INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION SAFETY EVALUATION OF CERTAIN FOOD ADDITIVES WHO FOOD ADDITIVES SERIES: 42 Prepared by the Fifty-first meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) World Health Organization, Geneva, 1999 IPCS - International Programme on Chemical Safety SAFETY EVALUATION OF ALIPHATIC ACYCLIC AND ALICYCLIC alpha-DIKETONES AND RELATED alpha-HYDROXYKETONES Mrs M.F.A. Wouters and Dr G.J.A. Speijers National Institute of Public Health and the Environment, Center of Substances and Risk Assessment, Bilthoven, The Netherlands Evaluation Introduction Estimated daily per capita intake Absorption, metabolism, and elimination Application of the Procedure for the Safety Evaluation of Flavouring Agents Consideration of combined intakes from use as flavouring agents Conclusions Relevant background information Explanation Intake Biological data Absorption and metabolism Toxicological studies Acute toxicity Short-term and long-term studies of toxicity and carcinogenicity Genotoxicity Other relevant studies References 1. EVALUATION 1.1 Introduction The Committee evaluated a group of 22 flavouring agents (Table 1) that includes aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones, using the Procedure for the Safety Evaluation of Flavouring Agents (Figure 1, p. 222, and Annex 1, reference 131). One member of the group, diacetyl, was evaluated at the eleventh meeting of the Committee; however, because of lack of data, no ADI was allocated (Annex 1, reference 14). 1.2 Estimated daily per capita intake In the United States, aliphatic acyclic and alicyclic alpha-diketones and alpha-hydroxyketones are generally used as flavouring agents up to average maximum levels of 200 ppm. The total annual volume of the 22 substances in this group is approximately 44 000 kg in Europe (International Organization of the Flavor Industry, 1995) and 56 000 kg in the United States (US National Academy of Sciences, 1970, 1982, 1987). In both Europe and the United States, more than 95% of the total annual volume is accounted for by three substances: acetoin (No. 405: 19 000 kg/year in Europe and 9200 kg/year in the United States), diacetyl (No. 408: 18 000 kg/year in Europe and 42 000 kg/year in the United States), and methylcyclopentenolone (No. 418: 4700 kg/year in Europe and 3700 kg/year in the United States). Two of these, acetoin and diacetyl, account for more than 90% of the total annual volume in the United States (US National Academy of Sciences, 1987). Nineteen of the 22 aliphatic acyclic and alicyclic alpha-diketones and alpha-hydroxyketones have been identified as natural components of a variety of foods, including fruits, vegetables, cocoa, and coffee (Maarse et al., 1994). Quantitative data have been reported for the natural occurrence of six of these substances, which indicate that the intake as natural components of food is greater than that from their use as flavouring agents (Stofberg & Kirschman, 1985; Stofberg & Grundschober, 1987), with one exception (diacetyl). 1.3 Absorption, metabolism, and elimination In rats and mice, orally administered aliphatic alpha-diketones are rapidly absorbed from the gastrointestinal tract (Gabriel et al., 1972). It is anticipated that at low levels of exposure, humans will metabolize aliphatic acyclic alpha-diketone principally by alpha-hydroxylation and subsequent oxidation of the terminal methyl group to yield the corresponding ketocarboxylic acid. The acid may undergo oxidative decarboxylation to yield carbon dioxide and a simple aliphatic carboxylic acid, which may be completely metabolized in the fatty acid pathway and citric acid cycle. At high concentrations, another detoxification pathway is used which involves reduction to the diol and subsequent conjugation with glucuronic acid (Westerfeld & Berg, 1943; Williams, 1959; Gabriel et al., 1972; Otsuka et al., 1996). Acyclic alpha-diketones and alpha-hydroxyketones without a terminal methyl group and alicyclic diketones and hydroxyketones are mainly metabolized by reduction to the corresponding diol, followed by glucuronic acid conjugation and excretion (Mills & Walker, 1990; Ong et al., 1991). 1.4 Application of the Procedure for the Safety Evaluation of Flavouring Agents Step 1. According to the decision-tree structural class classification (Cramer et al., 1978), all 22 alpha-diketones and related hydroxyketones are in class II. Step 2. Studies in vitro and in vivo have demonstrated two major routes of metabolism for diacetyl(2,3-butadione) and acetoin, involving complete oxidation to carbon dioxide and reduction to a diol, but data were not available on the higher linear homologues or cyclic analogues. Alcohol dehydrogenases, aldehyde reductase, and carbonyl reductase are widely distributed enzymes with broad substrate specificities; studies conducted in vitro show that 1,2-cyclo-hexandione is a better substrate than diacetyl for aldehyde reductase and carbonyl reductase. In consequence, it was consi-dered that the data on the extensive reduction of acetoin can be extrapolated to other members of the group. The alpha-diketone group is polar, and it would be expected that all members of the group evaluated would be eliminated by a combination of oxidation when the carbonyl group is adjacent to a methyl group, reduction of the carbonyl group, and excretion of the parent compound and metabo-lites in urine. The products of these metabolic pathways are not of concern. Step A3. The daily per capita intakes ('eaters only') of the substances in Europe and the United States are below the human intake threshold for class II (540 µg/person per day), indicating that they pose no safety concern when used at current levels of estimated intake as flavouring agents. The intakes of acetoin (2800 µg/person per day in Europe; 1800 µg/person per day in the United States), diacetyl (3300 µg/person per day in Europe; 8000 µg/person per day in the United States), and methylcyclopentenolone (890 µg/person per day in Europe; 710 µg/person per day in the United States) are, however, greater than 540 µg/person per day. Step A4. Acetoin and diacetyl occur endogenously in humans (Kawano, 1959; Zlatkis & Sivetz, 1960; Gabriel et al., 1972), but methyl-cyclopentenolone does not. Step A5. A NOEL of 500 mg/kg bw per day was reported for methylcyclo-pentenolone in a six-month study of toxicity in rats (Dow Chemical Co., 1953). A safety margin of > 10 000 exists between this NOEL and the estimated daily per capita intake ('eaters only') of 15 µg/kg bw. In addition, methylcyclo-pentenolone gave negative results in tests for genotoxicity (reverse mutation and unscheduled DNA synthesis; Heck et al., 1989). This information indicates that methylcyclo-pentenolone would not be expected to be of safety concern. The stepwise evaluations of the 22 aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones used as flavouring agents are summarized in Table 1. 