEs

GLP: YES [ ]

NO [v]

Test Results: A clear dose-related positive was obtained in the absence of S-9 (24-hour treatment) at 10-300mg/ml) (9, 19.9 and 24.2 SCEs per cell). Effects were less marked in the presence of S-9 but were still considered positive (8.8, 9.7, 11.1 and 9.8 SCEs cell at 0.1, 1, 10 and 100mg/ml).

Positive both in presence and absence of S-9.

Reference: Evaluation of the SCE method in mammalian cells as a screening system for carcinogens. In Evaluation of Short-Term Test fo Report of an International Collaborative programmer Carcinogens Perry, P. E., Thomsen, E. J. Progress in Mut. Research, 1 (1981) p560-569

Test Substance: Sodium ascorbate

Type of Cell Used: CHO and human lymphocytes

Method (e.g. OECD, others): Induction of SCEs

GLP: YES [ ]

NO [v]

Exogenous metabolic activation - none.

Test Results: Sodium ascorbate produced a clear dose-related increase in CHO cells over the range 0.1 - 10mM with effects being seen at all concentrations (9.8 ± 0.5 SCEs - >28.7 ± 1.8 SCEs per cell at 0.1 - >10mM respectively compared to 8.1 ± 0.5 in the controls).

Similarly a dose-related increase in SCEs was seen in human lymphocytes investigated over the concentration range 0.54mM - 5.4mM (10.2 ± 0.9 - 18.1 ± 0.9 SCEs per cell compared to 9.2 ± 0.6 in the controls).

Activity in CHO cells was markedly reduced by the addition of catalase. These results suggest that the SCE induction by sodium ascorbate in the absence of an exogenous metabolic activation system was due to hydrogen peroxide production via the oxidation of high concentrations of ascorbic acid, and consequent DNA damage by reactive oxygen species.

Reference: Vitamin C is positive in the DNA synthesis inhibition and SCE tests. Galloway, S. M., Painter, R. B. Mut. Research, 60 (1979) p321-327

6.5.3 Non-Bacterial Test In-Vivo

Test Substance: L-Ascorbic Acid

Test Species/Strain: Chinese Hamster

Method (e.g. OECD, others): In-vivo sister-chromatid exchange (SCE)

GLP: YES [ ]

NO [v]

Test Results: L-Ascorbic Acid dosed at 200 to 10,000 mg/kg p.o. and i.p. had no effect on SCE rate or cell proliferation in the bone marrow.

Lowest Dose Producing Toxicity: -

Effect on Mitotic Index or P/N Ratio: -

Genotoxic Effects:

+ ? -
    X

Comments: In contrast with cell cultures it appears that the mammalian organism is protected against damage by peroxide radicals or that such radicals are not formed by L-Ascorbic Acid in body fluid.

Reference: The SCE-Inducing Capacity of Vitamin C: Investigations In-Vitro and In-Vivo Speit, G.M. Mutation Research 78 p273-278

Test Substance: L-Ascorbic Acid

Test Species/Strain: CD - Mice

Method (e.g. OECD, others): Micronucleus test. Groups of 4 mice (2 male; 2 females) given daily doses of 80, 160 or 320mg/kg ip and examined 6-hours later for micronuclei induction (top dose 50% LD50).

GLP: YES [ ]

NO [v]

Test Results

No evidence for any increase in micronuclei at any dose level.

Reference: Activity of coded compounds in the micronucleus test.

In Evaluation of Short-Term Test for Carcinogens

Report of an International Collaborative programme

Tsuchimoto, T., Matter, B. E.

Progress in Mut. Research, 1 (1981) p705-711

Test Substance: L-Ascorbic Acid

Test Species/Strain: B6C3F1 Mice

Method (e.g. OECD, others): Micronucleus test. In first experiment groups of 4-10 mice given doses of 80mg/kg ascorbic acid 24-hours apart and bone marrow cells harvested at 24, 48 and 72 hours after the last dose.

