SPECIFICATIONS FOR IDENTITY AND PURITY
AND TOXICOLOGICAL EVALUATION
OF FOOD COLOURS
Geneva, 8-17 December 1964
Food and Agriculture Organization of the United Nations • World Health Organization 1988 1
1. THE NEED FOR AND VALUE OF SPECIFICATIONS FOR PURITY l
At the eighth meeting of the Expert C0111Dittee on food additives it was again emphasized that there was a need to establish at an international level specifications for the identity and purity of food additives. It was noted that for many years national phannacopoeias and similar compendiums have contained specifications for the more important drugs and drug components, while the International Phannacopoeia, published by WHO, has provided similar data at the international level. In recent years, certain governments have begun to prepare specifications for the chemicals that enter the national food supply. There is a great need to prepare such data at an international level.
1.1 Value in protecting the consumer
Whereas a natural food m~ vary in composition, sometimes to a considerable degree or in undefined w~s, considerations of public health dictate that, as a matter of principle, additives to food should be of known composition and purity. In fact, modern methods make it possible to produce chemicals of greater purity and unifonnity by synthesis than is usually achieved by derivation from substances of natural origin. The adoption of official specifications for food additives would give assurance to the cons\llling public that substances meeting established standards of purity are available for use in food.
1.2 Value for regulatory purposes
At the present time most food legislation merely indicates by name the substances which m~ be used in a particular food. It is a well-lmown fact that chemicals are produced in a variety of technical and refined grades. Toxicological evaluation, which is a costly and time-consuming procedure, must be related to the particular grade or quality of chemical intended for use in food. The adoption of specifications for purity for food additives would provide a 11111an. of accurate identification of the additive tor regulatory purposes and would limit the known undesirable ingredients or contaminants to acceptable tolerance levels.
1 This section is reproduced, with slight modifications, from Specifications for identity and purity of food additives. Vol. II. Food colors, Rome, Food and Agriculture Organization of the United Nations, 1963, pp, ,:s. FAO Nutrition Meetings Report Series No. 38B WHO/Food Add./66.25 'Ei,~lisi. on91
SPECIFICATIONS FOR IDENTITY AND PURITY AND TOXICOLOGICAL EVALUATION OF FOOD COLOURS
The content of this document is the result of the deliberations of the Joint FAO/WHO ~ert Committee on Food Additives 1,2,3
1 Fourth Report of the Joint FAO/WHO Expert Committee on Food Additives; subsequently revised and published as Specifications for Identity and Purity of Food Additives, Vol. II Food Colours, Rome, FAO 1963. 2 Eighth Report of the Joint FAO/WHO Expert Committee on Food Additives. Wld Hlth Org. techn. Rep. Ser. 1965, 309, FAO Nutr. Meetings Rep. Ser. 1965, No. 38 3 Tenth Report of the Joint FAO/WHO Expert Committee on Food Additives. To be published in the Wld Hlth Org. techn. Rep. Ser., and FAO Nutr. Meetings Rep. Ser. CONTENTS l. The need for and value of specifications for purity l 1.1 Value in protecting the consumer •• l 1.2 Value for regulatory purposes l 1.3 Value to industry ••••••• 2 1.4 Value in detennining safety for use 2 l. 5 Source and nature of impurities 3 2. General considerations on food colours 4 2.1 General principles governing the establishment of specifications for identity and purity. 4 2.1.l Natural colours. • • • • • • • • • • • • 4 2.1.2 Synthetic organic food colours 5 2.1.3 Substances studied •••••• 5 2.1.4 Note on the specifications in the monographs. '6 2.2 General principles governing the toxicological evaluation • • . . . . • • • • • . . • • • • . . • . 8 2.2.l Classification of colours in accordance with their toxicological evaluation 11 2.2.2 Conclusion 12 3. Note to the reader ••• 13 4. Classifications of colours 15 5. Monographs on Colours in Toxicological Categories A, B and Cl • • • . . • • • • • • • . • . • • • • • • • • • • 21 6. Specifications for Colours in Toxicological Categories CII, CIII and D Which are Used in Some Countries 103 Appendix A Methods 154 Appendix B Test Solutions ••••••••••• 169 Appendix C Index by Colour Index Number 1956 •••••• 172 Appendix D Alphabetical Index ••••••••• 191 l
1, THE NEED FOR AND VALUE OF SPECIFICATIONS FOR PURI'l'll l
At tbe eighth meeting of the Expert Committee on food additives it was again emphasized that there was a need to establish at an international level specifications for the identity and purity of food additives. It was noted that for many years national pharmacopoeias and similar compendiums have contained specifications for the more important drugs and drug components, while the International Pharmacopoeia, published by WHO, has provided similar data at the international level, In recent years, certain governments have begun to prepare specifications for the chemicals that enter the national food supply, There is a great need to prepare such data at an international level.
1.1 Value in protecting the consumer
Whereas a natural food may vary in composition, sometimes to a considerable degree or in undefined ways, considerations of public health dictate that, as a matter of principle, additives to food should be of known composition and purity. In fact, modern methods make it possible to produce ohemicals of greater purity and uniformity by synthesis than is usually achieved by derivation from substances of natural origin, The adoption of official specifications for food additives would give assurance to the consuming public that substances meeting established standards of purity are available for use in food.
1,2 Value for regulatory purposes
At the present time most food legislation merely indicates by n81118 the substances which may be used in a particular food, It is a well-known faot that chemicals are produced in a variety of technical and refined grades. Toxicological evaluation, which is a costly and time-consuming procedure, must be related to the particular grade or quality of chemical intended for use in food. The adoption of specifications for purity for food additives would provide a meana of accurate identification of the additive for regulatory purposes and would limit the known undesirable ingredients or contaminants to acceptable tolerance levels,
1 This section is reproduced, with slight modifications, from Specifications for identity and purity of food additives. Vol. II, Food colors, Rome, Food and Agriculture Organization of the United Nations, 1963, PP• }-6. 2
1,3 Value to industry
The existence of specifications, agreed upon by qualified specialists, serves to ensure a degree of reproducibility and of conformity to criteria of quality which are acceptable to both chemical manufacturers and food processors. Furthermore, established specifications might well act as a guide in the developnent of new chemicals of a quality suitable for food use,
It is important that specifications for identity and quality of a food additive should be no more stringent than necessary to accomplish their purpose and that they should be reasonably attainable by the producing industries. Otherwise, the consuming public would ultimately bear an unnecessary additional cost of production and control,
1,4 Value in determining safety for use
One of the most important areas in which specifications for purity would be of particular value is in the determination of the safety for use of food additives, It is essential to know the identity and concentration of the major component or components of a food additive before carrying out an effective toxicological investigation of its properties, Even small differences in composition of a compound may materially alter the results of toxicity tests, The investigator must also know the nature and quantity of the important impurities, Toxicologists have frequently emphasized that impurities or minor constituents may have a significance far greater than their amounts might indicate, Information relating to physical properties, such as solubility, is also essential,
In many animal tests, particularly with some of the relatively inert food additives, large amounts of the chemical are required and therefore the investigator must be certain that he has sufficient material of a uniform nature or a reliable source of the material of the same composition. In certain instances, years of animal studies have been discarded because the composition of the food additive changed during the test period, Furthermore, even if tests demonstrate beyond any reasonable doubt that a particular substance is safe for use, their value is impaired when the food additive used commercially differs significantly from the material tested.
The results of a single investigation are not likely to answer for all time the question of the safety for use of a particular material, The Joint FAO/WHO Expert Committee on Food Additives stated at its first session that permitted additives should be subjected to continuing observation for possible deleterious effects under changing conditions of uae and should be reappraised whenever indicated by advances in knowledge. Specifications based on the material used in previous teats would therefore be of great value in ll&k1ng certain that a ~omparable product was employed in such reappraisals. The divergent results which are occasionally encountered in the toxicological investigation of the same product mq conceivably be due to variations in the composition of batches of the material under test.
1.5 Source and nature of impurities
The purity of a food additive, as the term is here •ployed, refers to its freedom from substances other than those named in specifications. "Foreign substances• or "impurities" not included in the specifications mq be, for eXU1ple, simple inorganic salts or other substances not necessarily deleterious from the functional or safety standpoint.
Impurities mq arise from the raw materials used in the manufacture of chemicals (especially when they are complex natural substances), from substances used in processing steps, from solvents used in extraction or crystallization, and from equipnent. They mq also be unreacted intermediates or by-products formed in the course of processing, such as incompletely esterified acids or isomeric derivatives. Products of decomposition during storage, such as 111q result from oxidation, hydrolysis, or polymerization, are likewise regarded as impurities. However, the constituents of polymeric or other mixtures of reproducible composition are not regarded as impurities if they contribute to the.functional properties of the substance as a whole and are not deleterious.
Obviously contaminants like dirt, soot, rust, lubricants and insect frag111ents must be avoided in manufacture, packaging, and storage of food additives. Whereas their presence is generally revealed in the application of the tests given in the specifications, no specific tests for the detection and identification of these contaminants are included.
From the foregoing discussion it is obvious that, depending on the original materials and manufacturing procedure, impurities mq be volatile or nonvolatile, organic or inorganic, deleterious or nondeleterious. The important factors to be considered are:
(a) Is the illlpurity one which might Jeopardize the safe use of the food additive? (b) Is the amount of impurity sufficient to affect the activity or usefulness of the food additive? (c) Can the impurity be reduced in uount or avoided by good manufacturing practice? (d) Is the illlpurity ot autticient consequence to Justify a limitation?
Whereas food additives are usually employed in relatively small quantities and traces of impurities may pose no serious health hazards, prudence dictates that reasonable limits be established for impurities, consistent with good manufacturing practice as Judged by modem standards.
2. GENERAL CONSIDERATIONS ON FOOD COLOURS
2.1 General principles governing the establishment of specifications for identity and purity
2.1.l Natural colours Natural colours have been used in food over a long period of time and have been accepted for such use without supporting toxicological evidence in much the same manner as vegetables and cereal products. In considering such food colours, the Coamittee was faced with serious problems owing to the lack of published information relating to adequate identification and chemical composition. It was noted that natural colours may be available in different forms; in the case of botanical products both the powdered plant and the extract of the powdered plant have been used as colouring agents for many years. Also, because of differences in soil, climatic conditions, age of plant, and tille of harvest, the nature and proportions of the coloured~ other components of the same species of the saae plant may vary widely. Such products may contain a large percentage of substances which have not been defined. Morphological and histological examination of such botanicals has been used for a long time for the identification and quality evaluation of these 111&terials, but in the opinion of the C011111ittee the information obtained in this way is inadequate.
For certain products of other than plant origin - tor example, natural and synthetic ultramarines, cochineal and caramel - similar problems of chemical identification have been encountered. For this reason. the Coamittee concluded that it was impossible to prepare adequate specifications for these products.
The Coamittee considers it essential that 111ethods of analysis, including adequate identification and quality data, should be provided whenever any 5 toxicological evaluation is undertaken on colouring matters of natural origin. It is hoped that future studies Ifill provide such information.
2.1.2 Synthetic organic food colours
Identification of dyes is a difficult task, particularly in the oase of the water-soluble sulfonated dyes. Most of the colours for which specifications were drawn up are of this type. Dyes in commerce may be diluted for convenience of use or other reasons. The Conmittee made no attempt to soec1fy limits of purity for the large number of such mixtures marketed.
It should be noted that specifications were not recommended for certain synthetic organic food colours because there were not sufficient analytical data at hand to define good manufacturing practice. However, the Co111nittee recommends that any synthetic organic colour used in food should meet specification requirements similar to those set forth for such colours as Amaranth, Sunset Yellow and Tart.razine. Furthermore, specifications for colours synthesized from non-sulfonated aromatic amines should limit such impurities to not more than 0.02~. It should be emphasized that in no circumstances may known carcinogenic amines be used as starting materials 1n the production of food colours.
The CoDDittee agreed that the availability of substances as reference materials for the synthetic organic food colours is essential for their identification and assay.
2.1.} Substances studied
The Conmittee had before it a provisional list of food colours for which as much information as possible had been collected. The Conmittee examined this list from the standpoint of chemical specifications and classified the colours into one of the following four categories:
I. Food colours for which the Conmittee prepared specifications. II. Food colours for which the Conunittee was not ready to prepare specifications at this time, since toxicological studies are known to be in progress on substances of defined composition. III. Food colours for which the chemical information available to the C0111111ittee was not adequate to permit the preparation of a completely satisfactory specification. 6
IV. Food colours for which the Committee did not attempt to prepare specifications either because the toxicological data were totally lacking or because the colours were found to be harmful and their use in foods was not considered desirable.
On page 15 are shown the common names of the colours, the 1956 Colour Index numbers, and the categories into which the colours were placed.
2.1.4 Note on the specifications in the monographs
The specifications for identity and purity included in the monographs have been prepared in accordance with the general principles set out in the report of the Joint PAO/WHO Expert Committee on Food Additives which met in Geneva from 8-17 December 1964.
The specifications for synthetic food colours differ slightly in presentation from those for the natural food colours and those given in the manual on Antimicrobials, Preservatives and Antioxidants.1 Tests for identity have therefore not been included in the monographs; instead descriptions for procedures that can be applied are given in Appenau A. Colours are in each case defined by the terms of specifications. The specifications also imply reference to the purity tests detailed in Appendix A. The specifications, including minimum colour content, are meant to indicate the concentration of the colour that is obtainable under conditions of good manufacturing practice.
Nomenclature. The title of each monograph contalllS the name by which the substance is most coamonly known in food manufacture. Where other recognized names exist, they are listed as synonyms. For the chemical names, the recoanendations of the International Union of Pure and Applied Chemistry (IUPAC) and the usages of the various national chemical societies have been taken into consideration. Where the structure or cOD1position of a food additive has not been clearly elucidated, a description of its chemical nat\D'e is given instead of the chemical name.
Class and colour. The chemical class to which the principal colouring C011pound belongs and its colour are indicated in each monograph.
1 Specifications for identity and purity of food additives Volume I Antimicrobials, Preservatives and Antioxidants, PAO, Rome 1962. 7
Formulas. A chemical formula is given for each inorganic substance and empirical and structural formulas are shown, where lmown, for each organic substance. All fonnulas represent the pure compounds and serve only a descriptive purpose.
Molecular weight. For purposes of information and description, the molecular weights of the compounds are given where known. These have been calculated from the table of atomic weights approved by the Conunission on Atomic Weights of the International Union of Pure and Applied Chemistry.
Temperature. All temperatures are given in degrees centigrade.
Solubility. The solubilities of the substances contained in the monographs are given without reference to possible chemical changes. Unless otherwise stated, the standards adopted are room temperature and normal atmospheric pressure.
Solvents. Unless otherwise stated, reference to water in the monographs presumes distilled water. The term "ethanol" is used as referring to 95% v/v ethyl alcohol, and the term "absolute ethanol" for solvent containing not less than 98.8% v/v ethyl alcohol. Other v/v concentrations of ethanol may be specified in individual cases.
Assays and dye content. The methods described are those which are considered to be the most suitable. All results are reported on the basis of the total material assayed. A second method is included if the preferred procedure requires instruments not widely available.
Subsidiary dyes and intermediates. Maximum tolerances for subsidiary dyes and intermediates are given in the monographs dealing with synthetic organic colours. In the co11111ercial production of food colours under conditions of good manufacturing practice it is not practicable to use absolutely pure raw materials, or to eliminate all reaction products other than the main component. The commercial product therefore contains, from one or more sources, organic compounds other than the main component. Those that are coloured are termed "subsidiary dyes". Those that are not coloured are considered to be intermediate$.
Methods. General methods applicable to a number of colouring matters are presented in Appendix A. 8
Test solutions. All reagents referred to are assumed to be analytical grade, unless otherwise specified.
Test solutions (TS) are given in alphabetical order in Appendix B; the indication PbT means that the test solution must be free from lead.
Solutions in general are brought to approximate normality or to a simple multiple or fraction of normality. When the normality or percentage composition of acids is not indicated, the concentrated form should be used.
Where the use of a test solution as indicator is specified 1n a test or assay, three drops of the solution shall be added, generally as the end-point is approached, unless otherwise directed.
2.2 General principles governing the toxicological evaluation
The second and fifth reports of the Joint FAO/WHO Expert Conmittee on Food Additives have laid down the fundamental principles on which the evaluation of the acceptability of food additives should be based.
Advances in scientific knowledge and experience in the application of these principles have served to emphasize their importance. Certain aspects of the toxicological data required for adequate evaluation have been further elaborated in the sixth and seventh reports of the Joint FAO/WHO Expert Committee on Food Additives.
In applying these recoD111endations to the evaluation of food-colouring agents, the Committee had to take into consideration the fact that the use of this class of additives in food is considered by many to be unnecessary. At a meeting of the Joint FAO/WHO Expert Committee on Food Additives in 1956 it was agreed "that there are cases in which the use of food colours is justified and that the best method for regulation is the establishment of a list of permitted colours which have been tested adequately by animal experd.mentation. 11 1 On general grounds it is now felt that there should be insistence on the fulfilment of the complete requirements deemed necessary for reliable evaluation.
The decisions of the Committee have been reached solely on the basis of toxicological and related information. Questions concerning the need for food colours or their technical suitability are the province of other
1 FAO Nutrition Meetings Report Series, 1961, No. 29, p.25; Wld Hlth Org. techn. Rep. Ser., 1961, ggQ, 25. 9 bodies - on the intemational level, the Codex Alimentarius Conmittee on Food Additives.
It is uaeful to list here the main headings under which information was soughtsl,2,3,4
Chemical and physical specifications (second report, p. 7; fifth report, pp. 7-8) Acute toXicity (second report, p. 9) Short-term toxicity (second report, p. 10) Long-term toxicity (second report, pp. 11-12) Carcinogenicity studies (fifth report, pp. 8-14 and 22-23) Metabolic studies (second report, pp. 13-14; sixth report, p. 8).
In evaluating food colours the main emphasis must be placed on studies of metabolism and long-term toxicity. In the past, many investigations of a long-term nature were tests for carcinogenicity. Often, the n1.111ber of animals employed was small and comparisons with control groups were not made. Many of the reports contained no mention of the majority of the observations eonsi4ered to be an essential part of long-term toxicity studies.
The criteria for long-term studies that are especially relevant in the case of food colours are:
(a) Growth curves and associated data (second report, pp. 11-12) (b) Studies of haemopoietic function (c) Studies of hepatic and renal function, and, where indicated, the function of other organs (d) Organ weights at autopsy (e) Histopathological investigations (fifth report, pp. 15-16). The effects on reproduction and the foetus need to be studied (second report, p. 12).
1 FAO Nutrition Meetings Report Series, 1961, No. 29; Wld Hlth Org. techn, Rep. Ser., 1961, ggQ. 2 FAO Nutrition Meetings Report Series, 1962, No. 31; Wld Hlth Org. techn. Rep. ~·, 1962, 228.
3 FAO Nutrition Meetings Report Series, 1964, No. 35; Wld Hlth Org, techn. Rep. ~·, 1964, 281. 4 This section is reproduced, with slight modifications, from Specifications for identity and purity of food additives. Vol. II. Food colors, Rome, Food and Agriculture Organization of the United Nations, 1§63, pp. 3=6, 10
It should be added that, even when such studies are carried out in adequate detail, the final reports are often not presented in a fonn that permits independent assessment of the validity of the conclusions reached. Statistical treatment of the results is also often lacking. It must be stressed that a reliable evaluation can be reached only if detailed infonnation is available conceming the experimental results (fifth report, Annex 2, p. 33).
It is regrettable that a substantial proportion of the available infonnation is to be found only in unpublished reports. In the past, this has been attributed in part to the lack of suitable media tor the publication of results in this field. This situation has now been remedied by the foundation of specialist periodicals, such as "Toxicology and Applied Pharmacology" and "Food and Cosmetics Toxicology". One of these even undertakes to summarize unpublished data, to submit the sunmary to the authors for approval and to arrange for prompt publication. In view of these changed circ1111stances, it m~ in future be undesirable to assess acceptable daily intakes solely on the basis of unpublished work.
With regard to the number of animal species required, the Coamittee endorsed the recoanendations for short-term toxicity studies given in the second report, namely that at least two species, one of them a non-rodent should be used.
In the case or long-term toxicity studies, the requirements of the second report (p. 12) provide for the use of at least two species, in tests extending over the major part of their life-span. For the evaluation of carcinogenic hazard the fifth report (p. 22) recomnends "the acceptance of a life-span feeding study in two species of animal (e.g., rats and mice) carried out along the lines reconnended in the second report of the Joint FAOj\iHO Expert Con111ittee on Food Additives as a minimum safeguard." Thus, if these recoamendations are followed, both toxicity and carcinogenicity can be assessed in the same study.
The second and sixth reports (p. 13 and p. 8 respectively) have stated very clearly why metabolic studies are necessary for purposes of evaluation. In the past, some food additives have been considered acceptable in the absence of adequate information under this heading. For food colours, however, there must be insistence on a knowledge of metabolism, since many of these compounds, by their chemical nature, are liable to give rise to potentially toxic degradation products, either by the action of intestinal micro-organisms or by metabolic transformations within the body. 11
For the evaluation of the.acceptability of food colours derived from natural sources, the same criteria must be applied as for synthetic colours. Here the position is rendered difficult, not only by the paucity of biological studies but also by the lack of knowledge of composition and the variation in composition according to the source and the method of preparation employed.
This situation is particularly unhappy in the case of colours that may be extracted from edible vegetable products cons\lDed in relatively large amounts, e.g., tomatoes, carrots, beetroot, green leafy vegetables, and fruits. Nevertheless the C011111ittee felt that, in the absence of specifications (see section 2.1.1) and experimental data, the principles set forth in previous reports preclude the possibility of making an evaluation.
In view of the fact that no satisfactory specifications could be established for carbon black, ultramarine, iron oxides, metallic al1.U11ini\lD, silver and gold and that no toxicological data relevant to the use of these substances in food were available, no attempt was made at their toxicological evaluation.
In reviewing the controversial problem of the production of subcutaneous sarcomas at the sites of repeated injection of food colours, the Committee could find no adequate grounds on which to depart from the views expressed in the fifth report (pp. 16-17 and 25). Although the fifth report (p. 17) drew attention to the need for more research in this area, the question of the significance of the experimental findings remains unresolved. In the Monographs those colours producing more than 10-15% of local sarcomas in the animals injected have been indicated by a footnote, but this has not been allowed to influence the classification arrived at on the basis of the other experimental data.
The significance of conditional zones of acceptability has been explained in the sixth report (p. 10) and in the seventh report (p. 10) of the Joint FAO/WHO Expert Coauoittee on Food Additives. Having regard to all the circumstances, it was felt that in the case of food colours such latitude is neither desirable nor required.
2.2.1 Classification of colours in accordance with their toxicological evaluation
The following categories have been laid down:
Categoey A. Colours found acceptable for use in food. For these colours a maxiJnum acceptable daily intake has been stated. 12
Classificati'on of colours in this category should not be interpreted as signifying that further research on them is Wlllecessary. More work is called for, especially in view of the possibility that new advances in science may provide more delicate and accurate means of toxicological assessment. Additional investigations are particularly needed into the effects on reproduction and the foetus. The following colours have been placed in Category A:
c.I. No. Acceptable daily intakes for man in mg per kg body-weight Unconditional Conditional
Amaranth • ...... 16 185 0 - 1.5 Canthaxanthine...... - 0 - 12.5 12.5 - 2.5 Beta-Apo-8'-Carotenal . . . . - 0 - 2.5* ·2.5 - 5,0* Beta-Carotene (synthetic) . . - 0 - 2.5* 2.5 - 5.0* Beta-Apo-8'-Carotenoic Acid, Methyl or Ethyl Ester . . . - 0 - 2.5* 2.5 - 5.0* Sunset Yellow FCF 15 985 0 - 5.0 Tartrazine...... 19 140 0 - 7.5
* Expressed as total carotenoids by weight
Category B. Colours for which the available data are not!entirely sufficient to meet the requirements for Category A. Category C.I. Colours for which the available data are inadequate for evaluation but for which a substantial amount of detailed information is available concerning results or long-term tests (see the criteria on page 9). Category C.II. Colours for which the available data are inadequate for evaluation, and for which virtually no information on long-term toxicity is available. Long-term tests for tumour formation unaccompanied by other long-term studies are considered as falling within this category. Category C.III. Colours for which the available data are inadequate for evaluation but indicate the possibility of harmful effects. Category D. Colours for which virtually no toxicological data are available. lJ Category E. Colours found to be harmful and which should not be used in food:
Colour C.I. No. ~ C.I. No. Auramine 41 000 Oil Orange XO 12 140 Butter Yellow 11 020 Oil Yellow AB . . . . . 11 380 Chrysoidine 11 Z70, Oil Yellow OB 11 390 ...... 11 270B Ponceau 3R 16 155 Guinea Green B. 42 085 Ponceau SI . . . . . 14 700 Magenta 42 510 Sudan I •• 12 055 Oil Orange SS ...... 12 100 2.2.2 Conclusion The Couunittee wishes to reiterate here the point made in the fifth report (pp. 19-20) that evaluation of acceptability is the function of toxicologists, oncologists and food scientists. The application of their findings is the proper function of the authorities responsible for food legislation and public health. It is the duty of these authorities to ensure that the product used confonns to the appropriate specification and that analytical methods are available for effective control. The first report of the Joint FAO/WHO Expert Couunittee on Food Additives (p. 14) states that 11 the amount of an authorized additive used in food should be the minimum necessary to produce the desired effect". The present Couunittee emphasized the need to implement this limitation on levels of use, in accordance with good manufacturing practice. On page 15 of the same report attention is drawn to the need "to avoid a gradual and unrecognized increase in the amounts present in the total diet to a point at which an adequate margin of safety is no longer ensured". The Committee therefore points out that for future toxicological work in this field it is essential to have available an adequate specification for each substance. In order to obtain a satisfactory specification it may be necessary to define the starting material from a botanical standpoint, as well as to describe the colouring matter in terms of chemical composition. This will probably involve the extraction and purification of the active principle or principles. A general toxicological evaluation can be based ohly on a knowledge of the biological properties of the chemically defined compound or compounds. The Couunittee recommends that, when the chemical data become available, a group of experts, which should include a pharmacognosist as well as chE1Dists, should reconsider the possibility of drawing up specifications for food colours obtained from natural sources. 14
3. NOTE TO THE READER
Any comments relating to specifications should be addressed to
Food Science and Teclmology Branch, Nutrition Division, Food and Agriculture Organization, Rome, 1 taly. Any comments relating to biological data and their evaluation in these monographs, and any request for copies of the monographs not appearing in this publication should be addressed to i Food Additives, World Health Organization, Avenue Appia, Geneva, Switzerland. 15
4. CLASSIPICA'l'ION OP COLOURS
Chemical S9BIOII naae C.I. No. Toxicologicalb Page olaaaification! classification-
Acid Past Violet BG 42 561 IV D Acid Puchaine PB ~685 III C II 104 Acilan Brilliant Blue PPR ~ 7'5 IV D Acilan Past Green 10 G ~ 170 IV D Acridine Orange IH .lf6 005 IV D Alizarin. 58 000 IV D Alizarin Blue 67 410 IV D Alkali Blue ~ 750 III C II Alkanet and Allwlnin 75 520, 75 5:,0 III ... ! Alwainiua 77 000 III ... ! Amaranth 16 185 I A 22 Annatto, Bixin and 75 120 III ... ! Norbixin Antbocyanina ... III ... ! Auruine 41 OOO IV E Azo Rubins 14 720 I C II 105 Beet Red and Betanin ... III ... ! Benzopurpurine \ B 23 500 IV D Benzyl Bordeaux B 14 910 IV D Benzyl Violet 4B .\2 6lt0 I c III!!. 107 BisuNk Brown 21 OOO IV D Black 798.\ ... III C II 109 Blue VRS ~ 045 I C II~ uo Bordeaux B 16 LSO IV D Bruilwood 75 280 IV ... ! Brilliant Black BN 28 .\4o I C II ll2 "Specially Pure" Brilliant Blue PCP ~090 I #, 27 (Biological Stain) Brilliant Croceine 27 290 IV D
* For notes see end of table, page 19, 16
CLASSIFICATION OP COLOURS (continued)
c&.. 1.,a1 ToXicologica,, Comon MM C.I_. Ro. Page clusU1l)ation! claaaification-
Brilliut Green Crystals 112 o~ IV D Brilliant Milling Green B IJ2 100 IV D Brown n: included but II C II 114 not nmbered Butter Yellow 11 020 IV B Cantbuanth\l.De ... I A 31 Capautbine ... IV ... ! Capaorubine ... IV ... ! C-1 included but IV D not nUllbered Carbo Vegetabilia ... II ... ! Meclicina!ia Carbon Black 77 266 II ... ! Beta-Apo-8'-Carotenal ... I A 34 Beta-Apo-8'-Carotenoic ... I A 43 Acid Methyl or £'thyl Ester Carotene (natural) 75 13() III ... ! Beta-Carotene (synthetic) ... I A 38 Cartballua 751~ IV .. ! Cbloro~ll 75 810 III ... ! CblorophJll Copper 75 810 III ... ~ Complex Cblon.sol Sq Blue PF 21J IJlO IV D Chocolate Brown PB included but IV D not nmbered , Chocolate Brown H'1' 20 285 I C II 115 'Cbryaoidine 11 270,11 270 B IV E jChryaoine U270 I C II ll'l jCbryaoin sox"specially U275 III C II Pure" I I I ;.Citrus Red No. 2 12 156 I I C I Cochineal and Carminic 75 IJ70 III ... ! Acid I i .17
CLASSIFICATION OP COLOURS (oont1nued)
Cb•ioal To:ld.oologio~ Pap c-n JWN C.I. Ho. oluaitication! oluaitication-
Congo Red 22 120 III C II Coomusie N&VJ Blue ON 26 "°° IV D Crystal Ponceau 6R 16 250 IV D Direct Blue 2 B 22 610 IV D Direct Brown BC '5 o6o IV D Eosine "5'80 III C II 119 Eoa1ne B "5 "°° IV D Erytbroa1ne "5 4,0 I B 50 Past Green PCP 4205:, I #, 56 Put Red A 15 620 IV D Put Red E 16 0"5 I C II 120 Put Yellow 1, 1'5 IV D Put Yellow AB 1, 015 I C II 122 Pluoreace1n \'5 '50 III C III Puatio 75 2110. 75 66o IV ... ! Gold 77 "8o III ... ! Guinea Green B 42 o85 IV E lfall8a Yellow G ll 68o IV D Hel1ndon Pink BN 7' '75 IV D Heliogen Blue o 7\ 100 IV D !Hello Red RL 12 120 II C II Indanthrene Blue RS 69 8oo I C I 60 Ind1got1ne 7' 015 I B 63 Induline Spirit Soluble 50 "°° IV D Indul1ne Water Soluble 50 "°5 IV D Iron ozidea 77 ~9. 77 ~l III ... ! 77 -~. 77 499 Licorice ... IV ... ! Light Green SF 42 095 I C 111!! 125 Yellowish Litbol Rub1ne BK 15 850 III C II 18
CLASSIFICATION OF COLOURS (continued)
Che!Rioal Tox1colog1calb COIIIDOn name Pagt C.I. No. classification! classification-