1.5 Consideration of combined intakes from use as flavouring agents In the unlikely event that all 22 aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones were consumed simultaneously on a daily basis, the estimated combined intake would exceed the human intake threshold for class II; however, all 22 substances are expected to be efficiently metabolized and would not saturate the detoxification pathways. On the basis of the evaluation of the collective data, there is no safety concern about combined intake. Table 1. Summary of results of safety evaluations on aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones Step 1: All of the substances in the group are in structural class II. The human intake threshold for class II is 540 µg/day. Step 2: All of the substances in this group are metabolized to innocuous products. Substance No. CAS No. Estimated Step A3 Step A4 Step A5 Conclusion per capita Does intake Endogenous? Adequate based on intake exceed NOAEL for current Europe/USA intake substance intake (µg/day) threshold? or related substance? Acetoin 405 513-86-0 2800/1800 Yes Yes N/R1 No safety concern2-Acetoxy-3-butanone 406 4906-24-5 0.03/23 No N/R2 N/R2 No safety concern
Butan-3-one-2-yl-butanoate 407 84642-61-5 0.02/0.95 No N/R2 N/R2 No safety concern
Table 1. (continued) Substance No. CAS No. Estimated Step A3 Step A4 Step A5 Conclusion per capita Does intake Endogenous? Adequate based on intake exceed NOAEL for current Europe/USA intake substance intake (µg/day) threshold? or related substance? Diacetyl 408 431-03-8 3300/8000 Yes Yes N/R1 No safety concern
3-Hydroxy-2-pentanone 409 3142-66-3 ND/0.10 No N/R2 N/R2 No safety concern
2,3-Pentanedione 410 600-14-6 220/80 No N/R2 N/R2 No safety concern
4-Methyl-2,3-pentadione 411 7493-58-5 0.48/2 No N/R2 N/R2 No safety concern
Table 1. (continued) Substance No. CAS No. Estimated Step A3 Step A4 Step A5 Conclusion per capita Does intake Endogenous? Adequate based on intake exceed NOAEL for current Europe/USA intake substance intake (µg/day) threshold? or related substance? 2,3-Hexadione 412 3848-24-6 13/10 No N/R2 N/R2 No safety concern
3,4-Hexadione 413 4437-51-8 33/0.76 No N/R2 N/R2 No safety concern
5-Methyl-2,3-hexadione 414 13706-86-0 2/6 No N/R2 N/R2 No safety concern
2,3-Heptanedione 415 96-04-8 2/5 No N/R2 N/R2 No safety concern
Table 1. (continued) Substance No. CAS No. Estimated Step A3 Step A4 Step A5 Conclusion per capita Does intake Endogenous? Adequate based on intake exceed NOAEL for current Europe/USA intake substance intake (µg/day) threshold? or related substance? 5-Hydroxy-4-octanone 416 496-77-5 0.02/0.76 No N/R2 N/R2 No safety concern
2,3-Undecadione 417 7493-59-6 0.02/0.01 No N/R2 N/R2 No safety concern
Methylcyclopentenolone 418 80-71-7 890/710 Yes No Yes. The dose of No safety 500 mg/kg bw per concern day that had no adverse effects (Dow Chemical Co., 1953) is > 10 000 times the intake.
Table 1. (continued) Substance No. CAS No. Estimated Step A3 Step A4 Step A5 Conclusion per capita Does intake Endogenous? Adequate based on intake exceed NOAEL for current Europe/USA intake substance intake (µg/day) threshold? or related substance? Ethylcyclopentenolone 419 21835-01-8 50/23 No N/R2 N/R2 No safety concern
3,4-Dimethyl-1,2-cyclopentadione 420 13494-06-9 47/2 No N/R2 N/R2 No safety concern
3,5-Dimethyl-1,2-cyclopentadione 421 13494-07-0 55/29 No N/R2 N/R2 No safety concern
3-Ethyl-2-hydroxy-4-methylcyclopent-2-en-1-one 422 42348-12-9 ND/0.17 No N/R2 N/R2 No safety concern
Table 1. (continued) Substance No. CAS No. Estimated Step A3 Step A4 Step A5 Conclusion per capita Does intake Endogenous? Adequate based on intake exceed NOAEL for current Europe/USA intake substance intake (µg/day) threshold? or related substance? 5-Ethyl-2-hydroxy-3-methylcyclopent-2-en-1-one 423 53263-58-4 ND/0.38 No N/R2 N/R2 No safety concern
2-Hydroxy-2-cyclohexen-1-one 424 10316-66-2 0.08/0.76 No N/R2 N/R2 No safety concern
1-Methyl-2,3-cyclohexadione 425 3008-43-3 2/8 No N/R2 N/R2 No safety concern
2-Hydroxy-3,5,5-trimethyl-2-cyclohexen-1-one 426 4883-60-7 2/2 No N/R2 N/R2 No safety concern
N/R1, not required for evaluation because consumption of the substance was determined to be of no safety concern at Step A4 of the procedure. N/R2, not required for evaluation because consumption of the substance was determined to be of no safety concern at Step A3 of the procedure. N/D, no intake data reported. 1.6 Conclusions The 22 aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones evaluated do not pose a safety concern at current levels of intake as flavouring agents. No data on toxicity were required for application of the procedure to 21 of the aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones. The Committee noted that the available data on toxicity were consistent with the results of the safety evaluations using the procedure. 2. RELEVANT BACKGROUND INFORMATION 2.1 Explanation This group of substances was selected on the basis of the criteria that all members of the group are aliphatic and contain functional groups (alpha-diketones, alpha-hydroxyketones, and esters of a methylcyclopentenolone hydroxyketones) which have similar chemical reactivity and participate in common pathways of metabolic detoxification. Acyclic and alicyclic alpha-diketones exist to varying degrees in equilibrium with unsaturated alpha-hydroxyketones (i.e. the ketoenolic form). Therefore, 17 of the 22 substances in this group exist as mixtures of alpha-diketone and unsaturated alpha-hydroxyketone. Three (Nos 405, 409, and 416) other members of the group are aliphatic alpha-hydroxyketones which may be formed in vivo by simple reduction of one of the two ketone functions in the corresponding alpha-diketone. The two remaining members of the group (Nos 406 and 407) are aliphatic esters of the alpha-hydroxyketone acetoin (No. 405). Hydrolysis of these esters in vivo yields acetoin (No. 405) and simple aliphatic carboxylic acids. Consequently, it can be assumed that all of these flavouring agents either are, or can readily form, alpha-diketones or alpha-hydroxyketones (see Figure 1). In view of the close chemical and biochemical relationships between these substances and the consistent data on toxicity, they are evaluated here as a group of structurally related compounds. 2.2 Intake The total annual production volume of the 22 substances in this group is approximately 44 000 kg in Europe (International Organization of the Flavor Industry, 1995) and 56 000 kg in the United States (US National Academy of Sciences, 1970, 1982, 1987). Production volumes and intake values for each substance are reported in Table 2. On the basis of the annual volumes reported in Europe and the United States, the total estimated daily per capita intake ('eaters only') of the 22 flavouring agents in this group is 120 µg/kg bw per day in Europe and 180 µg/kg bw per day in the United States.
