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WHO FOOD ADDITIVES SERIES: 52

ANNEX 6

SUMMARY OF THE SAFETY EVALUATION OF SECONDARY COMPONENTS
OF FLAVOURING AGENTS WITH MINIMUM ASSAY VALUES OF LESS THAN 95%

No.

Flavouring agent

Minimum assay value (%)

Secondary components

Comments on secondary components

A. Alicyclic, alicyclic-fused and aromatic-fused ring lactones

1158

(+/-) 3-Methyl-gamma-decalactone

94% (sum of cis and trans isomers)

1-2% heptan-1-ol

The Committee has evaluated heptan-1-ol (No. 94) and concluded it was of no safety concern at current levels of intake.

1160

Tuberose lactone

45%

28-35% gamma-Dodecalactone; 22-30% 2(3H)-Furanone, dihydro-5-(2-octenyl)-(Z)

The Committee has evaluated gamma-dodecalactone (No. 235) and 2(3H)-furanone, dihydro-5-(2-octenyl)-(Z) (No. 249) and concluded they were of no safety concern at current levels of intake.

1164

(+/-)-(2,6,6,-Trimethyl-2-hydroxycyclohexylidene) acetic acid gamma-lactone

90%

3.5-4.5% 2,9-Dimethyl 3,8-decanedione; 3.5-4.5% 4-Hydroxy-5,6-oxo beta-ionone

2,9-Dimethyl-3,8-decadione has not been evaluated by the Committee However, the NOEL for another diketone, 3,4-hexandione (No. 413) was >17 mg/kg bw per day in a 90-day study in rats (Posternak et al., 1969). A NOEL of 10 mg/kg bw per day was reported for the structurally related substance, beta-ionone (No. 389), in a 90-day study in rats (Gaunt et al., 1983). Another 90-day study reported NOELs of 11 and 13 mg/kg bw per day for males and females, respectively (Oser et al., 1965).

B. Aliphatic, alicyclic, linear alpha,beta-unsaturated, di- and trienals and related alcohols, acids and esters

1179

(E,E)-2,4-Heptadienal

92%

2-4% (E,Z)-2,4-isomer;
2-4% 2,4-heptadienoic acid

Both secondary components are expected to share the same metabolic fate as the primary material. The (E,Z) isomer is expected to be converted to the (E,E) form by the action of 3-hydroxy acyl CoA epimerase and oxidized to 2,4-heptadienoic acid by aldehyde dehydrogenase (ALDH) (Feldman & Weiner, 1972). 2,4-Heptadienoic acid is a substrate of the fatty acid cycle and is metabolized and excreted primarily as carbon dioxide and water (Nelson & Cox, 2000). A 98-day study with the structurally related material 2,4-hexadienal showed a NOEL of 15 and 60 mg/kg bw for male and female rats, respectively (National Toxicology Program, 2001b).

1180

(E,E)-2,4-Octadien-1-ol

94%

2-4% (E,Z)-2,4-isomer

The (E,Z) isomer is expected to share the same metabolic fate as the (E,E) isomer: conversion to the corresponding carboxylic acid by alcohol dehydrogenase (ADH) (Pietruzko et al., 1973) and ALDH (Feldman and Weiner, 1972) and entry into the fatty acid cycle where it is metabolized and excreted primarily as carbon dioxide and water (Nelson and Cox, 2000).
A 98-day study for the structurally related material 2,4-hexadienal showed a NOEL of 15 and 60 mg/kg bw per day for male and female rats, respectively (National Toxicology Program, 2001a).
A 90-day study for the structurally related material (E,E)-2,4-decadienal identified a NOEL of 100 mg/kg bw per day (National Toxicology Program, 1997).

1183

2,4-Nonadien-1-ol

92%

4-5% 2-nonen-1-ol

2-Nonen-1-ol is scheduled to be evaluated by the Committee in 2004. It is expected to be oxidized to the corresponding acid and metabolized in the fatty acid cycle and excreted primarily as carbon dioxide and water (see Nos 1179 and 1180 above).

1185

2,4-Nonadienal

89%

5-6% 2,4-nonadien-1-ol;
1-2% 2-nonen-1-ol

2,4-Nonadien-1-ol (No. 1183) has been evaluated by the Committee. It is expected to be oxidized and completely metabolized in the fatty acid cycle (see No. 1179 and 1180 above).
2-Nonen-1-ol, see No. 1183 above.

