18  METHYL ANTHRANILATE grandparent (8 ). Some dominant loci for this cultivar are very possible in light of this informatinn, despite the fact that i t contains no measurable M A . The cultivar V. 50061 does have some V. labrusca in its ancest. ry, but no high MA o r TVE seedlings were observed in the family from which it was selected (Fuleki. personal communication), so a completely recessive genotype is justifiahle when considering t. his fact. The genotypic formulae are also consistent with the segregation ratios obt. ained (Tables 1 and 2 ) and the information derived from Tables 5 and 6. Using this same logic. i t is suggest. ed that, for TVE in family V. 7218, V. 54077 (TVE = 21 ppm) is of the genotype VuEe, while B. S. 5563 (TVE = 3 ppm) is Vuee o r w E c . For V. 7219, V. 54077 would b, e the same as just indicated, while V. 50061 (WE = 1 ppm) would have a similar genotype to B. S. 5563. The significant F value in Table 6 suggests that these two pollen parents may be genotypically different to some degree. Correlation between these and other characters: Regression analysis indicated that concentrations of MA and TVE were not correlated in any way (data not shown). A significant r? value of 0.6780 for family V. 7219 could be explained by outliers in the data. Neither character could be correlated with "Brix either, al though the misconception is still present that cultivars of labrusca flavor character are consistently low OBrix, high acid t. ypes. A correlation could also not be found between either flavor component .and vigor of winter hardiness (data not shown); this too has oft. en been the concern of many grape breeders, who have felt. that selections with labrusca character are the most vigorous and most winter hardy.

CONCLUSIO~ S Our results have shown that the labrusca flavor character is a complex one genetically, involving five dominant complementary genes plus modifiers. It must be pointed out that this is still an oversimplification, since many other compounds are likely responsible for this flaklor character. From a plant. breeding strindpoint, it is a difficult trait to breed against. Knowledge of the ancestry of the potential parental cultivars, coupled with efficient selection technigues (such as the utilization of the VGFI) appear to be effective weapons in combatting this problem. This study was enrisioned as being a preliminary invest. igation which could provide a base for further st. udy on the genetics of labrusca flavor. The study was 1imit. ed by the lack of availability of entirely adequat. e genetic mat. eria1. Future researchers in this area would do well to hybridize cultivars extremely rich in both MA and TVE with V. vinifera cultivars, which contain none of the former compound and little of the latter. The segregation ratios obtained from such crosses should yield valuable information. The examination of prog enies from selfed high VGFI cultivars should also be of great interest. Finally, much work is still required on the biosynthe sis of MA, TVE, and other flavor compounds in grapes. Nnt u n t i l more details are provided on the biochemical aspects of these flavor constituents w i l l the genetics of grape flavors be comp1et. ely elucidated.

LITERATURE ClTED I . Rradt, 0. A. The grape in Ontario. Ont. Min. Agrir. Food 2. Casimir. D. .J.. d: C. hhyer. and L. R. Mattick. Fluorometric determination of methyl anthranilate in C~ ncord grape juice. J. A. O. A. C. 59: 269 72 (1976). Puhl. 467 (1972).

3. Clore. 1%. J.. A. M. Neuberl, G . H. tarter, D. W. Ingalsbe, and V. P. Brummnnd. composition of Washington produced Con. cord grapes and juices. Wash. Agric. Expt. Sta. Tech. Bull. 48 (19651.

4. Fuleki. T. Changes in the chemical composition of Concord grapes grown in Ontario during ripening in the 19'70 season. Can. J. Plant Sci. 52: 863 7 (1972).

5. Fuleki. T. Methyl anthranilat. e, volatile esters, and antho. cvanin content of grape varieties grown in Ontario. Can. Hort, Council: Report of the Cmt. e. on Hort Res. 153 (19741.

6. Fuleki. T. A new objective index for the earl? screening of grape seedlings based on flavor character. Can. Hort. Council: Report of the Cmte. on Hort. Res. 179 80 (1975). 7. Fuleki. T. Vineland Grape Flavor index  a new objective measure for the early screening of grape seedlings based on flavor character. Can. Hort. Council: Report of the Cmte. on Hort. Res. 1E2 3 (1976). .

8. Galet. P. Precis d'Ampelographie Pratique. Tome H I . De.

9. Hedrick. t'. P., N. 0. Booth. 0. M. Taylor. R. Wellington. han, hlonrpellier (1976).

and M. J. Dome?. The Grapes of New York. Fifteenth Ann. Report, :

X. T. S. Agric. Expt. Sta., Vol. 3, Part 11. .J. B. Lyon, Albany. N. Y.! (1908).

10. Hill. Lj. T. Colorimetric determination of fatty acids and 11. Holley. R. W., B. Stoyla, and A. D. Hollev. The identifica 'esters. Ind. Eng. Chem. (Anal. Ed.) 1fX17 9 (19461.

tion of some volatile constituents of Concord grape juice. Food Res. 2@ 326 31 (19.55). 12. h'elson. R. R., T. E. Acree, C. Y. Lee. and R. M. Butts. hfethyl anthranilate as an aroma constituent in American wine. J. Food Sci. 4257 9 (19i7). 13. Xeudoerffer, T. S., S. Sandler, E. Zubeckis, and M. D. Smith. Detection of an undesirable anomaly in Concord grape by gas chromatography. J. Agric. Food Chem. 13584 8 (1965).

14. Ontario Crop Protection Committee. Fruit Production Rec ommendations: Ont. Min. Agric. and Food Publ. 360 (1980). 15. .Power, F. B.. and V. K. Chesnut. Examination of authentic grape juices for methyl anthrani1at. e. J. Agric. Res. 23: 47 53 (1923). 16. Reynolds. A. G. The inheritance of methyl anthranilate and total volatile esters in Vitis spp. MSc Thesis. Univ. of Guelph, Guelph. Ontario (1980).

13. Robinson. W. B., N. J. Shaulis, and C. S. Pederson. Ripening studies ofgrapes grown in 1948 for juice manufacture. Fruit Prod. J. and Amer. Food Manuf. 29( 2): 36 7 (19491..

18. Sale. J. W.. and J. B. N'ilson. Distribution of volatile flavor in grapes and grape juices. J. Agric. Res. 33: 301 10 (1926). 19. Stern, D. J., A. Lee, W . H. McFadden, and K. L. Stevens. Volatiles from grapes  identification of volatiles from Concord essence. J. Agric. Food Chem. 151100 3 (19671. 20. Stevens. K. L., A. Lee, W. H. hlcFadden. and X. Teranishi. yolatiles from grapes. I. Some volatiles from Concord essence. J. Food Sci. 3 0 1106 5 (1965).

Am. J. Enol. 'Vitic.. Vol. 33, No. 1, 1982 METHYL ANTHRANILATE  19 descendance de Vilis tini/ rra. Colloque C. N. R. S. 'Facteurs et Regulation de la Maturiti. des Fruits.": 335 9 11974). . 25. Wagner. R., N. Dirninger, V. Fuchs, and A. Rronner. Study of the intervariet. al differences in the concentration of volatile constituents (linalool and geraniol) in the arnrna of the grape. Interest of such analyses for the appreciation of the quality of the harvest. Int. Symp. Qual. Vintage: 137 42 (1977). cw2 26. Wagner. R., N. Dirninger, V. Fuchs. and A. Bonner. Premiers resultats concernant ]'etude gPn6tique de ronstifuants volatils im portants de l'irome des raisins dans deux descendances de Vitis L! inift= ra L. I P Symp. CbnPtique AmClior. Vigne: 419 34 (1978).

Am. J. Enol. Vitic., Vol. 33, No. 1, 1982 1024. :E programmed to 165'C at 2" C/ min, with a helium gas flow of 5ml/ min withoutsplitting. %

5 .: and the total ion trace wasrecorded at 20 eV, whilst the mass spectra were run at 70 eV and 50 p ~, s tea stair The technique5 6 did not employ an internal standard. Sel del= 2.4. Identification of components const The compoundswere identified by comparison of their g. c. retentim times and mas spectra with I more those of commercial samples and with those reported by other authors.*+* prese i. . injector valve temperature was 220° C. The separator and m. s. s~ nce were maintained at 2 6 0 %~ 1 four I I .__

.. ..  ....

4

.. i 3. Results and discussion The compounds identifipd are listed in Table I , together with the number of the chromatographic peak in which each was found. The relative amounts in the headspms of new and aged wine can Table 1. Compounds identified in red wine heafspace Identification Peak no. Formula Component Rcfernce r. t. ms.

i CH40 Methanol 1 CZH~ O Ethanol 1 X C3H a 0 n Propanol I CiHpO i Propanol I CCHaOz E t h y l a c e t a t e 1 X CsHmOz n P r o p y l a c e t a t e 1 x . CSHIOOZ i P r o p y l a c e t a t e 1 X C6H1202 Ethyl n butyrate 1 X C: H1402 Ethyl i valerate . I X CeHs Indene . 1 .x X 3 C6HltO n Hexanal 1 X C7H1402 . i Pentyl acetate I X 4 CsHlzO 3 Methyl I butanol I  x X CsHlzO 2 Methyl I butanol 1 X 5 CaHlsOz . Ethyl caproate . 1 X X X : 2 C4H 100 3" ethyl I propanol 1  x .

X X X X X X X X X 6 CeHlsOz n Hexyl acetate 1 8 C5Hl003 E t h y l l a c t a t e I 9 CsH14O I Hexanol .. I X .. 7 C; HloOz t< Ethoxymethyl) furaa X C6H 1 2 0 trans 3 Hexeno I X X CsH 1 2 0 I1 cis 3 Hexenol .. I CaHtsO n Nonanal 13 3 X X 16 CloHzoOz Ethyl octanate  I CTHIOO 17 Benzaldehyde I x . X CIOHZOO Decunal 3 19 I X X CIIHZZOZ Ethylpelargonate . .

22 I CiH803 fihyI 2 furoare X 23 1 GHnO Aremphenone . .

24 1 CeH1oOz Ethyl Cenzoate 1 . 25 C1zH~ 402 Ethyl caprate I x 26 CsH 1 4 0 4 Diethyl sureinate 1 X CI nHs Napthalene 29 I 32 T~ ethyldihydronaphthafene CloH1202 EtIplpheny7aretate. X 34 I  .x ClnH~ rOz IPhene~ hyl acefure X . 35 1  x CnHsNOz Aicrhyl attthranilate 36 4 C14HZeO: Ethyl lawme 37 1 CsHlnO .'b~ hcny&? thanol . . 1 X X X X 1 .

.)8 CloH1sO Linalool x . X x X X X X X x X X 28 . C13H16 X X The componenls found for the first time in wine by headspace tecknique. are given in italia. The components found for the fifst time in wine. are in bold. I. &<& li a d R. \'& ni Sithout splitting. Thc maintained at 26O. c n at 70 eV and 50 FA,

md mass spectra with ' the chromatographic :w and aged wine can entitication ?x sd' x X X x X X X X X X X .x x X ' X X x X X x ' X X ,x ..

. ' , x x msoc Rdesco 1973 I i 2 ! i i Rirbesco 1966 "J Figure 2. G. c. traces of. 1973 and 1966 wines headspaces. Among &e constituents identified I ,I .6 trimethyl l ,2 dihydronaphthalene (dehydroionene) and 2( ethoxymethyl) furan, were found for the first time in wine aroma. 1,1,6 TrimethyI l, 2 dihydro naphthalene had been previously described in strawberry, l2 peach 13 and tobacco leaves, 14 and in .

rum, 15 passion fruit, I6 and peach fruit. '; Since we were unabie to detect it in Swiss Garnay and Pinot Noir red wines, 18 its presence in the aroma together with other leaf constituents such as cis and frans 3 hexenols, could be explained by differences in some operations of wine making. The ethyl ether, 2( ethoxymethyl) furan: already known in white bread ar0rna1~. 20. has a fruity, aromatic and slightly pungent smell. The confinement of this compound io aged wine suggests that ageing processes involving ethanol reactions occur not only by esterification of carboxylic acids, as other authors have observed6J1* 21.22 hut also by etherification of Maillard formed alcohols, such as 2 furfuryl alcohol. Further work is in progress on the genesis of these compounds. i f 4. Conclusion i Headspace analysis, in which one measures only the volatile constituents present in the atmosphere of the glass in their natural equilibrium conditions, could become the technique of choice for 1 following the evolution of wine "bouquet" and thus supplement the information on the total aroma obtained by standard solven't extraction techniques. i Acknowledgements 1 We thank Professor G. Montedoro for useful discussions, and Dr G. Philippossian for the synthesis of ethyl furfuryl ether. . j References 1 1. 2. 3. 4. .5. 6. 7. 8. 9. 10.

, 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. c; TAB C. USlFDA REGISTRATION OF METHYL. ANTHRANILATE AS A GENERALLY RECOGNIZED AS SAFE (GRAS) MATERIAL.

TABLE OF CONTENTS 1. 21 CFR 1182.60  Synthetic flavoring substances andadjuvants;

2. 21 CFR 184.1021  Benzoic Acid Methyl anthranilate (methyl 2 aminobenzoate) 8 182.40 21 CFR ChA (4 1 91 Edition)

§ 182.40 Natural extractives (solvent free) used in conjunction with spices, seasaninp,

Natural extractives (solvent free) used in conlunction with spices. seasonings. and flavorings that are generaflyrecognized, as safe for their intended use, within the meaning of section 409 of the Act, are as follows: and flavorings.

~

canmon name Botankal m of plant saum W t kemel (persic oil) PlUOUS MneniBC8 L Peach kernel (persic air), .................".... ". f" penica %b. 81 % Peanut stearine . ............................... ..~.".....~ Arachis hvpooa~ L Persic o i l (s e e apicot kwnel and peach kemdL". ~uince seed ........................................ ......" cvdonia OMOnpa Mil&.

E42 F R 14640, Mar. 15.1977. as amended at 47 FR 47375. Oct. 26,19821 I182.50 Certain other spices, seasoninge. essential oils, oleoresins, and natural extracts. Certain other spices, seasonings, essential oils, oleoresins. and natural extract8 that are generally recognized 8s d e for their intended use. within the meaning of section 409 of the Act, are as follows:

B 182.60 Synthetic flavoring substances Synthetic `flavoring substances and adjuvants that are .generally recog nized as safe for their intended use, within the meaning of section 409 of the Act, are as follows: Acetaldehyde (ethanal). Acetoin (acetyl methylcarbinol). Anethole (parapropenyl anhole). Benzaldehyde (benzoic aldehyde). N Butyric acid (butanoic acidl. d or I Carvone tcarvol). Cinnamaldehyde (cinnsmtc aldehyde). Citral (2.6 dimethyloctadien 2.6 aZ 8. gem Decanal (N decylaldehyde, capraldehyde. nial. nerd).

c 10). capric aldehyde, caprinaidehyde. aldehyde and adjuvants.

Ethyl acetate. Ethyl butyrate. 3 Methyl 3 phenyl glycfdic acid ethyl ester (ethyl methyl Dhenul fclucidate. so called strawberry aldkhyde. 6 1 6 aldehyde). Ethyl vanillin. Geraniol (3.7 dfrnethyl 2,6 and 3,6 octadien 1 02). Geranyl acetate (geraniol acetate).. Limonene (d, I, and dl). Linalool (linalol, 3.7 dimethyl 1.6 octadien 3 02). Linalyl acetate (bergamol).. Methyl anthranilate tmethyl 2 aminoben

Piperonal (3.4 methylenedioxy be& d*

Vanfwn. E42 FR 14640. Mar. 15. 1977, as amended at 43 FR 47724. Oct. 17.1978: 44 FR 3963. Jan.

51907, Nov. 15. 1983: 54 FR 7402. Feb. 21, 19, 1979: 44 F'R 20656. Apr. 6. 1979; 48 F'R 19891 ff 182.70. Substances migrating from, cotton and cotton fabrics' used in dry food packaging. Substances migrating to foodfrom cotton and cotton fabrics used in drY food packaging that are generally r y ognized as safe for their intended use, within the meaning of section 409 of the Act. are as follows: Beef tallow. Carboxymethylcellulose. Coconut oil, refined. Cornstarch. Gelatin. Japan wax. Lard. Lard oil. Oleic acid. mate).

.. hyde, heliotropin).

404 Food a Pearrut c Potato s sodium sodium I sodium I sodium 1 SOY Talc Tallow (1 T ~O W n Tapioca 8 Tetrssod me!& st zinc m a E42 FR 1 43 FR 1 May IS, FR 27801 Nov. 7. 1' FR 51909 52445, 5; May 7.1s f4 18290 . fram s u m in food paper a recogniz use, wit1 of the A Alum <do 12SAlum moniun Aluminlu Aluminm Cellulose CaSeil3.

Diatomac cornstarc Ethyl cell Ethyl van Glycerin. oleic add Potsssiun Silicon d i t sodiuni al sodium cl Sodium hj Sodium h: sodium PI Sodium si sodium sc Sodium tr SorbitoL Soy protei Starch ac Starch. pr starch. ut Talc.

