First draft prepared by

Professor I.G. Sipes
Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA

and Professor A.G. Renwick
Clinical Pharmacology Group, University of Southampton, Southampton, England

The Committee evaluated a group of 39 flavouring agents that includes aliphatic acyclic secondary alcohols and ketones and esters derived from aliphatic secondary alcohols (see Table 1), using the Procedure for the Safety Evaluation of Flavouring Agents (see Figure 1). The Committee had not previously evaluated any of these agents.

Table 1. Summary of results of the safety evaluations of aliphatic secondary alcohols, ketones and related esters used as flavouring agents^a

Twenty-six of the 39 substances in this group of flavouring agents (Nos 1118, 1120, 1122–1132, 1134–1136, 1139, 1142, 1144, 1147, 1148, 1150–1152, 1154 and 1156) have been reported to occur naturally in foods. They have been found in fruits, juices, spices, vegetables, cocoa, coffee and tea.

The total annual volume of production of the 39 aliphatic secondary alcohols and ketones in this group is approximately 3900 kg in Europe (International Organization of the Flavor Industry, 1995) and 1100 kg in the USA (Lucas et al., 1999). Approximately 73% and 47% of the total annual production volume in Europe and the USA, respectively, is accounted for by 6-methyl-5-hepten-2-one (No. 1120) and 1-octen-3-ol (No. 1152). The daily per capita intake of 6-methyl-5-hepten-2-one (No. 1120) and 1-octen-3-ol (No. 1152) was 120 µg and 290 µg in Europe and 44 µg and 23 µg, respectively, in the USA, respectively.

In general, the aliphatic esters in this group hydrolyse to the corresponding secondary alcohols. The secondary alcohols and their corresponding ketones are interconvertible under physiological conditions. In the principal excretion pathway, the ketones are reduced to the corresponding secondary alcohols, which are subsequently conjugated with glucuronic acid and excreted.

When the ketone carbonyl function is located at the 2-position (i.e. a methyl ketone), the methyl group may undergo alpha,beta-hydroxylation and subsequent oxidation, to yield a corresponding ketocarboxylic acid. The ketoacids are intermediary metabolites (e.g. alpha,beta-ketoacids), which can undergo oxidative decarboxylation to yield carbon dioxide and simple aliphatic carboxylic acids. The acid may be completely metabolized in the fatty acid pathway and citric acid cycle. When the substance is an alpha,beta-unsaturated ketone (Nos 1124–1139 and 1147–1149) or secondary alcohol (Nos 1140–1141 and 1150–1154) which is oxidized to an alpha,beta-unsaturated ketone, it can conjugate with glutathione. The glutathione conjugates are converted to the corresponding mercapturic acid derivatives (N-acetylcysteine derivative) and excreted.

As the alpha,beta-unsaturated carbonyl group is a structural alert for toxicity, the Committee, at previous meetings, has devoted considerable attention to the safety of flavouring agents containing this reactive moiety. The Committee concluded at its fifty-seventh meeting (Annex 1, reference 154) that the presence of protective processes in cells provides adequate detoxication capacity at the low doses associated with use of such compounds as flavouring agents. With respect to alpha,beta-unsaturated ketones, these protective processes include reduction of the ketone to the corresponding alcohol (followed by conjugation of the alcohol with glucuronic acid) and conjugation with glutathione. These processes operate for the aliphatic ketones used as flavouring agents.

Table 1 summarizes the evaluation of aliphatic secondary alcohols, ketones and related esters used as flavouring agents.

In the unlikely event that all the flavouring agents in this group were to be consumed concurrently on a daily basis, the estimated combined intake would not exceed the human intake threshold for structural class I or II.

The Committee concluded that none of the flavouring agents in this group of 39 aliphatic secondary alcohols, ketones and related esters would raise a safety concern at the current estimated levels of intake. Other data on the toxicity of aliphatic secondary alcohols, ketones and related esters are consistent with the results of the safety evaluation.

This section summarizes the data relevant to the safety evaluation of 39 aliphatic ketones, secondary alcohols and related esters (see Table 1). The group of flavouring agents includes three saturated ketones (Nos 1118, 1155 and 1156), 24 unsaturated ketones (Nos 1119–1139 and 1147–1149), seven secondary alcohols (Nos 1140, 1141 and 1150–1154) and five esters formed from saturated secondary alcohols (Nos 1142–1146). The esters in this group are readily hydrolysed to the corresponding secondary alcohols. After hydrolysis, the resulting secondary alcohols, ketones and other secondary alcohols in the group are readily absorbed. Secondary alcohols are interconvertible with the corresponding ketones in vivo (McMahon, 1982). Therefore, all the substances in this group are expected to participate in common routes of absorption, metabolism, distribution and excretion and have similar toxicological profiles.