In a second experiment groups of mice given single doses of 40 and 80mg/kg and bone marrow cells harvested at 36, 48 and 60 hours post-dose.

GLP: YES [ ]

NO [v]

Test Results: No increase in micronuclei at any harvest time in the first study. (Incidence 0.08, 0.22 and 0.12% compared to 0.13% in the historic controls.)

A slight increase was seen in the second study at 80mg/kg with 0.24, 0.27 and 0.9% cells containing micronuclei at each time interval compared to 0.13% in the historic control. Result was equivocal. Overall L-Ascorbic Acid was negative in this study.

Reference: Mutagenic activity of 41 compounds in the in vivo micronucleus assay.

In Evaluation of Short-Term Test for Carcinogens

Report of an International Collaborative Programme

Ed. deSerres, F., Ashby, J., Salamone, M. F., Heddle, J. A., Katz, M.

Progress in Mut. Research, 1 (1981) p686-697

Overall Assessment of Mutagenicity of L-Ascorbic Acid

The weight of evidence indicates that L-Ascorbic Acid does not produce gene mutation in Salmonella typhimurium nor in mammalian cells using the mouse lymphoma assay. Positive results were consistently obtained in in vitro assays for SCE and UDS induction, indicating the ability to produce DNA damage under certain conditions. However, activity was seen in CHO cells in the absence of an exogenous metabolic activation system and was markedly inhibited by catalase. It was likely to have been due to formation of hydrogen peroxide and reactive oxygen species by the oxidation of high concentrations of L-Ascorbic Acid and would not be expected to be expressed in vivo under normal conditions. L-Ascorbic Acid has no structural alerts with regard to alkalylation. Negative results were obtained in in vivo assays, using high dose levels, for the induction of micronuclei and SCEs in bone marrow.

Thus the in vivo data although limited to one tissue (bone marrow) were consistently negative. Although activity was seen in certain in vitro assays for genotoxicity (UDS and SCEs but not gene mutation), the mechanism involved would suggest that activity was unlikely to be seen in vivo. It can be concluded that L-Ascorbic Acid is not an in vivo mutagen.

6.6 Carcinogenicity

Test Substance: L-Ascorbic Acid

Test Species/Strain: F344/N Rats

B6C3F1 Mice

Method (e.g. OECD, others): Groups of 50 male and 50 female mice and rats received 0, 2.5% and 5.0% dietary concentrations of L-Ascorbic Acid for 103 weeks. Complete histopathological examination was carried out on all animals.

GLP: YES [ ]

NO [v]

Test Results: L-Ascorbic Acid dosed at 2.5% or 5.0% dietary concentrations for 103 weeks was neither toxic nor carcinogenic for male and female F344/N rats or male and female B6C3F1 mice.

No compound-related clinical signs other than slight lowering of mean bodyweight in females were observed in mice and rats and survival was good (70-82% at 103 weeks).

Reference: No Evidence of Carcinogenicity for L-Ascorbic Acid in Rodents Douglas, J.F. et al. J.Toxicol. Environ. Health 1984 14(4) p605-9

6.7 Reproductive and Development Toxicity

6.7.1 Reproductive Toxicity

6.7.2 Teratogenicity/Developmental Toxicity

Test Substance: L-Ascorbic Acid

Test Species/Strain: (a) Rat dose levels 150, 250, 500, 1,000mg/kg

(b) Rat dose levels 150, 250, 500, 1,000mg/kg

(c) Mouse dose levels 250, 500, 1,000mg/kg

Method (e.g. OECD, others): 19-27 animals per group were dosed orally as indicated above for the following periods during pregnancy:

(a) day 6 to day 15

(b) day 0 precoitus to day 21 post-partum

(c) day 6 to day 15

GLP: YES [ ]

NO [v]

Test Results: The high doses of L-Ascorbic Acid showed no adult toxic of fetotoxic effects and was not teratogenic. It had no influence on the embryonic, foetal and post-partum development of the young animals and no effect on the pregnancy, parturition, breeding instinct and lactation capacity in the mother animals.

