QCVN-4-10-2010-BYT-Food-additives-Colours

NATIONAL TECHNICAL REGULATION

QCVN 4-10:2010/BYT

ON FOOD ADDITIVES - COLOURS

Foreword

QCVN 4-10:2010/BYT is developed by the Drafting Board for National technical regulation on food additives and food processing aids, submitted by the Vietnam Food Administration for approval and promulgated together with the Circular No. 27/2010/TT-BYT dated May 20, 2010 of the Minister of Health.

NATIONAL TECHNICAL REGULATION ON FOOD ADDITIVES - COLOURS

I. GENERAL

...

This National technical regulation (hereinafter referred to as “the Regulation”) provides for specifications and regulatory requirements for quality, hygiene and safety of colours used as food additives.

2. Regulated entities

This Regulation applies to:

2.1. Importers, exporters, producers, traders and users of colours used as food additives (hereinafter referred to as “entities”).

2.2. Relevant regulatory bodies.

3. Interpretation of terms and acronyms:

3.1. “colour” means a food additive used for the purpose of adding or restoring color in a food product.

3.2. JECFA monograph 1 - Vol. 4 (JECFA monographs 1 - Combined compendium of food additive specifications; Joint FAO/WHO expert committee on food additives; Volume 4 - Analytical methods, test procedures and laboratory solutions used by and referenced in the food additive specifications; FAO, 2006) is developed by JECFA and was published by FAO in 2006.

3.3. “C.A.S number” (Chemical Abstracts Service) refers to registry numbers of chemical substances identified by the American Chemical Society.

...

3.5. “ADI” stands for Acceptable daily intake.

3.6. “INS” stands for international numbering system.

II. SPECIFICATIONS, TESTS AND SAMPLING

1. Specifications and tests for flavour enhancers specified in the annexes to this Regulation:

1.1. Annex 1: Specifications and tests for curcumin

1.2. Annex 2: Specifications and tests for riboflavin

1.3. Annex 3: Specifications and tests for tatrazine

1.4. Annex 4: Specifications and tests for quinoline

1.5. Annex 5: Specifications and tests for sunset yellow FCF

...

1.7. Annex 7: Specifications and tests for carmoisine

1.8. Annex 8: Specifications and tests for amaranth

1.9. Annex 9: Specifications and tests for ponceau 4R

1.10. Annex 10: Specifications and tests for erythrosine

1.11. Annex 11: Specifications and tests for red 2G

1.12. Annex 12: Specifications and tests for allura red AC

1.13. Annex 13: Specifications and tests for indigotine

1.14. Annex 14: Specifications and tests for brilliant blue FCF

1.15. Annex 11: Specifications and tests for chlorophylls

...

1.17. Annex 17: Specifications and tests for chlorophyllins, copper complexes sodium and potassium salts

1.18. Annex 18: Specifications and tests for green S

1.19. Annex 19: Specifications and tests for caramel

1.20. Annex 20: Specifications and tests for brilliant black PN

1.21. Annex 21: Specifications and tests for brown HT

1.22. Annex 22: Specifications and tests for synthetic β-carotene

1.23. Annex 23: Specifications and tests for carotenes, vegetable

1.24. Annex 24: Specifications and tests for annatto extracts

1.25. Annex 25: Specifications and tests for β-Apo-carotenal

...

1.27. Annex 27: Specifications and tests for grape skin extract

1.28. Annex 28: Specifications and tests for titanium dioxide

1.29. Annex 29: Specifications and tests for iron oxides

1.30. Annex 30: Specifications and tests for fast green FCF

2. 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.

3. 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 colours shall be declared in accordance with the regulations set out in this Regulation.

...

2. Inspection of colours

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

IV. RESPONSIBILITIES OF ENTITIES

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 entitled to import, export, produce, sell and use colours only 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 CURCUMIN

1. Synonyms

Turmeric yellow, Kurkum

INS: 100i

ADI = 0 – 3mg/kg bw.

2. Definition

Curcumin is obtained by solvent extraction of turmeric i.e., the ground rhizomes of Curcuma longa L. (Curcuma domestica Valeton). In order to obtain a concentrated curcumin powder, the extract is purified by crystallization. The product consists essentially of curcumins; i.e. the colouring principle 1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-1,6-diene3,5-dione (synonyms: Curcumin, Diferuloylmethane, CI Natural Yellow 3, CI (1975) 75300) and its desmethoxy- and bis-desmethoxy-derivatives in varying proportions. Minor amounts of oils and resins naturally occurring in turmeric may be present. Only the following solvents may be used in the extraction and purification: acetone, methanol, ethanol, isopropanol, hexane and ethyl acetate. Supercritical carbon dioxide may also be used in the extraction.

Chemical names

...

1,7-Bis-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene-3,5-dione

II.

1-(4-Hydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene3,5-dione

III.

1,7-Bis-(4-hydroxyphenyl)-hepta-1,6-diene-3,5-dione

C.A.S. number

...

II.

33171-16-3

III.

33171-05-0

Chemical formula

C₂₁H₂₀O₆

II.

...

III.

C₁₉H₁₆O₄

Structural formula

R₁= R₂=OCH₃

II.

R₁= OCH₃; R₂=H

...

R₁= R₂=H

Formula weight

368.39

II.

338.39

III.

308.39

...

3. Description

Orange-yellow crystalline powder.

4. Functional uses

Colour.

5. Specifications

5.1. Identification

Solubility

Insoluble in water and in diethyl ether; soluble in ethanol and in glacial acetic acid.

...

A solution of the sample in ethanol is characterized by a pure yellow colour and a light green fluorescence. Addition of this ethanol extract to concentrated sulfuric acid produces a deep crimson colour.

Thin Layer Chromatography

Passes test (described in “Tests”).

5.2. Purity

Residual solvents

Acetone:

Not more than 30 mg/kg

Hexane:

...

Methanol:

Ethanol:

Isopropanol:

Ethyl acetate:

Lead

Not more than 2 mg/kg

5.3. Content

...

6. Tests

6.1. Identification

Colour reactions

Treat an aqueous or dilute ethanolic solution of the sample with hydrochloric acid until a slightly orange colour begins to appear. Divide the mixture into 2 parts and add some boric acid powder or crystals to one portion. A marked reddening will be quickly apparent, best seen by comparison with the portion to which the boric acid has not been added. The test may also be made by dipping pieces of filter paper in an ethanolic solution of colouring matter, drying at 100º, and then moistening with a weak solution of boric acid to which a few drops of hydrochloric acid have been added. On drying, a cherry-red colour will develop.

Thin Layer Chromatography

Spot 5 µl of test solution (0.01 g of sample in 1 ml of 95% ethanol) on a TLC (Microcrystalline cellulose, 0.1 mm) plate. Develop the plate in a chamber containing a mixture of 3-methyl-1-butanol/ethanol/ water/ammonia (4:4:2:1) as solvent and allow the solvent front to ascend 10-15 cm. Examine under daylight and under UV-light and observe:

Two or three yellow spots with Rf between 0.2 and 0.4 under daylight and UV-light.

Spots with R_f about 0.6 and 0.8 under UV-light

...

6.2 Purity

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. Method of assay

Accurately weigh about 0.08 g of the sample in a 200-ml volumetric flask and dissolve by shaking with ethanol. Make up to volume with ethanol and mix. Pipette 1.0 ml of solution into a 100-ml volumetric flask and make up to volume with ethanol.

Determine the absorbance (A) at 425 nm in a 1-cm cell. Calculate the total colouring matters content of the sample using the following equation:

...

Where:

A = absorbance of sample

W = weight of sample

1607 = specific absorbance of the curcumin standard in ethanol at 425 nm

The determination must be performed without delay, because the colour fades.

ANNEX 2

SPECIFICATIONS AND TESTS FOR RIBOFLAVIN

1. Synonyms

...

INS: 101i

ADI = 0 – 0.5 mg/kg bw.

2. Definition

Chemical names

Riboflavin; 3,10-dihydro-7,8-dimethyl-10-[(2S,3S,4R)-2,3,4,5- tetrahydroxypentyl]benzo-[g]pteridine-2,4-dione; 7,8-dimethyl-10-(1'-Dribityl)isoalloxazine.

C.A.S. number

83-88-5

Chemical formula

...

Structural formula

Formula weight

376.37

3. Description

Yellow to orange-yellow crystalline powder, with slight odour.

4. Functional uses

Colour.

5. Specifications

...

Solubility

Very slightly soluble in water; practically insoluble in alcohol, chloroform, acetone and ether; very soluble in dilute alkali solutions.

Spectrophotometry

Using the aqueous solution from the Assay, determine the absorbance (A) at 267 nm, 375 nm and 444 nm.

The ratio A₃₇₅/A₂₆₇ is between 0.31 and 0.33.

The ratio A₄₄₄/A₂₆₇is between 0.36 and 0.39.

Specific rotation

[a]20, D: Between -115^o and -140^o.

...

Passes test (described in “Tests”).

5.2. Purity

Loss on drying

Not more than 1.5%.

Sulfated ash

Not more than 0.1%.

Subsidiary colouring matters

Passes test (described in “Tests”).

...

Not more than 100 mg/kg calculated as aniline.

Lead

Not more than 2 mg/kg.

5.3. C₁₇H₂₀N₄O₆content

Not less than 98%.

6. Tests

6.1. Identification

Specific rotation

...

Colour reaction

Dissolve about 1 mg of sample in 100 ml of water. The solution has a pale greenish-yellow colour by transmitted light, and by reflected light has an intense yellowish-green fluorescence which disappears on the addition of mineral acids and alkalis.

6.2. Purity

Loss on drying

At 105^o, 4 h

Sulfated ash

- Tested as directed under JECFA monograph 1 - Vol. 4.

- Test 2 g of the sample

...

Prepare the standard for this test for the absence of lumiflavin by diluting 3 ml of 0.1 N potassium dichromate with water to 1000 ml. Pour some chloroform through an alumina column to remove any ethanol. To 10 ml of this chloroform add 35 mg of the sample, shake for 5 min and filter. The colour of the filtrate should be no more intense than that of 10 ml of the standard when viewed in identical containers.

Lead

Tested as directed under 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 Annex 1: General tests, “Instrumental Methods.”

6.3. Method of assay

Carry out the assay in subdued light. In a brown glass 500 ml volumetric flask, suspend 65.0 mg of the sample in 5 ml of water, ensuring that it is completely wetted, and dissolve in 5 ml of 2 N sodium hydroxide solution. As soon as dissolution is complete, add 100 ml of water and 2.5 ml of glacial acetic acid and dilute to 500.0 ml with water. Place 20.0 ml of this solution in a brown glass 200 ml volumetric flask, add 3.5 ml of a 1.4% w/v solution of sodium acetate and dilute to 200.0 ml with water. Measure the absorbance (A) at the maximum at 444 nm.

% Riboflavin = [(A×5000) / (328×W)] × 1,367

Where:

...

W = weight of sample in g.

ANNEX 3

SPECIFICATIONS AND TESTS FOR TARTRAZINE

1. Synonyms

CI Food Yellow 4, FD&C Yellow No. 5; CI (1975) No. 19140

INS: 102

ADI = 0 - 7.5 mg/kg bw.

2. Definition

...

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

Trisodium4,5-dihydro-5-oxo-1-(4-sulfophenyl)-4-[(4-sulfophenyl)azo]- 1H-pyrazole-3-carboxylate

C.A.S. number

1934-21-0

Chemical formula

C₁₆H₉N₄Na₃O₉S₂

Structural formula

...

534.37

3. Description

Light orange powder or granules.

4. Functional uses

Colour.

5. Specifications

5.1. Identification

Solubility

...

Identification of colouring matters

Passes test.

5.2. Purity

Loss on drying at 135 °C

Not more than 15% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

Not more than 0.2%.

Lead

...

Subsidiary colouring matters

Not more than 1%.

Organic compounds other than colouring matters

Not more than 0.5% sum of 4-hydrazinobenzenesulfonic acid, 4- aminobenzenesulfonic acid, 5-oxo-1-(4-sulfophenyl)-2-pyrazoline-3- carboxylic acid, 4,4'-(diazoamino)dibenzenesulfonic acid, tetrahydroxysuccinic acid.

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

Ether extractable matter

Not more than 0.2%.

...

Not less than 85% total colouring matters

6. Tests

6.1. Purity

Lead

- Tested as directed under 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, Volume 4, “Instrumental Methods.”

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

...

Organic compounds other than colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use HPLC under the following conditions: HPLC elution gradient: 2 to 100% at 2% per min.

6.2. Method of assay

Proceed as directed under Total Content by Titration with Titanous Chloride (in Annex 1: General tests), using the following:

Weight of sample: 0.6 - 0.7 g;

Buffer: 15 g sodium hydrogen tartrate;

...

ANNEX 4

SPECIFICATIONS AND TESTS FOR QUINOLINE

1. Synonyms

CI Food Yellow 13, CI (1975) No. 47005,

INS: 104

ADI = 0 – 10 mg/kg bw.

2. Definition

Prepared by sulfonating 2-(2-quinolyl)-1,3-indandione or a mixture containing about two-thirds 2-(2-quinolyl)-1,3-indandione and one-third 2- [2-(6-methyl-quinolyl)]1,3-indandione; consists essentially of sodium salts of a mixture of disulfonates (principally), monosulfonates and trisulfonates of the above compounds and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components.

...

Chemical names

Disodium 2-(1,3-dioxo-2-indanyl)-6,8-quinolinesulfates; disodium 2-(2- quinolyl)-indan-1,3-dionedisulfonates (principal component)

C.A.S. number

8004-72-0 (Unmethylated disulfonic acids)

Chemical formula

C₁₈H₉NNa₂O₈S₂ (principal component)

Structural formula

Principal component

...

8 salt: R₁, R₂= SO₃H

Formula weight

477.38 (Principal components)

3. Description

Yellow powder or granules.

4. Functional uses

Colour.

5. Specifications

5.1. Identification

...

Solubility

Soluble in water; sparingly soluble in ethanol

Identification of colouring matters

Passes test.

5.2. Purity

Loss on drying at 135°C

Not more than 30% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

...

Lead

Not more than 2 mg/kg.

Zinc

Not more than 50 mg/kg.

Subsidiary colouring matters

Not more than 4 mg/kg: of 2-(2-quinolyl)-1,3-indandione and 2-[2-(6- methylquinolyl)]-1,3-indandione

Organic compounds other than colouring matters

Not more than 0.5%, sum of 2-methylquinoline, 2-methylquinolinesulfonic acid, phthalic acid, 2,6-dimethylquinoline, 2,6-dimethylquinolinesulfonic acid

Unsulfonated primary aromatic amines

...

Ether extractable matter

Not more than 0.2%.

5.3. Content

Not less than 70% total colouring matters. Quinoline Yellow prepared from 2-(2-quinolyl)-1,3-indandione (only) shall have the following composition:

- not less than 80% shall be disodium 2-(2-quinolyl)-indan1,3-dionedisulfonates;

- not more than 15% shall be sodium 2-(2-quinolyl)-indan-1,3- dionemonosulfonates;

- not more than 7% shall be trisodium 2-(2-quinolyl)-indan-1,3- dionetrisulfonate;

6. Tests

6.1. Purity

...

Lead

Tested as directed under 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, Volume 4, “Instrumental Methods.”

Subsidiary colouring matters

Limit test for 2-(2-quinolyl)-1,3-indandione and 2-[2-(6-methyl-quinolyl)]- 1,3-indandione.

Use the apparatus and ether quality described in Ether Extractable Matter and carry out an extraction following the details given there under. Wash the ether extract with two 25-ml portions of water. Evaporate the ether extract to about 5 ml and then transfer it to an oven at 105^o to remove the remaining ether. Dissolve the residue in chloroform, and dilute the solution to exactly 10 ml. Determine the absorbance at the wavelength of maximum absorption (approximately 420 nm) using chloroform as the reference solution. The absorbance corresponding to the limit figure of 4 mg/kg 2-(2-quinolyl-1,3-indandione is 0.27. Any 2-[2-(6-methylquinolyl)]- 1,3-indandione is assessed as 2-(2-quinolyl)-1,3-indandione.

Organic compounds other than colouring matters

Organic compounds other than colouring matters in Quinoline prepared from 2-(2-quinolyl)-1,3-indandione (only).

Determine by liquid chromatography, using the following conditions:

Instrument: High performance liquid chromatograph fitted with a gradient elution accessory.

