2. Specifications and tests for sodium nitrate and potassium nitrate used as preservatives are specified in QCVN 4-5 :2010/BYT promulgated together with the Circular No. 22/2010/TT-BYT dated May 20, 2010 of the Minister of Health.

3. Specifications specified in this Regulation are tested under JECFA monograph 1 - Vol. 4, except for some specific tests described in the annexes. The tests provided in this Regulation are optional. Other equivalent tests may be used.

4. Sampling adheres to the guidelines in the Circular No. 16/2009/TT-BKHCN dated June 02, 2009 by the Ministry of Science and Technology and relevant regulations of law.

III. REGULATORY REQUIREMENTS

1. Declaration of conformity

1.1. Conformity of preservatives shall be declared in accordance with the regulations set out in this Regulation.

1.2. Methods and procedures for declaration of conformity shall comply with the Regulation on certification and declaration of conformity with regulations and standards under the Decision No. 24/2007/QĐ-BKHCN dated September 28, 2007 of the Minister of Science and Technology and regulations of laws.

2. Inspection of preservatives

The quality, hygiene and safety of flavour enhancers must be inspected in accordance with the regulations of law.

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1. Entities must declare conformity according to the specifications mentioned in this Regulation, register their declarations of conformity at the Vietnam Food Administration and ensure the quality, hygiene and safety as declared.

2. Entities are only entitled to import, export, produce, sell and use flavour enhancers as declared after their completion of registration of declarations of conformity and their compliance with regulations of law on quality, hygiene, safety and labeling.

V. IMPLEMENTATION

1. The Vietnam Food Administration shall preside over and cooperate with competent authorities concerned to provide guidance on and organize the implementation of this Regulation.

2. The Vietnam Food Administration shall, according to its managerial duties, suggest amendments to this Regulation to the Ministry of Health.

3. In the cases where any of the international guidelines for tests and regulations of law referred to in this Regulation is amended or replaced, the newest one shall apply.

ANNEX 1

SPECIFICATIONS AND TESTS FOR ACID SORBIC

1. Synonyms

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INS 200

ADI = 0 – 25 mg/kg bw

2. Definition

Chemical names

Acid sorbic; acid 2,4-hexadienoic; acid 2-propenylacrylic

C.A.S. number

110-44-1

Chemical formula

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...

Structural formula

Formula weight

112.12

3. Description

Colourless needles or white free flowing powder, having a slight characteristic odour.

4. Functional uses

Antimicrobial preservative, fungistatic agent

5. Specifications

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Solubility

Slightly soluble in water, soluble in ethanol.

Melting range

Between 132 and 135^oC (the melting apparatus should be preheated to 125º before introducing the sample).

Spectrophotometry

A 1 in 400,000 solution in isopropanol solution shows absorbance maximum at 254±2 nm

Test for double bond

Passes test.

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Water

Not more than 0.5%.

Sulfated ash

Not more than 0.2%.

Aldehydes

Not more than 0.1% (as formaldehyde).

Lead

Not more than 2 mg/kg.

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Not less than 99.0% calculated on the anhydrous basis.

6. Tests

6.1. Identification

Test for double bond

Shake about 0.02 g of the sample with 1 ml bromine TS; the colour disappears

6.2. Purity

Water

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 - Tested according to JECFA monograph 1 - Vol.4.

Sulfated ash

- Test 2 g of the sample.

- Tested according to method I, JECFA monograph 1 - Vol.4.

Aldehydes

To 1 ml of a saturated aqueous solution of the sample, add 0.5 ml of Schiff's reagent TS and allow to stand for 10-15 min. Compare the colour with that produced by 1 ml of formaldehyde solution (containing 2 µg) with the same amount of Schiff's reagent under the same conditions. The colour of the test solution should not be more intense than that of the formaldehyde solution.

Lead

- Tested according to JECFA monograph 1 - Vol.4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol.4, “Instrumental Methods.”

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Dissolve about 0.25 g of the sample, accurately weighed, in 50 ml of anhydrous methanol previously neutralized with 0.1 N sodium hydroxide, add phenolphthalein TS, and titrate with 0.1 N sodium hydroxide to the first pink colour which persists for at least 30 sec.

Each ml of 0.1 N sodium hydroxide is equivalent to 11.21 mg of C6H8O2.

ANNEX 2

SPECIFICATIONS AND TESTS FOR POTASSIUM SORBATE

1. Synonyms

 Potassium sorbate

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ADI = 0 – 25 mg/kg bw

2. Definition

Chemical names

Potassium sorbate, potassium salt of trans, trans-2,4-hexadienoic acid

C.A.S. number

24634-16-5

Chemical formula

C6H7KO2

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Formula weight

150.22

3. Description

White or yellowish-white crystals or crystalline powder or granules.

4. Functional uses

Preservative

5. Specifications

5.1. Identification

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Solubility

Freely soluble in water; soluble in ethanol.

Test for potassium

Passes test.

Melting range of sorbic acid derived from the sample

132-135^oC.

Test for unsaturation

Passes test.

5.2. Purity

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Loss on drying

Not more than 1%.

Acidity or alkalinity

Not more than about 1% (as sorbic acid or potassium carbonate).

Aldehydes

Not more than 0.1% (as formaldehyde).

Lead

Not more than 2 mg/kg.

5.3. C6H7KO2 content

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6. Tests

6.1. Identification

Test for potassium

Tested according to JECFA monograph 1 - Vol.4.

Melting range of sorbic acid derived from the sample

Acidify a solution of the sample with dilute hydrochloric acid TS. Collect the precipitated sorbic acid on a filter paper, wash free of chloride with water and dry under vacuum over sulfuric acid.

Test for unsaturation

To 2 ml of a 1 in 10 solution of the sample, add a few drops of bromine TS. The colour of the bromine disappears.

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Loss on drying

Tested according to JECFA monograph 1 - Vol. 4. (105º, 3 h).

Acidity or alkalinity

Dissolve 1.1 g of the sample in 20 ml of water and add 3 drops of phenolphthalein TS. If the solution is colourless, titrate with 0.1 N sodium hydroxide to a pink colour that persists for 15 sec. Not more than 1.1 ml should be required.

If the solution is pink in colour titrate with 0.1 N hydrochloric acid. Not more than 0.8 ml should be required to discharge the pink colour.

Aldehydes

Prepare a 0.3% solution of the sample, adjust the pH to 4 with 1N HCl and filter. To 5 ml of the filtrate add 2.5 ml of Schiff's reagent TS and allow to stand for 10 - 15 min. Compare the colour with that produced by 5 ml of a control solution containing 15 µg of formaldehyde instead of the sample. The colour of the test solution should not be more intense than that of the control solution.

Lead

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- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol.4, “Instrumental Methods.”

6.3. Method of essay

Weigh, to the nearest 0.1 mg, 0.25 g of the sample, previously dried at 105º for 3 h. Dissolve in 36 ml of glacial acetic acid and 4 ml acetic anhydride in a 250-ml glass-stoppered flask, warming to effect solution. Cool to room temperature, add 2 drops of crystal violet TS and titrate with 0.1 N perchloric acid in glacial acetic acid to a blue-green end point which persists for at least 30 sec. Perform a blank determination and make any necessary correction.

Each ml of 0.1 N perchloric acid is equivalent to 15.02 mg of C6H7KO2.

ANNEX 3

SPECIFICATIONS AND TESTS FOR CALCIUM SORBATE

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 Calcium sorbate

INS 203

ADI = 0 - 25 mg/kg bw

2. Definition

Chemical names

Calcium sorbate; calcium salt of trans, trans-2,4-hexadienoic acid.

C.A.S. number

7492-55-9

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C12H14CaO4

Structural formula

Formula weight

262.32

3. Description

Fine white crystalline powder not showing any change in colour after heating at 105^oC for 90 min.

4. Functional uses

Preservative

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5.1. Identification

Solubility

Soluble in water; practically insoluble in ethanol.

Test for calcium

Passes test.

Melting range of sorbic acid derived from the sample

132-135oC.

Test for unsaturation

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5.2. Purity

Loss on drying

Not more than 3%.

Fluoride

Not more than 10 mg/kg.

Aldehydes

Not more than 0.1% (as formaldehyde).

Lead

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5.3. C12H14CaO4 content

Not less than 98% and not more than 102% at the dried basis.

6. Tests

6.1. Identification

Test for calcium

Tested according to JECFA monograph 1 - Vol.4.

Melting range of sorbic acid derived from the sample

Acidify a solution of the sample with dilute hydrochloric acid TS. Collect the precipitated sorbic acid on a filter paper, wash free of chloride with water and dry under vacuum over sulfuric acid.