The vast majority of aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones have been reported to occur in traditional foods (Maarse et al., 1994). Quantitative data on the natural occurrence of these substances, with the exception of diacetyl, demonstrate that they are consumed predominantly from traditional foods (i.e. consumption ratio > 1) (Stofberg & Kirschman, 1985; Stofberg & Grundschober, 1987). 2.3 Biological data 2.3.1 Absorption and metabolism Aliphatic acyclic and alicyclic alpha-diketones participate in an enol-keto equilibrium with the corresponding ketoenol (see Figure 1). The enolic form predominates in alicyclic diketones, especially cyclopentyl alpha-diketones (Gordon & Ford, 1972), essentially 100% of which exist in the ketoenolic form; 50-80% of cyclohexyl alpha-diketones exist in the ketoenolic form at equilibrium. Increasing pH would be expected to shift the equilibrium in favour of the ketoenolic form (i.e. alpha-hydroxyketone). Therefore, at physiological pH, an increase in the alpha-hydroxyketone (i.e. enol) form is expected. In rats and mice, orally administered aliphatic alpha-diketones are rapidly absorbed from the gastrointestinal tract (Gabriel et al., 1972). Carbon dioxide produced primarily from methyl-substituted diketones (e.g. diacetyl) is eliminated in expired air. Thus, after injection of [2,3-14C]-acetoin to whole albino rats, 14C-carbon dioxide appeared in expired air, with an average 12-h production of 15% (Gabriel et al., 1972). In rats, 54-82% of orally administered radiolabelled 2,3-butanedione was excreted as carbon dioxide. At increasing doses, the percent of the dose excreted as carbon dioxide decreased, while urinary excretion of radiolabel increased, suggesting that saturation occurs at higher doses. Chromatographic analysis of urine samples yielded three major 14C-labelled components, one of which co-eluted with uric acid. Analysis with ß-glucuronidase or sulfatase decreased the amount of radiolabel present in one peak, with a corresponding increase in the amount present in another major component. Identification of urinary metabolites was not pursued owing to the extensive excretion of 14C as carbon dioxide (Dix & Jeffcoat, 1997). In general, esters are hydrolysed to their corresponding alcohols and carboxylic acids. Hydrolysis is catalysed by classes of enzymes recognized as carboxylesterases or esterases, the most important of which are the B-esterases. Acetyl esters are the preferred substrates of C-esterases (Heymann, 1980). These enzymes occur in most mammalian tissues (Heymann, 1980; Anders, 1989) but predominate in hepatocytes (Heymann, 1980). 2-Acetoxy-3-butanone (No. 406) and butanon-3-one-2-yl butanoate (No. 407) are expected to be metabolized in humans to acetic acid and butanoic acid, respectively, and to acetoin. Aliphatic acyclic diketones The metabolic fate of acyclic aliphatic diketones depends primarily on the position of the carbonyl function and on chain length. Aliphatic acyclic diketones and alpha-hydroxyketones that contain a carbonyl function at the 2 position (i.e. methyl ketones) may undergo alpha-hydroxylation and subsequent oxidation of the terminal methyl group to yield corresponding ketocarboxylic acids. The ketoacids are intermediary metabolites (e.g. alpha-ketoacids), which may undergo oxidative decarboxylation to yield carbon dioxide and a simple aliphatic carboxylic acid. The acid may be completely metabolized in the fatty acid pathway and citric acid cycle. Alternatively, the methyl-substituted diketones may be successively reduced to the corresponding hydroxyketones and diols, which are excreted in the urine as glucuronic acid conjugates. This pathway is favoured at high concentrations in vivo, especially for longer-chain ketones. If the carbonyl function is located elsewhere on the chain, reduction is the predominant detoxification pathway. alpha-Hydroxyketones and their diol metabolites may be excreted as glucuronic acid conjugates. For example, the glucuronic acid conjugate of cyclohexanol was detected in the urine of humans exposed to cyclohexanone (Ong et al., 1991), indicating that alpha-hydroxylation followed by reduction of the ketone function occurs in humans. Acetoin is metabolized primarily via oxidation at low concentrations in vivo and by reduction to 2,3-butanediol at high concentrations. It has been estimated that 1 g of rat liver can oxidize 86 µg (1 µmol) acetoin per day (Gabriel et al., 1972). Production of carbon dioxide at low levels and of 2,3-butanediol at high levels is associated with the slower rate of ketone reduction (Williams, 1959). Oxidation of the terminal methyl group may result in formation of an alpha-ketoacid, which undergoes cleavage to yield carbon dioxide and a carboxylic acid fragment. Alternatively, methyl group oxidation may yield a ß-ketoacid which undergoes ß-cleavage to yield two-carbon fragments. To a minor extent, these two-carbon fragments can act as acetyl donors for acetylation of para-aminobenzoic acid (Westerfeld & Berg, 1943). A total dose of 78 g of acetoin was administered to a dog over two months, both orally in a 3-4% solution and subcutaneously in a 20% solution. Urine was collected under toluene from the beginning of treatment up to 40 h after the last dose. 2,3-Butanediol was the major urinary excretion product, representing 5-25% of the dose. The remainder of the dose was completely metabolized (Westerfeld & Berg, 1943). In liver preparations obtained from rats and rabbits, more than 95% of the radiolabel of [2,3-14C]-acetoin was detected as a mixture of stereoisomers of 2,3-butanediol (Gabriel et al., 1971). Although reduction of diacetyl and acetoin has been observed in animals in vivo and in animal tissue preparations in vitro at high concentrations, it appears that oxidation of diacetyl is a major endogenous metabolic pathway. Reduction of ketones is mediated by alcohol dehydrogenase and