Zinc hydn Vanillin Zinc sulfa E42 FR 141 0 184.1011 tc) The ingredient is used as a flavor ing agent as definedin H 170.3( 0)( 12) of this chapter: leavening agent as de fined in 8 170.3( 0)( 17) of this chapter: and pHcontrol agent as defined in 0 170.3( 0)( 23) of this chapter. (d) The ingredient is used in foods at levels not to exceed current good man ufacturing practice in accordance with 0 184. UbXl). Current good manufac turing practice results in maximum levels, as served, of 0.05 percent for baked goods as defined in g 170.3tnM1) of this chapter; 0.005 percent for non alcoholicbeverages as defined in 0 170.3( n)( 3) of this chapter; 5.0 per cent for condiments and relishes as de fined in § 170.3tn)( 8) of this chapter: 0.45 percent for dairy product analogs as defined in 0 170,3tn)( lO) of this chapter; 0.3 percent for fats and oil as defined in H 170.3tn1( 12) of this chap ter; 0.0004 percent for frozen dairy desserts as defined in f 170.3tn)( 20) of this chapter: 0.55 percent for gelatin and puddings as defined in 8 170.3tn)( 22) of this chapter; 0.1 per cent for gravies as defined in § 170.3tn)( 24) of this chapter; 0.3 per cent for meat products as defined in P 170.3tn)( 29) of this chapter: 1.3 per cent for snackfoods as defined in § 170.3( n)( 37) of this chapter; and 0.02 percent or less for all other food cate gories. (e) Prior sanctions for adipicacid different from the uses established in .this section do not exist or have been waived. E47 FR 27810. June 25.19821 I 184,1011 Alginicacid. (a) Alginic acid is a colloidal, hydro philicpolysaccharide obtained from certain brown algae by alkaline extrac tion. (b) The ingredient meets the specifi cations of the Food Chemicals Codex,, 3d Ed. (1981). p. 13. which is incorpo rated by reference. Copies are avail able from the National. Academy Press, 2101 Constitution Ave. NW., Washington, DC20418, oravailable ' for inspection at the Office of the Fed . eral Register, 1100 L St. NW., Wash. ington. DC 20408. k ) In accordance with 8 184.1( b)( 2), the ingredient is used in food only within the followingspecific limita. '

tions:

soup and mlxea ?P 5 170.3( n) (40) Of this chapter.

(dl Prior sanctions for this ingredi ent different from, the use established in this section do not existor have been waived.

C47 F'R 41375, Oct. 26.19821 I 184.1021 Benzoicacid.

zenecarboxylicacidtC7He0,). occur ring in nature in free and combined forms. Among the foods in which ben zoic acid occurs naturally are cranber ries, prunes, plums, cinnamon, ripe doves, and most berries. Benzoic acid is manufactured by treating molten phthalic anhydride with steam in the presence of a zinc oxidecatalyst. by :

the hydrolysis of benzotrichloride, or by the oxidation of toluene with nitric acid or sodium bichromate or with air in the presence of a transition met@ salt catalyst. (b) The ingredient meets the specifi cations of the "Food Chemicals Codex." 3dEd. ( 19811, p. 35, which is incorporated by reference. Copies may be obtained from the National Acade my Press, 2101 Constitution. Ave. NW.. Washington. DC20418, or mayex amined at the Office of the Federal Register, 1 100 L St. ' NW.. Washington,

tc) The ingredient is used as an anti microbial agent as defined in 170.3( 0)( 2) of this chapter, and as a flavoring agent and adjuvant as de fined in 0 170.3( 0)( 12) of this chapter. (d) The ingredient is used in food at levels not to exceed good manufactur ing practice. Current usage results in a maximum level of 0.1 percent in food. tThe Food and Drug Administration (a) Benzoic acid is the chemical ben .

DC 20408.

422 Food has cant1 woul (e) ent lishe that ter, 142 F 49 m § 184 CAS (a cal I ered fatt Pare fern tion in c (b cati Cod incc be c Wa ami Reg DC (C ing 0 1: ((

acc not Pra ser go( Ch: fin 0.0 fin f 0 1 0 1 tin 5 1 pr' t h f i I O.( f iI fo er OUS pra ar 91 Edition)

cific limita

his ingredi established st or have ?mica1 ben 3A occur combined .which ben re cranber .mon, ripe snzoic acid ng molten ?a m in the ltalyst, by :1loride. or with nitric 3r .".;$ h air .. which is 'opies may .la1 Acade Ave. NW., .lay be ex le' Federal uslhington,

a, an anti fined in .d n d a s a nt as de 5 qhapter. in1 food at .Wfactur 3 S ~l t s in a nibtration t., b food. Food end Qrug Administration, HHS has not determined whether signifi cantly different conditions of use would be GRAS). (e) Prior sanctions for this ingredi ent different from those uses estab lished in this section, or different from that set forth in part 181 of this chap ter, do not exist or have been waived.

142 mZ 14653, Mar. 15. 1977, as amended at 49 FR 5610. Feb. 14.19841 I181.1025 Caprylic acid. (a) Caprylic acid CCH,( CH,), COOR, CASReg. No. 124 07 21 is the chemi cal hame for octanoic acid. It is consid ered to be a short or medium chain fatty acid. It occurs normally in vari ous foods and is commercially pre pared by oxidation of n octanol or by fermentation and fractional distilla tion of the volatile fatty acids present in coconut oil. (b) The ingredient meets the specifi cations of the "Food Chemicals Codex," 3d Ed. (1981). P. 207. which is incorporated by reference. Copies may be obtained from the National Acade my Press, 2101 Constitution Ave. NW.. Washington, DC 20418, or may be ex amined at the Office of the Federal Register, 1100 L St. NW., Wasfiington, DC 20408. <e) The ingredient is used as a flavor ing agent and adjuvant as defined in 8 170.3( 03( 123 of thischapter. (dl The ingredient is used in foods in accordance with § 184 1( b)( l), at levels not to exceedgood manufacturing practice. Current good manufacturing practices result in maximum levels, as served, of: 0.013 .Percent for baked goods as defined in 0 170.3( n)( l) of this chapter; 0.04 percent for cheeses as de fined in § 170.3tn)( 5) of this chapter; 0.005 percent for fats and oils as de fined in H 170.3( 11)( 12) of this chapter, for frozen dairy desserts as defined in 9 170.3tnX20) of this chapter, for gela tins and puddings as defied in H 170.3( n)( 22) of this chapter. for meat products BS defined in 9 170.3tn)( 29) of this chapter, and for soft candy as de fined in P 170.3tnX38) of this chapter; 0.016 percent for snack foods as de fined in 5 170.3( n)( 37) of this chapter; and 0.001 percent or less for all other (e) Prior sanctions for this ingredi ent different from the uses established ' food categories. in this sewon a0 noz exist or nave been waived.

[43 FR 19843. May 9,1978, as arnt FR 5611, Feb. 14.19841 I 184.1027 Mixed carbohydrase w e enzyme product. fa) hlixed carbohydrase and protease enzyme product is an enzyme tion that. includes carbohydrase and the culture filtrate resulting xrom a pure culture fermentation c * ~ ~~ ~

1 pathogenic strain of B. lichen (b) The ingredient meets the specifi cations of the Food Chemici 3d Ed. (1981). p. 107. which is incorpo . rated by reference. Copies are avail able from the National Academy

Press, 2101 Constitution A" ""

I Washington, DC 20418, or available eral Register, 1100 L St. NV ington. DC 20408.

the ingredient is used in fooc' 1 limitation other than currt manufacturing practice. The tion of this ingredient as generally rec ognized as safe as a direct human food ingredient is based upon the following current good manufacturi" conditions of use: (1) The ingredient is used as an this chapter, to hydrolyze protelns or carbohydrates. (21 The ingredient is used in the, fol lowing foods at levels not to exceed current good manufacturing    ~

alcoholic beverages, as defined in 0 170.3( n)( 2) of this chapter, candy. nutritive sweeteriers, and protein hy drolyzates.

148 F R 240, Jan. 4.19833 8 184.3061 Lactic acid. (a) Lactic acid (C3H603. CAS Reg. Nos.: DL mixture, 598 82 3; L isomer, 79 33 4; 0 isomer, 10326 41 7). the chemical 2 hydroxypropanoic acid. occurs naturally in several foods. It is produced commercia"" mentation of carbohydrates glucose. sucrose, or lactose, or by a protease activity. It is obtai:  J I for inspection at the Office of "

1 (c) In accordance wnn 3 18% 1( b)( i),

* '  I enzyme, as defined in § 170. I lL'  ~ e .. TABLE OF CONTEWS 1 . Fenaroli'sHandbook of FlavorIngredients, Second Edition,

2. Perfume Synthetics and Isolates Volume 2 I \

,

! t FENAROLI'S HANDBOOK FLAVOR INGREDIENTS Second Edition Volume 2 Edited, Translated, and Revised bY THOMAS E. FURIA d NICOU) BEUANCA QVtl47p 1 Palo Alto. Culifortzia Published by Adapted from the Italian language works of PROF. DR. GIOVANNI FENAROLI Director, Center for Srudies of Aromatic Substances University of Milano. Milano. Italy CRC PRESS. Inc. 18901 Cranwood Parkway Ckveland. OhioU128 .. . ..

..

e"

. ' , I' €46 Fenaroii'r Handbook of Flavor Ingredients Stmture Pbysical, 'chemial characteristicsJ APP= iUKC *= Y Moleculu weight Melting point Wii point Congealing point SpeciBc gravity Refractive index Cresol mtmt Solubility Organokpiic char acteristics SyMwsis Natural OCCII~~ CLKC Regulatory status P c m ~l methyl ether pMethoxy toluene Methyl pcrcsol Methyl ptolyl ether Methyl 2 aminoknzarre Methyl u aminoknzoate (?" CH, .

CH,

Colorless liquid 122.17 0.96do. 970 st i5/ 25'C2 1.5100 1.5130 at 20'C2 Not more than 0.5 0iz Fungent odor suggestive of ylang ylang By methylation of pcresol Reported found in the oils of ylang ylang cananga, and 0 t h ~~  .

Non alcoholic beverages 1.7 ppm Ice crram. ices etc. 2.7 pprn kked goods 7.6 Ppm jehtins and puddings 0. W. o ppm hdiments 2.0 ppm A 121.1164; FEMA No. 2681 h M i Y 4.8 pprn iYrUPS 8.0 pprn REFERENCES ,

For References 1 5, Jee end. of Part 111. 6. Mattick et d.. 1. Agric. fmd Chrm.. 4. 331. 1963. 7. Roger, Food Technof.. 6.309. 1961. v t Colorless to pale yellow Itquid uith bluish 98O, mln' 151.17 24 25 C i32'Cat I? mm Hg . 23.8. C (24 C )

t. 161 1.169 at IS .25 C: ' 1.16.$ 0 at 25 6 1.5820 1.5810 at 20 C: ' 1.5802 at I5 C fluorescence Characteristic orange flower odor. and dlghtly brtter. pungent taste By heating anthranilic acid and meth> i and subsequent distiliatlon alcohd in the preKnce of sulfzric s:: d Reported found inxw al essential oil*: neroli. orange. bergamot. lemon. man darin. jasmine. tuberose, gardenla. champaca. ylang ylang. and o t h m : also in the juice and oil of I :1t5 iabrusrrr*~ '

Non alcoholic beverages 16 ppm Alcoholic beverapcs lce cream. ices. etc. 0.20 ppm Candy II ppm Baked goods 56 pprn Selatins and puddings 13 ppm IO ppm Cheuinggum 2.200 ppm FDA GRAS: FEMA No. 2682 . *

I ' . ..

a I and IsoZates BY PAUL 2. BEDOUK14W, PH. D. Conlpagnic Parenlo, Inc.

D. VAN XOSTRAND  COMPANY, INC. *NEW VORK , p NEW TORK I). Van Nodrand Company, Inc., 250 Fourth Avenut, New Yort 3 TORONTO D. Van Sostrand Company (Canada), Ltd., a8 Bloor Street, Toronto .3f~ crnillan'& Company, Ltd., St. Martin's Street, London, W. C. 2 LOXWN ANTHRANILATES Methyl Anthranilate N Methyl Methyl Anthranilate,

CfJHfrO2N 3101. Weight 151.08 Dimethyl Anthranilate C* II,, O~ X Siol. Weight 165.09 Although Fritzsche's discovery of anthranilic acid dates hack to 1841, the importance of its esters, especially the methyl ester, was not r e a l h a until 1899 dien Walbaum noted its occurrence in neroli oil. Methyl anthra nilate has since been found iu .a number of other essential oils and bas attained !onsiderableimportance as a perfumery material. Although it possesses a powerful and pleasant odor, the use of anthraniiic acid and its esters in perfumes is limited, however, because the amino group reacts most readily with various aldehydes, giving highly colored Schiff bases which are undesirable. Large amounts of methyl, anthranilate or other esters a n n o t therefore ' b e used in most cases, as for example in cosmetics and soaps where discoloration is a serious problem. Derivatives of anthranilates where the amino group ia bound say, with an acetyl group are usetes for perfumery purposes, since they are either extremely weak in odor or odorless. Large quantities of methyl anthranilate are manufactured annually for the flavor industry, most popular grape sodas depending mainly upon methyl anthranilate for the characteristic grape flavor.

Occurrence. XethyI anthranilate has been found in a nunlber of essential oils, being first rrported in neroli oil by Walbaum. 1 This oil is obtained from the flowers of the bitter orange tree (Citrus biguradiu R.). It has also been identified in the oil from jasmine .flowers cultivated in France. 2 Practically all jasmine oils from flowers cultivated in other countries contain SIWIII quantifies of methyl anthranilate. Other source include : f Mol. Weight 165.09 Oil of rcrcir Bowers (Robiniu psc* doocacia L.) 8 Oil of tuberoe (Polyunlhcr tutcrotu L.) ' oil of nalfbov; en (Chciranfhw cheiri L.)@ oil of gardenia dowen * Oil from the leaves of the bergamot tree' Orange oil ' 3fetlq l antbranilat; has also been found in grape juice.) X methyl methyl antbranilate is known to occur in mandarin mendah leaf oil, ll oil of Koenlfctia efhclia," oil of rue,!! and in the oil of hyacinthflowers (Byacinlhtcs oricnfulis L.)."

Preparation of Anthranilic Acid. Anthranilic acid was discovered in the course of studies on indigo. In 1841, Fritzsche treated indigo with concentrated solution of potassium hydroxide at elevated temperatures and obtained a greenish yellow solution. This solution on acidibcation gave a dark brown precipitate which was later identified as chrysanilic wid. The latter decomposed on warming with mineral a&, and one of the products crystallized out. This product, which remairied unknown to the perfume industry for more than fifty years, was anthranilic acid. Nethyl anthranilate was prepared synthetically shortly after its dis covery in various essential oils. The literature on methods of preparing anthranilic acid is qnik volum inous and a fd discussion of the various rnethocb cannot be undertaken here. The acid has been prepared from various bewnt derivatives having an alkyl and nitro grouping in the ortho position. For example, it can be obtained by treating ortho nitro benzyl alcohol with sodium hFdroxide solution, 1' ortho nitro benzoic acid with zinc and hydrochloric acid? ' or by reducing orthonitro benzaldehgde with zinc dust and alkaliJ8  An unusual q ntbesis of anthranilic acid consists of treating ortho nitro "

8 Elre, Ckm. 2rg. 34, 814 (1910). 4 El, Bcr. 36, 1465 (1903). 6 Kummcrt, Ckm. ttg. 35, 667 (1911). * Pmroor. Bod. d i m . form. 41, 489 (1902). 7 GuDii .t% Chcmut and mgki 60, 1995 (1m). 8 Pam. Chcmuf 0 d Druggut 56, 462 and 2 2 (1MO). 0 Power, J . Am. Chtm. Sor. 13. 375 (1921). 10~ Tdbtom, J . pdt. a r m . I?]. 62, 136 (lOU0). n" anbot, Coappt. red. US, 580 (IS=). I* Goddig mud Roberta, J . C h m . SOC. 101, 316 (1915). 18 Shimme1 t Ce. Report, October (1901). 47. 14Hoejenbor and Coppenr, Rev. morquu parfum. rt (1931), 588.

I C c l r r e , BUZZ roc. dim. Pranm 131, 33.1161 (190% 1? Belktain and Kublberg, AU 163, 138 (1872). I* Frr~ ndlcr. BUR me. d i m . Prance [SI, 31, 450 (1904). 1sFlit+ e, 1. p 0 L C . C b t 2 3 , 67 (1841). ' 56 11 I nilic acid. One molecular quantity of the phthalimide is dissolved in three molecular quantities of aqueous alkali solution. To the cooled solution are added two molecular proportiolls of sodium hypochlorite with constant agitation. After several hours the oxidation 'is brought to completion by heating the mixture for half an hoar. The mixture is then acidified and tbt precipitated anthranilic acid subjected to purification. The yields obtained by this process are of the order of 'IO per cent,=

of anthranilic acid by the reduction of ortho nitrobenzoic acid with sulfuric acid and iron o t copper metal. This process, however, does not seen1 ta haye fannd commercial application. ., More recently a patent has been obtainedm describing the production I  AXTRRc with alkali. The re common method Y V I . I 50 PERFUYERF sY1` STIIETICS A S D ISOLATES '

f The above procedures u l alcoholysis do not work well with tertiarr i slcohols and an interesting variation is emplopd ill the preparation of such esters as Iinalyl anthranilate {IX). In this case, linatyl formate is i sodium alcoholate of linalool. Thus, 1 mole of litlalyI formate (VIII), 1 1 mole of methyl anthranilate (IV), 0.05 mole of linalool, and 0.05 atom of sodium are heated for several boun, and the methyl' formate formed dlowd to distill O~ T.~ ' The followingreaction takes place. 1 reacted withmethyl anthranilate in the presence of a small amount of : i IX AU of the above mentioned derivatives were esters 02 the carboxyl group in anthtaniIic acid. The other active group, namely, the amino group of anthranilic acid, is equally capabie of undergoing reactions and giving interesting derivatives. . 8 Methyl methyl anthranilate, which, (LS mentioned previously, wcum in se~ cral essential oih can be prepared by treating anthranilic acid (I)

Congding Point 18% 1S. C. 1cc. I2. C. 1O. C. 60 PERFD'YERP SrNTBETICS AKD ISOLATES Chemical Properties. Bn~ hranilic acid reacts with simpler aldehydes in a definite manner. Thus, 1 mole of formaldehyde condenses with 2 moles of anthranilic acid."