...

Column: 250 x 4 nm, Nucleosil C18, 7 µm.

Solvent system:

A: Acetate Buffer pH 4.6: water (1:10) (Acetate Buffer is 1M NaOH:1 M acetic acid:water (5:10:35).

B: (A):methanol (20:80).

Sample concentration: 1% weight/volume in Solvent A.

Gradient:

Min

...

100

...

100

Flow rate: 1 ml/min

6.2. Method of assay

...

Proceed as directed under Total Content by Spectrophotometry (JECFA monograph 1 - Vol. 4).

Solvent: pH 7 phosphate buffer

Dilution of solution A: 10 ml to 250 ml

Absorptivity (a): 86.35

Approximate wavelength of maximum absorption: 415 nm

Determination of the percentages of di-, mono- and trisulfonates in Quinoline prepared from 2-(2-quinolyl)-1,3-indandione (only): Use the HPLC conditions prescribed in the Determination of Organic Compounds other than Colouring Matters with a sample solution of concentration 0.05% in HPLC Solvent A in place of the sample solution of concentration 1%. Express the results as percentages of the Total colouring matters present.

...

SPECIFICATIONS AND TESTS FOR SUNSET YELLOW FCF

1. Synonyms

CI Food Yellow 3, FD&C Yellow No. 6; CI (1975) No. 15985

INS: 110

ADI = 0 - 2.5 mg/kg bw.

2. Definition

Consists essentially of disodium 6-hydroxy-5-(4-sulfonatophenylazo)-2- naphthalene-6-sulfonate and subsidiary colouring matters together with NaCl and/or Na₂SO₄as the principal uncoloured components.

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

...

C.A.S. number

2783-94-0

Chemical formula

C₁₆H₁₀N₂Na₂O₇S₂

Structural formula

Formula weight

452.38

3. Description

...

4. Functional uses

Colour.

5. Specifications

5.1. Identification

Solubility

Soluble in water; sparingly soluble in ethanol.

Identification of colouring matters

Passes test.

...

Loss on drying at 135°C

Not more than 15% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

Subsidiary colouring matters

Not more than 5%.

...

Organic compounds other than colouring matters

Not more than 0.5%, sum of 4-amino-1-benzenesulfonic acid;

3-hydroxy2,7-naphthalenedisulfonic acid;

6-hydroxy-2-naphthalenesulfonic acid;

7- hydroxy-1,3-naphthalenedisulfonic acid;

4,4'- diazoaminodibenzenesulfonic acid;

6,6'-oxydi-2-naphthalenesulfonic acid.

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

...

Not more than 0.2%.

5.3. Content

Not less than 85% total colouring matters

6. Tests

6.1. Purity

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

...

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions: Developing solvent: No. 4; Height of ascent of solvent front: approximately 17 cm.

Organic compounds other than colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Proceed as directed under Determination by High Performance Liquid Chromatography using an elution gradient of 2 to 100% at 4% per min followed by elution at 100%.

6.2. Method of assay

Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following:

...

Buffer: 10 g sodium citrate;

Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl₃: 11.31 mg.

ANNEX 6

SPECIFICATIONS AND TESTS FOR CARMINES

1. Synonyms

Cochineal carmin, CI Natural Red 4, CI (1975) No. 75470,

INS 120

A group ADI of 0-5 mg/kg bw for carmines, as ammonium carmine or the equivalent of Ca, K and Na salts.

...

Obtained by aqueous extraction of cochineal, which consists of the dried bodies of the female insect Dactylopius coccus Costa; the colouring principle is a hydrated aluminium chelate of carminic acid in which aluminium and carminic acid are thought to be present in the molar ratio 1:2.

In commercial products the colouring principle is present in association with ammonium, calcium, potassium or sodium cations, singly or in combination, and these cations may also be present in excess. Products may also contain proteinaceous material derived from the source insect, and may also contain free carminate or a small excess of aluminium cations.

Chemical names

Hydrated aluminium chelate of carminic acid (7-beta-D-glucopyranosyl3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-anthracene-2-carboxylic acid)

C.A.S. number

1390-65-4 (carmine)

1260-17-9 (carminic acid)

Chemical formula

Carminic acid: C₂₂H₂₀O₁₃

...

Carminic acid

Aluminium complex of carminic acid

Formula weight

Carminic acid: 492.39

...

Red to dark red, crumbly solid or powder

4. Functional uses

Colour

5. Specifications

5.1. Identification

Solubility

The solubility of carmine preparations varies depending on the nature of the cations present. Products where the major cation is ammonium (ammonium carminate) are freely soluble in water at pH 3.0 and pH 8.5. Products where the major cation is calcium (calcium carminate) are very slightly soluble in water at pH 3.0 but freely soluble at pH 8.5.

Colour reaction

...

5.2. Purity

Loss on drying

Not more than 20%.

Total ash

Not more than 12%.

Protein

Not more than 25%.

Matter insoluble in dilute ammonia

...

Lead

Not more than 2 mg/kg.

Microbiological criteria

Salmonella: Negative per test.

5.3. C₂₂H₂₀O₁₃content

Not less than 50% of C₂₂H₂₀O₁₃on the dry basis.

6. Tests

6.1 Identification

...

Colour reactions

- Make a solution of the sample slightly alkaline by adding 1 drop of 10% sodium hydroxide or potassium hydroxide solution. A violet colour is produced.

- Add a small sodium dithionite (Na₂S₂O₄) crystal to acid, neutral or alkaline solutions of the sample. The solutions are not decolourized.

- Dry a small quantity of the sample in a porcelain dish. Cool thoroughly and treat the dry residue with 1 or 2 drops of cold sulfuric acid TS. No colour change occurs.

- Acidify a dispersion of the sample in water with 1/3 volume of hydrochloric acid TS and shake it with amyl alcohol. Wash the amyl alcohol solution 2-4 times with an equal volume of water to remove hydrochloric acid. Dilute the amyl alcohol solution with 1-2 volumes of petroleum ether (40-60^o) and shake with a few small portions of water to remove colour. Add, dropwise, 5% uranium acetate, shaking thoroughly after each addition. A characteristic emerald-green colour is produced.

6.2 Purity

Loss on drying

- Tested as directed under JECFA monograph 1-Vol. 4.

...

Total ash

- Tested as directed under JECFA monograph 1-Vol. 4.

- Test 1 g of the sample as directed in the test for Ash.

Protein

- Tested as directed under JECFA monograph 1-Vol. 4.

- Proceed as directed under Nitrogen Determination (non-ammonia N x 6.25)

Matter insoluble in dilute ammonia

Dissolve about 0.25 g of the sample, previously dried and accurately weighed, in 2.5 ml of dilute ammonia solution (160 ml of strong ammonia TS, made up to 500 ml) and dilute to 100 ml with water: the solution is clear. Filter through a sintered glass filter. Wash with a 0.1% ammonia solution and dry to constant weight at 105º.

Lead

...

6.3. Method of assay

Weigh accurately about 100 mg of the sample, dissolve in 30 ml of boiling 2N hydrochloric acid and cool. Transfer quantitatively to a 1000-ml volumetric flask, dilute to volume with water, and mix. Determine the absorbance of the solution in a 1 cm cell at the wavelength of maximum absorbance (about 494 nm) using water as the blank. Calculate the percentage of carminic acid in the sample analysis using the formula:

(100 × A × 100) / (1,39 × W)

where:

A = absorbance of the sample solution;

W = weight, in mg, of the sample taken; and

1.39 = absorbance of a solution of carminic acid having a concentration of 100 mg per 1000 ml;

...

ANNEX 7

SPECIFICATIONS AND TESTS FOR CARMOISINE

1. Synonyms

CI Food Red 3, Azorubine; CI (1975): 14720

INS: 122

ADI = 0 - 4 mg/kg bw.

2. Definition

Consists essentially of disodium 4-hydroxy-3-(4-sulfonato-1-naphthylazo)- 1-naphthalenesulfonate and subsidiary colouring matters together with NaCl and/or Na₂SO₄ as the principal uncoloured components.

...

Chemical names

Disodium 4-hydroxy-3-(4-sulfonato-1-naphthylazo)-1-naphthalenesulfonate.

C.A.S. number

3567-69-9

Chemical formula

C₂₀H₁₂N₂Na₂O₇S₂

Structural formula

Formula weight

...

3. Description

Red powder or granules.

4. Functional uses

Colour.

5. Specifications

5.1. Identification

Solubility

Soluble in water; sparingly soluble in ethanol.

...

Passes test.

5.2. Purity

Loss on drying at 135 °C

Not more than 15% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

...

Not more than 1%.

Organic compounds other than colouring matters

Not more than 0.5% of 4-Amino-1-naphthalenesulfonic acid and 4-Hydroxy-1-naphthalenesulfonic acid together.

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

Ether extractable matter

Not more than 0.2%.

5.3. Content

Not less than 85% total colouring matters.

...

6.1 Purity

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions: Developing solvent: No. 4; Height of ascent of solvent front: approximately 17 cm.

Organic compounds other than colouring matters

...

- Use HPLC under the following conditions: HPLC elution gradient: 1 to 100% at 2.0% per min.

6.3. Method of assay

Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following:

Weight of sample: 0.5 - 0.6 g;

Buffer: 15 g sodium hydrogen tartrate;

Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl₃: 12.56 mg

...

SPECIFICATIONS AND TESTS FOR AMARANTH

1. Synonyms

CI Food Red 9, Naphtol Rot S.; CI (1975): 16185

INS: 123

ADI = 0 - 0.5 mg/kg bw.

2. Definition

Consists essentially of trisodium 3-hydroxy-4-(4-sulfonato-1-naphthylazo)- 2,7-naphthalenedisulfonate and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components.

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

...

C.A.S. number

915-67-3

Chemical formula

C₂₀H₁₁N₂Na₃O₁₀S₃

Structural formula

Formula weight

604.48

3. Description

...

4. Functional uses

Colour.

5. Specifications

5.1. Identification

Solubility

Soluble in water; sparingly soluble in ethanol

Identification of colouring matters

Passes test.

...

Loss on drying at 135 °C

Not more than 15% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

Subsidiary colouring matters

Not more than 3%.

...

Not more than 0.5%, sum of 4-amino-1-benzenesulfonic acid;

3- hydroxy-2,7-naphthalenedisulfonic acid, 6-hydroxy-2-naphthalenesulfonic acid, 7-hydroxy-1,3-naphthalenedisulfonic acid and 7-hydroxy-1,3,6- naphthalene-trisulfonic acid.

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

Ether extractable matter

Not more than 0.2%.

5.3. Content

Not less than 85% total colouring matters.

6. Tests

...

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions: Developing solvent: No. 4; Height of ascent of solvent front: approximately 17 cm, then 1 h further development.

Organic compounds other than colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

...

6.2. Method of assay

Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following:

Weight of sample: 0.7 - 0.8 g;

Buffer: 10 g sodium citrate;

Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3: 15.11 mg.

ANNEX 9

...

1. Synonyms

CI Food Red 7, Cochineal Red A ; New Cochineal;

CI (1975): 16255

INS: 124

ADI = 0 - 4 mg/kg bw.

2. Definition

Consists essentially of trisodium d-2-hydroxy-1-(4-sulfonato-1- naphthylazo)-6,8-naphthalenedisulfonate, and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components.

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

...

C.A.S. number

2611-82-7

Chemical formula

C₂₀H₁₁N₂Na₃O₁₀S₃.1,5H₂O

Structural formula

Formula weight

631.51

3. Description

...

4. Functional uses

Colour.

5. Specifications

5.1. Identification

Solubility

Soluble in water; sparingly soluble in ethanol.

Identification of colouring matters

Passes test.

...

Loss on drying at 135 °C

Not more than 20% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

Subsidiary colouring matters

Not more than 1%.

...

Not more than 0.5% of sum of: 4-amino-1-naphthalenesulfonic acid, 7-hydroxy-1,3- naphthalenedisulfonic acid, 3-hydroxy-2,7-naphthalenesulfonic acid, 6- hydroxy-2-naphthalenesulfonic acid, 7-hydroxy-1,3,6- naphthalenetrisulfonic acid.

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

Ether extractable matter

Not more than 0.2%.

5.3. Content

Not less than 85% total colouring matters

6. Tests

6.1. Purity

...

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions: Developing solvent: No. 3; Height of ascent of solvent front: approximately 17 cm, then 1 h further development

Organic compounds other than colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use HPLC under the following conditions: HPLC elution gradient: 2 to 100% at 2% per min.

...

Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following:

Weight of sample: 0.7 - 0.8 g;

Buffer: 10 g sodium citrate;

Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl₃: 15.78 mg.

ANNEX 10

SPECIFICATIONS AND TESTS FOR ERYTHROSINE

...

CI Food Red 14, FD&C Red No. 3; CI (1975): 45430

INS 127

ADI = 0 - 0,1 mg/kg bw

2. Definition

Consists essentially of disodium salt of 9-(o-carboxyphenyl)-6-hydroxy2,4,5,7-tetraiodo-3-isoxanthone monohydrate and subsidiary colouring matters together with water, sodium chloride and/or sodium sulfate as the principal uncoloured components.

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

Disodium salt of 9-(o-carboxyphenyl)-6-hydroxy-2,4,5,7-tetraiodo-3- isoxanthone monohydrate

C.A.S. number

...

Chemical formula

C₂₀H₆I₄Na₂O₅.H₂O

Structural formula

Formula weight

897.88

3. Description

Red powder or granules.

4. Functional uses

...

5. Specifications

5.1. Identification

Solubility

Soluble in water, slightly soluble in ethanol

Identification of colouring matters

Passes test.

5.2. Purity

...

Not more than 13% together with chloride and sulfate calculated as sodium salts.

Inorganic iodides

Not more than 0.1% calculated as sodium iodide.

Water insoluble matter

Not more than 0.2%.

Zinc

Not more than 50 mg/kg.

Lead

Not more than 2 mg/kg.

...

Not more than 4% (except fluorescein).

Fluorescein

Not more than 20 mg/kg.

Organic compounds other than colouring matters

Tri-iodoresorcinol: Not more than 0.2%

2-(2,4-dihydroxy-3,5-di-iodobenzoyl) benzoic acid: Not more than 0.2%

Ether extractable matter

From a solution of pH not less than 7, not more than 0.2%.

5.3. Content

...

6. Tests

6.1. Purity

Inorganic iodides

Weigh 1.0 g of the sample into a 100-ml beaker. Add 75 ml distilled water and the magnetic follower. Stir to dissolve. Immerse a iodide specific electrode and a reference electrode in the solution and set a suitable millivoltmeter to read the potential of the system in millivolts.

Add 0.001 M silver nitrate solution from a burette initially in 0.5 ml aliquots, reducing these to 0.1 ml as the end-point approaches as indicated by an increasing change in potential for each addition. After allowing time for the reading to stabilize, record the millivolt readings after each addition. Continue the titration until further additions make little change in the potential. Plot the millivolt readings against the volume of silver nitrate solution added. The equivalent point is the volume corresponding to the maximum slope of the curve.

The percentage of sodium iodide in sample is: Titre x 0.015%.

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

...

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions: Developing solvent: No. 5; Height of ascent of solvent front: approximately 17 cm. Note: Take special care not to allow the chromatograms to be exposed to direct sunlight.

Organic compounds other than colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Proceed as directed under Column chromatography, using, for example, the following absorptivities:

Tri-iodoresorcinol: 0.079 mg L^-1cm^-1 at 233 nm (acidic).

2(2,4-dihydroxy-3,5-di-iodobenzoyl)benzoic acid: 0.047 mg L^-1cm^-1 at 348 nm (alkaline).

Fluorescein

...

The fluorescein is separated from the sample by TLC and compared with a standard chromatogram prepared from fluorescein at the concentration corresponding to the limit figure.

Solvent:

Methanol+water+ammonia (s.g. 0.890) (500 ml+400 ml+100 ml).

Sample:

Weigh 1.0 g of the sample, dissolve in about 50 ml solvent and dilute to 100 ml in a volumetric flask.

Standard:

Weigh an amount of fluorescein, previously purified by recrystallisation from ethanol, equal to 1 g x the colouring matter content of the sample as determined under Assay. Dissolve in water (or in water with 10 ml ammonia s.g. 0.890 if fluorescein-free acid is being used) and dilute to 100 ml. Make further sequential dilutions as follows:

1 ml to 100 ml with water.