c Logwood 75 290 IV ... - Lycopene 75 125 III ... ~ Madder 75 330, 75 340 IV ... ~ 75 350, 75 }'fO 15 410, 75 II~ Magenta 42 510 IV E Malachite Green 42 OOO III C III Metanil Yellow 13 o65 III C II Methyl Eosine 45 385 IV D Methyl Violet 42 535 III C II 127 Methyl Violet 5 B 42 536 IV D Napbthol Black 3 B 27 260 IV D Naphthol Blue Black 20 470 III C II Naphthol Green B 10 020 IV D Napbthol Yellow S 10 316 I C III 128 Nigrosine 50 420 III C III Oil Orange SS 12 100 IV E 011 Orange XO 12 140 IV E 011 Red O 26 125 IV D 011 Red 2R 12 170 IV D Oil Yellow AB 11 }Bo IV E Oil Yellow JG 21 230 IV D Oil Yellow OB 11 390 IV E Oil Yellow XP 12 740 IV D Opal Blue 42 775 IV D Orange I 14 600 I C I~ 67 Orange II 15 510 I C II Orange G 16 230 I C II 130 Orange GGN 15 980 I C II 133 Orange RN 15 970 IV D Orohil and Orcein included but III ... 2. not nwnbered 19
CLASSIFICATION OP COLOURS (continued)
Ch•ical Coaaon name C.I. No. Toxicologic~ Pagt classification! classitication-
Patent Blue A 45 052 IV D Patent Blue V 42 051 I C I 71 Persian Berries 75 640, 75 430 IV ... ! 75 650, 75 670 75 690, 75 700 75 695 Phloxine 45 405 IV D Phthalocyanine Green 74 26o IV D Ponceau 2 R 16 150 I C III 135 Ponceau} R 16 155 IV E Ponoeau 4 R 16 255 I C I 74 Ponceau 6 R 16 290 I C II 138 Ponceau SX 14 700 IV E Quercetin and Quercitron 75 670 III C I 77 Quinoline Yellow 47 005 I C I 80 Red 6 B 18 055 IV D Red 10 8 17 200 I C II 140 Red PB 14 780 IV D Red 2 0 18 050 I C II 143 Resorcine Brown 20 170 III C II Rhodamine B 45 170 I C III! 146 Rhodamine o 45 150 IV D Rhodamine 6 O 45 16o III c II! Riboflavin ... I ... ! Rose Bengale 45 4}5 IV D Sattron, Crocin and 75 100 III ... ! Croce tin Sandal Wood 75 510, 75 540 IV D 75 550, 75 56o Scarlet ON"specially 14 815 I C II 148 Pure"
Scarlet R 16 020 IV D Silver 77 820 III ... ! 20
CLASSIFICATION OF COLOURS (end)
Ch•ioal Toxioologio&li, Comon name C.I. Ho. olaaaitioation! olaaaitioation- Pag1
Spirit Koaine "5'86 IV D Sudan I 12 055 IV E Sudan Blue II inoluded but IV D not nmbered Sudan III 26 100 III C II Sudan IV. 26 105 III C III Sudan O ll 920 III C II 150 Sudan Re O 12 150 III C II 151 Sunset Yellow FCP 15 985 I A 83 'l'artruine 19 no I A 88 'l'biaaine Brown R 20 220 III c II!! TitaniU11 Dioxide 77 891 I C I 93 'hnlerio (Curo•ln) 75 :,00 III ... ! 'l'arquoiae Blue BB ~ 035 IV D Ultruarine 77 O 5. MONOGRAPHS ON COLOURS IN TOXICOLOGICAL CATEGORIES A, B, AND CI.! Page Page Amaranth • • • • • • • • • • • 22 Indigo tine 63 Brilliant Blue FCF • • 2:7 Orange I. 67 Canthaxanthine • • • • 31 Patent Blue V 71 Beta-Apo-8'-Carotenal 34 Ponceau 4R •• 74 Beta-Carotene • • • • 38 Quercetin and Quercitron •• 77 Beta-Apo-8'-Carotenoic Acid, Methyl Quinoline Yellow. 80 or Ethyl Ester 43 Sunset Yellow FCF 83 Citrus Red No. 2. 46 Tartrazine ••• 88 Erythrosine • • 50 Titaniwn Dioxide. 93 Fast Green FCF •• 56 Wool Green BS • 99 Indanthrene Blue RS 60 ! See section 2.2.1 page 11 22 AMARANTH This colour has been classified in categories r!. and A£ Synonyms C.I. Food Red 9; FD and C Red No. 2; L-Rot 3; E.E.C. Serial No. E 12~ Bordeaux S; Amaranthe Monoazo Red Code Numbers C.I. (1956) No. 16185 C.I. (1924) No. 184 Schultz (1931) No. 212 Chemical Name Trisodium salt of l-(4-sulfo-l-naphthylazo)-2-naphthol-3,6-disulfonic acid Empirical Fonnula Structural Fonnula Molecular Weight 604.48 !. Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5. £classification of colours in accordance with their toxicological evaluation, see section 2.2.l page 11. .2, Description Amaranth is a water-soluble food colour cc lforming to the following specifications. Identification Tests See Appendix A. Purity Tests Dye content: Not less than 85% by titration with titanous chloride. Weigh 0.850-0.900 g of Amaranth and proceed as in App. A. Use 10 g of trisodium citrate as buffer. 1 ml of O.l N titanous chloride is equivalent to 0.01511 g of c H N o s Na • 20 11 2 10 3 3 Loss on drying at 135° total not more than Chlorides and sulfates calculated as sodium salts 15% Water-insoluble matter: not more than 0.2% Ether-extractable matter: not more than 0.2% Lead (as Pb): not more than 10 mg/kg Arsenic (as As): not more than 3 mg/kg Subsidiary dyes: not more than 4% Intermediates: not more than 0.5% Biological data Biochemical aspects Adult rats were given intravenous injections of the colour. The bile was collected for 6 hours and an average of 53% (43-79%) of the quantity of the colour administered was found.l Six adult rats were given a single oral dose of 100 mg as aqueous solution per animal. Only 0.45% of the dose administered was found in the faeces collected over a period of 48 hours. A single oral dose of 50 mg per animal was administered to 4 rats. Only 2.8% was absorbed from the gastro-intestinal tract; the metabolites in the urine and bile were predominantly products resulting from the reductive fission of the azo-linkage, such as l-amino-4-naphthalene sulfonic acid and l-amino-2-hydroxy- 3,6-naphthalene disulfonic acid. The former compound was found also in the faeces. The liver enzyme that reduces azo-linkages plays little part in the metabolism, as was shown in experiments in which the colour was given by intrasplenic infusion. R~uction of the compound is therefore most probably effected by the intestinal bacteria. A~ter a prolonged administration of a daily dose of 50 lllf!/rat the vitamin A content of the liver showed a threefold to fourfold decrease. 'lbe glutathione content of the liver ar.d spleen was !ncreased.3 24 Acute toxicity !&so References per kg body-weight Rat intraperitoneal >lg 4 Rat intravenous >lg 4 Special studies The colour was tested for mutagenic action in a concentration of 0.5 g/100 ml in cultures of Escherichia coli. No mutagenic effect was found.5 Short-term toxicity !!!!• A group of 5 young rats were given the colour subcutaneously twice daily for 3 days. The rats were killed on the fourth day. The colour was administered in aqueous solution at a level of 250 mg per kg body-weight each injection. No estrogenic activity (normal uterine weight) was detected in comparison with a cont1·ol group. Ne;, other abnormalities were found. 6 Guinea-pig. In experiments with guinea-pigs, it was found that this colour had no sensitization activity.7 Cat. The te~t for Heinz bodies was negative after injecting 4 cats with a 5% aqueous solu~ion of this colour at a level of lg on the first day and O.l g on the ninth and on the eighteenth day.4 Long-term toxicity Mouse. Twenty mice were fed 15-20 mg of this colour 5 days a week for periods up to 477 days. Autoposies were conduc~ed on 18 of the mice. No lesions were observed 1n the one liver examined. Feeding studies were conducted with C3Hf and C57BL mice; 100 of each strain were fed at 1.0 and 2.0%, and 200 of each strain served as controls. No tumours were observed in the mice of either strain. Rat. Three groups of 15 male and 15 female rats were given diets containing 0.03%-;-o.3% and 1.5% of the colour for 64 weeks. The mortality of the rats was the same as that in a similar control group. At a level of 1.5% a significant decrease in growth rate was found in female rats but not in male rats. This was attributed to an effect on food utilization rather than on food consumption. Female rats fed the colour at O.}% and 1.5% showed an increase in the weight of the liver. At the higher concentration there was also an increase in kidney weight. No influence on food intake, histopathology and blood-picture and no significant difference in t1.111our incidence was found.lo Ten rats were fed the colour at a level of 0.2% in the diet. Each animal received an average of 0.1 g/kg body-weight per day for 417 days. The total intake of the colour was 11.g/animal. The observation period was 830 days. One intestinal carcinoma was observed. 4 No tumours were noted in 11 rats subjected to the subcutaneous injection of 0.5 ml of a 1% solution twice weekly for 365 days. The ~bservation period was 879 ~. The total dose administered was 0.5 g/animal. At a level of 4% in the diet the colour was fed to 5 male and 5 female rats for periods up to 18 months. Gross staining of the glandular stomach and small intestine was observed. Granular deposits were noted in the stomach, small intestine and in some cases in the colon. A lymphosarcoma was observed. No twnours occurred in SO control animals surviving 20 months or more.11 Thie colour was injected subcutaneously into 18 rats of both sexes for 94 to 99 weeks, In general 1 ml of a 2% - ~ solution was injected weekly for 693 days. No tWDOurs were observed.12 Groups of 24 weanling rats were fed the colour at 0.5%, 1.0%, 2.0% and 5.0% in the diet. A similar group served as controls. '!he animals on 5,0% showed slight growth inhibition. Gross and microscopic examinations revealed a questionable increase in the number of m811111ary tumours; two tumours were observed in the control group and 3, 3, 6, and 4 tumours respectively in the groups receiving the colour. Additional feeding studies for 2 years were carried out to repeat this result with Osbome-Mendel and Spragul-Dawley rats at 0,0, 1.0, and 2,0%, with 100 animals of each strain. '!here was no statistically significant influence on the formation of tumours,10 ~· A seven-year toxicity study was carried out on female beagles. Five dogs were fed 2% of the colour in the diet, 3 dogs were used as controls. No histopathological or other abnormalities were found, 9 Convnents on experimental studies reported Several comprehensive long-term studies have been carried out in rats. Long term studies have also been done in mice and dogs. Adequate data are available to rule out carcinogenicity of this colour in rats and mice, Biochemical studies in rats show that a major portion of the colour is reduced at the azo-linkage apparently, by intestinal bacteria. The long-term studies in rats provide a basis for evaluation. Evaluation Level causing no significant toxicological effect in the rat 0,3% in the diet, equivalent to 150 mg/kg body-weight. Estimate of acceptable daily intake for man O - 1. 5 mg/kg body-weight Further work considered desirable Metabolic studies in several animal species Reproduction studies in several animal species Research on the combined effect with Sunset Yellow FCF and Tartrazine would be valuable. REFERENCES 1. Ryan, A. J. & Wright, S. E. (1961) J. Phann. Phannacol., !z, 492 2. Radomski, J. L. & Mellinger, T. J. (1962) J. Pharmacol. and Exp. 'lber., ~. 259 3. Galea, v., Ariesan, M. & Luputiu, G. (1962) Farmacia {Bue), 10, 531 (rer. C.A. 2§_, 1963, 1o6ll8b) 4. Deutsche Forschungsgemeinschaft, Bad Godesberg, Federal Republic or Gennany, Farbstorr Kommission (1957) Mitteilung 6 5. RUck, a. & Rickerl, E. (1960) Z. Lebensmitt.-Untersuch., 112, 157 6. Graham, R. c. B. & Allmark, M. G. (1959) Toxicol. appl. Pharmacol., !, 144 7. Bllr, F. & Griepentrog, F. (1960) Med,, u. Erpllhr., !, 99 8. Cook, J. W., Hewett, C. L., Kennaw&¥, E. L. & Kennaway, N. M. (1940) Amer. J. ~· 40, 62 9. United States Food and Drug Administration, Unpublished report submitted to WHO April 1964 10. Mannel, W. A., Grice, H. c., Lu, F. C. & Allmark, M. G. (1958) J. Phann. Phannacol., 10, 625 11. Willheim, R. & Ivy, A. c. (1953) Gastroenterology, gz, 1 12. Nelson, A. A. & Hagan, E. C. (1953) Fed. Proc., 12, 3'Tf BRILLIAN'l' :BLUE FCF (Biological stain) Thie colour baa been classified in categories ~ and ~,i!. S;ynoll.YIDS c.I. Food Blue 2; FD &: C Blue No. lJ Bleu brillant FCF. lli!.! Triarylmethane Colour Blue Code Numbers c.I. (1956) No. 42090 C.I. (1924) No. 671 Schultz (19,1) No. 770 Chemical Name Dieodium salt or 4-[.L'"4- Structural Formula + 2 Na Molecular Weif!bt 792.86 !: Classification or colours from the standpoint or chemical specifications, see section 2.1., page 5. R Classification or colours in accordance with their toxicological evaluation. see section 2.2.1 page 11. ~ Colours producing more than 10-1~ or local sarcomas at the site or repeated subcutaneous injection. 28 Description Brilliant Blue FCF is a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A. Purity Tests Dye contenta Not less than 8~ by titration with titanous chloride. Weigh o.aoo-o.900 g of Brilliant Blue FCF and proceed as in Appendix A. Use 15 g sodium hydrogen tartrate as buffer. 1 ml of 0.1 N titanous chloride is equivalent to 0.03964 g of c -/" N o s Na • 3 34 2 9 3 2 Loss on drying at 135° ~ total not more than l~ Chlorides and sulfates calculated as sodium salts 1 Water-insoluble mattera not more than o.2% Ether-extractable mattera not more than 0.27' Lead (as Pb)a not more than 10 mg/kg Arsenic (as As) a not more than 3 mg/leg Chromium (as Cr)a not more than 20 mg/kg Subsidiary dyesa not more than ,% lntermediates1 not more than o.~ Biological Data Biochemical aspects The colour was administered orally to rats as a 2'~ aqueous solution at a level of 200 mg per rat. Almost the entire amount was excreted unchanged in the faeces within 40 hours after administration. In a later investigation, the presence of the colour in the bile was observed in rats, rabbits and dogs after oral administration In the case of the dog, the amount did not exceed "ff, of the dose administered. ,2,3 Acute toxicity 1R50 per kff bod.y-weight Reference Rat oral >2.0 g 4 Short-term toxicity R!!:i• Eighty-five rats were fed a diet containing 0.1% of the colour. The daily intake was 10-15 mg. The duration of the exoeriment was not mentioned. No tumours were found.5 Groups of 5 young rats were given injections of the colour subcutaneously twice daily for, days. The rats were killed on the fourth day. The colour was administered in aqueous solution at a level of 250 mg per kg bo~-weight each injection. No oestrogenig activity (uterine weight) was detected. No other abnormalities were found. Two groups of rats, l of 25 and the other of 40 animals, were given respectively l ml of a 0.741> solution of the colour and l ml of a 2.0J' solution twice weekly for 4,4 and for ,1, ~s. The groups were kept under observation for 49, and 4,a ~s respectively. During this time 5 animals died in the first group and 15 animals in the second group. In addition, 12 fibrosarcomas were found at the site of injection in the rats receiving the 0.741> solution and 24 fibrosarcomas in the rats receiving the 2.0J' solution. Cat. A negative test for Heinz bodies was obtained on the administration to 4 cats';-as a~ solution in water, of 1.0 g on the first day and O.l g on the ninth and the eighteenth day.5 ~· Studies were conducted on beagle dogs fed diets oontaining l.OJ' and 2.0J' of th' colour for one year. No gross or microscopic changes due to treatment were seen. Long-term toxicity Mouse. A group of 100 mice (57 male and 43 female) were given l mg per day in the diet. The observation period was 500 to 700 days. No evidence of carcinogenic action was found.a !!:!• The colour was injected subcutaneously into 18 rats of both sexes for 94 to 99 weeks. In most cases, l ml of a 2% or~ solution was injected weekly. Fourteen subcutaneous fibrosarcomas appeared at the site of injection. 9 In 2 groups of rats, l of 48 and l of 27 animals, solutions containing respectively 0.741> and 2.0J' of the colour were injected subcutaneously at the same site twice weekly, l group being given 14.a mg and the other 40 mg weekly until sarcomas appeared or the animals died. This colour, which was highly purified produced sarcomas in 10 out of 48 and in 20 out of 27 rats after 20 months.l6 The colour was fed in a corcentration of~ of the diet to 5 male and 5 female rats for 600 days. They observed gross staining of the glanduJ:F stomach and some granular deposits in the stomach. No tumours were observed. Groups of 24 weanling Osborne-Mendel rats, evenly divided by sex, were fed the colour at levels of o.o, 0.5, 1.0, 2.0 and 5.0J' for 2 years. No untoward effects were observed during the experiment. Gross and microscopic examination revealed only incidental lesions, unrelated to treatment, with no effect on tumour production. The statistical evaluation revealed no changes in organ weights, mortality, or growth. Haematology was normai.7 Four groups of ,0 rats (15 of each sex) were given diets containing o.o, o.o~, o.;;; and 3.0J' of the colour for 75 weeks. No depression of growth, food consumption or food efficiency was found. In the~ group there was an increase in mortality. Death was due to respiratory infection. Since this was a prevalent cause of death among all groups, it was not considered to be related to the administration of the compound. No abnormalities in blood composition were found.12 A group of 20 young rats (10 male and 10 femalf) were given weekly subcutaneous injections of 0.5 ml of a If!, solution of the colour in isotonic saline. Each rat received 20 mg of the colour per week. Another group was given isotonic saline as control. Totally, 45 injections, i.e., 900 mg of the colour, were given. The experiment was teminated after 71 weeks. The mortality was as in the control group. No tumours were found.13 Comments on experimental studies reported Extensive long-tem studies are available in several species. At the highest levels used no pathological change or other adverse effect was found. Local sarcomas were produced on repeated subcutaneous injection. Paucity of biochemical data makes it impossible to carry out an evaluation of the acceptable daily intake in man. Further work required Metabolic studies in different animal species. REFERENCES 1. Hess, s. M. & Fitzhugh, o. G. (1953) Fed. Proc., Jl, 330 2. Hess, s. M. & Fitzhugh, o. G. (1954) Fed. Proc., JJ., 365 3. Hess, s. M. & Fitzhugh, o. G. (1955) J. Pharmacol. exp. Ther., .!!!, 38 Lu, F. c. & Lavallee, A. (1964) Canad. Pham, J., ~ (12), 30 Deutsche Forsohungsgemeinschaftt Bad Godesberg, Federai Republic of Germany, Farbstoff Kommission (1957} Mitteilung 6 6. Graham, R. c. B. & Allmark, M. G. (1959) Toxicol. appl. Pharmacol., l, 144 United States Food and Drug Administration, Unpublished report submitted to WHO in April 1964 8. Waterman, N. & Lignac, G. o. E. (1958) Acta physiol1 pharmacol. neerl., 1, 35 Nelson, A. A. & Hagan, E. c. (1953) Fed, Proc., Jl, 391 10. Gross, E. (1961) z, Krebsforsch., §.4, 287 11. Willheim, R. & Ivy, A. c. (1953) Gastroenterologv, .ll, l 12. Mannell, w. A., Grice, H. c. & Allmark, M. G. (1962) J. Pham. Pharmacol., y, 3· 13. Mannell, w. A. & Grice, H. c. (1964) J 1 Pham, Pharmacol., 16, 56 31 CANTHAXANTHINE a b This colour bas been classified in categories I- and A- Carotenoid Orange to red in oils and organic solvents Code Numbers DGF Lebensmittel-Orange 79 EEC No. E 1619 Chemical Name Trans-canthaxanthine Empirical Formula Structural Formula 0 0 Molecular Weight Definition Canthaxanthine contains not less than 96% of c cJ1 o2 and conforms to the following specifications. 4 52 Description Deep violet crystals. The articles of coDDDerce may be solutions in oil, fat or organic solvents or water-dispersible forms such as powders, granules or capsules, and are orange to red in colour. A Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5. R Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. J2 Identification Tests A. Solubility Water: Insoluble Ethanol: Insoluble Vegetable oils: Slightly soluble (about o.005i) Chloroform: S.luble B. Melting range: about 210° with decomposition c. Absorption: in solution in cyclohexa.ne the absorbance maximum is at 470 nm. D. The colour of a solution of canthaxantbine in acetone disappears after the successive additions of a 5i solution or sodium nitrite andl normal su.U'uric acid. E. A solution of cantbaxantbine in chloroform turns blue when one adds an excess or Carr-Price reagent TS. Purity Tests Arsenic: Not more than J mg/kg Heavy metals: Not more than 10 mg/kg Sulfated ash: Not more than o.1i (Same as ,-apo-8'-carotenal with the exceptions noted below). Note: The assay should be carried out rapidly, avoiding exposure to air and light. Weigh exactly approximately 100 mg of canthaxanthine in a loo.mJ. volumetric flask and dissolve by shaking briefly with 20 ml pure, acid-free chloroform. Measure spectrophotometrically the extinction at the absorption maximum (about 470 nm) comparing "!.1 with the solvent (cyclohexa.ne) and utilising cuvettes of 1 cm path-length. Calculate the content using E(1i, 1 cm)= 2200 for pure canthaxanthine. Method for water-dispersible materials Measure the extinction at the absorption maximum (about 470 nm) with cyclohexane as reference solvent and utilising cuvettes of 1 cm path-length. Calculate the content of canthaxantbine using E(1i, 1 cm)= 1970. This theoretical val1,1t1 corresponds to the proportion of isomers normally present in these water-dispt,rsible products. Biological De.ta Natural occurrence This substance was detected fort~ first time in an edible mushroom, the chanterelle (Cantharellus cinnabarinus)2 It was subsequently shown to be present in the plumage and organs of flamingoes ,J and various exotic birdsl such as the scarlet ibis (Guara rubra) and the roseate spoonbill (Ajaie aiaJa). It has recently been detected in various crustacea and fish (trout, salmon). )3 Biochemical aspects Cantbaxantbine does not exhibit provitamin A activity. 5 Acute toxicity LD50 Reference mg/kg bodY-weight Mouse Oral ) 10 OOO 5 Short-term toxicity l!g,g. Groups of three male and three female dogs received o, 100 and 4(;1;) mg/kg daily of canthaxanthine for 15 weeks. No significant effect wa8/5 noted on body weight of control or test groups or on their general health.· Long-term toxicity I!!S· A three-generation study using O ppm and 1000 ppm in the diet revealed no adverse effect in any generation.5 In another experiment, 25-30 male and 25-30 female rats received 0%, 0.5%, 2% and 5% canthaxanthine in their diet for 93-98 weeks. No adverse effect was noted on food consumption and weight gain, Mortality and tumour incidence were not increased. 5 Comments on experimental studies reported This compound has been adequately tested in the rat and has no provitamin A activity. Evaluation is therefore based on the toxicological information provided above. Evaluation Level causing no toxicological effect in the rat 5% in the diet, equivalent to 2500 mg/kg body-wight/day. Estimate or acceptable dailY intake for man mg/kg body-weight Unconditional 0 - 12.5 Conditional 12.5 - 25 REFERENCES 1. Haxo, F. (1950) Botan. Gaz., ~' 228 2. Fox, D. L. (1962) Comp. Biochem. Ph.Ysiol., 2, 1 3. Thommen, H. & Wackernagel, H. (1963) Biochem, Biophys. Acta, .22, 3f!"l 4, Fox, D. L, (1962) Comp. Biochem. Physiol., 2, 305 5, Hoffmann-La Roche (19€6) Unpublished report submitted to WHO 34 BETA-AF0-8'-CAROTENAL This colour bas been classified in categories 1.! and AR. Carotenoid Orange to red in oils and organic solvents Code Numbers DFG Lebensmittel-Orange 8 DC No. E 160 e Chemical Name Trans-beta-apo-81 -carotenal lp.pirical formula Structural Formula CHO Molecular Weight 416,65 Definition Apocarotenal contains not less than 96% of C rJI,;,O and conforms to the following specifications. 3 Description Deep violet crystals with metallic lustre. The articles of commerce may be solutions in oil, fat or organic solvents or water-dispersible forms such as powders, granules or capsules, and are o!'IIDge to red in colour • .a Classification of colours from the standpoint of chemical specifications, see section 2,1,3 page 5 9. Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. J5 Identification Tests .&. Solubility Waters Insoluble Ethanol1 Soluble Vegetable oils1 Soluble Chloroform: Soluble B. Melting range1 136-l.40crC C. Absorption1 in solution in cYclohexane the absorbence ratio A488/A461 is between 0.80 and 0.84. D. The colour of a solution of apocarotenal in acetone disappears after the successive additions of a 5% solution of sodium nitrite and sulfuric acid (1 normal). E. A solution of apocarotenal in chloroform turns blue when one adds an excess of Carr-Price reagent TS. Purity Tests .Arsenic1 Not more than 3 mg/kg Heavy metals1 Not more than 10 mg/kg Sultated ash1 Not more than 0.1% Note1 The assay should be carried out rapidlY, avoiding exposure to air and light. Weigh exactly approximately 80 mg of apocarotenal in a 100-ml volumetric flask and dissolve bY shaking briefly with 20 ml pure, acid-free chloroform. Make sure that the solution is clear. Make up to volume bY the addition of pure cYclo hexane. Pipet 5.0 ml of the solution into a 100-ml volumetric flask and make tn volume with the cyclohexane. Similarly, dilute 5.0 ml of this solution to 100 ml and measure spectrophotometrically the extinction at the absorption maximum (about 461 nm) comparing it with the solvent (cyclohexane) and utilising cuvettes of 1 cm path-length. Calculate the content using E(J~, 1 cm)= 2640 for pure apocarotenal. In certain commercial products such as the water-dispersible powders, apocarotenal is finely dispersed in an appropriate medium e.g. gelatin. In such preparations all the apocarotenal must be separated from the medium before making the spectrophotometric assay, and a correction must be applied to take account of the difference in absorption of the isomers. Method for such products Weigh exactly a quantity of the powder equivalent to about 10 mg free apocarotenal into a thick-walled glass cylinder. Add 10 ml distilled water, displace the air with nitrogen and heat the tube on a steam-bath at 60° stirring it from time to time with a glass stirring rod, in such a way that the powder is entirely moistened and so that the particles do not stick to the walls of the tube. After about 10 minutes a homogeneous suspension of the powder should be obtained. Transfer the contents of the tube to a 250-ml volumetric flask and add 50 ml of absolute alcohol; part ot the alcohol should be'used to rinse the glass stirring rod and the tube. Add 100 ml of etbYl ether (free from peroxides) rinsing the J6 stirring rod and test tube again, shake for J minutes make up to volU111e with ethyl ether and mix thoroughly. Let stand for 15 minutes in the absence of light so that the insoluble particles can precipitate. Pipet 5.o;ni of the above solution into a flask, replace the air with nitrogen and evaporate to dryness at about 50° in a rotary evaporator. After cooling, dissolve the residue in pure cyclohexane, transfer the solution quantitatively into a 100-ml volU111etric flask and adjust the vol\lllle with cyclohexane. Measure the extinction at the absorption maximum (about 4(:1) nm) with cyclohexane as reference solvent and utilising cuvettes of 1 cm path-length. Calculate the content of apocarotenal using E(~, 1 cm)= 2400. This theoretical value corresponds to the proportion of isomers normally present in these water-dispersible products. Biological Data Natural occurrence Beta-apo-8'-carotenal ls t'ound in abundance in the vegetable kingdjm, e.g. in the pulp and skin of citrus fruits3 and in various fodder plants. · Biochemical aspects After administration to rats as the only dietary carotenoid, some beta-apo-8'-carotenal accumulates in the liver together with Vitamin A and beta-apo-8'-carotenoic acid.1,2 Almost all of an excessive dose is excreted in the faeces, only a small fraction being absorbed ordinarily.J Dogs absorb it poorly from the gastrointestinal tract and excrete it in the urine together with beta-apo -8'-carotenoic acid.4 Hypervitaminosis A has not been seen in these dogs. The body fat and livers of monkeys show an orange discoloration after oral feeding of beta -apo-8'-carotenal, and it is stored in the liver together with an unidentified carotenoic acid. Laying hens excrete some into yolk ( Only 4'I, of dietary carotenal is converted to Vitamin A in the gut of Vitamin A-deficient rats, compared with 10% of beta-carotene. Carote~s·are easily oxidised to carotenoic acids and less readily reduced to alcohols in vivo, pointing to metabolic pathways other than beta-oxidation.J,7 Acute toxicity LD-5P Reference mg/kg bodY-weight Mouse oral ) 10 OOO 8 Short-term toxicity II!!.· Groups of 16 male rats received o, 100 or 500 mg/kg of carotenal intragastrically 5 days per week for J4 weeks. No adverse effects were seen on body weight gain, general health, survival, liver and kidney function and organ weights. The testicular weight or the high level test group was significantly lower than that of the controls. 'ICacroscopic findings were normal except for granular pigment deposition in the liver and kidneys of test animals. Fertility, as shown by monthly mating of 4 females, was not affected.8,9 l!gg. Groups of 2-J female and J-4 male dogs received o, 0.1 or 1.0 g of carotenal daily per animal during 14 weeks. All remained healthy and no significant effect was noted. No pathological lesions related to the test 37 substance were seen at post-mortem. Peripheral blood picture, liver function tests, serum enzymes and blood urea were normal. Vitamin A and carotenal levels of serum, liver, kidney, adrenal and mesenteric fat were estimated. The kidney level of Vitamin A ws 3-5 times that of controls. The serum level of carotenal was elevated in the group receiving l.O g and there was an occasional trace in the group on O.l g. Tissue amounts were variable. The only microscopic finding was pigmentation of the adipose tissue, kidney and adrenal cortex. Organ weights were norma1.lO Long-term toxicity Rat, A three-generation study in rats at O ppm, 1000 ppm.1. 2000 ppm and 5000 ppm for 2 years showed no adverse effect in any generation.o Comments on experimental studies reported Beta-apo-8'-carotenal has been adequately tested in rats. The use of this substance is unlikely to result in an increased level of Vitamin A although the conversion rate for man is not known. Similarly to beta-ca,otene, this substance is poorly absorbed from the gastro-intestinal tract when present in large amounts. It can, therefore, be evaluated on the .same basis as beta-carotene, Evaluation Estimate of acceptable daily intakes in man mg/kg bodY-weight/daY* Unconditional 0 - 2.5 Conditional 2.5 - 5.0 *Assum of total carotenoids. REFERENCES l. Thommen, H. (1962) ~., "2, 517 2. Brubacher, G., Gloor, U. & Wiss, O. (1960) Chimia, M:, 19 3, Wiss, O. & Thommen, H. (1963) Dtsch. Gesellsch. Ern&'hrung., ~' 179 4. Bagdon, R, E., Zbinden, G. & Studer, A. (1960) Toxic. Appl. Pharm., ~' 223 5. Tiews, J. (1963) Dtsch. Gesellsch. Ern&'hrung, ~' 236 6. Thommen, H. (1961) Chimia, !2, 433 7. Glover, J, (1960) Vit. Horm., .§, 371 e. Hoffmann-La Roche (1966) Unpublished report 9. Hoffmann-La Roche (1962) Unpublished report 10. Bagdon, R. E., lmpellizzeri, C. & Osadca, M. (1962) Toxic. Appl. Pharm., ,, 444 J$ BETA-CARO'l'ENE (SYNTHETIC) Thie colour baa been classified in categories I~ and All (Carotenoid) Colour Yellow Chemical Npe ,-carotene Bllpir:l.cal Fonula Structural Po:naula KoleoularWeiflht Definition Synthetic ~tene contains not leas than 98J' and not more than the equivalent of 101" of ,-carotene (C ) and oonto:naa to the following specifications. 4 ~ Cl.uaification of coloura from the standpoint of chelllioal speoifioations, aee section 2.1.~ J1869 5. lcl.&saifioation of ooloura in accordance with their toxicological evaluation, aee section 2.2.1 page 11. 39 Identification Teet• A. Solubili;tz Water• Ineoluble Bthanola Practicall.7 ineoluble Kethanola Practicall.7 insoluble Vegetable oilet Sparingl.7 soluble B. Melting ranpa 176° to 182° with decomposition c. Detemine the absorbance of Sample Solution B (prepared for the !11&7) at 455 • and at 48:5 .. The ratio .1 , is between 1.14 and 1.18. 451148 D. Detemine the absorbance of Sample Solution B at 455 11111 and that of Sample Solution .l at :,40 mp. The ratio .1 x 10/A, is not lower than 15; 455 40 Purib Testa Arsenic ( as .la)• IJot more than :, mgf'q HeaV7 metals, Not more than 10 mg/kg Sulfated asha Not more than o.~ Aeay (Provisional) IJotea Carry- out all work in low-actinic glasnare and in subdued light. Sample Solution A. Weigh 50.0 mg into a 100-ml volumetric flask, diaeolTe in 10 ml of acid.-free chloroform, :lmmediatel.7 dilute to volume with cyclohexane, and mix. Pipet 5 ml or this solution into a second 100-ml volmetric flask, dilute to TOlume with cyclohexane and mix. Sample Solution B. Pipet 5 ml or Sample Solution A into a 50-ml volumetric flask, dilute to TOlume with cyclohexane and mix. Procedure. Detemine the abaorbance of Sample Solution B in a 1-cm cell at the wave length of maximum absorption at about 455 ml,l, with a aui table spectrophotometer, using cyclohexane as the blank. Calculate the q~tity, in mg, of C iJl.56 in the sample taken by the formula 20,0001/250, on which A is the absorbance4of the solution, and 250 is taken to be the absorptiTity of pure J-carotene. IJote. The above ass~ procedure was considered to be proTiaional by the Committee due to the unavailability of an article of standardised purity to serve as a control upon the procedure and the instrumentation. 40 Biological Data Biochemical aspects In man 30 - 90:( of ingested beta-carotene is excreted in the faeces and concomit,.nt intake of fat does not improve absorption. Excessive doses depress the 'fitamin A activity or the absorbed fraction. Only small amounts a-ppear in the sel')DII. Beta-carotene dissolved in oil is much better absorbed, 10 - 41i for adult~, 50 - SO',' tor children.1,2 Vitamin Eis neces~ar,y to prevent enzymatic destruction and bile acids are necessar,y for absorption.J Other vorters quote ui absorption by adults and 2.6j by babies or a dose or 20 mg carotene. The major fraction or absorbed beta-carotene passes unchanged through the gut wall and reaches the liver via the portal system and to a small extent via the lymphatics. Sou,ie beta-carotene is stored in the liver but is never released as such, and some is converted to Vitamin A in jejunum, liver, lung, muscle and serum and stored in the liver as Vitamin A. Large doses lead to a rise in serum carotene and subsequent eeposition in organs and the horny layer or the skin without necessarily raising the Vitamin A serum level.5 Gastrointestinal and liver and renal disease, diabetes mellitus and phosphor poisoning reduce the conversion to Vitamin A and J111XOedema blocks conversion. Infants and small children hav, a smaller capacity for this conversion.3 Beta-carotene is transmitted in milk.6 In infants a single administration of 20 mg of beta-carotene in milk produces a rise in the beta carotene blood level, peaking in 24 hours and a rise in the Vitamin A ester blood level but practically no change in the Vitamin A alcohol blood level. H;n,ercarotenem1e Hypercarotenemia·l!!!:...!.! is hsrmless and causes no symptoms nor is there nol'llally any evidence or bype~~osis A. It disappears it excess intake or beta-carotene is discontinued. , No bypervitaminosis A was noted in volunteers given beta-carotene over extended periods.9 The possibility of producing bypervitaminosis A in animals by byper carotenemia bas never been completely proven because or the poor absorption and slow conversion. No effects have been shown in rats given the equivalent of 40 OOO - 70 OOO IU Vitamin A esters/day either i.v., i.p. or orally. However, oral or s.c. doses equivalent to 1500 IU Vitamin A/day gaused accelerated epithelial growth in adult rats and changes in oestrus. In another experiment, 20 mg/kg body-weight of beta-carotene vas fed to JO young rats on a Vitamin A deficient diet for 6-ll months without any deleterious effects, particularly without evidence or bypervitaminosis A or liver damage. High doses of beta-carotene reduce liver storage of labelled dl-gamma tocopherol acetate to 70{..lO Acute toxicit;y LD50 Reference mg/kg bodY-weight Rat i.m. (oil) > 1000 5 Dog oral > eooo 9 Short-term toxicity . Several short-terr studies in rat and dog showed the same results as the human studies reported. 41 H!!l• Fifteen subjects received 60 mg beta-carotene daily for 3 months. Serwn carotene levels rose from 128 Ilg to a maximum of 308 tLg/100 ml after one month while Vitamin A levels remained unchanged. No clinical signs of hyper vitaminosis A were seen.12 Other subjects ate several pounds of raw carrot~ daily, resulting in some skin discolouration. Beta-carotene appeared in the milk. Long-term toxicity ~· A four-generation study at dietar,y levels of O ppm and 1000 ppm of beta-carotene for 110 weeks showed no adverse effects in any of the generations.9 Coppents on the experimental studies and observations reported Results of short-term toxicity studies in rats 4Dd dogs have shown that even excessively high doses (up to 1000 mg/kg body-weight/day during 100 days in rats) failed to produce toxic effects. In multi-generation tests in rats the highest level investigated was 1000 ppm and no adverse effects were seen. Beta-carotene is a normal constituent of the human diet and is cODDnonly ingested over the entire life-span of man. Its biological importance rests on the provitamin A function. Concerning the known clinical syndrome of hyper vitaminosis A in man, evidence from human experience indicates that high dietar,y levels or beta-carotene do not affect serwn Vitamin A levels because of its poor absorption from the gastrointestinal tract. Evaluation With these substances it appears justifiable to apply a safety factor of 10 or 20 to the no-effect level established in long-term studies. Level'causing no toxicological effect in the rat 0.1% in the diet equivalent to 50 mg/kg body-weight/day. Estimate of acceptable daily intakes in man mg/kg bodY-weight/da.Y* Unconditional 0 - 2.5 Conditional 2.5 - 5.0 *As sum of total carotenoids. REFERENCE.S l. Fraps, G. s. & Meinke, W.W. (1945) Arch. Biochem., 2, 323 2. Bernhard, K. (1963) Wiss. Veroff. Btsch. Gesellsch. Erna1lrung, ~, 169 3. Wagner, K. (1962) Wien. Kl. Wschr., ~' 909 4. Ku"bler, w. (1963) Wiss. Veroft. Dtsch. Gesellsch. Ernahrung., ~' 222 5. Zbinden, J. & Studer, A. (1958) z. Lebensm. Unters. Forsch., 108, 114 6. Auckland, G. (1952) Brit. Med. J., ~, 267 7. Abrahamson, I. A. Snr., & Abrahamson, I. A., Jr., (1962) Arch. Ophth., 68, 4 42 8. Nieman, C. & Klein Obbink, H.J. (1954) Vit. Honn.,~' l:J) 9. Bagdon, R. E., Zbinden, G. & Studer, A. (1960) Toxic. Appl. Pbarm., ~' 223 10. Brubacher, G,, Scharer, K., Studer, A, & Wiss, O. (1965) z. Erna"hrw., 2, 190 11. Hoffmann-La Roche (1960) Unpublished Report submitted to WHO 12. Greenberg, R., Cornbleet, T, & Jeffay, A. I. (1959) J. Invest. Dermatol., ~' 599 BETA-.AP0-8'-CAJIOTmOIC ACID, METHYL OR ETHYL ESTER a b This colour has been classified in categories I- and A- Carotenoid Yellow to orange in oils and organic solvents Code Nwnbers DFG Lebensmittel-Orange 9 EEC No. E 160 f Chemical Name Trans-beta-apo-8'-carotenoic acid, methyl or et~l ester (c30) Empirical Formula H41+o (ethyl ester) or H (methyl ester) c32 2 c31 42o2 Structural Formula 0 l \ OR Where R = methyl or etti¥l ester Molecular Weight 460.70 (ethyl ester) or 41+6.70 (methyl ester) Definition Beta-apo-8'-carotenoic acid ethyl or methyl ester contains not less than 96i of c3~41+o2 or c31H42o2 respectively and both esters conform to the following specifications. Description Red crystals. The articles of commerce may be solutions in oil, fat or organic solvents or water-dispersible forms such as powders, granules or capsules, and are yellow to orange in colour. ~ Classification of colours from the standpoint of chemical specificatiClns, see section 2.1.3 page 5. -2 Classification of colours in accordance with their toxicological evaluation, see section 2.2.l page 11. 