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To 2 ml of a 1 in 10 solution of the sample, add a few drops of bromine TS. The colour of the bromine disappears.

6.2. Purity

Loss on drying

Tested according to JECFA monograph 1 - Vol. 4. (over sulfuric acid in vacuum, 4h).

Fluoride

Tested according to method I, JECFA monograph 1 - Vol.4 (weigh 5 g of the sample to the nearest mg and proceed as directed in the Fluoride Limit Test (Method I or III).

Aldehydes

Prepare a 0.3% solution of the sample, adjust the pH to 4 with 1N HCl and filter. To 5 ml of the filtrate add 2.5 ml of Schiff's reagent TS and allow to stand for 10 - 15 min. Compare the colour with that produced by 5 ml of a control solution containing 15 µg of formaldehyde instead of the sample. The colour of the test solution should not be more intense than that of the control solution.

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- Tested according to JECFA monograph 1 - Vol.4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol.4, “Instrumental Methods.”

6.3. Method of essay

Weigh to the nearest mg, 0.25 g of the dried sample. Dissolve in 35 ml of glacial acetic acid and 4 ml of acetic anhydride in a 250-ml glass-stoppered flask, warming to effect solution. Cool to room temperature, add 2 drops of crystal violet TS and titrate with 0.1 N perchloric acid in glacial acetic acid to a blue-green end point which persists for at least 30 sec. Perform a blank determination and make any necessary correction.

Each ml of 0.1 N perchloric acid is equivalent to 13.12 mg of C12H14CaO4.

ANNEX 4

SPECIFICATIONS AND TESTS FOR BENZOIC ACID

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Benzoic acid

INS 210

ADI = 0 – 5 mg/kg bw

2. Definition

Chemical names

Benzoic acid, benzenecarboxylic acid, phenylcarboxylic acid

C.A.S. number

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Chemical formula

C7H6O2

Structural formula

Formula weight

122.12

3. Description

White crystalline solid, usually in the form of scales or needles, having not more than a faint characteristic odour.

4. Functional uses

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5. Specifications

5.1. Identification

Solubility

Slightly soluble in water, freely soluble in ethanol

Melting range

121^oC -123^oC

Test for benzoate

 Passes test.

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About 4.0 (solution in water)

5.2. Purity

Loss on drying

Not more than 0.5%.

Sublimation test

Passes test (described in “Tests”).

Sulfated ash

Not more than 0.05%.

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Not more than 2 mg/kg.

Readily carbonizable substances

Passes test (described in “Tests”).

Chlorinated organic compounds

Not more than 0.07% (as Cl2).

Readily oxidizable substances

Passes test (described in “Tests”).

5.3. C7H6O2 content

Not less than 99.5% on the dried basis.

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6.1. Identification

Test for benzoate

- Tested according to JECFA monograph 1 - Vol.4.

 - Use 0.1 g of the sample with 0.1 g of calcium carbonate and 5 ml of water.

6.2. Purity

Loss on drying

- Tested according to JECFA monograph 1 - Vol.4.

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Sublimation test

Place a small amount of the sample in a dry test tube. Wrap the test tube about 4 cm from the bottom with moistened filter paper. Heat the test tube over a low flame. Benzoic acid sublimes and crystals deposit in the colder part of the test tube leaving no residue at the bottom.

Lead

- Tested according to JECFA monograph 1 - Vol.4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

Readily carbonizable substances

- Tested according to JECFA monograph 1 - Vol.4.

- Dissolve 0.5 g of the sample, weighed to the nearest mg, in 5 ml of sulfuric acid TS. The colour produced should not be darker than a light pink (Matching Fluid Q).

Chlorinated organic compounds

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Readily oxidizable substances

Add 1.5 ml of sulfuric acid to 100 ml of water, heat to boiling and add 0.1N potassium permanganate in drops, until the pink colour persists for 30 sec. Dissolve 1 g of the sample, weighed to the nearest mg, in the heated solution, and titrate with 0.1N potassium permanganate to a pink colour that persists for 15 sec. Not more than 0.5 ml should be required.

6.3. Method of essay

Weigh, to the nearest mg, 2.5 g of the dried sample. Dissolve in 15 ml of warm ethanol previously neutralized using phenol red TS as indicator. Add 20 ml of water and titrate with 0.5N sodium hydroxide, using phenolphthalein TS as indicator.

Each ml 0.5N sodium hydroxide is equivalent to 61.06 mg of C7H6O2.

ANNEX 5

SPECIFICATIONS AND TESTS FOR SODIUM BENZOATE

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Sodium benzoate;

INS 211

ADI = 0 - 5 mg/kg bw

2. Definition

Chemical names

Benzoic acid, benzenecarboxylic acid, phenylcarboxylic acid

C.A.S. number

532-32-1

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C7H5NaO2

Structural formula

Formula weight

144.11

3. Description

White, almost odourless, crystalline powder, flakes or granules

4. Functional uses

Antimicrobial preservative

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5.1. Identification

Solubility

Freely soluble in water, sparingly soluble in ethanol.

Test for sodium

Passes test.

Test for benzoate

Passes test.

5.2. Purity

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Loss on drying

Not more than 1.5%.

Acidity or alkalinity

Passes test (described in “Tests”).

Readily carbonizable substances

Passes test (described in “Tests”).

Chlorinated organic compounds

Not more than 0.07% (as Cl2).

Readily oxidizable substances

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Lead

Not more than 2 mg/kg.

5.3. C7H5NaO2 content

Not less than 99.0% calculated on the anhydrous basis.

6. Tests

6.1. Identification

Test for sodium

- Tested according to JECFA monograph 1 - Vol.4.

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- Tested according to JECFA monograph 1 - Vol.4.

- Use a 10% solution of the sample

6.2. Purity

Loss on drying

- Tested according to JECFA monograph 1 - Vol. 4.

- 105^oC, 4 h.

Acidity or alkalinity

Dissolve 2 g of the sample, weighed to the nearest mg, in 20 ml of freshly boiled water. Not more than 0.5 ml of either 0.1N sodium hydroxide or 0.1N hydrochloric acid should be required for neutralization, using phenolphthalein TS as indicator.

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Dissolve 0.5 g of the sample, weighed to the nearest mg, in 5 ml of sulfuric acid TS. The colour produced should not be darker than a light pink ("Matching Fluid Q") (Tested according to JECFA monograph 1 - Vol. 4).

Chlorinated organic compounds

- Tested according to JECFA monograph 1 - Vol.4.

- Test 0.25 g of the sample using 0.5 ml of 0.01N hydrochloric acid in the control.

Readily oxidizable substances

Add 1.5 ml of sulfuric acid to 100 ml of water, heat to boiling and add 0.1N potassium permanganate, dropwise, until the pink colour persists for 30 sec. Dissolve 1 g of the sample, weighed to the nearest mg, in the heated solution, and titrate with 0.1N potassium permanganate to a pink colour that persists for 15 sec. Not more than 0.5 ml should be required.

Lead

- Tested according to JECFA monograph 1 - Vol.4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

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Weigh, to the nearest mg, 3 g of the sample previously dried for 4 h at 105^oC and transfer to a 250-ml Erlenmeyer flask. Add 50 ml of water to dissolve the sample. Neutralize the solution, if necessary, with 0.1N hydrochloric acid, using phenolphthalein TS as indicator. Add 50 ml of ether and a few drops of bromophenol blue TS, and titrate with 0.5N hydrochloric acid, shaking the flask constantly, until the colour of the indicator begins to change. Transfer the lower aqueous layer to another flask. Wash the ethereal layer with 10 ml of water, and add the washing and an additional 20 ml of ether to the separated aqueous layer. Complete the titration with the 0.5N hydrochloric acid, shaking constantly the flask.

Each ml of 0.5N hydrochloric acid is equivalent to 72.05 mg of C7H5NaO2.

ANNEX 6

SPECIFICATIONS AND TESTS FOR POTASIUM BENZOATE

1. Synonyms

Potasium benzoate;

INS 212

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2. Definition

Chemical names

Potassium benzoate, potassium salt of benzenecarboxylic acid, potassium salt of phenylcarboxylic acid

C.A.S. number

582-25-2 (anhydrous)

Chemical formula

C7H5KO2.3H2O

Structural formula

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Formula weight

160.22 (anhydrous C7H5KO2)

214.27 (trihydrate C7H5KO2.3H2O)

3. Description

White crystalline powder

4. Functional uses

Antimicrobial preservative

5. Specifications

5.1. Identification

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Solubility

Freely soluble in water, soluble in ethanol.