NADPH-dependent cytosolic carbonyl reductases (Bosron & Li, 1980). Reduction of the endogenous substances acetoin (No. 405) and diacetyl (No. 408) is catalysed by the substrate-specific enzymes diacetyl reductase and acetoin reductase, respectively. In rat liver slices, diacetyl, acetoin, and 2,3-butanediol are interconvertible (Gabriel et al., 1972). In male Wistar albino rats, a single oral dose of diacetyl at 5 mmol/kg bw (430 mg/kg bw) was metabolized by reduction to acetoin, which was present at high concentrations in major organs 1 h after dosing. The subsequent reduction product, 2,3-butanediol, was detected in liver, kidney, and brain (Otsuka et al., 1996). Only a 10-min incubation is required to convert 10 nmol (9 × 10-4 mg) diacetyl to 3.7 nmol (3 × 10-4 mg) acetoin and 6.3 nmol (6 × 10-4 mg) 2,3-butanediol in rat liver homogenate. Diacetyl was reduced by NADH- and NADPH-dependent diacetyl reductase isolated from a homogenate of male Wistar rat liver. The organ-specific reductase activity was greatest in the liver and least in the brain (Otsuka et al., 1996). Acetoin was given either orally in a 3-4% solution or subcutaneously in a 20% solution to rats, multiple doses being spaced equally throughout each day. The acetoin used was obtained as the polymer (H, 15), which apparently reverts to an optically inactive monomer in aqueous solution. No diacetyl was detected in the urine of the rats; acetoin was not excreted to any appreciable extent in the urine, and the major excretion product was 2,3-butanediol (Westerfeld & Berg, 1943). In dogs, acetoin is excreted in the urine, in part as 2,3-butanediol. Most of an oral dose disappeared and was presumed to be completely metabolized (Westerfeld & Berg, 1943). In another study, 2,3-butanediol readily conjugated glucuronic acid (Neuberg & Gottshchalk, 1971). Diacetyl and acetoin are endogenous in humans (Kawano, 1959; Zlatkis & Sivetz, 1960). They are formed when pyruvate is converted to acetoin and diacetyl by pyruvate decarboxylase (Gabriel et al., 1972). Mean fasting blood concentrations of approximately 100 µg acetoin per 100 ml blood have been reported (Dawson & Hullin, 1954). Pyruvate also forms diacetyl in vitro in rat liver preparations (Järnefelt, 1955) and in microorganisms (Juni & Heym, 1956). Alicyclic alpha-diketones and alicyclic dicarbonyls In general, alicyclic alpha-diketones are metabolized via a reduction pathway (Williams, 1959). In humans, structurally related alicyclic monoketones are reduced to the corresponding alcohols or undergo alpha-hydroxylation and reduction to yield diols, which are excreted as the glucuronic acid conjugates. For example, the glucuronic acid conjugate of cyclohexanol is present in the urine of humans exposed to atmospheres containing cyclohexanone at concentrations of 2-30 ppm (Ong et al., 1991). A mixture of diols, including cis- and trans-1,2-cyclohexanediol,1,3-cyclohexanediol, and 1,4-cyclohexanediol, was detected in the urine of infants exposed to cyclohexanone present in dextrose solutions used for intravenous feeding. Presumably, the cyclohexanone undergoes alpha-hydroxylation and then reduction of the ketone function to yield the corresponding alpha-cyclohexanediols (Mills & Walker, 1990). On the basis of the studies described above, it is anticipated that humans will metabolize low concentrations of aliphatic acyclic methyl ketones principally by oxidation of the terminal methyl group. At higher concentrations, reduction to the diol and subsequent conjugation with glucuronic acid form a competing detoxification pathway. Other aliphatic acyclic alpha-diketones and alpha-hydroxyketones and alicyclic diketones and hydroxyketones are reduced, conjugated with glucuronic acid, and excreted. 2.3.2 Toxicological studies 2.3.2.1 Acute toxicity The results of studies of the acute toxicity of seven of the 22 diketones, hydroxyketones, and alicyclic dicarbonyls have been reported and are summarized in Table 3. The low acute toxicity of the group is demonstrated by oral LD50 values in the range 990 to > 8000 mg/kg bw. Table 2. Most recent annual usage of aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones as flavouring substances in Europe and the United States Substance (No.) Most recent Per capita intakea annual volume (kg) µg/day µg/kg bw per day Acetoin (405) Europe 19 000 2800 46 United States 9 000 1800 29 2-Acetoxy-3-butanone (406) Europe 0.2 0.03 0.0005 United States 120 23 0.4 Butan-3-one-2-yl-butanoate (407) Europe 0.1 0.02 0.0003 United States 5 0.95 0.02 Diacetyl (408) Europe 18 000 3300 56 United States 42 000 8000 133 3-Hydroxy-2-pentanone (409) Europe NR ND 0 United States 0.5 0.10 0.002 2,3-Pentanedione (410) Europe 1 100 220 4 United States 420 80 1 4-Methyl-2,3-pentadione (411) Europe 2.5 0.48 0.01 United States 12 2 0.04 2,3-Hexanedione (412) Europe 70 13 0.22 United States 50 10 0.2 3,4-Hexanedione (413) Europe 170 33 0.5 United States 4 0.76 0.01 5-Methyl-2,3-hexanedione (414) Europe 9 2 0.03 United States 30 6 0.10 Table 2. (continued) Substance (No.) Most recent Per capita intakea annual volume (kg) µg/day µg/kg bw per day 2,3-Heptanedione (415) Europe 8 2 0.03 United States 26 5 0.08 5-Hydroxy-4-octanone (416) Europe 0.1 0.02 0.0003 United States 4 0.76 0.01 2,3-Undecadione (417) Europe 0.03 0.01 0.0001 United States 0.1 0.02 0.0003 Methylcyclopentenolone (418) Europe 4 700 890 15 United States 3 700 710 12 3-Ethylcyclopentenolone (419) Europe 260 50 0.8 United States 120 23 0.4 3,4-Dimethyl-1,2-cyclopentanedione (420) Europe 250 47 0.8 United States 13 2 0.04 3,5-Dimethyl-1,2-cyclopentanedione (421) Europe 290 55 0.9 United States 150 29 0.5 3-Ethyl-2-hydroxy-4-methylcyclopent-2-en-1-one (422) Europe NR ND ND United States 0.9 0.17 0.003 5-Ethyl-2-hydroxy-3-methylcyclopent-2-en-1-one (423) Europe NR ND ND United States 2 0.38 0.01 2-Hydroxy-2-cyclohexen-1-one (424) Europe 0.4 0.08 0.001 United States 4 0.76 0.01 