1189

(E,E)-2,4-Decadien-1-ol

92%

3-5% (E,Z) isomer

The (E,Z) isomer is expected to share the same metabolic fate as the (E,E) isomer. The alcohol is converted to the corresponding carboxylic acid by ADH and ALDH and then enters the fatty acid cycle where it is metabolized and excreted primarily as carbon dioxide and water (see Nos 1179 and 1180).

1190

2-trans,4-trans-Decadienal

89%

3-4% mixture of cis cis, cis trans, and trans cis 2,4-decadienals; 3-4% acetone plus trace of isopropanol; 0.5% unknown.

The (Z,Z), (Z,E) and (E,Z) isomers are expected to share the same metabolic fate as the (E,E) isomer. The aldehyde is converted to the corresponding carboxylic acid by ALDH and then enters the fatty acid cycle where it is metabolized and excreted primarily as carbon dioxide and water (See Nos 1179 and 1180 above).
The NOEL for (E,E)-2,4-decadienal was 100 mg/kg bw per day (National Toxicology Program, 1997) and 33.9 mg/kgbw per day (Damske et al., 1980) in separate 90-day studies.
Acetone (No. 139) and isopropanol (No. 277) have been evaluated by the Committee. Both substances were concluded to be of no safety concern at current intake levels.

1191

Methyl (E)-2-(Z)-4-decadienoate

93%

5-7% (E,E) isomer

Readily hydrolysed to methanol and (E,E)-2,4-decadienoic acid which is a substrate for the fatty acid cycle (See No. 1180 above).
Separate 90-day studies on the related substance (E,E)-2,4-decadienal identified NOELs of 100 mg/kg bw per day (National Toxicology Program, 1997) and 33.9 mg/kgbw per day (Damske et al., 1980).
Methanol is oxidized in vivo to formic acid. The Committee has evaluated formic acid (No. 79) and concluded that it was of no safety concern at current intake levels. A NOEL of >400 mg/kg bw per day for formic acid was identified in a 2-year study in rats (Malorny, 1969).

1192

Ethyl trans-2-cis-4-decadienoate

90%

5-10% ethyl trans-2,trans-4-decadienoate

Readily hydrolysed to ethanol and (E,E)-2,4-decadienoic acid which is a substrate for the fatty acid cycle (see No. 1180 above).
Separate 90-day studies on the related substance (E,E)-2,4-decadienal identified NOELs of 100 mg/kg bw per day (National Toxicology Program, 1997) and 33.9 mg/kg bw per day (Damske et al., 1980).
Ethanol is oxidized in vivo to acetic acid. The Committee has evaluated acetic acid (No. 81) and concluded that it was of no safety concern at present intake levels. In a 63-day study in rats, the NOEL for acetic acid was 350 mg/kg bw per day (Pardoe, 1952).

1196

trans,trans-2,4-Dodecadienal

85%

11-12% 2-trans-4-cis isomer

The (E,Z) isomer is expected to share the same metabolic fate as the (E,E) isomer. The alcohol is converted to the corresponding carboxylic acid by ADH and ALDH and then enters the fatty acid cycle where it is metabolized and excreted primarily as carbon dioxide and water (see Nos 1179 and 1180).
NOELS for the related substance (E,E)-2,4-decadienal were 100 mg/kg bw per day (National Toxicology Program, 1997) and 33.9 mg/kg bw per day (Damske et al., 1980) in separate 90-day studies.

1198

2-trans-4-cis-7-cis-Tridecatrienal

71%

14% 4-cis-7-cis-tridecadienol; 6% 3-cis-7-cis-tridecadienol; 5% 2-trans-7-cis-tridecadienal; 3% 2-trans-4-trans-7-cis-tridecatrienal

All secondary materials are expected to oxidized to the corresponding acids and enter the fatty acid cycle where they will be metabolized and excreted primarily as carbon dioxide and water (See Nos 1179 and 1180 above).
NOELS for the related substance (E,E)-2,4-decadienal were 100 mg/kg bw per day (National Toxicology Program, 1997) and 33.9 mg/kg bw per day (Damske et al., 1980) in separate 90-day studies.

C. Aliphatic branched-chain, saturated and unsaturated alcohols, aldehydes, acids, and related esters

1209

2-Methyl-2-pentenal

92%

1.5-2.5% propionaldehyde; 3.5-4.5% propionic acid

Propionaldehyde (No. 83) and propionic acid (No. 84) have been evaluated by the Committee. It was concluded that both substances were of no safety concern at current intake levels.