Reference: Reproduction Toxicology Studies with Ascorbic Acid in Mice and Rats Frohberg, V. H., Gleich, J., Kieser, H. Arzneimforsch 23 (1973) p1081-1082

6.8 Specific Toxicities (Neurotoxicity, Immunotoxicity, etc.)

A review of possible side-effects of large oral intakes of L-Ascorbic Acid in man concludes that ingestion of up to 10g/day does not constitute a health risk.

Reference: Vitamin C

Ed. Counsell, J. N., Hornig, D. H.

The Safety of High Vitamin C Intakes in Man

Hornig, D. H., Moser, U.

Applied Science Publishers (1981) p225-248

6.9 Toxicodynamics, Toxicokinetics

Reference should be made to human data.

7. Experience with Human Exposure (give full description of study design, effects of accidental or occupational exposure, epidemiology)

Clinical experience with L-Ascorbic Acid is widely reported and reviews in the following standard publications are attached as Appendices.

Appendix 3 Martindale - The Extra Pharmacopoeia

29th Edition 1989

pp 1254-1256

Appendix 4 The Pharmacological Basis for Therapeutics

Goodman and Gilman

7th Edition

pp 1567-1572

An extract from a ROCHE Vitamin Information Status paper is also attached:

Appendix 5 Vitamin Safety

Marks, J.

7.1 Biological Monitoring (including clinical studies, case reports, etc.)

See Appendices 3 and 4.

8. Recommended Precautions, Classification (use and/or transportation) and Safety Data Sheets

L-Ascorbic Acid is not classified as hazardous for use (EEC) or dangerous for transport (UN). A Safety Data Sheet for L-Ascorbic Acid is attached (Appendix 2).

9. Availability and Reference(s) for Existing Review(s)

Copies of all references are available for review.

10. Name of Responder

Mr J S Maple

Roche Products Limited

P O Box No 8

Welwyn Garden City

Hertfordshire

AL7 3AY

Telephone: (0707) 328128 Ext. 2215

Facsimile: (0707) 338297

Appendix 6

Safety Information Data Set on L-Ascorbic Acid

L-Ascorbic Acid (Vitamin C) is a naturally occurring chemical essential for the life and well-being of man and animals. It is synthesised in most plants and by many animals but not by man, primates, invertebrates, fish and certain species such as the fruit bat and guinea pig. Species which do not naturally synthesise Vitamin C must obtain it from diet.

Vitamin C acts as a redox (oxidation-reduction) agent and participates in the following essential functions:

1. Hydrolysis of certain amino acids required for protein metabolism.

2. Formation of collagen essential for body growth, tissue repair and wound healing.

3. Synthesis of the hormone adrenaline and the hydroxylation of anti-inflammatory steroids in the adrenal gland.

4. Iron and copper metabolism.

5. Folic acid metabolism.

It has very low toxicity and although the minimum dietary requirement in man is generally thought to be 40-100mg per day an intake up to 100 times this quantity has no harmful effect.

Its presence is distributed uniformly throughout the environment, the concentration in different plant species being typically in the range of 200-500mg/kg and as high as 20,000mg/kg in acerola cherries. Although grass is not the normally recognised dietary source of Vitamin C the following calculation illustrates the degree to which Vitamin C is present in the environment.

Typical concentration of Vitamin C in grass: 500mg/kg (0.5kg/tonne)

Area of agricultural grass in the UK: 11.9 mio hectares

Typical annual yield of grass per hectare: 15 tonnes

Typical annual grass production in the UK: 178,500,000 tonnes

Quantity of Vitamin C in agricultural grass production per annum in the UK: 89,250 tonnes

For most cultivated crops and for much natural vegetation a similar calculation could be made to confirm the degree to which Vitamin C is naturally present in the environment. Throughout the world the presence of naturally occurring Vitamin C in the environment is many orders of magnitude above the manufacturing capacity for this chemical. The impact of the manufactured product on the environment, irrespective of its method of use is therefore considered to be negligible.