1-Methyl-2,3-cyclohexadione (425) Europe 11 2 0.03 United States 44 8 0.1 Table 2. (continued) Substance (No.) Most recent Per capita intakea annual volume (kg) µg/day µg/kg bw per day 2-Hydroxy-3,5,5-trimethyl-2-cyclohexenone (426) Europe 10 2 0.03 United States 8 2 0.03 Total Europe 44 000 United States 56 000 NR, not reported; ND, not determined a Intake (µg/day) calculated as follows: [(annual volume, kg) × (1 × 109 µg/kg)]/[population × 0.6 × 365 days], where population (10%, 'eaters only') = 32 × 106 for Europe and 32 × 106 for the United States; 0.6 represents the assumption that only 60% of the flavour volume was reported in the surveys (US National Academy of Sciences, 1970, 1982, 1987; International Organization of the Flavor Industry, 1995). Intake (µg/kg bw per day) calculated as follows: [(µg/day)/body weight], where body weight = 60 kg. Slight variation may occur from rounding off. Table 3. Studies of acute toxicity with aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones used as flavouring substances Substance No. Species Sex Route LD50 Reference (mg/kg bw) Acetoin 405 Rat NR Oral > 5000 Moreno (1977) Butan-3-one-2-yl 407 Mouse, rat NR Oral > 8000 Pellmont (1969) butanoate Diacetyl 408 Rat M Gavage 3400 Colley et al.(1969) F 3000 Rat NR Gavage 1580 Jenner et al.(1964) Guinea-pig NR Gavage 990 Jenner et al.(1964) 2,3-Pentanedione 410 Rat NR Oral 3000 Moreno (1977) 2,3-Hexanedione 412 Rat NR Oral > 5000 Moreno (1977) 5-Methyl-2,3-hexanedione 414 Rat NR Oral > 5000 Moreno (1979) Methylcyclopentenolone 418 Mouse NR Gavage 1350 Givadaun Corp.(1952) Rat NR Oral 1850 Moreno (1976) Guinea-pig NR Gavage 1400 Dow Chemical Co. (1953) Mouse NR Oral 1350 Leberco Labs (1952) NR, not reported; M, male; F, female 2.3.2.2 Short-term and long-term studies of toxicity and carcinogenicity The toxicity of nine of the 22 substances has been studied. The results are summarized in Table 4 and described below. Acetoin (No. 405) Groups of 15 male and 15 female CFE rats were given acetoin in their drinking-water at concentrations of 0 (control), 750, 3000, or 12 000 mg/kg (equivalent to 0, 85, 330, or 1300 mg/kg bw per day (US Food & Drug Administration, 1993)). No animals died during the study, and their condition and appearance were normal. The body weights of males at 12 000 mg/kg in drinking-water decreased significantly from week 5, and at weeks 2, 6, and 13 the relative weight of the liver was statistically significantly greater in these animals than in controls. A similar effect was seen in female rats, but only after 13 weeks. Haematological examination conducted at 13 weeks showed a small (4-8%) but statistically significant (p < 0.05) decrease in haemoglobin concentration and erythrocyte counts in animals of each sex at the high dose, but these changes were not accompanied by a decrease in haematocrit. Urinalysis and blood chemical determinations performed at the end of the study on all animals revealed no statistically significant differences between treated and control groups. Histopathological examination also revealed no adverse effects. The authors suggested that the increased relative liver weights were a reaction of the liver to an increased metabolic load resulting from the high intake of acetoin. The NOEL was 3000 ppm, equivalent to 330 mg/kg bw per day (Gaunt et al., 1972), which is greater than 10 000 the daily per capita intake ('eaters only') of 46 and 29 µg/kg bw from its use as a flavouring agent in Europe and the United States, respectively (see Table 2). Diacetyl (No. 408) Groups of 15 male and 15 female weanling specific pathogen-free-derived CFE rats were given 5 ml/kg bw of an aqueous solution containing 0, 0.2, 0.6, 1.8, or 11% diacetyl by gavage for 90 days, providing daily doses of diacetyl calculated to be 0, 10, 30, 90, and 540 mg/kg bw. Body weight, food intake, and water consumption were recorded weekly. Growth retardation and increased water consumption were observed in animals at the high dose, the effects being more pronounced in males. Haematological and urinary parameters and enzyme activities were measured. Anaemia and increased polymorphonuclear leukocytosis were observed in rats at the high dose but were attributed to haemorrhage, infections, and ulcers of the stomach. The relative weights of the liver, kidney, and pituitary and adrenal glands were increased in these animals, and the increases were greater than could be accounted for by the reduction in body weight. Macroscopic and microscopic examination of all major organs revealed ulcers in the squamous and glandular regions of the gastric mucosa. The lesions observed in the squamous part of the stomach were associated with hypertrophy or intercellular oedema. No histological changes were seen in animals treated at lower doses. No other significant differences were observed between treated and control animals. The NOEL was 90 mg/kg bw per day (Colley et al., 1969), which is 1500 times the daily per capita intake ('eaters only') of 56 µg/kg bw from its use as a flavouring agent in Europe and 500 times the intake of 130 µg/kg bw from its use as a flavouring agent in the United States (see Table 2). 