The total annual volume of use of the 39 secondary alcohols, ketones and related esters is approximately 3900 kg in Europe (International Organization of the Flavor Industry, 1995) and 1100 kg in the USA (Lucas et al., 1999) (see Table 2). In Europe and the USA, approximately 73% and 47% of the total annual production volume are accounted for by 6-methyl-5-hepten-2-one (No. 1120) and 1-octen-3-ol (No. 1152), respectively. Production volumes and intake values for each flavouring agent are reported in Table 2.

Table 2. Annual volumes of production and intake of aliphatic secondary alcohols, ketones and related esters used as flavouring agents in Europe and the USA

Twenty-six of the 39 flavouring substances in this group have been reported to occur naturally in foods. They have been detected in a variety of foods, including avocado, raspberry, mango, kiwi fruit, grapefruit juice, orange juice, cocoa, tea, tomato, asparagus, potato chips and rum (Maarse et al., 1999). Quantitative data on natural occurrence and consumption ratios have been reported for 10 of the substances: 6-methyl-5-hepten-2-one (No. 1120); 6,10-dimethyl-5,9-undecadien-2-one (No. 1122); 2,6,10-trimethyl-2,6,10-pentadecatrien-14-one (No. 1123); 3-penten-2-one (No. 1124); 3-octen-2-one (No. 1128); 4-methyl-3-penten-2-one (No. 1131); 1-penten-2-one (No. 1147); 1-octen-3-one (No. 1148); 1-penten-3-ol (No. 1150) and 1-octen-3-ol (No. 1152). The data demonstrate that their intake occurs predominantly from the consumption of traditional foods (i.e., consumption ratio > 1) (Stofberg & Kirschman, 1985; Stofberg & Grundschober, 1987) (see Table 2).

2.3.1 Biochemical data

(a) Hydrolysis

The aliphatic esters, such as 2-pentyl butyrate (No. 1142), are rapidly hydrolysed to the parent secondary alcohol and the component aliphatic carboxylic acids by classes of enzymes recognized as carboxylesterases (Ford & Moran, 1978; Heymann, 1980; Anders, 1989; White, 1990), the most important of which are the beta-esterases. Esters of aliphatic secondary alcohols were hydrolysed to their corresponding alcohols and carboxylic acids when incubated with liver homogenates obtained from male Wistar albino rats (Longland et al., 1977) or intestinal mucosal homogenates obtained from pigs (Leegwater & van Straten, 1974b). The ester of the secondary alcohol, 3-methyl-2-pentanol, and acetic acid were completely hydrolysed in intestinal mucosal homogenate within 2 h (Leegwater & van Straten, 1974b). Esters exposed to artificial intestinal fluid and simulated gastric juice were also hydrolysed, but more slowly. Partial hydrolysis (15–40%) was reported 2 h after incubation of 2-propyl butyrate or 4-methyl-2-pentyl acetate with simulated intestinal fluid (Leegwater & van Straten, 1974a). Rat liver homogenates were found to be between 8 and > 25 000 times more efficient than artificial pancreatic juice in hydrolysing a wide variety of aliphatic esters, and hydrolysis in simulated intestinal fluid with pancreatin was much faster than in simulated gastric juice (Longland et al., 1977). The authors concluded that tissue homogenates provide a good model for the situation in vivo. Thus, it is anticipated that the esters (Nos 1142–1146) in this group rapidly hydrolyse to secondary alcohols and carboxylic acids, which are then readily absorbed.

(b) Absorption, distribution, metabolism and excretion

After oral administration, aliphatic secondary alcohols and ketones are absorbed through the gastrointestinal tract and subsequently rapidly eliminated from the blood. Peak blood concentrations normally occur within 1–2 h after dosing (Lehman et al., 1945; Nordmann et al., 1973; Bonte et al., 1981). Although ketones and secondary alcohols are readily interconverted, reduction of the ketones by cytosolic carbonyl reductases (Felsted & Bachur, 1980) is favoured in vivo, yielding the corresponding secondary alcohols, which are excreted in the urine mainly as glucuronic acid conjugates (Kasper & Henton, 1982; Annex 1, reference 138).

In the case of a methyl ketone, the terminal methyl group may undergo oxidation, eventually yielding alpha-ketoacid, which is the substrate for further oxidation in the fatty acid pathway and citric acid cycle (Annex 1, reference 138). In the case of alpha,beta-unsaturated ketones, glutathione may conjugate at the beta-position, yielding cysteine and mercapturic acid derivatives, which are also excreted primarily in the urine as the conjugated secondary alcohol (Williams, 1959; Portoghese et al., 1989).