...

Chromatography solvent:

n-Butanol+water+ammonia (s.g. 0.890)+ethanol (100 ml+44 ml+1 ml+ 22.5 ml).

Procedure:

Spot 25 µl of the sample and standard solutions side by side on a cellulose plate. Develop for 16 h in the chromatography solvent. Allow the plate to dry. View under a UV light source and compare the fluorescence of the standard with the fluorescence of the corresponding area on the chromatogram of the sample. The intensity of the latter shall not be greater than that of the former.

Note: Take special care not to allow the chromatograms to be exposed to direct sunlight.

6.2. Method of assay

Dissolve about 1 g of the sample, accurately weighed, in 250 ml of water, transfer to a clean 500-ml beaker, add 8.0 ml of 1.5 N nitric acid and stir well. Filter through a sintered glass crucible (porosity 3, diameter 5 cm) which has been weighed containing a small glass stirring rod. Wash thoroughly with 0.5% nitric acid until the filtrate gives no turbidity with silver nitrate TS, and then wash with 30 ml water. Dry to constant weight at 135±5º, carefully breaking up the precipitate by means of the glass rod. Cool in a desiccator and weigh

...

Determination of Hydrochloric Acid-insoluble Matter in Erythrosine Lake

Reagents:

Concentrated hydrochloric acid

Hydrochloric acid 0.5% v/v

Dilute ammonia solution (dilute 10 ml ammonia, s.g. 0.890 to 100 ml with water).

Procedure:

Accurately weigh approximately 5 g of the lake into a 500-ml beaker. Add 250 ml water and 60 ml concentrated hydrochloric acid. Boil to dissolve the alumina while the Erythrosine converts to its "free acid" form, which is insoluble in acid. Filter through a tared No. 4 sintered glass crucible. Wash the crucible with a small amount of hot 0.5% hydrochloric acid and then with some hot distilled water. Remove the acid filtrate from the filter flask, replace the crucible and wash with hot dilute ammonia solution until the washings are colourless. Dry the crucible to constant weight at 135º. Express the residue as a percentage of the weight taken.

...

ANNEX 11

SPECIFICATIONS AND TESTS FOR RED 2G

1. Synonyms

CI Food Red 10, Azogeranine; CI (1975): 18050

INS: 128

ADI = 0 - 1 mg/kg bw.

2. Definition

Consists essentially of disodium 8-acetamido-1-hydroxy-2-phenylazo3,6-naphthalenedisulfonate and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components.

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

...

Disodium 8-acetamido-1-hydroxy-2-phenylazo-3,6-naphthalenedisulfonate

C.A.S. number

3734-67-6

Chemical formula

C₁₈H₁₃N₃Na₂O₈S₂

Structural formula

Formula weight

509.43

...

Red powder or granules.

4. Functional uses

Colour.

5. Specifications

5.1. Identification

Solubility

Freely soluble in water; sparingly soluble in ethanol.

Identification of colouring matters

...

5.2. Purity

Loss on drying at 135 °C

Not more than 20% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

Subsidiary colouring matters

...

Organic compounds other than colouring matters

Not more than 0.3% of sum of 5-acetamido-4-hydroxy-2,7-naphthalene2,7-disulfonic acid and 5-Amino-4-hydroxy-2,7-naphthalene-2,7- disulfonic acid.

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

Ether extractable matter

Not more than 0.2%.

5.3. Content

Not less than 80% total colouring matter.

6. Tests

...

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions: Developing solvent: No. 4; Height of ascent of solvent front: approximately 17 cm.

Organic compounds other than colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

...

6.2. Method of assay

Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following:

Weight of sample: 0.6 - 0.7 g;

Buffer: 15 g sodium hydrogen tartrate;

Weight: (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl₃: 12,74 mg

ANNEX 12

...

1. Synonyms

CI Food Red 17, FD&C Red No. 40; CI (1975): 16035

INS: 129

ADI = 0 - 7 mg/kg bw.

2. Definition

Consists of disodium 6-hydroxy-5-(2-methoxy-5-methyl-4- sulfonatophenylazo)-2-naphthalenesulfonate and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components.

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

Disodium 6-hydroxy-5-(2-methoxy-5-methyl-4-sulfonatophenylazo)-2- naphthalenesulfonate

...

25956-17-6

Chemical formula

C₁₈H₁₄N₂Na₂O₈S₂

Structural formula

Formula weight

496.43

3. Description

Dark red powder or granules.

...

Colour.

5. Specifications

5.1. Identification

Solubility

Soluble in water; sparingly soluble in ethanol.

Identification of colouring matters

Passes test.

5.2. Purity

...

Loss on drying at 135 °C

Not more than 15% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

Subsidiary colouring matters

Not more than 3%.

Organic compounds other than colouring matters

...

Not more than 0.2% of 4-amino-5-methoxy-2-methyl-benzene-sulfonic acid;

Not more than 1.0% of disodium 6,6'-oxybis(2-naphthalene-sulfonate);

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

Ether extractable matter

Not more than 0.2%.

5.3. Content

Not less than 85% total colouring matters

6. Tests

...

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions: Developing solvent: No. 4; Height of ascent of solvent front: approximately 17 cm.

Organic compounds other than colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

...

6.2. Method of assay

Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following:

Weight of sample: 0.5 - 0.6 g;

Buffer: 15 g sodium hydrogen tartrate;

Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl₃: 12.41 mg.

ANNEX 13

...

1. Synonyms

Indigocarmin; CI Food Blue 1, FD&C Blue No. 2; CI (1975): 73015

INS: 132

ADI = 0 - 5 mg/kg bw.

2. Definition

Consists essentially of a mixture of disodium 3,3' -dioxo-[delta^2,2'- biindoline]-5,5'-disulfonate (principal component) and disodium 3,3'- dioxo-[delta^2,2'-biindoline]-5,7'-disulfonate (isomer) and subsidiary colouring matters together with NaCl and/or Na2SO4 as the principal uncoloured components.

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

Disodium 3,3'-dioxo-[delta^2,2'-biindoline]-5,5'-disulfonate (principal component)

...

860-22-0 (5,5' isomer)

Chemical formula

C₁₆H₈N₂Na₂O₈S₂

Structural formula

Formula weight

466.36

3. Description

Blue powder or granules.

...

Colour.

5. Specifications

5.1. Identification

Solubility

Soluble in water; sparingly soluble in ethanol.

Identification of colouring matters

Passes test.

5.2. Purity

...

Loss on drying at 135 °C

Not more than 15% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

Subsidiary colouring matters

Not more than 1% (except disodium 3,3'-dioxo-[delta^2,2'-biindoline]- 5,7'- disulfonate).

Organic compounds other than colouring matters

...

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

Ether extractable matter

Not more than 0.2%.

5.3. Content

Not less than 85% total colouring matters.

Not more than 18% of disodium 3,3'-dioxo-[delta^2,2'-biindoline]-5,7'- disulfonate

6. Tests

6.1. Purity

...

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions: Developing solvent: No. 3; Height of ascent of solvent front: approximately 17 cm.

Note 1. The 5,7' isomer is separated as a wide blue zone just in front of the main blue band. Do not include this zone in the subsidiary colouring matter zones which are cut out and measured.

Note 2. The 15 ml sodium hydrogen carbonate solution used in the general procedure is replaced by 15 ml 0.05 N hydrochloric acid in order to avoid the decomposition which the sulfonated indigo undergoes in alkaline solution.

Organic compounds other than colouring matters

...

- Use liquid chromatography under the following conditions: HPLC elution gradient: 2 to 100% gradient followed by elution at 100%.

Ether extractable matter

- Tested as directed under JECFA monograph 1-Vol. 4.

- Weigh accurately about 2 g sample instead of the 5 g stated in the general methods.

6.2. Method of assay

Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following:

Weight of sample: 1.0 - 1.1 g;

...

Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl₃: 23.32 mg.

Isomer content by paper chromatography:

Refer to the conditions for the determination of subsidiary colouring matters. Cut the isomer band from the chromatogram in the manner detailed for the subsidiary bands, extract into solvent and measure the absorbance at its Smax. Measure the absorbance of the corresponding blank at the same wavelength. As a standard use 0.1 ml of an 0.20% solution of the sample applied to the 18 cm x 0.7 cm rectangle.

Isomer expressed as a percentage of the sample

% = [A / A_s] x 20% x [D / 100]

where:

A is the net absorbances (of the corresponding blank) of the isomer.

A_s is the net absorbances (of the corresponding blank) of the standard.

D is the total colouring matters content of the sample.

...

The 5,7' isomer separates under the HPLC conditions detailed above for the separation of subsidiary colouring matters, and the amount present can be quantified.

ANNEX 14

SPECIFICATIONS AND TESTS FOR SUNSET BRILLIANT BLUE FCF

1. Synonyms

CI Food Blue 2, FD&C Blue No. 1; CI (1975): 42900

INS: 133

ADI = 0 - 1 mg/kg bw.

2. Definition

...

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

Disodium 3-[N-ethyl-N-[4-[[4-[N-ethyl-N-(3-sulfonatobenzyl)- amino]phenyl](2-sulfonatophenyl)methylene]-2,5-cyclohexa-diene-1- ylidene]ammoniomethyl]-benzenesulfonate.

Disodium I-[4-(N-ethyl-3-sulfonatobenzylamino)phenyl]-I- [4-(N-ethyl-3- sulfonatobenzyliminio)cyclohexa-2,5-dienyli-dene]toluene-2-sulfonate (an alternative chemical name).

C.A.S. number

3844-45-9

Chemical formula

C₃₇H₃₄N₂Na₂O₉S₃

Structural formula

...

Formula weight

792.86

3. Description

Blue powder or granules.

4. Functional uses

Colour.

5. Specifications

5.1. Identification

...

Soluble in water; sparingly soluble in ethanol.

Identification of colouring matters

Passes test.

5.2. Purity

Loss on drying at 135 °C

Not more than 15% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

Not more than 0.2%.

...

Not more than 2 mg/kg.

Chromium

Not more than 50 mg/kg.

Subsidiary colouring matters

Not more than 6%.

Organic compounds other than colouring matters

Not more than 1.5%, sum of 2-, 3- and 4-formylbenzenesulfonic acids.

Not more tan 0.3% 3-[[N-ethyl-N-(4-sulfophenyl) amino] methyl] benzenesulfonic acid.

Leuco base

...

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

Ether extractable matter

Not more than 0.2%.

5.3. Content

Not less than 85% total colouring matter.

6. Tests

6.1. Purity

...

- Tested as directed under JECFA monograph 1-Vol. 4.

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions: Developing solvent: No. 4; Develop chromatogram for approximately 20 hours.

Organic compounds other than colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Proceed as directed under Column chromatography, using, for example, the following absorptivities:

3-formylbenzenesulfonic acid: 0.0495 mgL^-1/cm^-1 at 246 nm in dilute HCl.

...

Leuco base

Tested as directed under JECFA monograph 1-Vol. 4. Weigh accurately 120±5 mg sample and proceed as directed under Leuco Base in Sulfonated Triarylmethane Colours (Vol. 4) Absorptivity (a) = 0.164 mgL^-1cm^-1at 629 nm Ratio = 0.9706.

6.2. Method of assay

Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following:

Weight of sample: 1.8 - 1.9 g;

Buffer: 15 g sodium hydrogen tartrate;

Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl₃: 39.65 mg.

...

ANNEX 15

SPECIFICATIONS AND TESTS FOR CHLOROPHYLLS

1. Synonyms

Magnesium chlorophyll, magnesium phaeophytin;

Clorophyllin; C.I. Natural Green 3; C.I. (1975): 75810;

INS 140

ADI “Not limited”.

2. Definition

Obtained by solvent extraction of grass, lucerne, nettle and other plant material; during the subsequent removal of solvent, the naturally present coordinated magnesium may be wholly or partly removed from the chlorophylls to give the corresponding phaeophytins; the principal colouring matters are the phaeophytins and magnesium chlorophylls. The extracted product, from which the solvent has been removed, contains other pigments such as carotenoids as well as oils, fats and waxes derived from the source material. Only the following solvents may be used for the extraction: acetone, dichloromethane, methanol, ethanol, propan-2-ol and hexane.

...

The major colouring principles are:

Phytyl (13²R,17S,18S)-3-(8-ethyl-13²-methoxycarbonyl-2,7,12,18-tetramethyl13'-oxo-3-vinyl-13¹ -13² -17,18-tetrahydrocyclopenta [at]-prophylrin-17- yl)propionate, (Pheophytin a), or as the magnesium complex (Chlorophyll a).

Phytyl (13²R,17S,18S)-3-(8-ethyl-7-formyl-132 -methoxycarbonyl-2,12,18- trimethyl-13'-oxo-3-vinyl-13¹ -13²-17,18-tetrahydro-cyclopenta [at]-prophylrin17-yl)propionate, (Pheophytin b), or as the magnesium complex (Chlorophyll b).

C.A.S. number

Phaeophytin a, Magnesium complex: 479-61-8

Phaeophytin b, Magnesium complex: 519-62-0

Chemical formula

Phaeophytin a Magnesium complex (Chlorophyll a): C₅₅H₇₂MgN₄O₅

Phaeophytin a: C₅₅H₇₄N₄O₅

...

Phaeophytin b: C₅₅H₇₂N₄O₆

Structural formula

where:

X = CH3 for the "a" series

X = CHO for the "b" series.

Loss of Mg converts chlorophyll into the corresponding phaeophytin

Formula weight

Phaeophytin a magnesium complex (Chlorophyll a): 893.51

...

Phaeophytin b magnesium complex (Chlorophyll b): 907.49

Phaeophytin: 885.20

3. Description

Waxy solid ranging in colour from olive green to dark green depending on the content of co-ordinated magnesium.

4. Functional uses

Colour.

5. Specifications

5.1. Identification

...

Insoluble in water; soluble in ethanol, diethyl ether, chloroalkanes, hydrocarbons and fixed oils.

Thin-layer chromatography

Passes test (described in “Tests”).

5.2. Purity

Residual solvents

Acetone, methanol, ethanol, propan-2-ol, hexane: Not more than 50 mg/kg, singly or in combination.

Dichloromethane: Not more than 10 mg/kg

Arsenic

...

Not more than 3 mg/kg.

(Tested as directed under JECFA monograph 1-Vol. 4 - Method II).

Lead

Not more than 5 mg/kg.

(Tested as directed under 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.”

5.3. Content

Content of total combined phaeophytins and their magnesium complexes is not less than 10%.

6. Tests

6.1. Identification

...

Thin-layer chromatography

The 5% ethanol in the solvent mixture is in addition to this), until the solvent ascends to a point 15 cm above the initial spots. Allow the solvent to evaporate, then visually examine the separated spots and identify the components of interests by their R_f values and colours. Approximate R_f values and colour of the spots are as follows:

Phaeophytin a: 0.77; grey/brown

Phaeophytin b: 0.75; yellow/brown

Chlorophyll a: 0.50; blue/green

Chlorophyll b: 0.63; yellow/green

In addition spots may be visible for b-carotene at Rf 0.81 and xanthophyll at R_f 0.47 and 0.23.

...

Residual solvents

Tested as directed under JECFA monograph 1-Vol. 4.

Determine by gas chromatographically using either the method of entrainment distillation (Determination of Residual Solvents) or headspace analysis (Limit Test for Solvent Residues).

Arsenic

- Tested as directed under JECFA monograph 1-Vol. 4.

- Method II.

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

...

6.1. Method of assay

Accurately weigh about 100 mg of the sample and dissolve in diethyl ether, making the volume to 100 ml. Dilute 2 ml of this solution to 25 ml with diethyl ether. The concentration of the sample should not give an absorbance at 660.4 nm that is in excess of the working range for Absorbance measurements, i.e., not in excess of 0.7.

Measure the absorbances of the solution in a 1 cm cell against a diethyl ether blank at 660.4 nm, 642.0 nm, 667.2 nm and 654.4 nm. (These being the absorbance maxima in diethyl ether for chlorophyll a, chlorophyll b, phaeophytin a, and phaeophytin b, respectively). In addition measure at 649.8 nm and 628.2 nm. To the remaining diluted solution add one crystal of oxalic acid and after dissolution and mixing, remeasure the absorbances at the same wavelengths. "dA" is the difference between the absorbances between the absorbance at the respective wavelengths, before and after addition of oxalic acid.