XI rY XI1 The amine group can be acetylated by treating the acid with acetic anhydride.= Although esters of anthranilic acid are stable to heat, anthranific acid itself decomposes to aniline and carbon dioxide on heating to 210' C. a It h interesting to note that in nature methyl anthranilate and indole are very often found together. Skatole (XIII) can be oxidized to acetyl anthranilic acid (SIV) uith potassium permanganate. N XI11 I I XIV .... 7 .: ao H e 6 and Fieuelmnnn, ARK 324, 119 (1902). 81 Ticdke, Bcr. 42, 611 (1909). 32Kaufmann, Bcr. 42, 3455 (1909). as Fritrrhe, Inn. 39, 86 (1511).

as Qtr. Pat. 137,008, Norembcr 29, 190?, ;I 8 , Jackson, Ber. .14,885 (1881). A3Th /

........ ............. .. . ... ...... .. ... ..... ..

.......... ......... , ...". .. .I ...~ Ifany 6UCh interrelationships haw been studied in detail since they rre of considerable importance in the spthesis of indigo and other dpstuf! r I 1.68 8 1.76 6 5.11 S ~

 The anthranilates are shipped in glass or aluminum containers. Tb lined containers are also used occssionally. They should be stored away from light; otherwise they undergo considerable disco~ oratioa ... ..................... __ .:,. .......... .. ....... . . j .  ... I................... I __ ... ...... ....... ......... , :> .,_...._ ~

...... :.,: ......... .. .. p t y TAB E U. S. DEPARTMENT OF COMMERCE SCIENTIFIC \ 'k b' LITERATURE REVIEW OF ANTHRANILATES IN FLA? 7QR USAGE. VOL. I. fntrouduction and Summary Tables of Data, Bibliography.

TABLE OF CONTENTS 1. 2. 3. 4. 5 . ..

6 . 7. 8. Toxicity to mouse, rat and Guinea pig. Chemical identity and physical properties Pharmacological and toxicological ef€ ects Natural occurrance Flavor and Extract Manaufacturers' Association (FEMA) and Addendum to Table IV 2 Bibliography Data guide. National Acadamy of Science (NAS) use levels. Scientific Literature Review of Anthranilates in Flavor Usage. Volume 1. .Introduction and Summary Tables of Data, Bibliography flavor and Exltod Manufactunrs' Association of .the Unitd States, Washington, DC fwd and Drug Administration, Washington, DC Nor 10 291112 SCIEMIFIC LITERATURE REVIEW ' OF AMMRANILATES IN FUVOR USAGE . ..

.. SCIWlFIC LITERATURE REVIM OF ANlliRANIUTES IN FLAVOR =AGE BIBLIOGRAPHY bY SCIEhTIFIC LITERATURE REVIEW OF A8TIiRAs 1 LATES IN FLAVOR US ACE PIgC 1 3 4 8 sEc? IcN 1 .

A. S. ..

., ,

9 12 13 17 SECTION 11. SUBSTANCES REVIEWED Ncrrsriul Designation urd Chemic81 Identity . (Table 11 1) Alphabetical Cnus Reference List of Nbms and Synonym (T r b l ~. 11 2) Physical Proporties l. fable 11 5) A. E.

C. 18 24 .

SECTICH 111.

A.

SECTIW IV.

A. 1. 39 42 4 5 73 SECffON V. 74 79 SECTIQJ VI. 60 96 V O W 11. COPIES OP .WICLES CInD IN SIwI( ART SECTION SEcTIOH I .A.

This revieu is a prssentatioa of det? p e r t i n e n t t o the safety evalua

ti= of mthraailrtss wed as flavor ingredients, fhe 14 substances uere selected for renew Y 8 gmup because of their close chemical relationships on tho asrumption that the) would f o l l w s i d l u metabolic 3rthwryr, and havo sim5la. r physiological effects in tho mamnialian organism. The list of sdrtmces rovicrwtd urs compilerr fma sevtrlj sources. I t includes flavoring caapo\ pl& tistad by tho Food urd k g Admiairtratiacr (FM) in ,Ticlo 2: of the Cod. or' Fed0181 Regulations ,. md rdrtaaces clurifird u Gonorrlly Recognirsd u Safe (GRS) by the Expert Pmal of the Flavcr 8ad Extract Mmufacturers' k s o d a t i o n ( 1 urd recognAred by FpA;. md ury additional flavor rdsturces roportod by. rtsponden: r t o a aatiaul s w r y conducted by RUA Bnd the Natian. 1 Acat3r. r of Sciences/ riational Resuucb Council !NAT; lNNRC) in 1970 1971. C; uid. linor for the typos and ammt o f data t o be gatherod yet. developed . .

in consultrtioa utth the EqorC Puul o f RClA (4 5 1, a d in ucordutc6uitb tho ctftorir pub: irhed by tho bod Proteetian Colrittw o f .the Natiaul A u d y of Sciences/ Nationrl Res Cormcil (WRVRC) (% 1.

Ih8 bulk of the infomtioa on biologic81 properties YU &t8ined froll a soarch of the sciearific'literrturi frolr 3930 thm 1977, hy articles, revim md texts wet. well Lnavn urd had been utilized in oulier evalwtionr. Using t h i chemical aIyr (u wtll u the c n ae1# .md ryno nyrs of gach r\ rbrtanw) as key wor&, ca independent orgmixatisn urrid out t h i s search by locating a l l refe~ encer t o litersturn pertaining t o phyriologiui, yhWCOiOgiC8ls toxicologfal, metabolic or o t h t r b i o l o l i u l . fniorvtion or. &use substmces. This litemture s w d ) oxtended back t o 1920 for'the aeuch p u r a +t e r r t o s i c i t y and metrboliu. hppmximteLv 40 nformci8 uom 10cated in this reuth, but only thwe a r t i c l e s conttidq drtr relevant t o safety o~ alurrtlon r o n selected for inclwioa in this =v i r . S w i u of a l l OC tho rrlovmt articles Y e n then w r i t t e n urd are presented in Section 111. A .corrpleto bibliogrtphy o f a l l references lwsted,. whuther or not the articles were usod, 1s also included (Section v),

1 Iho informtion included on the natural 0ccurrcn; e of :he reviared sd c: uIces i n food *PI provided thxwgh food i n b t l v sources, and includes I I W ~O U references t o the open l i t e m t u r e . This natura: occurrence infor s t i o n is pxcsanted i n table foi t in Section :V. A. Physical data we= a l s o obtainod ftor the industry as v e l 1 as v a t L o u coapendia. Ihesa data are pre santod in S r c t i k I I . 0. t. on the usage of thcso srrbrturce u flavor ingre d i e n t s uoro 0ttuxa. d fro8 tho a8tioul surveys o f wrgcs cmducted by both FEW and by N W N R C in 1970; 1. Theso data aert coPqri1. d by the M I N X , a d revoml calculations, includirg ai: e s t h t e d possible daily intake value, are pr* rurted and d w c r i k d in dotril ia Soctioa 1V. B.

Ihr uniqueness of the types m d amorat of data presented, u uoll u the f o n u t for iu p n s o a t r t i o n , i? i d i c t a t e d by the f a c t thst flrvorinp sub

stances c o a r t i t u t o I spodal s d o r o u ~ a i chemical c a u t i t r u n t r of fwd which 1 this problem has been tpprlached by coruidering the substances i? r gxyups or classes of structurally related c ~~o u n d s ,' a s well as individwlly. nit approach is j u t t i t i e d becaucsr o f t h e i r r e l a t i v e l y simple che8ical nature and the fact that the great o r j o r i t y f i * r e r d i l y i n t o w e l l e r t d l i r h c d pathways of metabolis8 describd proviowly i n W ' s Scientific Litcraturo k v i u of Alipbtic P r i p r r y Alcohols, AlMydes, ki.& md Estozr t 14 1. For theso muons, uch of the r e i o n t i f i c 1 itar8turo' is 8 b O org8nixed by groups of s t n a c t u n l l y r t l r t e d coapounds. A l l o f tho above arterial can be fomd as indicated ia t h o Table of Catents md is arranged as follows. F i r s t , imedi8toly follawing this Intro &tion is 8 rupury of tho biologic81 properties and other irponrnt data fros tho oatirc, reVi."

Slrctiorr 11 of t h i s roviw iacludos tho fallwing: Tablo 11 1 lists ouh subst. aco includod i n tho ropolct, statu i t s chemical i d m t i t y , u. d pro vides 8 SpMifiC ~~r i C 8 1 d~ si@ UtiOA for *&; Tab1811 2 Cmt8im . alpha betical cmrs mfenr; ce list of n r and sy" 3nyu for o r h coqomd; . .

TJ1o 11 3 givor tho physical p t o p o r t i ~ o f tho substances unhr roviw. Section .I t ! contains tha dotailod biological data for each iadividurf compourd, p r r r o n t e d i n tdlw containing abstracts o f all the individurl paprs frcm which thae dat8 wore t8. k. n. h b l o 111 preseats i d b r u t i o a on ~u m c o l o g i c 8 l a d t o x i c o l o j f u l offrctr, md .st& o1ism of'the substaaces. Sctioa IV is devoted t o tho pFuaF. ce urd amounts of 08ch of the CM

potads i n tho food supply. Table IV 1 i s drvotod t o natural occurrwtce urd frblo IV 2 p k v i b r infomtioa c o l f o c t ~d by R34A urd NASORC with respect t o

tho US8g. 10VOlS O f 0. d SubSmCO.

Section V is caaplotr biblioarrphy of a l l articles and papers con ' s i b r e d 'for us0 i n this rwim,. including a r t i c t u aot deemed' refovaat u well u r r t i c l o r f r o m uhidr d8ta wen r c t w l l y takea. n o l a t t o r 8 noted by ut asterisk precodily tho ontry. Tha find section of tho report, Section VI, pr0vid. r quick nfer~ nco to tho ammats and typu of d8tr pxormtrd for each s& st. nco in this r e v i a . : " . "

SECTION I. B.

Eleven simple esters o f o n t h r m i l i c u i d , 2 esters of H methylurthnnilic acid and othyl N cthylaathrurilic acid are reviewed here for thc*. r flavor use,

All 14 substances are used a t lcru levels i n food a t average maximum we .levels .

(50 pps, with the exception of UJO i n chewing gum (where wen high we !evels r e s u l t i n very lw ingestion) as follous: methyl m t h m n i h t c (No, 1)  1583 ppm; ethyl anthrmilate (No. 2)  116 ppm; .ethyl X methylanthranilate (No, 12)

 91. S ppm. The "possible rvozqe daily intakes'' for these throo rdstmces f r o m cherinp gum US. havo bwa calculated (seo Tablo IV 2) t o be 0.08, 0.91 and 0.005 q, respectively. nesa l o r usage levels are reflected in the lw mnua1 volumes for flavor US. which rage fmm 9 pounds for isobutyl N ncthylmthrurilate (No, 13) t o 2056 pounds for mthyl 'X nethylmthmilate (No. 12) and 75,300 paurds for wthyl anthranilrtr (No. 1). Iho calculated (sea intruduction to Table IV 2) per wit0 intakes aro 0.02 rg/& y or ~w o r for 811 except for 0.7 mg/& y for eethyi anthra nilrt..

ents of Iood ($ eo Tab10 IV I). Wrthyl m t h r u r i l a t r (No, 11 occuA in a d e r of f r u i t s : reported 1ovels tcing 17A ppa i n grape juico and 33 p p i n Concord grapes. Theso levo18 r n coqu8blo. to tho$@ resulting f t a flavor use. Ethyl rnthr8nilrto (NO. 2) i s also 8 component of grrprs. &thy1 N 8athyl. nthrdlate (No. 12) has boen roportd t o c o a s t i t u t o 0.7s) of bergamot o i l urd O .X \ of bitter oranzr oil. The th. rro largest voluw substaacer covered hen are 811 natural constitu

iaTABOLISM k h v boon discussed befon [F. E. M. A. 1974. Ref. No, 141, tho 14 esters dcid O? N 8ikyl i n t h m i l i c studies on methyl anthrrnilato be hydrolyze4 t o an alcohol and .tither anthranilic acid. This sssmption is supported by in Vik. 0 (No. i) vhich, *hilo only slowly hydrolyzing i n 4 .

a r t i f i c i a l g a s t r i c or pancreatic juice, is readily hydralyted i n r a t liver homogenate (50% i n 27 ainutes) and rapidly hydro1y: ed i n r a t small intestinc mucosa (SO\ in 2 .5 minutes) itongland, e t a l . 1977. Ref. So. 34 1. F'ethyl anthrani. late has also be en^ show t o be coqlercly hydrolyzed (, 99% in 2 hours) i n p i g , liver homogenate but is more r e s i s t a n t t o hydrolysis i n pig jejunum homogenate and completely unhydro1y: ed by pancreatic solution [CnmCschober. 1977. Hef. !a. 23). I n a d d i t i o n , lpcthyl K sethylmthranilate (Yo, 12) has been shown t o be hydrolyzed on oral adair. ia. tration to a hwur and a r a t [F. D. R. L. ulpubl. Wep. 1963. Ref. No. 18 J. A small amount of N Canethylatian occurs i n both c a e s . In an i n :u i t m StUdyD methyl S rwhylanthrrnilatt uas resistant to hydrolysis by p a n c r e a t i n o r p i g jejuI. um homqenatc but's993 hydxlyted i n 2 . ,

hourr by p i g l i v e r homogenate [Cnmdschober. 1977. Ref. So. 23 1. I t has also been s h w n t h a t methyl & 8cthylanthr&, il8te w a s ra2121Y rbso* d f r o m the small intestin8 of guine8 pigs, corpletely hydrolyzed a t 10u cimcmtntionr 8nd par t i q l l y hydrolyzed at higher cor., entrationr [Pelting, e t a1. Unpubl, Rep. ' !? ef,. so. 46 1. Prompt ditappearsrce f r o m the blood of .these guinea Figs indicated rapid Met8bolfS8. In supypxy, it appears t h a t t h e a n t h m i l a t e s are not hydrolyzed t o any a p p r e c i a b l e e l t e n t i n the StOmaChD but eXtenSiYe if not COBplete hydrolysis takes place o n a b s o r p t i o n t h r o u g h t h e i n t e s t i n a l u r l l s a t tht concentrations at which these substances aro used. Any remaining uahydmlyrd u t e i t a l can be hydrolysed by t h e liver. Excretion of anthranilic acid occurs primarily as o minahippuric 8cid and t o 8 lesser extent u anthmnilic reid glucuronide i n u. n [Brown. and Price. 1956. Ref. 80. 5 1, rabbits and. rats (Oarrcoanct tiarJing, e t 81. 1933. R e f . 80. 8 1.

nJXICIl7

!n l i @t of the above, it is obvious that 8 discussion of the toxicity of the 8 n t h r u r f h t e esters m u s t also i n c l u d e o n t h r 8 n i l i c a c i d d a t r , k r t h r 8 n i l i c r c i d is a nom1 Betrbolite i n man and is c x c r e t d i n t h a urine u 0 88inohip p u r i c acid (27 u# les/& y) and && ranitic acid alucutonid+ (6 vwXes/ day). Ihir is equiV8h1t t o tot81 rvetrge d 8 i l y excretion of &art f a# urthturilic a c i d p e r day [Ria. e t 81. 19%. bf. NO. 47 1. . hlr liritcd d8t8 a r e 8 v t i l .b l e on t h e toxicity of a n t h r a n i l i c a c i d ; however, the Or81 tbso of 4S49 mglkg i n r a t s 1R. T. E. C. S: 1977. Ref. SO. 48 ] indicates low ,toxicity. It ha been reported 5 ..

[Et and Strombeck. 1949. Ref. Xo. I?] that O.?\ in the diet of rats pro

duces blodd.. r papilloma^ on long feeding. The oral iDjO values for the esters are also quite h i f i for t h i s class o f compounds, vllues ranging fnPl 2250 m&/ kg for methyl S methvlanthrsn: 'atc (SO. 1:) 13 guinea pigs t o >SG30 mdl'kp in rats for several o f the &t e s (see Table A:).

The two anthranilates uith highest eaposurc have botl; h e n studied more extensively. Methyl anthranilate (ertinroted as ?xplained in. the Introduction t o Table I V 2 t o have a l e r qitct daily intake o f 0.31 8g/ kg for a t4 kg person) h* s been added t o t h e d i e t of r a t s at levels ;rf ?OOO. and 10,000 ppm trpproximtely equivalent t o an averape.& rfly intake of SO and 500 mg/ kg for an adult rat). f o r . 13 ueeks u i t h no Ldverst effects .( Haoan, e t 81. 1967. Ref. So. 27 j. 'kthyl X methylanthranilrtcr (No. 12) (estimated per qitc: daily intake of .C. OOO, t ag/ kg f o r a 60 kg'pcrson) added t o the d i e t of rats a t levels o f 300, 1230 and 3604 ppm (approximately 1s. LO and 120 ag/ kg/& y for 8n a d u l t r a t ) for 90 days, [Cauirt, e t 81. 1970. Ref. No. 23 f resulted i n 8 s l i g h t b u t s i g n i f i c a n t leukocytopenia and anemia a t t;. e 2 higher levels urd i n c r r u c d kidnc; ' w i g h t s i n tLe u l e s .