44 Identification Tests A. Solubllity Waters Insoluble Ethanols Insoluble Vegetable oilss Soluble Chloroforms Soluble B. Melting ranges 1J4-1J8°C C. Absorp"tion1 in solution in c7clohexane the absorbance ratio A475/A449 is between 0.82 and 0.86. D. The colour of a solution of apocarotenoic acid ester in acetone disappears after successive additions of a 5% solution of sodium nitrite and in sulturic acid (1 normal). E. A solution of apocarotenoic acid ester in chloroform turns blue when one adds an excess of Carr-Price reagent TS. l'llrit;y Tests Arsetdcs Not more than J mg/kg HeaV7 metals s Not more than 10 mg/kg Suli'ated ashs Not more than 0.1% (Salle as for beta-apo-8'-carotenal with the exceptions noted below). 1 ••••• measure spectrophotometrically the extinction at the absorption maximum (about 449 nm) comparing it with the solvent (cyclohexane) and utilising cuvettes of 1 cm path-length. Calculate the content using E(l%, 1 cm)= 2550 for pure apocarotenoic acid, ethyl or methyl ester. 2. In Method for water-dispersible powders. water-dispersible materials Measure the extinction at the absorption maximum (about 449 nm) with cyclo hexane as reference solvent and utilising cuvettes of 1 cm path-length. Calculate the content of beta-apo-8'-carotenoic acid, ethyl or methyl ester using E(l%, 1 cm}= 2320, This theoreticel value corresponds to the proportion or isomers normal17 present in these water-dispersible products. !dditional criteria for the commercial products The composition or the article of co11111erce can be specified as to the methyl or ethyl eater content. Biological Data Biochemica1 aspects The acid as well as the esters are normal metabolites of apocarotenal. Onl7 a minor fraction of the methyl ester, if given in large doses to rats, is absorbed, most being excreted in the faeces,l The elimination of ester from the blood of human infants is proportional to the blood concentration and is very rapid.2 45 Acute toxicit:y ~50 Reference mg/kg bodY-weight Mouse oral ) 10 OOO J Short-term toxicit:v ~. Groups or 16 rats received o, 100 or 500 mg/kg or methyl ester daily, 5 days per week, tor 34 weeks. No adverse effects on mortality or weight gain were noted, but the male rats receiving 500 mg/kg body-weight showed reduced testicular weights compared with controls, and granular pigment deposits in the liver and kidney. No deleteri~f effect on spermatogenesis was noted and their general health was satisfactory. ' .Long-term toxicity !11• In a tour-generation study, test groups or male and female rats or 20-40 animals each and control groups or 28-39 each were fed diets containing o, 0.1, 0.2 and 0.5 and 1.o,g or methyl ester tor periods of 52-104 weeks. No difference was observed between test and control groups regarding food consumption and general health; mortality was unrelated to the test substance.J In another experiment, 15 male rats were fed the ethyl ester at l~ or their diet tor 2 years, with a control group on 7500 IU of Vitamin A per day per animal and another control group on the basic diet. Mortality and weight gain were identical in test and control groups except tor the group fed the Vitamin A supplement. Fertility was similarly unaffected except in the group given Vitamin A. No adverse effects were noted on general health.) Comments on experimental studies reported Although the toxicological information on each individual ester is incomplete, long-term studies have been reported in rats. The use or these substances is unlikely to result in an increased level or Vitamin A, although the conversion rate tor man is not known. Similarly to beta-carotene, these substances are poorly absorbed from the gastrointestinal tract especially when present in large amounts. They can, therefore, be evaluated on the same basis as beta-carotene. Evaluation Estimate or acceptable daily intakes in man mg/kg bocb'-weight/da:y* Unconditional 0 - 2.5 Conditional 2.5 - 5.0 *As sum or total carotenoids. REFERENCES 1. Wiss, o., & Thommen, H. (1963) Dtsch. Gesellsch. Ernahrung., ~, 179 2. Kubler, W. (1963) Wiss. Vero!!. Dtsch. Ges. ErniJ.'hrung., ~' 222 J. Hoffmann-La Roche (1966) Unpublished report 4. Hoffmann-La Roche (1962) Unpublished report CITRUS RED No. 2 '!'his colour has been classified in categories 1.! and c11 llonoazo ~ Reddish-orange Code Numbers c.1. (1956) No. 12156 Chemical NBM l-L\2,5-Dimetho:xyphenyl)az£7...2~naphthol Empirical Formula Structural Formula llolecular Weight Description Citrus Red No. 2 is an oil-soluble food colour conforming to the following specifications. Identification Tests See .Appendix ..l, 1 • .! Classification of colours from the standpoint of chemical specifications, see section 2.1.~ page 5 • .l? Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. Purity Tests Dye contents Not less than 9e.' by titration with titanous chloride. Weigh 0.300..0.350 g of Citrus Red No. 2 and proceed as in Appendix A, a, except that the sample is dissolved in 50 ml of 95% alcohol and diluted with 50J' alcohol to a volume of 150 ml. Then add 15 g of sodium hydroe;en tartrate as buffer. l ml of 0.1 N titanous chloride is equivalent to 0.007709 g of c a N o • 18 16 2 3 Water-soluble mattert not more than o.,% Matter insoluble in carbon tetrachloridet not more than 0.5% Leadt not more than 10 mg/kg Arsenics not more than 1 mg/Jcg Dimethox;yaniline j not more than 0.05% combined Beta naphthol Methods of analysis available from the producer. Subsidiary dyest not more than :!'J'. Assay Spectrophotometric method1 Weigh 0.2500 g of the well ground sample and transfer quantitatively to a 250 ml beaker using 150 ml (graduated cylinder) of toluene to complete the transfer. Place the beaker on a hot plate in the hood. Heat until boiling to assure the completeness of solution. When solution is complete, cool and transfer quantitatively to a 250 ml volumetric flask, using toluene to effect the transfer. Cool to room te~perature, dilute to volume with toluene and mix well. This is solution A ( 1000 mg pP.r litre). Pipet 10 ml of Solution A into a 100 ml volumetric flask and dilute to volume with toluene and mix well. This is solution D (100 mg per litre). Pipet 25 ml of Solution B into a 200 ml volumetric flasl; and dilute to volume with toluene and mix well. This is Solution C (12.5 mg per litre). Using a General Electric Recording Spectrophotometer, or any other instrt:lllent capable of recording in the 400-700 my range, obtain the transmission curve of Solution C in matched 1.00 cm Corex cells using toluene P.s the ::-eference liquid. Evaluate the strength at the absorption maxim1w. at about 506 my relative to the curve of the reference standard. Biolold.cal Data Biochemical aspects After single oral doses of 2-20 r.!(S in corn oi.l solution to rats, 'j-7;', of the administered colour w:JS recovered fr the faeces in 48 hours. \7hen the do::;e was reduced to 0.5 mg no colour was found. \'Then admir.istered as a o.55i dry mixture, 26.3'.'!'. we.s found in the faeces. Rabbits and docs s::owed si1~ilar results. Sm1=tll anounts of l-aoino-2-naphthyl sulfate v,ere iuentified in the urine of rats. This r'emonstrates that the colour is reduced at the azo linka.::;e in the anir.al body. After a single dose small amounts of t:le colour were found in the bod:, fat. Repeated administration resulted in cradual reduction of concentration of the colour in the fo.t P.nd finally it disappeared al togethP.r after 7-10 days. In vitro incubation of emulsions of ti1e colour with the intestinal contents of the rat, rabbit and do1, resulted in destrllction of fae colour by the bacterial flora. 1 Acute toxicity No data available. Short-term toxicity J?gg. Nineteen beagle dogs received O, 50 and 200 mg of the colour per kg body-weight per day by capsule, 6 days per week for 76 or 104 weeks. Clinical studies did not show differences between control group and the group with 50 mg/kg/day. The animals of the group of 200 mg/kg/day showed in the beginning decreased activity and periods of poor appetite. The animals progressed in about 26 weeks and seemed comparable with the control animals in their appearance and behaviour. A decrease in cell volume, haemoglobin and total erythrocyte co\Dlt was fo\Dld in this group after 26 weeks. Five of the 6 animals of the 200 mg/kg/day showed an increase in total of leucocytes. No biochemical values and urine analyses showed abnormalities. Four dogs of the 50 mg group and 3 ontrol groups were sacrificed after 76 weeks. The other dogs were sacrificed after 104 weeks. No significant eTOss or microscopic pathological abnormalities were fo\Dld in the control animals and the animals of the 50 mg/kg/day. The organs, liver, kidneys and spleen of the animals with 200 mg/kg/day were significantly increased in weight. Histologically no abnormalities were fo\Dld, except an evidence of haemopoietic hyperplasia.2 Long-term toxicity R!i• Seven h\Dldred and eighty rats received dietary levels of O, 0.05, 0.1, 0.5, 1.0, 3.0 and 5.oj. of the colour for 2 years. The animals of the groups with 3.0 and 5.0% showed a significant inhibition of the erowth. These animals were killed after 31 weeks. The animals of the 0.05 and O.]% exhibited normal appearance and behaviour. Some rats of o.~ and of higher levels developed subcutaneous oedema-like swelling of the head, neck, thoracic region and forelimbs. Hydrothorax was revealed in a number of these animals. At 31 weeks clinfoal studies were performed and in the female animals at the 5.0}~ level the plasma-protein, haemoglobin and haematocri t values were significantly lowered. The male animals showed only the decreased plasma-protein levels. The ratio liver weight/body-weight of the animals of the o.~~ and higher was increased. 'i'he bistopathological examination of the oreans of the animals of 0.05 and 0.1~{. did not show abnormalities. At o. '.>, 1.0, 3.0 and 5.0% cardiac c:ianees com:isting of interstitial oedema and fibroblastic cell proliferation were found. Two erol,ps of 50 male and 50 female rats were given once a week subcutaneously respect l vely 0.2 ml tricaprylin and l,.2 ml tricaprylin containing 20 mg of the colonr for 2 years. The injections were 1:;iven alternated in the axillary and inguinal regions. These injections cave small subct•taneous deposits or nodules located at the injection sites. During the experimento the nodules persisted but tbere was no evirience of active enlargement. These nodules showed hif'tolotically the encapsulated injected material with a thin fibrous wall. No tumours were found.2 Comments on experimental studies reported The long-texm studies are extensive and have been carried out in a rodent and a non-rodent. The metabolic studies are incomplete and yet this aspect is of particular importance for a colour of this chemical type. Therefore, the data are insufficient to permit toxicological evaluation of this colour. Further work required Biochemical studies, and toxicological testing of the metabolites. REFERENCES 1. Radomski, J. L. (1961) J. Pharmacol, exp. Ther., ill, 100 2. Paynter, o. E. & Scala, R. A., Unpublished report of Hazleton Laboratories, Falls Church, Virginia, submitted to WHO, May 1964 ERYTHROSINE These colours have been classified in categories 1.!. and :eS Synon,yms c.1. Food Red l4f FD & C Red No. 31 LB-Rot l. Class Xanthene Red Code Numbers c.1. (1956) No. 45430 c.1. (1924) No. 113 Schultz (1931) No. 887 Chemical Name Disodium or dipotassium salt of 21 ,4',5',7' tetrs,.iodofluorescein Empirical Formula Structural Formula I I NaoA--1-LJ-o~I0 ~0 COONa I 6-~ Molecular Weight .!. Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5• ..!? Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. 51 Description Erythrosine is a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A, l. Purity Tests Dye contenta Not less than 8~ by the method described below. Dissolve 1.000 g of Erythrosine in 250 ml of water, transfer to a clean 500-ml beaker, add a.o ml 1.5 !i 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 o.~ nitric acid until the filtrate gives no turbidity with silier Qitrate T.s. and then wash with 30 ml water. Dry to constant weight at 135 .± 5, carefully breaking up the precipitate by means of the glass rod. Cool in dessicator and weigh. J weight of residue x 105 1 3 7" dye content• weight of the sample Loss on drying at 135° ~ total not more than l~ Chlorides and sulfates calculated as sodium salts j Water-insoluble mattera not more than 0.2% Ether-extractable matter (from alkaline solutions only )1 not more than 0.2% Lead (as Pb)a not more than 10 mg/kg Arsenic (as As) 1 not more than 3 mg/kg Inorganic iodides: not more than 1000 m~/kg as sodium iodide Reagents. The reag~nts shall be of a recognized analytical reagent quality. Distilled water of at least equal purity shall be used. (a) Silver nitrate - 0.1 N solution, accurately standardized. (b) Nitric acid - 1.5 Ii solution. Apparatus. Potentiometric titration apparatus, with silver indicator electrode, calomel reference electrode and saturated potassium sulphate bridge. Procedure. Accurately weigh about 3 g of the dye sample into a 500 ml beaker. Dissolve in 300 ml of water and add 200 ml of the nitric acid. Filter through a Grade 3 sintered glass filter!: of diameter preferably 2.5 in, but not less t>!an 1.5 in a.nd wash with 2 50 ml portions of water. Place the silver electrode jn the combined filtrate and washings and connect the calomel electrode to this solution by means of the saturated potassium stdphatc i,ridge. 'i'itrate the solution with the 0.1 N silver nitrate solution to the followjng end points in succession: zero mV, the iodide end point, +275 mV, the chloride end point. 1 ml of 0.1 N AgNO} solution • 0.0150 g of Na.I 1 ml of 0.1 H Agtl03 solution = 0.00585 g of llaCl .! B.S.1752, 'Sintered disk filters for laboratory use' Calculations. Iodide as sodiwn iodide, per cent. by weight • A xwl,SO Chloride, as sodi\D chloride, per cent. by weight .(W) : 0.5S5 where A• volume, in millilitres, or 0.1 N silver nitrate solution required for the iodide end point B • vol\De, in millilitres, of 0.1 N silver nitrate solution required for the chloride end point and 1f • weight, in grams, or dye sample taken. Subsidiary dyes• not more than 41, Intermediates• not more than O·"'· Biological Data Biochemical aspects The metabolic behaviour and excretory pattern for erythrosine have been studied in adult rats. The colour was given by stomach-tube in log-spaced doses from 0.5 to 500 mg ;,er kg body-weight. In 5 days the recovery in the excreta was 102". After an intravenous application or ~ mg per kg body-weight the urine and bile for the initial 2 to 4 hours was collected, an average of 5"' (50.4-sa.()J') or the administered quantity was found in the bile. In the urine 1.~ (o.s-1.9"). No glucuronic acid conjugation is found. The basic fluorescein structure is stable and the higher halogenated derivatives as erythrosine are metabolically inert.l This colour was found to be largely excreted in the faeces by rats (55-72%) and despite the presence of 2 groups capable of undergoing conjugations, no colour could be identified in the urine. A small amount of the colour is excreted in the bile (0.4-1.7").2 When cherries coloured with erytbrosine are stored in plain cans, fluorescein is readily formed by interaction of the tin-iron couple present. This does not occur in lacquered cans. The production of fluorescein (with 4 J atoms} from erythrosine occurs in presence of metallic iron and/or tin and free organic acid (result or electro-chemical reduction in the can}.~ It was found that this colour in a concentration or 200 to 400 ipg/litre, inhibited the action of pepsin but had no effect on lipase activity.4 It was also found that this colour had in vivo as well as in vitro haemolytic effect. In the in vivo studies the mouse was used.5 5, Acute toxicitz ~o Reference per kg body-weight Mouse intravenous 370 mg 5,6 Rat intraperitoneal 300 mg 6 Rat oral >2.0 g 6,7 Babbit intravenous 200 mg 6 Special studies '!'his colour was tested for mutagenic activity and showed a very slight but statistically significant mutagenic effect on Escb@richia coli in concentrations of 0.5 g/100 ml. It was found that xanthene molecule itself was the causative factor and that the substituent groups only modify the effect.8,9 Short-term toxicitz !4!:1. A group of 5 young rats were given subcutaneous injections twice daily for 3 days. The rats were killed on the fourth day. The colour was administered in aqueous solution at a level of 250 mg per kg bo~-weight each injection. No estrogenic activity (normal uterine weight) was detected.10 Guinea-pig. In experiments with guinea.-pigs it was found that this colour had no sensitization activity.11 B• Two-year toxicity studies were conducted with 2 male and 2 female beagles at dosage levels of 0.5, 1.0 and 2.0J'. The control group contained 6 doge. All animals survived the experiment and no systemic effects attributable to the drugs were noted.12 MBA. Iodide goitre has been observed to opcur following daily doses of several 100 mg of iodide administered for years.13 Goitre and varying degrees of tiniothyroidism developed in 13 patients with an aee ranging between 5 and 17 years, who had taken iodide (as syrup of hydriodic acid, potassium iodide and Lugol's solution) for 2 to 5 yeafS for control of asthma. After discontinuation of iodide and administration of desiccated thyroid or sodium 1-thyroxine, the thyroid regressed in size to normal. It was concluded that in some individuals prolonged iodide therapy ~ have an antithyroid action similar to that of thiouracn.14 Long-term toxicitz Mouse. A total of 122 male and female mice produced by mixed breeding from 5 different strains were given a diet containing 1 mg per animal per day of the colour. Mice at the age of 50 to 100 days were used. a m111ber of the animals were sacrificed after an observation period of 500 days and the survivors of the rest after 700 days. A total of 168 mice was used as control. Positive control groups which were given 0-a.minoazotoluene and dimethylamino azobenzene, were also included. In these groups the formation of liver tumours was noted after approximately 200 days. The incidence of tumours in mice receiving the colour was not significantly greater than in the controls.15 Chronic feeding studies were conducted with mice. Seventy mice were fed at 1.0 and 2.0%. Because of the small number of animals surviving the expe'riment and the small number of tumours found, no effect of tumour formation could be attributed to the colour.12 Rat. Twenty rats were subjected to weekly subcutaneous injections of 1 ml of a ~aqueous solution for 596 days. The total quantity of colour administered was 2.65 ftanimal. Seven rats survived ~00 days or more. No tUJr.ours were observed. Groups of 24 weanling rats, evenly divided by sex, were fed this colour at O, 0.5, 1.0, 2.0 and 5.0}(, for 2 years. Slight growth depression was observed on the animals at the 5.0% level, and those above o.~ had distended caectmts but microsco;iically the distended caecums showed normal histology. The statistical evaluation of the rat study revealed no changes in organ weights at the highest level. (Evaluations of ~ower levels have not been finished.) There was no difference in survival.1 Forty rats were fed 1% of the colour for 2 years. Six animals died between time. No tumours were found.6 The colour was fed et a level of 4% of the diet to 5 male and 5 female rats for periods up to 18 months. Gross staining was observed in the glandular stomach and small intestine and gTSllular deposits in the stomach, small intestine and colon. Hepatic cirrhosis was noted in 1 case out of 4 rats living up to 12 months. Fifty control animals obs!rved for 20 months or more failed to develop tumours, or hepatic cirrhosis.·, Eighteen rats were injected subcutaneously 1 ml of a 2-p or 3% solution per week for periods of 94 to 99 weeks. No tumours either at the site of the injections or in the other parts of the body were observed.18 CoDUDents on experimental studies reported Despite the fairly extensive studies on metabolism and long-term toxicity, it is considered that the data are not fully adequate to permit evaluation. Further work required More information on metabolism in various animal species. Particular attention to thyroid in young growing animals. 55 REFERENCES 1. Viebb, J. M., l''onda, .M. & Brouwer, E. A. (1962) J. Pharmacol. exp. Ther., ill (2), 141 2. Daniel, J. w. (1962) Toxicol. appl. Pharmacol., .! (5), 572 3. Dickinson, D. & Raven, T. w. (1962) J. Sci. Food Af,Tic., ll, 650 4. Diemair, w. & Hausser, D. (1951) z. Lebensmi tt.-Untersuch., -2£, 165 5. Waliszewski, T. (1952) Acta Pol. pharm., .2 (2), 127 6. Deutsche Forschungsgemeinschaft, Bad Godesberg, Federal Republic of Germany, Farbstoff Kommission (1957) !.litteilung 6 1. Lu, F. c. & Lavallee, A. (1964) Canad. pharm. J., !zl, 30 6. LU.ck, H., Wallntlfer, P. & Bach, H. (1963) Path. et Micro biol. (Basel), 26, 206 9. LU.ck, H. & Rickerl, E. (1960) z. Lebensmitt.-Untersuch., .!!£, 157 10. Graham, R. c. D., Allmark, M. G. (1959) Toxicol. appl. Pharmacol., !, 144 11. Bgr, F. & Griepentrog, F. (1960) Med. u. Ernlhr., ! (5), 99 12. United States Food and Drug Administration, Unpublished report submitted to WHO, April 1964 13. Saxena, K. M., Chapman, E. M. & Pryles, Ch. v. (1963) Science,~. 430 14. Turner, H. H. & Howard, R. B. (1956) J. clin. Endocr., 16, 141 15. Waterman, N. & Lignac, G. o. E., as summarized by H. van Genderen (1958) Acta physiol. pharmacol. neerl., 1, 35 16. Umeda, M. (1956) ~. Al, 51 17. Willheim, R. & Ivy, A. c. (1953) Gastroenterology, £l, 1 18. Nelson, A. A. & Hagan, E. c. (1953) Fed. Proc.,]£, 391 FAST GREEN FCF This colour has been classified in categories I~ and ~,.2 Synonyms C.I. Food Green 3; FD & C Green No. 3; Vert Solide FCF. ~ Triarylmethane Green Code NtUDber c.r. (1956) No. 42053 Chemical Name Disodi\Ull salt of ,t..<"L4,-(N-ethyl-p,,,sulfobenzylamino)-phenyl7-(,t..hydroxy- 2-sulfoni1.UDphenyl)-methylen~-f:i"-(N-ethyl...N-p-sulfobenzyl)-~2,5-cyclohexadienimin!7 Empirical Formula Structural Formula C2H5 C2H5 I I H2C/N,o~ ~I/N'cH2 ~ (+) ~ ~ c ,:;::? SO::- l 503 3 ~ OH i4olecular Weight 806.86 ~ Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5 • .!? Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11 • .2 Colours producing more than 10-1% of local sarcomas at the site of repeated subcutaneous inJection. 57 Description Fast Green~ is a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A. Purity Tests Dye content: Not less than 8~ by titration with ti tanous chloride. Weigh o.aoo..0.900 g of Fast Green FCF and proceed as in Appendix A. Use 15 g of sodium hydrogen tartrate as buffer. 1 ml of 0.1 N titanous chloride is equivalent to 0.04044 g of c -1' o N s Na • 3 34 10 2 3 2 Loss on drying at 135° ~ total not more than l~ Chlorides and su!fates calculated as sodium salts} Water-insoluble mattert not more than o.~ Ether-extractable mattert not more than o.~ Lead (as Pb): not more than 10 mg/kg Arsenic (as As)t not more than 3 mg/kg Chromium (as Cr): not more than 20 mg/kg Subsidiary dyes: not more than]% Intermediates: not more than o.~ Biological Data Biochemical aspects Rats and dogs were given orally 200 mg of the colour. In the rats the urine and faeces were collected for 36 hours and analysed. In the dogs a bile fistula was installed and the bile was analysed. It was found that in the faeces of the rats almost all the administered colour was excreted unchanged (>90'fo). In the urine no colour was found. In the bile of the dogs the amount of the colour never exceeded ~ of the given dose. After feeding, the colour was found in the bile of rats and rabbits, but not in their urine. It was concluded that the quantity found in the bile provides a reasonable estimate of the amount absorbed from the gastro-intestinal tract.1,2,3 Acute toxicity 1%0 Reference per kg bod,v-weip..ht Rat oral >2.0 g 4 Short-term toxicity !mi• Two groups of 16 female rats (control group of 10 rats) were given subcutaneous injections of a 0.5 ml of a 3 and 81' solution. {The rats received with each injection respectively 15 and 30 mg.) The used colour was certified as 92% pure and was supplied as the disodium sulfonate salt. The 10 control rats were given distilled-water injections. At first, injections of 81' were given 3 times a week; after 17 weeks it became necessary to reduce the dose to "1,. Thereafter both groups were given injections of YI, twice weekly for 9 weeks·. The rest of the time, 22 weeks, both groups were injected usually once a week, occasionally 2 injections were tolerated. Growth inhibition was found. Thirteen out of 16 animals receiving 81' of the colour had fibrosarcomas. The animals given ',I, showed also fibrosarcomas (10 out of 12). The controls did not show neoplastic tissue at the site of injection.5 ~· Dog feeding studies were completed. Four beagles per group, equally divided by sex, were fed at o, 1.0 and 2.0J' for 2 years. Histopathology attributable to the colour was limited to green blobs of pigment in the renal cortical tubular epithelial cytoplasm of a male dog on a high dose level; a female dog on a high dosi level showed slight interstitial nephritis and slight bone marrow hyperplasia. Long-term toxicity Mouse. Chronic mouse feeding studies were conducted; 2 groups of 100 mice each were fed for 2 years at 1.0 and 2.~, and 200 mice served as controls. Histopathology is complete. Microscopic ex~nation revealed no lesions that were attributed to the feeding of the colour. fu!!• Eighteen rats of both sexes were injected this colour subcutaneously for 94 to 99 weeks. In general l ml of a 2% or~ solution was injected weekly. Subcutaneous fibrosarcomas appeared at the site of the injection in 15 of the animals.7 The colour was fed at a level of 4% in the diet to 5 male and 5 female rats for periods from 18 to 20 months. This procedure resulted in gross staining of the forestomach, glandular stomach, small intestine and colon. Granular 8 deposit.a were noted in the otomach. No tlDDours were observed. Groups o.f 50 littcr-roati,d ·,;,Ni.n.ling, Osborne-Mendel rats, evenJ..y divided by se:x:, were fed the colour at D, 0,.. ';, l.O, 2.0 mid 5.(% for 2 years. No effocts on growth or mortality were ol;sc-•:'ifed. Microscopic exa.min!J,tit,n revealed no lesionn tha~ were att:r-:ibuiect to i.t10 fecd:ing of the colou.1~,b Comments_on p-,ryer:i.ment;..l ~itudie~~ rnpyrtrecl Long-,term r,tud.iefl arc nvo.iJabJo in S<~veral species. Local r,arcomas ,mre prod.rn:ed un l'ep~a ted. G ttbcuto.necn.:, :i.n,jection. Paucity of rd och<'.mi.ca] tla \;a makes .i.t impos~dble to carry out an 2vul11ation of the acce-p~abJ,~ dail,y ir,1;,,.1;,· ·t.n m:>.n.:. 59 REFE,iENCES 1. Hess, s. M. & Fitzhugh, o. G. (1955) J. Pharme.col. exp, Ther., 1!! (1), 38 2. Hess, s. m, & Fitzhugh, 0, G. (1953) Fed, Proc., 12, 330 3. Hess, s. M. & Fitzhugh, O. G. (1954) Fed. Proc., ll, 365 4. Lu, F. c. & Lavallee, A. (1964) Canad, pha:rm. J., ~. 30 5. Hesselbach, M. L. & O•Gara, R. w. (1960) J. nat. Cancer Inst.,~. 769 6. United States Food and Drug Administration, Unpublished report submitted to 1"110, April 1964 7. Nelson, A. A. & Hagan, E, C, (1953) Fed. Proc., 12, 397 a. Willheim, R. & Ivy, A, C. (1953) Gastroenterology, £l, 1 INDANTHRENE BLUE BS These colours have been classified in categories I~ and c1lz Swon.yms C.I. Vat Blue 41 L-Blau lJ Indanthrene Blue BS.lJ Bleu SolanthreneJ E.!l.C. Serial No. E 130. Class .lnthraquinone Blue Code Nunbers c.1. (1956) Bo. 69800 c.1. (1924) No. 1106 Schultz (1931) Bo. 1228 Chemical Name N,B~dro.l,l,1~21-anthraquinone-azine Empirical Formula Structural Formula c t llolecularWeiffht ~ Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5. l2. Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. 61 Description Indanthrene Blue RS is a food colour insoluble in water and edible oils, conforming to the following specifications. Identification Tests See Appendix A. Purity Tests Dye content1 not less than 99%. (Method required) Loss on drying at 135° ~ total not more than 1% Chlorides and sulfates calculated as sodium salts ) Ether-extractable matter1 not more than 0.2% Lead (as Pb)1 not more than 10 mg/kg Arsenic (as As)1 not more than 3 mg/kg Biological Data Biochemical aspects No data available. Acute toxicity LD50 Reference mg/kg body-weight Rat Oral 2000 1 Short-term toxicity 1111· This colour was fed toe; rats at a level of 0.1% in the diet. The animals received a daily dose of 10 to 15 mg. No in~reased tumour incidence was observed. The observation period was not mentioned. Twenty-one rats were fed a diet containing 0,1% of the colour for 184 days. Eight rats which survived for more than 400 days (432-683 days) showed no abnormality. Twelve rats received monthly subcutaneous injections of 2 ml of a 2.5% aqueous solution of the colour, Six rats survived.for 417-570 days. No tumours were seen at the site of injection.3 Guinea-pig. In experiments with guinea-pigs it was found that this colour had no sensitization activity.4 Qa!:. Cats received daily doses of 0.1 g/kg of colour for seven ctays. No increase in Heinz bodies was noted in the blood of the test animals,5 Long-term toxicity I!a1, Twenty-one rats were given 0.1% of the colour in their diet for 683 days. Thirteen animals di~d before the end of the experiment. No increased tumour incidence was observed, 62 Twelve rats were given 2 ml of a 2.5% suspension subcutaneously monthly. The observation period was 570 days. Six animals died b~fore the end of the experiment. No increased tumour incidence was observed. Groups of 20 male and 20 female rats or more were fed diets containing O ppm and 10 OOO ppm of the colour for 2 years. A similar test group was formed from the first filial generation and was fed the 10 OOO ppm level for a similar period of time. No effect of the diet was noted in the test groups, and gross and microscopic examination of the animals disclosed no changes attributable to the test di;t. There was no significant difference in tumour incidence between the groups. Comments on experimental studies reported The available data are not fully adequate for evaluation but a substantial amount of detailed information is available concerning the results of long-term tests. Further work required Long-term tests in another species. Metabolic studies in animals and man. REFERENCES 1. Lu, F. C. & Lavallee, A. (1964) Canad. pharm. J., 22:, 30 2. Deutsche Forschungsgemeinschaft, Bad Godesberg, Federal Republic of Germany Farbstoff Kommission (1957) Mitteilung 6 3. Umeda, M. (1956) 9mn, il, 51 4. Bar, F. & Griepentrog, F. (1960) Med. u. Ernahr., l (5), 99 5. Oettel, H., Frohberg, H., Nothdurft, H. & Wilhelm, G. (1965) Arch. fiir. Toxicol., 21, 9 INDIGOTINE These colours have been classified in categories r!: and~ Synon.yms c.r. Food Blue 1; FD and c Blue No. 2; 1-Blau 2; Indigo Carmine; E.E.C. Serial No. E 132 £!!!.! Indigo id Blue Code Numbers c.r. (1956) No. 73015 c.r. (1924) No. 1180 Schultz (1931) Ho. 1309 Chemical Name Disodium salt of Indigotin-5,51-disulfonic acid Empirical Formula Structural Formula Molecular Weight ~ Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5. ~ Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. Definition Indigotine is a mixture ot Indigotin aultonic acids consisting princi~ ot Indigotin 5,5 1-diaultonic acid with appreciable amounts ot Indigotin 5, 7•.. diaultonic acid; Description Indigotine is a water-soluble food colour conforming to the following spscitioations. Identification Testa See Appendix .1, l. Purity Testa Dye content• Not leas than s'JI, by titration with titanoua chloride. Weigh 0.500-0.600 g ot Indigotine and proceed as in Appendix .1, s. Ose 15 g ot sodium hydrogen tartr&te as butter. l ml of 0.1 titanoua chloride is equivalent to 0.02332 g of C B o s Ba • !. 1(!18 2 8 2 2 Loss on drying at 135° } total not more than l'JI, Chlorides and aulfates calculated as sodium aalte Water-iDBoluble matter1 not more than o.i,r, Bthe:r-extractable matter• not more than 0.2" Lead ( as Pb)• not more than 10 mg/kg .lrsenio (as .ls)• not more than 3 mg/q Subsidiary dyes a not more than ~ (other than the 5, 71 isomer) !satin aultonic acida not more than 1" Prepare a column of cellulose as directed in the method tor intermediates, in Appendix .1. Prepare the eluting solution by adding 10 ml of hydrochloric acid to 2 litres of 2'JI, UUDOnillB aulfate solution. Weigh a sample of 0.4 g and proceed as directed for •intermediates" Preparation ot !satin aultonic acida Dissolve l got Indigotine in 25 ml of water and add 0.5 ml of aulfuric acid. Heat the solution to about 90° on a steam bath. Add to the hot solution small portions of potassium psrsulfate until the blue colour disappears. About l g will be required. Evaporate the solution to about 10 Ill and add l g ot solid potassium chloride. Allow the solution to cool to room temperature. Collect the potasaiua !satin aultonate by tiltrat1on, waah with a very little ioe-oold water. Dry at 100°. , Biological Data Biochemical aapecta llicroobemioal methods gl.ve evi4enoe that filterable indigotine in the blood plasma ot rabbits is excreted through the glomeruli. The filterable traction is about l'JI, while 7,!, of the colour ia excreted by the tubules. The lmina of the tubules and BOWIIIUl1a capsules were injected with solutions ot indigotine. 10 mg par cent. waa the lowest concentration which leaves detectable traces ot the colour in the kidneys. When an increased dose of colour is injected and the interval between the injection and the excision of the kidney shortened, the colour can be detected in the intracapeular spaces. The tubular excretion of the indigotine resembles quantitatively that of phenol red.l Following the administration of s}5 labelled colour to rats in single oral doses of 5 to 50 mg, 60 to 7,!, was excreted in the faeces and 1 to 2% in the urine within 72 hours. After a single intravenous injection of a dose of 0.6 to 1.5 1llll.lkst bo4T-weight, f:IJfo was excreted in urine and 1()% was excreted in the bile.lr ·~ Acute toxicity ~o Reference per kg body-weight Rat oral 2.0 g Special studies The colour was tested for mutaeenic effect in a concentration of 0.5 g/100 ml in cultures of Escherighia goli. No mutagenic effect was found.4 Short-term toxicity ~· A group of 5 young rats was given the colour subcutaneously twice daily for} days. The rats were killed on the fourth day. The colour was administered in aqueous solution at a level of 250 mg per kg body-weight each injection. No estrogenic activity (normal uterine weight) was detected. The animals lost weight.5 Twenty rats were given first 1 ml of a ~ solution of the colour subcutaneously. Later the dose was reduced and 0.5 ml of a 0.5% solution given. Over a period of 7 months a total of 55 injections were administered. Only 1 tumour in the axillary region was found.6 Forty rats were given a diet containing 1" of the colour. Seven animals died before the end of the experiment. No tumours developed. 1'he duration of the experiment was not mentioned. 6 Guinea-pig. In experiments with guinea-pigs it was found that this compound had no sensitization activity.7 1?2£. Dog feeding studies were conducted on beagles fed 1.0 and 2.(% of the colour in their diet for 2 years. Two dogs served as controls. After 2 dogs on the high-dose level died, 2 more were placed on the 2.(% ciet and 1 on control. There were no clinical symptoms, gross lesions, or microscopic pathology attributed to the colour. The treated dogs were more susceptible to canine distemper and canine hepatitis than were the controls, ?.!though this was not statistically significant because of the small number of animals used.a Long-tenn toxicity ~. Groups of 24 weanling Osborne-Mendel rats, equally divided by sex, were fed the colour at O, 0.5, 1.0, 2.0 and 5.C% for 2 years. 'l'he statistical evaluation showed growth inhibition of the males at 2.0 and 5.C%. There was no ch&ll89 in mortality, organ weights, or haematology, nor any gross or microscopic pathology related to treatment.a 66 Comments on experimental studies reported Long-term studies are available in several species. Paucity of biochemical data makes it impossi,.le to carry out an evalue.tion oi' the acceptahle daily intake in man. Further work reouired Metabolic studies in different ani;;:al species. 