Calculate the concentration of the individual compounds in mmol/L from the following equations:

Chlorophyll a = 17.7 d A (660.4 nm) + 7.15 d A (642.0 nm) Chlorophyll b = 19.4 d A (642.0 nm) - 2.92 d A (660.4 nm) Phaeophytin a = -4.89 A (649.8 nm) + 0.0549 A (628.2 nm) +18.7 A (667.2 nm) + 0.0575 A (654.4 nm) - chlorophyll a Phaeophytin b = -71.0 A (649.8 nm) + 2.51 A (628.2 nm) - 13.5 A (667.2 nm) + 84.3 A (654.4 nm) - chlorophyll b

Convert the figures in mmol/L to percentages using the following equations:

...

ANNEX 16

SPECIFICATIONS AND TESTS FOR CHLOROPHYLLS, COPPER COMPLEXES

...

Copper chlorophyllin, copper phaeophytin

C.I. Natural Green 3; C.I. (1975) No. 75810;

INS: 141i

ADI = 0 - 15 mg/kg bw.

2. Definition

Obtained by addition of an organic salt of copper to the substance obtained by solvent extraction of grass, lucerne, nettle and other plant material; the product, from which the solvent has been removed, contains other pigments such as carotenoids as well as fats and waxes derived from the source material. The principal colouring matters are the copper phaeophytins. Only the following solvents may be used for the extraction: acetone, dichloromethane, methanol, ethanol, propan-2-ol and hexane.

Chemical names

The major colouring principles are:

[Phytyl (13²R,17S,18S)-3-(8-ethyl-13² -methoxycarbonyl-2,7,12,18- tetramethyl-131 -oxo-3-vinyl-13¹ ,13² ,17,18-tetra-hydrocyclopenta [at]- prophyrin-17-yl)propionate] copper (II) (Copper chlorophyll a)

...

C.A.S. number

65963-40-8

Chemical formula

Copper phaeophytin a: C₅₅H₇₂CuN₄O₅

Copper phaeophytin b: C₅₅H₇₀CuN₄O₆

Structural formula

where:

X = CH₃ for the "a" compound

...

Formula weight

Copper phaeophytin a: 932.75

Copper phaeophytin b: 946.73

3. Description

Waxy solid ranging in colour from blue green to dark green depending on the source material.

4. Functional uses

Colour.

5. Characteristics

5.1. Identification

...

Solubility

Insoluble in water; soluble in ethanol, diethyl ether, chloroalkanes, hydrocarbons and fixed oils.

Spectrophotometry

A (1%, 1 cm) at 405 nm in chloroform is not less than 54.

Thin-layer chromatography

Passes test (described in “Tests”).

5.2. Purity

Residual solvents

...

Dichloromethane: Not more than 10 mg/kg.

Free ionizable copper

Not more than 200 mg/kg.

Total copper

Not more than 8% of the total copper phaeophytins.

Arsenic

Not more than 3 mg/kg.

Lead

Not more than 5 mg/kg.

...

Not less than 10% of total copper phaeophytins.

6. Tests

5.1. Identification

Thin-layer chromatography

Apply a 1 in 20 solution of the sample in chloroform as a band of the length of 2 cm to a Silica 60 TLC plate. After drying, develop the plate by a mixture of 50% hexane, 45% chloroform and 5% ethanol (General purpose reagent grade chloroform is supplied with 2% of added ethanol as a stabilizer. The 5% ethanol in the solvent mixture is in addition to this), until the solvent ascends to a point 15 cm above the initial spots. Allow the solvent to evaporate, then visually examine the separated spots and identify the components of interests by their R_f values and colours. Approximate R_f values and colour of the spots are as follows:

Copper phaeophytin a: 0.5; green

Copper phaeophytin b: 0.73; yellow/green

In addition spots may be visible for b-carotene at R_f 0.81 and xanthophyll at R_f 0.47 and 0.23.

...

Residual solvents

- Tested as directed under JECFA monograph 1-Vol. 4.

- Determine by gas chromatographically using either the method of entrainment distillation (Determination of Residual Solvents) or headspace analysis (Limit Test for Solvent Residues).

Free ionizable copper

Accurately weigh about 1 g of the sample and dissolve in 20 ml of arachid oil, with the aid of gentle heat. Add exactly 200 ml of water, stir mechanically, and adjust to pH 3.0 by careful addition of 0.5 N hydrochloric acid (avoid overshooting). Allow the mixture to stand for 10 min. If necessary readjust to pH 3.0 by careful addition of 0.5 N hydrochloric acid. Transfer to a separating funnel and allow to stand for about 20 min. Filter the aqueous phase through a No. 50 Whatman filter paper, rejecting the first 10 ml.Subject this solution to analysis for copper by atomic absorption spectrometry - JECFA monograph 1-Vol. 4.

Total copper

Ignite about 0.1 g, accurately weighed, of the sample contained in a silica dish, at a temperature not exceeding 500º, until all carbon is removed; moisten with one or two drops of concentrated sulphuric acid and re-ash. Dissolve the ash by boiling with 3 portions (each of 5 ml) of 10% (w/w) hydrochloric acid, filtering each addition through the same small filter paper into a 100 ml volumetric flask. Cool, and make up to volume with purified water. Subject this solution to analysis for copper by atomic absorption spectrometry - See JECFA monograph 1-Vol. 4.

Arsenic

...

- Method II.

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

6.3. Method of assay

Measure the absorbances (A) of the solution in a 1 cm cell against a diethyl ether blank at 667.2 nm, 654.4 nm, 649.8 nm and 628.2 nm. (The latter two wavelengths being the absorbance maxima in diethyl ether for copper phaeophytin a and copper phaeophytin b respectively).

Calculate the concentration of the individual compounds in (mol/L from the following equations:

...

Copper phaeophytin b = -8.46 A (649.8nm) + 20.7 A (628.2nm) - 1.69 A (667.2nm) + 5.13 A (654.4nm)

Convert the figures in mmol/L to percentages using the following equations:

ANNEX 17

SPECIFICATIONS AND TESTS FOR CHLOROPHYLLINS, COPPER COMPLEXES SODIUM AND POTASSIUM SALTS

...

Sodium copper chlorophyllin; potassium copper chlorophyllin

Chlorophyllins, C.I. (1975) No. 75810;

INS 141ii

ADI = 0 - -15 mg/kg bw.

2. Definition

The alkali salts of Copper Chlorophyllins are obtained by the addition copper to the product obtained by the saponification of a solvent extraction of grass, lucerne, nettle and other plant material; the saponification removes the methyl and cyclophytol ester groups and may partially cleave the pentenyl ring; after addition of copper to the purified chlorophyllins, the acid groups are neutralised to form the salts of potassium and/or sodium; the commercial products may be presented as aqueous solutions or dried powders. Only the following solvents may be used for the extraction: acetone, dichloromethane, methanol, ethanol, propan-2-ol and hexane.

Chemical names

The major colouring principles are:

3-(10-Carboxylato-4-ethyl-1,3,5,8-tetramethyl-9-oxo-2-vinylphorbin-7- yl)propionate, copper complex (Copper chlorophyllin a)

...

Depending on the degree of hydrolysis the cyclopentenyl ring may be cleaved with the resultant production of a third carboxyl function.

Chemical formula

Copper phaeophytin a: C₃₄H₃₂CuN₄O₅

Copper chlorophyllin b (acid form): C₃₄H₃₀CuN₄O₆

Structural formula

where:

X = CH₃ for the "a" series

X = CHO for the "b" compound.

...

Formula weight

Copper chlorophyllin a: 640.20

Copper chlorophyllin b: 654.18

Each may be increased by a 18 Daltons if the cyclopentenyl ring is cleaved.

3. Description

Dark green to blue/black powder or dark green solution.

4. Functional uses

Colour.

5. Specifications

...

Solubility

Soluble in water; very slightly soluble in lower alcohols and ketones and diethyl ether; insoluble in chloroalkanes, hydrocarbons and fixed oils.

Spectrophotometry

Passes test (described in “Tests”).

Test for copper

Passes test (described in “Tests”).

Test for sodium

Passes test.

...

Passes test.

5.2. Purity

Basic dyes

Passes test (described in “Tests”).

Residual solvents

Acetone, methanol, ethanol, propan-2-ol, hexane: Not more than 50 mg/kg, singly or in combination.

Dichloromethane: Not more than 10 mg/kg

Free ionizable copper

...

Total copper

Not more than 8% of the total copper phaeophytins.

Arsenic

Not more than 3 mg/kg.

Lead

Not more than 5 mg/kg.

5.3. Content

Not less than 95% total copper chlorophyllins after drying (100º, 1 h).

6. Tests

...

Spectrophotometry

A (1%, 1 cm) of the sample, dried at 100º for 1 h, at 405 nm in pH 7.5 phosphate buffer is not less than 540.

Test for copper

Dissolve the sulfated ash of the sample (using 1 g of sample, Method I) in 10 ml of dilute hydrochloric acid TS by heating on a water bath. Filter if the solution is not clear, and dilute to 10 ml with water. Use this solution as the test preparation for the following tests:

To 5 ml of the test preparation add ammonia TS to make the solution alkaline. Blue colour should appear.

To 5 ml of the above test preparation add 0.5 ml of a 1 in 1000 sodium diethyldithiocarbamate solution. A brown precipitate should be formed.

Test for sodium

Tested as directed under JECFA monograph 1-Vol. 4 - Test the solution described under Tests, identification tests Test for copper.

...

Tested as directed under JECFA monograph 1-Vol. 4 - Test the solution described under Tests, identification tests Test for copper.

6.2 Purity

Basic dyes

To 5 ml of a 0.5% aqueous solution of the sample in a test- tube add 1 ml of 1 N hydrochloric acid and add 5 ml of diethyl ether. Mix thoroughly and allow to separate. The ether layer should be no darker than pale green.

Residual solvents

Determine by gas chromatographically using either the method of entrainment distillation (Determination of Residual Solvents) or headspace analysis (Limit Test for Solvent Residues). (Tested as directed under JECFA monograph 1-Vol. 4)

Free ionizable copper

Accurately weigh about 1 g of the sample and mix in 20 ml of arachid oil, with the aid of gentle heat. Add exactly 200 ml of water, stir mechanically, and adjust to pH 3.0 by careful addition of 0.5 N hydrochloric acid (avoid overshooting). Allow the mixture to stand for 10 min. If necessary readjust to pH 3.0 by careful addition of 0.5 N hydrochloric acid. Transfer to a separating funnel and allow to stand for about 20 min. Filter the aqueous phase through a No. 50 Whatman filter paper, rejecting the first 10 ml. Subject this solution to analysis for copper by atomic absorption spectrometry (see JECFA monograph 1-Vol. 4).

...

Arsenic

Tested as directed under JECFA monograph 1-Vol. 4- Method II

Lead

Tested as directed under 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 assay

Weigh accurately about 1 g of the sample, dried previously at 100º for 1 h, then dissolve in 20 ml Phosphate Buffer Solution (pH 7.5) and dilute to 1000 ml with distilled water. Dilute 10 ml of this solution to 100 ml with Phosphate Buffer Solution (pH 7.5). Measure the optical density of the final solution (0.001% w/v) in a suitable spectrophotometer, using a 1 cm cell and slit width of 0.10 mm at 403-406 nm, recording the maximum within this range.

...

Optical density ´ 10⁴

--------------------------------------

565 ´ weight of sample (g)

This formula was derived on the assumption that 100% pure sodium copper chlorophyllin has a specific absorbance of 565.

ANNEX 18

SPECIFICATIONS AND TESTS FOR GREEN S

1. Synonyms

CI food green 4, food green S; CI (1975): 44090

...

ADI “Not specified”.

2. Definition

Consists essentially of sodium N-[4-[[4-(dimethylamino)phenyl](2- hydroxy-3,6-disulfo-1-naphthalenyl)-methylene]-2,5-cyclohexadien-1- ylidene]-N-methylmethanaminium and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured compounds.

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

Sodium N-[4-[[4-(dimethylamino)phenyl](2-hydroxy-3,6-disulfo-1- naphtalenyl)-methylene]-2,5-cyclohexadien-1-ylidene]-N-methylmethanaminium;

or Sodium 5-[4-dimethylamino-I-(4-dimethyliminiocyclohexa-2,5- dienylidene) benzyl]-6-hydroxy-7-sulfonato-naphthalene-2-sulfonate.

C.A.S. number

860-22-0

...

C₂₇H₂₅N₂NaO₇S₂

Công thức cấu tạo

Formula weight

576.63

3. Description

Dark green powder or granules.

4. Functional uses

Colour.

...

5.1. Identification

Solubility

Soluble in water; slightly soluble in ethanol.

Identification of colouring matters

Passes test.

5.2. Purity

Loss on drying at 135 °C

...

Water insoluble matter

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

Chromium

Not more than 50 mg/kg.

Subsidiary colouring matters

Not more than 1%.

Organic compounds other than colouring matters

...

Not more than 0.1% of 4,4'-Bis (dimethylamino) benzophenone.

Not more than 0.2% of 3-Hydroxynaphthalene-2,7-disulfonic acid.

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

Leuco base

Not more than 5.0%.

Ether extractable matter

Not more than 0.2%.

5.3. Content

...

6. Tests

6.1. Purity

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions: Developing solvent: No. 2; Height of ascent of solvent front: approximately 17 cm.

...

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use liquid chromatography under the following conditions: HPLC elution gradient: 2 to 100% at 2% per min.

Leuco base

- Tested as directed under JECFA monograph 1-Vol. 4.

- Weigh accurately 110±5 mg of sample and proceed as directed under Leuco Base in Sulfonated Triarylmethane Colours. Absorptivity: (a) = 0.1725 mg L^-1 cm^-1 at approximately 634 nm. Ratio = 0.9600

6.2. Method of assay

Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following:

...

Buffer: 15 g sodium hydrogen tartrate;

Weight (D) of colouring matters equivalent to 1.00 ml 0.1 N TiCl3: 28.83 mg.

ANNEX 19

SPECIFICATIONS AND TESTS FOR CARAMEL

1. Synonyms

Caramel colours are divided into four classes. The synonyms for each class are:

Class I: Plain caramel, caustic caramel; INS No. 150a

Class III: Ammonia caramel; INS No. 150c

...

ADI for class I “Not specified”;

ADI for class III = 0-160 mg/kg bw

ADI for class IV = 0-200 mg/kg bw (0-150 mg/kg on solids basis)

2. Definition

Complex mixtures of compounds, some of which are in the form of colloidal aggregates, manufactured by heating carbohydrates either alone or in the presence of food-grade acids, alkalis or salts; classified according to the reactants used in their manufacture as follows:

Class I: Prepared by heating carbohydrates with or without acids or alkalis; no ammonium or sulfite compounds are used.

Class III: Prepared by heating carbohydrates with or without acids or alkalis in the presence of ammonium compounds; no sulfite compounds are used.

Class IV: Prepared by heating carbohydrates with or without acids or alkalis in the presence of both sulfite and ammonium compounds.

In all cases the carbohydrate raw materials are commercially available foodgrade nutritive sweeteners consisting of glucose, fructose and/or polymers thereof. The acids and alkalis are food-grade sulfuric or citric acids and sodium, potassium or calcium hydroxides or mixtures thereof.

...

Where sulfite compounds are used they are one or any of the following: sulfurous acid, potassium, sodium and ammonium sulfites and hydrogen sulfites.

Food-grade anti-foaming agents may be used as processing aids during manufacture.

3. Description

Dark brown to black liquids or solids having an odour of burnt sugar.

4. Functional uses

Colour

5. Specifications

5.1. Identification

...

Miscible with water

Identification of colouring matters

Passes test.

Classification

Class I: Not more than 50% of the colour is bound by DEAE Cellulose and not more than 50% of the colour is bound by Phosphoryl Cellulose.

Class III: Not more than 50% of the colour is bound by DEAE Cellulose and more than 50% of the colour is bound by Phosphoryl Cellulose.

Class IV: More than 50% of the colour is bound by DEAE Cellulose and it exhibits an Absorbance Ratio of not more than 50.

See description under TESTS.

5.2. Purity

...

Solid content

Class I: 62-77%

Class III: 53-83%

Class IV: 40-75%

Colour intensity

Class I: 0.01-0.12

Class III: 0.08-0.36

Class IV: 0.10-0.60

Total nitrogen

...