3,4-Hexanedione (No. 413) Groups of 10-16 male and female Charles River CD rats were housed in pairs of the same sex and fed for 90 days on a basal diet alone or supplemented with 3,4-hexanedione for an average daily intake of 17 mg/kg bw. Body weight and food consumption were measured daily and were considered normal. Haematological examinations and blood urea determinations were performed on 50% of the animals at week 7 and at the end of treatment: no significant differences were seen between treated and control animals. At necropsy, the liver and kidney weights were normal, and gross and histological examination performed on a wide range of organs revealed no dose-related lesions. The NOEL was 17 mg/kg bw per day (Posternak et al., 1969), which is more than 30 000 the daily per capita intake ('eaters only') of 0.5 and 0.01 µg/kg bw from its use as a flavouring agent in Europe and the United States, respectively (see Table 2). Methylcyclopentenolone (No. 418) Groups of 15 male and 15 female rats aged four to five weeks (strain not specified) were placed on a diet containing 0 or 1% methylcyclo-pentenolone, equivalent to a daily intake of 0 or 500 mg/kg bw, for six months. Each animal was weighed twice weekly and observed frequently for gross appearance and behaviour. The tissues of most animals that died or were killed during the course of the study and of all those kiled at the end of the study were examined grossly. Tissues of representative animals from the control and treated groups (numbers unspecified) were examined microscopically, and the weights of the lungs, heart, liver, kidneys, spleen, and testes were recorded. Haematological parameters were measured in representative animals from the control and treated groups (numbers not specified) at the end of the experiment. There were no statistically significant differences between treated and control animals in any of the parameters measured. The NOEL was 500 mg/kg bw per day (Dow Chemical Co., 1953), which is more than 30 000 the daily per capita intake ('eaters only') of 15 and 12 µg/kg bw from its use as a flavouring agent in Europe and the United States, respectively (see Table 2). 3-Ethyl-2-hydroxy-2-cyclopenten-1-one (No. 419) Groups of 10 male and 10 female Charles River CD rats were fed diets providing 3-ethyl-2-hydroxy-2-cyclopenten-1-one at doses of 0, 100, 200, or 400 mg/kg bw per day for 91 days. No adverse effects were seen on behaviour, body weight, food consumption, haematological, urinary, or ophthalmological parameters, or gross or histopathological appearance. The NOEL was 400 mg/kg bw per day (King et al., 1979), which is more than 50 000 the daily per capita intake ('eaters only') of 0.8 and 0.4 µg/kg bw from its use as a flavouring agent in Europe and the United States, respectively (see Table 2). In a combined study of developmental toxicity and carcinogenicity, three successive generations of male and female Charles River CD-COBS rats received 3-ethyl-2-hydroxy-2-cyclopenten-1-one in the basal diet at doses of 0 (untreated control), 0 (propylene glycol control), 30, 80, or 200 mg/kg bw per day. The F0 generation was entered into the study after weaning and was mated 64 days later. These animals were treated for 12 months, during which time the females were given a supplemental diet throughout gestation and lactation. The F1 generation was initially exposed in utero, subsequently via the dams' milk until weaning, and then treated for two years and bred twice (at days 99 and 155) to produce F2a and F3a generations. The F0 and F2 generations consisted of 40 animals of each sex in the untreated control group and 20 of each sex in the propylene glycol control and 3-ethyl-2-hydroxy-2-cyclopenten-1-one-treated groups. In the F1 generation, there were 100 animals of each sex in the untreated control group and 50 of each sex in the propylene glycol control and 3-ethyl-2-hydroxy-2-cyclopenten-1-one-treated groups. Survival, clinical symptoms, food consumption, reproductive perfor-mance, and haematological and clinical chemical parameters were not adversely affected in the F0 or F1 generations. Slightly depressed growth was reported in F1 females receiving the highest dose, but the effect was not significant when compared with the growth of controls receiving propylene glycol. Gross pathological and histopathological examination of animals of these generations revealed no significant treatment-related effects. An increased incidence of bile-duct hyperplasia observed in these groups was not statistically significant when compared with the incidence of in controls. The incidence of benign or malignant tumours in treated animals was similar to that in controls. The NOEL was 200 mg/kg bw per day (King et al., 1979), which is more than 200 000 the daily per capita intake ('eaters only') of 0.8 and 0.4 µg/kg bw from its use as a flavouring agent in Europe and the United States, respectively (see Table 2). In a three-generation study of toxicity in Charles River CD-COBS rats with 3-ethyl-2-hydroxy-2-cyclopenten-1-one, 120 F0 animals were entered into the study just after weaning and were mated after 64 days, providing the F1 generation. Animals of the F0 generation were selected at random to be maintained as controls or were given 3-ethyl-2-hydroxy-2-cyclopenten-1-one in the diet. The F1 generation was maintained on the diet for 755 days, during which time they were mated twice (at days 99 and 155). The first mating provided the rats for the F2 generation, and the second mating provided rats which were sacrificed after weaning. The F2 generation was treated for 84 days and then mated to produce the F3 generation. Rats of all generations were maintained on a basal diet with 3-ethyl-2-hydroxy-2-cyclopenten-1-one at doses equal to daily intakes of 0, 30, 80, or 200 mg/kg bw. Interim clinical chemical analyses were performed on the F0 generation at months 4 and 6. Ophthalmological, clinical chemical, and haematological examinations were performed on the F1 generation at months 6, 12, 18, and 24. At termination, both the F0 and the F1 generations were submitted to clinical chemical, haematological, and histopathological examinations. F2 adult animals were killed without necropsy at the time of weaning of their offspring. No evidence of treatment-related effects was found, and the general health of the rats was unaffected throughout treatment. Mammary swellings in females and other subcutaneous nodules were considered not to be related to treatment because they were equally distributed among control and treated groups. No effects were seen on food consumption, weight gain, or clinical chemical or haematological parameters. In the F0 generation, there were no pathological findings at the 12-month necropsy or at histopathological examination. In the F1 generation, there were no macroscopic observations that could be related to the treatment. Histologically, bile-duct hyperplasia was seen in all groups, including controls; however, there was no statistically significant difference between treated and control groups and the findings were considered not to be related to treatment. Only tumours usually found in rats of this strain and age were observed, indicating the absence of treatment-related effects. The NOEL in this study was 200 mg/kg bw per day (King et al., 1979), which is more than 100 000 the daily per capita intake ('eaters only') of 0.4 µg/kg bw from its use as a flavouring agent in the United States (see Table 2). 