No effects were seen at 300 ?pm. A drily intake of 19.9 q /k g (males) and 22.2 &kg (femles) by .rats for 90 days caused no 8dvem. e effects [Oscr, e t a l . 1965.

Ref. So. 441.

* Both methyl anthranilate (No. I ) urd einnuyl anthrrailate( No, IO) in tricapxylin solution uem infoetad intrrperitonerlly 3 tines per week for I weeks a t the saxfur tolemted des. (WD) 8nd a t 0.2 tire the WfD in me mice uhich were maintsined for 15 rdditiorvl uetks betom. sacrifice and autopsy, Methyl a n t h n n i h t e w u injected i n repante doses of 0,09 d k g (0.2 m) a d 0.47 g/ bg (WTD) for 8 total doso of e i b r 2.25 or 11.2 g/ kg t o 20 f c u l e i a t s ' .

each. A t t h e end of t h e 24 ueek test period (I week administration and 16 ueek observation) 3 of tho 18 survivors (ln) at' tho 1 dose md S of the 19 sw\* ivon (26%) a t t h e m3 had developed lung tumors. C i n n q l a n t h r a n i l a t e MU injms d i n separate doses of 0.1 g/ kg (0.2 MTD) and 0.5 g/ kg (MD) for (L total dose of . e i t h e r 2.4 or 12.0 o/ kg t o IS male urd IS ftmale tats each. In t h i s case, a l l male rats 8nd IS f e u l o r8ts survived a t either dose. Sev? males (47E3 and 6 f e u l e s (468) a t tho low dose urd 14 v l c s ~~(Y 3 t ) and 7 f d e s (S4%] a t the'wTD developed lung ttrwr,. O f the 80 m l a urd 8C femslc controls injected similarly with tricaprylin, 77 8810s and t? females survived the test petioa. Of these z8t of tha males md 20\ .of the fe= lm developed lung tmm [Stoner, et 1973. Ref. No. 531. .c ... ,>

6 .. ..

PI: ,"" . . . " e.. ." . ~

L l k 1. ,/

.,

1 ,

With. tha exbcpticns of isobutyl S methy1anthr: milatc (So. 13! and ethyl .N e: hylanthranilat~ (No. 14). which have never bees submitted to the Pane!, 311 o f the anthranilates discussed here nave been reviewed by t h e Exper, oanel of F. E. N. A. and found to be Generally Recoyrlired as Safe. (CRAS) under the condi tions o f intended use as flavor ingredients (F. E. M. A. 1965 1978. Ref. No, 15 1 . lhis determination w a s based on a review of a l l available. data as well as the rruonable analogies which the Panel juciged could be drawn.

.# ..

..

c! ': . se+^ so " S O Substance Spec ios Rout e   L Werhyl anthrazilatc hb us e Xntrapeti toneal Rat Intraperitoneal ). bcs e Ora 1 kat (fasted) Oral Guinea pig Oral (fasted)  17 30 1 7 30 30 Oral Oral 2 Ethyl anthrmilate Mouse Rat 17 42 4 Sutyl anthranilate Rat Oral 38 Oral  4 1 I! . .. '..?. ... <.. .. Phenethyl anthranilate Rat Oral 43 Oral >S g/ kg 39 10 Cinnamyl anthranilate Rat 12 Methyl N mtthyl Rat (fasted) anthranilate ,. ,Rat Or81 Oral 23 2.2s

3.7 ml/ kg 3 .3 g/ kg 40 8 SECTIN 11. A.

NUMERICAL DESIGNATION AND CHEMICAL IDENTITY TAEE 11 1 This table lists the prirpary names, chemical structures, molecular formulae and molecular weights of the substances included i n this review. These Corupoun~ 8 listed by chmical stxucture as explained bclw, and am assigned I numberby which they a b referred thmughout th8 review. In order t o mom easily sake comparisons between metabolically nlated substances, the compounds are organired md n d e r o d according t o chemical structure pro gressing f r o m r t q l e s t t o . more complex. . A cross rsference of the restances and t h e i r s p o n p s is listed alphabetically in Table 11 2. Additional inclusions in Table 11 1 are: a) 'the n h e r assigned t o t h e substance by the Flavor and Extract Manufactu~ e~ s~ cIJsoci8tion (€ Em) in its prtblicatioas I f ] listing substances judged by the Expert Psnel of FEMA t o be generally recognized. u safe conditions 'of intanded use; b) the section o f 2 1 Cod8 o f Federal R e p l s t i o n r i n which tho r r ~s t a n c c l have been l i s t e d by' the Food and Drhg Mn$ strrtion '( FDA) ; c) the QIdca1 Abstracts registry number. I hl rr)

bn bn N b c.

..

L. l rr)

m L. l 0 r 4 CJ 0 Y L1 "

e4 0 11 c; m N .. e 5 .4 =m L. l m u Y r( e a Y c .a c.

c x w Y 10 N .. .:

L m m hl c4 .II R 0 2 Q v s: Fi Q 1 I wl (Y Q hl N 2 Q wl n" yr LI *! ' 4 u n" h = i c . u u"

c I CI e Y d e Y 8 C L. 4 t s 5 0 *,

d a * a a " .d 4 & CI Y rl 3 x 3 L 0 d LI 2 x Q i a 9 Q Y,

cy t I I I I I 2 m 9 L. .I n N m  N rn N 9 8

N 0 n" 2 *a U U ? h 8" In =e4 U" r.

0 Y L..

r. r. SECTION 11. b".

ALPHABkTIUL CROSS REFERENE LIST OF SAME5 AND SySoNy). Ls TAB& 11 2 Thrr tablo .lists a l p h i b e t i d l y the principal names (i n ' c a p i t a l e t t e a ) a d synonyms [in louer case lotters) of the substances i n t h i s roviw. A l l synonyms am l i s t e d a f t e r o a A principal name u uoll. Iho ndcrs refor to tho rtnrctunl 1is. tiag i a T d l o 11 1 and indiuto tho ordering of tho tub

ttmccu i a a11 othor tdlos.

mi3 cmrr nteGacr list inc1dws tho30 synonyms most cauDnly found in tho literatwo md synonym, including soma trado names, frequently wed in tho flavor indwtry. In a11 c u .3 thr. rtder is rearred t o the cqitalired nasa u h i d is the n l w used throug. ho* t h i s review. No

3 4 LO i 6 TABLE 11 2 AX nt tu.\ I LATE s Substvrse Si; ron \IS .

~l l y l 2 rpinobentoate [ALLYL &W I L I \T E n o . 31 A l l y l o iuninobntoate [A l l Y L "HRNIUTE no. 31 ALLY t A S l I i i i X I UTE A I l y l o. uninobcururrr Allyl 2 aminobcnrootc 2 Propenyl ?; lrPinobentoatt : Propenyl anthranilate Vinyl carbinyl anthruri: rta 6 Amino methyl benzoate [WTHYL A!! WIUTE no. 11 Butyl o uinobsnzocrte [BLIIYL ~1 L A l E n o . 41 iw' htyl o aminobenroato [ISOSUlYL MTHUNiUTE no. 5;

Butyl 2 rninobcnzonte [BUIYL MMWILATE no. 41 ..

BUTYL AKMRANIUTE Rttgl 2 aminobcntoate Wltyl o. cuinolenrorte b k t y l &n t h ~i h t . 1 I S 8 V n L AMMRANIUTE M). S]

CiMIII) Il o adnoburroat. iC1 t NIH~ IU'TE T~ O. 10)

Cinnuyl 2 ainobmzoate [CIMUNfL AMHRANIUTE no. 10)

c INNAMYL mRAN1 UTE C i n n u y l 2& imbennrorto Cinnuyl o ainobcnroate 3 Phenyl 2 propenyl 2 amino benzoate 3 P h ~y I t p m p ~t 8nthIWlil8te Cyclohexyl 2 uinobentoato [CTC XYL AKIHIUNfUTE m. 6j CYCLOHEXYL ANTHRANIUTE Cyclohexyt 2 aminobcntorte ..

14

i_ No " Subrtmc. Synonps Diethyl unthranihtc [E M L X LTHk'UhiIWIUTE no. 141 Dimethyl anthranilate (WTHYL S. WMU, XWlUTE no. 121 .

3,7 DiP~ thyl l, O octa~ ien 3~ 1 2 ullincbur: oate JUUAYL ApiTHaASlUTE no. 7 )

$, 7 Pi~ thy1 1,6 octrCica J~ l urthrrnilato ~L l W Y L AN'MWlUTE no. t ]

Ethyl o dnobenzoatr [ETHYL ANWXLATE no. :!

Ethyl 2 dnobrnroate [ETHYL ~l U T ' E n a . 2)

Ethyl o adnobenzoate Ethyl 2+ miaobmrolitr 5 Diothyl .nthturilrto Ethyl 2 ethyluinobenroate Ethyl (2 ethyluiaophcnyl)  8ethuxnto Isobutyl 2 uinobratorto (ISWJlYL A ~' M I U T E n o . 5)

Isobutyi 2~ ethyluinobenrmtr [ ISO% VTrt N IIEMYUMHRANIUTE ao. 131 Isobutyl 2 methylmino

24ethylpmpyl 2 wthyl kntoatr uinobaitmt~

tinalyl 2 uinobenroato (UN& Yt ANTHRANIIATE no. 71 7 1 it 11 Subs t mce Synonyms Linalyl c rminobcnroati [LIsMYL ANHMNItAI'F. no. ?]

LINALYf. &T1iRA! iILATE 3,~ Diwrhyl 1,6 octadicn

3.7 Dimethyl i ,+ oitadien

Linalyl 2 aminobenzoate t i h ~&1 y . c aninobtnroatr 3 yl 2 ainoben: oate 3 71 rnth1urilate p)( mthhr l m tyl 2 uinobonroate ja TERPINYL &7lWh( IUTE no. 81'

p l4enth l. rr l y1 ~thraniIgte [a TERPINYL N7HRANI'JTE no. 81 2 lbthtluriao wthyl bonroate [) 4EIliYL N lETHY~ RMIUTE no. 12)

m Y L AKIHRANIIAX o kino w t h y l bcnroa: e kbthyl o uinobenzoate Methyl 2 uinobcnroate Dimethyl 8nthr. nilrto 2 libthyluino rtthyl bontoate Matby1 2 rwthyluinobentoate Wthyl o methyluinobenroate 2 Naphthyl o: rinobaroato [# NAPWHY1 ANMRANItAfE no. 111 2 Naphthyl 6 uinobenzoato 2 Nqhthyl mthrmilrto 2 Naphthyl m t h r u r i l a t o f8 U" HYI. uJTHRAxIUTE no. 111 16 . No Substance  Synonyms 6 Phencthyl o~ minobcn: o; rte 2 Pharylethyl mthrmilate 2 Phenylethyl anthranilate (PHENETHTL Ah'l? lRAi. YIUTE no. 01 3 Phrnyl 2 propenyl 2 u i n o t a t o o t e [CINNQIAHI'L ANMRMfUTE rro. 101 8 a TERPINYL ANTHWIUTE  "_. ." . . "

1 SECTION I i .C.  .. I I I t should further be erqhuird that the data and descri? tion of sub

stances contamed within this table r're those lasted in t h t referenced source and no o t t e q t Lss been made t o c l a r i f y these listings. ;or eurnplc. although sone substances ore listed as color! esr; i t should not be asslmpcd that com pounds not so designated are colord. As another exaprple, the d, ifference between sOlubl8 and 8isciblr w u not aluays clearly defined.

Abbreviations Used in This Tablt alc I ethyl alcoho:

s. 6 solubl. far insoluble rls slightly soluble l i q  liquid mr 6 melting point bp = boiling point rpg = specific gravity d = density doc 6 decowsu 19 I i I " " "  1 i L I +

I  .*  9 4 1 "

" b i a 3 .. a  f 2 . 3 f "' I .. I c)

t 4 YI 22 .................. ........ ............. ...... ... .... ....... . . ................ ............. .. ........ : >.:.. , .:. ...., ..._ I_ ". i i z t P W ..

2.4 SECTION I If TAELE 111 lhis tablo contains inforvtion en tho biological yroperties and .stabolism of the substances included in this review. Tho bulk of this inferntion uu derived f r o m t r t i c l o s gt* rined Cram t coaprshcnsive survey of tho s d o n t i f i c l i t o r r t u m published betkcen 1.920 and 1977. This survey v u carriod out by an indepeaant orgurizstioa, bfomticsD lac. of RocLVillU, Cluy1 d. ..

hfomatics, Inc. u r d the chaical rima, c o m n naaos md syaonym of each substance as key uords t o locato reforcrncer t o tho publish& l i t e r a t u r e pertaining t o tho p h r r k o l o g i u l urd toxicological daQ on thoso substances. Summaries of a11 the portinont articles fro. this survey worn then written and are pnsentod i n this tal.. Othor data c o l l e c t e d f m a o u l i o r litera

two md privrtr industry SOUIICOS 8ro also s izod .and includod h m i n . Iho tablo is orgaairod by subttaac. with the simplost c h u i c a l stnac t u o s procoding a0 .on C l.% D u in oqor roctions of this rovim. me nuberr rftor tho nmo of oach substurco uo the same f o i a11 the other tablos in the riviou. rithia or& substurco. gmup. tho entries ue.~ orpnized <..

! 25 .. bferencc KO. : 3.1 (Longland, W. C., e t 01. Toxicology. 1> 77)

b t i I y 1 a n t h r a n i l a t e uas i n c u b a t e d w i t h a r t i f i c i a l g a s t r i c juice aJ a r t i  ficial Fancreat ic j u i c e at 37. for up to J hours. The time required for SO\ hydrolysis bas 5950 and 4150 minutes, respectively.

i n SO$ hydrolysis in 27 and 2, s minutes, respectively, Incubation hith rat l i v e r homogenate and small i n t e s t i n e mucosa. resulted Reference No.: 25 (Gradschober, F. Toxicology. 1977)

Methyl a n t h r a n i l a t e a t a cowentrarion of 100 or 250 u l /l w a s incubated i n 0.5M phosphate buffer (pH 7.5) at 37. w i t h e i t h e r p a n c r e a t i n , pig jejunum homogenate o r p i g l i v e r homogen8te for 2 hours. C U u d l y s i s r e v e r l d 0, 1s urd ,998 hydrolysis, respectively, of t h e ester. I , N m of Substance: !EB'zHyi AKl? iRANXLATf (1)

Reference No.: 17 (F k Unpubl. Rep. 1977)

Species: Nouse No. /Croup : 10 Duration: Acute Route : Intraperitoneal Vehicle: Sot Specified Control 0 Sot Specified Tho intraperitonoa: LD f o r methyl anthtanilato i n l i c e u u reported t o be 1.04 el/ kg (958 C. L. Oy 1.19 W k g ).

Referonce No.: 30 (Janner, Z .M ., e t 81. Food Cosaet. loxicol. 1964)

Spocies : .House No./ Group: Not Specified [kration: Acuto (14 day obremtiou) Route: otrl (intub8tion) \'ahicle: Nert Control: Not Sprcifid.

Tho om1 LD (Litchfield Wtlcoxon) for methyl anthnnilato in mice w a s calculattd t o beSg90O 8 d k g (OS8 C. L. 3260 4680 &kg). Symptom of toxicity included depression and death uithin 4 18 hours.

i i . c * k& J \

Roforenco No. : 53 (Stoner, C. D., e t a1. Cancer Res. 1075)

Species: )louse (W e ) Rout. : Xntrrp. riton& l No./ Crwp: 20 (F) Vehicle: Trieapxylin Duration: 8 w e +L t (24 week obsonation) Control : Tricqrylin alone Mica wore injected inttrperiteneally uith 24 d a m , cithar 0.09 g/ kg or 0.47 g/ kg.; of methyl a n t h r 8 n i l i t o i n t r i c r p x y l i n 3 tims per week for 8 weeks f o r 8 t o t a l dose of eithor 2.23 or 11.2 fig. A t the. higher dose, 19/ 20 feaalos sunivad the treatment, while a t the lower dam .the survivors wero 18/ 20. A t the ond of the 24 week test period, 3 (268) had develoyd luly tumrs a t tho high dose and 5 (In) had a t t h e lower dolo. Y i t h tricapryfin alone them utre 77/ 60 male and 77180 female survivort of which 28* of the males and 20% of the females developed lung tmors.

!27 Species : bat .io. /Croup : 10 Duration: Acute Kefertnce So.: 1 7 (Hapan, E .C ., e t a l . Food CoSmct. Toxicol. 1967)

Species : Kat (Osborne Sendel) Route: Oral So./ Croup: 20 (10M L 10F) Vehicle: Diet Duration: 13 weeks Control: Diet 810m ' . Methyl anthranilate was administered a t diotrry concentrations of 1,000 *qd 10, COO ppm (approximately equivalent t o 8 daily intake of 50 and 500 q /k g i n a d u l t r a t s ) t o weanling Osborne Mendel r a t s f o r 13 weeks. Xo effect ras seen on g n n t h or hesatology, .IS determined by an examination of white and ("?< I. . red blood c e l l count, Iwrtoglobin 8nd hematocrit, i n e i t h e r group. So gross ;$ tissue changes uere found i n e i t h e r group, nor uere any microscopic changes d d observed i n t h o high dose gmup.