Class III: 1.3 -6.8%

Class IV: 0.5-7.5%

Total sulfur

Class I: max 0.3%

Class III: max 0.3%

Class IV: 1.4-10.0%

Sulfur dioxide

Class I: -

...

Class IV: max 0.5%

Ammoniacal nitrogen

Class I: -

Class III: max 0.4%

Class IV: max 2.8%

4-Methylimidazole (MEI)

Class I: -

...

Class IV: max 1000 mg/kg & max 250 mg/kg on an equivalent colour basis

2-Acetyl-4-tetrahydroxybutylimidazole (THI)

Class I: -

Class III: max 40 mg/kg & max 25 mg/kg on an equivalent colour basis.

Class IV: -

Arsenic

Not more than 1 mg/kg

Lead

...

6. Tests

6.1. Identification

Classification/Colour bound by DEAE Cellulose

For the purposes of this specification, colour bound by DEAE cellulose is defined as the percentage of decrease in absorbance of a caramel colour solution at 560 nm after treatment with DEAE Cellulose.

Special reagent:

DEAE (diethylaminoethyl) Cellulose of 0.7 meq/gram capacity, e.g. Cellex D from Bio-Rad or equivalent DEAE Celluloses of higher or lower capacities in proportionately higher or lower quantities.

...

Prepare a caramel colour solution of approximately 0.5 absorbance unit at 560 nm by transferring an appropriate amount of caramel colour into a 100- ml volumetric flask with the aid of 0.025 N hydrochloric acid. Dilute to volume with 0.025 N hydrochloric acid and centrifuge or filter, if solution is cloudy. Take a 20 ml aliquot of the caramel colour solution, add 200 mg of DEAE Cellulose, mix thoroughly for several min, centrifuge or filter, and collect the clear supernatant. Determine the absorbance of the caramel colour solution and the supernatant in a 1-cm cell at 560 nm, with a suitable spectrophotometer previously standardized using 0.025 N hydrochloric acid as reference. Calculate the percentage of Colour Bound by DEAE Cellulose by the formula:

where:

A₁ is the absorbance of the caramel colour solution at 560 nm

A₂ is the absorbance of the supernatant after DEAE Cellulose treatment at 560 nm.

Classification/Colour bound by phosphoryl cellulose

For the purposes of this specification colour bound phosphoryl cellulose is defined as the percentage of decrease in absorbance of a caramel colour solution at 560 nm after treatment with Phosphoryl Cellulose.

Special reagent:

...

Procedure:

Transfer 200-300 mg of caramel colour into a 100-ml volumetric flask, dilute to volume with 0.025 N hydrochloric acid, and centrifuge or filter, if solution is cloudy. Take a 40 ml aliquot of the caramel colour solution, add 2.0 g Phosphoryl Cellulose and mix thoroughly for several min. Centrifuge or filter, and collect the clear supernatant. Determine the absorbance of the caramel colour solution and the supernatant in a 1-cm cell at 560 nm, with a suitable spectrophotometer previously standardized using 0.025 N hydrochloric acid as reference. Calculate the percentage of Colour Bound by Phosphoryl Cellulose by the formula:

where:

A₁ is the absorbance of the caramel colour solution at 560 nm

A₂ is the absorbance of the supernatant after Phosphoryl Cellulose treatment at 560 nm.

Classification/ Absorbance ratio

...

Procedure:

Transfer 100 mg of caramel colour into a 100-ml volumetric flask with the aid of water, dilute to volume, mix and centrifuge if solution is cloudy. Pipet a 5.0 ml portion of the clear solution into a 100-ml volumetric flask, dilute to volume with water, and mix. Determine the absorbance of the 0.1% solution in a 1-cm cell at 560 nm and that of the 1:20 diluted solution at 280 nm with a suitable spectrophotometer previously standardized using water as reference. (A suitable spectrophotometer is one equipped with a monochromator to provide a band width of 2 nm or less and of such quality that the stray-light characteristic is 0.5% or less.) Calculate the Absorbance Ratio of the caramel colour by dividing the absorbance units at 280 nm multiplied by 20 (dilution factor) by the absorbance units at 560 nm.

6.2. Purity

Solid content

The solids content of Caramel Colour is determined by drying a sample upon a carrier composed of pure quartz sand that passes a No. 40 but not a No. 60 sieve and has been prepared by digestion with hydrochloric acid, washed acid-free, dried and ignited. Mix 30.0 g of prepared sand accurately weighed with 1.5-2.0 g Caramel Colour accurately weighed and dry to constant weight at 60o under reduced pressure 50 mm/Hg (6.7 kPa). Record the final weight of the sand plus caramel. Calculate the % solids as follows:

% solids =

where:

...

m₂ = weight of sand (g)

m₁= weight of caramel initially added (g)

Calculation on a solids basis

The contents of Total Nitrogen, Total sulfur, Ammoniacal nitrogen, sulfur dioxide, 4-MEI and THI are expressed on a solids basis. The concentration (Ci) of each impurity is determined on an "as is" basis; the concentration (C_S) on a solid basis is then calculated using the formula:

C_i ´ 100

% solids

C_{s =}

...

For the purpose of this specification, Colour Intensity is defined as the absorbance of a 0.1% (w/v) solution of Caramel Colour solids in water in a 1 cm cell at 610 nm.

Procedure:

Transfer 100 mg of Caramel Colour into a 100 ml volumetric flask, dilute to volume with water, mix and centrifuge if the solution is cloudy. Determine the absorbance (A610) of the clear solution in a 1 cm cell at 610 nm with a suitable spectrophotometer previously standardized using water as a reference. Calculate the Colour Intensity of the Caramel Colour as follows:

A₆₁₀ ´ 100

% solids

Colour intensity =

Determine % solids as described under Solids content.

...

C_S ´ 0,1

Colour intensity

Equivalent colour basis =

where:

C_S = concentration on a solids basis.

This gives content expressed in terms of a product having a Colour Intensity of 0.1 absorbance units.

Total nitrogen

...

Total sulfur

In the largest available casserole that fits in an electric muffle furnace, place 1-3 g MgO or equivalent quantity of Mg(NO₃)₂·6H₂O (6.4 - 19.2 g), 1 g powdered sucrose, and 50 ml HNO3. Add 5-10 g caramel colour. Place same quantities of reagents in another casserole for blank. Evaporate on steam bath to paste. Place casserole in cold electric muffle (25º) and gradually heat until all NO2 fumes are driven off. Cool, dissolve and neutralize with HCl (1+2.5), adding excess of 5 ml. Filter, heat to boiling, and add 5 ml 10% BaCl₂·2H₂O solution dropwise. Evaporate to 100 ml, let stand overnight, filter, wash, ignite, and weigh the BaSO₄. Correct result for BaSO₄ obtained in blank and report as mg S/100 g. Commercial instruments that analyse for total sulfur such as, the Leco-Combustion/Titration procedure can also be used and are recommended for sample amounts of about 200 mg.

Sulfur dioxide

Apparatus

Use a modified Monier-Williams apparatus (available from 5GA Scientific, Inc., Bloomfield, N.J., USA) for the determination of sulfurous acid, or construct the apparatus as shown in the figure. The assembly consists of a 1000-ml three-neck round-bottom distillation flask having 24/40 standardtaper ground-glass joints. A 30-cm Allihn condenser is attached in the reflux position to an outer neck of the flask, and the other end of the condenser is connected with 1/4-inch. Tygon or silicon tubing (preboiled with 1 in 20 hydrochloric acid solution and rinsed with water) to the absorption tube assembly (having 35/20 ball joints or the equivalent). Connect the centre neck of the flask with a 125-ml cylindrical separator, and attach a piece of tubing to a short U-tube inserted through a rubber stopper in the neck of the separator. Attach a curved glass inlet tube, reaching nearly to the bottom of the flask, to the other outer neck of the flask, and connect the inlet tube to a 250-ml gas-washing bottle with a piece of the tubing. The gas-washing bottle, in turn, is connected by tubing to a nitrogen cylinder.

...

To each U-tube of the absorption tube assembly add the following: two pieces of 8-mm glass rod about 25 mm in length, 10 ml of 3-mm glass beads at the exit side, 10.0 ml of 3% hydrogen peroxide solution, and 1 drop of methyl red TS.

Assemble all pieces of the apparatus, and check for leaks by blowing gently into the tubing attached to the neck of the separator. While blowing, close the stopcock of the separator. Let stand for a few min; if the liquid levels in the U-tubes equalize, reseal all connections and test again. If the system is airtight proceed as directed below.

Procedure:

Disperse about 25 g of the sample, accurately weighed, in 300 ml of recently boiled and cooled water, and transfer the slurry to the flask with the aid of water, using a large-bore funnel. Dilute to about 400 ml with water, and reseal the separator. Add 90 ml of 4 N hydrochloric acid to the separator, and force the acid into the flask by blowing gently into the tube in the neck of the separator. Close the stopcock of the separator. Unclamp the tubing on both sides of the gas-washing bottle, and start the nitrogen flow at a steady stream of bubbles. Heat the distilling flask with a heating mantle to cause refluxing in approximately 20 min. When steady refluxing is reached, apply the line voltage to the mantle and reflux for 1.75 h. Turn off the water in the condenser, and continue heating until the inlet joint of the first U-tube shows condensation and slight warming. Remove the separator and turn off the heat.

When the joint at the top of the condenser cools, remove the connecting assembly and rinse it into the second U-tube, leaving the crossover tube attached to the exit joint of the first U-tube but disconnected from the entrance of the second U-tube. Rotate the crossover tube until the free end almost touches the entrance of the first U-tube. Add 1 drop of methyl red TS to the first U-tube, and titrate with 0.1 N sodium hydroxide just to a clear yellow colour, mixing with a gentle rocking motion. After titrating the first Utube, remove the crossover tube, attach it to the second U-tube exit, and titrate similarly. Record the sum of the two titers as V_t, in ml.

Perform a blank determination, and record the volume of 0.1 N sodium hydroxide required as V_o. Calculate the percentage of sulfur dioxide in the sample by the formula

Where:

m is the weight of the sample taken, in g.

...

Add 25 ml of 0.1 N sulfuric acid to a 500-ml receiving flask, and connect it to a distillation apparatus consisting of a Kjeldahl connecting bulb and a condenser such that the condenser delivery tube is immersed beneath the surface of the acid solution in the receiving flask. Transfer about 2 g of caramel colour, accurately weighed, into an 800-ml long-neck Kjeldahl digestion flask, and to the flask add 2 g of magnesium oxide (carbonatefree), 200 ml of water, and several boiling chips. Swirl the digestion flask to mix the contents, and quickly connect it to the distillation apparatus. Heat the digestion flask to boiling, and collect about 100 ml of distillate in the receiving flask. Wash the tip of the delivery tube with a few ml of water, collecting the washings in the receiving flask, then add 4 or 5 drops of methyl red indicator (500 mg of methyl red in 100 ml of alcohol), and titrate with 0.1 N sodium hydroxide, recording the volume, in ml, required as V_t. Conduct a blank determination, and record the volume, in ml, of 0.1 N sodium hydroxide required to neutralize as V_o. Calculate the percentage of ammoniacal nitrogen in the sample by the formula:

Ammoniacal nitrogen

Where:

m is the weight of the sample taken, in g.

4-Methylimidazole

Note: Information on an improved method is sought.

The following materials and reagents are required (the reagents should be ACS grade or equivalent where applicable).

...

Pyrex glasswool, 22 x 300 mm chromatography column with PTFE stopcock (e.g. Kimax 17800); 150 ml polypropylene beaker (e.g. Nalge 1201); 250 ml round-bottom flask (e.g., Pyrex 4320); 75 mm powder funnel; 5 cm spatula; rotary vacuum evaporator; hot plate; pan for water bath; disposable Pasteur pipets; 5 ml volumetric flask.

Reagents

Acetone; Celite 545; methylene chloride; sodium hydroxide; and tetrahydrofuran.

Procedure:

After thoroughly mixing the caramel colour sample by shaking or stirring, weigh a 10.00 g aliquot into a 150 ml polypropylene beaker. Polypropylene is considered superior to glass because of its hydrophobic surface which facilitates quantitative sample transfer. A 5.0 g portion of 3.0 N NaOH is added and thoroughly mixed to ensure that the pH of the entire sample exceeds 12. A 20 g portion of Celite 545 is added to the beaker, and the contents are mixed until a semi-dry mixture is obtained. This normally requires approximately 2 to 3 min. With samples of unusually high water content, the resultant caramel colour-Celite 545 mixture may be overly wet. In such cases, a 5.00 g aliquot of caramel colour may be mixed with 2.5 g of 3.0 N NaOH and 15 g of Celite 545 and carried through the remainder of the analysis.

A plug of Pyrex glasswool is placed in the bottom of a 22 x 300 mm chromatographic column with PTFE stopcock. The caramel colour-Celite 545 mixture is placed in the column with the aid of a 75 mm powder funnel. The column contents are settled by repeatedly allowing the column to fall vertically about 10 cm to a padded surface. When properly settled, the caramel colour-Celite 545 mixture should occupy approximately the lower 250 mm of the column. Care should be exercised at this point to avoid a column bed which is either too loosely or too tightly packed. Loose packing will allow too rapid elution of the methylene chloride and possibly incomplete extraction. A too tightly packed column, e.g., the result of tamping down the column contents, can result in regions of the bed which are relatively inaccessible to the extraction solvent. This can also result in incomplete extraction.

With the stopcock open, the column is filled with methylene chloride poured from the sample beaker. When the solvent reaches the glasswool plug, the stopcock is closed and the solvent is allowed to stand in contact with the bed for 5 min. The stopcock is then opened and the column is further eluted with methylene chloride until 200 ml have been collected in a 250 ml roundbottom flask. A 1.00 ml aliquot of 2 MEI internal standard solution (50.0 mg of 2 MEI/50.0 ml of methylene chloride) is added to the collected eluate. The 2 MEI is well separated from the 4 MEI under the GLC conditions employed and has not been found in caramel colour.

The bulk of the solvent is then removed from the eluate on a rotary vacuum evaporator operated at 45-50 kPa and with the round-bottom flask maintained at 35^o in a water bath. The extracted residue is transferred quantitatively to a 5 ml volumetric flask with a disposable Pasteur pipet, by rinsing the round-bottom flask several times with small (ca. 0.75 ml) portions of either tetrahydrofuran or acetone. Both solvents have been used with equal success. After mixing the contents thoroughly by several inversions of the flask, the extract is ready for GLC analysis. The extracts should be analysed as soon as possible after their preparation, because stability problems have occasionally been encountered with extracts more than 1 day old.

The GLC analysis is carried out using a gas chromatograph equipped with a hydrogen flame detector. The column is glass, 1 mm x 6 mm o.d. x 4 mm i.d., filled with 7.5% Carbowax 20M + 2% K0H on 90/100 mesh Anakrom ABS. The GLC parameters are as follows: carrier, nitrogen, 50 ml/min; hydrogen, 50 ml/min; oxygen, 80 ml/min; injection port, 200^o ; column isothermal, 180^o ; detector, 250^o ; sample size, 5 µl. All quantitation is done by using the internal standard technique.

...

Note: Information on an improved method is sought.

THI is converted into its 2,4-dinitrophenylhydrazone (THI-DNPH). This derivative is separated from excess reagent and carbonylic contaminants by HPLC on RP-8, then determined by its absorbance at 385 nm.

Procedure:

Caramel colour (200-250 mg) is weighed accurately, then dissolved in water (3 ml). The solution is transferred quantitatively to the upper part of a Combination Column. Elution with water is started, and a total of about 100 ml of water is passed through the columns.

The upper column is then disconnected. The lower column is eluted with 0.5 N HCl. The first 10.0 ml of eluate are discarded, then a volume of 35 ml is collected.

The solution is concentrated to dryness at 40^o and 15 torr. The syrup residue is dissolved in carbonyl-free methanol (250 µl) and the 2,4- dinitrophenylhydrazine reagent (250 µl) is added. The reaction mixture is transferred to a septum-capped vial and stored for 5 h at room temperature. A volume of 5 µl (but also from 1 to 25 µl) is injected onto a LiChrosorb RP-8 (10 µm) HPLC column. The mobile phase is MeOH/0.1 M H3PO4 50/50 (v/v). Adjustments in mobile phase composition may be needed as column characteristics vary, depending upon the manufacturer. (Use of LiChrosorb RP-8, 10 µm, 250 x 4 mm "Vertex" column manufactured by Knauer, Bad Homburg, F.R.G. is strongly recommended). At a mobile phase flow rate of 2 ml/min and column dimensions of 250 x 4.6 mm, THI-DNPH is eluted at about 6.3±0.1 min. It is detected at 385 nm and the peak height is measured. The amount is calculated from a calibration curve prepared with THI-DNPH in methanol.