Test for potassium

Passes test.

Test for benzoate

Passes test.

5.2. Purity

Loss on drying

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Acidity or alkalinity

Passes test (described in “Tests”).

Readily carbonizable substances

Passes test (described in “Tests”).

Chlorinated organic compounds

Not more than 0.07% (as Cl2).

Readily oxidizable substances

Passes test (described in “Tests”).

Lead

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5.3. C7H5KO2 content

Not less than 99.0% calculated on the anhydrous basis.

6. Tests

6.1. Identification

Test for potassium

- Tested according to JECFA monograph 1 - Vol. 4.

- Use a 10% solution of the sample.

Test for benzoate

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- Use a 10% solution of the sample.

6.2. Purity

Loss on drying

- Tested according to JECFA monograph 1 - Vol. 4.

- 105^oC, 4 h.

Acidity or alkalinity

Dissolve 2 g of the sample, weighed to the nearest mg, in 20 ml of freshly boiled water. Not more than 0.5 ml of either 0.1N sodium hydroxide or 0.1N hydrochloric acid should be required for neutralization, using phenolphthalein TS as indicator.

Readily carbonizable substances

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- Dissolve 0.5 g of the sample, weighed to the nearest mg, in 5 ml of sulfuric acid TS. The colour produced should not be darker than a light pink (Matching Fluid Q).

Chlorinated organic compounds

- Tested according to JECFA monograph 1 - Vol. 4.

- Test 0.25 g of the sample using 0.5 ml of 0.01N hydrochloric acid in the control.

Readily oxidizable substances

Add 1.5 ml of sulfuric acid to 100 ml of water, heat to boiling and add 0.1N potassium permanganate, dropwise, until the pink colour persists for 30 sec. Dissolve 1 g of the sample, weighed to the nearest mg, in the heated solution, and titrate with 0.1N potassium permanganate to a pink colour that persists for 15 sec. Not more than 0.5 ml should be required.

Lead

- Tested according to JECFA monograph 1 - Vol. 4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

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Weigh to the nearest 0.1 mg, 2.5 to 3 g of the dried sample, and transfer to a 250-ml Erlenmeyer flask. Add 50 ml of water to dissolve the sample. Neutralize the solution, if necessary, with 0.1N hydrochloric acid, using phenolphthalein TS as indicator. Add 50 ml of ether and a few drops of bromophenol blue TS, and titrate with 0.5N hydrochloric acid, shaking the flask constantly, until the colour of the indicator begins to change. Transfer the lower aqueous layer to another flask. Wash the ethereal layer with 10 ml of water, and add the washing and an additional 20 ml of ether to the separated aqueous layer. Complete the titration with the 0.5N hydrochloric acid, shaking constantly the flask.

Each ml of 0.5N hydrochloric acid is equivalent to 80.11 mg of C7H5KO2.

ANNEX 7

SPECIFICATIONS AND TESTS FOR CALCIUM BENZOATE

1. Synonyms

Monocalcium benzoate;

INS 213

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2. Definition

Chemical names

Calcium benzoate

C.A.S. number

2090-05-3

Chemical formula

Anhydrous: C14H10CaO4

Monohydrate: C14H10CaO4.H2O

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Structural formula

Anhydrous

Formula weight

Anhydrous: 282.31

Monohydrate: 300.32

Trihydrate: 336.36

3. Description

White or colourless crystals, or white powder.

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Antimicrobial preservative

5. Specifications

5.1. Identification

Solubility

Sparingly soluble in water

Test for calcium

Passes test.

Test for benzoate

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5.2. Purity

Loss on drying

Not more than 17.5%.

Water insoluble matter

Not more than 0.3%.

Acidity or alkalinity

Passes test (described in “Tests”).

Fluoride

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Lead

Not more than 2 mg/kg.

Chlorinated organic compounds

Not more than 0.07% (as Cl2).

Readily oxidizable substances

Passes test (described in “Tests”).

5.3. C14H10CaO4 content

Not less than 99.0% calculated on the anhydrous basis.

6. Tests

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Test for calcium

Tested according to JECFA monograph 1 - Vol.4.

Test for benzoate

Tested according to JECFA monograph 1 - Vol. 4.

6.2. Purity

Loss on drying

- Tested according to JECFA monograph 1 - Vol. 4.

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Water insoluble matter

Dissolve 10 g of the sample, weighed to the nearest mg, in 100 ml of hot water. Filter through a Gooch crucible, tared to an accuracy of ±0.2 mg, and wash any residue with hot water. Dry the crucible for 2 h at 105ºC. Cool, weigh and calculate as percentage.

Acidity or alkalinity

Dissolve 2 g of the sample, weighed to the nearest mg, in 20 ml of freshly boiled water. Not more than 0.5 ml of either 0.1N sodium hydroxide or 0.1N hydrochloric acid should be required for neutralization, using phenolphthalein TS as indicator.

Fluoride

- Tested according to JECFA monograph 1 - Vol. 4,  Method I or III.

- Weigh 5 g of the sample to the nearest mg.

Lead

- Tested according to JECFA monograph 1 - Vol. 4.

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Chlorinated organic compounds

Test 0.25 g of the sample using 0.5 ml of 0.01N hydrochloric acid in the control.

Readily oxidizable substances

Add 1.5 ml of sulfuric acid to 100 ml of water, heat to boiling and add 0.1N potassium permanganate in drops, until the pink colour persists for 15 sec. Dissolve 1 g of the sample, weighed to the nearest mg, in the heated solution, and titrate with 0.1N potassium permanganate to a pink colour that persists for 15 sec. Not more than 0.5 ml should be required.

6.3. Method of essay

Weigh accurately 0.6 g of the dried sample, dissolve in a mixture of 20 ml of water and 2 ml of dilute hydrochloric acid TS, and dilute to 100 ml with water. While stirring (preferably with a magnetic stirrer) add about 30 ml of 0.05M disodium ethylenediaminetetraacetate from a 50-ml buret, then add 15 ml of sodium hydroxide TS, 40 mg of murexide indicator preparation (an alternative indicator is hydroxynaphthol blue, of which 0.25 g is used - in this case the naphthol green TS is omitted) and 3 ml of naphthol green TS, and continue the titration until the solution is deep blue in colour.

Each ml of 0.05M disodium ethylenediamine tetraacetate is equivalent to 14.116 mg of C14H10CaO4.

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SPECIFICATIONS AND TESTS FOR ETHYL p-HYDROXYBENZOATE

1. Synonyms

Ethyl p-oxybenzoate; Ethylparaben

INS 214

ADI= 0 – 10 mg/kg bw

2. Definition

Chemical names

Ethyl p-hydroxybenzoate, ethyl ester of p-hydroxybenzoic acid

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C.A.S. number

120-47-8

Chemical formula

C9H10O3

Structural formula

Formula weight

166.18

3. Description

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...

4. Functional uses

Preservative

5. Specifications

5.1. Identification

Solubility

Freely soluble in ethanol, ether and propylene glycol.

Melting range

115^oC -118^oC.

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...

Melting range of p-hydroxybenzoic acid derived from the sample is 212- 217^o.

5.2. Purity

Loss on drying

Not more than 0.5%.

Sulfated ash

Not more than 0.05%.

Acidity

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p-Hydroxybenzoic acid and salicylic acid

Passes test (described in “Tests”).

Lead

Not more than 2 mg/kg.

5.3. C9H10O3 content

Not less than 99.0% calculated on the anhydrous basis.

6. Tests

6.1. Identification

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To 0.5 g of the sample add 10 ml of sodium hydroxide TS. Boil for 30 min and concentrate to about 5 ml. Cool, acidify with dilute sulfuric acid TS, collect the precipitate on a filter, and wash thoroughly with water. Dry in a desiccator over sulfuric acid. Determine the melting range of phydroxybenzoic acid so obtained.

6.2. Purity

Loss on drying

- Tested according to JECFA monograph 1 - Vol. 4.

- Not more than 0.5% (80º, 2 h).

Sulfated ash

- Tested according to JECFA monograph 1 - Vol. 4,  Method I.

- Test 2 g of the sample.