1. Kempton, R. T., :1ott, P. A. & Rici,ards, A. N. (19}7) Amer. J. Ano.t., 61 (:,), 505 2. Lethco, E. J. & Webb, J. M. (1964) led. Proc., ll, 406 :,. Lu, F. C. & Lavallee (1964) Canad. pharm. J., :II., :,o 4. LUck, H. & Rickerl, E. ( 1960) z. Le' ensmitt.-Untersuch., 112, 157 5. Uraham, R. c. H. & Allm,,.rk, t.i. G. (1959) 'i'oxicol. appl. Pharmacol., .!, 144 6. Deutsche r'orschungs1:,cmeinschaft, :;,id Godesi,er,;, t•'ederal Republic of Germany, Farbstoff Kommfasion (1957) :·,:ittei11,ng 6 7. Bff.r, F. & Griepentrog, F'. (1960) Med. u. Ern;.:hr., 1 (')), 99 a. Unj ted States Food and Drue i\dminjstraLion. Unpublished report submi tted to WHO, April 1964 67 ORANGE I This colour has been classified in categories r.! and CI~,St Synon.yms Naphthol Orange; Ext DC Orange No. 3; c.r. Acid Orange 20. Monoazo ~ Orange Code Numbers c.r. (1956) No. 14600 c.r. (1924) No. 150 Schultz (1931) No. 185 Chemical Name Sodium salt of R -L(4-ohydroxy"J.,.naphthyl)az,g]benzene sulfonic acid Empirical Formula Structural Formula OH Molecular Weight 350.33 .! Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5. ! Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11 • .Q. Colours producing more than 10-1~ of local sarcomas at the site of repeated subcutaneous injection. Description Orange I is a wate!'-soluble food colour conforming to the following specific& tiona. Identification Tests See Appendix A, 1. Purity Testa Dye content a Not leas than 8~ by titration with ti tanoua chloride. Weigh 0.250..0.}00 g of Orange I and proceed as in Appendix A, e. Use 15 g of sodium hydrogen tartrate as buffer. Loss on drying at 135° l total not more than l~ Chlorides and sulfates calculated as sodium salts~ Watel'-inaoluble mattera not more than o.2% Ethel'-extractable mattera not more than o.2% Intermediatesa not more than o.~ Subsidiary dyesa not more than l{fo Leada not more than 10 mg/kg Araenica not more than 3 q/kg Biological Data Biochemical aspects In dog it was found that 6-41% of the administered colour was excreted in the faeces. No colour was found in the urine. Incubating the colopr with a dog's fresh intestinal content destroyed SOJ' of the colour in l hour.1,2 Acute toxicitz 11%0 Reference per kg bod.y-weight Rat intraperitoneal 1000 mg l Short-term toxicitz Rat. Ten rats were fed this colour at a level of ,ff, in the diet. The animalslived only a few months. Gross staining of the glandular stomach and the small intestine, and granular deposits in the stomach and small intestine were observed., Weanling male rats were fed diets containing 1, 2 or ~ of the colour for 16 weeks. The animals with the 2 and"' died all within the first 2 weeks of the feeding period. A mortality of 80J' occurred in the rats fed 1% and the growth rate of the surviving animals in this group was markedly retarded. The inclusion of the colour in the diet caused severe diarrhoea, enlargement of the spleen and an anaemia.4 69 122£. '!'his colour had a significant cathartic effect when given 100 to 200 mg to groups of 5 dogs.2 !f!!!· '!'his colour induces also a cathartic action in lll8ll (80 mg). The intact molecule appears to be the active cathartic agent.5 H\818D vol\Dlteers who ate candy containing o.o-r.' or this colour exhibited diarrhoea upon ingestion of 1 to 8 pieces of the candy. 6 Skin tests with this colour showed in patients sensitive to paraphenylene diamine no reaction.7 Long-term toxl.citz !!2J!l!• Twenty mice were fed 15-20 mg per week or this colour for periods up to 409 ~ five da,ys a week. No tumours were observed which could be attributed to the colour.a Rat. This colour was fed to 85 rats at a level of 0.1% in the diet. The dailyintake varied from 10 to 15 mg for a period of 400 days. No tumours were observed.1 Groups of 24 weanling rats, equally divided by sex, were fed the colour at O, 0.5, 1.0 and 2.0J' in the diet. No rats fed at 2.0J' survived beyond the fifth week, observations are confined too, 0.5 and l.OJ' levels. Body-weight and food intake were recorded weekly for 2 years; blood co\Dlts were taken 4 times. Gross findings at 1.0J' were marked increase of mortality, enlargement of spleens, leukocytosis and anaemia, diarrhoea, and growth depression. .At o.,% kidneys of test rats showed more chronic congestion than those of controls and there was some splenic enlargement. Microscopically, spleens showed uniformly chronic congestion and less often slight hyperplasia and increased pigmentation. Kidneys showed nephritis of the t~ common in older rats with no difference among the groups except in incidence.~,10 Eighteen young rats were given subcutaneous injections, 20 mg of the colour per week (2" aqueous solution) for 2 years. In 6 cases fibrosaroomas were found. The controls did not get t1.111ours.ll )22&. The colour was given to 14 dogs at 4 levels in daily quantities by capsule ranging froll about 0.02% to 1.0J' in the diet. The dogs on the lowest level survived for 5 years without shoring any effect. .At higher levels (0.2" or more of the diet) effects were variable; some dogs survived only for short periods, others showed little or no effect for long periods. Pathological changes were generally non..specificJ some animals were found dead with little to explain the ~athJ others were emaciated and showed orgs.n changes chiefly of inanition.9,1 Comments on experi11&ntal studies reported Long.term experiments have been carried out in 3 animal species. Sarcoma have been produced aa a result of repeated subcutaneous injections. Information on the metabolism of the colour is lacking. The data are insufficient to permit toxicological evaluation of this colour. Purther work required Metabolic studies in several animal species. See general principles governing the toxicological evaluation, section 2.2 pap e. 70 REFERENCF:3 1. Deutsche Forschlmg&gemeinschaft Bad Godesberg, Federal Republic of Ge~, Farbstoff Kommission (1957) Mitteil1U1g 6 2. Vos, B. J., Radomski, J. L. & Fu,at, H. N, (1953) Fed. Proc., Jl, 376 3. Willheim, R. & Ivy, A. c. (1953) Gastroenterology, £2., l 4. Hallesy, D. w. & Doull, J. (1956) J 1 Pharmacol. exp, Ther., 116, 26 5. Radomski, J. L. & Deichmann, H. B. (1956) J, Pharmacol. exp. Ther., fil, 322-327 6. United States Food and Drug Adllinistration, Department of Health, Education and Welfare (1955) Title 21, Food and Drugs, Part 135, Federal Register, lQ, 8492 7. Baer, R. L., Leider, K. & ~er, R. L. (1948) Proc. Soc. exp, Biol., fil.., 489 8. Cook, J. w., Hewett, c. L., Kennaway, E. L. & Kennaway, N. M. (1940) Amer. J. Cancer, AQ, 62 9. Bourke, A. R., Nelson, A. A. & Fitzhugh, o. G. (1956) Fed, Proc., .!2, 404 10. United States Food and Drug Administration, Unpublished report submitted to WHO, April 1963 11. Belson, A. A. & Davidow, B. (1957) Fed. Proc., 16, 367 71 PATENT BLUE V This colour has been classified in categories :c.! and c1k SynonYIIIS C.I. Acid Blue 3J ~Blau 3J Bleu Patent4 V; E.E.C. Serial No. E 131 Triarylmethe.ne Colour Blue Code NU11bers c.1. (1956) No. 42051 c.1. (1924) No. 712 Schultz (1931) No. 826 Chemical Name Calcium salt of the disulfonic acid of m.hydroxy-tetra.-ethyl diaminotriphenyl carbinol anhydride Empirical Formula Structural Formula (+) Ho;c H~ 2 Molecular Weight so; .! Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5. ll Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. Description Patent Blue Vis a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A. Purity Tests Dye contentt Not less than 85% by titration with titanous chloride. Weigh 1.000 g of Patent Blue V and proceed as in Appendix A. Use 15 g of sodium hydrogen tartrate as buffer. 1 ml of 0.1 N titanous chloride is equivalent to 0.02898 g of (c H N ) ca. 27 31 2ot52 2 Loss on drying at 135° } total not more than 15% Chlorides calculated as calcium salts Water-insoluble matter, not more than 0.5% Ether-extractable mattert not more than o.2% Lead (as Pb)a not more than 10 mg/kg Arsenic (as As)a not more than 3 mg/kg Chromium (as Cr)a not more than 20 mg/kg Subsidiary dyes: not more than 1" Intermediatest not more than 0.5% Biological Data Biochemical aspects After intravenous injection into the rat of 0.5 ml of an aqueous solution containing 5 g per 100 ml the colour is excreted in the urine which is coloured blue during the 12 hours following the injection. Findings are similar in the case of man when the colour is injected subcutaneously for the detectiyn of peripheral lymphatic trunks and nodes for the purpose of lymphography. · Acute toxicitz Am!!! ~o References ~ J?!r kg bo!}.I-weil?iht Mouse oral >3.0 g 1 Mouse intravenous 1.2 g 1 Rat oral >5.0 g 1 Rat intravenous 5.0 g 1 Short-term toxicity Cat. Three cats were given daily a"§, aqueous solution of the colot..r orally doses'"'or 0.25, 0.50 and 0.75 g. No abnormalities were found.l Long-term toxicity .!!!!• Sixty rats, half males and half females were given in their diet 10 OOO ppm of the colour for their life-span. The used colour was pure (no impurities were found by paperchromatography studies). The average life-span of treated animals was 24 months and the last animal died at an age of 37 months, 2 weeks. The average in~e of the colour was approximately 80 g. Forty animals were used as controls. The average life-span of these animals was 22 months, 2 weeks. For the second experiment 30 rats, half males and half females, were given the same diet with 10 OOO ppm and also for 15-19 months once a week a subcutaneous injection of 1 ml of al" aqueous solution. The average life-span of the animals was 18 months. The last animal died at an age of 30 months. Twenty-six rats were used as controls and were given 1 ml of distilled water subcutaneously for the same period as the treated animals. The average life-span of these animals was 19 months. In both experiments it was found that growth, blood composition, reproduction and the 3 generations of animals which were bred were not influenced. Also, no abnormal ~istopathological findings or sarcomas at the site of injection were observed. Comments on experimental studies reported Long-term experiments have been carried out only in 1 species, namely the rat. Information on the metabolism of the colour is lacking. The data are insufficient to permit toxicological evaluation of this colour. Further work required See general principles governing the toxicological evaluation, section 2,2 page a. REFERENCES 1. Truhaut, R. (1962) Aliment. et Vie, .2Q, 77 7- PONCEAU 4R This colour has been classified in categories 1!: and c1h S;ypOIIYIDs C.I. Food Red 7J t,.Rot 4; Coccine Nouvelle; Cochineal Red AJ E.E.C. Serial No. E 124. llonoazo Red Code Numbers c.1. (1956) No. 16255 c.1. (1924) No. 185 Schultz (1931) No. 213 Chmnical Nue Trisodium salt of' 1.,.(4-sulf'o,.,l-naphthylazo) ..2-naphthol..6,S-disulf'onic acid Empirical Formula Structural Formula Molecular Weight !; Classification of' colours from the standpoint of' chemical specifications, see section 2.1.3 page 5. -b Classification of' colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. 75 Description Ponceau 4R is a water-soluble food colour conforming to the following specifications. Identification Testa See Appendix A. Purit:y Tests Dye contenta Not less than 8~ by titration with titanous chloride. Weigh o. 700-0.800 g of Ponceau 4R and proceed as in Appendix A. Use 10 g of trisodium citrate as buffer. Loss on drying at 135° ~ total not more than lSJ' Chlorides and sulfates calculated as sodium salts j Water..insoluble mattera not more than o.~ Ether-extractable mattera not more than o.~ Lead (as Pb)a not more than 10 mg/kg Arsenic ( as As) a not more than 3 mg/kg Subsidiary dyesa not more than 2.o,( Intermediatesa not more than o.~ Biochemical aspects Rats were injected the colour intravenously and the bile was collected for 6 hours and analysed. The recovery of the colour was an average of 34% (30-4~) of the administrated quantity.I Acute toxicit;r ~o References per leg bocb;-weight Rat intraperitoneal approx. 2.0 g 2 Rat intravenous 1.0 g 2 Special studies This colour was tested for mutagenic effect in a concentration of 0.5 g/100 ml in cultures of Escherichia goli. No mute.genie effect was round.3 Short-term toxicity !!!:!· Seventy-five rats were fed the colour at a level of 0.1% of the diet. Each animal received 10 to 15 mg per ~ • No t\DDours were observed. Similar results were obtained with 10 rats when fed at a level of 0.2" of the diet, each animal receiVing 20 to 30 mg per day. (The duration of the experiments are not mentioned.)2 Four groups of 20 rats (10 male and 10 female) were given diets containing O, 0.03, 0.3 and }.OJ' of the colour for 64 weeks. The mortality of the male and female rats was not influenced. Female rats receiving ',I, in the diet had a lower food consumption throughout the experiment than did the controls. This produced a significant decrease in bo~-weight at 16 weeks and 64 weeks. There was an increase in the relative weights of heart, liver and kidney for the females on the ',I, level. No effects were found on histopathology and haemoglobin levels.4 Guinea.-pig. In expsriments with guinea.-pigs it was found that this colour had no sensitization activity.5 £11• A negative test for Heinz bodies was obtained after administering this colour in a 'JI, aqueous solution by stomach-tube to 4 cats.2 Loy-term toxicity 11!:1• Ten rats were given 0.27' of the colour in their diet for 417 days, the daily intake being about 0.1 g psr kg body-weight. The total intake was 11.0 g/animal. The total duration of the experiment was 1011 days. One rat died during the expsriment. No tumours were found.2 Eleven rats were given 1.0J' of the colour in their drinking-water for 216 ~. The observation psriod was 791 days. The daily intake of the colour was about l g per kg bo~-weight. The total intake was 52 g/animal. Two animals died during the experiment and in l animal a sarcoma was found in the liver at the junction of the liver and gall bladder.2 Thirteen rats were given twice weekly subcutaneous injections of 0.5 ml of l.OJ' solution of the colour for 365 days. The observation period was 857 days. Five animals died during the experiment. No tumours were found.2 Cownts on experillental studies reported Long.term expsriments have been oarried out only in l species, nsmely the rat. Information on the metabolism of the compound is lacking. The data are insufficient to permit toxicological evaluation of this colour. Further work required See general principles governing the toxicological evaluation, section 2.2 page a. REFERENCES 1. Ryan, A. J. &, Wright, s. E.. (1961) J. Pham. Pba:rmacol., .ll, 492 2. Deutsche Forschungsgemeinschaft, Bad Godesberg, Federal Republic of German;,, Farbstoff Kommission (1957) Mitteilung 6 }. LUck, H. &, Rickerl, E. (1960) z. Lebensmi tt.-Untersuch., ill, 157 4. Allmark, K. G., llannell, W. A. & Grice, H. c. (1957) J 1 Pbarm. Pha:rmacol., i, 622 5• Bir, F. & Griepsntrog, F. (1960) lled. u. Erpllr.. , .! (5), 99 77 QUERCETIN AND QUERCITRON These colours have been classified in categories III~ and crll. Synonyms c.r. Natural Yellow 10; T...C:elb bzw. GrUn 1. Flavone Yellow Code Numbers c.r. (1956) No. 75670 Chemical Name 3,3',4',5,7-Pentahydroxyflavone 3-rhamnoside, and its aglycon, quercitin Empirical Formula Structural Formula ( Querci trin) HO OH Molecular Weight Quercitrin 44a.39 .! Classification of colours from the standpoint of chemical specificatior.s, see section 2.1.3 page 5, .£ Classification of colours in accordance with their toxicological evaluation, see section 2e2.l page 11. Biological Data Biochemical aspects After oral administration of quercetin to rabbits substantial amounts are absorbed, metabolized and excreted in the urine within 24 hours. The following metabolites were found1 3,4""dfhydroxyphenylacetic acid (>25%), 3-hydroxyphenylacetic acid and its glucuronide and 3-methoxy-4-hydroxyphenylacetic acid.1,2 Protocatechuic acid has been found to be a metabolic produce of this colour in rat kidney.3 Twelve hours after oral administration of c14-labelled quercetin, 44% of the radioactivity was still found in the intestinal tract and 15.1% was recovered in respiratory carbon dioXide. Small amo\Dlts were fo\Dld in the blood and kidney and an appreciable amo\Dlt in the lungs and in the wall of the gastro-intestinal tract. No other organs contained detectable amounts of radioactivity. The urine contained about 4% of the administered activity.4 In the urine radioactive phloroglucinolcarboxylic acid, phloroglucinol and protocatechuic acid could be identified in ether extracts of lyophilized stomach and its contents. A fourth radioactive compound not identified and a radioactive "quercetin-like" compo\Dld were also noted. It was not unchanged quercetiD• Incubation of quercetin with a porcine gastric mucin gave the same results., Following intraperitoneal injection of labelled quercetin, then probably vanillic acid was found in the urine. Quercetin may possibly be degraded in the rat by at least 2 metabolic pathways, but it was fo\Dld that protocatechuic acid, phloroglucinolcarboxylic acid and phlor>glucinol were artefacts arising during the chemical extraction procedures.4, Acute toxicity ~o References per kg bodv-weight Babbit intravenous 100 mg* {quercetin) 7 Rabbit intravenous 270 mg* {quercitron) 1 given in 2 doses * No toxic signs were seen. Short-term toxicity BA1· When 20 or 100 mg of commercial quercetin was given by stomach-tube to rats as a suspension, either in a single dose or in 4 daily doses of 5 mg or 25 mg respectively, 50J' of the animals developed cataracts within 10 weeks. No bilateral cataracts were observed. Chromatographically pure quercetin did not cause cataracts under similar conditions.a · Seven groups of 10 weanling rats, 5 of each sex, were placed on the following diets, O, 0.25, 0.5 and 1.0J' of quercetin or quercitrin, for 410 days. No evidence of abno?'ll&l.itiea or injury as judged by growth, food consumption, blood composition, organ weights and histopathological examination could be found.7 79 Long-tenn toxicity No data available Comments on experimental studies reported The production or cataracts in rats with conmercial quercetin is difficult to assess without further evidence on the nature or the responsible impurity and information on its distribution in commercial samples or quercetin and quercitron. It has not been found possible to prepare a specification or identity and purity for this colour. Consequently no toxicological evaluation can be carried out. Further work required See general principles governing the toxicological evaluation, section 2.2, page 8. REFERENCES 1. Murray, C. w., Booth, A.N., De Fds, F. & Jones, F. T. (1954) J. Amer. Phal'II. All·, t.l, J61 2. Booth, A. N., Murray, C. w., Jones, F. T. & De Fds, F. (1956) J. Biol, Chem., m, 251 J. Douglass, c. D. & Hogan, R. (1958) J. biol. Chem.,~' 625 4. Petrakis, P. L., Kallianos, A. G., Wender, s. H. & Shetlar, M. R. (1959) Arch. Biochem., ~' 264 5. Kallianos, A. G., Petrakis, P. L., Shetlar, M. R. & Wender, s. H. (1959) Arch. Biochem., §!, 430 6. Masri, M. s., Booth, A. N. & De Fds, F, (1959) Arch, Biochem., ~' 284 7, Ambrose, A. M., Robbins, D. J, & De Fds, F. (1952) J. Amer. pharm. Ass.,"'' 119 6. Nakagawa, I., Shetlar, M. R, & Wender, s. H. (1961) Proc. Soc. exp, Biol., !Q§, 401 8o QUilfOLINE lELWlf Thie colour hae been olaeeified in categories 1.! and CI!! S;ypon.yu c.I. Food Yellow 13s L.Gelb 3J Jaune de Quinolline1 E.E.c. Serial Ho. B 104. Quinophthalone 22!m Yellow Code 1,-bera c.1. (1956) 10. 47005 c.1. (1924) lo. 801 Sohults (1931) lo. 918 Chemical Bw Dieodiua salt of diaulfonic acid of 2-(2..quinolyl)-l,,..indandione BO!Sa Technical euaplea are autures or mono..,di-, and tri-sulfonic acid. Bapirical Pormula - --- (SOfa)2 I 0 B .! Claeeitication of colours from the standpoint or chemical specifications, ... section 2.1.3 118&9 5. lt Claeeification of ooloure in accordance with their toxicological evaluation, aee section 2.2.1 paae 11. Molecular Weight 477.38 Description Quinoline Yellow is a water-soluble food colour conforming to the following specifications. Purity Tests Dye content: Loss on drying: information required Chlorides and.-sulfates: Water-insoluble matter: not more than O.~ Ether-extractable matter: not more than O.~ Lead (as Pb): not more than 10 mg/kg Arsenic (as As): not more than 3 mg/kg Biological Data Biochemical aspects Ho data available A9ute toxicity .Ll250 Reference mg/kg bodY-weight Rat oral 2000 l Special studies This colour was tested for mutagenic effect in a concentration of 0.5 and 1.0 g/100 ml in cultures of Escherichia coli. No mutagenic effect was found. 2 Short-term toxicity Rat. Groups of 5 male and 5 female rats were fed diets containing o, 0.25, 0.5, l.O, 2.0 and 5.o,g for 90 days. No effe~t on body-weight, food intake, blood cell counts and organ weights were observed. Guinea-pig. In experimznts with guinea-pigs it was found that this colour had no sensitization activity. Cat. Cats received daily doses of 0.1 g/kg of colour for 7 dQYs. No increase in Heinz bodies in the blood of the test animals was noted.5 Long-term toxicity !!!!, Groups of 20 male and 20 female rats or more were ,fed diets containing O ppm and 10 OOO ppm of the colour for 2 years. A similar test group was formed from the first filial generation and was fed the 10 OOO ppm level for a similar period of time. No effect of the diet was noted in the test groups and gross and microscopic examination of the animals disclosed no changes attributable tn +.11e test diet. There was no significant difference in tumour incidence between the groups.5 Twenty male and female rats were given a total of 55 subcutaneous injections of 1 ml of 2% aqueous solution of the colour over a period of seven months,then observed until death. No local tumours developed and total tumour incidence , was less than in control groups given similar injections of glucose or salt solutions.· Comments on experimental studies reported The available data are not fully adequate for evaluation, but a substantial amount of detailed information is available concerning results of long-term tests. Further work reguired Long-term teats in !lnother species. Metabolic studies in animals and man. REFERENCES 1. Lu, F. C. & Lavallee, A. (1964) Canad. pharm. J., Jl, 30 2. Luck, H. & Rickerl, E. (1960) z. Lebensmitt.-Untersuch., 112, 157 3. Hansen, W. H., Wilson, D. C. & Fitzhugh, O. G. (1960) Fed. Proc., Jj (1), 390 4. Bar, F. & Griepentrog, F. (1960) Med. u. Ernahr., ! (5), 99 5. Oettel, H., Frohberg, H., Nothdurft, H. & Wilhelm, G. (1965) Arch. fur. Toxicol. , Z!, 9 8:, SUNSET YELWR FCF This colour has been classified in categories I!; and A'il. SY?IO!IYlllS C.I. Food Yellow 31 FD&C Yellow No. 6; !,..Orange 2; Jaune Soleil; Jaune Orange Si E.E.C.Serial No. E 110. Monoazo Orange Code Numbers c.r. (1956) No. 15985 Chemical Name Disodium salt of 1-(4-sulfophenylazo)-2-naphthol-6-sulfonic acid Empirical Formula Structural Formula HO Molecular Wei€,ht .!!: Classification of colours from t~e st'Uldpoint of ~hemi~al specifl~ations, see section 2.1.3 page 5. ~ Classification of colours in accordance with their t0Ki9ological evaluation, see section 2.2.1 page 11. Description Sunset Yellow FCF is a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A. Purity Tests Dye contenta Not less than 85% by titration with titanous chloride. Weigh 0.600-0. 700 g of Sunset Yellow FCF and proceed as in Appendix A. Use 10 g of trisodium citrate as buffer. Loss on drying at 135° ~ total not more than 15% Chlorides and sulfates calculated as sodium salts 1 Water-insoluble mattert not more than o.2% Ether-extractable mattera not more than 0.2" Lead (as Pb)t not more than 10 mgjkg Arsenic (as As)t not more than 3 mg/kg Subsidiary eyesa not more than 4% Intermediatesa not more than 0.5% Biological Data Biochemical aspects When rabbits were fed 0.5 g/kg boey-weight of the colour the following ·metabolites could be identified in 48 hours urine; Sunsent Yellow (2%), sulfanilic acid (54%), p-acetamido benzenesulfonic acid (2,%), l-amino-2-naphthol-6..sulfonic acid (55% in 24 hours).l Rats were given a single oral dose of 100 mg of the colour per animal. Only o.~ of the dose administered was found in the faeces. After a single oral dose of 50 mg only 3.&J' was absorbed from the gastro-intestinal tract. The metabolites in the urine were predominantly products resulting from the reductive fission of the azo-linkage. The liver enzyme that reduces azo linkages plays little part in the metabolism. Reduction of the colour by the intestinal bacteria is therefore the most probable way to aromatic amines and aminosulfonic acids; these are then partly absorbed by the intestinal tract.2 Rats were injected the colo;..:r intravenously. The bile was collected for 6 hours and analysed. Th~ average recovery of the colour was 22% (20-3~) of the administered quantity., Acute toxicity ~o Reference per kg bod.v-weight Rat oral >2.0 g 4 Special studies The colour was tested for mutagenic effect in a ccncentration of 0.5 g/100 ml in cultures of Escherichia coli. No mutagenic effect was found.5 Short-tenn toxicity H!i• Groups of 10 young rats were given the colour subcutaneously twice daily for 3 days. The rats were killed on the fourth day. The colour was administered in aqueous solution at a level of 250 mg per kg body-weight each injection. No estrogenic activity {uterine weight) was detected.6 Twenty rats were given subcutaneously 1 ml of a 1% solution of the colour twice a week for 7 months. Totally 55 injections were given. Only 1 intraperitoneal tumour was found. (The observation period was not mentioned.)7 The colour was given to a group of 16 rats as a 2% solution in the drinking water for 10 months. The diet of these rats was suboptimal in vitamin B2• In comparison with a group without the colour, it was noticed that the colour accelerated the growth of the young rats and improved the survival rate of these animals. No histopathological changes in the liver were observed.a Guinea-pig. A skin test with the colour showed in patients sensitive to para-phenylenediamine eczematous hypersensitivity produced by cross-sensitization. This reaction is explained by the ease of its transformation into compounds of quinone structure and upon the ability of the quinone compound to couple with certain body constituents.9 In experiments with guinea-pigs it was found that this colour had no sensitization activity.10 Long-term toxicity Mouse. J. group of 30 mice were given o.o~ of the colour in their drinking-water for 52 weeks. The animals were kept for their life-span. The weekly ingestion of the colour was about 17 mg and totally 884 mg/mouse. Nine lymphomas and 1 benign intestinal tumour were found in 7 survivors. In the controls, 5 lymphomas and 1 intestinal tumour were found in 13 survivors. There were 60 mice in the control groups at the beginning of the experiment.11 Two year feeding studies were conducted with this colour using 2 strains of mice; C black and CH at levels of 1 and 2%. One hundred animals of each strain w4te fed the coiour at both dosage levels and 200 animals of each strain were fed a control diet. There was no effect on tumour formation.12 !l!:1• Five male and 5 female rats were given the colour at a level of 4% in the diet for periods up to 18 months. There was some staining of the glandular stomach and small intestine and in some anirials granular deposits were also observed in these organs. No tumours were observed.13 Four groups of 15 male and 15 female rats were given diets with O, 0.03, 0.3, and l.~ of the colour for 64 weeks. The mortality of the rats was as in the control group. No inflUP.nce on food intake, growth, organ-weights, histopathology and blood picture was found. No significant difference in tumour incidence was found.14 Groups of 24 litter-mated Osborne-Mendel rats, evenly divided by sex, were fed the colour at O, 0.5, 1.0, 2.0 and 5.0J'. There was a statistically in significant increase in the number of mammary tumours. The number of tumours that were found in the different groups were respectively 2, l, 6, 3 and 6. 86 Additional feeding studies were conducted with Osborne-Mendel and Sprague-Dawley rats; 100 animals of each strain were fed at 1.0 and 2.0J', and 200 of each strain were placed on the control level. Gross and microscopic pathology showed no effect on tumour formation.12 ;Qg&. Feeding studies with dogs were conducted; 4 beagles were fed the colour at each level of 1.0 and 5.0J'. Two of the 4 animals at 5.0J' and 1 animal at 1.0J' lost weight progressively and had to be sacrificed after 2-~ months. In general, 5.0J' in the diet of dogs was moderately, and l.OJ' slightly toxic. Weight loss and diarrhoea were the chief clinical effects. Gross and microscopic pathological changes were present but were not characteristic. Five female beagle dogs were fed the colour at 2.0J' in the diet for 7 years. No histopathology was reported.12 Comments on experimental studies reported Several long-term studies have been carried out in rats. Long-term studies have also been carried out on mice and dogs. The biochemical studies indicate that in the rat this colour is reduced at the azo linkage by bacteria present in the intestine and that some of the breakdown products are absorbed and then excreted in the urine. In rats the no-effect level was found to be r:11, and in the long-term studies in dogs the no-effect level was 2.0J' in the diet. The long-term studies in dogs provide a basis for evaluation. Evaluation Level causing no significant toxicological effect in the dog Two per cent. in the diet, equivalent to 500 mg/kg body-weight. Bstimate of acceptable daily intake for man 0-5.0 mg/kg body-weight. Further work considered desirable Metabolic studies in several animal species. Reproduction studies in several animal species. Research on the combined effect with Amaranth and Tartrazine would be valuable. 87 REFERENCES 1. Daniel, J. w. (1962) Toxicol. appl. Pharmacol., ! (5.), 572 2. Radomski, J. L. & Mellinger, T. J. (1962) J. Pharmacol., .122, 259 3. Ryan, A. J. & Wright, S. E. ( 1961) J. Pharm. Pharmacol., ll, 492 Lu, F. c. & Lavallee, A. (1964) Canad. pharm. J., !ll.. (12), 30 LUck, H. & Rickerl, E. (1960) z. Lebensmitt.-Untersuch., l,!g_, 157 6. Graham, R. c. B. & Allmark, M. G. (1959) Toxicol. apol. Pharmacol., .!, 144 Deutsche Forschungsgemeinschaft, Bad Godesberg, Federal Republic of Germany, Farbstoff Kommission (1957) Mitteilung 6 8. Manchon, Ph. & Lowy, R. (1964) Food cosmet. Toxicol., £ (4), 453 Baer, R. L., Leider, M. & Mayer, R. L. (1948) Proc. Soc. exp. Biol. (N.Y.), fil., 489 10. Bir, F. & Griepentrog, F. (1960) Med. u. Ernlihr., .!, 99 11. Bonser, G. M., Clayson, D. B. & Jull, J. w. (1956) Brit. J. Cancer, 10, 653 12. United States Food and Drug Administration, Unpublished report submitted to WHO, April 1964 13. Willheim, R. & Ivy, A. c. (1953) Gastroenterology, £l, l 14. Mannell, W. A., Grice, H. C., Lu, F. C. & Allmark, M. G. (1958) J. Pharm. Pharmacol., 10, 625 88 TARTR1ZINE a b This colour has been classified in categories I- and 1.- S;ynon.yms c.r. Food Yellow 4; FD&C Yellow No. 5; L-Gelb 2f E.E.c. Serial No. E 102. Monoazo Yellow Code NU11bera c.r. (1956) No. 19140 c.r. (1924) No. 640 Schultz (1931) No. 737 Chemical Name Trisodium salt of 5-hydroxy-i..p,,.aulfotbenyl-ie(P-sulfophenylazo) pyrazo]... 3-carboxylic acid Empirical Formula Structural Formula IoDf -0-·-·- - -COONa 11 11 HO/~ Molecular Weight 534.37 ~ Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5• .k Classifio!f..tion of colours in accordance with their toxicological evaluation see section 2.2.1 page 11. 89 Description Tartrazine is a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A. Purit;r Tests Dye content, Not less than 8~ by titration with titanous chloride. Weigh 0.750..0.800 g of Tartrazine and proceed as in Appendix A. Use 15 g of sodium hydrogen tartrate as buffer. Add 1.0 ml of a 1" aqueous solution of Light Green SF Yellowish as indicator near the end of titration. Carry out a blank determination omitting only the Tartrazine sample. Loss on drying at 135° ~ total not more than l~ Chlorides and sulfates calculated as sodi\UD salts j Water-insoluble mattert not more than o.2% Ether-extractable mattert not more than o.2% Lead {as Pb) a not more than 10 mg/kg Arsenic (as As)t not more than 3 mg/kg Subsidiary ~est not more than ~ Intermediatesa not more than o.~ Biological Data Biochemical aspects Rats were injected the colour intravenously. The bile was collected for 6 hours and analysed. The recovery of the colour was an average of~ (0..2%) of the administered quantity.l The low biliary excretion a_ppears from these results to be associated with a free carboxyl group in the 3-position of the pyrazolone ring. Metabolites that might be produced by the reductive fission, have not so far been identified in the bile. A conjugated form of tartrazine appears rapidly in the urine following intra.peritoneal injection but also here no reductive fission products are detected. These results ~how that this colour is not a true azo compound but a substituted phercy-lhydrazone. Rats were given by intra.peritoneal injection 2.4 mg labelled tartrazine per kg bo~-weight and by stomach tube 5 mg/animal. A rabbit was given intraperitoneally 2.4 mg/kg bo~-weight. Two rabbits received respectively 1 g orally and l g intraperi toneally. Four men took approximately 100 mg of the colour orally in gelatine capsules. In all, species urine was collected for 48 hours and examined for tartrazine and metabolites. The urine of rate given the colour intraperitoneally contained only unchanged colour. Conjugated colour and amines derived from the reduction of the colour in the urine were not present. After oral doses sulfanilic acid {presumably the N-acetate) was found in the urine. This was the case in rats, rabbits and men. The 90 results show that tartrazine can be reduced in vivo, when given orally and not when given intraperitoneally. The reduction is therefore carried out by the gastro-intestinal flora. This finding seemed to indicate the possible absence of a true azo-linkage and it was demonstrated by physical methods that tartrasine was the keto..hydrazone tautomer. Cleavage of this tautomer by the flora of the alimentary canal of animals appears to have its counterpart in man because experiments revealed no unchanged colour in the urine.3,4 When rabbits were fed 0.5 g/kg body-weight of the colour the following metabolites could be identified in 48 hours'urinea tartrazine (~), sulfanilic acid (7~), p..acetamidobenzenesulfonic acid (22").5 A concentration of 200 to 400 mg/litre of this· colour was able to inhibit the action of pepsin, but not that of lipase. Acute toxl.ci tz ~o References ~ J:!!r y bo!t[-weist Mouse oral 12.75 g 8 ( vehicle ~ gma arab.) Rat intraperitoneal >2.0 g 7 intravenous >l.O g 7 SJ:!!cial studies The colour was tested for mutagenic effect in a concentration of 0.5 g/100 ml in cultures of Eacherichia coli. No mutagenic effect was found.9 Short-term toxicity Bll• A group of 5 young rats was given the colour subcutaneously twice daily for 3 days. The rats were killed on the fourth day. The colour was administered in aqueous solution at a level of 250 mg per kg body-weipt each injection. No estrogenic activity (uterine weight) was detected. No other abnormalities were found.10 The colour was fed to 85 rats at a level of o.~ in the diet. The animals received 10-15 mg per day. No tumours were observed. (The duration of the experiment was not mentioned.) 1 Guinea-pig. In experimeyrs with guinea-pigs it was found that this colour had no sensitization activity. Cat. A negative test for Heinz bodies was obtained on 2 cats, the one fed o~.l gfkg for 8 days and the other 0.005 g/kg for 35 days.7 ~· Twelve 6-month old beagles, divided into 3 groups of 2 males and 2 females were given O, 1.0 and 2.0% of the colour in their diet for 2 years. No clinical symptoms of toxicity or haematologic abnormalities were noted during the study. No gross lesions were noted at autopsy. The histopathology was limited to the usual incidental lesions which, with the possible exception of pyloric gastritis in 1 dog with 2" of the colour.12 91 !i!roB-term toxicitz Mouse. A total of 117 male and female mice produced by mixed breeding from 5 difTereiit strains was given a diet containing 1 mg per animal per day of the colour. Mice at the age of 50 to 100 days were used. A number of the animals was sacrificed after an observation period of 500 days and the survivors of the rest after 700 days. A total of 168 mice was used as negative control. Positive control groups which were given o-aminoazotoluene and dimethylaminoazobenzene, were also included. In these groups the formation of liver tumours was noted after approximately 200 days. The incidence of tumours in mice reitiving the colour was not significantly greater than in the negative controls.' Jl&1• The colour was fed to 5 male and 5 female rats at a level of Ill, in the diet for appl'IOXimately 18 months. Gross staining of the forestomach and some staining of tlie glandular stomach, small intestine and colon was observed. Granular deposits were also noted in the stomach, small intestine and colon. Bo tumours were observed.14 This colour was injected subcutaneously into 18 rats of both sexes for 94-99 weeks. In general, 1 ml of a 2" or,!, solution was injected weekly. lfo tumoure were observed, either at the site of injection or in other parts of the boq.15 Ten rats nre fed a diet containing 0.2" of the colour for 417 days. The daily intake was 0.1 g/kg boq-weight and the total intake 11 g/animal. The animals nre kept under observation for periods up to 922 days. lfo tumours were observed. Two animals died between time.7 Eleven rats were subjected to subcutaneous injections of 0.5 ml twice weekly for a year of a 2" solution of the colour until a total of 1.0 S/'animal had been adllinistered. The animals were kept under observation for periQds up to 856 days. No tumours were noted. Four animals died between time.1 Four groups of 15 male and 15 female rats were given diets with O, 0.03, 0.3 and l.