Materials:

- 2,4,-Dinitrophenylhydrazine hydrochloride reagent: Commercial 2,4- dinitrophenylhydrazine (5 g) is added to concentrated hydrochloric acid (10 ml) in a 100-ml Erlenmeyer flask, and the latter is gently shaken until the free base (red) is converted to the hydrochloride (yellow). Ethanol (100 ml) is added and the mixture is heated on a steam bath until all the solid has dissolved. After crystallization at room temperature, the hydrochloride is filtered off, washed with ether, dried at room temperature and stored in a desiccator. On storage the hydrochloride is slowly converted to the free base. The latter can be removed by washing with dimethoxyethane. The reagent is prepared by mixing 0.5 g of 2,4-dinitrophenylhydrazine hydrochloride in 15 ml of 5% methanol in dimethoxyethane for 30 min. It should be stored in the refrigerator and be checked periodically by HPLC.

...

- Cation-exchange resin (weak): Amberlite CG AG 50 I, H+ , (100-200 mesh). (Sediment two or three times prior to use).

- Methanol, carbonyl-free: Methanol is prepared after Y. Peleg and C.H. Mannheim, J. Agr. Fd. Chem, 18 (1970) 176, by treatment with Girard P reagent.

- Dimethoxyethane: If impure, dimethoxyethane is purified by distillation from 2,4-dinitrophenylhydrazine in the presence of acid and redistilled from sodium hydroxide. Immediately prior to use it is passed through a column of neutral aluminium to remove peroxides.

Apparatus:

Combination Columns: Similar to the set-up described in J. Agr. Fd. Chem. 22 (1974) 110. The upper column (150 x 12.5 mm, filling height max. 9 cm, or 200 x 10 mm, filling height max. 14 cm, with capillary outlet of 1 mm i.d.) is filled with weakly acidic cation-exchanger; bed height, approx. 50-60, or 80-90 mm, respectively. The lower column (total length 175 mm, i.d. 10 mm, with capillary outlet and Teflon stopcock) is filled with strongly acidic cationexchanger to a bed-height of 60 mm. As a solvent reservoir, a dropping funnel (100 ml) with Teflon stopcock is used. All parts are connected by standard ground-glass joints (14.5 mm).

HPLC: With the column specified above and an ultraviolet detector capable of reading at 385 nm.

Calibration: THI-DNPH is dissolved in absolute, carbonyl-free methanol (about 100 mg/l; calculated concentration of THI: 47.58 ng/µl). A portion of this solution is diluted tenfold with methanol (4.7 ng THI/µl). THI-DNPH standard solutions are stable for at least twenty weeks when stored in the refrigerator.

Arsenic

...

- Tested as directed under JECFA monograph 1-Vol. 4.

- Method II.

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

ANNEX 20

SPECIFICATIONS AND TESTS FOR SUNSET BRILLIANT BLUE FCF

1. Synonyms

...

INS: 151

ADI = 0 - 1 mg/kg bw.

2. Definition

Consists essentially of tetrasodium 4-acetamido-5-hydroxy-6-[7-sulfonato-4- (4-sulfonato-phenylazo)-1-naphthylazo]-1,7-naphthalene-disulfonate and subsidiary colouring matters together with sodium chloride and/or sodium sulfate as the principal uncoloured components.

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

Tetrasodium 4-acetamido-5-hydroxy-6-[7-sulfonato-4-(4-sulfonatophenylazo)- 1-naphthylazo]-1,7-naphthalene-disulfonate

C.A.S. number

2519-30-4

...

C₂₈H₁₇N₅Na₄O₁₄S₄

Structural formula

Formula weight

867.69

3. Description

Black powder or granules

4. Functional uses

Colour.

...

5.1. Identification

Solubility

Soluble in water; sparingly soluble in ethanol

Identification of colouring matters

Passes test.

5.2. Purity

Loss on drying at 135 °C

...

Water insoluble matter

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

Subsidiary colouring matters

Not more than 4%.

Organic compounds other than colouring matters

Not more than 0.8%, sum of 4-Acetamido-5-hydroxy-1,7-naphthalenedisulfonic acid, 4-Amino-5-hydroxy-1,7-naphthalenedisulfonic acid, 8-Amino-2-naphthalenesulfonic acid, Sulfanilic acid, 4,4'-Diazoaminodi(benzenesulfuric acid)

Unsulfonated primary aromatic amines

...

Ether extractable matter

Not more than 0.2%.

5.3. Content

Not less than 80% total colouring matter.

6. Tests

6.1. Purity

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

...

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions:

Developing solvent: Chromatogram (i): No. 1.

Chromatogram (ii): No. 4.

Height of ascent of solvent front: (i): approximately 17 cm

(ii): approximately 17 cm

Organic compounds other than colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

...

6.2. Method of assay

Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following:

Weight of sample: 0.6 - 0.7 g;

Buffer: 15 g sodium hydrogen tartrate;

Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl₃: 10.86 mg.

ANNEX 21

...

1. Synonyms

CI Food Brown 3, Chocolate brown HT, CI (1975) No. 20285

INS: 155

ADI = 0 - 1,5 mg/kg bw.

2. Definition

Consists essentially of disodium 4,4'- (2,4-dihydroxy-5-hydroxymethyl-1,3- phenylene-bisazo) di-1-naphthalene-sulfonate and subsidiary colouring matters together with NaCl and/or Na₂SO₄ as the principal uncoloured components.

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

Disodium 4,4'-(2,4-dihydroxy-5-hydroxymethyl-1,3-phenylene-bisazo di-1- naphthalenesulfonate.

...

4553-89-3

Chemical formula

C₂₇H₁₈N₄Na₂O₉S₂

Structural formula

Formula weight

652.57

3. Description

Brown powder or granules.

...

Colour.

5. Specifications

5.1. Identification

Solubility

Soluble in water; insoluble in ethanol.

Identification of colouring matters

Passes test.

5.2. Purity

...

Loss on drying at 135 °C

Not more than 30% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

Subsidiary colouring matters

Not more than 10%.

Organic compounds other than colouring matters

...

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

Ether extractable matter

Not more than 0.2%.

5.3. Content

Not less than 70% total colouring matter.

6. Tests

6.1 Purity

...

- Tested as directed under JECFA monograph 1-Vol. 4.

Subsidiary colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Use the following conditions:

Prepare the standard in the following manner: Dilute 1.0 ml of the 1% dye solution to 100 ml with water and mix well. Transfer 0.10 ml of this solution to a test tube; add 5.0 ml of water: acetone (1:1 by vol.) and then 14.9 ml of 0.05 N sodium hydrogen carbonate solution and shake the tube to ensure mixing. Determine the net absorbance (A_S) of the standard.

Developing solvent: No.6.

Develop chromatogram for approximately 14 h.

Organic compounds other than colouring matters

...

- Use liquid chromatography under the following conditions: HPLC elution gradient: 2 to 100% at 2.0% per min.

6.2. Method of assay

Proceed as directed under Total Content by Spectrophotometry (in JECFA monograph 1-Vol. 4), using the following conditions:

Solvent: pH 7 phosphate buffer;

Dilution of solution A: 10 ml to 250 ml.

Absorptivity (a): 40.3

Approximate wavelength of maximum absorption: 460 nm.

...

ANNEX 22

SPECIFICATIONS AND TESTS FOR β-CAROTENE, SYNTHETIC

1. Synonyms

CI food Orange 5; CI (1975): 40800

INS: 160ai

ADI = 0 - 5 mg/kg bw.

2. Definition

These specifications apply to predominantly all trans (Z) isomer of b-carotene together with minor amounts of other carotenoids; diluted and stabilized forms are prepared from b-carotene meeting these specifications and include solutions or suspensions of b-carotene in edible fats or oils, emulsions and water dispersible powders; these preparations may have different cis/trans isomer ratios; the analytical methods described for the parent colour are not necessarily suitable for the assay of or determination of impurities in the stabilized forms (appropriate methods should be available from the manufacturer).

Chemical names

...

C.A.S. number

7235-40-7

Chemical formula

C₄₀H₅₆

Structural formula

Formula weight

536.88

3. Description

...

4. Functional uses

Colour.

5. Specifications

5.1. Identification

Solubility

Insoluble in water; practically insoluble in ethanol; slightly soluble in vegetable oils; soluble in chloroform.

Spectrophotometry

Determine the absorbance of the sample solution C (See Method of Assay) at 455 nm and 483 nm.

...

Determine the absorbance of the sample solution C at 455 nm and that of sample Solution B (See Method of Assay) at 340 nm.

The ratio A₄₅₅/A₃₄₀ is not lower than 15.

Test for carotenoid

Passes test (described in “Tests”).

Carr-Price reaction

A solution of the sample in chloroform turns blue on addition of an excess of Carr-Price reagent TS.

5.2. Purity

Sulfated ash

...

Subsidiary colouring matters

Carotenoids other than b-carotene: Not more than 3% of total colouring matters.

Lead

Not more than 2 mg/kg.

5.3. Content

Not less than 96% total colouring matters, expressed as b-carotene.

6. Tests

6.1. Identification

...

The colour of a solution of the sample in acetone disappears after successive additions of a 5% solution of sodium nitrite and 1 N sulfuric acid.

6.2. Purity

Sulfated ash

- Tested as directed under JECFA monograph 1-Vol. 4.

- Test 2 g of the sample (Method I)

Subsidiary colouring matters

Carotenoids other than b-carotene

Dissolve about 80 mg of sample in 100 ml chloroform. Apply 400 µl of this solution as a streak 2 cm from the bottom of a TLC-plate (Silicagel 0.25 mm). Immediately develop the chromatogram with a solvent mixture of 95 parts dichloromethane and 5 parts diethyl ether in a saturated chamber, suitably protected from light, until the solvent front has moved 15 cm above the initial streak. Remove the plate, allow the main part of the solvent to evaporate at room temperature and mark the principal band as well as the bands corresponding to other carotenoids.

...

Remove the silicagel adsorbent that contains the combined bands corresponding to the other carotenoids, transfer it to a glass-stoppered, 50 ml centrifuge tube and add 20.0 ml chloroform (solution 2).

Shake the centrifuge tubes by mechanical means for 10 min and centrifuge for 5 min. Dilute 10.0 ml of Solution 1 to 50.0 ml with chloroform (solution 3). Determine, with a suitable spectrophotometer, the absorbances of Solutions 2 and 3 in 1-cm cells at the wavelength maximum about 464 nm, using chloroform as blank.

Calculation:

Calculate the percentage of carotenoids other than b-carotene (%) =

where:

A₂ = absorbance of Solution 2

A₃ = absorbance of Solution 3

Lead

...

6.2. Method of assay

Proceed as directed under Total Content by Spectrophotometry in JECFA monograph 1-Vol. 4 using the following conditions:

W = 0.08 g

V₁ =V₂ =V₃ = 100 ml

v₁ = v₂ = 5 ml

A^1%_1cm = 2500

...

ANNEX 23

SPECIFICATIONS AND TESTS FOR CAROTENES, VEGETABLE

1. Synonyms

Natural b-carotene, carotenes-natural; CI Food Orange 5, mixed carotenes; CI (1975) No. 75130; CI (1975) No. 40800 (b-Carotene).

INS 160aii

ADI "acceptable", provided the level of use does not exceed the level normally found in vegetables.

2. Definition

Carotenes (vegetable) are obtained by solvent extraction of carrots (Daucus carota), oil of palm fruit (Elaeis guinensis), sweet potato (Ipomoea batatas) and other edible plants with subsequent purification. The main colouring principles are a- and b-carotenes of which b-carotenes account for the major part. Minor amounts of g- and d-carotenes and other pigments may be present. Besides the colour pigments, this substance may contain oils, fats and waxes naturally occurring in the source material. The only solvents used for the extraction are acetone, methanol, ethanol, propan-2-ol, hexane, carbon dioxide and vegetable oils.

...

Class

Carotenoid

C.A.S. number

7235-40-7

Chemical formula

C₄₀H₅₆ (-β carotene)

Structural formula

All-trans-b-carotene

...

536.88 (b-Carotene)

3. Description

Red-brown to brown or orange to dark orange solid or liquid.

4. Functional uses

Colour.

5. Specifications

5.1. Identification

Solubility

...

Spectrophotometry

A cyclohexane solution of the sample (1 in 200,000) shows maximum absorptions at 440-457 and 470-486 nm.

Colour reaction

Passes test (described in “Tests”).

5.2. Purity

Residual solvents

Not more than 50 mg/kg, singly or in combination, of acetone, hexane, methanol, ethanol and propan-2-ol.

Lead

...

5.3. Content

Content of carotenes (calculated as b-carotene) is not less than declared.

6. Tests

6.1. Identification

Colour reaction

A spot of a solution of the sample in toluene (about 400 µg /ml of ß-carotene) on a filter paper turns blue 2-3 min after application of a spray or drop of 20% solution of antimony trichloride solution in toluene.

6.2 Purity

...

Tested as directed under 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 assay

Proceed as directed under Total Content by Spectrophotometry in JECFA monograph 1-Vol. 4 using the following conditions:

W = amount of the sample to obtain adequate absorbance (g)

V₁ =V₂ =V₃ = 100 ml

v₁ = v₂ = 5 ml

A^1%_1cm = 2500

...

ANNEX 24

SPECIFICATIONS AND TESTS FOR CARAMEL EXTRACTS

1. Synonyms

Annatto F; Orlean; Terre orellana; R. Orange; CI (1975) No. 75120 (Natural Orange 4).

INS 160bii

ADI = 0 - 12 mg/kg bw (for bixin);

ADI = 0 - 0.6 mg/kg bw (for norbixin and its salts)

The colouring matters bixin and norbixin derived from annatto extracts (solvent-extracted bixin; solvent-extracted norbixin; aqueous-processed bixin; alkali-processed norbixin, acid-precipitated; and alkali-processed norbixin, not acid-precipitated) are included in the ADIs for bixin and norbixin.

...

Alkali-processed norbixin (acid-precipitated) is prepared by removal of the outer coating of the seeds of the annatto tree (Bixa orellana L) with aqueous alkali. The bixin is hydrolysed to norbixin in hot alkaline solution and is acidified to precipitate the norbixin. The precipitate is filtered, dried and milled to give a granular powder.

Alkali-processed norbixin contains several coloured components; the major colouring principle is cis-norbixin, a minor colouring principle is trans-norbixin; thermal degradation products of norbixin may also be present as a result of processing.

Products supplied to the food industry may be formulated with appropriate carriers of food grade quality.

Chemical names

cis-Norbixin: 6,6'-Diapo-Ψ,Ψ-carotenedioic acid

cis-Norbixin dipotassium salt: Dipotassium 6,6'-diapo-Ψ,Ψcarotenedioate

cis-Norbixin disodium salt: Disodium 6,6'-diapo-Ψ,Ψ-carotenedioate

C.A.S. number

cis-Norbixin: 542-40-5

...

cis-Norbixin disodium salt: 33261-81-3

Chemical formula

C₂₄H₂₈O₄; C₂₄H₂₆K₂O₄; C₂₄H₂₆Na₂O₄

Structural formula

Formula weight

380.5 (acid), 456.7 (dipotassium salt), 424.5 (disodium salt)

3. Description

Dark red-brown to red-purple powder.

...

Colour.

5. Specifications

5.1. Identification

Solubility

Soluble in alkaline water, slightly soluble in ethanol.

UV/VIS absorption

The sample in 0.5% potassium hydroxide solution shows absorbance maxima at about 453 nm and 482 nm.

Thin-layer chromatography

...

5.2. Purity

Arsenic

Not more than 3 mg/kg.

Lead

Not more than 2 mg/kg.

Mercury

Not more than 1 mg/kg.

5.3. Content

...