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Heat 750 mg of the sample with 15 ml of water at 80o for 1 min, cool, and filter. The filtrate should be acid or neutral to litmus. To 10 ml of the filtrate add 0.2 ml of 0.1 N sodium hydroxide and 2 drops of methyl red TS. The solution should be yellow without even a light cast of pink.

p-Hydroxybenzoic acid and salicylic acid

Dissolve 0.5 g of the sample, accurately weighed, in 30 ml of ether, add 20 ml of a 1 in 100 sodium hydrogen carbonate solution, shake, and separate the water layer. Wash the water layer with two 20 ml portions of ether, add 5 ml of dilute sulfuric acid and 30 ml of ether, and shake. Separate the ether layer, and shake with about 10 ml of water. Filter the ether layer, and wash the vessel and the filter with a small amount of ether. Combine the washings and the filtrate, evaporate ether on a water bath, and dry the residue over sulfuric acid to constant weight. The weight of the residue should not exceed 5 mg. Dissolve any residue in 25 ml of water, heat to about 70o , filter, and add a few drops of dilute ferric chloride TS. No violet to reddish violet colour should be produced.

Lead

- Tested according to JECFA monograph 1 - Vol. 4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

6.3. Method of essay

...

...

...

Each ml of N sodium hydroxide is equivalent to 166.18 mg of C9H10O3.

ANNEX 9

SPECIFICATIONS AND TESTS FOR METHYL p-HYDROXYBENZOATE

1. Synonyms

Methyl p-oxybenzoate; Methylparaben

INS 218

ADI = 0 - 10mg/kg bw

2. Definition

...

...

...

Methyl p-hydroxybenzoate, methyl ester of p-hydroxybenzoic acid

C.A.S. number

99-76-3

Chemical formula

C8H8O3

Structural formula

Formula weight

...

...

...

3. Description

Almost odourless, small, colourless crystals or a white, crystalline powder.

4. Functional uses

Preservative

5. Specifications

5.1. Identification

Solubility

Slightly soluble in water; freely soluble in ethanol and propylene glycol; soluble in ether.

...

...

...

125oC -128oC.

Test for p-hydroxybenzoate

Melting range of p-hydroxybenzoic acid derived from the sample is 212- 217o.

5.2. Purity

Loss on drying

Not more than 0.5%.

Sulfated ash

Not more than 0.05%.

...

...

...

Passes test (described in “Tests”).

p-Hydroxybenzoic acid and salicylic acid

Passes test (described in “Tests”).

Lead

Not more than 2 mg/kg.

5.3. C8H8O3 content

Not less than 99.0% calculated on the anhydrous basis.

6. Tests

6.1. Identification

...

...

...

Test for p-hydroxybenzoate

To 0.5 g of the sample add 10 ml of sodium hydroxide TS. Boil for 30 min and concentrate to about 5 ml. Cool, acidify with dilute sulfuric acid TS, collect the precipitate on a filter, and wash thoroughly with water. Dry in a desiccator over sulfuric acid. Determine the melting range of phydroxybenzoic acid so obtained.

6.2. Purity

Loss on drying

- Tested according to JECFA monograph 1 - Vol. 4.

- Over silica gel, 5 h.

Sulfated ash

Tested according to JECFA monograph 1 - Vol. 4,  Method I (Test 2 g of the sample).

...

...

...

Heat 750 mg of the sample with 15 ml of water at 80o for 1 min, cool, and filter. The filtrate should be acid or neutral to litmus. To 10 ml of the filtrate add 0.2 ml of 0.1 N sodium hydroxide and 2 drops of methyl red TS. The solution should be yellow without even a light cast of pink.

p-Hydroxybenzoic acid and salicylic acid

Dissolve 0.5 g of the sample, accurately weighed, in 30 ml of ether, add 20 ml of a 1 in 100 sodium hydrogen carbonate solution, shake, and separate the water layer.

Wash the water layer with two 20 ml portions of ether, add 5 ml of dilute sulfuric acid and 30 ml of ether, and shake. Separate the ether layer, and shake with about 10 ml of water.Filter the ether layer, and wash the vessel and the filter with a small amount of ether. Combine the washings and the filtrate, evaporate ether on a water bath, and dry the residue over sulfuric acid to constant weight. The weight of the residue should not exceed 5 mg. Dissolve any residue in 25 ml of water, heat to about 70o , filter, and add a few drops of dilute ferric chloride TS. No violet to reddish violet colour should be produced.

Lead

- Tested according to JECFA monograph 1 - Vol. 4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

6.3. Method of essay

...

...

...

Weigh, to the nearest mg, 2 g of the dried sample and transfer into a flask. Add 40 ml of N sodium hydroxide and rinse the sides of the flask with water. Cover with a watch glass, boil gently for 1 h and cool. Add 5 drops of bromothymol blue TS and titrate the excess sodium hydroxide with N sulfuric acid, comparing the colour with a buffer solution TS (pH 6.5) containing the same proportion of indicator. Perform a blank determination with the reagents and make any necessary correction.

Each ml of N sodium hydroxide is equivalent to 152.2 mg of C8H8O3.

ANNEX 10

SPECIFICATIONS AND TESTS FOR SULFUR DIOXIDE

1. Synonyms

INS 220

ADI = 0 - 0.7 mg/kg bw

2. Definition

...

...

...

7446-09-5

Structural formula

SO2

Formula weight

64.07

3. Description

Colourless, non-flammable gas, with strong, pungent, suffocating odour. Its vapour density is 2.26 times that of air at atmospheric pressure and 0^oC. The specific gravity of the liquid is about 1.436 at 0^o/4^oC. At 20^o the solubility is about 10 g of SO2 per 100 g of solution. It is normally supplied under pressure in containers in which it is present in both liquid and gaseous phases.

Caution: Sulfur dioxide gas is intensely irritating to the eyes, throat, and upper respiratory system. Liquid sulfur dioxide may cause skin burns, which result from the freezing effect of the liquid on tissue. Safety precautions to be observed in handling of the material are specified in technical brochures from liquid sulfur dioxide manufacturers, suppliers or organizations of gas manufacturers or suppliers.

4. Functional uses

...

...

...

5. Specifications

5.1. Identification

Solubility

Soluble in water (36 v in 1 v) and ethanol (114 v in 1 v) (tested according to JECFA monograph 1 - Vol. 4).

Test for sulfurous substances

Passes test.

Oxidizing activity

Passes test.

...

...

...

Water

Not more than 0.05%.

Non-volatile residue

 Not more than 0.05%.

Selenium

Not more than 20 mg/kg.

Lead

Not more than 5 mg/kg.

...

...

...

Not less than 99.9 % SO2 by weight.

6. Tests

6.1. Identification

Test for sulfurous substances

The sample blackens filter paper moistened with mercurous nitrate TS.

Oxidizing activity

Expose a filter paper, treated with potassium iodate and starch TS, to the sample. A blue colour is developed that fades on continued exposure.

6.2. Purity

...

...

...

Water

- Tested according to JECFA monograph 1 - Vol. 4.

- Transfer about 50 ml of liquid sulfur dioxide into a Karl Fischer titration jar, determine the weight of the sample taken, and determine the water content by Karl Fischer Method.

Non-volatile residue

Measure out 200 ml of sulfur dioxide (288 g) into a 250-ml Erlenmeyer flask, and determine the weight of sample taken by the loss in weight of the sample bomb. Evaporate to dryness on a steam bath, and displace the residual vapours with dry air. Wipe the flask dry, cool in a desiccator, and weigh.

Selenium

- A 2.0-ml portion of the Sample Solution meets the requirements of the Selenium Limit Test, Method II.

- For sampling and sample preparation, see “Tests”.

Lead

...

...

...

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

6.3. Sampling

Samples of sulfur dioxide may be safely withdrawn from a tank or transfer lines, either of which should be equipped with a 1-cm nozzle and valve. Samples should be taken in bombs constructed of 316 stainless steel, designed to withstand 7 MPa (1000 psig) and equipped with 316 stainless steel needle valves on both ends. To draw a sample, the bomb is first flushed with dry air to remove any sulfur dioxide, remaining from previous sample drawings, and then attached to the tank or transfer lines with a solid pipe connection. A hose is connected to the other end of the bomb and submerged in either a weak caustic solution or water. Any gas in the bomb is discharged into the caustic or water by first opening the valve at the pipe end, followed by slowly opening at the valve at the hose end. When all of the gas is dispelled and liquid sulfur dioxide begins to emerge into the solution, the valve at the hose end is blocked off. The other valves are then tightly closed, and the bomb is detached from the pipe connecting it to the tank or transfer line Approximately 15% of the liquid sulfur dioxide in the bomb is then discharged into the water or caustic solution. The bomb is then capped at its end and transferred to the laboratory for analysis.

Caution: The bomb should never be stored with more than 85% of the total water capacity of the bomb.