1211

2,4-Dimethyl-2-pentenoic acid

92% (sum of isomers)

5-7% 4-methyl-2-methylenevaleric acid

4-Methyl-2-methylenevaleric acid has not been evaluated previously. A 90-day study of oral administration in rats identified a NOEL of >2500 mg/kg bw per day for the structurally related material, isovaleric acid (No. 259) (Amoore, 1978).

1219

dl-Citronellol

90% (of total alcohols as C10H20O)

5-8% di-unsaturated and saturated C10 terpene alcohols; 1% citronellyl acetate; 1% citronellal

Geraniol, a terpene alcohol, exhibited NOELs of >1000 and >100 mg/kg bw per day in 16- and 28-week studies in rats, respectively (Hagan et al., 1967).
A NOEL of 2000 mg/kg bw per day was reported when rats were fed a mixture of 71% geranyl acetate and 29% citronellyl acetate for 2 years (National Toxicology Program, 1987b). This corresponds to an estimated daily dose of 580 mg/kg bw for citronellyl acetate.
In a 2-year study, the NOEL for citral, an agent that is structurally related to citronellal, was 100 mg/kg bw per day in male and female rats (National Toxicology Program, 2001b).

1220

Citronellal

85% of aldehydes as C10H18O

12-14% mixture of terpenoid materials: mainly 1,8-cineole, 2-Isopropylidene-5-methylcyclohexanol, linalool, citronellyl acetate and other naturally occurring terpenes

1,8-Cineole (eucalyptol, No. 1234) has been evaluated by the Committee. In a 28-day study in rats, the NOEL for 1,8-cineole was 300 and 1200 mg/kg bw per day for males and females, respectively (National Toxicology Program, 1987a). In an 80-week study in mice a NOEL of 32 mg/kg bw per day was reported (Roe et al., 1979).
The Committee has evaluated 2-isopropylidene-5-methylcyclohexanol (isopulegol, No. 755) and concluded that it was not of safety concern at current intake levels. In a 14-day study of oral toxicity in rats, isopulegol was reported to have a NOEL of 250 mg/kg bw per day (Imaizumi et al., 1985).
Linalool (No. 356) has been evaluated by the Committee. It was concluded that linalool was not a safety concern at current intake levels. In an 84-day study in rats, the NOEL for linalool was >50 mg/kg bw per day (Oser, 1967).

A NOEL of 2000 mg/kg bw per day was reported when rats were fed a mixture of 71% geranyl acetate (No. 58) and 29% citronellyl acetate (No. 57) for 2 years (National Toxicology Program, 1987b). This corresponds to an estimated daily dose of 580 mg/kg bw for citronellyl acetate.

1221

3,7-Dimethyl-6-octenoic acid

90%

5-8% citronellal, citronellyl, neryl, and geranyl acetate esters and other naturally occurring terpenes

Citronellal (No. 1220) has been evaluated by the Committee. In a 2-year study, the NOEL for the structurally related material citral, was 100 mg/kg bw per day in male and female rats (National Toxicology Program, 2001b). The naturally occurring terpenoid esters are expected to hydrolyse in vitro to the acetic acid and the corresponding terpene alcohols citronellol, nerol, and geraniol. See No. 1220 above for geranyl and citronellyl acetate. Neryl acetate, being the cis isomer of geranyl acetate is expected to follow similar metabolic pathways and exhibit similar toxicologic potential.

1222

Rhodinol

82% (of total alcohols as C10H20O)

15-17% terpenoid esters: mainly citronellyl, neryl, and geranyl acetate esters and other naturally occurring terpenes

See No. 1221 above.

1223

Geraniol

88% (of total alcohols as C10H18O)

8-10% terpene esters: mainly citronellyl, neryl, and geranyl acetate esters and other naturally occurring terpenes

See No. 1221 above.

D. Aliphatic and aromatic ethers

1233

1,4-Cineole

75%

20-25% 1,8-cineole

1,8-Cineole (eucalyptol, No. 1234) has been evaluated by the Committee. In a 28-day study in rats the NOEL for 1,8-cineole was 300 and 1200 mg/kg bw per day for males and females, respectively (National Toxicology Program, 1987a). In an 80-week study in mice a NOEL of 32 mg/kg bw per day was reported (Roe et al., 1979).