28 Reference No. : 30 (Jenner, P. M., e t a l . Food Coraet. Toxicol, 1964)

Species : Rat (Qsborne libndal) Route: Oral (intubation). No. 'Group: 10 (SM 6 SF) Vehicle: Seat Duration: Acute (14 day Obsenration) Control: Not Specifid f u t d r a t s up3 8 h l a t .d to bo 7910 &kg (9S\ C. L. 1500 3400 'mgjkg). Syaptom of toxicity includod depression, c o u upd death vithia 1 2 days. The oral LD (titchficld WilcoxQn) for. methyl anthranilato in 18 hour Reference No.: . 30 (Jenner, P. N., e t a1. F c o d Cosset. Toxicol. 1964)

Tho oral LD (tiidfield Nilcoxon) for thy1 mthnnilato in 18 hour fast& pucnea piat YU calculated t O be 2780 rg/ kg (9SI C. L. . 2210 3500 8g/ kg). SpptoM of toxicity included depression, gasping, rapid respiration, sutro i n t e s t i n a l i r r i t a t i o n and death vithin 4 hourr t o 4 days.

29 Sme of Substance: E W L ILYIHRAh'liAfE [2 ]

Reference KO.: 17 (FW Cnpubl. Rep., 1977)

Specjer: Nouse. .

ro./ Cloup: 10 (W 6 F) Durrtron: Acute &l i t e :. Oral (intubation) Vehicli. Not Specified Control : Not Specifrd The oral LD for ethyl anthranilate in mice w a s reported t o be 3.37 ml/ kg (958 C. L. ao2.78 4.60 ml/ kg) .

Referencr No. : 42 (Opdykc. D. L. J. F& d Cosoct. l o x i c o l . 1976)

spiciss : Rat Route: Oral No./ Group: Not Specified Vehicle: . Sot Specifid Ouration: Acute ., Contrcl : Sot Specif id &'kg (958 C. L. 'g. Y 4.18 g/ kg). The oral LD for ethyl mthrmilote i n r8: s YU reported to be S, 7t ,. 3 0 '. X'irme of Substance: ETHYL AXRiRAI; I! ATE {2 )

Reference KO.: 1 7 (FDA. Unpubl, Rcp. 1977)

.Species: PWse %o./ C, roup: 10 (N 6 F] Durrtlon: Acute Route:. Oral (intubation) V2hicI. a. Not Specified Control : Not S p e c i f i d The or81 LO for ethyl anthranilate in mice u u reported t o be 3.57 d /k g (958 C. 1. 3° 2.78 1.60 81/ kg).

Referenca. No.: 42 {Opdyke, D. L. J. FoodXosaet: Toxicol. 1976)

spocia: Rat Route: O r a l No./ Croup: Not SpUlfied Vehicle: Sot Spwified (kntion: Acute t o n t x l : Sot Sptcif id me oral LD for achy1 urthrmilate in r8: s v u reported t o be 3.75 &kg (95% C. L. sg. 32 4.18 g// kg).

30 i N .u of %brturco: S u n & AMHRAN: uT€ [4]

Reference No.: 38 (Opdyke, D. L. J. Food 6 % Spec ics : Rat. No./ Crouy: Not Specified Owation: Acute ~h r oral for butyl anthranilate than 5 p/ kg. Cosmet . Toxicol. 1975)

Rout e : Crol Vehicle: trot Specified , Control : Not Sp ified in rats vi& reported t o be greater Samo o f Substance: LIMLYL LiNlXIHRASIUTE [7 j Reference tio. : 41 (Opdyke, D .L .3 . Food Comet. foxicol. 19'6)

Species: Rat SG. iftoup: >kt Specified Dura ion : Ac ut e Route: Oral Vehicle: ,Uot Specified Control: Nor SyccifirZ '. L'

I 32 I Naae of Substmci: PHENRhYL ANTHRASIUTE 191 Reference No.: 43 (OyrtyLc, D. L. J. Food Cosmet. Toxicol. 1976)

Species : Rat No./ Group: tiot Specified &ration : Acute Route: Or01 Vehicle: " Kat Specified .Control : Sot Specified oral LD for phenethyl anthturilateain rats w a s reported t o be greater than 5 24,. I I 1 Nsme o f Subrtuce: ClNNAHYL AlSTHSWIUTE [lo)

Reference do.: S j (Stoner, G .D ., etoal. Cexer b s . 1973)

Species : Mose (NHe) Route: Intraperitoneal Duration: 8 weeks (24 week observation) Contml: Tricaprylin alone N o .i C ~p : :# O (194 I 1SF) Vehicle: Triuprylin Mice were injected intraperitoneally with 24 doses, either 0.1 g/ ky or 0. S g/ kg, of cinnllpyl ant). zonirate i n t r i c a p x y l i a 3 times Fer week for 8 weeks ior a t o t a l dose of either 2.4 or 12.0 dkg. At the higher dcse, 1S/ 15 mala and 13/ 15 fe les sunived the treatment, bhile a t the lower dose the survivors wero 13/ 15 males and 13/ 15 females. A t the end of the 24 week test period, I4 males (93t) and 7 females (S48) had developed lung twrs a t the high dose and 7 (47%) moles 1nd. 6 (468) females had af t h e lower dose. K i t h t r i c a p y l i n alone there were 77/ 80 male ann 77/ 80 'female survivors of which28% of the .

des urd 20% of the females developed lung tumors.

Reference No.: .39 (Updyko, D. C. J. Food' Cosmef. Toxicol. 1975)

Species : Rat No./ Croup: Not Specified Ruation: Acute Route: Oral . Vehicle: Not Specified Controi: Not Specified I Name of Substance: WEIIML I\" MET" niRAKIUTE [12]

Reference No.: 18 (F. D. R. L. 'hpubl. Sep. 19s;) I I One a d u l t r a t each was adainistercd .orally (stomach tube) a 1.0, 5.0 or SO mg dose of methyl N methylar! tnranilate. Analysis of the 24 hour urine revealed unsytcified amounts 0'' S merhylanthranilic acid ar. d anthranilic acid i n a r a t i o of approximately :O i l . 7he authors concluded tfiet ingestlot c f methyl N mathyl. anthnnilote is followed promptly b) d e e s t e r i f i c a t i o n w i t h t h e urinary elimination principally of the X methylated acid.

Reference No. : 46 (Pelting, e t 01. Unpuol. Rep. )

Metnyl H ut thylanthranilate a t concentrations ranging' from 25 t o  260 p p ~ i n physiological saline nas i n j e c t e d i n t o t h e duodenal lmcn of male guinea pigs a t a dose volume o f 5 .ml/ kg boiyweight a t o rate o f 6 mllmin. A ligature was t i e d around t h e duodenvnr n e a r t h e p y l o n s t o p r e v e n t r e g u r g i t a t i o n i n t o the, stoaaach. Samples of portal blood were taken a t 2, 5 ,. ?O. 20 and 30 minutes and analyz ed for unhydrolyzed e s t e r . TBe r e s u l t s i n d i c a t e t h a t . methyl fz methyl a n t h r m i l a t e is rapidly .absorbed a t a l l c o n c e n t r a t i o n s and i n a conp: ctcly hydrolyzed form at 25 ppo. So unhydrolyzed e s t e r wits detectable 10 minutes a f t e r i n f e c t i o n of the 40 pym solution and 20 ainutes after injection of *he 120 ppm s o l u t i o n . w i t h t h e 260 ppm solution a peak concentration of ?.? 6 ug ester/ rpl o f blood uas r e t c h e d a t t h e 3 minute sampling and a concentration of 0 .~6 ulJm1 remained a t t h e 30 minute sampling.

Reference No.: 25 (Grundschober, F. Toxicology. 1977)

 Methyl N methyla& lirurilate at a concentration of 15 o r 250 ul/ i.. was i n c u b a t e d i n 0. SU phosphate buffer (pH 7.5) at with tither pancreatin, p i g 3ejunwn homogenate or p i g liver homogenate for 2 hours. G U x a l y s i s revealed 0 , IS and ,99% hydrolysis, respectively, of t h e ester.

35 . .

Name of Substance: MERfYL N CIE'MYWJRRANILATE [I t ]

Reference Yo. : 18 (F. Q. R. L. U~ publ. Rep. 1963)

One human volunteer vas administered orally a single 150 mg dose of methyl X methylanthranilate and urine w a s c o l l e c t e d a t 7 hours following treatment. Analysis revealed unspecified amounts of N methylantkranilic acid and anthra n i l i c a c i d i n a r a t i o of approximately 20 :l. The authors concluded t h a t inges t i o n of methyl N methylant5ranilate is foliowed promptly by deesterification with the urinary elimination principally of the N mccnylated acid,

. . 36 Reference No.: 4 (8ar, F. and F. Griepetltrog. Xed. Ernachr. ,196;)

Species: Rat Ho./ Group: Not Specified Duration: 12 weeks Rout e : Oral Vehicle: Sot Specified Conrro 1 : tiot Specif id For. 12 weeks, rats uere administered daily a 20.3 mg/ kg dose of methyl N methylanthranilate by gastric intubation. No adverse toxic effects uere aoted.

Reierence No.: 23 (Gaunt, I .F., et a l . FoodCosmet. Toxicol. 1970)

Species: Rat (CFE). .Route: Oral No./ Croup: 30 (15H 6 15F) Vehicle: Diet Duration: 90 days Control : Ciet alone MetSyl N met? tiylanthrar. ilate w a s added t o t h e d i e t of rats at levels of either 300, l? 5U or 3600 ppa (apprOXimiitely equivalent t o a J8ily intake of 15, bo or 180 mgjkg) f o r 90 days. Measurements of bodyweight urd food intakes were recorded regularly and no significant differences between t e s t and con t r o l s were noted. ilematological examination revealed a slight but significant leukocytopenia and anemia i n animals receiving 1200and 3600 ppo a t week 6, !. ut not at 90 days. The r e s u l t s o f u r i n a l y s i s conducted a t week 4 and again with blood Chemical deteminations 8t week 13 were within the normal limits. Measurements of organ weights revealed a statistically significant increase i n the kidaey weights i n aales receiving 1200 and 3600 ppm. Gross findings showed no evldencer of agent nlated lesions.

,,

' I Reference No. : 23 (Gaunt, I. F., e t 81. Food Cosmet. Toxicol, 1970)

Species: Rat (CFE) Route: Oral . No./ Group: 4 (F) Vehicl e: Weat '

Duration: Acutr (7 day observ8tior,) Cantml: Not Specified The Or81 LD f o r methyl N mtthylanthranilate in fasted rats w a s repottcd t o be 2.25 3.38 39kg. Symptoms of intoxication included increased exploratory behavior for 15 minutes, decreased motcr, activity at 4 24 hours, non responsive ness to painful stimuli, piloerection, t~ loody nasal discharge, loss of con sciousness and death within 18 48 hours. Cross cxaminaticu raveold 3 slight reddening o f pulmonary tissue. .. I Saae of Substance:

Reference No.: 40 (% dyke, D. L. J. Fcod Cosmet. Toxicol. 1975)

Species: Rat R c u t e : C r a l So./ Croup: Sot Specified Vehicle: No: Specified Puration: Acuto Control : Not Specified 3.7 ml/ kg. The oral LOso f o r methyl N methylanthrPnilatt in r a t s v u ' e y o r t d t o be Reference No.: 44 (her,

Species: Rat (FDRL) No./ Group: 30 (ISM 4 1SF) &ration : 90 days B. L., e t 01. Fwd Cos met .

Rout e : Vehicle: Control: foxicol. .196S)

Oral Diet Net alone Nethyl N methylurthrrnilate v u added t o t h e d i e t of r a t s a t :cvels cal c u l a t e d t o r e s u l t i n approximate d r i l y i n t a k e s o f 19.9. mg/ kg (M) a d 22.2 mg/ kg (F) for 90 days. Neasurunents of bodyweight and food consumption uere recorded regularly and nc significant differences between test and control r a t s were seen. Hematological examinations and blood chemical determinations .conducted a t weeks 6 and 12 revealed noma1 values. Liver and kidney weights at autopsy were n o r u l , and .hirtopCthology revealed no dose related lesions. I' I I SECTION 1V. A.

~r b 1 e IV I sumaarites references in the scientific literature t o the natural occurrenee of the substuncas i n foods. For each substance which YU found t o occur i n foods, the tab10 indica% the food source, .h o t h o d of detoctiar, level of concentratioa i n t h o food (tf ruportodl , and other portinent coIMllts. Ihe nfonnco nudor folloring oath entzy i n tho tablo raforr t o the bibliography nubeg usignod t o the article i n which that d8ta m u . fomd. Copies of the articlos citod w i t h English tru~ shtiont' Of foreign lmgurgo 8fliClM are in cluded i n Voluw 111. As previously s t a t e d i n S o c t i o a I , tho natural occulldnce of a substance in fwds indicrtes that tho substurco has bean cogmod b) humans for centuries. Where q u m t i t a t t n d a t a on t h o hV. 1 of COnCentr8tia1 are awilrble, tho level of CoWrnptiOU CIO bo ~~t i m t e d ; hheta 80 qrturtit8tiVO &t8 UOZG f m d , the o c c m n c a of a substance in a variety, of foods usually indicates that mom than . traco q& titios have bem eonsrrwd.

tho~ flavor indusuy. Altho the tables do'not necessuily represent 8 em plcte l i s t i n g of a l l references t o n a t u r a l ocrurrencs, which e x i s t i n t h e l i t e r a  ture, 8n a t t e q t u8s asdo t o include roferenccr t o n a t u r r l occurrence of each substmco in u .m n y d i f f e m t food sources u possible. Tho d a t ~ i n t h i s t a b l e wero gathered primarily f r o m inforution pmvided by When it is s t a t e d t h r t a substance wu detected by isolation, t5is means t h r t the rCtw1 SUbStWC,. Or 8 S i q i e derivrtive, W U isolrted i n 8 relrtivcly pure torr. TIm torr "chdcaily characterized" mew that identificr; ion YU obtdnod by UI `rppropri@ to dtcdcal dotaction method. For conveni~ cr in pre paring tho tablo, 8 e t h e using physical wuu of deteectim, such u mixed mltiw points, wen included under t h i s heading.

39 . .. . Abbreviations Used In l h i s fable i : CLC gas liquid chromatography IR infrared spectroscopy W = ultraviolet spectroscopy HHR * nuclear magnetic resonance spettmcopy TLC thin layer fixautography PC .= paper chromatography E6 = mass spectroscopy 40 1 u ? 8 P 0 I 0 R ." c4 a ._ U c u U 0 u e o *c c o o n " C 0 ..

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t SECTIQN I V. 6.

. USAGE EVES FRM SURVEYS BY fE) lrr Ah0 NU TABLE IV 2

This tablo is primrily a c o l l o c t i ~ of the most ponincnt results published in a series o f tables by tho Nationul &a& q of Sciencm md avail. blo frorr the National Technics1 Infomation Semi.. T h e tables contain results calculated from data obtained f r o m several sources, princi pally f r o m tho 1970 71 surveys of usage conducted by the Flavor and Extract "frctumrr' Associatioa (=. MI m d t h e Nation81 Ac of Scioncos, Utimal Rosoarch Council (WINRC). The mothods of compiletion md coqutation aro oxplainad in dotail i n tho Addendum t o Tab12 IV 2 b d in sources roforrtd t o i n that Addendum.

'Ihcso documents 8%. 8vril. hlo f r o m the National Tochnic8l Infom8tion Sewice. ..

Tho notes below uhich explain the entries i n this t a b l o r e f e r t o tho ~

Not.. 1  This colran lists she nuL. of tho substmco and i n brackets, srople page that follows.

tho nubor by dich t h o sub, stmco i s l i s t e d in all othor t& hs in this rrviw. Bonoatb tho n u o i s tho t o t a l urnwl poundage us& in flavorr. u reprtod on both tho FEN4 survey ud the NM/ NRt survey. Tho NECSINRC sub c o d t t e e o s t .i r t 8 d thrt tho datr reported ropnsentcd between 608 ud I O t of .tho actus1 pomd8ge rdd8d t o tho nition's ,food supply 8nausllym To astipate per argita daily intako in mg for any r3st. nce. mw the docinl point of the pom& p figure five places t o the left. TU trtrrr into r c c o ~t conwrsian factors and rn estimated b o % coverage. Ihm, 19 annual age of 73,300 l b s f o r =thy1 m t h m i l a t e is r p p r ~x l u t e l y equivalent t o a daily p 8 r +tu intake of 0.73300 ag. . .

Note 2  Tho food crtegorisr for which .usage u l t . reported aro listed Noto 3  Tho n\ Pbcr of fim raportinp usage in ea& foal eitaaozy it in this colraa. Sea thq Adden t o t h i s table for further eqlurrtiarr. 1 ..

l i s t e d hem.. An asterisk (*) indicates 3 OY f or firrr reported us8gom i "

Note 4  These columns l i s t :he weighted means of t k *usual a d r u x i u levels of use reported on the survey of usage. ' Usage 'levels were reighted wag* level reported by each f i m on a given substance w 1 s muJ. tiplied by a ratio ca: culated by dividing the t o t a l pounds reportad by the firm by the total reported by a i l fims. n o levels thus weighted uere s~ lppyd t o ob t a i n tho weighted mans. "occoadiny t o t h e annual poundage reported as being used i n foods. Each Note 5  This lists the age gmup for uhich food consumption data were Note 6  These colums list the possible urd potential intakes of the wad t o calculate tho possible drily intake values.

designnrrrd substance for. each category by aye group. The t o t a l f o r a l l categories is presented a t the top o f each c o l m on a level w i t f r the nue of the substance. These intake lovels WIT calculated, a t described in tho Addenb, from usage lavolt and food COnSullptim data obtained h m surveys conductod by the Ynrket Research Corporation of h r i c a and United States Deputnent of Agriculture and are highly inflated as 8 r e s u l t of the m l t i p l e exaggercrtions b u i l t into the calculations. first; the (wslmrption is made ,,.~.. .. \: ~i? 4 that if a substance i s used i n one food i n I cotegozy, it is used i n a l l lurri foods in that categozy. . For cxnqle, if one mvfacturer reported the w e I YU i n a l l bread, cakes, cookios, etc. in the baked goods crtegozy a t the , of a substance i n cheese blintzs, i t would be assumed t h a t the ,substance s a n lovel. Secondly, i t i s usurred that the 8verrpc person eats food fmm .; 1 food categories rvry diy. This would result .in a' d a i l y i n t & of oyer . .