Sample Solution for the Determination of Lead, and Selenium: Measure out 100 ml of sulfur dioxide (144 g) into a 125-ml Erlenmeyer flask, and determine the weight of sample taken by the loss in weight of the sample bomb. Evaporate to dryness on a steam bath, add 3 ml of nitric acid and 10 ml of water to the dry flask, and warm gently on a hot plate for 15 min. Transfer the contents of the flask to a 100-ml volumetric flask, dilute to volume with water, and mix. Transfer a 10.0-ml aliquot into a second 100-ml volumetric flask, dilute to volume with water, and mix.

Note: The tests in which this solution is to be used will be accurate assuming a 144 g sample has been taken; if not, the weight of sample actually taken must be considered in the calculations.

6.4. Method of essay

...

...

...

ANNEX 11

SPECIFICATIONS AND TESTS FOR SODIUM SULFITE

1. Synonyms

Disodium sulfite;

INS 221

ADI=0 – 0.7 mg/kg bw as SO2

2. Definition

Chemical names

...

...

...

C.A.S. number

7757-83-7

Chemical formula

Na2SO3

Formula weight

126.04

3. Description

White powder with not more than a faint odour of sulfur dioxide.

4. Functional uses

...

...

...

5. Specifications

5.1. Identification

Solubility

Freely soluble in water; sparingly soluble in ethanol.

Test for sodium

Passes test.

Test for sulfite

Passes test.

...

...

...

pH

8.5 - 10.0 (1 in 10 soln).

Thiosulfate

Not more than 0.1%.

Iron

Not more than 10 mg/kg.

Lead

Not more than 2 mg/kg.

...

...

...

Not more than 5 mg/kg.

5.3. Na2SO3 content

Not less than 95.0%.

6. Tests

6.1. Identification

Test for sodium

- Tested according to JECFA monograph 1 - Vol. 4.

Test for sulfite

...

...

...

6.2. Purity

Thiosulfate

A 10% solution of the sample should remain clear on acidification with sulfuric or hydrochloric acid.

Iron

- Tested according to JECFA monograph 1 - Vol. 4.

- Use 0.5 ml of Iron Standard Solution (5 µg Fe) in the control.

Lead

- Tested according to JECFA monograph 1 - Vol. 4.

...

...

...

Selenium

Reagents:

Hydrochloric acid, hydrazinium sulfate, standard selenium solution (100 µg Se/ml)

Procedure:

Weigh 2.0 ± 0.1 g of sample and transfer to a 50-ml beaker. Add 10 ml water, 5 ml hydrochloric acid and boil to remove SO2.

Into a second beaker, weigh 1.0 ± 0.1 g of sample, add 0.05 ml standard selenium solution and proceed as above.

To each beaker add 2 g hydrazinium sulfate and warm to dissolve. Let stand for 5 min. Dilute the contents of each beaker to 50 ml in a Nessler tube and compare the colour of the two solutions. The sample should be less pink than the sample with the added standard.

6.3. Method of essay

...

...

...

Weigh 250 mg of the sample, add to 50.0 ml of 0.1 N iodine in a glassstoppered flask, and stopper the flask. Allow to stand for 5 min, add 1 ml of hydrochloric acid and titrate the excess iodine with 0.1 N sodium thiosulfate, adding starch TS as the indicator.

Each ml of 0.1 N iodine is equivalent to 6.302 mg of Na2SO3.

ANNEX 12

SPECIFICATIONS AND TESTS FOR SODIUM HYDROGEN SULFITE

1. Synonyms

Sodium hydrogen sulfite;

INS 222

ADI =0 - 0,7 mg/kg bw as SO2

2. Definition

...

...

...

Chemical names

Sodium hydrogen sulfite, sodium bisulfite

C.A.S. number

7631-90-5

Chemical formula

NaHSO3

Formula weight

104.06

3. Description

...

...

...

4. Functional uses

Antibrowning agent, antioxidant, preservative

5. Specifications

5.1. Identification

Solubility

Freely soluble in water; sparingly soluble in ethanol

Test for sodium

Passes test.

...

...

...

Passes test.

5.2. Purity

Water insolubles

Passes test (described in “Tests”).

pH

 2.5 - 4.5 (1 in 10 soln).

Iron

Not more than 10 mg/kg.

...

...

...

Not more than 2 mg/kg.

Selenium

Not more than 5mg/kg.

5.3. SO2 content

Not less than 58.5% and not more than 67.4% of SO2

6. Tests

6.1. Identification

Test for sodium

...

...

...

Test for sulfite

Tested according to JECFA monograph 1 - Vol. 4.

6.2. Purity

Water insolubles

Dissolve 20 g of the sample in 200 ml of water. The solution should be clear with only a trace of suspended matter.

Iron

- Tested according to JECFA monograph 1 - Vol. 4.

- Use 0.5 ml of Iron Standard Solution (5 µg Fe) in the control.

...

...

...

- Tested according to JECFA monograph 1 - Vol. 4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

Selenium

Reagents:

Hydrochloric acid, hydrazinium sulfate, standard selenium solution (100 µg Se/ml)

Procedure:

Weigh 2.0 ± 0.1 g of sample and transfer to a 50-ml beaker. Add 10 ml water, 5 ml hydrochloric acid and boil to remove SO2.

Into a second beaker, weigh 1.0 ± 0.1 g of sample, add 0.05 ml standard selenium solution and proceed as above.

To each beaker add 2 g hydrazinium sulfate and warm to dissolve. Let stand for 5 min. Let stand for 5 min. Dilute the contents of each beaker to 50 ml in a Nessler tube and compare the colour of the two solutions. The sample should be less pink than the sample with the added standard.

...

...

...

Weigh 0.2 g of the sample, to the nearest mg, add 50.0 ml of 0.1 N iodine in a glass-stoppered flask, and stopper the flask. Allow to stand for 5 min, add 1 ml of hydrochloric acid, and titrate the excess iodine with 0.1 N sodium thiosulfate, adding starch TS as the indicator.

Each ml of 0.1 N iodine is equivalent to 3.203 mg of SO2.

ANNEX 13

 SPECIFICATIONS AND TESTS FOR SODIUM METABISULFITE

1. Synonyms

Sodium metabisulfite;

INS 223

...

...

...

2. Definition

Chemical names

Sodium disulfite, disodium pentaoxodisulfate, disodium pyrosulfite

C.A.S. number

7681-57-4

Chemical formula

Na2S2O5

Formula weight

...

...

...

3. Description

White crystals or crystalline powder having an odour of sulfur dioxide.

4. Functional uses

Antibrowning agent, antioxidant, flour treatment agent, preservative

5. Specifications

5.1. Identification

Solubility

Freely soluble in water; sparingly soluble in ethanol.

...

...

...

Passes test.

Test for sulfite

Passes test.

5.2. Purity

Water insolubles

Passes test (described in “Tests”).

pH

 4.0 - 4.5 (1 in 10 soln).

...

...

...

Not more than 0.1%.

Iron

Not more than 10 mg/kg.

Lead

Not more than 2 mg/kg.

Selenium

Not more than 5 mg/kg.

5.3. Na2S2O5 content

Not less than 90.0%.

...

...

...

6.1. Identification

Test for sodium

- Tested according to JECFA monograph 1 - Vol. 4.

Test for sulfite

- Tested according to JECFA monograph 1 - Vol. 4.

6.2. Purity

Water insolubles

...

...

...

Thiosulfate

A 10% solution of the sample should remain clear on acidification with sulfuric or hydrochloric acid.

Iron

- Tested according to JECFA monograph 1 - Vol. 4.

- Use 0.5 ml of Iron Standard Solution (5 µg Fe) in the control.

Lead

- Tested according to JECFA monograph 1 - Vol. 4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

Selenium

...

...

...

Hydrochloric acid, hydrazinium sulfate, standard selenium solution (100 µg Se/ml)

Procedure:

Weigh 2.0 ± 0.1 g of sample and transfer to a 50-ml beaker. Add 10ml ml water, 5 ml hydrochloric acid and boil to remove SO2.

Into a second beaker, weigh 1.0 ± 0.1 g of sample, add 0.05 ml standard selenium solution and proceed as above.

To each beaker add 2 g hydrazinium sulfate and warm to dissolve. Let stand for 5 min. Let stand for 5 min. Dilute the contents of each beaker to 50 ml in a Nessler tube and compare the colour of the two solutions. The sample should be less pink than the sample with the added standard.

6.3. Method of essay

Weigh 0.2 g of the sample, to the nearest mg, add 50.0 ml of 0.1 N iodine in a glass-stoppered flask, and stopper the flask. Allow to stand for 5 min, add 1 ml of hydrochloric acid and titrate the excess iodine with 0.1 N sodium thiosulfate, adding starch TS as the indicator.