1253

Benzyl butyl ether

93%

2-5% benzyl alcohol

Benzyl alcohol (No. 25) has been evaluated by the Committee, which concluded that it was not a safety concern at current intake levels. A 13-week and a 2-year study in rats identified NOELs of 100 and >200 mg benzyl alcohol/kg bw per day, respectively (National Toxicology Program, 1989).

F. Linear and branched-chain aliphatic, unsaturated, unconjugated alcohols, aldehydes, acids and related esters

1271

3-Hexenal

80% (total of cis and trans isomers)

18-20% trans-2-Hexenal

trans-2-Hexenal is scheduled to be evaluated by the Committee in 2004. A 13-week study of oral administration in rats identified a NOEL of 30 mg/kg bw per day for this material (Gaunt, 1971).

1279

3-Hexenyl 2-hexenoate

86%

6-8% 3-Hexenyl-3-hexenoate; 4-6%, 0-1%, and 0-0.5% of isomers 1, 2, and 3 of hexenyl hexenoate, respectively

cis-3-Hexenyl-cis-3-hexenoate (No. 336) has been evaluated by the Committee, which concluded that it was not a safety concern at current intake levels. 3-Hexenyl-3-hexenoate and its isomers are expected to hydrolyse in vivo to mono unsaturated hexenol and mono unsaturated hexenoic acid. Regardless of the position of unsaturation, the resulting alcohol is oxidized to the corresponding acid that participates in normal fatty acid metabolism.
The Committee has evaluated 3-hexen-1-ol (No. 315) and 3-hexenoic acid (No. 317) and concluded that they are of no safety concern at current intake levels.
A NOEL of 120-150 mg/kg bw per day was reported for cis-3-hexen-1-ol in a 98-day study of oral administration (Gaunt et al., 1969).

A NOEL of >400 mg/kg bw per day was reported for 10-undecenoic acid (No. 331), a material that is structurally related to 3-hexenoic acid, in a 6-month study in rats (Tislow et al., 1950).

1282

(Z)-5-Octenyl propionate

93%

2-3% (E)-5-Octenyl propionate; 0.5-1% (Z)-5-Octenol

Like the Z isomer, the E isomer is expected to hydrolyse in vivo to 5-octenol and propionic acid.
The Committee has evaluated cis-5-octen-1-ol (No. 322) and concluded that it was of no safety concern at current intake levels.
The Committee has evaluated propionic acid (No. 84) and concluded that it was of no safety concern at current intake levels.

1284

(E)-3,(Z)-6-Nonadien-1-ol

92%

6% (E,E) isomer

In a 28-day study in rats, a NOEL* of 2.06 mg/kg bw per day was reported for the structurally related material (E,Z)-2,6-dodecadienal (No. 1197) (Edwards, 1973).
*material was administered as part of a mixture.

G. Simple aliphatic and aromatic sulfides and thiols

1293

4-Mercapto-4-methyl-2-

48-50% pentanone

48-50% 4-methyl-3-penten-2-one

The Committee has evaluated 4-methyl-3-penten-2-one (No. 1131) and concluded that it posed no safety concern at current intake levels. A 14-day study in rats identified a NOEL of >10 mg/kg bw per day for the structurally related substance 5-methyl-5-hexen-2-one (No. 1119) (Gill & van Miller, 1987).

REFERENCES FOR ANNEX 6:

Amoore, J.E., Gumbmann, M.R., Booth, A.N. & Gould, D.H. (1978) Synthetic flavors: efficiency and safety factors for sweaty and fishy odorants. Chemical Senses and Flavour. 3, 307-317.

Damske, D.R., Mecler, F.J., Beliles, R.P. & Liverman, J.L. (1980) 90-Day toxicity study in rats. 2,4-Decadienal. Private communication to the Flavor and Extract Manufacturers Association of the United States. Submitted to WHO by the Flavor and Extract Manufacturers Association of the United States.

Edwards, K.B. (1973) Biological evaluation of 2,6-dodecadienal and 2,4,7-tridecatrienal. 4-week feeding study in rats. Unpublished report.

Feldman, R.I. & Weiner, H. (1972) Horse liver aldehyde dehydrogenase. Journal of Biological Chemistry. 247(1), 260-266.

Gaunt, I.F., Butler, W.H. & Ford, GP (1983) The short-term (90 day) toxicity of alpha- and beta-ionones in rats. Performed by the British Industrial Biological Research Association (BIBRA). Unpublished report to the International Organisation of the Flavor Industry (IOFI).