6. Tests

6.1. Identification

Thin-layer chromatography

Activate a TLC plate (e.g. LK6D SILICA GEL 60 A (layer thickness: 250 μm, size: 5 x 20 cm)) for 1 h at 110˚. Prepare a 5% solution of the sample in 95% ethanol and apply 10 µl to the plate. Allow to dry and develop using a mixture of n-butanol, methyl ethyl ketone and 10% aqueous ammonia (3:2:2 by volume) until the solvent front has ascended about 10 cm. Allow to dry. Bixin and norbixin appear as yellow spots with R_f values of about 0.50 to 0.45, respectively. Spray with 5% sodium nitrite solution and then with 0.5 mol/l sulfuric acid and the spots immediately decolourise.

6.2 Purity

Arsenic

- Tested as directed under JECFA monograph 1-Vol. 4.

...

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

- Determine using an AAS ICP-AES technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the methods described in JECFA monograph 1-Vol. 4.

Mercury

- Tested as directed under JECFA monograph 1-Vol. 4.

- Determine using cold vapour atomic absorption technique. Select sample size appropriate to the specified level.

6.3. Method of assay

...

Solvent: 0.5 % potassium hydroxide

Absorbance at l_max: about 482 nm.

Specific absorbance A^1%_1cm = 2870.

ANNEX 25

SPECIFICATIONS AND TESTS FOR β-APO-CAROTENAL

1. Synonyms

CI food Orange 6; CI (1975) No. 40820

INS 160e

...

2. Definition

Specifications apply to predominantly all trans (Z) isomer of b-apo-8'-carotenal together with minor amounts of other carotenoids; diluted and stabilized forms are prepared from b-apo-8'-carotenal meeting these specifications and include solutions or suspensions of b-apo-8'-carotenal in edible fats or oils, emulsions and water dispersible powders; these preparations may have different cis/trans isomer ratios; the analytical methods described for the parent colour are not necessarily suitable for the assay of or determination of impurities in the stabilized forms (appropriate methods should be available from the manufacturer).

Chemical names

b-apo-8’-Carotenal, 8’-apo-b-carotenal

C.A.S. number

1107-26-2

Chemical formula

C₃₀H₄₀O

Structural formula

...

Formula weight

416.65

3. Description

Deep violet crystals with metallic lustre or crystalline powder; sensitive to oxygen and light and should therefore be kept in a light-resistant container under inert gas.

4. Functional uses

Colour.

5. Specifications

5.1. Identification

...

Insoluble in water, slightly soluble in ethanol, sparingly soluble in vegetable oils, soluble in chloroform.

Spectrophotometry

Determine the absorbance of the sample solution (See Method of Assay) at 461 nm and 488 nm.

The ratio A₄₈₈/A₄₆₁ is between 0.80 and 0.84.

Carotenoid

Passes test (described in “Tests”).

Carr-Price reaction

A solution of the sample in chloroform turns blue on addition of an excess of Carr-Price reagent TS.

5.2. Purity

...

Sulfated ash

Not more than 0.1%.

Subsidiary colouring matters

Not more than 3% of total colouring matters.

See description under TESTS.

Lead

Not more than 2 mg/kg.

5.3. Content

Not less than 96% of total colouring matters.

...

5.1. Identification

Carotenoid

The colour of a solution of the sample in acetone disappears after successive additions of a 5% solution of sodium nitrite and 1 N sulfuric acid.

6.2. Purity

Sulfated ash

- Tested as directed under JECFA monograph 1. Vol.4.

- Test 2 g of the sample (Method I)

...

Dissolve about 80 mg of sample in 100 ml chloroform. Apply 400 µl of this solution as a streak 2 cm from the bottom of a TLC-plate (Silicagel 0.25 mm). Pretreat the thin-layer plate by soaking in a tank with 3% KOH in methanol so that it is completely wetted. Then dry the plate for 5 min in the air and activate for 1 h at 110^o in an oven. Let cool over CaCl2 and keep in a desiccator over CaCl₂.

Immediately after applying the carotenoid solution to the plate, develop the chromatogram with n-hexane/chloroform/ethylacetate (70+20+10) in a saturated chamber suitably protected from light, until the solvent front has moved 10 cm above the initial streak. Remove the plate, allow the main part of the solvent to evaporate at room temperature and mark the principal band as well as the bands corresponding to other carotenoids

Remove the silicagel adsorbent that contains the principal band, transfer it to a glass-stoppered 100 ml centrifuge tube and add 40.0 ml chloroform (solution 1).

Calculation:

Carotenoids other than b-apo-8'-carotenal:

where:

...

A₃ = absorbance of Solution 3

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

6.3. Method of assay

Weigh accurately about 80 mg of the sample and proceed as directed under Total Content by Spectrophotometry in JECFA monograph 1-Vol. 4 with the following conditions.

Absorptivity (a) = 2640

...

ANNEX 26

SPECIFICATIONS AND TESTS FOR β-APO-8'-CAROTENOIC ACID ETHYL ESTER

1. Synonyms

CI food Orange 7; CI (1975) No. 40825

INS 160f

ADI = 0 - 5 mg/kg bw.

2. Definition

Specifications apply to predominantly all trans (Z) isomer of ß-apo-8'- carotenoic acid ethyl ester together with minor amounts of other carotenoids; diluted and stabilized forms are prepared from ß-apo-8'-carotenoic acid ethyl ester meeting these specifications and include solutions or suspensions of ßapo-8'-carotenoic acid ethyl ester in edible fats or oils, emulsions and water dispersible powders; these preparations may have different cis/trans isomer ratios. The analytical methods described for the parent colour are not necessarily suitable for the assay of or determination of impurities in the stabilized forms (appropriate methods should be available from the manufacturer).

...

b-Apo-8'-carotenoic acid ethyl ester, ethyl 8'-apo-b-caroten-8'-oate.

C.A.S. number

1109-11-1

Chemical formula

C₃₂H₄₄O₂

Structural formula

Formula weight

460.70

...

Red to violet-red crystals or crystalline powder; sensitive to oxygen and light and should therefore be kept in a light-resistant container under inert gas.

4. Functional uses

Colour.

5. Specifications

5.1. Identification

Solubility

Insoluble in water, very slightly soluble in ethanol, slightly soluble in vegetable oils, soluble in chloroform.

Spectrophotometry

...

The ratio A⁴⁷⁵/A⁴⁴⁹ is between 0.82 and 0.86.

Carotenoid

Passes test (described in “Tests”).

Carr-Price reaction

A solution of the sample in chloroform turns blue on addition of an excess of Carr-Price reagent TS.

5.2. Purity

Sulfated ash

Not more than 0.1%.

...

Not more than 3% of total colouring matters.

Lead

Not more than 2 mg/kg.

5.3. Content

Not less than 96% of total colouring matters.

6. Tests

5.1. Identification

Carotenoid

...

6.2. Purity

Sulfated ash

Tested as directed under JECFA monograph 1-Vol. 4 - Method I, test 2 g of the sample

Subsidiary colouring matters

Carotenoids other than ß-apo-8'-carotenoic acid ethyl ester: Dissolve about 80 mg of sample in 100 ml chloroform. Apply 400 µl of this solution as a streak 2 cm from the bottom of a TLC-plate (Silicagel 0.25 mm). Pretreat the thin-layer plate by soaking in a tank with 3% KOH in methanol so that it is completely wetted. Then dry the plate for 5 min in the air and activate for 1 h at 110^o in an oven. Let cool over CaCl2 and keep in a desiccator over CaC_l2.

Remove the silicagel adsorbent that contains the principal band, transfer it to a glass-stoppered 100 ml centrifuge tube and add 40.0 ml chloroform (solution 1).

...

Calculation:

Carotenoids other than b-apo-8'-carotenoic acid ethyl ester (%):

where:

A₂ = absorbance of Solution 2

A₃ = absorbance of Solution 3

Lead

- Tested as directed under JECFA monograph 1-Vol. 4.

...

Weigh accurately about 80 mg of the sample and proceed as directed under Total Content by Spectrophotometry in JECFA monograph 1-Vol. 4 with the following conditions:

Absorptivity (a) = 2550

l_max ~ 449 nm.

ANNEX 27

SPECIFICATIONS AND TESTS FOR GRAPE SKIN EXTRACT

1. Synonyms

...

INS 163 (ii)

ADI = 0 - 2.5 mg/kg bw.

2. Definition

Obtained by aqueous extraction of grape skin or marc after the juice has been expressed from it; contains the common components of grape juice, namely: anthocyanine, tartaric acid, tannins, sugars, minerals, etc., but not in the same proportions as found in grape juice. During the extraction process, sulphur dioxide is added and most of the extracted sugars are fermented to alcohol; the extract is concentrated by vacuum evaporation during which practically all the alcohol is removed; a small amount of sulphur dioxide may be present.

Chemical names

The principal colouring matters are anthocyanins, glucosides of anthocyanidins (2-phenylbenzopyrylium salts) such as peonidin, malvidin, delphinidin, and petunidin.

Chemical formula

Peonidin : C₁₆H₁₃O₆ X

Malvidin : C₁₇H₁₅O₇ X

...

Petunidin : C₁₆H₁₃O₇ X

X: acid moiety

Structural formula

Peonidin : R = OCH₃ ; R’= H

Malvidin : R, R’= OCH₃

Delphinidin : R, R’= OH

Petunidin : R = OCH₃ ; R’= OH

X: acid moiety

...

Purplish-red liquid, lump, powder or paste, having a slight characteristic odour.

4. Functional uses

Colour

5. Specifications

5.1. Identification

Solubility

Soluble in water

Spectrophotometry

...

Colour reaction

Passes test (described in “Tests”).

5.2. Purity

Sulfur dioxide

Not more than 0.005% per 1 colour value.

Basic colouring matters

Passes test (described in “Tests”).

Other acidic colouring matters

...

Lead

Not more than 2 mg/kg

5.3. Content

The colour intensity is not less than declared.

6. Tests

6.1. Identification

Colour reaction

Add 0.1 g of the sample to 50 ml of water and shake thoroughly. Filter if necessary. The solution shows red to purplish-red colour and it turns to blue or dark green on the addition of sodium hydroxide TS.

...

Sulfur dioxide

Distil 1 g of the sample with 100 ml of water and 25 ml of phosphoric acid solution (2 in 7) in a distilling flask with the Wagner tube (Figure 1). In an absorption flask, place 25 ml of lead acetate solution (1 in 50) previously prepared. Insert the lower end of condenser into lead acetate solution in the absorption flask. Distil until the liquid in the absorption flask reaches about 100 ml and rinse the end of the condenser with a little amount of water. To the distilled solution add 5 ml of hydrochloric acid and 1 ml of starch TS, and titrate with 0.01 N iodine.

Each ml of 0.01 N iodine is equivalent to 0.3203 mg of SO₂

Figure 1 : Wagner Tube

Basic colouring matters

Add 1 g of the sample to 100 ml sodium hydroxide solution (1 in 100) and shake well. Take 30 ml of this solution and extract with 15 ml of ether. Extract this ether extract twice with each 5 ml of dilute acetic acid TS. The acetic acid extract is colourless.

...

Add 1 ml of ammonia TS and 10 ml of water to 1 g of the sample and following the directions Chromatography place 0.002 ml of the solution on the chromatographic sheet and dry it. Use a mixture of pyridine and ammonia TS (2:1 by volume) as developing solvent and stop the development when the solvent front reaches about 15 cm height from the point where the sample solution was placed.

No spot is observed at the solvent front after drying under daylight. If any spot is observed, it should be decolourized when sprayed with a solution of stannous chloride in hydrochloric acid (2 in 5).

Lead

Tested as directed under 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 assay

In the absence of an assay method, a measurement of colour intensity by the following method may be used.

Prepare approximately 200 ml of pH 3.0 citric acid - dibasic sodium phosphate buffer solution: Mix 159 volumes of 2.1% citric acid solution and 41 volumes of 0.16% dibasic sodium phosphate solution, and adjust the pH to 3.0, using the citric acid solution or Na₂HPO₄solution.

...

Colour value =

m = weight of sample (g)

ANNEX 28

SPECIFICATIONS AND TESTS FOR TITANIUM DIOXIDE

1. Synonyms

Titania, CI pigment white 6, CI (1975) No. 77891

Titanium dioxide

INS 171

...

2. Definition

Titanium dioxide is produced by either the sulfate or the chloride process. Processing conditions determine the form (anatase or rutile structure) of the final product.

In the sulfate process, sulfuric acid is used to digest ilmenite (FeTiO₃) or ilmenite and titanium slag. After a series of purification steps, the isolated titanium dioxide is finally washed with water, calcined, and micronized.

In the chloride process, chlorine gas is reacted with a titaniumcontaining mineral under reducing conditions to form anhydrous titanium tetrachloride, which is subsequently purified and converted to titanium dioxide either by direct thermal oxidation or by reaction with steam in the vapour phase. Alternatively, concentrated hydrochloric acid can be reacted with the titanium-containing mineral to form a solution of titanium tetrachloride, which is then further purified and converted to titanium dioxide by hydrolysis. The titanium dioxide is filtered, washed, and calcined.

Commercial titanium dioxide may be coated with small amounts of alumina and/or silica to improve the technological properties of the product.

C.A.S. number

13463-67-7

Chemical formula

TiO₂

...

79.88

3. Description

White to slightly coloured powder

4. Functional uses

Colour

5. Specifications

5.1. Identification

...

Insoluble in water, hydrochloric acid, dilute sulfuric acid, and organic solvents. Dissolves slowly in hydrofluoric acid and hot concentrated sulfuric acid.

Colour reaction

Passes test (described in “Tests”).

5.2. Purity

Loss on drying

Not more than 0,5%. At 105°, 3h.

Loss on ignition

Not more than 1.0% on the dried basis. At 800°.

...

Not more than 2%, either singly or combined

Acid-soluble substances

Not more than 0.5%; Not more than 1.5% for products containing alumina or silica.

Water-soluble matter

Not more than 0,5%.

Impurities soluble in 0.5 N hydrochloric acid

Antimony

Not more than 2 mg/kg

...

Not more than 1 mg/kg

Cadmium

Not more than 1 mg/kg

Lead

Not more than 10 mg/kg

Mercury

Not more than 1 mg/kg

5.3. Method of assay

Not less than 99.0% on the dried basis (on an aluminium oxide and silicon dioxide-free basis)

...

6.1. Identification

Colour reaction

Add 5 ml sulfuric acid to 0.5 g of the sample, heat gently until fumes of sulfuric acid appear, then cool. Cautiously dilute to about 100 ml with water and filter. To 5 ml of this clear filtrate, add a few drops of hydrogen peroxide; an orange-red colour appears immediately.

6.2. Purity

Acid-soluble substances

...

Suspend 5 g of the sample in 100 ml 0.5 N hydrochloric acid and on steam bath for 30 min with occasional stirring. Filter through a Gooch crucible fitted with a glass fibre filter paper. Wash with three 10-ml portions of 0.5 N hydrochloric acid, evaporate the combined filtrate and washings to dryness and ignite at a dull red heat to constant weight.

Water-soluble matter

Proceed as directed under acid soluble substances (above), using water in place of 0.5 N hydrochloric acid.

Impurities soluble in 0.5 N hydrochloric acid

Antimony, arsenic, cadmium and lead

Tested as directed under JECFA monograph 1-Vol. 4: General tests.

...

Determine antimony, cadmium, and lead using an AAS/ICP-AES technique appropriate to the specified level. Determine arsenic using the ICP-AES/AAS-hydride technique. Alternatively, determine arsenic using Method II of the Arsenic Limit Test, taking 3 g of the sample rather than 1 g. The selection of sample size and method of sample preparation may be based on the principles of the methods described in JECFA monograph 1-Vol. 4: general tests.

Mercury

Determine using the cold vapour atomic absorption technique. Select a sample size appropriate to the specified level. Tested as directed under JECFA monograph 1-Vol. 4.

Aluminium oxide

Reagents and sample solutions

0.01 N Zinc Sulfate

Dissolve 2.9 g of zinc sulfate (ZnSO₄·7H₂O) in sufficient water to make 1000 ml. Standardize the solution as follows: Dissolve 500 mg of high-purity (99.9%) aluminium wire, accurately weighed, in 20 ml of concentrated hydrochloric acid, heating gently to effect solution, then transfer the solution into a 1000-ml volumetric flask, dilute to volume with water, and mix. Transfer a 10 ml aliquot of this solution into a 500 ml Erlenmeyer flask containing 90 ml of water and 3 ml of concentrated hydrochloric acid, add 1 drop of methyl orange TS and 25 ml of 0.02 M disodium ethylenediaminetetraacetate (EDTA) Add, dropwise, ammonia solution (1 in 5) until the colour is just completely changed from red to orange-yellow. Then, add:

...