...

...

...

ANNEX 14

 SPECIFICATIONS AND TESTS FOR POTASSIUM METABISULFITE

1. Synonyms

Potassium metabisulfite;

INS 224

ADI=0 - 0.7 mg/kg bw as SO2

2. Definition

Chemical names

...

...

...

C.A.S. number

16731-55-8

Chemical formula

K2S2O5

Formula weight

222.33

3. Description

Colourless free-flowing crystals, crystalline powder, or granules, usually having an odour of sulfur dioxide.

4. Functional uses

...

...

...

5. Specifications

5.1. Identification

Solubility

Soluble in water; insoluble in ethanol.

Test for potassium

Passes test.

Test for sulfite

Passes test.

...

...

...

Water insolubles

Passes test (described in “Tests”).

Thiosulfate

Not more than 0.1%.

Iron

Not more than 10 mg/kg.

Lead

Not more than 2 mg/kg.

...

...

...

Not more than 5 mg/kg.

5.3. K2S2O5 content

Not less than 90.0%.

6. Tests

6.1. Identification

Test for potassium

Passes test.

Test for sulfite

...

...

...

6.2. Purity

Water insolubles

Dissolve 20 g of the sample in 200 ml of water. The solution should be clear with only a trace of suspended matter.

Thiosulfate

A 10% solution of the sample should remain clear on acidification with sulfuric or hydrochloric acid.

Iron

- Tested according to JECFA monograph 1 - Vol. 4.

- Use 0.5 ml of Iron Standard Solution (5 µg Fe) in the control.

...

...

...

- Tested according to JECFA monograph 1 - Vol.4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

Selenium

Reagents:

Hydrochloric acid, hydrazinium sulfate, standard selenium solution (100 µg Se/ml)

Procedure:

Weigh 2.0 ± 0.1 g of sample and transfer to a 50-ml beaker. Add 10 ml water, 5 ml hydrochloric acid and boil to remove SO2.

Into a second beaker, weigh 1.0 ± 0.1 g of sample, add 0.05 ml standard selenium solution and proceed as above.

To each beaker add 2 g hydrazinium sulfate and warm to dissolve. Let stand for 5 min. Let stand for 5 min. Dilute the contents of each beaker to 50 ml in a Nessler tube and compare the colour of the two solutions. The sample should be less pink than the sample with the added standard.

...

...

...

Weigh 250 mg of the sample, add to 50.0 ml of 0.1 N iodine in a glass stoppered flask, and stopper the flask. Allow to stand for 5 min, add 1 ml of dilute hydrochloric acid TS and titrate the excess iodine with 0.1 N sodium thiosulfate, using starch TS as the indicator.

Each ml of 0.1 N iodine is equivalent to 5.558 mg of K2S2O5.

ANNEX 15

SPECIFICATIONS AND TESTS FOR POTASSIUM SULFITE

1. Synonyms

Potassium sulfite;

INS 225

...

...

...

2. Definition

Chemical names

Potassium sulfite

C.A.S. number

10117-38-1

Chemical formula

K2SO3

Formula weight

...

...

...

3. Description

 White, odourless, granular powder.

4. Functional uses

Antibrowning agent, antioxidant, preservative

5. Specifications

5.1. Identification

Solubility

Freely soluble in water; slightly soluble in ethanol.

...

...

...

Passes test.

Test for sulfite

Passes test.

5.2. Purity

Alkalinity

Between 0.25 and 0.45% as K2CO3.

Iron

Not more than 10 mg/kg.

...

...

...

Not more than 2 mg/kg.

Selenium

Not more than 5 mg/kg.

5.3. K2SO3 content

Not less than 90.0%.

6. Tests

6.1. Identification

Test for potassium

...

...

...

Test for sulfite

Tested according to JECFA monograph 1 - Vol. 4.

6.2. Purity

Alkalinity

Dissolve 1 g of the sample in 20 ml of water and add 25 ml of 3% hydrogen peroxide, previously neutralized to methyl red TS. Mix thoroughly, cool to room temperature, and titrate with 0.02 N hydrochloric acid. Perform a blank determination using 25 ml of neutralized hydrogen peroxide solution. Each ml of 0.02 N hydrochloric acid is equivalent to 1.38 mg of K2CO3.

Iron

- Tested according to JECFA monograph 1 - Vol. 4.

- Use 0.5 ml of Iron Standard Solution (5 µg Fe) in the control.

...

...

...

- Tested according to JECFA monograph 1 - Vol.4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

Selenium

Reagents:

Hydrochloric acid, hydrazinium sulfate, standard selenium solution (100 µg Se/ml)

Procedure:

Weigh 2.0 ± 0.1 g of sample and transfer to a 50-ml beaker. Add 10 ml water, 5 ml hydrochloric acid and boil to remove SO2.

Into a second beaker, weigh 1.0 ± 0.1 g of sample, add 0.05 ml standard selenium solution and proceed as above.

To each beaker add 2 g hydrazinium sulfate and warm to dissolve. Let stand for 5 min. Let stand for 5 min. Dilute the contents of each beaker to 50 ml in a Nessler tube and compare the colour of the two solutions. The sample should be less pink than the sample with the added standard.

...

...

...

Weigh accurately about 0.75 g of the sample and dissolve in a mixture of 100 ml of 0.1 N iodine and 5 ml of dilute hydrochloric acid TS. Titrate the excess iodine with 0.1 N sodium thiosulfate, adding starch TS as the indicator.

Each ml of 0.1 N iodine is equivalent to 7.912 mg of K2SO3.

ANNEX 16

SPECIFICATIONS AND TESTS FOR NISIN

1. Synonyms

INS 234

...

...

...

Nisin is a mixture of closely related antimicrobial polypeptides produced by strains of Lactococcus lactis subsp. lactis under appropriate fermentation conditions.

Nisin preparation consists of nisin and sodium chloride with an activity of not less than 900 units per mg. Nisin preparation is stable at ambient temperatures and upon heating under acid conditions (maximum stability at pH 3).

C.A.S. number

1414-45-5

Chemical formula

C143H230N42O37S7

Structural formula

...

...

...

Dhb=dehydrobutyrine

Formula weight

Ca. 3354

3. Description

White to light brown micronized powder

4. Functional uses

Antimicrobial preservative

5. Specifications

5.1. Identification

...

...

...

Solubility

Soluble in water and insoluble in non-polar solvents

Differentiation from other antimicrobial substances

Passes test.

5.2. Purity

Loss on drying

Not more than 3.0% (105^oC , 2 h)

Lead

...

...

...

Microbiological criteria

Salmonella species: Absent in 25 g of sample

Total coliforms: Not more than 30 per gram

Escherichia coli: Absent in 25 g sample

5.3. Content

Not less than 900 IU of nisin per milligram and not less than 50% sodium chloride

6. Tests

6.1. Identification

...

...

...

- Stability to acid: Suspend a 100-mg sample in 0.02 N hydrochloric acid as described in “Standard stock solution” under Method of Assay. Boil this solution for 5 min.

Using the method of assay described below, determine the nisin activity. No significant loss of activity is noted following this heat treatment. The calculated nisin concentration of the boiled sample is 100% +/- 5% of the assay value. Adjust the pH of the nisin solution to 11.0 by adding 5N sodium hydroxide. Heat the solution at 65° for 30 min, and then cool. Adjust the pH to 2.0 by adding hydrochloric acid dropwise. Again determine the nisin concentration using the assay method described below. Complete loss of the antimicrobial activity of nisin is observed following this treatment.

- Tolerance of Lactococcus lactis to high concentrations of nisin: Prepare cultures of Lactococcus lactis (ATCC 11454, NCIMB 8586) in sterile skim m (<1% fat) milk by incubating for 18 h at 30°. Prepare one or more flasks containing 100 ml of litmus milk, and sterilize at 121° for 15 min. Suspend 0.1 g of sample in the sterilized litmus milk, and allow to stand at room temperature for 2 h.  Add 0.1 ml of the L. lactis culture, and incubate at 30° for 24 h. L. lactis will grow in this concentration of sample (about 1000 IU/ml); however, it will not grow in similar concentrations of other antimicrobial substances. This test will not differentiate nisin from subtilin.

6.2. Purity

Lead

- Tested according to JECFA monograph 1 - Vol. 4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

6.2. Method of essay

...

...

...