5,000 Calories, obviously an exrggoration.

of thr usage data and the inherent ikccurrcies that exist in the. tdle. To fully understand and apprtcirte the finitations of this data, thd Addendum should be nvisved c u e f l l l y , I Iho foregoing discussion. is only a brief sumnary of t h t significance .. i ..

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' ... . .' 61 ." 6 _."" """" .. " 0 I fie purpose of t h i s ciiscussion iS to revlev in 8 cursory, m n t s h i c r l way, tire background, purpose, VIIUCS, and l i n i t a t i m s of a w j o r e f f o r t to gather data relating to the w e of food ingredients wtrich are generally recognized as. safe ('* GUS* '). Not only t h i s sumnary discussion. but the f u l l report should 'be read carefully by those intending to d e .any use of the data.

1. Background and Purpose A review of the safety and appropriate classification of substances ' ~e n e r a l l y flecognized 2s fafe" .( '',,,, 'I ('I for their intended use in food, re@ res (1) identifying each substance, A d (21 gathering for each substance all available data on its toxicity and occurrence in food:. goth kinds of dot8 are necessary, since the safety of 8 substance is detemined 3y consid ering both its toxicity how little or grtat. its inhertnt capacity, to cause h m a n d the amount of the substance t o vhich) uc are exposed. Determining exposwe requires two primry c. ypes of informtion: First, ,

hos arch of each GRAS substpnca is wed in each clearly defined food category? Second, how mch is eaten of each food category in chich thc CFUS s& stance is wed?

2. Survey Procedun AS part o i thm GRAS review, a special subcorittcc of the htion. 1 Academy 'of Sciences conducted a .survey designed t o provide informtion an the probable and possible intake of such food ingredients. The subcornittee US& both its own f a c i l i t i e s and those of sever81 ifkiustxy groups.. nit took the forr of a co. plex, comprehensive survey of the chemical and food processing industries t o deterrino the exact identity +nd specifications of the subttances aided to various foods. The suney obtained confidential inforntion fro. each participating firm on erch CRM ingredient used, the f a d categories in which ~

used, and the usual a d 8aximm levels of use in e8ch food category. Respoading firms kdic8tcd the importance they attached t o each ingredient, the errliest date of use, and the pomd8ge of the ingredients they eolritted to the food (') See Glossary of Tcru f o r d e f i n i t i o n of underlined words. 2

supply. The survey'provlded much additional data or1 toxicology, natural Occurrence, and other information relevant to the evaluation o f safety. n, e Comaittee made use of other surveys bhich covered specis1i: ed ingredient areas., such as flavors, the ingredienrs used in chewing gwn and certain candies, and brewed oevtrayes. A separate survey covered infant foods. Thew usage data vera reviewed for consistency, accuracy, and appropriate ness, referred t o the originators for correction or :orfirmatier, *here necessary, tabulated i n a way t o preserve confidentiality and collated to provide informa tion of more direct value in safety evaluation. In order that information on the amounts of each ingredient used in food be applied to the estimation of possible human intake, the Committee obtained from t h e Market Research Corporation of Anri'ca (bRCA) data from a survey of frquency of consumption of a l l food i t e m eaten In or away froa home. nese data were r5classified to f i t wizhin the food categories crployed in the Academy survey of ingredient usage by the food industry. Data on portion size obtained from a US9A survey were employed t o complete t h e c3lculation of ..

"average" and "high" coalsumption of each fooci cakcgory used in the survey. me data finally present. ed on each GUS substance included weighted Lcans of the wuai and maximm &e in each food category, potential "average," and high intakes from each focd category and the tot81 diet by age, total yorradage used in the food supply, technical effects for which used, and industrial importance.

3. Scope of the Survey Within the limits of 8vailible tire and funds, the Comittee concludcd t h a t t h e survey achieved its intended purpose. Consistent and apparently valid information was obtained from firms responsible f o r 60 to 70 percent of the nat. ion's processed food supply (andeven a higher percentqe for flavors utd certain specialty foods:.

4. Valuer  mere s w e y s provided the first n l i 8 b l e d a t a on the levels and r b c r of use of oost ingredients on the original FD4 'White: Listn and those used by prior sanction. I t generated ve ry useful detailed information, including re .

checks, on :he highest wages of ingredients, and the foods in which such @". ,.

k j" . r*.+$ >, high levels ocrwrred.

.?" '. L. 63.

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The survey shows uhether a, ri ingredient was used by m y f i m s o r only a few; or used i n many or few food categories. A related concluslon to which the surveys point is the familiar, but frequently ignored, need to achieve variety and balance in and t o avoid an excess The surveys also on t h e amounts used in use can be estimated. is an extremely useful of other data. the diet as t h e best way to insure adequate nutrition of any ingredient, whether naturally occurring or added. provided data on the importance of each ingredient, and the total f& d supply fromwhich average per capita While not directly valuable in safety evaluation, this bench mark against which to compare the reasonableness These efforts provided inforubatio; l which is less than pcrfsct; but the best available on food consumption by current categories of processed food andby age. Furthermore, these new results 2rovide consumption data for those individuals who, "eating in a rut," consume a ?articular food category more often than 90 percent of the population. The proccdtxes wed. in these surveys have resulted in several 'benefits. Among these are a consistent, comprehensive clas. sification of "technical effects" for which GRAS substances and food additives are &. xi, and the , definition of food categories mre adapted t o modern processed foods than the traditional nutritional. or commodity classifications. The results of the survey will be of substantial value in providing for industry guides t o good manur'act\ uir, g practice i n ingredient usage. Finally, it is c l e a r t h a t t h i s aust be'a recurrent, orderly process, and these sur

veys have set a useful pattern for the future. . ,

5. Limitations, Cautions, and Restraints Though valuable, these survey data a r e cocsplex and subject t o a number of uncertainties. To mderstand their values and 'limitations and avoid mi$

,interpretation and invalid conclusions, those who wish to use the information in the exhibits or the tables should first read thoroughly the report urd exhibits prepared by the Committee. The most inportant caution t o be observed is t o k g n i z e t h e cumulative effect of the several conservative assumptions mdt in the coqilation of t h e  data. The result is a substantial over estimate of possible intake. The reported data indicate usage in some food rarely a11 foods in a food cotegQry.

i,, '

I 64 . 4

Such d a t a a r e v a l i d o n l y when and i f the ingredient is acxual! y used i n  t h a t category; yet the calculations conservatively assume usage app; iet to a l l foods in a' category. Furthermore, an ingredient may be partially lost in processing due to evaporation, washing away, or other causes.

The survey can only indicate indirectly whether a particular ingredient is used i n many foods within a given food catsgory, in uhich case there would usually be mjny responses on that ingredient, or is *sed only in a feu foods (usually specialty foods). Fdr example, the use of sulfur dioxide is conftned t o sow d r i e d f r u i t s . I t is not used in fresh, canned, o r f r o z e n f r u i t s or juices. Caramel is used o n l y i n some gravies, not in cream and other sauces; yet its level qf use, only in those fords in which it is used, could be taken t o a p p l y to the whole sauce and gravy category. Such e r r o r s should be avoided. Uhether an ingredient is used i n many or feu foods within a category depends upon t h e a v a i l a b i l i t y of alternative ingredients. NO, or few, a l t e r  natives mean wide use. Many alternatives rean g r e a t l y r e s t r i c t e d we, although t h i s is not clearly apparent since the reported level of use sppears :o apply t o a l l f c o d s i n a category e v a though t h i s would seldom, if ever, be the east.

A d i s t i i l c t i o n must be drawn between those tables based on the frcquency with which foods are consumed by the average of the total populatiar ('Total Sample") as compared with those tables (" Eaters o n ) which are based solely on those who consume the food a t some time withir. the suwey period. The l a t t e r d i s r e g a r d s t h o s e i n the population who d i d n o t consume the food during t h a t p e r i o d . (Consumption of any one food category reduces the consumption of other food categories due t o r e s t r i c t i o n s rn t o t a l a l o t i e intakt.) Thus, levels of conslmtption based on "Eaters Only" can be valid only for s i n g l e o r a few food categories. "Eaters Only" intake figures cannot b, e added across a l l categories, since no one eats everything. Such an assumption rould r e s u l t in a wholly impossible total calorie intake. n e same l o g i c makes the levels in t h e "E a t e r s W y " t a b l e s n o t u s u a l l y a p p l i c a b l e even to a l l foods within a single food category. From t h e l e v e l o f each ingredient used in at least .some foods within each food category, the frequency with which each category is consumed, and the portion sire when consumed, the tables calculate the possible d a i l y i n t a k e of the ingredient through each food category. The sua of such possible intakes fTW a11 food categories Frovides an estimate of possible

65 . .. t o t a l d a i l y intake. For the reasons just discussed, t h i s estimate is u s u l l y hizhly inflated. The extent of such inflation can be gathcred by comparing that figure of possible d a i l y intake with the per capita daily intake obtarned fTOP the .poundage figures committed to food, the coqletencss factor of t. le survey (a t l e a s t 60 percent), and the population.") Tht ratio is low i .e .,

. tha exaggeration of possible daily intake compared with the per capita is satall) uhcre an additive is broadly used and where there are feu alternatives.

The r a t i o (the exaggeration) is high where an additive is u s e d . infrequently, as only in specialty foods, o r where there are many alternatives for obtaining the same technical effect. h e cannot take the possible or potential daily intake, even khat is perhaps misleadingly called the flaverage, l' from the "total sample" table, a l t i p l y by the population and the days per yeor, and expect to obtain the annual food us0 of an ingredient. This is an impermissible us8 of the data because of the multiple exaggerations reviewed above. The intake tables inevitably reflect s a c p o i n t a t the upper tail of the distribution curve. From thi's it follows that the intake values labeled "high" or '% e.:,

high" could be obtained only as a result of a highly improbable combination of circumstances; selection fror food Category of arly those foods contain ing a particular ingredient, no toga or loss o f the ingredient, cons tion of the food category at m t u w l l y high frequency QI high levels of llsa of the ingredient or both, and no 1st of alternative ingredients. Irrprobable in itself for one fwd categor/, this is virtually inpossible for more than a feu food categories simltaneowly. Because one could not possibly seek a11 possible technical effects in .

a single hod, and because of the multiple alternatives frequently available, one cannot add a11 substances used in any f+ category to obtain the totof amount of substances potentially occurring a t one time in any t00d or category.

lack s t a t i s t i c a l s o l i d i t y and refiect very limited, therefore mrnetim. S extreme, we. The data base for some food categories and Copulation GOUPS is also narrower than the i d u l . The figures indicated by asterisk t o signify three responses or less This is the NAS 11/# 2 ratio in Exhibit. 58. .... .. ......

"7' '7 ......

0

Is is clear there is nc! final Lay to be absolutely certain of either accuracy, or completeness i n t h e survey. Overall, the survey is as complete as uas practical because of the diminishing returns €or add'itional effort expended.

6 . Qgortmities Tkis survey points out the desirability of having (1) better food eon

sumption rather than use or disappearance data, (2 ) more exrensive analysis of foods to establish what colPponents remain i n them r e l a t i v e t o what was introduced, (3) determination of the effects of preparatim and p l a t e s a s t e on ingredient loss, (4) better understanding of 'food category use by specific population groups, ( 5) estimates o f intake from a l l sources, natural and added, : and (6 ) future surveys t o r e f i n e amd *date these data, The present inforiation deserves substantial additional analysis. A cursory inspection reveals food c o n s q t i o n patterns in processed foods uhich are age dependent o r o f n u t r i t i o n a l i n t e r e s t . I t seems probable t h a t t h e .

ingredients used for different technical effects tend to group within definable wage and t o t a l a n n u l poundage levels. As examples, salt m d t h e t h r e e , sueeten. ers sucrose, dextrose, and corn syrup make up the four most .u s e d GRAS substaxes. Flavors constitute the bulk of those as& i n least voluae.

A better appreciation of such patterns of w e could lead to an aderstanding .

of where it would be desirable from e i t h e r . an economic or a public health point of view to concentrate research on new alternatives.

67 (; LOSSXRY ACCEPTAPLE DAILY INTALE .( AD?)

Daily dose of a chemical that appears to bc without appreciable risk on the basis of a l l the facts knom a t thc time. "Without appreciable risk" is taken t o mean the practical certamty t h a t injury will not resuit even a f t e r a lifetime of exposure.

ANNUAL POUNDAGE The t o t a l number of pounds used per year by'each participating firm or ih t o t a l by a l l r e p r t i n g firms. ..

AVERAGE DAILY INTAKE >

Estimated amount of a ($ AS substance that may be ingested daily through consumption of foods to which the substance has been added.

CONSWION LEVEL The level, i n grams per QY, of a food category consumed by the rveragh person (" total sample") or consumed only by those who, within the sur vey period, ate food from that category at least once (" eaters only").

DAILY INTAKE See CONSfp( PTI0N LEVEL.

FAO/ nm> JOINT EXPERT ' C W f l T E E A committee of toxicologists and o t h e r s c i e n t i s t s .appointed for their individual qualifications and not representing government agencies or industries. Brought together =der the joint sponsorship of the Food and Agriculture 'Organization 'and the World Health Organization for the purpose of defining specifications for food additives, appraising their safety in we, and setting such limitations or requirements for further information as seem appropriate t o them.

FEMA Flavor and Extract Manufacturers' Association. ..

FpW GRAS LIS Set GRAS LIST. .. a . f" 8

The food at consumed distinguished fro8 intermtdiate and incoaplete stages of processing or preparation, such as dry atixes, concentrated syrups, ray meat or vegetables, etc.

FLAVORING ADJUNCT A substance which does not itself contributc flavor, but which is used in association u i t h flavoring ingredients t o improve their effectiveness in use. This includes .solvents, fixatives, hntioxidantt, etc.

FLAVORING ADJWAHT Synonym for FLAVORING ADJUNCT FUVORINC INGREDIENT oft& simply called a "flavor," is any substatce added to food, ;Irugs, or other products taken i n the mouth, the clearly predominant purpose and effect of which is to prqvide a particular flaror in the final product.

"1. a: miterial consisting essentially of protein, carbohydrate, a d fat used in the body of an organism t o sustain growth, repair, and v i t a l processes and t o furnish energy; also: such food together l l f h supple mentary substances (as minerals, vitamins, and condiments)" This is used here in the sense of human fodd. In the law, the term ** food**. includes beverages, chewing p, and the componenu of ail food articles.

FOOD ADDITIVE General Definition ~n y m i i g r e d i c n t added t o food, or residues ofwhich are found in fwd, resulting fro. its use to achieve a particular technical effect. (2) . . Leg81 Definition  ** Any substance the intended use of which results or m y reasonably bc expected t o r e s u l t r d i r e c t l y o r i n d i r e c t l y , in its becomiag 8 corponcnt or othemise affecting the characteristics of YIY focd (including my substance intended f o r use in producing, manuf8cturing, packing, pro cessing, preparing, treating, packaging, transporting, or holding f&; and bcluding any source of radiation intended for any such use), if such substance fr not generally recognized, among experts qualified by scientific training and experience t o evaluate its safety, as having been adequately shown through sciemific procedures (or, in the case of a stance used in food p r i o r t o January 1, 1958, through either s c i a i t i f i c procedures OT experience based on co n use in food) t o be safe under the conditions of its intended use; ... .'*

(l) Webster'$ Seventh New Colltgiate Dictionary, p 324. Springfield, Massachusetts: G. 6 C. Merriaa Company (1969). 9

AJI act of Congress, parsed in 1958. amending the federal Food, Drug, acd Cosmetic Act. The amendment established' requirements for premarket clear a?:=; f o r safety and functionality o f . a11 substances intended for use in food h i t h certain exceptions. The exceptions include substances that qualifjcd scientists generally recognize as safe, other substances covered by a '. prior sanction" (q. v. 1, color additlves, and pesticidal residues,

f OCQ cATE03RY One of 34 categories into which a l l foods haye been classified. for the purpxe of t h i s survey. Categories consist of foods closely related i n purpo,~, structure, process, composition, 01 propcrties.

GOOD MANUFACTJRXNG PRACTICE Procedures, facilities, quipment, and personnel training, whichtoken togethex and properly applied, define the conditions for safe and effec tive processing of food.

GRAS The acronym for *'generally recognized as safe." it is a s l i g h t ad; pta tion and incomplete rendering of the language of the Food Additives hzdment of 1958 to the Federal Food, Drug, and Cosmetic Act. I t r e i r r r to I*. . . t substance . . . generally recognized, among experts qualified by scientific training and experience to evaluate its safety, w having been aCequacelY shown . . t o be safe mder the conditions of its incended use; . . ." '17s total, much longer definition. is gwen i n Section 201 of the Act. (See also FOOO ADOITIVE.)