~ of the colour for 64 weeke. The mortality of the rats was as in the control group. Bo influence on food intake, growth, organ weights, histopathology and blood pic!ure was found. Bo significant difference in tumour incidence was found.1 Five groups of 24 rats, evenly divided by sex, were fed O, 0.5, 1.0, 2.0 and 5.0J' of the colour for 2 years. Growth effect was negligible and there was no effect on survival, haematology or organ weight. At the 2 and ~ dosage level diarrhoea was noticed, but not at the~ level. The incidence of tumours and of the common incidental diseases was unaffected. lfo org1,n pathology attributable to treatment was noted except at the highest dosage level, where the feeding of the colour mq have influenced \he deposition of small amounts of gritt7 material in the renal pelvis of some of the surviving male rats.12 Cownts on experimental studies reported Several comprehensive long-term studies have been carried out in the rat. Long-term studies have also been carried out on mice and dogs. The biochemical studies in rats and rabbits are fairly extensive. In rats the no-effect level was found to be l.~ in the diet, in dogs the no-effect level was at least 2" in the diet. The long-term studies in rats provide a basis for evaluation. Evaluation Level causing no significant toxicological effect in the rat l.~ in the diet, equivalent to 750 "'1/k8 bo~-weight. Estimate of acceptable daily intake for 111&11 0-7.5 mg/kg bo~-weight. Further work considered desirable Metabolic studies in aore animal species. Reproduction studies in several animal species. Research on the combined effect with Amaranth and Sunset Yellow FCP would be valuable. 1. )b'an, A. J. a. Wright, s. E. (1961) J. Pharm. Pbal'lllacol., .ll, 492 Byan, A. J. & Wright, S. E. (1962) ~. J.22, 1009 3. Wright, s. E. (1963) .lust. J'ed. Manuf'., ~ (7), 32 (ref. Bibra, 19631 (e), 514) Jones, R., Ryan, A. J. a. Wright, s. E. (1964) Food cosmet, Toxicol., l (4), 447 Daniel, J. w. (1962) Toxicol. appl. Pharmacol., i, 572 Diemair, w. & Hausaer, D. (1951) z, Lebensmitt,-Unterauch., 3£, 165 Deutsche Forachungagemeinachaftt Bad Godesberg, Federal Republic of Gf·rinany, Farbstoff KoDlllission (1957} llitteilung 6 e. lational Institute of Hygienic Sciences, Department of Pha.rmacoloa, .Japan, Unpublished data subai tted to WHO, September 1964 LUck, H. a. Rickerl, E. (1960) z. Lebenami tt.-Unterauch., .ill, 157 10. Graham, R. c. B. a. Allmark, JI. G. (1959) Toxicol. appl. Pharmacol., !, 144 11. Bir, F. & Griepentrog, F. (1960) lled. u. Ernlhr., ! (5), 99 12. Davies, K. J., Fitzhugh, o. G. & Nelson, A. A. (1964) Toxicol. appl. Pharmacol., .2, 621 13. Wateraan, N. a. L:l:gnac, G. o. E. (1958) Acta phYaiol. pharmacol. neerl., 1, 35 14. Willheim, R. & Iv;y, A. c. (1953) Gaatroenterology, ~. 1 15. Belson, A. A. & Hagan, E. C. (1953) Fed. Proc., _g, 397 16. Mannell, W. A., Grice, H. C., Lu, F. C. & Allmark, M. G. (1958) J. Pharm. Pharmacol., 12., 625 9, TITANHII DIOXIIE This colour has been classified in categories 1!: and cil Synon:yms c.I. Pigment White 6; Bio:xyde de titaneJ LB-Pigment No. 31 E.E.C. Serial No. E 171. Inorganic colouring matter Colour White Code Numbers C.I. (1956) No. 77891 C.I. (1924) No. 1264 Schultz (1931) No. 1418 Chemical lf!!m Titanium dioxide Chemical Formula Molecular Weight 79.90 Definition Titanium dioxide contains not less than 9~ of Ti0 and conforms to the following specifications. 2 Description Titanium dioxide is a white, tasteless, odourless, infusible powderJ insoluble in water, and dilute acids • .! Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5 • .h Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. Identification Tests J.. Peroxide reaction• Fuse titanium dioxide with potassium hydrogen sulfate and dissolve in water. J. yellow colour is produced upon addition of hydrogen peroxide. B. SpectrographYa The two most characteristic lines for titanium are at 4981.733 and 3653.496 l. c. Melting ranpa Heat-resistant to at least 500°. Puri t;y Tests Acid-soluble substances• Suspend 5 g of titanium dioxide in 100 ml of 0.5 N hydrochloric acid and heat on a water bath with occasional stirring for 30 minutes. Filter through a Gooch crucible in which the mat has been built up in 3 layers using first medium coarse asbestos; second, pulped filter paper; sn$1 finally, fine asbestos. The asbestos should previously have been acid washed. Wash with 3 10 ml portions of 0.5 N hydrochloric acid. Evaporate the filtrate to dryness in a platinum dish and ignite at a dull red heat to constant weight. The weight of the residue should not exceed 0.0175 g. Antimon:n not more thsn 100 mg/kg. (a) Digest 0.10 g of titanium dioxide with 30 ml of sulfuric acid containing 3 g of anhydrous sodium sulfate \Dltil it has almost completely dissolved; cool, dilute to 100 ml with water, and cool; transfer 50 ml to a wide-mouthed bottle of 200 ml capacity, fitted with a rubber bung and a delivery tube containing a rolled cylinder of lead paper, and 50 ml of water and 10 g ot granulated zinc; quickly replace the b\Dlg snd pass the liberated gas for 16 hours by mesns of a capillary jet 1 cm from the base of the glass, into a 50 ml Nessler glass containing an absorption solution prepared by adding to 25 ml of silver sulfate solution (0.2" w/v, in water) 2 ml of starch mucilage T.S., followed by 0.5 ml of a 0.01% w/v solution of tartaric acid in water, 0.25 ml of N sulfuric acid and 1 ml of methyltrihydroxyfluorone solution (O.Ol"w/v ot methyltrihydroxyf'luorone in 95'% alcohol containing 0.2" v/v of sulfuric acid). Any colour which develops is not deeper than that produced by 10 ml of antimony, standard T.S. when similarly treated, commencing with the digestion with the sulfuric acid and anhydrous sodium sulfate. (b) J.ltemate methoda Melt a mixture of 100 mg titanic dioxide, 5 g potassium hydrogensulfate JKHS04) snd 10 mg glucose in a porcelain crucible until the mixture is limpi4. Allow to cool partly, add 10 ml sulfuric acid (96-.~) to the partly solidified mass and warm the mixture \Dltil: the mass is dissolved. Allow to cool. Place in a 100 ml calibrated flask 50 to 75 ml 30J' hydrochloric acid and add the contents of the crucible by small portions into the flask, continually mixing to prevent local overheating which would cause turbidity. Cool and fill up to the mark with 30% hydrochloric acid snd mix t• sample solution). Place in a separatory tunnel• 7 ml ~,b)'dTOchloric acid, 10 ml of sample solution snd about 2 mg solid eerie sulfate LCe(S04)2.4Bi.Q7 and mix. After 5 minutes add 15 ml diisopropyl ether by mesns of a burette, shake slowly, close the tunnel and shake for halt a minute. Add 7 ml of water, mix snd cool the contents of the f\Dlnel to room temperature. After 10 minutes shake for halt a minute, allow the 1.qers to separate completely and discard the aqueous phase. 95 Add 5 ml Rhodamine-B-solution (0.01% in 0.5 N hydrochloric acid), shake and allow the layers to separate. If the ether layer is violet, discard the aqueous phase and transfer the ether layer into a centrifuge tube with a glass stopper and centrifuee. Determine light absorption in a 1 cm cell at 555 m The absorption should not be brighter than the absorption of the standard. Prepare a standard colour by placing in a separation funnela 7 ml 30% hydrochloric acid, 10 ml of dilute antimony standard solution ('. 0.01 mg Sb), and add to that the mixture as described above. The antimony standard solution utilized here is made~~ dissolving 274 mg of potassium antimonyl tartrate ~ (SbO) C4H4o6.l/2H22{ to 100 ml in 30% hydrochloric acid, and diluting this solution 1 1 1000 with 30% hydrochloric acid immediately before use fj. ml• lpg Sb (III..l7. Zinc I not more than 50 mg/kg. Heat 2.0 g of titanium dioxide with 10 ml of sulfuric acid PbT and 5 ml of nitric acid Pl:11' in a long-necked flask; continue the heating and add, dropwise, nitric acid PbT until the solution is clear and pale yellow in colour; cool, add 5 ml of water, evaporate to low bulk, cool, add 5 ml of water, again evaporate to low bulk, cool, and dilute to 100 ml with water; add 10 ml of citric acid solution (10% w/v of citric acid PbT in water), 5 ml of sulfurous acid PbT and 0.2 ml of thymol blue solution T.S., and make just alkaline with ammonia, PbT; extract with successive portions, each of 5 ml of diphenylthiocarbazone PbT until the extract is not britilt red in colour; wash the combined extracts with 5 ml of water, reject the washings, and extract with 2 portions, each of 5 ml of 0.1 N hydrochloric acid, dilute the combined extracts to 100 ml with water. (a) To 25 ml of the acid solution add O.l ml of thymol blue T.S. and make just alkaline with ammonia, dilute PbT; add 10 ml of borax buffer PbT and titrate with diphenylthiocarbazone solution PbT by adding the reagent in small portions, shaking vigorously after each addition, allowing to separate, and wi thdrawin;; the lower layer; the end point is reached when the chloroformic layer remains purple. (b) To 50 ml of the acid solution add 0.1 ml of thymol blue T.S. and make just alkaline with ammonia, dilute PbT; add 5 ml of potassium cyanide solution PbT and 10 ml of borax buffer PbT and titrate with diphenylcarbazone solution PbT as described in the determination of zinc. (c) Prepare a control by repeating the above operation replacing the sample by 20 ml of zinc sulfate, standard T.S. and 4 ml lead, standard T.s., using a total volume of nitric acid PbT equal to that required for the digestion of the sample. (d) From the amount of reagent required for the determination of zinc (paragraph a) subtract half the amount used in the determination of lead (paragraph b); the amount of diphenylthiocarbazone solution PbT is not more than that required when 25 ml of the acid solution derived from the control is similarly treated, and half the amount used in the determination of lead in the control has been deducted. At the same time, if the amount of diphenylthiocarbazone solution PbT used in the determination of lead (paragraph b) is not more than that required when 50 ml of the control is similarly treated, the analyzed sample meets the tolerance for lead. 96 Soluble barium compoundsa not more than 5 mgfkg. Heat the residue obtained in the "acid-soluble substances" test in a bath of boiling water with 20 ml slightly acidified water (HCl) and filter. To the clear filtrate add 1 ml of diluted sulfuric acid. No turbidity or precipitate is produced after allowing to stand for 30 minutes. ~t not more than 20 mg/kg. Place 2 g of titanium dioxide in a platinum dish and add 5 ml of 70-7':!fo perchloric acid. Evaporate on a hot plate with 2 successive 5-ml portions of hydrofluoric acid until white fumes appear. Add 5 ml of hydrofluoric acid and heat until solution is effected. Cool, dilute with water to 100 ml and mix thoroughly. Prepare a standard solution by adding 4 ml of standard lead T.s. to 0.5 ml of 70-7':!fo perchloric acid and 1.5 ml of hydrofluoric acid. Evaporate in a platinum dish until the appearance of white fumes, and dilute to 10 ml. Carry 10 ml of the sample solution and the entire standard solution through the lead limit test {Appendix A, 10), using 6 ml of ammonium citrate T.S., 2 ml of potassium cyanide PbT, and 2 ml of hydroxylamine hydrochloride solution. The colour of the final carbon tetrachloride extract of the sample should have no more red colour than the standard solution corresponding to not more than 20 me;/kg. Arsenica not more than 5 mg/kg. Place 1 g of titanium dioxide in a platinum dish and heat with a mixture of 5 ml of sulfuric acid and 5 ml of nitric acid until white fumes are given off. Heat until the appearance of white fumes on a hot plate with 3 successive 5-ml portions of hydrofluoric acid. Cool and dilute cautiously with distilled water to 25 ml. A 5-ml portion of this solution meets the requirements of the test for arsenic (Appendix A, 11). Assay Fuse 0.300 g of titanium dioxide with about 3 g of potassium hydrogen sulfate in a platinum dish, cool and dissolve in 150 ml of dilute sulfuric acid T.S. Activate a suitable zinc amalgam T.S. "reductor'' by passing through the column 100 ml of dilute sulfuric acid T.S. followed by 100 ml of water. Pass through the column 200 ml of N sulfuric acid, followed by 100 ml of water, and collect in a receiver containing 50 ml of a 15% solution of ferric ammonium sulfate in a 25% solution of dilute sulfuric acid T.S.; titrate the mixture with O.l N potassium permanganate. Reactivate the column with 100 ml of N sulfuric acid. Pass through the column the titanium solution, followed by 100 ml of N sulfuric acid and 100 ml of water, collect in a receiver containing 50 ml of the acidified solution of ferric ammonium sulfate, and titrate the mixture with 0.1 N potassium permanganate. The difference between the titrations represents the amount of potassium permanganate required by the titanium dioxide. Each ml of 0.1 N potassium permanganate is equivalent to 0.00799 g of titanium dioxide. For this method of assay, the "reductor" should be frequently recharged with freshly amalgamated zinc. Biological Data Biochemical aspects From an experiment with rats given for 7 days a diet containing 2000, 10 OOO and 20 OOO ppn titanium dioxide, it was concluded that no colour was absorbed from the gastro-intestinal tract.l 97 Rats were given 0.25'% of Titanium dioxide in the diet for a week. Ninety two per cent. of the administered colour was excreted with the faeces. It was concluded that no absorption took place.2 Acute toxicity ~o References per kg body-weight Rat oral >12.0 g (as barium titanate, bismuth titanate, calcium titanate and lead titanate) Rat intraperitoneal 2.0-5.3 g (as barium titanate, bismuth titanate, calcium titanate and lead titanate) Short-term toxicity Rat. Rats were given 0.66 g of the colour per kg body-weight for 15 days. No titanium was found in blood, liver, kidney and urine (sensitivity of the method 10 µg).l Technical grade Titanium dioxide was fed to 2 guinea-pigs, 2 rabbits, 2 cats and 1 dog for 390 days. The dog received 9 g/day, rabbits and cats 3 g/day, guinea-pigs 0.6 g/day. Two additional cats received 3 g of the colour daily for 175 and 3oO days respectively. No toxic effect was seen during the experiment, and histopathological examination revealed no abnormality. There was less than 5 µg of titanium in the liver bile, heart, spleen and skeletal muscle.4 1?2g. Three groups of 2 dogs were given orally at the beginning of the experiment 0.05, O.l and 0.15 g of titaniwn dioxide. Each 5.days the dose was increased with the same quantity. One dog of each group for 1 month, the other dog for 2 months. No toxic effects were seen.5 Three dogs received weekly subcutaneous injections of a suspension of the colour in oil; an initial dose of 500 mg which was progressivel,Y raised to 3 g over 7 weeks. A fourth dog received an initial dose of 250 mg/kg rising to 2 g/kg body-weight. Three of the dogs survived without adverse effect. The fourth died of natural causes.5 Comments on experimental studies reported Toxicological and other studies employing soluble compounds of titanium (ionic titanium) are not included here. It is useful to note, however, that although there was absorption of titanium ion into the body, no toxic effect was observed. When titanium dioxide was administered in the diet no significant tissue storage took place. The long-term studies and other investigations are inadequate to permit evaluation of an acceptable daily intake. Further work required See general principles governing the toxicological evaluation, section 2.2 page 8. 98 REFERENCES 1. Fournier, P. (1950) C.R. Acad. Sci. (Paris), Lil, 1343 2. Lloyd, L. E., Rutherford, B. E. & Crompton, E.W. (1955) J. Nutr., ~' 265 3. Brown, J. R. & Mastromatteo, E. (1962) Industr, Med. Surg., l!, 302 4. Lehman, K. B. & Herget. L. (1927) Chem. Ztg., .2!, 793 5. Vernetti-Blina, L. (1928) Rif. med., ~. 1516 99 WOOL GREEN BS a b This colour has been classified in categories I- and CI- S;ynon.yms c.1. Food Green 4J Vert acide brillant ES; Green S; Wool Green B. £.!!!!. Triarylmethane Green Code Numbers c.1. (1956) No. 44090 c.1. (1924) No. 737 Schultz (1931) No. 836 Chemical Name Monosodium salt of 4,4-bis(dimethylamino)-diphenylmethylene- ,[2'-naphthol,,,3, 6-disulfonic aci~ Empirical Formula Structural Formula Molecular Weight 576.63 .! Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5• .R Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11~ 100 Description Wool Green BS ie a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A, 1 Purity Tests Dye contents Not less than 82" by titration with ti tanous chloride. Weigh o.600-0. 700 g of Wool Green BS and proceed as in Appendix A, a. Use 15 g of sodium hydrogen tartrate as buffer. Loss on drying at 135° l total not more than 19" Chlorides and sulfates calculated as sodium salts 1 Water-insoluble matters not more than 0.2" Ether-extractable matters not more than 0.2" Lead ( as Pb) 1 not more than 10 mg/q Arsenic (as As)s not more than 3 mg/kg Chromium (as Cr) s not more than 20 mg/kg Subsidiary dyess not more than 2.°" Intermediatess not more than 0.5% Biological Data Biochemical aspects No data available. Acute toxicity ~o Reference per ls6 bod.v-weight Rat oral >2.0 g 1 Short-term toxicity ~· Twenty rats were given 10 ml of milk daily containing 0.15 mg of the colour/litre for 3 months. Another 20 rats were given 10 ml of milk containing 30 mg of the colour/litre for 5 weeks. No abnormalities were observed in food intake and growth. No pathological changes were found. The gastric and intestinal mucosa of the animals on the highest dose level showed a green colour. No colour was found in the muscles, liver and kidneys.2 Pig and calf. Feeding pigs and calves with milk containing 25 mg of the colour per litre, no absorption of the colour was found. .lll was excreted in the faeces within 5 days and 1-2 weeks respective1y.2 101 Lope;-tera toxioitJ bi• The colour was fed at a level of 2" in the diet pt 20 male and 20 female Wistar rats for 2 years. Twenty-four of the animals so treated were aljjve at the end of this time. No tumo\ll'S were observed and deaths occurring the second ~ar were attributable to l'Wl8 infection which occurs also in control animals. Two samples of the colour were used containing respectively 0.02" of ether extractable material and o.'"' ot ether extractable material. Ten doses of 20 mg per rat (2" solution) were followed by 50 doses of 30 mg per rat (&% solution). The maximum total dose in each case was 1.7 gm per rat. Twenty four male and 24 female Wistar rats were tested with each sample making a total of 96 animals. In 5 rats local sarcomata developed at the site of injection. Fifty-four were still living at 2 years. Five of the females developed lll8llllllarY and 2 uterine tumours. No subcutaneous sarcomata were seen in control rats (24 male and 24 female rats were dosed with distilled water in parallel as controls). Twenty-six out of 48 control rats were still alive at the end of 2 years. In the female controls, 1 mammary, 1 ovarian and 4 uterine tumours were found.3 Forty rats were given a diet containing 2" of the colour for 730 days. The total intake was 146 g/animal. Sixteen animals died within 730 d~s. No tumours were found.3 Six male and 6 female Slonaker rats were also given the same doses as above and controls injected with distilled water were included as before. Of the 12 rats dosed all were alive at 500 d~, 9 at 600 days and 1 at 700 days. No tumours developed at the injection site in any of these rats. Three mammary tumours were found in the tested group. One female control rat was found to have a 11181D1118,ry tU1Dour.} A group of 20 yo'Wl8 rats, 10 males and 10 females were given weekly subcutaneous injections of 0.5 ml of a'"' solution of the colour in isotonic saline. Each rat received 20 mg of the colour per week. Another group was given isotonic saline as control. Totally 45 injections, i.e. 900 mg of the colour, were given. The experiment was teminated after 71 weeks. The mortality was as in the control group. No tumours were found.4 Thirty-five Wistar rats, 15 males and 20 females, age 2 months, were given the colour in a dose level of 10 OOO ppm during the first 2 months. After this period the dose level was increased to 20 OOO ppm and given to the animals for their life-span. Fifteen animals (5 male and 10 female) were used as control. The average life-span of the test animals was 21 months and for the control animals 24 months. The last animal died in both groups at an age of 31 months. The colour was found to have no effect on behaviour of the ani,mals, growth, reproduction and the offspring. No histopathological abnormalities were found except a benign tumour in the small intestine of a female animal in the test group.5 Sixty rats, half male and ha!f female, age 2 months, were given a diet containing 20 OOO ppm for their life-span. Thirty animals were used as controls. The average life-span of the test animals was 30 months, the control animals 24 months. The last animal of the 2 groups died after 42 and 31 months respectively. No influence of the colour on growth, blood picture, reproduction and the offspring was found. One female animal in the test group had a benign tumour in the small intestine and in 2 female control animals, a benign mammary tumour was found.5 102 Fifty Wistar rats, half male and half female, age 2 months, were given 20 OOO ppm of the colour for their life-span. These animals received a subcutaneous injection of l ml of a 1'% aqueous solution each week. Twenty-five rats, 13 male and 12 female, were used as controls. These animals received subcutaneously 1 ml of distilled water weekly. The average life-span of the treated and control animals was 29 months and the last animal died after 36 months in both groups. No influence of the colour on growth, blood picture, reproduction and on the offspring was noticed. In 3 female test animals and in l female control animal a subcutaneous tumour was fo\Uld.5 One hundred Wistar rats, half male and half female, age 2 months, were given subcutaneous injections of 1 ml of a YI, colour solution in saline {each injection contained about 30 mg) twice weekly for 30 weeks. The total amo\Ult given was about 1.6 g. Fifty control animals, half male and half female were given subcutaneous injections of 1 ml saline in the same way. The animals were observed for their life-span (about 2 years). In the control animals, no tumours were found on the site of injection while in 3 test animals, 2 male and~ female, sarcomas were induced.5 Comments on experimental studies reported Long-term experiments have been carried out only in 1 species, namely the rat. Information on the metabolism of the colour is lacking. The data are insufficient to permit toxicological evaluation of this colour. Further work required See general principles governing the toxicological evaluation, section 2.2 page e. 1. Lu, F. c. & Lavallee, A. (1964) Canad. pharm. J., j]_, 30 2. Dalgaard-Mikkelsen, s. W. & Rasmussen, F. (1962) 16th International Dairy Congress, 465 3. Imperial Chemical Industries, Unpublished report submitted to WHO, 4 December 1964 4. Mannell, w. A. & Grice, H. C. (1964) J. Pharm. Pharmacol., 1:.2, 56 5. Truhaut, R., Unpublished report dated 7 December 1964, submitted to WHO 10:, 6. SPECIFICATIONS FOR COLOURS IN TOXICOLOGICAL CATEGORIES CII, CIII AND 'rf! WHICH ARE USED IN SOME COUNTRIES Page Page Acid Fuchsin FB...... 104 Naphthol Yellow S •• 126 Azo Rubine ...... 105 Orange G . 130 Benzyl Violet 4B 107 Orange GGN 133 Black 7964 ...... 109 Ponceau 2R 135 Blue VRS . . 110 Ponceau 6R 136 Brilliant Black BN Specially Pure 112 Red lOB , , 140 Brown FK ...... 114 Red 2G . 143 Chocolate Brown HT 115 Rhodamine B •• 146 Chrysoine. 117 Scarlet GN Specially Pure 146 F.osine . 119 Sudan G , •• , 150 Fast Red E . . . . . 120 Sudan Red G • , , 151 Fast Yellow AB 122 Violet 5 BN • , 152 Light Green SF Yellowish 125 Yellow 27 175N Specially Pure. 153 Methyl Violet. , • , , • 127 ~ See section 2,2,1 page 11 104 ACID FUCHSIHE FB a b This colour baa been claaaified in categories III- and CII- Synony11a Acid Magenta; Acid Magenta II; Fuchaine acide; C.I. Acid Violet 19. Triaryblethane Red Code Naber c.1. (19$6) No. 1,268$ C.I. (1924) No. 692 Schultz (1931) No. 800 Chemical Name A lld.xture of the sodiUIII or ammonium salts of the disulfonic and trisulfonic acids of pararonsaniline and rosaniline. For example, di.sodium salt of 2-amino-.S - (4-amino-3-sulfophenyl) • «S - (4-:llld.no-3-sulfo-2,S-cyclohexadien-1-ylidene) -3,S-,o'lenesulfonic acid Empirical Foraula For eXS11ple C N Na o s 2Ji-l.7 3 2 9 3 Structural Fol'IIIUla ( For example) Molecular Waight ! Classification of colours from the standpoint of chemical specifications, see section 2.1.3 pages. ~ Classification of colours in accordance with their toxicological evaluation see section 2.2.1 page n. 10, AZO RUBINE a b This colour has been classified in categories I- and CII- Synonyms C,I, Food Red 3J E,E,C, Serial No, E 122; Carmoisine Monoazo Red Code Nmbers C.I. (1956) No. 1-\720 C,I, (1924) No, 179 Schultz (1931) No. 208 Chemical Name Disodium salt of 2-(4-sulto-l-naphthylazo)-l-naphthol-4-sultonic acid Empirical Formula Struct\D'al Fon1ula OH 1ao s 3 Molecular Weigh_l 502,44 ! Classification of colours from the standpoint of chemical specifications, see section 2.1 • .:, page 5, ~ Classification of colours in accordance with their toXicological evaluation, see section 2,2,l page 11, 1o6 Description Azo Rubine is a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A. Purity Tests Dye content: Not less than 85% by titration with titanous chloride. Weigh 0.750-0.850 g of Azo Rubine and proceed as in App, A. Use 15 g of sodiwn hydZ"Ogen tartrate as buffer. 1 ml of 0.1 N titanous chloride is equivalent to 0.01256 g of C20Hl2N20~2Na2. Loss on drying at 135° total not more than Chlorides and sulfates calculated as sodium salts 15% Water-insoluble matter: not more than 0,2% Ether-extractable matter: not more than 0.2% Lead l as Pb) t not more than 10 mg/kg Arsenic (as As): not more than 3 mg/kg Subsidiary dyes: not more than 2% Intermediates: not more than 0,5% 107 BENZYL VIOIE'l' This colour has been classified in categories 1!. and CIII.!?,S Synonyms c.I. Food Violet 2; FD & C Violet No. l; Violet Acide 5B; Benzyl Violet 4B. ~ Triarylmethane ~ Violet Code Numbers c.I. (1956) No. 42640 c.r. (1924) No. 697 Schultz (1931) No. 805 Chemical Name !l.onosodi um salt of 4-/L4-.!!i-ethyl-p-sulfobenzylamino )-pheny1J-.l4-(!!-ethyl.. p-sulfoniumbenzylamino)-pheny~- methylene} - (!,!-dimethyl~,5-cyclohexadienimine) Empirical Formula Structural Formula + Na .! Classification of colours fron the standpoint of chemical specifications, see section 2.1.3 page 5 • .!? Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 paee 11. g Colours producing more than 10-15'}~ of local sarcomas at the site of repeated subcutaneous injection. lo8 Molecular Weight Description Benzyl Violet is a water-soluble food colour conforming to the following specification,,. Identification Tests See Appendix A. Purity Tests Dye contents Not less than 8"' by titration with titanous chloride. Weigh 0.400-0.500 g of Benzyl Violet and proceed aa in App. A. Use 15 g of sodium hydrogen tartrate as buffer. 1 ml of 0.1 N titanous chloride is equivalent to 0.0}670 g of 1,0~ Na. c,,;7140 2 Loss on drying at 1}5° ) total not more than Chlorides and sulfates calculated as sodium salts) 1"' Water-insoluble matter• not more than o.2% Ether-extractable matter1 not more than o.2% Lead (as Pb)t not more than 10 mg/kg Arsenic ( as As) 1 not more than } mg/kg Chromium (as Cr)1 not more than 20 mg/kg Subsidiary dyes1 not more than "' Intermediates• not more than o.~; 109 BLACK 7984 a b This colour has been classified in categories III- and CII- S;ynODY11111 I,,.Schwarz 2J E.E.C. Serial No. E 152. £.!!!!. Diaazo Black Chelllical NBM Tetraaodium salt of 6-amino,..+-bydro~-i.-[".ff-sulfo-4--.iR-aulfophenyl) azi/ -L-naphth7.l7 azi/-2,7-naphthalenedisulfonic acid Zllpirioal Ponula Structural Ponula Molecular Weight ~Claasifioation ot colours from the standpoint of chemical specifications, see section 2~1.} page 5. ~ Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. llO BLUE VllS This colour has been classified in categories 1.! and CII~,2 SYnon.yms c.1. Food Blue}; c.1. Acid Blue 1. ~ Triarylmethane Colour Bright blue Code NUDbers c.r. (1956) No. 42045 c.1. (1924) No. 672 Schultz (19}1) No. 769 Chemical Name Sodium salt of /.4 - fa: -Ji - (diethylamino)phenyY-2,4 -disulfobenzylideny 2, 5-cyclohexadien-1-ylideni7 die thylammoni um hydroxide, inner salt Empirical Formula S true tural Formula Molecular Weigh~ 566.68 .! Classification of colours from the standpoint of chemical specifications, see section 2.1.} page 5 • .R Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. £ Colours producing more than 10-1~ of local sarcomas at the site of repeated subcutaneous injection. lll Description Blue VRS is a water soluble food colour conforming to the following specifications. Identification Tests See Appendix A, 1. Purit:y Tests Dye content, Not less than 82"1' by titration with titanous chloride. Weigh 1.000-1.200 g of Blue VBS and proceed as in Appendix A, a. Use 15 g sodium hydrogen tartrate as buffer. 1 ml of 0.1 titanous chloride is equivalent to o.02a34 g of -/1 N 0i3,J1a• !'! c2 31 2 Loss on drying at 135° ) Chlorides and sulfates calculated as sodium salts} Total not more than 18% Water-insoluble matter, not moi:e than 0.2% Ether-extractable matter, not more than o.2% Chromine ( expressed as Cr) 1 not more than 20 mg/kg Intermediates, not more than 0.5% Subsidiary ~esa not more than 2% Leada not more than 10 mg/kg Arsenioa not more than 3 mg/kg. 112 BRILLIANT BLACK Blf "SPBCULLl PURE" Thie oolour bae been claseit'ied in categories I~ and CII~ Smon.yma c.r. Food Black 1, L-Sohwartz 1, Noir Brillant Blf. :S.E.C. Serial No. B 151. Diaazo Colour Black Code Number c.r. (1956) No. 2a440 Chemical Name Tetrasodium salt of 2-Li-(P-sulfophenylazo)-7-aulfo-l,..naphthylaz!] -8- acetamino-]...naphtholw3,5-disulfonic acid Empirical Formula Structun.l Formula Molecular Weight !:. Classification of colours from the standpoint of chemical specificationa, see section 2.1.3 page 5. ~ Classification of oolours in accordance with their toxicological evaluation, see section 2.2.1 page 11. Description Brilliant Black BN is a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A. Puritz Tests Dye contenta Not less than 7ofo by titration with titanous chloride. Weigh 0.600-0.700 g or Brilliant Black BN and proceed as in Appendix A. Use 15 g of sodium hydrogen tartrate as buffer. Loss on drying at 135° ~ total not more than 30% Chlorides and sulfates calculated as sodium salts 1 Water-insoluble matters not more than o.2% Ether-extractable matters not more than o.2% Lead (as Pb)s not more than 10 mg/kg Arsenic ( as As) a not more than 3 mg/kg Subsidiary dyess not more than 1~. (The presence of the subsidiary dyes, among which the deacetylated compound has been identified, is essential to give the correct shade.) Intermediatess not more than 1%e BROIIN FIC a b These colours have teen classified in categories II- and CII- SypO?IYIIIS Brun FK; c.I. Food Brown 1. Disazo ~ Yellowish brown Code Numbers Included in Colour Index, 1956, but not numbered. Chemical Names Mixture of disodium salt of 4,4'- L\4,6-diamin-phen;ylene) bis.iazoJ] dibenzenesulfonic acid and sodium salt of :e-.Lr4,6-diamin0"!!'-tol.yl)az2{ benzenesulfonio acid Empirical Formula C1aH1l6Na20f2 (1) c u N Na o s (2) 13 13 4 3 (1) •-•--O•o,,, and . =·-O·o, .. Molecular Weifdtts 520.46 (1) 32a.33 (2) ~ Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5• .l:! Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. u, CHOCOLATE BRClfN Hr This colour has been classified in categories I~ and cu.!! Sypon.vms Brown RS; c.r. Food Brown 3. Disazo Reddish brown Code NU111bers c.r. (1956) No. 202a5 Chemical Name Disodi\lD salt of 4,4'-ffi,4-dieydro:Q"-5" (eydrox;ymeteyl)1-phe~len.!7 bis (azo.l7di,,,l,,.naphthalenesulfonic acid Empirical Formula Structural Formula Molecular Weight Description Chocolate Brown HT is a water soluble food colour conforming to the following specifications • .! Classification of colours from the standpoint of chemical specifications, see section 2.1~3 page 5• .2 Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. ll6 Identification Tests See Appendix A, 1 Purity Tests Dye content, Not less than 80% by titration with titanous chloride. Weigh 0.350,.0.450 g of Chocolate Brown HT and proceed as in Appendix A, e. Use 15 g of sodium hydrogen tartrate as buffer. 1 ml of 0.1 .!! titanous chloride is equivalent to o.ooe14 g of c f o Na 2 18Nl2 9 2 Loss on drying at 135° total not more than 20]' Chlorides and sulfates calculated as sodium salts Water-soluble matters not more than 0.2% Ether-extractable mattera not more than 0.2% Inte1111ediatess not more than 0.5% leads not more than 10 mg/kg Arsenica not more than 3 mg/kg 117 CHRYSOINE This colour has been classified in categories 1!: and CIIb S;ynonYDl8 c.I. Food Yellow 8; L,.Gelb 4; Jaune de resorcine; Chrysoine S; Chrysoine Extra; E.E.C. Serial No. E 103. Monoazo Yellow Code Numbers C.I. (1956) No. 14270 c.1. (1924) No. 148 Schultz (1931) No. 184, 186 Chemical Name Sodium salt of 4-(4-sulfophenylazo)~resorcinol Empirical Formula Structural Formula Molecular Weight 316.27 Description Chrysoine is a water-soluble food colour oonforming to the following specifications. !: Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5• .l? Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. Identification Tests See Appendix A. Purity Tests Dye content: Not less than 701> by titration with titanous chloride. Weigh 0.250-0.300 g of Chrysoine and proceed as in Appendix A. Use 10 g of sodium hydrogen tartrate e.s buffer. 1 ml of 0.1 ! titanous chloride is equivalent to 0.00790 g of C ,J!tj1 o sNa. 1 2 5 Loos on drying at 135° ~ total not more than 30% Chlorides and sulfates calculated as sodium salts j Water-insoluble matter, not more than o.2% Ether-extractable matter: not more than o.2% Lead (as Pb): not more than 10 mg/kg Arsenic (as As)t not more than 3 mg/kg Subsidiary dyest not more than 4% Intermediatest not more than o.'JI, Biological Data Biochemical aspects No data available. Acute toxicity ~o Reference per kg body-weight Rat intraperitoneal >l.O g 1 Rat intravenous >l.O g 1 Special studies The colour was tested for muta.genic effect in a eoncentratio~ of 0.5 g/100 ml in cultures of Eseherichia coli. No mutagenie.effeet was found. !!!!• Ten rats were given in their diet 25~ of the colour for 167 days. The daily intake was 1.5 g per kg body-weight and the total intake 50 g per animal. The observation poriod was 545 days. No tumours were found.l Guinea-pig. In experiments with guinea-pigs it was found that this compound had no sensitization activity.3 Long-term toxicity ~· A total of 64 male and female mice produced by mixed breeding from 5 different strains were given a diet containing 1 mg per animal per day of the colour. J.'dee at the age of 50 to 100 days were used. A number of the animals were sacrificed after an observation period of 500 days and the survivors of the 119 E Yellowish pink Code Numbers c.r. (1956) No. 45360 c.r. (1924) No. 766 Schultz (1931) No. 661 Chemical Name Disodium or dipotassium salt of 2',4',5',7'-tetrabromofluorescein Empirical Formula Disodium salt• C NaO O=BrI o-?r o Br ~ C~Br 6COONa Molecular Weight Disodiuma 691.ee Dipotassiumt 632.20 .! Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5• .2 Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. 120 FAST. REDE This colour has been classified in categories I~ and c11:la S:ynonY1DS c.1. Food Red 41 Rouge solide E. Monoazo Red Code Numbers c.1. (1956) No. 16045 c.1. (1924) No. 182 Schultz (19}1) No. 210 Chemical Nam Disodium salt of 1-(4-sulpho-l-naphthylazo)-2-naphthol,,,6..sulfonic acid Empirical Formula Structural Formula HO Molecular Weight ~ Classification of colours from the standpoint of chemical specifications, see section 2.1.} page 5. :la Classification or colours in accordance with their toxicoloffical evaluation, see section 2.2.l page 11. 121 Description Fast Red E is a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A. Purity Tests Dye contents Not less than 8~ by titration with titanous chloride. Weigh 0.240-0.300 g of Fast Red E and proceed as in Appendix A. Use 15 g of sodium hydrogen tartrate as buffer. No indicator is needed. 1 ml of 0.1 N titanous chloride is equivalent to 0.01256 g of H N 0-t3,,!1a c20 12 2 2 Loss on drying at 135° ~ total not more than l~ Chlorides and sulfates calculated as sodim salts j Water-insoluble matters not more than o.2% Ether-extractable matters not more than 0.2% Lead ( as Pb )a not more than 10 mg/kg Arsenic (as As}s not more than 3 fD8/kg Subsidiary d,yes1 not more than 4% Intermediatess not more than o.