Determination of nisin activity (Based on the method of Friedman and Epstein, J. Gen. Microbiol. 5: 830, 1951)

Preparation of the test organism:

Lactococcus lactis sbsp .cremoris (ATCC 14365, NCDO 495) is subcultured daily in sterile separated milk by transferring one loopful to a McCartney bottle of litmus milk and incubating at 30^o. Prepare inoculated milk for the assay by inoculating a suitable quantity of sterile skim milk with 2 percent of a 24 h culture, and place it in a water-bath at 30^o for 90 min. Use immediately.

Standard stock solution:

Dissolve an accurately weighed quantity of standard nisin in 0.02N hydrochloric acid to give a solution containing 5 000 units/ml. Immediately before use, dilute the solution further with 0.02N hydrochloric acid to give 50 units/ml. (NOTE: Nisin preparation containing 2.5 % nisin, minimum potency of 10 6 IU/g, obtainable from Sigma, St Louis, USA or Fluka, Buchs, Switzerland, may be used for the Standard Stock Preparation, as well as, the preparation under the name of Nisaplin, available from Danisco, Copenhagen, Dennmark).

Sample solution:

Weigh an amount of sample sufficient to ensure that corresponding tubes of the sample and standard series match, i.e. within close limits, the nisin content in the sample and standard is the same. Dilute the sample solution in 0.02 N hydrochloric acid to give an estimated concentration of 50 units of nisin per ml.

...

...

...

Prepare a 0.0125% solution of resazurin in water immediately before use.

Procedure:

Pipet graded volumes (0.60, 0.55, 0.50, 0.45, 0.41, 0.38, 0.34, 0.31, 0.28, 0.26 ml) of the 50 unit per ml sample and standard solutions into rows of 10 dry 6-inches x 5/8-inch bacteriological test-tubes. Add 4.6 ml of the inoculated milk to each by means of an automatic pipetting device. The addition of inoculated milk is made in turn across each row of tubes containing the same nominal concentration, not along each row of ten tubes. Place the tubes in a water-bath at 30^o for 15 min, then cool in an ice water bath while adding 1 ml resazurin solution to each. The addition is made with an automatic pipetting device, in the same order used for the addition of inoculated milk. Thoroughly mix the contents of the tubes by shaking. Continue incubation at 30^o in a water-bath for a further 3-5 min.

Examine the tubes under fluorescent light in a black matt-finish cabinet. The sample tube of the highest concentration which shows the first clear difference in colour (i.e. has changed from blue to mauve) is compared with tubes of the standard row to find the nearest in colour. Make further matches at the next two lower concentrations of the sample and standard. Interpolation of matches may be made at half dilution steps. As the standard tubes contain known amounts of nisin, calculate the concentration of nisin in the sample solution. Obtain three readings of the solution and average them. Calculate the activity in terms of IU per gram of preparation.

NaCl

Accurately weigh about 100 mg of sample, and transfer to a porcelain casserole. Add 100 ml water, 2 ml 2% dextrin soln, and 1 ml 0.1% dichlorofluoroscein soln. Mix, and titrate with 0.1 N silver nitrate soln until the silver chloride flocculates and the mixture acquires a faint pink colour.

Sodium chloride,% (w/w):

...

...

...

Where:

V is the volume of silver nitrate solution consumed (ml)

N is the normality of the silver nitrate solution

58.5 is the formula weight of sodium chloride, and

W is the weight of the sample (mg).

ANNEX 17

SPECIFICATIONS AND TESTS FOR HEXAMETHYLENE TERTRAMINE

1. Synonyms

...

...

...

INS 239

ADI = 0 - 0.15 mg/kg bw.

2. Definition

Chemical names

1,3,5,7-tetraazatricylo[3.3.1.13,7]-decan; hexamethylen- tetramin

C.A.S. number

100-97-0

Chemical formula

...

...

...

Structural formula

Formula weight

140.19

3. Description

Nearly odourless, colourless lustrous crystals, or white crystalline powder.

4. Functional uses

Antimicrobial preservative

5. Specifications

...

...

...

Solubility

Freely soluble in water, soluble in ethanol.

Test for formaldehyde

Passes test.

Ammonium

Passes test.

5.2. Purity

...

...

...

Not more than 2.0%.

Sulfated ash

Not more than 0.05%.

Test for ammonia

Passes test (described in “Tests”).

Lead

Not more than 2 mg/kg.

5.3. C6H12N4 content

Not less than 99.0% on the dried basis.

...

...

...

6.1. Identification

Test for formaldehyde

Heat a 1 in 10 solution of the sample with dilute sulfuric acid TS. Formaldehyde is liberated, recognizable by its odour and by its darkening of paper moistened with silver ammonium nitrate TS.

Ammonium

Tested according to JECFA monograph 1 - Vol. 4.

6.2. Purity

Loss on drying

...

...

...

Sulfated ash

Tested according to JECFA monograph 1 - Vol. 4,  Method I (Test 2 g of the sample).

Test for ammonia

Add 1 ml of Nessler's reagent TS to 10 ml of a 5% solution of the sample. The mixture should not be darker than a mixture of 1 ml of the reagent in 10 ml of water.

Lead

- Tested according to JECFA monograph 1 - Vol. 4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

6.3. Method of essay

...

...

...

Weigh, to the nearest 0.1 mg, 1 g of the sample, previously dried for 4 h over phosphorus pentoxide. Transfer into a beaker and add 40.00 ml of N sulfuric acid. Boil gently adding water from time to time, if necessary, until the odour of formaldehyde is no longer perceptible. Cool, add 20 ml of water, and methyl red TS and titrate the excess acid with N sodium hydroxide.

Each ml of N sulfuric acid is equivalent to 35.05 mg of C6H12N4.

ANNEX 18

SPECIFICATIONS AND TESTS FOR DIMETHYL DICARBONATE

1. Synonyms

Dimethyl dicarbonate; DMDC, dimethyl pyrocarbonate

INS 242

2. Definition

...

...

...

Dimethyl dicarbonate

C.A.S. number

004-525-33-1

Chemical formula

C4H6O5

Structural formula

Formula weight

139.09

...

...

...

Colourless liquid.

Caution: Corrosive to eyes and skin and toxic by inhalation and ingestion; must be kept in a tightly sealed container to exclude moisture.

4. Functional uses

Preservative

5. Specifications

5.1. Identification

Solubility

Soluble in water with decomposition; miscible with toluene.

...

...

...

The infrared spectrum of the sample corresponds with the reference infrared spectrum below.

5.2. Purity

Dimethyl dicarbonate

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

5.3. C4H6O5 content

Not less than 99.8%.

...

...

...

6.1. Purity

Dimethyl dicarbonate

Apparatus:

Gas chromatograph with a flame ionization detector, equipped for capillary gas chromatography, and inlet part for the "on column" technique (e.g., Carlo Erba Fraktovap 4160, or Hewlett-Packard 5880 or 5792).

- Capillary: 50 m SE 30-D (internal diameter: 0,3 mm).

- Potentiometric recorder, 1 mV.

- Microliter syringes with quartz needles, contents 0,010 ml, suitable

Operating conditions

...

...

...

- Injection block: Room temperature (approximately 25^o)

- Detector: 200^o.

- Capillary Tube: Initial temperature 30^o; Preliminary phase 5 min, Rate of ascent 40^o/min; Final temperature 120^o, Final phase 5 min.

Carrier gas:

- Preliminary pressure: 3.0 bar (Helium)

- Capillary tubes: Approx. 11 ml/min

- Make-up gas: 28 ml/min

- Hydrogen: 35 ml/min

- Air: 300 ml/min

...

...

...

Reagents:

Methylisobutylketone, purest grade.

Procedure:

Accurately weigh about 10 g of the sample to the nearest mg (W1 mg) into a rolled rim vial. Add in a quantity of dimethyl-isobutylketone (W2 mg), corresponding to the anticipated dimethylcarbonate content. Seal the rolled rim vial, mix well and inject 0.002 ml.

Retention times:

dimethylcarbonate: Approx. 2 min

methylisobutylketone: Approx. 4.5 min

dimethyldicarbonate: Approx. 8 min

Typical Chromatogram in 7.2

...

...

...

Where:

F1: peak area of dimethylcarbonate  

F2: peak area of the standard

K: a Correction Factor for dimethylcarbonate

Remarks:

1. It should be noted the DMDC is sensitive to moisture and heat.

2. If so-called peak splits occur to some extent at the given gaschromatography conditions, then the peak areas are best determined by summation of the areas under both peaks.

3. The correction factor for dimethylcarbonate should be determined with corresponding test solutions in DMDC which is to the greatest extent possible free of dimethylcarbonate.

...

...

...