3,4-Dimethylcyclopentane-1,2-dione (No. 420) Groups of 10 male Charles River CD rats were fed diets containing 3,4-dimethyl-1,2-cyclopentadione at concentrations of 0, 400, 4000, or 13 000 mg/kg (equivalent to 0, 20, 200, and 640 mg/kg bw per day) for 90 days. Food consumption, body-weight gain, haematological parameters, organ weights, and gross and microscopic appearance were observed throughout the study. Animals at the intermediate and high doses showed a moderate but consistent depression in food intake throughout the experiment, and their final mean body weights were about 10% less than those of controls. There were no consistent indications of a profound disturbance of food conversion efficiency, although minor increases in the food conversion ratio were seen in some animals at the high dose. Haematological parameters, organ weights, and gross and microscopic appearance were similar in treated and control groups. The authors attributed the decreased body weight observed at higher doses to the unpalatability of the test substance. The NOEL was 200 mg/kg bw per day (Wheldon & Krajkeman, 1967), which is more than 200 000 the daily per capita intake ('eaters only') of 0.8 and 0.04 µg/kg bw from its use as a flavouring agent in Europe and the United States, respectively (see Table 2). 3,5-Dimethylcyclopentane-1,2-dione (No. 421) Groups of 10 male Charles River CD rats were fed 3,5-dimethyl-1,2-cyclopenthexadione in the diet for 13 weeks at concentrations of 0, 1000, or 10 000 mg/kg diet (equivalent to 50 and 500 mg/kg bw per day). A fourth group was fed a diet initially containing 12 000 ppm (equivalent to 610 mg/kg bw per day), but the level was increased to 24 000 mg/kg diet (equivalent to 1200 mg/kg bw per day) during week 6 through to the end of experiment. Haematological examinations were performed on five animals from each group just before the end of the 13-week period; organ weight analysis and gross and histopathological examinations at the end of the study revealed no significant differences between control and treated animals. The food consumption of animals at the two highest doses was reduced throughout the experiment, accompanied by decreased weight gain, which resulted in mean body weights that were 10-17% lower than those of controls at the end of the study. The authors attributed the decrease to the unpalatability of the test substance. The NOEL was 50 mg/kg bw per day (Wheldon & Krajkeman, 1967), which is more than 50 000 the daily per capita intake ('eaters only') of 0.9 and 0.5 µg/kg bw from its use as a flavouring agent in Europe and the United States, respectively (see Table 2). 2-Hydroxy-2-cyclohexen-1-one (No. 424) 2-Hydroxy-2-cyclohexen-1-one was added to the diet of 15 male and 15 female albino Wistar rats for 90 days at a concentration calculated to result in an average daily intake of 5 mg/kg bw. Detailed measurements of haematological parameters, blood chemistry, urine, gross and histopathological appearance, organ weights, body weight, and food consumption revealed no statistically significant difference between treated and control groups (Morgareidge et al., 1974). The NOEL was 5 mg/kg bw per day (Cox, 1974), which is more than 500 000 the daily per capita intake ('eaters only') of 0.001 and 0.01 µg/kg bw from its use as a flavouring agent in Europe and the United States, respectively (see Table 2). 1-Methyl-2,3-cyclohexadione (No. 425) Groups of 10 male Charles River CD rats were fed 1-methyl-2,3-cyclohexadione in the diet at concentrations of 0, 100, or 1000 mg/kg (equivalent to 0, 5, and 50 mg/kg bw per day) for 13 weeks. A fourth group was fed a diet initially containing 13 500 mg/kg diet (equivalent to 675 mg/kg bw per day), which was raised to 27 000 mg/kg bw (equivalent to 1350 mg/kg bw per day) during week 6. Observation of haematological parameters, organ weights, and gross and histopathological appearance revealed no difference between treated and control groups. The food consumption of rats at the high dose was reduced throughout the experiment, accompanied by decreased body-weight gain, and the effect was more marked when the dietary concentration was raised. The NOEL was 50 mg/kg bw per day (Wheldon & Krajkeman, 1967), which is more than 500 000 the daily per capita intake ('eaters only') of 0.03 and 0.1 µg/kg bw from its use as a flavouring agent in Europe and the United States, respectively (see Table 2). 2.3.2.3 Genotoxicity The available studies of genotoxicity are summarized in Table 5. Most of the studies were carried out with Salmonella typhimurium in the Ames test. Acetoin, diacetyl, and 1,2-cyclohexanedione showed some mtagenicity in strains TA100 and TA104. As the mutation frequencies were low and the positive results were always accompanied by negative results, the overall conclusion was that this group of substances does not induce gene mutation in bacteria in vitro. Diacetyl did not induce mutation in Saccharomyces cerevisiae (US Food & Drug Administration, 1974). Methylcyclopentenolone did not induce unscheduled DNA synthesis in rat hepatocytes (Heck et al., 1989). 