~ 122 ?AST YELLCNI AB This colour has been classified in categories r.! and cuE. Smon.yms C.I. Food Yellow 2; t.Gelb l; Jaune solide; Acid Yellow G; E.E.C. Serial No. E 105. Monoazo Yellow Code Numbers c.1. (1956) No. 13015 C.I. (1924) No. 16 Schultz (1931) No. 172 Chemical Name Disodium salt of 5-(4,osulfophenylazo)-2~aminobenzenesulfonic acid Empirical Formula Structural Formula Molecular Weigh' Description Fast Yellow AB is a water-soluble food colour conforming to the following specifications. ~ Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5. E. Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. 12, Identification Tests See Appendix A. Purity Tests Dye content• Not less than 8~ by titration with titanous chloride. Weigh 0.300-0.400 g of Fast Yellow All and proceed as in Appendix A. Use 10 g sodium hydrogen tartrate as buffer and 1.0 ml of a 1% solution of light Green SF as indicator. Loss on drying at 135° ~ total not more than l~ Chlorides and sulfates calculated as sodium salts 1 Water-insoluble matter• not more than o.2% Ether-extractable mattera not more than 0.2% Lead ( as Pb) 1 not more than 10 mg/leg Arsenic ( as As) 1 not more than 3 mg/kg Subsidiacy dyesa not more than ,% Intermediates• not more than o.~ Non-sulfonated aromatic amines and anilinea not more than 10 mg/leg A. Determination of 2-aminoazobenzene 1 and 4-aminoazobenzene Dissolve 20.0 g of Fast Yellow AB in 400 ml of water, and add 5 ml of N sodium hydroxide. ',hake 4 times with 50 ml of chlorobenzene in a separatocy funnel for 5 minutes. Wash the combined chlorobenzene extracts with 0.1 N sodium hydroxide in 400 ml portions until the upper aqueous layer remains colourless. Filter the chlorobenzene solution through a thick-folded filter paper and ) measure its extinction (E1 with a spectrophotometer against chlorobenzene in cells of suitable thickness (4i) at 414 m~. Calculations a E x 100 1_ • mg/kg o+P-aminoazobenzene. 0.397 x 4i Note a 1 mg/1 ml at 414 m ~for o-aminoazobenzene • 39. 7 111 cm for P-aminoazobenzene • 35.2 It is only possible to determine P-aminoazobenzene in amounts up to~. The separation of the°"' and P-COmpound can be achieved by the following method: Concentrate 100 ml of the chlorobenzene extract to about 20 ml on a water bath in a current of hot air and then transfer to an alumina column of suitable size. See Appendix l. Blute with chlorobenzene. The first 100 ml of the chlorobenzene eluate contain the o-compound; the P-COmpound is also further eluted with chlorobenzene. Bring both solutions to 100 ml. Measure the extinction of the o-compound at 414 d (E ), and the P-Compound at 376 ~(E ). 2 3 124 El mg/l ml 414 1111,1, for o-aminoazobenzene • 39.7 1 cm E1 me/l ml 376 mµ for p-aminoazobenzene • 110 1 cm E2 x lOO • mg/kg o-aminoazobenzene 0.397 x d2 E xlOO 3 • mg/kg p-aminoazobenzene 1.10 x d3 B. Determination of aniline Extract 75 ml of the residual chlorobenzene extract twice by shaking with 50 ml of 0.5 N hydrochloric acid and twice with 25 ml portions of water. Neutralize the combined aqueous extracts with 30% sodium hydroxide and acidify with 10 ml of 0.5 N hydrochloric acid. Then dissolve 1-2 g of potassium bromide in this solution. After cooling in iced water add about 20 drops of O.l N sodium nitrite (excess) and allow to stand for 10 minutes. Eliminate the excess of nitrite solution by adding aminosulfonic acid. Pour the solution into about 5 ml of a -,fo R-salt (•sodium salt of 2-naphthol-3,6- sulfonic acid) solution containing 10 ml of 2 N sodium hydroxide. Allow to stand for 15 minutes. Acidify the dyestuff solution to Congo red, T.s. and filter. The a.rainoazobenzene dyestuff, if present, will remain on the filter. Bring the filtrate to 200 ml and measure the extinction at 490 nµ (E ). 4 Calculate as follows: 266 E4 x = mg/kg aniline 2.26 x d4 El mg/l ml ~90 nµ for aniline= 226 1 cm 125 LIGHT GREEN SF lELLOIIBH This colour hu been classified in categories i.! and CI!Ii,.2. C.I. Food Green 2J FD & C Green No. 2J Vert LumUre SP. Triarylmethane Green Code Numbers c.I. (1956) No. 42095 c.I. (1924) No. 670 Schultz (1931) No. 765 Chemical NBM Disodil.Dll salt of 4"' {.Li-(l-ethyl.. p,.aulfobenzylamino).P4eny.!7 .. (4"' sulfoniumphenyl)-ethyleneJ·-Ll-(N..ethyi..&.p..sulfobenzyl).. "6,.2,5-cyclohe:x.. adieniminy Empirical Formula Structural Formula !: Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5• .l? Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11 • .£. Colours producing more than 10..1~ of local sarcomas at the site of repeated subcutaneous injection. Koleoular Weifht Description Light Gnen SF Yellowish is a water-soluble food colour conforming to the following apecificationa. Identification Testa See Appendix A. Purity Testa Dye contenta Bot leBB than 8'11' b7 titration with titanous chloride. Weigb o.aoo-0.900 g or Ligbt Green SF Yellowish and proceed as in Appendix A. Use 15 g of sodium hydrogen tartrate aa buffer. l ml of 0.1 If titanous chloride is equivalent to 0.03964 g of Ba • c3,f-,l2o/3 2 Losa. on drying at 135° j total not more than 1'11' Chlorides and aulfatea calculated as sodium aalta Water-insoluble matter• not more than 0.2" Ether-extractable matter• not more than 0.2" Lead (as Pb)a not more than 10 mg/kg Arsenic (as Aa)a not more than 3 mg/kg Chromi1.11 (as Cr)a not more than 20 mg/kg Subsidiary dyeaa not more than 1" Inte~diateaa not more than o.'11' l21 IETHYL VIOIE'l' a b These colo\11'8 ban been classified in categories III- md CII- SY1lOnJII! Jfe~l Violet 2Bf llethyl Violet B; Violet de Parisf Violet de •t~le Bf C.I. Basic Violet l. Claaa 'l'riphe~laethalle ~ Violet Code lfuabara c.1. (1956) No. 425~5 c.1. (1924) No. 680 Schultz (19~1) No. 78~ Chemical l'fw A mixture of the ~drochlorides of the more hishly methylated pararosanilines, containing principally the !- tetra- pent., and he:xame~l derivatives. Some or the characteristics or one of the simpler compounds in this mixture are given here. Empirical Poraula Structural Formula llolecular Weight a - Classification of colours from the standpoint of chemical specifications, see section 2.1.~ page 5• .k Classification or colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. 128 JWIB'l'BOL YEL:OOW S These coloure have been classified in categories 1.!. and CIII)! Ext D + C Yellow lo. 7 (sodium salt), Ext D + C Yellow No. 8 (potassium salt), Acid Yellowf Sulfur Yellow, Jaune naphthol s, C.I. :rood Yellow 1. Greenish yellow Code •-ben c.1. (1956) 10. 10316 c.1. (1924) 10. 10 Schultz (1931) lo. 19 Chnical .... Sodim or potassium salt of S..~dro2;1-5,7-clinitro..2..naphtbalenesulfonic acid !!Pirical Pomula C1df4',iM&208S C1dfl2•2°/ Stractural. Pomula Kolecular Weipt Ba salt - 358.20 I: ealt .. 390.42 .!. Claesitication of colours from the standpoint of chemical specifications, see section 2.1.3 pag9 5. b . - Claesitication of colours in accordance with their toxicological evaluation,. see section 2.2.1J18B911. Description laphthol yellow Sis a water-soluble food colour contoning to the following specifications. Purit;y 'l'esta Dye content, Hot less than a5.0J' b7 titration with titanous chloride. 1 ml of l ! titanous chloride is equivalent to 0.002955 g or c10Hl2o8sw~ l ml or 0.1 !! titanous chloride is equivalent to 0.003253 g of C Synon.yms Orange GG Crystal; D & C.Orange No. 3; Naphthalene orange solide GG; c.I. Food Orange 41 c.I. Acid Orange 10. Monoazo ~ Bright orange Code Numbers C.I. (1956) No. 16230 C.I. (1924) No. 27 Schultz (1931) No. 39 Chemical Name Disodium salt of l-phenylazo-2-naphthol-6, S..disulfonic acid Empirical Formula Structural Formula 0-·- HO Molecular Weight 452.37 .! Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5. R Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. Description Orange G is a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A, 1. Purity Tests Dye content, Not less than 8~ by titration with ti tanous chloride. Weigh 0.500 to o.600 Orange G and proceed as in Appendix A, a. Use 15 g of sodium hydrogen tartrate as buffer. No indicator is needed. Loss on drying at 135~ ~ total not more than l~ Chlorides and sulfates calculated as sodium salts j Water-insoluble matter, not more than 0.2% Ether-extractable matter, not more than 0.2% Aniline, not more than 0.02% A.l Apparatus. 'Spectrophotometer or photoelectric absorptiometer A.2 Reagents. The reagents shall be of a recognized analytical reagent quality. Distilled water or water of at least equal purity shall be used. (a) Benzene (b) Hydrochloric acid, approx.!• solution (c) Hydrochloric acid, approx. 31! solution (d) Potassium bromide, approx. 50% solution (e) Sodium carbonate, approx. 2! solution (f) Sodium h.ydroxide, approx. !. solution (g) Sodium hYdroxide, approx. O.lJ! solution (h) ·~· (2-naphthol-316-disulfonic acid, disodium salt), approx. 0.05J! solution (i) Sodium nitrite, approx. 0.5J! solution (j) Standard aniline solution. Into a small we~,t~ beaker weie:h 0.500 g of redistilled aniline, then wash it into a 1000 ml 1-mark volumetric flask, rinsing the beaker out several times with water. Add 30 ml of approx. 3! hydrochloric acid solution and dilute to the mark with water.at room temperature. Call this Solution A. Dilute 10.0 ml of Solution A with water to 100 ml in a 1-mark volumetric flask and mix well. Call this Solution B; 1 ml of this solution will be equivalent to 0.00005 g of aniline. Prepare Solution B freshly when required. A.} Procedure. (a) Preparation of calibration graph. Measure the following vol1.UDes of standard aniline Solution B into a series of 100 ml 1-mark volumetric flasksa 5.0 ml, 10.0 ml, 15.0 ml, 20.0 ml, 25.0 ml. Dilute to 100 ml with approx.! hydrochloric acid solution and mix well. Pipette 10.0 ml of each mixture into clean, dry test tubes and cool for 10 minutes by immersion in a beaker of ice/water mixture. To each tube add 1 ml of the potassium bromide solution and 0.05 ml of the sodium nitrite solution. Mix and allow to stand for 10 minutes in the ice/water bath. Into each of 5 25 ml volumetric flasks, measure 1 ml of the R salt solution and 10 ml of the sodium carbonate solution. Pour each diazotized aniline solution into a separate flask containing R salt solution, rinsing the test tubes with a few drops of water. Dilute to the mark with water, stopper the flasks, mix the contents well and allow to stand for 15 minutes in the dark. Measure the optical density of each coupled solution at 510 mµ in 1 cm cells using as a reference a mixture of 10.0 ml of.!! hydrochloric acid solution, 10.0 ml of the sodium carbonate solution and 2.0 ml of the 'R salt' solution, diluted to 25.0 ml with water. Plot a graph relating optical density to weight of aniline in each 100 ml of aniline solution. (b) Preparation and examination of test solution. Weigh, to the nearest 0.01 g, about 3.0 g of the dye sample into a separating funnel containing 100 ml of water, swill down the sides of the funnel with a further 50 ml of water, swirl to dissolve the sample and add 5 ml of.!! sodium hydroxide solution. Extract with 2 50 ml portions of benzene and wash the combined benzene extracts with 10 ml portions of O.lJ! sodium hydroxide solution to remove traces of dye. Extract the washed benzene with 3 10 ml portions of 3!!. hydrochloric acid solution and dilute the combined extracts to 100 ml with water. Mix well. Pipette 10.0 ml of this solution into a clean, dry test-tube, cool for 10 minutes by immersion in a beaker of ice/water mixture, add 1 ml of the potassium bromide solution and proceed as described above for the preparation of the calibration graph, starting with the addition of 0.05 ml of the sodium nitrite solution. Use as the reference solution in the measurement of optical density a solution prepared from 10.0 ml of the test solution, 10 ml of the sodium carbonate solution and 2.0 ml of the 'R salt' solution, diluted to 25.0 ml with water. Read from the calibration GI'aph the weight of aniline corresponding to the observed optical density of the test solution: A.4 Calculation. Percenta:;e of primary aromatic amine (as aniline) in dye sample a wt. of aniline x 100 wt. of dye sample taken Leada not more than 10 mg/kg Arsenica not more than 3 mg/kg Intermediatest not more than 0.5% Subsidiary dyesa not more than 2.~ i,, ORANGE GGN This colour has been classified in categories 1!: end cu! SY?lOnYIDS c.1. Food Orange 2; I,..Orsnge l; EEC Serial No. E 111. Class Monoazo Orange Code Numbers c.1. (1956) No. 15980 Chemical Name DisodiUID salt of 1-(3-sulfophenylazo)-2-naphthol-6-sulfonic acid Empirical Formula Structural Formula OH Molecular Weight ~ Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5. l2 Classification of colours in accordance with their toxicolgioal evaluation, see section 2.2.1 page 11. Description Orange GGN is a water-soluble food colour conforming to the following specifications. Identification Testa See Appendix A. Purity Testa Dye content• Not less than 70!, by titration with titanous chloride. Weigh o.eoo..o.900 g of Orange GGN and proceed as in Appendix A. Use 10 g of trisodium citrate as buffer. 1 ml of 0.1 titanous chloride is equivalent to 0.01131 g of H N 0,f> N~. l! c16 10 2 2 Loss on drying at 135° total not more than 30!, Chlorides and sulfates calculated as sodium salts Water-insoluble mattera not more than o.2% Ether-extractable matters not more than o.2% Lead (as Pb)s not more than 10 mg/kg Arsenic (as As}a not more than 3 mg/kg Subsidiary dyes s not more than ~ Intermediatesa not more than 0.5% 1J5 PONCEAU 2R These colours have been classified in categories I-a and CIII-b Synonyms Ponceau G; Ponoeau IIX; Ponceau R; Scarlet 2R; D&C Red No. 5; C.I. Food Red 5; c.1. Acid Red 26. Monoazo ~ Yellowish scarlet Code Numbers c.1. (1956) No. 16150 C.I. (1924) No. 79 Schultz (19}1) No. 95 Chemical Name Mixture of the disodium salts of }oohydroxy-4--(2,4 and 2,6-xylylazo)-2,7- naphthalenedisulfonio acid Empirical Formula Structural Formula Molecular Wei,s;ht .!. Classification of colours from the standpoint of chemical specifications, see section 2.1.} page 5. ~ Classification of colours in accordance with their toxicological evaluation, see section 2.2.l page 11. Description Ponceau 2R is a water-soluble food colour conforming to the following specifications. Identification Tests See Appendix A, 1. Purity Tests Dye content• Not less than 82% by titration with titanous chloride. Weigh 0.300-0.400 g of Ponceau 2R. Use 10 g of trisodium citrate dihydrate as buffer. No indicator is needed. Loss on drying at 135° ~ total not more than laJ' Chlorides and sulfates calculated as sodium salts j Water-insoluble matter• not more than o.2% Ether-extractable mattert not more than 0.2% m-Xylidinet not more than 0.02% Method for the determination of Primary Aromatic Amine (as m-Xvlidine) A.l Apparatus. Spectrophotometer or photoelectric absorptiometer A.2 Reagents. The reagents shall be of a recognized analytical reagent quality. Distilled water or water of at least equal purity shall be used. (a) Benzene (b) Hydrochloric acid, approx • .!! solution (c) Hydrochloric acid, approx. 3! solution (d) Potassium bromide, approx. 50% solution {e) Sodium carbonate, approx. 2.!i solution (f) Sodium hydroxide, approx • .!! solution (g) Sodium hydroxide, approx. O.J.! solution (h) 'R salt' (2-naphthol-3: 6-disulfonic acid, disodium salt), approic':" 0.051! solution (i) Sodium nitrite, approx. 0.51! solution (j) Standard m-xylidine solution. Into a small weighing beaker weigh 0.500 g of redistilled m-xylidine, then wash it into a 1000 ml 1-~ark volumetric flask7 rinsing the beaker out several times with water. Add 30 ml of approximately 3!!. hydrochloric acid solution and dilute to the mark with water at room temperature. Call this Solution A. Dilute 10.0 ml of Solution A with water to 100 ml in a 1-mark volumetric flask and mix well. Call this Solution B; 1 ml of this solution will be equivalent to 0.00005 g of m,-xylidine. Prepare Solution B freshly when required. A.3 Procedure. la) Pre'D&r&tion of calibration graph. lleasure the following volumes of standard !l-:xylidine Solution B into a series of 100 Ill l..mark volumetric flaska; 5.0 ml, 10.0 ml, 15.0 ml, 20.0 ml, 25.0 ml. Dilute to 100 ml with approximately! hydrochloric acid solution and mix well. Pipette 10.0 ml of each mixture into clean, dry test tubes, and cool for 10 minutes by immersion in a beaker of ice/water mixture. To each tube add 1 ml of the potassium bromide solution and 0.05 ml of the sodium nitrite solution. Mix and allow to stand for 10 minutes in the ice/water bath. Into each of 5 25 ml volumetric flasks, measure 1 ml of the 'R salt' solution and 10 ml of the sodium carbonate solution. Pour each diazotized m-:xylidine solution into a separate flask containing 'R salt' solution, rinsing the test tubes with a few drops of water. Dilute to the mark with water, stopper the flasks, mix the contents well and allow to stand for 15 minutes in the dark. Measure the optical density of each coupled solution at 510 mµ in 1 cm cells, using as a reference a mixture of 10.0 ml of! hydrochloric acid solution, 10.0 ml of the sodium carbonate solution and 2.0 ml of the 'R salt' solution, diluted to 25.0 ml with water. Plot a graph relating optical density to weight of amine in each 100 ml of .!!!-xylidine solution. (b} Preparation and examination of test solution. Weigh, to the nearest 0.01 g, about 3.0 g of the dye sample into a separating funnel containing 100 ml of water, swill down the sides of the funnel with a further 50 ml of water, swirl to dissolve the sample and add 5 ml of! sodium hydroxide solution. Extract with 2 50 ml portions of benzene and wash the combined benzene extracts with 10 ml portions of 0.1 ! sodium hydroxide solution to remove traces of dye. Bxtract the washed benzene with 3 10 ml portions of 3! hydrochloric acid solution and dilute the combined extracts to 100 ml with water. Mix well. Pipette 10.0 ml of this solution in a clean, dry test tube, cool for 10 minutes by immersion in a beaker of ice/water mixture, add 1 ml of the potassium bromide solution and proceed as described above for the preparation of the calibration graph, starting with the addition of 0.05 ml of the sodium nitrite solution. Use as the reference solution in the measurement of optical density a solution prepared from 10.0 ml of the test solution, 10 ml of the sodium carbonate and 2.0 ml of the 1R1salt'solution, diluted to 25.0 ml with water. Read from the calibration graph the weight of .!!!-xylidine corresponding to the observed optical density of the test solution. A.4 Calculation. Percentage of primary aromatic amine (as .!!!:'"lCYlidine) in dye sample, wt, of m-xylidine x 100 wt. of dye sample taken Subsidiary dyes, not more than }.°" Lead, not more than 10 mg/kg Arsenic, not more than 3 mg/kg PONCEAU 6R This colour has been classified in categories I~ and cu:l?. S:ynoll.Ylll8 C.I. Food Red 8; L-Rot 5; E.E.C. Serial No. E 126. Monoazo Red Code N1.DDbers c.1. (1956) No. 1629() c.1. (1924) No. 186 Schultz (19~1) No. 215 Chemical Name Tetrasodium salt of 1-(4-sulfo-l-naphthylazo)-2-naphthol-~,6,8-trisulfonic acid Empirical Formula Structural Formula Molecular Weight 706.52 ~ Classification of colours from the standpoint of chemical specificatior.s, see section 2.1.~ page 5• .!! Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. Description Ponceau 6R is a water-soluble food colour conforming to the following specifications~ Identification Tests See Appendix A. Puri b Testa Dye oontentt Not less than 60}b by titration with titanous chloride. Weigh o.600-0. 700 g of Ponceau 6R and proceed as in Appendix A. Use 10 g of trisodium citrate as buffer; no indicator is necessary. Loss on drying at 135° ~ total not more than 40% Chlorides and sulfates calculated as sodium salts j Water-insoluble mattert not more than 0.27' Ether-extractable mattert not more than 0.2% Lead (as Pb)t not more than 10 mg/kg Arsenic (as As)t not more than 3 mg/kg Subsidiary dyeSt not more than ,rl, lntermediatest not more than 0.5% l.lJO RBD lOB a b Thie colour has been classified in categories I- and en- S;yponm Fast Acid Magenta BJ Fut Acid Fuchsine BJ Acid Fuchsine DJ H~phthalene Red BJ DtC Red Ho. }:5J llagenta acide solide BJ c.r. Food Red 12. Konoaso Colour Bluish red Code Humbera c.r. (1956) Bo. 17200 c.r. (1924) Bo. :50 Schultz (19:51) Bo. }8 "Chemical Hue Disodium salt of 5-amino-4-h7dro:q-3e(phe~luo)..2,7-naphthalenediaulfonic acid Bllpirical Formula Structural Formula Molecular Weight .!l Classification of colours from the standpoint of chemical specifications, see section 2.1.} page 5. l? Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. Description led lOB is a water,,.aoluble rood colour contoraing to the rolloring apeciricationa. Identirication Teats. See Appendi.% A, 1 Purity Testa Dye content• Not leBB than 82" by titration with titanoua chloride. Weigh 0.350 to 0.400 got Red lOB. Uae 15 g or sodium hydrogen tartrate as buffer. Use a 1" solution or Light Green SI' Yellowish in water as indicator. 1 al or 0.1 B titanoua chloride solution is equivalent to 0.01169 g or c1~110.,S2... • LoBB on d171ng at 135° i total not more than 19" Chlorides and aulfatea or sodium Water-insoluble matter• not more than 0.2" Ether-extractable -ttera not aore than 0.2" Aniline• not aore than 0.2" Method for the determination or Primar;y Aromatic .Alline (as Aniline) A.l Apparatus. Spectrophotoaeter or photoelectric absorptiometer A.2 leapnta. The reagents shall be or a recognized analytical reagent quality. Distilled water or water or at least equal purity shall be used. (a) Benzene (b) IJdrochlorio acid, approx. ! solution (o) Bzdroohloric acid, approx. 3! solution (d) Potaasium bromide, approx. 50J' solution (e) Sodium carbonate, approx. 2! solution (r) Sodi• lgdroxide, approx. ! solution {g) Sodi1a hYdroxide, approx. O.)! solution (h) 'R salt' (2.. naphthol..3• 6..disulfonic acid. diaodiua salt), approx. o.05! solution (1) Sodium nitrite, approx. 0.5! solution {j) Standard aniline solution. Into a saall weighing beaker weigh 0.500 g or rediatilled aniline, then wash it into a 1000 ml 1-mark TOluaetrio flaak, rinaing the beaker out several time• with water. Add ,0 ml of apprca:. 3! hydrochloric acid solution and dilute to the mark with water at room temperature. Call this Solution A. Dilute 10.0 ml or Solution A with water to 100 ml in a 1--ma.rk -yol1aetric flask and mix well. Call this Solution BJ 1 ml or this solution will be equivalent to 0.00005 g of aniline. Prepare Solution B freshly when required. 142 A.} Procedure. (a) Preparation of calibration graph. Measure the following TOlumes ot etancl&rd aniline Solution B into a series of 100 al 1-marlt TOlumetric flasksJ 5.0 al, 10.0 11., 15.0 ml, 20.0 al, 25.0 Ill. Dilute to 100 Ill with approxillate:cy !,qclrochloric acid solutio11 ad mix well. Pipette 10.0 al ot each mixture into clean, dry test tubes ad cool for 10 minutes b7 immersion in a beaker of ice/water llixture. To each tu'!>& add 1 al of the potassium bromide solution and 0.05 Ill of the sodium nitrite solution. llix and allew to stand tor 10 minutes in the ice/wate.r bath. Into each of 5 25-ml Tolumetric flasks, measure 1111 of the 'R salt\1 solution and 10 ml of the sodim carbonate solution. Pour each diazotized aniline solution into a separate flask containing R salt solution, rinsing the test tubes with a few ~s ot water. Dilute to the uric with water, stopper the flasks, llix the contents nll ad allow to stand for 15 minutes in the darlc. Measure the optical density of each coupled solution at 510 11 in 1 cm cells, uing as a reference a mixture of 10.0 ml of ! h7drechloric acid solution, 10.0 al of the sodi1111 carbonate solution and 2.0 Ill of the 'R salt• solution, diluted to 25.0 al with water. Plot a graph relating optical density to weight of aniline in each 100 ml of aniline solution. (b) Preparation and emination of test solution. Weigh, to the nearest 0.01 g1 about }.O g of the qe sample into a separating tlmnel containing 100 al of water, swill down the sides of the flDIDel with a further 50 ml of water, swirl to diaaolft the seaple ud add 5 ml of ! sodiua h7drexide solution. Extract 111 th 2 50 ml portions of benzene and wash the combined benzene extracts with 10 ml portione ot 0.1 ! eodium ~xide solution to remove traces ot qe. Extract the washed benzene with} 10 al portions of}! )Qrdrochloric acid solution and dilute the combined extracts to 100 al 111 th water. llix nll. Pipette 10.0 ml of this solution into a clean, dry test tube, cool for 10 minutes b7 iJllaersion in a beaker of ice/water lllixture, add 1 ml of the potassium bromide solution and proceed as described above tor the preparation of the calibration graph, starting with the additioll of 0.05 ml of the sodium nitrite solution. Use as the reference solution in the •asurement ot optical deuity a solution prepare4 from 10.0 ml of the test solutions, 10 ml of the sodi1a oarbonate solution and 2.0 al of the 'll salt• eolution, diluted to 25.0 al with water. Read from the calibration graph the weight of aniline correspondillf to the obaened optical densi v of the test solution. A.4 Calculation. Peroentap ot prillar, aromatic amine (as aniline) in qe seaplea wt. ot apiline :ic 100 wt. of qe sample taken Intenetiatesa not more thBD o.'-" Subsidiary qest not aore thBD 2.0J' Lea4 (as Pb)t not more than 10 ms/kg Arsenic• not more than } q/kg n, mm 2G a b Thie colour hall been classified in categories I- and CII- Azogeramnef A11idonaphthol Red GJ Azogeramne BJ c.I. Food Red lOJ Ext DIC Red Bo. ll. Bri&bt red Code B1aber11 c.1. (1956) Bo. ~o c.1. (1924) Bo. ~l Schultz (19~1) Bo. 40 Chemical•- Dillod11a aal t of 5-acetamido-4-eydro:Q"•~(phe~lazo )-2, 7-naphthalene d111ulfonic acid Eapirical Formula Structural Fonula llolecular Weil!bt ~ Cla.eaification of colours from the standpoint of chemical specifications, see section 2.1.~ P889 5. lt Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. Description Red 2G is a water-soluble food colour confol'llling to the following specifications. Identification Teets See Appendix A, 1. Purity Teets Dye content• Not lese than 827' by titration with titanoue chloride. Weigh 0.350 to 0.400 g of Red 2G. Use 15 g of sodium hydrogen tartrate ae buffer. No indicator ie needed. 1 ml of 0.1 B titanoue chloride is equivalent to 0.01274 g of c18~ 3B30,fi2'Ba2• Loee on deying at 135° ~ total not more than 1- Chloridee and eulfates of sodium Water-insoluble matter• not more than 0.27' Bther-extractable matter• not more than 0.2" .Aniline• not more than 0.027' Method for the determination of Primaq Aromatic Ap1ne (ae Aniline) A.l Apparatuil~ Spectrophotometer or photoelectric absorptiometer A.2 Reagents. The reagents shall be of a recognized analytical reagent quality. Distilled water or water of at leaet equal purity shall be ueed. (a) Benzene (b) B;,drochloric acid, approx. !!. solution (c) Hydrochloric acid, approx. 31! solution ( d) Potaeeiua bromide, approx. 5<:JJ' solution (e) Sodiua carbonate, approx. 2! solution (f) Sodi\111 tgdroxide, approx. I. solution (g) Bodi• tgdroxide, approx. 0.11 solution (h) 'R salt' (2-naphthol-3•6-dieulphonic acid, disodium salt), approx. 0.051 solution (i) Sodim nitrite, approx. o.5J solution (j) Standard aniline solution. Into a small weighing beaker weigh 0.500 g of redietilled aniline, then waeh it into a 1000 ml 1-urk vol..-tric flask, rinsing the beaker out several times with water. Add 30 al of approximately 3! hydrochloric acicl. solution and dilute to the mark with water at room temperature. Call this Solution A. Dilute 10.0 ml of solution A with water to 100 Ill in a 1-.:rk volu.tric flaek and mix well. Call this Solution B; 1 ml of this solution will be equivalent to 0.00005 g of aniline. Prepare Solution B freshly when required. 1.\5 A.} Procedure. (a) Preparation of calibration graph. lfeaaure the follcnring Tolumea of standard aniline Solution B into a series of 100 ml 1-mark Tolumetric flaaka• 5.0 al, 10.0 ml, 15.0 al, 20.0 ml, 25.0 al. Dilute to 100 ml with approxima.teq ! hydrochloric acid solution and mu well. Pipette 10.0 al of each mixture into clean, d17 test tubes and cool for 10 minutes by immersion in a beaker of ice/water lli.xture. To each tube add 1 al of the potasaiua bromide solution and 0.05 ml of the sodium nit;i:ite solution. llix and allow to stand for 10 minutes in the ice/water bath. Into each of 5 25 ml volumetric flasks, Masure 1 ml of the 'R salt' solution and 10 ml of the sodit1111 carbonate solution. Pour each diazotized aniline solution into a separate flask containing R salt solution, rinsing the test tubes with ·a fn drops of water. Dilute to the mark with water, stopper the flasks, mix the contents well and allow to stand for 15 minutes in the dark. lleaaure the optical density of each coupled solution at 510 1111 in 1 cm cells, using aa a reference a mixture of 10.0 al of !,hydrochloric acid solution, 10.0 ml of the sodium carbonate solution and 2.0 ml of the •R salt' solution, diluted to 25.0 ml with water. Plot a graph relating optical density to weisht of aniline in each 100 ml of aniline solution. (b) Preparation and exaaination of test solution. Weish, to the nearest 0.01 g, about }.O g of the dye sample into a separating funnel containing 100 ml of water, swill down the sides of the funnel with a further 50 ml of water, swirl to diasolve the sample and add 5 ml of I sodium hydroxide solution. Extract with 2 50 ml portions of benzene and wash the combined benzene extracts with 10 ml portions of O.]l sodiU11 hydroxide solution to remove traces of dye. Extract the washed benzene with} 10 ml portions of}.!! hydrochloric acid solution and dilute the combined extracts to 100 ml with water. llix well. Pipette 10.0 ml of thia solution into a clean, d17 test tube, cool for 10 minutes by immersion in a beaker of ice/water mixture, add 1 ml of the potassium bromide solution and proceed aa described above for the preparation of the calibration graph, starting with the addition of 0.05 ml of the sodium nitrite solution. Use as the reference solution in the measurement of optical density a solution prepared from 10.0 ml of the teat solution, 10.0 ml of the sodium carbonate solution and 2.0 ml of the 'R salt' solution, diluted to 25.0 ml with water. Read from the calibration graph the weight of aniline corresponding to the obsel'Yed optical density of the test solution. 1.4 Calculation. Percentaee of primary aromatic amine (aa aniline) in dye samples wt. of aniline x 100 wt. of dye sample taken Interaediatea not more than o.5!,( Subsidi&17 dyeaa not more than 2" Leada not more than 10 mg/kg Arsenioa not more than} mg/kg 1"6 RBOJWIINE B a bo This colour has been classified in categories I- and CIII' Sznomma D&C Red No. 19; c.1. Food Red 15. £l!ll Xanthene Colour Bluish pink to reddish violet Code Nunbers c.1. (1956) No. 45170 c.1. (1924) No. 749 Schultz (1931) No. 864 Chemical Name L9,-~arboxyphenyl)-6..(diethylamino)-3!-xanthen-3-yliden!] diethylammoniun chloride Empirical Formula llolecular Wei@ht A Claasification of colours from the standpoint or chemical specifications, see section 2.1.3 page 5. l Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 pas- 11 • .£ Colours producing more than 10..1"" or local sarcOll88 at the site or repeated subcutaneous injection. Description Bhoduine B 18 a water-soluble food colour conforming to the following specificatione. Identification Testa See Appendix A, 1. Purity Tests Dye content• lot less than 90J' by titration with titanous chloride. Weigh 0.9()0,.1.000 g of Bhodamine Band proceed 18 in Appendix A, s. Use 15 g of sodium hydrogen tartrate 18 buffer. 1 ml of O.llf titanous chloride is equivalent to 0.02'595 g of 1 0,c1. c281,1 2 Loss on drying at 1}5° } total not more than lOJ' Chlorides and sulfates calculated 18 sodium salts Water-insoluble mattera not more than 0.2" Ethe;r-extractable matter• not more than 0.2" Intermediate• not more than o."' Diethy1-uenophenola not more than o.°"' Leada not more than 10 mgfke Arsenic• not more than '5 mg/kg 148 SCAIW!l'l' GN "SPEqALLY PURE" Thie colour ha8 been classified in categories 1A and cull Sznon.yms c.I. Food Red 2, L-Jlot 6, Bcarlate GNJ E.E.c. Serial No. E 125 llonoaso ~ Red Code NU11ber c.I. (1956) No. 14815 Chemical Name Dieodium salt of 2-{6-eulfo..2,~lyluo).J,.,aaphthoJ...5-eul.i'onic acid Empirical Formula Structural Formula en, OB BC~1'-1' ' - ~-. . so 11a 3 ~olecularWei&ht 400.43 Description Scarlet GB ii a -ter-eoluble food colour conforming to the following specifications. A Classification of colou:t11 :troa the standpoint of cheaical specifications, see section 2.1.3 J1889 5. li Classification of coloun in accordance 111 th their toxicological evaluation, see section 2.2.1 psae 11. Identification Tests See Appendix J.. Purity Tests Dye content, Bot less than 70,. by titration with titanous chloride. Weigh 0.300-0.400 g or Scarlet GB and proceed as in Appendix A. Use 10 g or sodium hydrogen tartrate as buffer. l ml or O.l B titanous,chloride is equivalent to 0.01201 g or ClBH • Ba • 14 2o~2 2 9 Loss on drying at 135 ~ total not more than 30,. Chlorides and sultates calculated as sodium salts Water-insoluble 111atter1 not aore than 0.2" Ether-extractable matter• not more than 0.2" Lead (as Pb)t not more than 10 mg/Jcg Arsenic (as As) t not more than 3 mg/kg Subsidiary czyesa not more than 4'I, Intermediates, not more than o."' 150 SUDAN G This colour has been classified in categories III§ and cu.2 SznollYIDS Sudan Orange; Oil Yellow G G; Soudan G; C.I. Food Orange 3. Monoazo Yellowish orange Code Numbers C.I. (1956) No. 11920 C.I. (1924) No. 23 Schultz (1931) No. 31 Chemical Name 4-(Phenylazo}resorcinol Empirical Formula 012H1012°2 Structural Formula Molecular Weight 214.23 § Classification of colours from the standpoint of chemical specifications, see section 2.1.3 page 5• .2 Classification of colours in accordance with their toxicological evaluation, see section 2.2.11)8€;e 11. 151 SUDAN RED G This colour has been classified in categories III~ and err!?. S:ynon:yms Sudan Red; Soudan R; c.r. Food Red 16. Monoazo Red Code Numbers c.r. (1956) No. 12150 c.r. (1924) No. 1n Schultz (19~1) No. 149 Chemical Name 1-&-Methoxyphenyl)az.2?-2-naphthol Empirical Formula S true tural Formula Molecular Weight ~ Classification of colours from the standpoint of chemical specifications, see section 2.1.~ page 5. !?. Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11. 152 VIOl&T 5 BN This eolour has been classified in categories III!. and cnk Sznonyms Acid Violet 6B; Acid Violet 4,m; Acid Violet; Fo~l Violet; Violete acide 6B; c.r. Food Violet 1. file!. Triarylmethane Bright bluish violet Code N\Dbers c.r. (1956) No. 42650 c.r. (1924) No. 698 Schultz (19}1) No. 806 Chemical NBM Sodium s~t of [i.,.£ ..fi:.. (diethylamino)PheJ1¥!7-~§thyl(m.-sulfobenzyl) amini/benzylideniJ.. 2, 5-cyclohexadien,,.l,.yliden.!{ethyl {m-sulfobenzyl) ammonium hydroxide, inner salt Empirical Formula Structural Formula llolecular Weight 761.94 !. Classification of colours from the standpoint of chemical specifications, see section 2.1.} page 5. b - Classification of colours in accordance with their toxicological evaluation, see section 2.2.1 page 11; lBLLOW 271751' "SPECIALLY PURE" !hia colour baa been classified in categoriea III! and cIIl!. Jaune 27175111 c.1. l'ood Yellow 7• ~ llonouo Colour Yellow Code laben c.1. (1956) •o. 1}445 Chuioal •ue Trisodium salt of }-emno-4-L<'a-sulfopheJQ"l) ui} -2, 7-naphthalenesulfonic acid Bgirioal l'oraul.a sola Structural Forwla llolecular Weipt :Biologloal Data Biochuical aspects •o data aT&ilable. A Classification of colours from the atandpoint of chemical specifications, aee section 2.1.} P8&9 5. lclaasification of colours in accordanc, with their toxicological evaluation, see section 2.2.1P8B911. 