GRAS LIST A a&, used, but. rnaccurate term f o r all substances used in food an the basis that they are GRAS, rather than regulated additives. There has Tot been .any single "GRAS List." The FDA published two intentionally incoaplete lists ('White Lists'3; the flavor and chewing gum industries pub1 i s h 4 8 series of lists (the "FEMA GX4S ?. ist* '), the brewing industry pmpared a coqrehensive unpublished list, the FDA presmed some sub stances t o be CRAS without publication urd issued "no objection letters," usually tmpub1ishcd. m feet other substances, and thert undoubtedly were, as the law allows, some private, rnputlished determinations that a use of a particular substance was WS. Aside from these groups, USDA and FDA had i n earlier y e n s approved Dy "prior sanction" a large number of substances not necessarily covered by thr; e l a t e r GUS actions,

IMPORTANCE Commercial importance in the sense o f how unique are the properties of the ingredient, and how easily can it be replaced by another ingredient or change of process. \ ...... I INGRED I ENT A component Or constituent 3f food. The term is usualiy U J ~ in the sense of an ingredient vhich is mtentionally made a part of food. .

Cozstituent often mans a naturally cxcurrkg component.

WIMJN USAGE LEVEL Highest level used in any product.

WEA? ' INSPECTION ACT kr act of Congress originally passCp in 1907, amended and extend4 to permit continuous inspection of neat slaughtering. and processing est& l i s b e n t s by employees of the Dcpar tmnt of Agriculture.

HEDUN USAGE LEVEL The usage level of a particular ingredient in a food categor), uhich f a l l s in tho middle of the reported uses; i. e.. half the wes are reported a t a lower level and half the uses, a t a higher level.. . "

NAS ,.

National Academy of Sciences.

NDN USER FIRMS Firms uhich. mnufacture or distribute, but do not cornit the kyr& ieat t o food.

Letters issued by FDA after 1958, by which FDA indicated that there would be no objection posed at the ti of the l e t t e r to the w e t; food of the ingredient or ingredients named therein in accordance  . with the. uses outlind.

Offensive to the sense of tsttt or me11 and therefore inedible; usually usel t o refer to 1 food which is spoiled or overflavord. ..

PER CAPITA DAILY INTAKE An average figure derived by simply dividing annual national food use of an ingredient by the population and days in a year.

PORTION SIZE A figure derived from estiuutes of nean food consumption using "total le'* (see def'nition o f CONSUMPTIC3 LEVEL) and average food additive use for each foot category, described in Tables i3A and B IS "aver: lge intake level. '' Bccause or' a series of conservative assumptions (see, discussion in report), t h i s reprtsents J pgssible intake that would only infrquently be achieved.

POTENTIAL MILY INTAKE A figure dcrivcd from estimates or' food c o a s q t i o n based on "eaters only" [see definition of COPISUHMION LEVEL), or the 90th percartile frequency of consurption, or higher levels of hod additive use., or some co. bination of these for each food category. These are described in tat tables as ''High A," ''High B," or "Very High," or "Eaters Only.** In addition t o t! w conservative assumptions inherent in the calculation .

intskes mlikely for .ore thar. one or a few food categories.

WL'TRY PRODOCM INSPECTIW A r c f . . of 'possible" if. tOkCS, these additional factors =de the potential 1 This 5 M 8 C t O f COngrCSS passed in 1957 t o provide for the inspec tion of poultry and poultry products and otherwise rcgulatiag the pro

.n." cessing and distribution of such articles in interstate copmerce. ;=.><

Ld f \., PRIOR SANCTION Action prior to 19S8, by the FDA or USDA. to p e d t 8 substance to bo usd in food der the Food, Drug, and Cosme: ic Act, the Poultry Products Inspectiat Act, or the M e a t Inspection Act.

SPECIALTT FO0OS Foods of iiritrd appeal, usually to particular ethnic, economic, or , geographic groups, often mote expensive and limited_ in distribution and conswption. Th. tern irplles lack of broad appeal or frquant .

US..

The rchitvicg of 8 desired characteristic in a food or food manufactw ing process. For euPpla, 8 desired nutritional content or. antioxidant in a food produet; ws* of removal f r o m a baking pan; or ease of extm sion for pasta product.

see cotlsw1oN LEVEL.

Capacity of the substance to produce injury. '&% e term includes capacity t o induce teratogenic, mutagenic, and carcinogenic effects.

72  12

Refers t o "Food Intake and Nutritive Value of Ilietr of h, b m , a d chihirea in t h e United States, Spring, 1965, '* a preliminaq report by' the Gaasuvr and Food Economics Research Division, b r i e u l t u r i l &sar& Service, hit4 States Department of Agriculture (sea Exhibit 328). ,.

USER FIM Nom1 or average concentration roughly weigh4 by amur1 prodwt VOIUC.

YEIQITU) MEAN USAGE LEVEL 'The average level a t which a p r t i c u l a r ingredient, if it is ~4 , is added to foods within a fwd category weighted in proport' &on to the VOIWC of the additive used by each firm; e. g. , a firpl Vhia wed a mil lion porurb of an additive coats in the average one Wrtd t i a c t =re thrn 8 fim which uses o l y lO,, OOO pounds. The bibliograpky presents a c ' q l e t c l i s t i n g o f a l l l i t e t q t u n a r t i c l e s and references used in the preparation of t h i s r c i e n t t z i c l i t e r a t u n r e v i a . me e n t r i e s i n t h i s s e c t i o n ire l i s t e d i n alphaLetica1 order, by nane a f the principal author: The bibliography include a l l a r t i c l e s 1cca: lti i n the literatwe sear&, whether o r n o t i n f o n n a t i m f r o m those arti: lrss w. u included in the literature review. Those references denoted by a tingle asterisk (*) are a r t i c l e s c i t e d within the literature review, ad references denoted with a double asterisk (0 0 ) are a r t i c l e s cited within the S m r y portion of the review, in Section I. Y d u m e t 11 and 111 contain copies of a l l c i t e d a r t i c l e s , inclvding . . .

English trans! o: ions o f f o r e i k language. articles.

.. 1.

2.

3.

4.

+* 5.

6.

7.

,. +* 8.

' 9 ,

10.

11.

** 12.

13.

+ 14. Andrcvs, B. D., A. J . Poyaton, and I. 0. Rat. 1972. Aromatic a i d e r . VII. Steric hiadrance to hydrogen bocding io o substituted acetanilides. Auat. 3 . Cheg. 15( 3): 639 646.

Arctander, S. 1969. Perfume and flavor chedcalr, S. Arctander, Moatelair, N. J. Vol. 1 and 11.

Barbaa, S. I., N. S. Karavya and U. S. Ilifnavy. 1971. Study of cue peel ailr of lemoa, lima and mandarin groviug i n Egypt. Ip. Prrfm. Corret . 86: 53 56.

Bar, ?., and e'. Gricpcatrog. 1967. Vherc & rtaad coacrrning the evalurtioa of flavoring rubrtanccr frcm the viewpoint of health. Ktd. Lruaehr. 6: 264 251.

Brom, P. E., m d J. X. Price. 1956. Quantitative studirr ah metabolite~ of tryptophaa in the urine of t h e d o 8 ,c a t , rat, and un. J. Diol. Cha. 219: 985 997. 1 i CaIvarano, 1. 1968. q t a l i u r p e t i t g r a i n o i l . 11. Eiiar. derrrd bergamot petitgrain oil.. Esreare Deriu, a r m . 33( 1 21: 3143.

Carcoavt, P., and I . florun, 1974. On the ecnporitim of the uma of black tea. V. Helv. Chim. Acta. 57( 25): 209 211.

Charcoanct Earding, f ., C. E. Dalgticrh, and A. ileuberger. 1953. The relation bctwea riboflavin and tryptophan rctabolirr, rtudied in the rat. Biocbrr. J. 53: S13 521.

D i C i a c w , A. 1967. Citrur errmtial oil;. XXIX. Riechrt., hr n, Koerprrpflrga. 17( f1): 460, 462.

Drauert, ?., urd A, von Rapp. .l% 6. The' componcotr of rurtr (grape j u i c e r ) and wincr. VIf. C u Cbra8tOgr8phiC invtrtigatioa of a r a a t i c r u b r t u ~c e r i n vi* and tbeir bi@ gencrir. Vitir. 5: 3S1 376 .

Drawert, ?., and A. voo Uapp. I%& Car chraatograghic malyri8 .

of plant araar. I. The enrichment, reparation, and identification of v o l a t i l a arm. rubatancer i n grape mrtr aid vines. Chromato g r q i d & 1: 446 457.

Ekman,. B., and J. P. Strabcck. 1% 9. The effect of some rplitproductr of 2, .3' acotoluene on t h e u r i n a r y b l a d d e r i n t h e rat and t h e i r excretion at wriour ,dictr. Acta Pathol. Xicrobial. Seand. 26: 467 471.

Sracntial O i l .Arroeiatioo of U. S. A., fnc. 1970. MIA rpteificatioor and rtandardr. #cu York, Reu York.

m, 1974. S c i e n t i f i c l i t e r a t u r e r e v i e v of aliphatic pr, imary aIcoholr, aldehyder, aeidr and id :era i n f l a v o r usage. Vol. 1 VLI, Publiahcd by 0. S. Food md Drug Admamiatration, Yarhington, D. C.

7s 15 16 17.

18.

19.

20 21 .

22 23 24.

25.

26 .

270 28 .

29 FEU G U S Subrtance8, 1965 18. Recent progrerr in the coaridtratioa of flavoring ingredients under the food additives onendment. CRAS rubrcancer. A series of 9 articles publirhed i a Food technol. 19( 2, part 2): 151, 1965; 2 f O ):?S , 197C; 2 O (5 i :3 5 , 1972; 27( 1): 64, 1973, 27( 11): 50, 1973; 26( 9): 76, 1974; 29( 8): 70, 1975; 31( 1): 65, 1977; 32( 2): 60, 1976.

Fieldr, E. K. 1971. Xethyl anthranilates. U. S. Pat. 3,625,389.

Food m d Drug Adminirtration. 1977. Swmrry of toxicity &ti. Unpublirhcd Repoct.

Food a d Drug Research Labotrtoticr, Ine. 1963. Hetabolic fate of methyl o methyl anthraailatc. No. 84919. Unpublirhed Report.

Food Citemicala Coder, 2nd ed. 1972. C ittee on specifications. Food Chemicals Codex of the Camittee of Food Protection, Uation. 1 Research Counc'il; National Academy of Sciences, Yashington, DC. 1039 p p o Poreiaa C pound Hctrbolia ia Mmmrlr. Vol. I. 1970. Vol. 11. 1972. Vol. 111. 197s. The Chemical Society. Burlington Roure. London.

Puria, t. B., and W. Btl~ mer, cd. 197s. Fcnaroli'r handbook of fl. vot ingrtdieats. 2nd ed. Vol. 1 11. The Chemical Rubber Company, Cleveland, Ohio.

Cabel, L. P., and U, R. J. Sirpron. 1972. Dialkyl  cliaubrtitutcd 4  ~~ hydroxyalkyl) amino) quinazoline nitrates. U. S. Pat. 3,637,699.

Gaunt, I, l!. .Sharratt, P. Crarro, and X.. Uri8br. 1970. Acute and rbort term toxicity of methyl U mcthpl anthranilate ia rats. rood Cornet. Toxieol. 8( 4): 359 368.

Civaudan Index, 1961. Specifications of ryntheticr m d isolate8 for perfmery. 2nd cd. Civaudaa Deliuiuna, fnc., New York. _.

431 pp*

Grundrchobcr, I, 1977. toxico~ ogic8l asrcrrocnt of flavotiry esters. T O I i C O l O u . 8: 3870390.

Cuentbar, t.. 19SO. The errentirl oils. Vol. I IV. D. Van Rortrrad Coaparf, Inc. Priacetoa, Rev Jcrrey.

Hagan, C. C., Si. %. Ranren, 0. 0. fitrhugh, P. W. Jcnner, W. I. Joner, J. ll.. TayIor, E. L. Long, A. A. Nelson, and J. B. Irouver. 1967. Food C08met. toxicol. 5: 141 157. .

euet, Eo 1968. The of citrus fruit juicer. Fruits. U( g): 493 471.

76 ** X.

31.

32.

33,

** 34.

35.

36.

3?.

** 38.

** 39.

** bo.

4' 41.

** 12.

** 03.

** 44.

b S e ** 66. Ienner, P. X., E. C. 88g. n~ J * X. Taylor, E. Lo Cook, and 0. C. Pitthugh. 1964. rood flavourings aad compouadr of r e l a t e d s t r u c t u ~e . X. .\ cute oral toxicity. Food Coract. Toxicol. 2: 327 343.

Kahn, J. H. (Rcvicv). 1969. Compound8 identified i n whiskey, vine, i n d beer: a tabulation. 3. Assoc. Off. Agric. Chca. 52( 6): 1166 1178.

Kicchncr, J . G., und 3. M. Hiller. 1953. Volatile oil conatituentt of grapefruit juice. J. Agric. Food Chem. 1( 7): 512 518.

Kugler, E.# and 3. Kovatr. 1963. Information w Handarin peel o i l (Citrus rcciculata Blanc0 or Citrus nobilir var. delicioza Suinglc "Xandarin"). Ilclv. Chim. Acta 46: 1480 1513.

Longland, R. C., W. P. Shilling, and S. D. Gangolli. 1977. The hydro, lysis of flavouring esters by a r t i f i c i a l g a s t r o i n t e s t i n a l j u i c e r md rat risrue preparation.. Toxicology. 8: 197 204.

Warck Index  An encyclopedia of chemicals a d drugs. 9th edition. 1916, Herck aad. Company, Inc. Rahvay, R. 3. 1313 pp. i National Ac8dcap of Scieacrs. 1970. Evrlwtioc! of food eheucalr. Wa8hiagtoa, D. C.

National Pomdrry XIP. 14th ad. 1975. Aaerican Phomaceutical Asrociation. Yashiagtou, D. C. ..

Opdykc, D. L. J. 19fSa. Xonographs oa fragrance raw rateriala. a Eutyl aathrMilrte. lood Coatcat. Toxicol. 13: 727 728,

Opdykc, De L. J. 197Sb. Mouographr on fragrance raw mat, erialr. Cinn. ay1 anthraailate. lo& Comet. Toxicol. 13: 731.

Opdyke, b. L. 3.. 1975~. Honogrrpha oa fragrance mu materials. Dimethyl aarhrmil8te. Food Comet. Toxicol. 133791.

r Opdyke, 0. L. J. 1976.. ~oaographs 00 fragrance rau materiala. C i a l y l aathrmihte. Pood Comet. Toxicol. 14( 5): 459.

Opdyka, D. L. J. l976b. l4onographa 021 fragrance raw materials. Ethyl mthr. nilate. rood Comet. Toxicol. 14: 7f9.

Opdyke, D. L. J. 1976~. Monographs on'frrgrancc raw materials. Phenyl ethyl urthtaaitrta. Food Comet * Toxicol. :& :831.

Oser, B. L., S. Carson, and X. Osct. 1365. Toxicological teet8 06 flavouring aatfer8. Food Cosmet. Toxicol. 3: 563 369.

'Oset, B. L., a d It. L. 8.11. 1977, Criteria employed by tbe . .

Expert P, aael of P. t. KA* for the GRAS evaluatioa of flavorizq ,rubstancta. h o d Comet. Toxicol. 15: 457 466.

pelli, b., lt. Longland, n. Dufltp, and S. D. C; n~ o; 1.5, *A . study of intestinn1 abrorptios of four flavouring caters in the guinea pig. ToxicologJl, (s u h i t t t d f o r p u b l i c a t i o n ).

77 .

*. ** 48. Registry of Toxic Effects of Chemical SubstJnces. 1977. U. S. Dept. of Heaith, Educ8tion and Welfare. Public Health Service Center f o r .

Disease C o n t r o l , N a t i o n a l I n s t i t u t e for Occupatimal Safety a d Health, Rackville, Maryland. 1296 pp.

49. Roger, N. F. 1961. The r e c o v e r y o f m e t h y l a n t h r t n i l a t e i n concord grape 1 essence. Food Technol, 13( 6): 309 314.  .

.. SECI'ION V I .

DATA GUIDE The following guide provides a slfmrmy each substance i n t h i s S c i e n t i f i c  Literat. of various data included for Review.

The principal name and FEMA number of the substance is foliowed by its synonyms. ?he Code of Federal Regulations (CFR) refereace is given for those sub

stances publibhed by the Food and Drug Administration.

?% e range of average usual and average maximum usage levels of the sub

stance is shown next, as u e i l as annual volumc i n pounds. These data are taken from tho 1970 71 NAS and FEW surveys. Tfie pur cupitq daily intakes are calculated as described i n t h e introduction t o Table IV 2 froa the annul MlUIDU.

I f the substance has been found as a natural component o f food, this is noted a f t e r the =age data, w i t h the number of'different foods in, which the substance is found indicated i n parenthesis. Finally, a sununary of the biological data available'for the substance is listed. . The references am t o the sunme of the principal author and the year of tho publication for each study. lhe bibliography can be consulted .for the complete journal referbncc,. This guide has been generated by computer and is therefore printed in capital letters, with the brief descriptions of the biological data being limited t o 60 characters. uhbro Greek l e t t e r i should occur, these 8 printed as t h e i r English equivalent followed by ui ampersand; .. i. 0. u AIL mc! A .DL. ,

J .