5. Hamilton syringes with Metal needles can result in a partial decomposition of the DMDC.

Lead

- Tested according to JECFA monograph 1 - Vol. 4.

- The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

6.2. Method of essay

Introduce about 70 ml of pure acetone into a 150-ml glass beaker. Using a disposable 2 ml syringe weigh 1.0-1.3 g of the sample to an accuracy of ±0.1 mg into the glass beaker. Pipette exactly 20 ml dibutyl amine solution (add chlorobenzene to 120 g dibutyl amine until the 1 L mark is reached) while stirring. Titrate the solution potentiometrically with 1N hydrochloric acid. Run a blank test.

Calculate the % of dimethyldicarbonate from:

Where:

...

...

...

V2 = amount of hydrochloric acid used for titration of the blank (ml)

t = normality of hydrochloric acid

W = weight of sample (g)

7. Spectrum and chromatogram

7.1. Infrared spectrum

7.2. Typical chromatogram

ANNEX 19

...

...

...

1. Synonyms

Propionic acid; propanoic acid; ethylformic acid; methylacetic acid

INS 280

ADI “Not limited”

2. Definition

Chemical names

Propionic acid

C.A.S. number

...

...

...

Chemical formula

C3H6O2

Structural formula

Formula weight

74.08

3. Description

An oily liquid with a slightly pungent odour

4. Functional uses

...

...

...

5. Specifications

5.1. Identification

Solubility

Miscible with water and ethanol.

Specific gravity

d2020 = 0.993 – 0.997.

5.2. Purity

...

...

...

138.5 - 142.5^o.

Non-volatile residue

Not more than 0.01% when dried at 140^o to constant weight

 Formic acid

Not more than 0.1%.

 Aldehydes

Not more than 0.2% (as propionaldehyde)

Lead

Not more than 2 mg/kg.

...

...

...

Not less than 99.5% on dried basis.

6. Tests

6.1. Purity

Formic acid

Dissolve 15 g of sodium hydroxide in 50 ml of water, cool, add 6 ml of bromine, stirring to effect complete solution, and dilute to 2,000 ml with water. Transfer 25.0 ml of this solution, into a 250 ml, glass-stoppered Erlenmeyer flask containing 100 ml of water, and add 10 ml of a 1 in 5 solution of sodium acetate and 10.0 ml of the sample. Allow to stand for 15 min, add 5 ml of a 1 in 4 solution of potassium iodide and 10 ml of hydrochloric acid, and titrate with 0.1 N sodium thiosulfate just to the disappearance of the brown colour.Perform a blank determination. The difference between the volume of 0.1 N sodium thiosulfate required for the blank and that required for the sample is not more than 4.4 ml.

Aldehydes

Transfer 10.0 ml of the sample into a 250-ml glass-stoppered conical flask containing 50 ml of water and 10.0 ml of a 1 in 8 solution of sodium bisulfite. Stopper the flask, and shake vigorously. Perform a blank determination. Allow the mixture to stand for 30 min, then titrate with 0.1N iodine to the same brownish yellow end-point obtained with a blank treated with the same quantities of the same reagents. The difference between the volume of 0.1N iodine required for the blank and that required for the sample is not more than 7 ml.

Lead

...

...

...

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

6.2. Method of essay

Mix 3 g of the sample, weighed to the nearest 0.1 mg, with 50 ml of water in a 250-ml flask. Add phenolphthalein TS, and titrate with 1N sodium hydroxide to the first appearance of a faint pink end-point which persists for at least 30 sec.

Each ml of 1N sodium hydroxide is equivalent to 74.08 mg of C3H6O2.

ANNEX 20

SPECIFICATIONS AND TESTS FOR SODIUM PROPIONATE

1. Synonyms

...

...

...

INS 281

ADI “Not limited”

2. Definition

Chemical names

Sodium propionate

C.A.S. number

137-40-6

Chemical formula

...

...

...

Structural formula

Formula weight

96.06

3. Description

White or colourless, hygroscopic crystals with not more than a faint characteristic odour.

4. Functional uses

Preservative, antimould and antirope agent.

5. Specifications

...

...

...

Solubility

Freely soluble in water, soluble in ethanol.

Test for sodium

Passes test.

Test for propionate

Passes test.

Test for alkali salt of organic acid

Passes test.

...

...

...

Loss on drying

Not more than 4.0%.

 pH

7.5 - 10.5 (1 in 10 soln).

Water-insoluble matter

Not more than 0.1%.

Iron

Not more than 50.0 mg/kg.

...

...

...

Not more than 5 mg/kg.

5.3. C3H6NaO2 content

Not less than 99.0% on the dried basis.

6. Tests

6.1. Identification

Test for sodium

Tested according to JECFA monograph 1 - Vol. 4.

Test for propionate

...

...

...

Test for alkali salt of organic acid

Ignite the sample at a relatively low temperature. The alkaline residue effervesces with acid.

6.2. Purity

Loss on drying

Tested according to JECFA monograph 1 - Vol. 4. (105º, 2 h).

Water-insoluble matter

Weigh 5 g of the sample to the nearest mg, transfer into a 100-ml beaker and add 50 ml of water. Stir until all the sample appears to be completely dissolved. Filter through a Gooch crucible, tared to an accuracy of ± 0.2 mg. Rinse the beaker with 20 ml of water. Dry the crucible with its contents in a 60º oven to constant weight. Cool in a desiccator, weigh, and calculate as percentage.

Iron

...

...

...

- Test 0.5 g of the sample as described in the Limit Test using 2.5 ml of Iron Standard Solution (25 µg Fe) in the control.

Lead

- Tested according to JECFA monograph 1 - Vol. 4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

6.3. Method of essay

Weigh, to the nearest mg, 3 g of the sample previously dried at 105º for 1 h, into a distillation flask and add 200 ml of 50% phosphoric acid. Boil for 2 h and collect the distillate. During distillation keep the volume in the flask at about 200 ml by adding water using a dropping funnel. Titrate with 1N sodium hydroxide using phenolphthalein TS as indicator to the first appearance of a faint pink end-point which persists for at least 30 sec.

Each ml of 1N sodium hydroxide corresponds to 96.06 mg of C3H5NaO2.

...

...

...

SPECIFICATIONS AND TESTS FOR SODIUM HYPOSULFITE

1. Synonyms

Sodium hyposulfite;

INS 539

ADI = 0 - 0.7 mg/kg bw

2. Definition

Chemical names

Sodium thiosulfate

...

...

...

7772-98-7

Chemical formula

Na2S2O3.5H2O

Formula weight

248.17

3. Description

Colourless crystals or coarse crystalline powder; deliquesces in moist air and effloresces in dry air above 33^o.

4. Functional uses

Antibrowning agent, antioxidant, sequestrant

...

...

...

5.1. Identification

Solubility

Very soluble in water; insoluble in ethanol

Reducing activity

Passes test.

Test for sodium

Passes test.

Thiosulfate

...

...

...

5.2. Purity

Loss on drying

32-37% (40-45^o , 16 h, under vacuum).

Iron

Not more than 10 mg/kg.

Selenium

Not more than 5 mg/kg.

Lead

...

...

...

5.3. Na2S2O3 content

Not less than 99.0% on the dried basis.

6. Tests

6.1. Identification

Reducing activity

To a 1 in 10 solution of the sample add a few drops of iodine TS; the colour is discharged.

Test for sodium

Tested according to JECFA monograph 1 - Vol. 4.

...

...

...

Tested according to JECFA monograph 1 - Vol. 4.

6.2. Purity

Iron

- Tested according to JECFA monograph 1 - Vol. 4.

- Use 0.5 ml of Iron Standard Solution (5 µg Fe) in the control.

Selenium

Reagents

Hydrochloric acid, hydrazinium sulfate, standard selenium solution (100 µg Se/ml).

...

...

...

Weigh 2 ± 0.1 g of sample and transfer to a 50-ml beaker.

Into a second beaker, weigh 1.0 ± 0.1 g of sample, add 0.05 ml standard selenium solution and proceed as above.

To each beaker, add 10 ml water, 5 ml hydrochloric acid and boil to remove SO2; then add 2 g hydrazinium sulfate and warm to dissolve. Dilute the contents of each beaker to 50 ml in a Nessler tube and compare the colour of the two solutions. The sample should be less pink than the sample with the added standard.

Lead

- Tested according to JECFA monograph 1 - Vol. 4.

- Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the method described in JECFA monograph 1 - Vol. 4, “Instrumental Methods.”

6.3. Method of essay

...

...

...

 Each ml of 0.1 N iodine is equivalent to 15.81 mg of Na2S2O3.