154 APPENDIX A METHODS The methods suggested can, in certain cases, be replaced by others conveniently selected by competent experts, taking into account possible development of techniques. I. ldentificatio11 of dyes The positive identification of a dye is often quite difficult. The identification of unsulfonated dyes of the azo class is not particularly difficult since they may be puri fied and handled in the classical manner of organic qualitati\'e analysis as well as by the chromatographic techniques described below. The more complex dyes and almost all of the sulfonated dyes however arc not easily obtained in chemically pure form and, in general, they have no precise melting points or boiling points, nor definite crystalline structure. Identification, therefore, is best obtained by comparison of observed properties with the properties of materials of known structure obtained through synthesis from known materials by unequivocal routes of preparation. The principal techniques in use are chromatography and spectrophotometry. Fre quently both techniques are required. The presence of subsidiary dyes may so affect the observed spectra, for example, that positive identification of the principal compo nent cannot be made. For this reason, it is advisable to chromatograph the dyes by either paper or column techniques before other means of identification are attempted. Paper chromatography is often very useful in identification of dyes and does not require expensive equipment. But it must be kept in mind that the Rr-value of a sub stance is only theoretically a constant. In practice, so many factors, most of which arc beyond control, have such an important influence that the Rr-values become a ,·ery unreliable quantity. Such more or less influential factors include: composition and age of the solvent mixture, quality of the paper, machine direction, kind and quality of subsidiary substances, concentration, pH-value of the solution, and temperature. Fer this reason, comparative chromatography should always be used. By simultaneous running of several substances of similar concentration a number of these factors are eliminated. Coincidence of migration distances with one solvent only should be looked upon only as one criterion of identity and further tests should be made for securing the finding. Assessment of the color shade should be made on the chromatograms still moist with solvent as well as dried. The shade should be assessed in incident and trans mitted daylight as well as in UV-light. In ultraviolet light many dyestuffs show char acteristic color changes. Furthermore it is thus often possible to trace colorlcss fluo rescent impurities. lf possible, two UV emitters which yield different wave lengths, should be used; one lamp should emit around 250 mµ. Tests with acids, alkalis and other suitable reagents, in order further to safeguard the results, should be made. All tests may be carried out with fine capillary pipettes on only one dyestuff-spot. 155 Appendix A The following requirements should be met when proving the identity of dyestuffs: equal migration distances in several solvents; equal shade in daylight and ultraviolet light; equal color changes with reagents. Spectrophotometric methods of examination are among the most useful means of identification of dyestuffs. Ultraviolet, visible, and infrared regions are all employed. The visible region of the spectrum is ordinarily examined as the first step in attempt ing to identify an unknown dyestuff. Many dyestuffs show characteristic absorption in the visible region while others do not. Spectra in the ultraviolet region may also be of use, and should be obtained if possible. Infrared absorption spectra often offer the best means of identification of various compounds, but there are some difficulties in the practical use of the methods. In the application of visible and ultraviolet spectrophotometry, spectra should always be obtained in more than one solvent, or if in a single solvent, under various conditions. Spectra of water solutions should be obtained under neutral (buffered with ammonium acetate), acid (0.1 N hydrochloric acid), and alkaline (0.1 N sodium hy droxide) conditions. Absorption spectra are ordinarily plotted as charts, showing the absorbance at all wave lengths. Examination of the resulting curves should include more than loca tion of the wave length of maximum absorption. The entire curve should be carefully inspected to determine the particular shape of the curve since " shoulders " or inflec tion points may be the most characteristic and useful features of the absorption spectra. These features often make it possible to distinguish between two or more dyes that have absorption maxima at the same wave length. Many dyes can be definitely charac terized by observing the extent to which the absorption maxima and other features of the absorption curve are changed by changes in pH or by other changes in the solvent. lnfrared spectra can be obtained in several ways; the more commonly used are: spectra of solutions of the material in suitable solvents; spectra of suspensions of the material in a suitable liquid; the potassium bromide pellet technique (a small amount of the dye, usually from 1 to 3 mg, is thoroughly mixed with pure, dry potassium bromide, the mixture transferred to a suitable die and pressed into solid form by exerting a pressure of 700 to 1400 kg/square cm). The spectra obtained are plotted in the usual way. Salient features of the result ing curves are the wave lengths at which absorption peaks occur, and the shape of the curves near the peaks. Detailed discussion of the interpretation of infrared spectra is beyond the scope of this brief statement. It must be pointed out, however, that care must be taken to ensure that absorption peaks due to inorganic contaminants are not considered as due to the dye. The crystal structure or other physical state of the sample may affect the spectra obtained from suspensions or potassium bromide pellets. It is necessary to make certain that the unknown material has been treated in exactly the same manner as was the standard or known sample. Water soluble dyes can often be handled by dis solving the materials in water, adding a little acetic acid, evaporating to apparent dryness, and then drying at about IOOo to remove the residual water. All materials to be tested should be free from water or other solvent before an infrared spectrum is obtained. Water and all organic solvents absorb infrared radiation. 156 Appendix A As an example of the use of infrared spectra, two of the dyes listed in the ml)no graphs may be mentioned. Sunset Yellow and Orange GGN have absorption spectra in the visible region and in the ultraviolet region so nearly identical that they cannot be distinguished through examination in these regions. Their infrared spectra, however, are quite different in the region of the spectrum in which the sulfonic acid groups absorb strongly. In some instances, chromatographic procedures, spectrophotometric procedures, and any combination of the two may fail to give positive identification. In such cases, the problem may often be solved by reducing the dyestuff or otherwise degrading the compound and identifying the resulting products. This technique is particularly applicable to azo dyes. The amino compounds resulting from the reduction can fre quently be readily identified by chromatographic and spectrophotometric techniques. Many other techniques have been applied to identification of dyes. Their descrip tion here will not be undertaken, but one example will be cited. Many pigments have definite crystalline structure and can be identified by X-ray diffraction patterns, or by optical crystallography. Some dyes can be converted 10 crystalline derivatives and similarly identified. Preparation of alumina chromatographic column a. Apparatm·: A glass tube of 200 mm / 7 mm inside diameter. b. Procedure: Close one end of the tube with a tight glass-wool plug I cm thick. Fill the tube with a suspension of adsorption alumina in benzene. Use standardized aluminum oxide absorbant, 80-200 mesh. The aluminum oxide after sedimentation should occupy two thirds of the tube. Attach the tube to a suction flask. Fill this column with benzene and adjust suction to about 30 drops per minute, continuing until the benzene layer is 2-3 mm above the alumina. Non:* It is advisable to place a glass-wool plug at the top of the alumina to prevent churning when solvent is added, and to apply a reservoir of solvent at the top of the column, which will exert a mild pressure on the chromatographic column. The suction must be carefully controlled to prevent channeling in the alumina. Avoid eluting the benzene or other solvent completely. It is necessary that the surface of the alumina be kept covered with a liquid layer. 2. Loss on drying at 135° Weigh 2.0-3.0 g of the dye sample in a tared weighing bottle fitted with a ground lid. A weighing bottle of squat form about 50 mm in diameter and 30 mm high is suitable. Heat at 135° ± 5° until a constant weight is obtained. Express the loss in weight as a percentage of the weight of sample taken. 3. Water-insoluble matter \V,:igh 4.5-5.5 g1 of the dye sample into a 250 ml beaker. Add about 200 ml of h, ,, water (80-900), stir to dissolve the dye and allow the solution to cool to room temperature. Filter the solution through a tared Grade 4 sintered glass filter (B.S. 1752, sintered disk filters for laboratory use) and wash with cold water until the washings are colorless. Dry the filter and residue at 135u until a constant weight is obtained. Express the weight of the residue as a percentage of the weight of sample taken. 1 Certain dyes, like Indigotine, Chrysoine, and Patent Blue V have solubilitics less than 5 g/200 ml. Smaller quantities of dyes should then be used. 157 Appendix A 4. Ether-extractable matter a. Apparatus: Upward displacement type liquid/liquid extractor with sintered glass distributor, working capacity 200 ml. A piece of bright copper wire is suspended through the condenser and a small coil of copper wire (0.5 g) is placed in the distillation flask. b. Procedure: (i) Alkaline ether extract. Weigh 5.0 g 1 of the dye sample, dissolve in 150 ml of water, add 2 ml of 2.5 N sodium hydroxide solution and transfer the solution to the extractor, diluting to approximately 200 ml with water in the process. Add 200 ml of ether • into the distillation flask and extract for 2 hours with a reflux rate of about I 5 ml/min. Reserve the dye solution. Transfer the ether extract to a separatory funnel and wash the ether extract with two 25 ml portions of 0.1 N sodium hydroxide and then with water. Distill the ether in portions from a tared 150 ml flask containing a clean copper coil, reducing the volume to about 5 ml. (ii) Acid ether extract: To the dye solution reserved from (i) add 5 ml of 3 N hydrochloric acid, mix and extract with a further quantity of the ether as in (i). Wash the ether extract with two 25 ml portions of 0.1 N hydrochloric acid and then with water. Transfer in portions to the flask containing the evaporated alkaline extract and carefully evaporate all the ether. Complete the drying in an oven at 85° for 20 minutes, then allow the flask to cool in a desiccator for 30 minutes and weigh. Repeat the drying and cooling until constant weight is obtained. NOTE: It is recommended that the tared flask be counterpoised by a similar flask and that both be subject to the drying and cooling operations. The increase in weight of the tared flask, expressed as a percentage of the weight of samples taken, is the " ether extract." 5. Subsidiary dyes METHOD A a. Principle: The subsidiary dyes are separated from the main dye by ascending paper chromatography and are extracted separately from the paper. The optical den sities of the extracts are measured at their wave lengths of maximum absorption in the visible spectrum and are used to calculate the content of subsidiary dyes as a per centage by weight of the sample. Because it is impracticable to identify each subsidiary dye in each coloring matter, an approximate method of expressing the subsidiary dyes as a percentage of the sample 1 Certain dyes, like Indigotine,Chrysoine, and Patent Blue V have solubilities less than 5 g/200 ml. Smaller quantnies of dyes should then be used. • Diisopropyl ether. Immediately before use the freshly distilled ether should be passed through a 30 cm column of chromatography grade aluminum oxide in order to remove peroxides and inhibitors. Test to ensure the absence of peroxides, as fol lows: Prepare a colorless solution of ferrous thiocyanate by mixing equal volumes of 0.1 N solutions of ferrous sulfate and ammonium thiocyanate 0.1 N and carefully discharging any red coloration, due to ferric ions, with titanous chloride. To 50 ml of this solution add 10 ml of the ether and shake the mixture vigorously for 2-3 min utes. No red color should develop. Appendix A has been adopted: the optical densities of the extrac~ are added together and the total is converted to a percentage of the sample by means of a graph relating optical den sity to weight, and which is prepared with the main dyestuff itself. This is considered to be a sufficiently close approximation for the determination of a minor component. b. Apparatus: (i) Chromatography tank and ancillary equipment: Suitable apparatus is shown in Figs. 2 and 3 and comprises: a glass tank (A) and cover (B); a supporting frame (C) for the chromatography paper sheets; a tray (D) for developing solvent; a secondary frame (E) supporting "drapes" of filter paper; sheets of chromatography grade paper (Whatman No. 1 Chromatography grade paper is suitable) not less than 20 cm x 20 cm. (ii) A microsyringe, capable of delivering 0.1 ml with a tolerance of± 0.002 ml. (iii) A spectrophotometer. -r--GlossI covtr(8} /,I / / Filler pope, <1rope Cromotogrophy paper FIGURI! 2. - Assembly of chromatography apparatus. 159 Appendix A S11pportin9 1,om, (C) F1GURF. 3. • Compo11enrs of chromatography apparatus. c. Procedure: (i) Not less than 2 hours before carrying out the determination arrange the filter paper drapes in the glass tank and pour over the drapes and into the bottom of the tank sufficient of an atmosphere-saturating solvent [a mixture of 700 ml ethyl methyl ketone, 300 ml acetone, 2 ml ammonia solution (sp. gr. 0.88-0.89), 300 ml water] • to cover the bottom of the tank to a depth of approximately 1 cm (see Table 1). Place the solvent tray (D) in position and fit the cover to the tank. (ii) Mark out a sheet of chromatography paper as shown in Fig. 4. Apply 0.10 ml of a 1.0 % aqueous solution of the dye as uniformly as possible within the confines of the 18 cm x 7 mm rectangle, holding the nozzle of the microsyringe stead ily in contact with the paper. Allow the paper to dry at room temperature for 1-2 hours, or at 50° for 5 minutes followed by 15 minutes at room temperature. Mount the sheet, together with a plain sheet to act as a blank, in frame (C). Pour sufficient of a devel· oping solvent (which is the same as the atmosphere-saturating solvent) into the tray (D) to bring the surface of the solvent about 1 cm below the base line of the chroma togram sheet. The volume necessary will depend on the dimensions of the apparatus and should be predetermined. Put frame (C) into position and replace the cover. Allow the solvent front to ascend a specified distance, to be determined for each color, above the base line (see Table 1), then remove frame (C) and transfer it to a drying cabinet at 500-60° for 10-15 minutes. Remove the sheets from frame (C). If required, sevc:ral chromatograms may be developed simultaneously. l.6o Appendix A 0 0 I 18(,n - 7~·. 8011 line ' i ···---·------ Figure 4. - Method of marking out chromatography paper. Table 1. Atmosphere-saturating and Height of climb of solvent Food Color developing solvent on the paper Amaranth} Methyl ethyl ketone 700 ml To the full height of the Orange I Acetone 300 ml paper and development to Orange G Water 300 ml be continued for 1 hour afterwards. Azo Rubine Methyl ethyl ketone 700 ml 17 ± O.S cm Naphthol Yellow ~ Acetone 300 ml orange U Concentrated ammonia 2 ml "' Water 300 ml Ponceau 4R Methyl ethyl ketone 700 ml To the full height of the Acetone 300 ml paper and development to Water 300 ml be continued for 1 hour afterwards. Sunset Yellow FGF Methyl ethyl ketone 700 ml 17 ± 0.5 cm Acetone 300 ml Concentrated ammonia 2 ml Water 300 ml Tartrazine Methyl ethyl ketone 700 ml 12 ± O.S cm Acetone 300 ml Concentrated ammonia 2 ml Water 300 ml Non. At the time of writing these methods of analysis, the precise conditions for chromatographic separation of subsidiary dyes have been established only for a limited number of the colorina matters. The reagents proposed, however. proved to be effective in most of the samples which have been studied and should be used unless it is stated to the contrary in the Specifications. 161 Appendix A (iii) Cut each subsidiary band from the sheet as a strip, and cut an equivalent strip from the corresponding position of the plain sheet. Place each strip, subdivided into a suitable number of approximately equal portions, in a separate test tube. Add 5.0 ml of extracting solvent (a mixture of equal volumes of acetone and water) to each test tube, swirl for 2-3 minutes, add 15.0 ml of 0.05 N sodium hydrogen car bonate solution and shake the tube to insure mixing. Filter the colored extracts and blanks through a 9 cm filter paper of open texture and determine the optical densi ties of the colored extracts at their wave lengths of maximum absorption, using 40 mm closed cells, against a filtered mixture of 5.0 ml of extracting solvent and 15.0 ml of the sodium hydrogen carbonate solution. Measure the optical densities of the extracts of the blank strips at the wave lengths at which those of the corresponding colored extracts were measured. Add the optical densities together, subtract the sum of the blanks, and convert the net optical density to a percentage by weight of the sample by means of a calibra tion curve established from the main dyestuff. METHOI) 8 P.11,, T a. Apparatus: I I A chromatographic tube - 2.1 x 45 cm (Fig. 5) J A rammer - stainless steel or brass plunger to fit the tube h. Phases: (i) Mobile phase. Mi.x 100 ml of n-butyl alcohol and ICO ml Ii I r1r of carbon tetrachloride in a separatory funnel. Ada 100 ml cf diluted hydrochloric acid as specified below: LJ '"" Color to be examined Hydrochloric acid dilution Ponceau 3R I+ 99 Amaranth I+ 19 Ponceau SX I+ 49 Tartrazine I -1 19 Sunset Ycllow FCF 1 + 49 Shake the mixture for 3 minutes, then let stand until the layers have separated. Use the bottom layer as the mobile phase. (ii) Stationary phase. Use the top layer from above as the stationary phase. FIGURE 5. - Chromatographic tube for subsidiary dyes. c. Procedure: Mix thoroughly, 10 g of Cclite 545 (or equivalent diatomaccous earth) with 5 ml of the stationary phase. Put the mixture into the tube and pack firmly with the rammer. Dissolve 0.050 g of the sample in 25 ml of the mobile phase and mix thoroughly a 5.0 ml aliquot of this solution with 10 g of Celite. Put the mixture into the column and pack firmly with the rammer. Elute with the mobile phase. The lower sulfonated subsidiary dyes are eluted first. Dilute the solution of the eluted color with the mobile phase to exactly 25 ml. In some cases further dilution may be required. 162 Appendix A Determine the amount of color eluted spectrophotometrically. It is preferable to use as a standard a purified sample of the particular subsidiary dye encountered. For practical purposes, no serious error is introduced when a solution of the principal color is used as the standard. (Brilliant Black BN is an exception). The standard must be prepared using a solvent of the same composition as the mobile phase. NOTE: When the amount of subsidiary dye is large, it may be necessary to use a small sample of the dye. This procedure has been used for a number of dyes not listed above. It is gener· ally applicable to separation of dyestuffs having different numbers of sulfonic groups. In application of this method to dyes not listed above, the determination should be carried out with reagents containing various concentrations of acid in order to determine the optimum concentration. 6. Chloride a. Apparatus: Potentiometric titration apparatus, with silver indicator electrode, calomel reference electrode, and saturated potassium sulfate bridge. b. Procedure: Weigh 0.5-1.0 g of the dye sample, dissolve in 100 ml of water, and acidify with 5 ml of 1.5 N nitric acid solution. Place the silver electrode in the dye solution and connect the calomel electrode to the solution by means of the saturated potassium sulfate bridge. The saturated potassium sulfate bridge may be eliminated by using a glass electrode as the reference electrode; this simplifies the apparatus con siderably, and the glass electrode is sufficiently constant to be used as a reference for this type of titration. Determine the chloride content of the solution by titration against the 0.1 N silver nitrate solution, and calculate the result as sodium chloride. I ml of 0.1 N silver nitrate solution = 0.00585 g of sodium chloride. Express the result as a percentage of the weight of sample taken. 7. Su/fates Weigh 5.0 g of dye sample, transfer it to a 250 ml conical flask and dissolve in about 100 ml of water by heating on a water bath. Add 35 g of sulfate-free sodium chloride, stopper the flask, and swirl at frequent intervals during I hour. Cool, trans· fer with saturated sodium chloride solution to a 250 ml measuring flask, and dilute to the mark at 20°. Shake the flask, and filter the solution through a dry filter paper. Pipette 100 ml of the filtrate into a 500 ml beaker, dilute to 300 ml with water and acidify with hydrochloric acid, adding I ml in excess. Heat the solution to boiling, and add an excess of 0.25 N barium chloride solution drop by drop, with stirring. Allow the mixture to stand on a hotplate for 4 hours or leave it overnight at room temperature and then bring it to about 80°, and allow the precipitate to settle. Filter off the precipitated barium sulfate, wash with hot water, and ignite at a dull red heat in a tared crucible until a constant weight is obtained. Carry out a blank determination, apply any necessary correction to the weight of barium sulfate found in the test, and calculate the result as sodium sulfate. Weight of sodium sulfate in sample = 2.5 x corrected wt of barium sulfate. Express the result as a percentage of the weight of sample taken. 16J Appendix A 8. Dye Content a. Apparatus: (i) Carbon dioxide generator, e.g., a Kipp apparatus. (ii) Titration apparatus, as shown in Fig. 6, comprising an aspirator and a burette fitted with a double-oblique tap and side arm. The aspirator is closed at the top by a rubber stopper with two holes, through one of which passes a glass tube connected to the carbon dioxide generator. Through the second hole passes a glass tube connected to the top of the burette. The side arm of the burette is connected to the bottom of the aspirator. To CO, source FIGURB 6. • Apparatus for storing titanous chloride solution. (iii) Carbon dioxide flasks or titanous reduction flasks (see Fig. 7) of 500 ml capacity. b. Procedure: (i) Preparation of 0.1 N titanous chloride solution. Measure into a large round flask a volume of 15% titanous chloride solution containing 15.5-16.5 g of titanous chloride for each liter of solution required. Add 100 ml of hydrochloric acid (sp. gr. 1.16-1.18) for each liter of solution required. Boil the mixture for 1-2 min utes and then pour it into cold water. Make up the required volume in the aspira tor, mix well, and preserve the solution in an atmosphere of carbon dioxide (see Fig. 5). Cover the solution with a layer of liquid paraffin (about 0.6 cm deep). (ii) Standardization of titanous chloride solution. Weigh 3 g of ammonium fer rous sulfate [(Nff.>tSO•. FeS0•. 6H10] into a carbon dioxide flask or titanous reduc tion flask, and pass a stream of carbon dioxide through the flask continuously until the end of the determination. Add SO ml of water and 25 ml of 10 N sulfuric acid solu- 164 Appendix A tion, then 30 ml of 0.1 N pota~:um dichromate solution, accurately standardized. Titrate with the titanous chloride solution until the calculated end point is nearly reached. Add 5 ml of a 20 % ammonium thiocyanate solution and continue the titra tion until the red color is discharged and the solution remains green. Carry out a FIGURE 7. - Titanous reduction flask. blank determination on 3 g of ammonium ferrous sulfale, using the same quantities of water, acid, and ammonium thiocyanate solution and passing a continuous current of carbon dioxide through the flask as before. The factor for 0.1 N titanous chloride so:ution is 30 F ml (corrected) of titanous chloride solution required (iii) Determination of dye content of sample. Accurately weigh the quantity of the dye sample specified in each monograph into a carbon dioxide flask or titanous reduction flask, and add 10 g of sodium citrate or 15 g of sodium hydrogen tartrate, as specified in each monograph, and 150 ml of water. Pass a stream of carbon dioxide through the flask, heat the solution to boiling, and titrate with standardized titanous chloride solution, maintaining a steady flow of carbon dioxide. Towards the end of the titration, add the titanous chloride solution dropwise to the end point. The dye will act as its own indicator unless otherwise stated in the appropriate monograph. Ax D x 100 x F The percentage of dye in sample = weight of sample where A is ml of 0.1 N titanous chloride solution required (corrected). D is weight of dye equivalent to 1.00 ml of 0.1 N titanous chloride solution (specified in each monograph). l.65 Appendix A 9. Limit test for Chromium A limit test is described which is designed to show whether the sample contains more or less than 20 mg/kg of chromium. Weigh 1.0 g of the dye into a quartz dish. Char the material, raising the temper ature slowly. Allow to cool, add 10 ml of a 25% magnesium nitrate solution, evap orate heating slowly until no more nitrous vapor evolves. Heat the material in an oven at 600° until all black particles have disappeared (l hour). Dissolve the residue by adding 10 ml of 4 N sulfuric acid and 20 ml of water. Heat on a water bath for about 5 minutes. Add 0.5 ml of 0.1 N potassium permanganate. Add more permanganate if the solution decoloriies. Cover with a watch glass and heat on a water bath for about 20 minutes. Add 5 % sodium azide solution, one drop every JO seconds, until the excess potassium permanganate has been removed (avoid excess of sodium azide, 2 drops are usually sufficient). Cool the solution in running water, and filter if man ganese dioxide is evident. Transfer the solution to a 50 ml volumetric flask. Add 2.5 ml of 4 M sodium dihydrogenphosphate, add 2 ml of diphenyl carbazide T.S. and fill to the mark with water. Measure the extinction at 540 m!t Y2 hour after adding the diphenyl carbazide T.S. A blank with the latter two reagents should show no color or only a slight purple color. At the same time run a parallel test with 1.00 ml of standard chromate T.S. (I ml = 20 µg Cr) and a few of saccharose placed into a second quartz dish. Treat the mixture exactly as the dye sample and measure the extinction at the same wave length. Calculate the chromium content of the dye from the two extinction values observed. 10. Limit test for Lead The limit test described for lead is designed to show if a sample contains more than 10 mg/kg or 20 mg/kg of lead. The sample is digested with nitric and sulfuric acids, and a clear solution of the digest is prepared. The same lead limit test is describ ed in the manual on antimicrobial preservatives and antioxidants, except that the addition of citrate is left out, because it is already added for dissolving the digest. a. Digestion. Weigh 1.0 g of sample for substances with 10 mg/kg limit or 0.5 g for those with 20 mg/kg limit. Place with 5 ml of water, 5 ml of 65 % nitric acid and 5 ml of sulfuric acid in a digestion flask. Warm slightly. If foaming becomes excessive add a little water. Evaporate the mixture. Maintain strongly oxidizing con ditions in the flask during digestion by adding cautiously small quantities of nitric acid whenever tlie mixture begics to turn brown or darken. Continue digestion until organic matter is destroyed and sulfur trioxide fumes are copiously evolved. The final solution should be colorless or at most slight straw color. To remove nitrosylsulfuric acid, after partial cooling transfer the residue to a dish, rinse with 25 ml of water, evaporate and heat again to fuming point. Add 25 ml of water and evaporate and heat again. b. Solution of digest. Allow to cool, add 20 ml of ammonium acetatecitrate solu tion T.S., and allow again to cool. Neutralize to about pH 7 with 25 % atT1monia and boil if necessary to dissolve calcium sulfate. Cool the clear solution. Shake the solution with 10 ml of carbon tetrachloride and discard the lower layer. c. Test with dithizone. To a 100 ml separatory funnel add 10 ml of 0.2 N ammo nium hydroxide, 2 ml of 10% potassium cyanide, 2 ml of 10% hydroxylamine hydro chloride solution and 2 ml of a 20 mg per liter solution of dithizone in carbon tetra chloride. Shake the separatory funnel for a few minutes and discard the carbon tetra chloride layer. Add 2 ml of carbon tetrachloride, shake and discard again the carbon tetrachloride layer. Add the solution of digest to the separatory funnel. Adjust the 166 Appendix A pH of the solution to about 10 (thymol blue paper) with ammonia. Again add 5 ml of dithizone solution in carbon tetrachloride and shake vigorously for a few minutes. The carbon tetrachloride layer becomes red according to the amount of lead present. Treat simultaneously a standard solution containing 10 µg of lead in the same manner as the solution of the digest in b. Evaluate the quantity of lead in the digest by comparing the colors of the carbon tetrachloride layers. Insure that the red color is due to lead by shaking the carbon tetrachloride layer obtained from the digest solution with JO ml of a buffer pH 2 T.S. The red color will turn green if it is due to lead. 11. Limit test for Arsenic The limit test described for arsenic shows whether a sample contains more than 3 mg/kg of arsenic. The test is described in the manual on antimicrobial preservatives and antioxidants. ~o ''"" FIGURE 8. a. Digestion of I g sample and removal of nitrosylsulfuric acid as described under Lead (10.a). Allow the digest, with 5 ml sulfuric acid, to cool. b. Distillation (according to Snyder). Pour the digest into the distillation flask (Fig. 8). Rinse the dish twice using each time 2.5 ml of water and add the water to the digest. Cool the solution. Add some drops of 0.1 N potassium permanganate solution until the red color persists. Add 0.250 g of freshly powdered ferrous sulfate, 2.5 ml of 38% hydrochloric acid and 0.1 ml of 20~{ potassium bromide solution. Close the flask. Place a reagent tube containing 8 ml of water in a beaker with water as shown in Fig. 8. Heat with microburner. In the beginning a slow stream of air bubbles appears, followed after 3-5 minutes by hydrochloric acid gas. Heat then a little stronger so that the solution boils. After bulb A has become very hot boil for 40 seconds. There- 167 Appendix A upon lower the reagent tube and remove the flame. The hydrochloric acid content of the distillate must be 9-10%; only then has arsenic been completely distilled over; moreover this acid concentration is required for the following test. c. Test with mercury bromide paper (Mayercon-Bergeret). The distillate is transferred to a conical flask of 25 ml capacity. The flask is closed with the device as shown in Fig. 9, containing a small disk of test paper. Add 3 pieces of aluminum strips (8 x 8 x I mm), 1 ml of tin (II) chloride solution (2% SnCl1.2H10 in 10% hydro chloric acid) and immediately close the flask with the stopper. Allow the flask to stand in a water bath of 25-300 for SO to 60 minutes. At the same time carry out parallel experiments using a solution of 2, 4, 6 or 8 µg of As in 10 ml of 10% hydrochloric acid in place of the test sample. Compare the color of the test papers for evaluating arsenic content of the sample. Test paper: Soak strips of filter paper in a saturated ethanolic solution of mercuric bromide and allow to dry. test paper cotton gauze soaked in 5% leod acetate solution and dried 50mm FIGURf. 9. 12. Intermediates a. Apparatus; Chromatographic tube (see Fig. 10). Suitable spectrophotometer for use in the ultraviolet range. b. Column preparation: Prepare a slurry of Whatman powdered cellulose in a 25% ammonium sulfate (very low in iron) solution. If other cellulose is used, the iron content must be very low. Test: Prepare column as directed and pass 200 ml of 25 % ammonium sulfate solution through it. The ultraviolet absorption of the solution must be sufficiently low to avoid interference with the intended analysis. Use about 75 g of cellulose to 500 ml of liquid. Place a small disk of stainless steel gauze in the con striction above the tip of the tube. Pour sufficient of the slurry into the tube to give a column to a height of about S cm in the mouth of the tube. Tap the tube occasionally to ensure a well-packed column. Wash the column with 200 ml of the eluant. c. Procedure; Place 0.200 g of the dye sample in a suitable beaker and dissolve in 20 ml of water. Add approximately 5 g of powdered cellulose. Add SO g of ammo nium sulfate to salt out the dye. Transfer the mixture to the chromatograph tube, 168 Appendix A rinse the beaker with the 25 % ammoni.1m sulfate solution and add washings to tube. Allow the column to drain until How ceases, or nearly so. Add the ammonium sulfate solution to the column at a rate equivalent to the rate of flow through the column. Collect the effluent in 100 ml fractions. Continue until twelve fractions have been collected. Reserve the column and contents until the last fraction has been examined. Mix each fraction well, and obtain the ultraviolet absorption spectra of each solu tion from 220 to 400 mµ. If the spectrum of the twelfth fraction shows the presence of any intermediate, continue collecting fractions until the intermediates present are eluted. 5.8 em 6.4 em 12.8em 2.6l em 2.3 em 4.1t em 2.8 em -L l.7em 0 0.8 ~ t cm 2.9em .l. FIGURE 10. - Chromatographic tube for intermediates. Usually only one intermediate is encountered. Identification and quantitative deter mination is accomplished by comparison of the absorption spectra of the eluted material with the spectra of solutions of the pure intermediates in the same solvent. When more than one intermediate is present in significant quantities in any frac tions, the spectrophotometric data will so indicate. In such case, the amounts of the various intermediates must be determined by the procedure customarily used in spec trophotometric analysis of mixtures of absorbing materials. Some samples contain small amounts of various materials, particularly inorganic salts, that contribute " background absorption." Correction for this is made as fol lows: Determine the amount of such absorption of the fraction collected from the column immediately before and of the fraction immediately following those fractions in which the intermediates are encountered. Subtract one half of the sum of these two determinations from the observed absorbance of the fractions containing the inter mediates. The remainder is taken as the absorbance due to the intermediate present. APPENDIX B TEST SOLUTIONS Ammonia, dilute, PbT• A solution containing approximately lo,( w/w of HH3 in water; it complies with the following test• to 20 ml add l ml of potassium cyanide-solution PbT, dilute to 50 ml with water, and add 2 drops of a 10% sodium sulfide solution in water. No darkening is produced. Anunonia. strong, TS• A solution containing approximately 2"' w/w of NH in water {approximately 15 !). 3 Ammonium cetate citrate TS PbT)• Dissolve 12.5 g of 811111lOnium acetate (CH3COONH4) and 12.5 of ammonium citrate C:s1f40H (COOH) (COONH4)v in water, add ammonia, strong, TS until the solution is alkaline to thymol blue paper and add water to 100 ml. Purify with a 0.002" w/v solution of dithizone in carbon tetrachloride, and finally shake the solution with carbon tetrachloride to remove excess of dithizone. Ammonium citrate TS (PbT}t Dissolve 40 g of citric acid in 90 ml of water. Add 2 or 3 drops of phenol red TS, then cautiously add strong ammonia TS until the solution acquires a reddish colour. Remove any lead that may be present by extracting the solution with 20-ml portions of dithizone extraction TS until the dithizone solution retains its orange-green colour. AntimOnY 1 standard. TS• Dissolve 2.742 g of antimony potassium tartrate in water, and dilute to 100 ml; dilute 5 ml of this solution to 500 ml with wi>.ter, and dilute 5 ml of the dilution to 500 ml with water. Each ml of the solution contains 0.001 mg of Sb. Borax buffer Pb'l'• Dissolve 3.0 g of borax in 90 ml of water, and extract with successive portions, each of 5 ml of 1 volume of diphenylthiocarbazone solution PbT and 4 volumes of chloroform, with vigorous shaking, tmtil the extract is blue or purple in colour; continue the extraction with successive portions, each of 10 ml of chloroform, tmtil the extract is colourless; reject the extracts, and dilute the solution to 100 ml with water. Buffer pH 2 TS• Combine 11.90 ml of 0.2 ! hydrochloric acid and aa.10 ml of 0.2 ! potassium chloride in a 200-ml volumetric flask and add water to volume. Chromate, standard. TS• Dissolve 0.0566 g potassium dichromate (K cr o in 2 2 ) 1000 111 of water; l ml contains 20 I.lg of Cr. 7 Congo red TS• Dissolve 0.,,10 g of Congo red• sodium ,Ldiphenyl-pp'-bis-2-azo..l naphthylamine-4,,-sulfonat~ (c-,.,J1.22•6ol'lfa2> in 20 ml of 90J' ethanol and add sufficient water to make 100 di. · Diphen:rlcarbazide TS• Dissolve 125 mg of diphenylcarbazide l(c6B5.NH.NH)2C!i/ in a mixture of 25 ml acetone and 25 111 water. To be prepared immediately before use. 170 Appendix B Di he lthiocarbazone solution PbT1 Extract 15 ml of a 0.1% w/v solution of diphen;ylthiocarbazone C&f 5.NaN.CS.NH-NH.C6'{5) in chloroform, with 2 successive portions, each of 50 ml of water containing 5 ml of dilute ammonia solution; acidif7 the combined extracts with hydrochloric acid, dilute PbT, and extract with• 100 ml of chloroform; wash the extract with 2 successive portions, each of 10 ml of water, and filter through a dry filter. Determine the approximate strength of this solution b7 the method for determination of zinc (see titanium dioxide monograph), using 5 ml of zinc sulfate, standard 'I'S diluted to 25 ml with water in place of the 25 ml of acid solution used in the determination; dilute with chloroform so that~ ml is approximately equivalent to each ml of zinc sulfate, standard TS. This solution must be freshly prepared. Dithizone 1 extraction, TS1 Dissolve ~O mg of dithizone in 1000 ml of chloroform, and add 5 ml of ethanol. Store the solution in a refrigerator. Before use shake a suitable volume of the dithizone extraction solution with about half its volume of~ nitric acid, discarding the nitric acid. Dithizone1 standard. TS1 Dissolve 10 mg of dithizone in 1000 ml of chloroform. Keep the solution in a glass-stoppered, lead-free bottle,.suitably wrapped to protect it from light, and store in a refrigerator. Hydrochloric acid 1 dilute, PbT1 A solution containing approximately lo:' w/w of HClJ it complies with the following testa make 10 ml alkaline with ammonia, dilute, PbT; and 1 ml of potassium cyanide solution PbT, dilute to 50 ml with water, and add 2 drops of a lOJ' sodium sulfide solution in water. No darkening is produced. H dr drochloride TS1 Dissolve 20 g of hydroxylamine hydrochloride HONH2.HCl in sufficient water to make approximately 65 ml. Transfer to a separatory funnel, add a few drops of thymol blue pH indicator, then add strong ammonia 'I'S until the solution assumes a yellow colour. Add 10 ml of a 4% solution of sodium diethyl dithiocarbamate, mix well, and allow to stand for 5 minutes. Extract this solution with successive 10 to 15-ml portions of chloroform until a 5-ml portion of the chloroform extract does not assume a yellow colour when shaken with a dilute cupric sulfate solution. Add diluted hydrochloric acid until the solution is pink, and then dilute with sufficient water to make 100 ml. Lead. standard. TS1 Dissolve 0.1598 g of lead nitrate /jt,(No-,,)27 in water to which has been added 1 ml of nitric acid and dilute to 1000 ml. Thi'n dilute 10 ml of this solution to 100 ml. Each ml contains 0.01 mg of lead. This solution must be freshly prepared. Potassium c:yanide TS (PbT)1 Dissolve 50 g of potassium cyanide in sufficient purified water to make 100 ml. Remove the lead by shaking with portions of the dithizone extraction solution. Part of the dithizone remains in the aqueous phase but can be removed, if desired, by washing with chloroform. The strong potassium cyanide solution is then diluted to a concentration of 10 gin 100 ml. Silver nitrate, TS1 A 4.2~ w/v solution of silver nitrate (AgNO~) in water {0.25 i). 