(b): 10 ml of diammonium hydrogen phosphate solution (150 g of diammonium hydrogen phosphate in 700 ml of water, adjusted to pH 5.5 with a 1 in 2 solution of hydrochloric acid, then dilute to 1000 ml with water).

Boil the solution for 5 min, cool it quickly to room temperature in a stream of running water, add 3 drops of xylenol orange TS, and mix. Using the zinc sulfate solution as titrant, titrate the solution to the first yellow-brown or pink end-point colour that persists for 5-10 sec. (Note: This titration should be performed quickly near the end-point by adding rapidly 0.2 ml increments of the titrant until the first colour change occurs; although the colour will fade in 5-10 sec, it is the true end-point. Failure to observe the first colour change will result in an incorrect titration. The fading end-point does not occur at the second end-point.)

Add 2 g of sodium fluoride, boil the mixture for 2-5 min, and cool in a stream of running water. Titrate this solution, using the zinc sulfate solution as titrant, to the same fugitive yellow-brown or pink end-point as described above.

Calculate the titre T of zinc sulfate solution by the formula:

T = 18,896 m / V

where:

T is the mass (mg) of Al₂O₃ per ml of zinc sulfate solution

m is the mass (g) of aluminium wire

V is the ml of the zinc sulfate solution consumed in the second titration

...

R is the ratio of the formula weight of aluminium oxide to that of elemental aluminium.

Sample Solution A

Accurately weigh 1 g of the sample and transfer to a 250-ml high-silica glass Erlenmeyer flask. Add 10 g of sodium bisulfate (NaHSO₄·H₂O). (Note: Do not use more sodium bisulfate than specified, as an excess concentration of salt will interfere with the EDTA titration later on in the procedure.) Begin heating the flask at low heat on a hot plate, and then gradually raise the temperature until full heat is reached.

(Caution: perform this procedure in a well ventilated area. ) When spattering has stopped and light fumes of SO3 appear, heat in the full flame of a Meeker burner, with the flask tilted so that the fusion of the sample and sodium bisulfate is concentrated at one end of the flask. Swirl constantly until the melt is clear (except for silica content), but guard against prolonged heating to avoid precipitation of titanium dioxide. Cool, add 25 ml sulfuric acid solution (1 in 2), and heat until the mass has dissolved and a clear solution results. Cool, and dilute to 120 ml with water. Introduce a magnetic stir bar into the flask.

Sample Solution B

Prepare 200 ml of an approximately 6.25 M solution of sodium hydroxide. Add 65 ml of this solution to Sample Solution A, while stirring with the magnetic stirrer; pour the remaining 135 ml of the alkali solution into a 500-ml volumetric flask.

Slowly, with constant stirring, add the sample mixture to the alkali solution in the 500-ml volumetric flask; dilute to volume with water, and mix. (Note: If the procedure is delayed at this point for more than 2 hours, store the contents of the volumetric flask in a polyethylene bottle.) Allow most of the precipitate to settle (or centrifuge for 5 min), then filter the supernatant liquid through a very fine filter paper. Label the filtrate Sample Solution B.

Sample Solution C

Transfer 100 ml of the Sample Solution B into a 500-ml Erlenmeyer flask, add 1 drop of methyl orange TS, acidify with hydrochloric acid solution (1 in 2), and then add about 3 ml in excess. Add 25 ml of 0.02 M disodium EDTA, and mix. [Note: If the approximate Al₂O₃ content is known, calculate the optimum volume of EDTA solution to be added by the formula:

...

Add, dropwise, ammonia solution (1 in 5) until the colour is just completely changed from red to orange-yellow. Then add10 ml each of Solutions 1 and 2 (see above) and boil for 5 min. Cool quickly to room temperature in a stream of running water, add 3 drops of xylenol orange TS, and mix. If the solution is purple, yellow-brown, or pink, bring the pH to 5.3 - 5.7 by the addition of acetic acid. At the desired pH, a pink colour indicates that not enough of the EDTA solution has been added, in which case, discard the solution and repeat this procedure with another 100 ml of Sample Solution B, using 50 ml, rather than 25 ml, of 0.02 M disodium EDTA.

Procedure

Using the standardized zinc sulfate solution as titrant, titrate Sample Solution C to the first yellow-brown or pink end-point that persists for 5-10 sec. (Important: See Note under “0.01 Zinc sulfate”.) This first titration should require more than 8 ml of titrant, but for more accurate work a titration of 10-15 ml is desirable.

Add 2 g of sodium fluoride to the titration flask, boil the mixture for 2-5 min, and cool in a stream of running water. Titrate this solution, using the standardized zinc sulfate solution as titrant, to the same fugitive yellow-brown or pink end-point as described above.

Calculation:

Calculate the percentage of aluminium oxide (Al₂O₃) in the sample taken by the formula:

% Al₂O₃ = 100 × (0,005VT)/m

where:

V is the number of ml of 0.01 N zinc sulfate consumed in the second titration,

...

m is the mass (g) of the sample taken, and

0.005 = 500 ml / (1000 mg/g × 100 ml).

Silicon dioxide

Accurately weigh 1 g of the sample and transfer to a 250-ml high-silica glass Erlenmeyer flask. Add 10 g of sodium bisulfate (NaHSO4 · H2O). Heat gently over a Meeker burner, while swirling the flask, until decomposition and fusion are complete and the melt is clear, except for the silica content, and then cool. (Caution: Do not overheat the contents of the flask at the beginning, and heat cautiously during fusion to avoid spattering.)

To the cooled melt add 25 ml of sulfuric acid solution (1 in 2) and heat carefully and slowly until the melt is dissolved. Cool, and carefully add 150 ml of water by pouring very small portions down the sides of the flask, with frequent swirling to avoid over-heating and spattering. Allow the contents of the flask to cool, and filter through fine ashless filter paper, using a 60 degree gravity funnel. Rinse out all the silica from the flask onto the filter paper with sulfuric acid solution (1 in 10). Transfer the filter paper and its contents into a platinum crucible, dry in an oven at 120°, and heat the partly covered crucible over a Bunsen burner. To prevent flaming of the filter paper, first heat the cover from above, and then the crucible from below.

When the filter paper is consumed, transfer the crucible to a muffle furnace and ignite at 1000^o for 30 min. Cool in a desiccator, and weigh. Add 2 drops of sulfuric acid (1 in 2) and 5 ml of concentrated hydrofluoric acid (sp.gr. 1.15), and carefully evaporate to dryness, first on a low-heat hot plate (to remove the HF) and then over a Bunsen burner (to remove the H₂SO₄). Take precautions to avoid spattering, especially after removal of the HF. Ignite at 1000^o for 10 min, cool in a desiccator, and weigh again. Record the difference between the two weights as the content of SiO₂ in the sample.

6.3. Method of assay

...

Accurately weigh about 150 mg of the sample, previously dried at 105^o for 3 hours, and transfer into a 500-ml conical flask. Add 5 ml of water and shake until a homogeneous, milky suspension is obtained. Add 30 ml of sulfuric acid and 12 g of ammonium sulfate, and mix. Initially heat gently, then heat strongly until a clear solution is obtained. Cool, then cautiously dilute with 120 ml of water and 40 ml of hydrochloric acid, and stir. Add 3 g of aluminium metal, and immediately insert a rubber stopper fitted with a U-shaped glass tube while immersing the other end of the U-tube into a saturated solution of sodium bicarbonate contained in a 500-ml wide-mouth bottle, and generate hydrogen. Allow to stand for a few minutes after the aluminium metal has dissolved completely to produce a transparent purple solution. Cool to below 50^o in running water, and remove the rubber stopper carrying the U-tube. Add 3 ml of a saturated potassium thiocyanate solution as an indicator, and immediately titrate with 0.2 N ferric ammonium sulfate until a faint brown colour that persists for 30 seconds is obtained. Perform a blank determination and make any necessary correction.

Each ml of 0.2 N ferric ammonium sulfate is equivalent to 7.990 mg of TiO₂.

ANNEX 29

SPECIFICATIONS AND TESTS FOR IRON OXIDES

1. Synonyms

Iron oxides;

Iron Oxide yellow: CI Pigment Yellow 42 and 43; CI(1975) No. 77492; INS No. 172(iii)

Iron Oxide Red: CI Pigment Red 101 and 102; CI (1975) No. 77491; INS No. 172(ii)

...

ADI = 0 - 0.5 mg/kg bw.

2. Definition

Iron oxides are produced from ferrous sulfate by heat soaking, removal of water, decomposition, washing, filtration, drying and grinding.

They are produced in either anhydrous or hydrated forms. Their range of hues includes yellows, reds, browns and blacks. The food-quality iron oxides are primarily distinguished from technical grades by their comparatively low levels of contamination by other metals; this is achieved by the selection and control of the source of the iron or by the extent of chemical purification during the manufacturing process.

Chemical names

Iron Oxide Yellow: Hydrated ferric oxide, hydrated iron (III) oxide

Iron Oxide Red: Iron sesquioxide, anhydrous iron (III) oxide

Iron Oxide Black: Ferroso ferric oxide, iron (II,III) oxide

C.A.S. number

...

Iron Oxide Red: 1309-37-1

Iron Oxide Black: 1317-61-9

Chemical formula

Iron Oxide Yellow: FeO(OH) ^. xH₂O

Iron Oxide Red: Fe₂O₃

Iron Oxide Black: FeO · Fe2O3

Formula weight

88.85 FeO(OH)

159.70 Fe₂O₃

...

3. Description

Yellow, red, brown or black powder

4. Functional uses

Colour

5. Specifications

5.1. Identification

Solubility

Insoluble in water and organic solvents; soluble in concentrated mineral acids.

...

Not more than 1,0%.

5.2. Purity

Loss on drying

Iron Oxide Red : Not more than 1.0% (105°, 4 h)

Arsenic

Not more than 3 mg/kg.

Cadmium

Not more than 1 mg/kg.

...

Not more than 10 mg/kg

Mercury

Not more than 1 mg/kg

5.3. Content

Not less than 60% of iron.

6. Tests

6.1. Purity

Arsenic, cadmium and lead

...

Mercury

- Tested as directed under JECFA monograph 1-Vol. 4.

6.2. Method of assay

Weigh accurately about 0.2 g of the sample, add 10 ml of 5 N hydrochloric acid and heat cautiously to boiling in a 200-ml conical flask until the sample has dissolved. Allow to cool, add 6 to 7 drops of 30% hydrogen peroxide solution and again heat cautiously to boiling until all the excess hydrogen peroxide has decomposed (about 2-3 min). Allow to cool, add 30 ml of water and about 2 g of potassium iodide and allow to stand for 5 min. Add 30 ml of water and titrate with 0.1 N sodium thiosulfate adding starch TS as the indicator towards the end of the titration. Each ml of 0.1 N sodium thiosulfate is equivalent to 5.585 mg of Fe (III).

ANNEX 30

...

1. Synonyms

CI food green 3,CI (1975): 42053

INSL: 143

ADI = 0 - 25 mg/kg bw.

2. Definition

Consists essentially of disodium 3-[N-ethyl-N-[4-[[4-[N-ethyl-N-(3- sulfonatobenzyl)amino]phenyl](4-hydroxy-2-sulfonatophenyl)methylene]-2,5-cyclohexadien-1-ylidene]ammoniomethyl]benzenesulfonate and isomers and subsidiary colouring matters together with water, NaCl and/or Na₂SO₄ as the principal uncoloured components.

May be converted to the corresponding aluminium lake in which case only the General Specifications for Aluminium Lakes of Colouring Matters applies.

Chemical names

Disodium 3-[N-ethyl-N-[4-[[4-[N-ethyl-N-(3-sulfonatobenzyl) amino] phenyl](4-hydroxy-2-sulfonatophenyl)methylene]-2,5-cyclohexadien-1- ylidene] ammoniomethyl]-benzenesulfonate;

...

C.A.S. number

2353-43-9

Chemical formula

C₃₇H₃₄N₂Na₂O₁₀S₃

Structural formula

Formula weight

808.86

3. Description

...

4. Functional uses

Colour.

5. Specifications

5.1. Identification

Solubility

Soluble in water; sparingly soluble in ethanol.

Identification of colouring matters

Passes test.

...

Loss on drying at 135°C

Not more than 15% together with chloride and sulfate calculated as sodium salts.

Water insoluble matter

Not more than 0.2%.

Lead

Not more than 2 mg/kg.

Chromium

Not more than 50 mg/kg.

...

Not more than 6%.

Organic compounds other than colouring matters

Not more than 0.5%, sum of 2-, 3-, and 4-Formylbenzenesulfonic acids, sodium salts.

Not more than 0.3%, sum of 3- and 4-[N-Ethyl-N-(4- sulfophenyl)amino]methylbenzenesulfonic acid, disodium salts.

Not more than 0.5% of 2-Formyl-5-hydroxybenzenesulfonic acid, sodium salt.

Unsulfonated primary aromatic amines

Not more than 0.01% calculated as aniline.

Leuco base

Not more than 5.0%.

...

Not more than 0.4%.

5.3. Content

Not less than 85% total colouring matter.

6. Tests

6.1. Purity

Lead

Tested as directed under 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.”

Subsidiary colouring matters

...

Note: In the calculation of the subsidiary colour, it is assumed that its absorptivity at its peak is the same as that of the parent compound at its peak. Standard solutions must be prepared and their absorptivities measured within 1 h. Spectrophotometric measurements of the extracted subsidiary colours must be made as promptly as possible.

Organic compounds other than colouring matters

- Tested as directed under JECFA monograph 1-Vol. 4.

- Proceed as directed under Column Chromatography, using, for example, the following absorptivities:

3-formylbenzenesulfonic acid: 0.495 mg L^-1 cm^-1 at 246 nm in dilute HCl

3-[(ethyl)(4-sulfophenyl)amino]methylbenzenesulfonic acid: 0.078 mg L^-1 cm^-1 at 277 nm in dilute ammonia

2-formyl-5-hydroxybenzenesulfonic acid: 0.080 mg L^-1 cm^-1 at 335 nm in dilute ammonia

Leuco base

- Tested as directed under JECFA monograph 1-Vol. 4.

...

Absorptivity (a) = 0.156 mg L^-1 cm^-1 at approx. 625 nm. Ratio = 0.971.

6.2. Method of assay

Proceed as directed under Total Content by Titration with Titanous Chloride (under JECFA monograph 1-Vol. 4), using the following:

Weight of sample: 1.9 - 2.0 g;

Buffer: 15 g sodium hydrogen tartrate;

Weight (D) of colouring matters equivalent to 1.00 ml 0.1 N TiCl₃: 40.45 mg.

Lưu trữ
Ghi chú
Ý kiến
Facebook
Email
In

Nguồn: https://thuvienphapluat.vn/TCVN/Cong-nghe-Thuc-pham/QCVN-4-10-2010-BYT-Food-additives-Colours-921582.aspx

QCVN-4-10-2010-BYT-Food-additives-Colours

Bài viết liên quan:

Tiêu chuẩn quốc gia TCVN 9364:2012 về Nhà cao tầng - Kỹ thuật đo đạc phục vụ công tác thi công

Công điện 23/CĐ-TTg 2025 đẩy nhanh tiêm chủng vắc xin phòng chống bệnh Sởi

Quyết định 607/QĐ-TTg 2025 Quy chế hoạt động của Khu Công nghệ cao sinh học Hà Nội

Quyết định 124/QĐ-QLD 2025 danh mục 699 thuốc được cấp giấy đăng ký lưu hành Đợt 216

Chỉ thị 07/CT-TTg 2025 đẩy mạnh triển khai Đề án phát triển ứng dụng dữ liệu về dân cư

Công văn 1791/BCT-TTTN 2025 điều hành kinh doanh xăng dầu

Công văn 128/CHQ-GSQL 2025 triển khai mô hình tổ chức mới của cơ quan Hải quan

Công văn 56/BHXH-TCCB 2025 bổ sung bố trí sắp xếp nhân sự sắp xếp tinh gọn tổ chức bộ máy

Quyết định 120/QĐ-QLD 2025 công bố Danh mục thuốc biệt dược gốc Đợt 1

Thông báo 105/TB-VPCP 2025 kết luận Đề án Phát triển Trường Đại học Y Hà Nội đến 2030