Gaunt. I.F., Colley, J., Grasso, P., Lansdown, A.B.G. & Gangolli, S.D. (1969) Acute (rat and mouse) and short-term (rat) toxicity studies on cis-3-hexen-1-ol. Food and Cosmetics Toxicology. 7, 451-459.

Gaunt, I.F., Colley. J., Wright, M., Creasey, M., Grasso, P. & Gangolli, S.D. (1971) Acute and short-term toxicity studies on trans-2-hexenal. Food and Cosmetics Toxicology. 9, 775-786.

Gill, M.W. & van Miller, J.P. (1987) Fourteen-day dietary minimum toxicity screen (MTS) in albino rats. Private Communication to the Flavor and Extract Manufacturers Association of the United States.

Hagan, E.C., Hansen, W.H., Fitzhugh, O.G., Jenner, P.M., Jones, W.I. & Taylor, J.M. (1967) Food flavourings and compounds of related structure. II. Subacute and chronic toxicity. Food and Cosmetics Toxicology. 5, 141-157.

Imaizumi K, Hanada K, Mawartari K, and Sugano M (1985) Effect of essential oils on the concentration of serum lipids and apolipoproteins in rats. Journal of Agricultural and Biological Chemistry. 49, 2795-2796.

Malorny G (1969) Acute and chronic toxicity of formic acid and formates. Z Ernaehrungswiss. 9, 332-339.

National Toxicology Program (1987a) Twenty-eight day gavage and encapsulated feed study on 1,8-cineole in Fischer 344 rats. NTP Chem. No. 15-NTP Expt. Nos 5014-02 and 5014-06; NCTR Expt. Nos 380 and 439.

National Toxicology Program (1987b) Carcinogenesis studies of food grade geranyl acetate (71%) and citronellyl acetate (29%). NTP-TR-252; NIH Publication No. 88-2508, Washington DC, US Government Printing Office.

National Toxicology Program (1989) Toxicology and carcinogenesis studies of benzyl alcohol in F344/N rats and B6C3F1 mice (gavage studies). NTP-TR-343; NIH Publication No. 89-2599, Washington DC, US Government Printing Office.

National Toxicology Program (1997) Final report on subchronic toxicity studies of 2,4-decadienal administered by gavage to F344/N rats and B6C3F1 mice and cellular and genotoxic toxicology tables. Study Nos 93022.01-93022.02.

National Toxicology Program (2001a) Draft Report: Toxicology and carcinogenesis studies of 2,4-hexadienal in F344/N rats and B6C3F1 mice (gavage studies). NTP-TR-509.

National Toxicology Program (2001) Draft report: Toxicology and carcinogenesis studies of citral (microencapsulated) (CAS No. 5392-40-5) in F344/N rats and B6C3F1 mice (feed studies). Technical Report Series 505, NIH Publication No. 01-4439. United States Department of Health and Human Services, Public Health Service, National Institutes of Health.

Nelson DL and Cox MM (2000) Lehninger Principles of Biochemistry. Worth Publishers, Inc., New York.

Oser BL, Carson S, and Oser M (1965)Toxicological tests on flavouring matters. Food Chemistry and Toxicology. 3: 563-569.

Oser BL (1967) Unpublished report via FAO Nutrition Meeting Report Series No. 44a.

Pardoe SU (1952) Renal functioning in lead poisoning. British Journal of Pharmacology. 7: 349-357.

Pietruszko R, Crawford K, Lester D (1973) Comparison of substrate specificity of alcohol dehydrogenases from human liver, horse liver, and yeast towards saturated and 2-enoic alcohols and aldehydes. Archives of Biochemistry and Biophysics. 159, 50-60.

Posternak JM, Linder A, Vodoz CA (1969) Summaries of toxicological data. Toxicological tests on flavoring matters. Food and Cosmetics Toxicology. 7, 405-407.

Roe FJ, Palmer AK, Worden AN, Van Abbé NJ (1979) Safety evaluation of toothpaste containing chloroform. I. Long-term studies in mice. Journal of Environmental Pathology and Toxicology. 2(3), 799-819.

Tislow R, Margolin S, Foley EJ and Lee SW (1950) Toxicity of undercylenic acid. Journal of Pharmacology and Experimental Therapeutics. 98(1), 31-32.












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       Toxicological Abbreviations