2.3.2.4 Other relevant studies 3-Ethyl-2-hydroxy-2-cyclopenten-1-one (No. 419) In the three-generation, 23-month study of toxicity described above, treatment had no effect on clinical signs, weight gain, food consumption, copulation rates, or mating behaviour of males or females, fertility index, length of gestation, parturition, litter size, or incidence of stillbirth. The growth and survival rates of pups during lactation were normal. Gross examination of all offspring from birth to weaning revealed no significant treatment-related malformations or lesions (King et al., 1979). Diacetyl (No. 408) Groups of 25-27 Syrian golden hamsters, 21-24 CD-1 mice, and 21-23 albino Wistar rats were given a solution containing 90% diacetyl by gavage on days 6-10 of gestation for hamsters and days 6-15 of gestation for mice and rats. The doses for all species were 16, 74, 345, or 1600 mg/kg bw per day. No effects were seen on maternal survival, weight, or reproductive parameters or on fetal survival or microscopic appearance of external, skeletal, or soft tissues (US Food & Drug Administration, 1973). 4. REFERENCES Aeschbacher, H.U., Wolleb, U., Loliger, J., Spadone, J.C. & Liardon, R. (1989) Contribution of coffee aroma constituents to the mutagenicity of coffee. Food Chem. Toxicol., 27, 227-232. Anders, M. W. (1989) Biotransformation and bioactivation of xenobiotics by the kidney. In: Hutson, D.H., Caldwell, J. & Paulson, G.D., eds, Intermediary Xenobiotic Metabolism in Animals, New York, Taylor & Francis, pp. 81-97. Table 4. Short-term and long-term studies of toxicity in rats with aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones used as flavouring agents Substance No. Sex No. groups/ Route Time NOEL Reference no. per group (days) (mg/kg bw per day) Acetoin 405 M/F 3/30 Drinking-water 90 330 Gaunt et al. (1972) Diacetyl 408 M/F 4/30 Gavage 90 90 Colley et al. (1969) 3,4-Hexanedione 413 M/F 2/10-16 Diet 90 17a Posternak et al. (1969) Methylcyclopentenolone 418 M/F 1/30 Diet 185 500a Dow Chemical Co. (1953) 3-Ethyl-2-hydroxy-2-cyclopenten-1-one 419 M/F 3/20 Diet 91 400a King et al. (1979) M/F 3/100 Diet 730 200 King et al. (1979) 3,4-Dimethyl-1,2-cyclopentadione 420 M 3/10 Diet 90 645 Wheldon & Krajkeman (1967) 3,5-Dimethyl-1,2-cyclopentadione 421 M 3/10 Diet 90 610a Wheldon & Krajkeman (1967) 2-Hydroxy-2-cyclohexen-1-one 424 M/F 1/30 Diet 90 5a Morgareidge et al. (1974) 1-Methyl-2,3-cyclohexadione 425 M 3/10 Diet 90 675-1350a Wheldon & Krajkeman (1967) M, male; F, female a The study was performed at a single or multiple doses that had no adverse effects; therefore, no NOEL was determined. The NOEL is probably higher than the dose reported to have no adverse effects. Table 5. Results of assays for the genotoxicity of aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones used as flavouring agents Substance No. End-point Test object Dose Results Reference Acetoin 405 Reverse mutation S. typhimurium TA100 < 4500 mg/plate Negativea Garst et al. (1983) Positiveb Reverse mutation S. typhimurium TA100 420 mg/plate Negativeb Kim et al. (1987) Diacetyl 408 Reverse mutation S. typhimurium TA100, Not reported Positivea,c Kato et al. (1989) (modified test) TA104; E. coli Reverse mutation S. typhimurium TA104 530 µg/plate Positived Marnett et al. (1985) (modified test) Reverse mutation S. typhimurium TA100 90 µg/plate Negativee Kim et al. (1987) Reverse mutation S. typhimurium TA104 5-500 µg/platef Positivea,c Shane et al. (1988) Negativeb Reverse mutation S. typhimurium TA102, 5-500 µg/platef Negativee Shane et al. (1988) TA100 Reverse mutation S. typhimurium TA100 152-950 µg/plate Negativeb Dorado et al. (1992) Reverse mutation S. typhimurium TA100 10, 100, 1000, Positivee Bjeldanes & Chew (1979) 10 000 µg/plate Reverse mutation S. typhimurium TA98 10, 100, 1000, Negativee Bjeldanes & Chew (1979) 10 000 µg/plate Reverse mutation S. typhimurium TA1535, 1% Negativee US Food & Drug (suspension test) TA1537, TA1538 Administration (1974) Reverse mutation S. typhimurium TA102 0.17-17 200 µg/plate Negativee Aeschbacher et al. (1989) Reverse mutation S. typhimurium TA98, 0.17-17 200 µg/plate Negativee Aeschbacher et al. (1989) TA100 Reverse mutation S. typhimurium TA100 2 or 4 mmol/plate Positiveg Suwa et al. (1982) Mutation S. cerevisiae Not reported Negativee US Food & Drug Administration (1974) 2,3-Pentanedione 410 Reverse mutation S. typhimurium TA100 105 µg/plate Negativeb Kim et al. (1987) Reverse mutation S. typhimurium TA100, 0.9-90 000 µg/plate Negativee Aeschbacher et al. (1989) 3,4-Hexanedione 413 Reverse mutation S. typhimurium TA100 228-4900 µg/plate Negativeb Dorado et al. (1992) Table 5. (continued) Substance No. End-point Test object Dose Results Reference Methylcyclopentenolone 418 Reverse mutation S. typhimurium TA1535, 10 000 µg/plate Negativee Heck et al. (1989) TA1537, TA1538, TA98, TA100 Unscheduled DNA Rat hepatocytes 500 µg/plate Negativea Heck et al. (1989) synthesis 2-Hydroxy-2-cyclohexen-1-one 424 Reverse mutation S. typhimurium TA100 10, 100, 1000, Positivee Bjeldanes & Chew (1979) 10 000 µg/plate Reverse mutation S. typhimurium TA98 10, 100, 1000, Negativee Bjeldanes & Chew (1979) 10 000 µg/plate Reverse mutation S. typhimurium TA100 112-1000 µg/plate Negativeb Dorado et al. (1992) a With metabolic activation b Without metabolic activation c Mutation frequency, 2-3 d Result based on authors' criterion for significant mutagenic effect, i.e. mutation frequency > 1.5 e With and without metabolic activation f Estimated from graph g Mutation frequency, < 2.5 Bjeldanes, L.F. & Chew, H. (1979) Mutagenicity of 1,2-dicarbonyl compounds: Maltol, kojic acid, diacetyl and related substances. Mutat. Res., 67, 367-371. Bosron, W.F. & Li, T.-K. (1980) Alcohol dehydrogenase. In: Jacoby, W.B., ed., Enzymatic Basis of Detoxication, New York, Academic Press, Vol. I, pp. 231-248. 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See Also: Toxicological Abbreviations