VGLUHE (L E I 73300

RAT CRAL HAGAN 6 7 FOA UR 77 r (continued on next page)

<I I N V I T R G GRUNDSCHOBER 77 VL'LUCIE 4431 1784

NArURAC GCCUHREhCt Ih FbkO ( 2)

.. REFEWENCE CPOYKE 16 . .. .

USE LEVEL (P P N , MAX 1.00 f O 2. so USUAL' 4 30 TO 2.00 S E M I 2020 dUlYL AklHRAhlLATE [J]

BUlYL 2 A* IhC& EhZCATE dlllYC O AMl hCdEN2l; ATE CFR 172 5lS FEHA# 2181 REFERENCE OPDYKE . 75 i" * 7 I .. 8 tULOG ICAL .OA rA NONE 38.00 33.33 REFERCNCE NO&€ CFR 1? 2 51S A& TERP INYL ANTHRAkIl4Tt [8 ] i FEPAr 3048 ~

TERPINYL 2 AFINUSEkLUATf 7EAPINYL O Af41Ni26EhLOAlEE TERPINYL ANLNThRAhILlTE "SO CALLtO' P MEhIHA l EN 8 YL 2 AHIhCBthZCAIE P MEN7H I Eh 8 YL AYTHRANlLAIE 8 lOLOCICA1 DATA kONE REEERENCE Noh€ . C F R 172.515 PER C A P I T A IFtTAKE (W /O A Y ) 0.00* 25 OPOY KE 7s 1 B I U L O G I C A L D A l A hUNL , R€ fERENC€ hONE ..

f ..

.. .: . I f/"# Lp I CFR 172a5LS VOLUME 118) PER CAPITA I N f A K € tl4G/ DA7) .

2036. 0.02036 NATURAL OCCURRENCE IFC FGOU [S)

BIOLOGICAL O A T A R A T MAL LO1 5 0 ) 2 25 3.38 GfUG REF ER€ NCE GAUNT 70 ,'

(continued on next page)

93 i .. I 4 .." ""_ "i .. . . . " . .  .. . .

USE L€ VEL IPPMJ : MAX 7.00 TO 12.00 USUAL 2.67 TO 6.00 6IOLOGICAl O A I A NGN E .. 95 fiEfERENC€

NOM . .. 1. Food Flavoring and Compounds of Related Structure. 1. Acute Oral 2. Acute Oral Toxicity and Repellency of 933 Chemicals to House and Toxicity.

Deer Mice.

Factors Affecting Absorption from Hamster Cheek Pouch. 4. The Acute Oral Toxicity, Repellency, and Hazard Potential of 998 Chemicals to One or More Species of Wild and Domestic Birds. 5. ComparisonofFish Toxicity Screening Data and QSAR Predictions for 48 Aniline Derivatives. , 3. Studies of Drug Absorption from Oral Cavity: Physico chemical ' I I Research Section Food Flavourings and Compounds of Related Structure I. Acute Oral Toxicity p. M. J, ENNER, E. C. HAGAN, JEAN M. TAYLOR, E. L. COOK and 0. G. FITZHUGH Di,. tsion o/ Toxicological Evaluation$ Food and Drug Administration, Vnifed States kpartment of Health. Education, and Wel/ m, Washington 25, D. C., U. S. A. (Received 11 MUF 1964)

Al& act" Oral dosages of 107 synthetic and naturai flavourings and structurally related corn ~~u n d s were administered by intubation to the mouse, rat or guinea pig. A n i d were ob served usually lor 2 weeks during which time the development of toxic si was fdlowed and tim of death r~~ rded. The acute oral LD,, of each compound was determined.

MRODUCTlON Substances w d as food flavourings have rcceived little attention from the toxicological vjewoint. Becaw of their extensive ux as food additives, the Food and Drug Administra tion has been investigating their toxicity. The initial step in our toxicity studies was the determination of the acute oral effects. This paper presents data on acute toxicity for a large number of flavouring matters. Similar data are reported for additionai compounds, not ncccssarily flavourings, but included as a means of correlating structure with toxicity. These relationships b v e been discussed by Taylor, Jenner & Jones (1964) and Hagan, JeMer, Jones & Fitzhugh Toxicology: Long, Brouwer & Webb Pathology (1964). Flavour additives include compounds with a wide variety of chemical structures, and mixtures of variable composition derived from plants and other natural sources. Some of the substances are synthetic, others are isolares or extracts of natural products. Since the purpose of the* studies was to evaluate the toxicity of thex materials in relation to their UK as food additives, a commercially available material was used. No attempt was made to secure chemically pure compounds.

METHODS Groups of 10 young adult Osborne Mendel rats evenly divided by sa were fasted for approximately 18 hr prior to treatment. Groups of guinea pigs consisting of both males and females were fasted for the same period. Mice were treated on full stomachs. Animals had access to water at all times, and the food was replaced in cages as soon as animals reccivcd their respective doses. A I 1 doses were given by intubation. All animals were maintained under clox observation for recording toxic signs and time of death. Such observation was continued until animals appeared normal and showed weight gain The usual observation period was 2 weeks; in a few cases, where no acute toxic signs were seen, the animals were observed for only one i* eek. LD, 's were computed by the method of titchfield & Wilcoxon (1949).

A 327 343 Condiments et Complexes de Strucfurc I'oisine. I. Tosiciti Ai@ par \'& Buccale ~Cwni On adrninistra par intubation des d m de complexes faits & 107 condimenu syn thetiqua et naturels, de structure chimique voisinc. i da souris, des rats et des cobayes. On obxrva habituellemnt ks anirnaua pendanI2 scmaines, durant lerquelks on suivit k divelog pcment de sigtus toxiqua et on nota la date de la mort. Pour chaquc compkAe on dClcrmina la dose ordelimite au deli de laquellc commence I'intoxication aigul!.

Lebensmittelg~ hmac~ zusatze und Verbindungen verwdter Struckt\ mn 1. Akute Onltoxititat Zu~ tnrnenf~~ smng" 107 synthetirhen un narorliche Gtk hmackuudtte und nrukturvcr wandte Vcrbingdungm wvrden durch. Intubalion an M a u ~, Ratten und hlecrschweinchen vcraixeicht. Die Tiere wurdcn gewohnlich 2 Wochen Ian8 unttr Beobachtung Iten. uahrend wekher Zcit die Entwicklung toxixhcr Sxmptome vcrfolgl und die &it da Tod exintritts rcpistrien wurde. Die alrute orale rn~ ltlcre Wliche Dosir j d e r Verbindung wurdc feslgcs~ ellt.

B J x .., < cy I 326 FORTHCOMING PAPERS, CORRICESDA I the same an initial description of the substance is followed by txamplcsof its likely use, the toxic dose andlor maximum allowable concentration (i f known) and likely pathological effects. Descriptionsof clinical findings in acute andchronicpoisoning arc related to laboratory and X ray examination, and an outline given of emergency treatment and prop nosis. The author has not hesitated t o use trade names uherc the composition of the formula. tion is no* obvious. This has added to the usefulness of the book, which is concise and cleal, yet sufficiently detailed possibly lo instruct those who may considerthcmselvcswell ac quainted with industrial poisons.

FORTHCOMNG PAPERS I t is hoped to publish the following papers in the nextissue of Food und Co.~ ntetir. t ToxicologJJ:

Food flavourings and compounds of related structure. I. Acute oral toxicity. By P. M. Jenner, E. C. Hapn, Jean hl. Taylor, E. L. Cook and 0. G. Fiuhugh.

The effect on rats of longyterm exposure of Guinea Green B and Benzyl Violet 4B. By W. A. Manncll, H. C. Grice and lsabelle Dupuis. Recherchcs dephysiologie cellulairc sur la losicitti de I'alcool tthlique. Par R. Low, et "1 :

I'olunte I (I963) .

I G. Griffaton.

Etudes sur I'activiti azoreductasique des surnageants d'hornogenat de foie de rat. Par ph. Manchon, S. Gradnauer et R. Low.

CORRIGENDA x , p. 229, linc 13: for E 214 p Hydrox_ vberuc? ic acid, and its sodium salt IMJ E 214 Lth! l 4 ester oip hydroxybenzoic acid and its sodium salt.

p. 57, lines 28 35 inclusive u. hich constitute 'Srction( 4) Resinous and Pol> neric coaliny. ' ,'

I were included in error and should be deleled. 1 i Volunte 2 (1964)

.

. , ' I 1 ...... .. ,. ::. L ........... .....  .. , ~bmd. Fiveindividualbioassayrepeliency or toxicity variables were estimated or determined for deer mice (Peromyscus mnniculafus) and house mice (MUS musculus) under laboratory conditions. ALDS (Approximate Lethal Doses) or LD" s of 2~ chcfnids to deer mice are presented, as are f& nduction (FR) values (3day feeding test as a 2 .~ treatment rate) for whitewheatseeds (Tri ticum aesfivum) for 6% chemicals and Douglas fu x& (Pseudofsuga menziesii) for 81 chemicals. A GmilN repellency evaluation (REP) using a Sday test with white wheat seeds at a 2.0%~ treatment rate was conducted with house mice and the results For 347 chemicals are presented. These toxicity and re pcltency data should be useful to those desiring to predict the potential for acme toxicity in wild mam mals following exposure to a wide variety of chem M S .

A calculation of the daily chemical dose ingested in mgkglday during the wheat test on deer mice and its resultant effects on mortality are also presented for most of the 696 chemicals. This calculated value, when used along with the ALD or LD5,. should permit a rough estimate o€ the potential sub acute toxicity of any tested chemical, on wild m a  mals for which both types of data are availablt.

A series of publications summarizing the results of opproximateiy 25 years of chemical research con ducted by the Denver Wildlife Research Center (UWRC), on wild or domestic birds and mammals has been initiated. The first publication presented wild avian toxicity or repellency results for 998 chemicals (Schafer et 01. 1983). This paper will present similar data for 933 chemicals tested on wild deer mice and white (house) mice. Our purpose is to make available these generally unpublished test results so that they tan be referenced or usedby the various public, private, and governmental groups that may require this information.

Methods ' h e chemicals included in & tests were technical of analucii. 1 grade pesticides and other commercially available or cxperi mental chemicals. Tbcy were purchased from various commer cial sources or conmbutcd by cooperating chemical cmnpanics. For presentation purpoxs. they have k e n prrangcd by Chemical Abstracts Registry Number (CASRN). and PIC idenlified by an accepted trade. coined. product or other chemical name that is penedl? ncrr included in thc 8th or Rh Collcctiw In&\ of the Chemical Absuacts Senice. ' Wild trapped house and deer mice or domestically bred house mice were used in dl test procedures which are dcscnid in deldil by Kvem (1954) and Kverno. a 01. (1965). Five bioassay tests were conducted. resulting in six basic data sets as f0llow. t: ..

Repellency k c rcpeileacy tests were conducted, two on dew mice and one on house mice. ?bc initid test used five individually caged deer mice. Each was offered 2S white wheat seeds treated with 2.0% (wtlwt) oftbe candidate chemical daily for 3 days. followed by 4 days d observation for gross subacute effects. An alternate less preferred food (laboratory d e n t pellets) and aater were .

Because of the length and complexity of Chemical Abstracts nomenclature. tbe names used to identify chemicals in Table 1 were extracted from several sources. Primary consideration uras given to the common name, but shorkned chemical names. code numbers, or registered trademarks were also used but may not be specifically identified.

..

............. ................ ...... ........... .. .. .... .. .. ......... 2.. ; t 12 Alloxan lremorine U re t h u K Bay 37341 DL Penicillamine pilocupint hydrochloride 3,& Diaminopyridim Baycr 37342 Fcnthion Tetraethylammonium chloride Tributyltin oxide Tiiutyltin acetote Dicthylstifkstcrd . '

Bis( phcnoxyarsiny1) oxide 2 Hydroxyquinoline 2 Bentoxm\ d Strychnine sulfate Dimethoate CNilroknzoic acid Sodium fluoroaceute c 1 Phcnylacetylurea 3 Mcthylphcnyluru I Phenylurca Physostigmine sulfate Tctracyciinc bydrochloride ~Aminosllicylic acid Bcnzoic acid Phena+ ine hydrochloride CydoHexamide CM~ zhylkntenesuKoaMlidc Endrin MestTIlrd N,~ Dipkn~ l. Cphcnylcnediamine p Nitrophcnylazoresorcind Ethyl mcrcrptra Isopropyl mercaptan ~~ H BuC~ I AnOXanti8 Auytripbenyltin Triphenyhiu hydroxide Diiurylcin dilaunte Dimcthyhydanloin Bis( tripheay1tin) sulfide . hiutyltia maleate Menaura Thiosemicarbazide 2 MelhacryIic acid Dapsone Tetramethylenedisdfotetmmine coumaphoz I Ethyb k 3,5 Dunelhylpynzole Dichlow~ thyl b i,.+. c . :.> x N Bu! y~~ su~ o~~ a. sulfa" anijidine ".,>,

i,.":. p ., i. :. TigIic acid +:_. Musk xyld Warfarin /'

 1" 50715 +1m 51730 +70.0 517% + 1150 52608 +S I . 52664 + 1230 54711  54966 + 1125 55378 t 347 s5389 461.5. S6348 + I250 .% 359 +Mo 56W 437.5 56531 +I 4 6 56572  58366 + 50.0 59314  59494  60413  60515 +375 61289

62748 263 63252 +6Qo 63989 +I 2 5 0 63990 + 1225 6410% +I238 64471

654% +1113 65850 + 1250 66057 +336 "9 125 67516  70553 .

72004 +a 72208 100 72333 +950 14317  74395  75081 +I238 62231

647s. +925 75332 + 1238 75661 +I 2 5 0 76244 + 1013 76631 68.8 76879 + 75.0 77587 +427 77714 i l 2 5 0 77805 +663 78046 +250 78579 +613 791% +317 79414 1238. 8oO80 80126 113 80228 +121 80591 +flu) 8 i m + 1213 81812 +1233 .. . . . . . , ,

'.. .. . . 0.00 8.00 1.60 94.4 20.7  10.0 72.3 83.0 95.0 97.0 84.0 97 .O 39.5 98.8 w. 0 98.0 0.00     70.0   79.0 s2. o 0.00 2.00. 1.00 .

 38.0 11.0 0.00 73.1 90.0   71 0 92.0 24 0 .  I .00 I .oo 0.00 19.0 89. q1.0095) 94.0 w. 0 0 .9 417.0 14.0. 80.0 51.1 71.7 88.0 0.00 1 00  91 0 1.00. 8.00 3.00 t 3 3 . . ... : '  ' .. .. . . .. . . ..... , .. 82439 + 1 125 82431 + 1225 a3012  83078 .+ SO0 a3u1 +u 3 M662  am + 1238 855%  s55983 + 1125 86533 +850 174'18 +313 87514 + 1243 87M1  1240 87887 +I 1 2 5 88Mo +425 88120 +lo90 88142 ?1225 60299 + 1038 89009 +I163 89021 +I 1 3 8 892s +I 2 5 0 893% + 1150 89394 +12M 89623  @I690 isso 90017 +I 2 3 0 91021 +8U 91634 +I176 92524 +450 92535 + I225 9jm  92842  93049 +825 93107 +925 93185 + 1213 93469 "

93754 +463 94091 94188  94520 +l l S 9 4 8 6 0 .

95034 +975 95647 +900 95692 +315 95716  95761 + 75.0 957w +. 843 9588s  96128 +I240 96242 +625 ,

96311 +I250 964s7 +I 2 5 0 96480 +I245 96504 +350 96537 +20.0 96968 +1175' 96991 +I250 925991  10.0 2.00  56.0 "5 98.4  I 00  10.0 32.0 75 0 0.5" 10.6 0. m 2.40 10.0 9.0 12.8 2.00 17.0 50.0 7.00 9.00 0.00 8. W 0.00  56.0 34 .O 64.0 . , 0.00 5.87.

2.00    u. 0 , . . 26.0 3.00  63 0 .

 5.00  22 0 28.0 70.0 94 0 31 0

 0.800 0.00 0 00 0.330 72.0 38.4 50.0 . .

6.00 0.00  .

Yellow sulfon chloride Genite +Chloro 2, Cdimethoxrrnilinc 2 Amino S azotolume Anantoin &nzenesulfonyl, chloride 4 Bromobenzenesulfonyl chloride &Chlorobenzenesulfonyl cbbridc >Nitroaniline Citrarinic acid SNitro 2 mclhylanili~ ZCNitro 2~ methoxyaniline . . 1.3 Dinitrobenzene Methylparaben Ethyl 4 nitrobtntoate Moslenc 4' Aminoacetopheno~ &Nitroaniline Isocinchomcronic acid KyanopyridiM Benzaldehyde 3€ yanopyridiw 3 F+ yridylcubinol 2 Cyanopyridine Hexamethylenetetramine h c n e +hind' nitrodiphenyI sulfide 2 Ethylpyridin1 ~Isopropylaminophenyllmine 4 Bcnzencazodiphcnylamine 4.4' Diaminophen). lmethanc U' Diaminqphen) l ethc: : Benzylpyridine h'. N" Di srr butyl p phca) fcnediunine Phinyluretbam 2.5 Dimethox) aniline kra Nitrostyrene  Hydroquinone monobenzyl ether Azobenzcne Benzyl succinate DiethyE3ox~ utarate lj Diethylthioulta F'mpyl butyrpte em01 CChIoroaniline 4Methplaniline l Methyl4piperidinol CChlorobenzenelhiol . '

Acrolein l propyl mercaptan Tetraethylpymphosphate >Mereaptopropionic acid Pyrazoxon 3 Methylthiophenol 2.4 Dimethylpyridine 2.6Dimethylpyridine Thiophenol w ;none . . ..