171 Appendix B Starch mucilap 'l'St 'lriturate 0.5 g of starch, or 0.5 g of soluble starch, with 5 ml of water, and add this, with constant stirring, to sufficient water to produce about 100 ml. Boil for a few minutes, cool, and filter. Starch mucilaee must be recently prepared. Sulfuric acid. dilu.te 1 'l'S1 Mix 57 ml of sulfuric acid (H~o , approximately 90}b w/w) with sufficient water to produce 1000 ml (approximately 2 f).4 Thyltol blue solution 'l'S1 Wa1'111 O.l g of thymol blue (Thymolsulfonephthalein1 C2,H}c,05S) with 4.~ ml of 0.05 ! sodium hydroxide and 5 ml of 90}b alcohol; after solution is effected, add sufficient 20J' alcohol to produce 250 ml. Zinc amalgamate 'l'S1 Add about 10 g of granulated zinc to sufficien~ mercury, about 20 ml to produce a liquid amalgam on cooling, end heat at 150 , with stirring, until the zinc is dissolved. Zinc amalgam ma;y be used repeatedly until the content of zinc is reduced to 0.2" w/g, as determ.ned by the following process. Fill a pycnometer with mercury at 0 25 ;t l, and wei&h. Repeat the operation, using the amalgam. Calculate the proportion of zinc from the fomulat Pe rcentaee w!.w o f zinc• · 13.534• - A 1 0 000875 where A'• wt of amalgam - 13.534 wt of mercury Zinc sulfate1 standard 'l'S1 Dissolve 0.440 g of zinc sulfate in sufficient water to produce 1000 ml, and dilute 5c ml of the solution to 1000 ml with water. Each ml of the solution contains 5 pg of zinc. 172 APPENDIX C IHIEX BY COLOUR Ilf1Bl[ NtMBER (1956) c.11 (1956) No. Common Name or S;monu 10020 c.I. Acid Green 1 10020 Naphthol Green B 10316 Acid Yellow 10316 c.r. Food Yellow 1 10316 Ext. DC Yellow No. 7 10316 Ext. DC Yellow No. 8 10316 Jaune Naphthol S 10316 Naphthol Yellow S 10316 Sulfur Yellow 11020 Bl7rTER YELLOI 11020 Jaune de beurre 11020 OIL YELLOI 11020 c.r. Solvent Yellow 2 11270, 11270 B Chrnoidine 11270, 11270 B Chrysoidine G 11270, 11270 B Chrysoidine GN 11270, 11270 B Chrysoidine Y 11270, 11270 B c.r. Basic Orange 2 11270, 11270 B C.I. Solvent Orange 3 11380 c.r. Food Yellow 10 11380 ~Yellow AB 11380 Yellow AB 11390 c.r. Food Yellow 11 11390 Jaune OB 11390 Oil Yellow OB 11680 Pigment Yellow G 11920 Oil Yellow GG 11920 Soudan R 11920 Sudan G 11920 Sudan Orange 12055 c.r. Solvent Yellow 14 12055 Oil Orange 17' Appendix C c.r. (1956) No. Common Name or Smonym 12055 Sudan 1 12055 Sudan J 12100 c.r. Solvent Orange 2 12100 Oil Orange SS 12100 Orange BN 12100 Orange SS 12100 PD + C Orange No. 2 12120 C.I. Pigment Red 3 12120 DC Red No. 35 12120 Helio Red RL 12120 Pigment Fast Red HL 12120 Rouge Helio 12120 Toluidine Red 12140 Ext. DC Red No. 14 12140 Oil Orange XO 12140 Oil Red XO 12150 c.r. Food Red 16 12150 Soudan R 12150 Sudan Red 12150 Sudan Red G 12156 Citrus Red No. 2 12170 c.r. Solvent Orange 7 12170 c.r. Solvent Red 4 12170 Oil Red 12170 Oil Red 2R 12740 c.r. Food Yellow 12 12740 c.r. Solvent Yellow 18 12740 Oil Yellow XP 13011 c.r. Food Yellow 6 13011 Jaune RFS 13011 Yellow RFS 13015 Acid Yellow G 13015 c.r. Food Yellow 2 13015 E.E.C. Serial No. E 105 13015 FAST YELLOW AB 13015 L-Gelb 1 174 Appendix C c.r. (1226} No. Common Name or Synonym 13015 Jaune Solide 13065 c.r. Acid Yellow 36 13065 Ext. DC Yellow Nos. 1 and 2 13065 Jaune de Metanil 13065 Metanil Yellow 13135 Acid Yellow R 13535 c.r. Acid Yellow 69 13135 FAST YELLC1t/ AB 13445 c.I. Food Yellow 7 13445 Jawie -.n17; li 13445 Yellow 27175 N Specially Pure 14270 Chrysoine 14270 Chrysoine Extra 14270 Chrysoine S 14270 c.r. Food Yellow 8 14270 E.E.C. Serial No. E 103 14270 Jaune de resorcine 14270 I.,..Gelb 4 14275 Chrysoin SGX Specially Pure 14330 c.r. Food Yellow 9 14330 Jaune RY u:no Yellow RY 14600 c.r. Acid Orange 20 14600 Ext. DC Orange No. 3 14600 Naphthol Orange 14600 Orange I 14700 c.r. Food Red 1 14700 FD and C Red No. 4 14700 Ponceau SX 14720 .AZO RUBINE 14720 CARMOISINE 14720 c.r. Food Red 3 14720 E.E.C. Serial No. E 103 14780 Chlorazol Pink Y 14780 c.r. Food Red 13 14780 Red FB 175 Appendix C C.I. (1956) No. Common Name or SrnonYID 14780 Rose chlorazol Y 14815 C.I. Food Red 2 14815 Ecarlate GN 14815 E.E.C. Serial No. E 125 14815 L-ROT 6 14815 Scarlet GN Specially Pure 14910 Benzyl Bordeaux B 14910 c.1. Acid Red 3 15510 C.I. Acid Orange 7 15510 D and C Orange No. 4 15510 Gold Orange 15510 Mandarin G Extra 15510 Naphthalene Orange G 15510 Orange II 15620 c.1. Acid Red 88 15620 Ext. DC Red No. 8 15620 FAST RED 15620 Fast Red A 15620 ROCCELINE 15850 c.1. Pigment Red 57 15850 D and C Red Nos. 6 and 7 15850 E.E.C. Serial No. E 180 15850 Lithol Rubine BK 15970 c.1. Acid Orange 12 15970 c.1. Food Orange 1 15970 Croceine Orangee 15970 Crocein Orange G 15970 Orange ENL 15980 Orange RN 15970 PONCEAU .4GB 15980 c.1. Food Orange 2 15980 E.E.C. Serial No. E 111 15980 L-Orange 1 15980 Orange GGN 15985 C.I. Food Yellow 3 15985 E.E.C. Serial No. E 110 176 Appendix C c.I. (1956) No. Common Name or SYJ10nY111 15985 J aune Orange S 15985 Jaune Soleil 15985 I-Orange 2 15985 Sunset Yellow FCF 16020 c.I. Acid Orange 17 16020 Ecarlate R 16020 Orange L 16020 Scarlet R 16045 c.I. Food Red 4 16045 FAST REDE 16045 Rouge Solide E 16150 c.r. Acid Red 26 16150 C.I. Food Red 5 16150 D and C Red No. 5 16150 Ponceau G 16150 Ponceau MlC 16150 Ponceau R 16150 Ponceau 2R 16150 Scarlet 2R 16155 c.r. Food Red 6 16155 FD and C Red No. 1 16155 Ponceau 3R 16180 Acid Bordeaux B 16180 Bordeaux B 16180 c.r. Acid Red 17 16180 FAST RED B 16185 Amaranth 16185 Amaranthe 16185 Bordeaux S 16185 c.I. Food Red 9 16185 E.E.C. Serial No. E 123 16185 FD and C Red No. 2 16185 L-ROT 3 16230 c.1. Acid Orange 10 16230 c.I. Food Orange 4 16230 D and C Orange No. 3 lTT .Appendix C C.I. (12~6} No. COIIIDIOD Name or SynOnYlll 162~0 Naphthalene Orange Solide G6 162~0 Orange G 1623() Orange GG Crystal 16250 C.I • .lcid Red 44 16250 Cr.ystal Ponceau 6R 16255 c.I. Food Red 7 16255 Coccine Nouvelle 16255 Cochineal Red A 16255 E.E.C. Serial E 124 16255 ~ROT 4 16255 Ponceau 4R 16290 C.I. Food Red 8 16290 E.E.C. Serial No. E 126 16290 ~ROT 5 16290 Ponceau 6R 17200 Acid Fuchsine D 17200 C.I. Food Red 12 17200 D and C Red No. :H 17200 Fast Acid Fuchsine B 17200 Fast Acid Magenta B 17200 Magenta acide solide B 17200 Naphthalene Red B 17200 Red 10 B 18050 .Amidonaphthol Red G 18050 Azogeranine 18050 Azogera.nine B 18050 C.I. Food Red 10 18050 Ext. D and C Red No. 11 18050 Lissamine Red 6B 18050 ~ 18055 Amidonaphthol Red 6B 18055 c.1. Acid Violet 7 18055 c.1. Food Red 11 18055 Ext. D and C Red No. 1 18055 Kiton Red 6B 18055 ~ 178 Appendix C C.I! (1226} No. Coounon Name or Synonvm 18965 C.I. Acid Yellow 17 18965 c.I. Food Yellow 5 18965 Ja\Ule 2G 18965 Yellow 2G 19140 E.E.C. Serial No. E 102 19140 FD Yellow No. 5 19140 Tartrazine 20170 Br\Ul de resorcine 20170 c.r. Acid Orange 24 20170 Light Brown 20170 Resorcine Brown 20170 Resorcine Brown R 20220 Br\Ul de Thiazin R 20220 c.r. Direct Brown 18 20220 Thiazine Brown R 20285 Brown RS 20285 Chocolate Brown HT 20285 C.I. Food Brown 3 20470 c.r. Acid Black I 20470 D and C Black No. 1 20470 Naphthol Blue Black 21000 BISMARCK BROWN 21000 Bismarck Brown G 21000 Bismarck Brown Y 21000 C.I. Basic Brown 1 21230 c.I. Solvent Yellow 29 21230 Oil Yellow 3G 22120 c.r. Direct Red 28 22120 Congo Red 22120 Rouge Congo 22610 c.r. Direct Blue 6 22610 Direct Blue B 22610 Direct Blue 2B 23500 Benzopurpin 4B 23500 BENZOPURPINE 4B 179 Appendix C c,1, (12~6) No. Common Name or S;m2!Vl! 23500 c.1. Direct Red 2 24410 Chlorozal SJst Blue F.F 24410 c.1. Direct Bluel 25105 C.I. Solvent Red 24 26100 c.1. Solvent Red 23 26100 D and C Red No. 17 26100 Soudan III 26100 Toney Red 26105 Scarlet Red 26105 D and C Red No. 18 26105 Sudan IV 26125 C.I. Solvent Red 27 26400 C.I. Acid Blue 120 26400 COOMASSIE NAVY BLUE GN 26125 Oil Red O 26905 C.I. Acid Red 66 26905 Wool Scarlet ~R 27260 c.1. Acid Black 3 27260 Tetracid Brilliant Black B 27260 Naphthol Black~ 27290 Brilliant Croceine 27290 Brilliant Croceine MOO 27290 C.I. Acid Red 73 27290 Crocein Scarlet MOO 27290 Ext. D and C Red No. 13 28440 Brilliant Black BN Special;!,'£ Pure 28440 c.1. Food Black 1 28440 E.E.C. Serial No. E 151 28440 L-Schwarz 1 28440 Noir Brilliant BN 35060 c.1. Direct Brown 39 35060 Direct Brown BC 35060 Direct Brown 3R 41000 Auramine 41000 Aura.mine O l.80 Appendix C c,1, (1956) Ho. Common Name or SYtlOnYDI 41000 Auramine 00 41000 c.r. Basic Yellow 2 42000 C.I. Basic Green 4 42000 Fast Green 42000 Malachite Green 42000 Vert Malachite 42035 Turquoise Blue BB 42040 Brilliant Green 42040 Brilliant Green C:cystals 42040 Brilliant Green Crystal Y 42040 c.r. Basic Green 1 42040 C.I. Pigment Green 1 42040 Emerald Green Crystal 42040 Vert Brilliant 42045 Blue V1lS 42045 C.I. Food Blue 3 42051 E.E.C. Serial No. E 131 42051 I-Blau 3 42051 Patent Blue V 42051 Bleu Patente V 42052 D and C Blue Nos. 7 and 6 42052 Patent Blue A 42053 c.r. Food Green 3 42053 FAST GREEN FCF 42053 FD and C Green No. 3 42053 Vert Solide FCF 42065 FD and C Green No. 1 42065 Vert Guinee B 42065 C.I. Food Green 42065 Guinea Green B 42090 Bleu Brilliant FCF 42090 Brilliant Blue FCF (Biological Stain) 42090 c.r. Food Blue 2 42090 FD and C Blue No. 1 42095 c.r. Food Green 2 42095 FD and C Green No. 2 181 Appendix C c.I. (1956) No. Common Name or S;mom 42095 Lis;bt Green §l Yellowish 42095 Vert Lumiere SF 42100 Brilliant Milling Gaen B 42100 C.I. Acid Green 9 42100 D and C Green No. 7 42100 PATENT GREEN OH 42510 C.I. Basic Violet 14 42510 Fuchsine 42510 Magenta 42170 Acilan Fast Green 100 42535 c.I. Basic Violet 1 42535 llethYl Violet 42535 Methyl Violet B 42535 Methyl Violet 2B 42535 Violet de methyle B 42535 Violet de Paris 42170 C.I. ACID GREEN 22 42536 Benzyl Violet IX:IC Crystals 42536 c.I. Basic Violet 13 42536 Methyl Violet 5B 42561 Acid Fast Violet BG 42561 .lcid Violet 4BN 42561 C.I. Acid Blue 34 42640 Benzyl Violet 42640 BENZYL VIOLET ~ 42640 c.I. Food Violet 2 42640 FD and C Violet No. 1 42640 VIOLET .lCIDE 5B 42650 .lcid Violet 42650 Acid Violet 6B 42650 Acid Violet 411> 42650 C.I. Food Violet 1 42650 Fo~l Violet 42650 Violet 5 BN 42650 Violete acide 6B 42685 Acid Magenta Appendix C C.I. (12~6} No. Common Name or Smo!!.m 42665 Acid Magenta II 42665 Acide Fuchsine FB 42665 C.I. ACID VIOLET 19 42665 FUCHSINE ACIIB 42735 Acilan Brilliant Blue FFR 42735 L-EX'l'. BLA.U 3 42750 Alkali Blue 42750 Bleu alkalin 42750 c.r. ACID BLUE 110 42755 Cotton blue 42755 Soluble Blue 42755 c.I. Acid Blue 22 42755 Water Blue I 42755 Water Blue 6 42755 Bleu soluble a 1•eau 42775 Aniline Blue 42775 C.I. Solvent Blue 3 42775 Opal Blue 42775 Spirit Blue 44045 C.I. Basic Blue 26 44045 Victoria Blue B 44090 C.I. Food Green 4 44090 Vert acide brilliant BS 44090 Wool Green B 44090 Wool Green BS 45100 C.I. Acid Red 52 45160 Rhodamine 6GDN 45100 Xylene Red B 45150 C.I. Pigment Red 62 45150 c.1. Basic Red 6 45150 Rhodamine G 45160 Rhodamine 6G 45160 C.I. Basic Red 1 45170 C.I. Food Red 15 45170 D and C Red No. 19 18:, Appendix C c.1. {12~6} No. Common Name or Synonym 45170 Rhodamine B 45190 c.I. Acid Violet 9 45190 Ext. D and C Red No. 3 45190 Violamine R (Biological Stain) 45350 c.r. Acid Yellow 73 45350 D and C Yellow No. 7 45350 Fluorescein 45350 Fluoresceine 45350 Uranine (sodium or potassium salt) 45380 c.r. Acid Red 87 45380 ~ 45380 c.r. Pigment Red 90 45380 D and C Red hos. 22 and 23 45380 Eosine A 45380 Eosine YS 45380 Eosine Y 45385 c.r. Solvent Red 44 45385 Methyl Eosine 45386 C.I. Solvent Red 45 45386 Eosine a l'alcohol 45386 Eosine S Spirit Soluble 45386 Spirit Eosine 45400 C.I. Acid Red 91 45400 EOSrnE B 45405 C.l. Acid Red 98 45405 Erythrosine BB 45405 Phloxine 45405 Phloxine P 45435 C.l. Acid Red 93 45430 C.I. Food Red 14 45430 E;Q'throsine 4'.>435 Ext. D and C Red Nos. 5 and 6 4':>430 FD and C Red No. 3 45435 Rose Bengale 45430 LB-ROI' 1 Appendix C c.11 {12~6) No. Common Name or S;mon~ 46005 c.1. Basic Orange 14 46005 ACRIDINE ORANGE DH 47005 C.I. Food Yellow 13 47005 Quinoline Yellow 47005 E.E.C. Serial No. E 104 47005 Jaune de Quinoline 50400 c.1. Solvent Blue 1 50400 c.1. Solvent Blue 1 50400 lnduline Spirit Soluble 50405 c.1. Acid Blue 20 50405 Fast Blue B 50405 Induline B 50405 Induline hydrosoluble 50405 lnduline Water Soluble 50420 C.I. Acid Black 2 50420 Nigrosine 58000 Alizarin 58000 Alizarine 50000 Alizarine IV 58000 c.I. Pigment Red 83 58000 D and C Orange No. 15 67410 Alizarin Blue 67410 HLEU d'Alizarine 69800 Bleu Solanthrene 69800 E.E.C. Serial No. E 130 69800 Indanthrene Blue RS 69800 Indanthrene Blue RSA 69000 L-Blau 1 73015 c.r. Food Blue 1 73015 E.E.C. Serial No. E 132 73015 Indigo Carmine 73015 Indigo tine 73015 L-Blau 2 13315 c.1. Pigment Red 86 13315 Helindon Pink BN Appendix C C.I. (12~6l No. Common Name or S;:l!!.O!m!! 73375 Rose helindono 73375 Thioindigo Pink A 74100 C.I. Pigment Black 16 74100 Helioi!l!n Blue G 74100 Phthalocyanine Blue 74260 L-Ext. GrUn 3 74260 Phthalocyanine Green 74260 c.r. Pigment Green 7 75100 c.r. Natural Yellow 6 75100 Saffron 75100 Safran 75100 Croce tin 75100 Croce tine 75100 Crocin 75100 Crocine I i i 75100 Crocus I' 75120 BIXIN 75120 BIXINE 75120 Annatto 75120 c.r. Natural Orange 4 75120 E.E.c. Serial No. E 160 b 75120 L-Orange 4 75120 Or lean 75120 Lycopene 75125 Lycopin 75125 Marigold 75120 NORBIXIN 75120 Norbixine 75120 ROCOU 75120 Terre Orcellana 75125 Calendulin 75130 Carotene (natural) 75130 alpha-Carotene 75130 beta,..Caro tene 75130 gamma.-Carotene 75130 C.I. Natural Brown 5 186 Appendix C c.r. (12~62 No. Common Ne.me or S;i'.!);on~.n 75140 Carthame 75140 Carthamin 75140 Carthe.mus 75140 c.r. Natural Red 26 75140 Safflower 75240 Bois Jaune 75240 c.r. Natural Yellow 8 75240 c.r. Natural Yellow ll 75240 Fus tic 75240 Old Fustic 75240 Yellow Wood 75280 BRAZILIN 75280 BRAZILWOOD 75280 BRESILINE 75280 c.r. Natural Red 24 75290 Campeche 75290 Campeachy Wood 75290 c.r. Natural Black land 2 75290 Haematin 75290 l!aematoxylin 75290 Logwood 75300 Indian Saffron 75300 L-Gelb 7 75300 c.r. !latural Yellow 3 75300 Curcumine 75300 E.E.C. Serial No. E 100 75300 Safran des Indes '75300 Turmeric (Curcumin) 15330 Garance 75330 Madder 75330 c.r. Natural Red 8 75340 Garance 75340 Madder 75340 c.r. Natural Red 8 75350 Garance 187 Appendix C C.I. (1256} No. Common Name or S:aion;m 75350 C.I. Natural Red 8 75370 Madder 75370 Garenoe 75370 Madder 75370 C.I. Natural Red 8 75410 Garenoe 75410 C.I. Natural Red 8 75410 Madder 75410 Garance 75420 Garance 75420 Madder 75420 C.I. Natural Red 8 75430 Graines de Perse 75430 C.I. Natural Yellow 13 75430 Persian Berries 75470 E.E.c. Serial No. E 120 75470 L-ROT 7 75470 Acide Carminique 75470 Carmine 75470 CARMINIC ACID 75470 C.I. Natural Red 4 75470 COCHINEAL 75470 COCHENILLE 75510 Bois de Santal 75510 c.r. Natural Red 22 75510 Sandal Wood 75510 Sanderswood 75520 Alcanna 75520 Alkanet 75520 Alkannin 75520 c.r. Natural Red 20 75520 Oroanette 75520 Oroanettine 75520 Orkanet 75530 Alcanna 75530 Alkanet 188 Appendix C c.I. (1956) No. Common Name or SYJLonym 75530 Alka.nnin 75530 C.I. Natural Red 20 75530 Orcanette 75530 Orcant c,1. (1956) No. Common Name or S;ynonYm 75670 '!'-Gelb bzw. Grlln 1 75690 C.I. Natural Yellow 13 75690 Graineo de Perse 75690 Persian Berries 75695 c.I. Natural Yellow 13 75695 Graines de Perse 75695 Persian Berries 75700 C.I. Natural Yellow 13 75700 Graines de Perse 75700 Persian Berries 75810 Chlorophyll 75810 Chlorophyll copper complex 75810 Chlorophyllin copper complex 75810 c.1. Natural Green 3 75810 E.E.C. Serial No. E 140 75810 E.E.c. Serial No. E 141 75810 I,.GrQn 1 75810 I,.GrQn 2 77000 Aluminium 77000 Aluminium Bronze 77000 Aluminium Flake 77000 C.I. Pigment Metal 1 77000 E.E.c. Serial No. E 173 77000 LB,.Pigment No. 5 77007 Bleu d'outremer 77007 C.I. Pigment Blue 29 77007 C.I. Pigment Violet 15 77007 L-EX'l'. BLAU 6 77007 Ultramarines 77007 Ultramarine Red 77013 c.1. Pigment Green 24 77013 Ultramarines 77266 Acetylene Black 77266 BEN'ZOL BLACK 77266 Carbon Black Appendix C c,1, {1956) No. Conunon Name or Synop,ym 77266 Carbo Medicinalis 77266 C.I. Pigment Black 6 and 1 77266 Gas Black 77266 Noir de carbon 77266 Noir de Fumee 77480 C.I. Pigment Metal 3 77480 E.E.C. Serial No. E 175 77480 ~ 77480 Gold li'lake 77480 Gold Leaf 77489 E.E.C. Serial No. E 172 77489 Iron Oxides 77491 c.I. Pigment Brown 6 and 7 77491 E.E.c. Serial No. E 172 77491 Iron Oxides 77491 LB-Pigment No. 4 77492 C.I. Pigment Brown 6 and 7 77492 E.E.C. Serial No. E 172 77492 c.I. Pigment Yellow 42 and 43 77492 Iron Oxides 77492 LB-Pigment No. 4 \ 77499 C.I. Pigment Black 11 77499 c.I. Pigment Brown 6 and 7 77499 E.E.C. Serial No. E 172 77499 Iron Oxides 77499 LB-Pigment No. 4 77820 ~ 17820 E.E.C. Serial No. E 174 77891 Bioxyde de titane 77891 LB-Pigment No. 3 77891. c.I. Pigment White 6 77891 Titanium Dioxide 191 Al'PEIIDIX J ALPRAllB'rICAL IlllEX COlllllOD name or smon:n> c.r. {1956) 10. Pf!I! No. Acetylene Black 77266 16 Acid Bordeaux B 16180 15 Acid Past Violet Ji!! 42561 15 Acid Puchaine D 17200 140 Acid Yellow G 1,015 122 Acid Yellow R 1'1:55 17 Acid Magenta 42685 104 Acid llagenta n 42685 104 Acid Violet 42650 152 Acid Violet 6B 42650 152 Acid Violet 4Blf 42561 15 Acid Violet 4l!S 42650 152 Acid Yellow 10}16 128 Acide Carm:inique 75470 16 Acide Fuchaine D 42685 104 Acilan Brilliant Blue Ill 427}5 15 Acilan Fast Green 100 42170 15 Ac£idine Orana DH 46005 15 Acridine Orange 2G 46005 15 .llcanna 75520 755}0 15 Alkali Blue 42750 15 Alkanet 75520 755}0 15 Ukannin 75520 755}0 15 Alisarin 58000 15 Alisarin Blue 67410 15 Alisarine 58000 15 Alisarine IV 58000 15 Aluminium 77000 1; AluminiUII Bronze 77000 15 Almdnium Flake 77000 15 § The Common Names are given in bold type. 192 .lppepdµ D Qomon tf!!!! oi: s;m<>n.vm! c111 (;!.2~6l Bo. Pap Bo. •ear,pth 16185 22 uaranthe 16185 22 Amidonaphthol Red 6B 18055 19 hidonaphthol Red G 18050 143 Aniline Blue 42775 18 lnnatto 75120 15 .lnthocyanea 15 Anthocnn1na 15 .lnthocyana 15 Auru:lpe 41000 15 Auramine O 41000 15 Auru:lne 00 41000 15 Azogeranine 18050 143 Azogeranine B 18050 143 J,ZO RUBIHE 14720 105 Baking Brown }5060 17 BEET RED 15 BBRZOL BUCK 77266 16 Benzopurpin 4B 2}500 15 BBlfZOPURPIIRINE ~ 23500 15 Benz;y:l Bordeaux J! 14910 15 Benzyl Violet 42640 107 1IENZYL VIOu:T di 42640 107 Benzyl Violet me Cryatala 425}6 18 BETANilf 15 Bioxyde de titane 77891 93 BISMARCK BR § The COIIIIDon Naaea are given in bold type. 193 4Ppep4:lx p Comaon Iw or SmoJ C.I. {1956) llo. Paa lo. Bleu alltal.in 42750 15 Bleu brilliant PCJ' 420,0 2:7 BUltJ d'alisarine 67410 15 Bleu d'outremer 7700, 20 Bleu Patent4 V 42051 71 Bleu Solanthrine 69800 60 Bleu soluble l 11eau 42755 20 Blue VBS 42045 110 Bordeau B 16180 15 Boie de aantal 75510 75540 19 75550 75560 Boie Jaune 75240 75660 17 Bordeaux S 16185 22 BRAZILill 75280 15 BRAZ1uroop 75280 15 BllESILINE 75280 15 Brilliant Black BX 28440 112 'Specialb Pure" Brilliant Blue 1CF 27 {Biolo~cal StainJ 42090 Brilliant Croceine 27290 15 Brilliant Crocein IIOO 27290 15 Brilliant Green 42040 16 Brilliant Green C£Utala 42040 16 Brilliant Green Crystal T 42040 16 Brilliant Ifill!!!& Green B 42100 16 BROD ff Included but not nuaber.4 114 Brown JIS 20285 115 Bl'IID de chocolate PB Included but not n•bered 16 Brun de riaorcine 20170 19 Brun de thiaain R 20220 20 BRUN PK Included but not n•bered 114 B11l'TER IELUllr 11020 16 A The Common N8118s are given in bold type. 194 Appendix D a Common Naae or SypollYllo c.I. (1956) No. Page No. Calendulin 75125 18 Campeche 75290 18 Campeachy Wood 75290 18 Can thaxan thine 31 CAPS ANT HINE 16 CAPSORUBINE 16 Caramel Included but not nU111bered 16 Carbon Black 77266 16 Carbo medicinalis 77266 16 Carbo VeS!tabilis Medicinalis 16 Carlline 75470 16 CARIIINIC ACID 75470 16 CARIIOISINE 14720 105 Carthame 75140 16 Carthamin 75140 16 Carthamua 75140 16 BETA-.AP0-8'-Carotenal 34 Carotene (natural) 75130 16 Alpha-Carotene 75130 16 Beta-Carotene 75130 16 Beta-Carotene (synthetic) 38 GSlllll&-Carotene 75130 16 Jleta.,,!ao..81-Carotenoic Acid EthYl Ester 43 Chlora.zol Pink Y 14780 19 Chloroph.vll 75810 16 Chloroam;ll COJUl!r Comalex 75810 16 Chlorophyllin Copper Complex 75810 16 Chlorozal Sty: Blue FF 24410 16 Chocolate Brown Included but not numbered 16 Chocolate Brown FB Included but not numbered 16 Chocolate Brown HT 20285 115 ~ The Common Names are given in bold type. 195 Appendix D gommon Name or S,mo!!,ym! c,I. (1956) No. ~ No. Chocolate NS Included but not numbered 16 Chrnoidine 11270 11270 B 16 Chr7soidine G 11270 11270 B 16 Chrysoidine GN 11270 11270 B 16 Chryaoidine Y 11270 11270 B 16 £hr;ysoine 14270 117 Chrysoine Extra 14270 117 Chrysoine S 14270 117 Ch.aaoin SGX SacialJ.'£ Pure 14275 16 c.I. Acid Black 1 20470 18 C.I. Acid Black 2 50420 18 c.I. Acid Black 3 27260 18 c.I. Acid Bluel 42045 110 C.I. Acid Blue 3 42051 71 c.I. Acid Blue 5 42052 19 C.I. Acid Blue 20 50405 17 C.I. Acid Blue 22 42755 20 C.I. Acid Blue 34 42561 15 c.I. Acid Blue 104 42735 15 c.I. ACID BLUE 110 42750 15 C.I. Acid Blue 120 26400 17 c.I. Acid Green 1 10020 18 c.I. Acid Green 9 42100 16 c.I • .&cm GRED 22 42170 15 c.I. Acid Orange 7 15510 18 c.I. Acid Orange .10 16230 lJO C.I. Acid Orange 12 15970 18 C.I. Acid Orange 17 16020 19 c.I. Acid Orange 20 14600 67 c.I. Acid Orange 24 20170 19 c.I. Acid Bed 3 14910 15 C.I. ACID Red 17 16180 15 .! The COIDDOn Names are given in bold type. 196 Appendµ D a Comaon lw or Bmon.yr- c,1, {1956) 10. Pye lo. C.I. Aciel Beel 26 16150 135 c.I. Aciel Bed 44 16250 17 c.I. Acid Bed 52 45100 20 C.I. Acid Bed 66 26905 20 c.I. ACID Bed 1, 27290 15 C.I. Acid Bed 87 45~0 119 C.I. Acid Bed 88 15620 17 C.I. Acid Beel 91 45400 17 C.I. Acid Bed 9, 454,5 19 C.I. Acid Red 98 45405 19 c.I. Acid Violet 7 18055 19 c.I. Acid Violet 9 45190 20 C.I. ACID YIOIBr 19 42685 104 C.I. Acid Yellow 17 18965 20 c.I. Acid Yellow ,6 1,065 18 C.I. ACID lELLOI 69 1,1,5 17 C.I. Acid Yellow 7' 45,50 17 c.1. Basic Blue 26 44045 20 C.I. Basic BrOIID l 21000 15 c.I. Basic Green 1 42040 16 c.I. Basic Green 4 42000 18 C.I. Basic Orange 2 11270 11270 B 16 c.I. Buio Orange 14 46005 15 C.I. Basic Red 1 45160 19 C.I. Ba.sic Red 8 45150 19 c.I. Basic Violet 1 425,5 127 c.I. Ba.eio Violet 1, 42536 18 c.I. Basic Violet 14 42510 18 c.I. Basic Yellow 2 41000 15 c.I. Direot Bluel 24410 16 C.I. Direct Blue 6 22610 17 c.1. Direct BrOWD 18 20220 20 c.I. Direct Brown '9 ,5060 17 A The Common laaea are given in bold type. 197 Appandix p COIIIIOD Bw or sm .! The Common llJaaes are given in bold type. 198 .lppendiJ D COIIIIOD •me or S:mon.ni! C.I. {1956) lo. Pye No. c.I. :rood B.d 16 12150 151 c.I. J'ood Violet l 42650 152 c.I. Food Violet 2 42640 107 c.I. J'ood Yellow l 10:516 128 c.I. l'ood Yellow 2 l:5015 122 c.I. l'ood Yellow :5 15985 83 c.I. J'ood Yellow 4 19140 88 c.I. J'ood Yellow 5 18965 20 c.I. J'ood Yellow 6 l:5011 20 c.I. J'ood Yellow 7 l:5445 153 c.1. J'ood Yellow 8 14270 117 C.I. J'ood Yellow 9 ld,~:50 74 C.I. J'ood Yellow 10 ll:580 18 c.I. J'oocl Yellow 11 ll:590 1e c.I. l'ood Yellow l2 12740 18 C.I. J'ood. Yellow l:5 47005 80 c.1. Batural Black land 2 75290 18 C.I. Batural Brown 5 751:50 1.6 c.1. Natural Brown 10 Included but not nabered 16 c.I. lfatural Green :5 75810 16 c.1. Batural Orange 4 75120 15 c.1. Natural Red 4 75470 16 C.I. lfatural Red 8 75:5:50 75:540 75:550 18 75:570 75410 75420 C.I. lfatu:ral Red 20 75520 755:50 15 C.I. lfatural Red 22 75510 75540 75550 19 75560 C.I. Natural Red 24 75280 15 C.I. Natural Red 26 75140 16 c.1. Natural Red 28 Included b!llt not numbered 18 C.I. Natural Yellow :5 75:500 20 c.I. Natural Yellow 6 75100 19 C.I. lfatural Yellow 8 75240 75660 17 § The Common James are given in bold type. 199 J.ppendix :p CCIIIIDOD B!!! or snoJ c,I. (1956) Bo. Pue No. c.I. Batural Yellow 10 75670 77 c.I. Batlll'&l Yellow 11 75240 75660 17 c.I. Batllral Yellow l} 754}0 75640 75650 19 75670 75690 75695 75700 c.I. Bataral Yellow 26 751}0 16 a.I. Batural Yellow 27 18 C.I. 1181181lt Blaolc 6 and 7 77266 )6 C.I. 1:1.pent Blaolc 11 77499 17 C.I. 1:1.pent Blaolc 16 74100 17 C.I. 1:1.pent Blue 29 77007 20 C.I. Pigment Brown 6 and 7 77491 77492 77499 l? C~I. Pipent Green 1 42040 16 a.I. 1:1.pent Gnen 7 74260 19 C~I. 1:1.pent Green 24 7701} 20 a.I. 1:1.pent lletal l 77000 15 C~I. Pipent lletal } 77480 17 C.I. Pipent Bed } 12120 17 c.r. 1:1.gaent Red 57 15850 17 c.r. Pipent Red e2 45150 19 C~I. Pi.pent Bed 8} 58000 15 C~I~ 1:1.gaent 86 7'}75 17 c.r. Pi.pent Red ,o 45}80 119 c.r. 1:1.gaent Tiolet 15 77007 20 c.r. 1:1.saent White 6 77891 93 c.r. Pi.pent Yellow l 11'80 17 o.r. Pipent Yellow 42 and 4} 77492 17 c.r. Sorbet Blue } 42775 18 c.I. Solvent Bl118 7 50400 17 c.I. Solvent Blue }5 Included but not n•bered 20 c.r. Soluble or.nee 2 12100 18 o.r. SGl.,.nt 0ranc- , 11270 11270 B 16 c.r. SolTent Or.nee 7 l2J.40 18 c.r. Solftnt lled 4 12170 18 c.r. SolTent Red 26100 20 C.I. 8o1Tent Red 25105 20 C.I. SolTeat Bed 27 26125 18 !l The Common ..... are g1 ven in bold type. APP!Bdiz P s-1-or8~ c 1 r 1 ,1!~§l Bo. lM! IP• C.I. Solvent Red 44 45'85 18 c.I. Solftnt Red 45 45'86 20 C.I. Solnnt Yellow 2 11020 16 c.r. Solvent Yellow 5 11,SO 18 c.r. Sol.,.nt Yellow 6 11}90 18 c.r. Solvent Yellow 14 12055 20 c.r. Solvent Yellow 18 12740 18 C.I. Solvent Yellow 29 212}0 18 c.r. Tat Blue 4 6,ec>O 60 Citna Red Bo1 2 12156 16 C90oine Bo1D9lle 16255 74 COCBJIIRlL 75470 16 Coobineal Red A 16255 74 COCBDILI& 75470 16 COJlfl! Bff, 22120 17 COOll&SSJI !J&TT BLUB GB 26400 17 Cotton Blue U7'5 20 Crocein Ol1111P 15970 18 Crooein Orange G 15970 18 Crooein Scarlet MOO 272,0 15 crooeun .mQ2 19 Croce tine 75100 19 ~ 75100 19 Crocine 75100 19 Crooua 75100 19 Crz:•Bl Ponoeau fl l.'250 17 Curouma 75}00 20 CuroUlline 75}00 20 D and C Black lo. l 20470 18 D end C Blue Boe. 7 md 8 42052 19 D end C Green lo. 7 42100 16 D and C Oranp Bo. } 162}0 130 .! The Common ..... are given in bold type. 201 .lppepdix p comaon Bm ot smJ c,1. (1956) Bo. Pao Bo. D and C On.np Bo. 4 15510 18 D and C Oranp Bo. 15 58000 15 D and C Bed lo. 5 16150 135 D and C Bed Boa. 6 and 7 15850 17 D and C Bed Bo. 17 26100 20 D and C Bed Bo. 18 26125 18 D and C Red Bo. 19 45170 146 D and C Bed Boe. 22 and 2, 45,ao 119 D and C Bed Bo. '' 17200 140 D and C Bed Bo. ,5 12120 17 D and C Yellow Bo. 7 45,So 17 Direct Blue B 22610 17 Direct Blue 2B 22610 l'i Direct Brown 1!12 ,5060 17 Direct Bl"0111l :,B ,5060 17 Ecarlate GB 14815 148 Bcarlate B 16920 19 B.B.C. Serial Bo. B 100 15,00 20 E.B.C. Serial Bo. B 101 19 B.B.C. Serial Bo. B 102 19140 88 B.B.O. Serial Bo. B ·10, 14270 117 E.B.C. Serial Bo. E 104 47005 80 E.B.C. Serial Bo. B 105 1,015 122 B.B.C. Serial lo. B 110 15985 83 E.E.C. Serial Bo. E 111 15980 133 B.B.C. Serial Bo. B 120 75470 16 B.B.C. Serial Bo. B 121 helmed but not n1abered 18 B.B.C. Serial Bo. E 122 14720 105 E.B.C. Serial Bo. B 12, 16185 22 B.B.C. Serial Bo. E 124 16255 74 B.B.C. Serial Bo. B 125 14815 148 E.B.C. Serial llo. E 126 16290 138 A The Common Bamee are giffn in bold type. 202 Appendix :p C01111gn ,Ill!! or smoJ c.r, {1956) 1'o. Pap Bo. B.B.C. Serial Bo. B l}O 698()0 60 E.E.c. Serial lo. E l}l 42051 71 E.E.c. Serial llo. E 132 7}015 63 E.E.C. Serial JJo. B 140 75810 16 B.E.C. Serial No. E 141 75810 16 E.E.C. Serial lo. E 150 Included but not numbered 16 B.E.c. Serial lo. E 151 28440 112 B.E.c. Serial lo. B 152 109 E.E.c. Serial lo. E 153 16 E.E.C. Serial No. E 160 b 75120 15 B.E.C. Serial Bo. B 160 c 16 E.B.c. Serial No. E 161 g 31 E.E.C. Serial No. E 162 15 E.B.C. Serial lo. E 16} a-f 15 B.E.C. Serial No. E 171 77891 93 E.B.C. Serial No. B 172 77489 77491 77492 17 77499 E.B.c. Serial Jfo. E 17} 77000 15 E.E.c. Serial lo. E 174 77820 19 B.E.C. Serial No. B 175 77480 17 E.E.c. Serial No. B 180 15850 17 Emerald Green Crystal 42040 16 ell! 45380 119 Eosine J. 45}80 119 ECSINB B 45400 17 Eosine lB 45}80 119 Eoeine a 11alcool 45}86 .io Bosine S Spirit Soluble 45J86 20 Eosine Y 45}80 119 Erzthrosine 454}0 50 Erythrosine D 45405 19 Ext D and C Orange lo. 3 14600 6'i .!!: The Common Names are given in bold type. 203 Appendix D a Coaaon !er or· SJ1lOD.1r c,1, (1956) lfo. Pye •o. Bxt D and C Red Bo. 1 18055 19 Bxt D and C Red lfo. :, 45190 20 Bxt D and C Red lfoe. 5 and 6 454:,5 19 Bxt D and O Red No. 8 15620 17 Ext D and O Jle4 lfo. 11 18050 143 Bxt D and c Rea Bo. 1:, 27290 15 Ext D and C Red No. 14 12140 18 Ext D and C Yellow Jfoa. 1 and 2 1:,065 18 Bxt D and C Yellow Jo. 7 10:,16 128 Ext D and C Yellow No. 8 10:,16 128 Ext D and C Yellow lfo. 9 11:,80 18 Ext D and C Yellow Bo. 10 11:,90 18 J'ast .lcid Fuoheine B 17200 140 Put .lcid Magen ta B 17200 140 Put Blue B 50405 17 Put Green 42000 18 FAST GREEN l'CF 4205:, 56 PAST RED 15620 17 Fast Red A 15620 17 FAST RED B 16180 15 PAST RED B 16045 l20 FAST lELLOI .lB 1:,015 122 PAST YELIIJW 1:,1:,5 17 PD and C Blue No. 1 42090 27 PD and. C Blue Jo. 2 7:,015 63 PD and C Green No. 1 42085 17 PD and C Green Bo. 2 42095 125 PD and C Green No. :, 4205:, 56 PD and C Orange No. 2 12100 18 PD uad C Red No. 1 16155 19 FD and C Red No. 2 16185 22 PD and C Red. lfo. :, 45430 50 !.!he Common JJ•ee are given in bold t1P9. 204 Appendix D goma011 Bame 01: smonJ C1I 1 (1956) lfo. Page No. PD and C Red No. 4 14700 19 J'J) and O Violet Bo. l 42640 107 PD and C Yellow lfo. 5 19140 88 l'J> and C Yellow lfo. 6 15985 83 J'l&TODDthin 20 nuonaoein 45,50 l? J'luonaoeine 45,50 17 J'o~l. Violet 42650 152 J'uobaine 42510 18 J'UCBSID .ICIJB 42685 10~ hatfc 75240 75660 l'i" Garanoe 75,,0 75,40 75,50 1e 15,10 75410 75420 Gu Blaok 77266 16 GlyoJrrbisa 17 CJold 77480 17 Gold Plake 77480 17 Gold Leat 77480 17 Cold Oruip 15510 18 Gl'&illea de Pene 754,0 75640 75650 19 75670 756,0 75695 75700 Gnen 8 440,0 99 Gllinea Gnen B 42085 17 Bae•tin 752,0 18 Buut~lia 75290 18 Pena• Yellow G 11680 17 Bel!!d011 Pink J! 7,,75 17 B1li24!n Bl!!! G 74100 17 Bello Red BL 12120 17 In4Pthnne Blue BS 69800 60 IIIUllthnne Blue BSA 69800 60 Intigo Canine 7,015 63 • ft• c-on 1.-a are given in bold type. 205 Appendµ p COlllllon Bame or Sm~ c,1, {1956) Bo. f!&! Bo. Indian Saffron 75:,00 20 Indiptine 73015 63 Induline B 50405 17 Induline IQ'droeoluble 50405 17 !!4ul i!I• 81111'1i SolubJ! 50400 17 In4uUa• W1ter SolubJ,e 50405 17 Iron Oxides 77489 77491 77492 17 77499 Jaune .&B 11380 18 Jaune de ..tanil 13065 18 Jaune de Quinoline 47005 80 Jaune de r4aoroim 14270 117 Jaune de beurre 11020 16 Jaune 2G 18965 20 Jaune 2717~ 13445 153 Jaune naphthol 8 10:,16 t28 Jaune Oil 11:,90 18 Jaune Oranp 8 15985 151 Jaune RPS 13011 20 Jaune JlT 14:,30 20 Jaune Soleil 15985 83 Jaune aolide 13015 122 Jriton Jled 6B 18055 19 Kr;Jptonnthin 20 Wlau 1 6'8()0 60 i...Blau 2 73015 63 'L-Blau :, 42051 71 I&.Pipeat lfo. :, 77891 93 14-Pipent Bo. 4 77491 77492 77499 17 Lll-Pipent Bo. 5 77000 15 LB,.JlO'.r 1 454:,0 50 L-BU BLltJ:, 42735 15 ~ !he Comon lues an gl.Ten in bold type. 206 .lppepclix p a COIIIIOD Bame or Snaomr- c,1, (1956) Ho. Pa,re Bo. L-EXT BLlU 6 77007 20 L-EXT BLlU 6 77007 20 L-BXT GR'Olf :5 74260 19 L-Gelb 1 1:5015 122 L-Gelb 2 19140 88 I..Gelb :5 47005 80 L-Gelb 4 14270 117 L-Gelb 6 19 I..Gelb 7 75:500 20 L-Gr1tn 1 75810 16 L-Grlll 2 75810 16 I.-Oranp l 15980 l33 I,..Orange 2 15985 83 ~4 75120 15 L-Orange 5 16 I,,,.llO'? :5 16185 22 L-ROT 4 16255 74 WO'!' 5 1629() 138 I,..ROT 6 14815 148 I,..ROl' 7 75470 16 L-Schwars l 28440 112 L,.SCDJBZ 2 109 L-Schwan 3 16 Le.ctoflann 19 Le.ctofl.avi.m 19 Licorice 17 Le.caua Included. but not n,abered. 18 Light Brom 20170 19 Li,mt Gresn §l YeJJ.me 42095 125 Liaauine Red. 6B 18055 19 Lithol l!l!a & 15850 17 Litaua Included. but not nuabered. 18 .! The Comaon ..... are given in bold type. 207 APP!P"1J p CHmOn I ... •£ §med Celt (1956) Bo. Pan ••• Ifflooi 752,0 1€ Lute in 20 LYoopene 75125 18 Ivcopin 75125 18 lladder 75:,:,0 75'40 75'50 18 75'70 75410 75420 lfyenta 42510 18 Magenta acide aolide B 17200 140 Jfalaghi te Green 42000 18 lfandarin G Extra 15510 18 llarigold 75125 18 lfetanil Yellow 1:,065 18 llethYl Eosipe 45'85 18 lletbYl Violet 425'5 127 Met}ql Violet B 425:55 127 Me~l Violet 2B 425:55 127 Met}ql Violet 5B 425'6 18 Baphthal.ene Red B 17200 140 Baphthalene Oranp G 15510 18 Baphth&lene Orange Solide GG 162,0 130 Ba2hthol B!!Qk ~B 27260 18 Ba2hthol Blue ;§lack 20470 18 1la1?!3~ol Gmn B 10020 18 laphthol <>ruse 14600 67 lfal?!3thol Yellow S 10:,16 128 Hiqosine 50420 18 IIOIR Brilliant Blf 28440 112 Hoir de oarbone 77266 16 Hoir de fuNe 77266 16 BOBBIXII 75120 15 lorbixi11e 75120 15 Oil Orange 12055 20 011 Orang ss 12100 18 A The Common ..... are given in bold type. 208 .lppenclix p gommon •m !E s-oaul' c.1, (1956) 10. Pye lfo. Oil Oranp XO 12140 18 Oil llecl 12170 18 Oil lled O 26125 18 Oil lled 21 12170 18 Oil lled XO 12140 18 OIL ?ELLc. 11020 16 Oil Yellow il 11,SO 18 Oil Yellow 3G 212,0 18 Oil Yellow 00 11920 150 Oil Yellow OB 11,90 18 Oil Yellow XP 12740 18 Old J'uatic 75240 75660 17 Opal Bl• 42775 18 Oranp I 14600 67 Oranp II 15510 18 Oranp B1f 12100 18 Orange DL 15970 18 Qrane g 162,0 13C Orange 00 C179tal 162,0 130 Orang GGlf 15980 133 Oranp L 16020 19 Qrane D 15970 18 Orange SS 12100 18 Orcanette 75520 755,0 15 Oroanettine 75520 755,0 15 Orce in Included but not n•bered 16 Oro4ine Included but not n•bered 18 18 ~ Included but not.n•berecl Orkanet 75520 755,0 1"5 Orlean 75120 15 Oraeille Included but not n•bered 18 Paprika extract 16 • 'fbe Common ..... an giTen in bold type. 209 Appendix P Comon Bue or Sm.oJ C111 (1956} Bo. Paa •o. Patent Blue A 42052 19 Patent Blue T 42051 71 PJ.TD'l GBBD (I[ 42100 16 Persian Berne• 754,0 75640 75650 19 75670 756,0 75695 75700 l)loxine 45405 19 PhlOXine p 45405 19 Phthal.oOJ&)line Blue 74100 17 !2!,tll1locl!l!iDe GjE!!D 74260 19 P:1.pent hat Red BL 12120 17 P:l.pent Yellow G 11680 17 Ponceau G 16150 135 Ponoeau 2G 19 POJICBlV ,4GB 15970 18 hnoea11. IIX 16150 135 Pcmceau :a 16150 135 Ponceau a 16150 135 Popoeau ,a 16155 19 Ponceau q 16255 74 l'onoeau 6B 162,0 138 Ponoeau SX 14700 19 Poupre de Pnnoe Included but not 111abend 18 QuproeUp 75670 77 Queroitron 75670 77 Am!!oline Iellow 47005 80 IU..§1 18055 19 Bed lQB 17200 140 !a..l! 14780 19 l!!.J! 18050 143 Rlgliaae 17 baoro!!!! Brown 20170 19 Reaoroi:ae Brown :a 20170 19 210 J.ppendix p S!OMOD •m or BnflDY'II. C.I, (1956) Bo. Pap Bo. lhoclu1De B 45170 146 IJ2od•w1n• G 45150 19 lhod•.,ne 6C 45160 19 Bhocluine 6CDB 45160 19 lho4oeentbiD 20 BI@LlYD 19 ll A 'l'he C0911on B... s are given in bold type~ -211- Appendix P a Common B!!!!! or §;mon.vm- c,1, (1956) 110. P..m No. Soudan IV 26105 20 Spirit Blue 42775 18 Spirit Boeine 45,86 20 Sudan I 12055 2l) Sudan III 26100 20 Sudan IV 26105 20 Sudan Blue II Included but not numbered 20 Sudan Cl ll920 150 Sudan J 12055 20 Sudan Ora.nee ll920 150 Sudan Bed 12150 151 Sudan Red G 12150 151 Sulfur Yellow 10,16 128 Sunset Yellow~ 15985 83 'l'artrazine 19140 88 'l'erre orellana 75120 15 'l'ertracid Brilliant Black B 27260 18 '!'-Gelb bzw. Grlln l 75670 77 Diazine Brown R 20220 20 'l'hioindisc, Pink J. 7,,75 17 'l'itanium )2!oxiy 77891 93 'l'oluidine Bed 12120 17 'l'one;y Bed 26100 20 'l'oumesol Included. but not numbered 18 'l'urmeric (ClU'Cumin) 75,00 20 %!!!:!IUOiae Blue JIB 420'5 20 l11.traurines 77007 7701, 20 Ul t:eamarine Red 77007 20 Ure.nine (sodium or 42,50 17 potaasi\DD salt) Vert acide brilliant BS 44090 95 Vert Guin4e B 42085 l'i Vert brilliant 42040 16 .! 'l'he Coaon llaes an sf.ven in bold t;rpe. 212 .lppenclix P a co-on Bame or S:ynonpr- c,r. {1956} Bo. Pap No. Vert Lmiilre s:r 42095 125 Vert llalachi te 42000 18 Vert Solide :rc:r 42053 56 Victoria Blue B 44045 20 Violamine I {Biolos;!,cal Stain) 45190 20 Viol.&xanthin 20 Violet 5B1f 42650 152 VIOIBl' .lCilE 5B 42640 107 Violete acide 6B 42650 152 Violet de methyle B 42535 127 Violet de Paris 42535 127 Vitudn B2 19 Water Blue I 42755 20 Water Blue 6 42755 20 Wool Green B 44090 99 Wool Greep BS 44090 99 Wool Scarlet 5Jl 26905 20 Xantbopbzlla 20 XYlene Red B 45100 20 Yellow .lB 11380 18 Yellow 2G 18965 20 Yellow 21115B SJ!!cil!;Lt Pure 13445 153 Yellow OB 11390 18 Yellow BPS 13011 20 Yellow BY 14330 20 Yellow Wood 75240 75660 17 Zeuanthin 20 .! 'fbe Common Bame• are given in bold type.