(i) Reduction of aliphatic secondary alcohols and acyclic ketones

Single high doses of a homologous series of aliphatic secondary alcohols and ketones were administered individually by gavage to rabbits, principally to identify the extent of urinary glucuronide metabolite formation. The level of urinary excretion of glucuronic acid conjugates was determined over 24 h. The results showed that secondary alcohols, either administered directly or formed via ketone reduction, are largely excreted as glucuronic acid conjugates (Kamil et al., 1953). The substances, doses, and average urinary output of glucuronide as a percentage of the dose administered are listed in Table 3.

Table 3. Urinary glucuronic acid conjugate after administration of aliphatic
secondary alcohols and ketones to rabbits by gavage

^a Calculated on the basis of a dose of 25 mmol/3 kg bw

alpha,beta-Unsaturated ketones are also reduced to the corresponding secondary alcohols. Cytoplasmic NADPH-dependent alpha,beta-unsaturated ketone reductase isolated from human liver catalysed the reduction of aliphatic alpha,beta-unsaturated ketones to the corresponding secondary alcohols (Fraser et al., 1967). The resulting alcohols can then be excreted as glucuronic acid conjugates (Williams, 1959). Glucuronic acid conjugates of substances like secondary alcohols are frequently the end-products of metabolism and are excreted in the urine or the bile (Kasper & Henton, 1982).

(ii) alpha-Oxidation of methylketones

Twenty aliphatic secondary ketones and related esters are methyl ketones (Nos 1119–1124, 1127, 1128, 1130–1132, 1134–1139 and 1149) or are secondary alcohols or related esters that are readily converted to methyl ketones in vivo (Nos 1140 and 1142) (see Table 1). Additional metabolic options are available for these substances. Methyl ketones undergo alpha-hydroxylation and subsequent oxidation of the terminal methyl group to eventually yield corresponding ketocarboxylic acids (Gabriel et al., 1972). The ketoacids are intermediary metabolites (e.g. alpha-ketoacids) that undergo oxidative decarboxylation to yield carbon dioxide and simple aliphatic carboxylic acids. The acids may be completely metabolized in the fatty acid pathway and citric acid cycle. The metabolism of alpha-hydroxyacids and alpha-ketoacids was recently reviewed (Annex 1, reference 144).

(iii) Glutathione conjugation of alpha,beta-unsaturated ketones

Twenty-six of the substances in this group are alpha,beta-unsaturated ketones (Nos 1124–1139, 1147–1149) or alcohols (Nos 1140, 1141, 1150–1154), which can be oxidized to alpha,beta-unsaturated ketones. In addition to reduction of the carbonyl function and, in some cases, alpha-oxidation of the methyl group, these substances may also react with free glutathione (GSH) in a nucleophilic Michael-type addition (Portoghese et al., 1989). GSH conjugates can be excreted in the bile or they can be converted enzymically to the mercapturic acid conjugate. It has been shown that alpha,beta-unsaturated carbonyl compounds have an affinity for sulfhydryl groups (Boyland & Chasseaud, 1967, 1968; Portoghese et al., 1989). These substances [e.g., ethyl vinyl ketone (No. 1147)] react with GSH through addition of a nucleophile (Nu^–) to the beta-carbon atom of the double-bond. This reaction is catalysed by glutathione-S-transferase but can also occur non-enzymatically (see Figure 1) (Esterbauer et al., 1975; Chasseaud, 1979; Portoghese et al., 1989).

Figure 1. Metabolic fate of aliphatic ketones and secondary alcohols

The conjugation of glutathione with alpha,beta-unsaturated ketones in vivo is catalysed by glutathione-S-tranferases. Up to 13 different forms of this enzyme exist in the human liver. These isoenzymes have many similarities including subunit size, immunochemical properties, catalytic activities, other binding activities and resistance to sulfhydryl reagents (van der Jagt et al., 1985). They also are identifiable by isoelectric point and haematin binding properties. The conjugation reaction may occur, however, even in the absence of glutathione-S-transferase, since many alpha,beta-unsaturated ketones are sufficiently electrophilic to react with glutathione. The reactivities of several alpha,beta-unsaturated ketones for glutathione addition were studied in the absence of glutathione-S-transferase (Portoghese et al., 1989). Included in this group were 2-octene-4-one (No. 1129), 4-methyl-3-penten-2-one (No. 1131) and 6-methyl-3,5-heptadien-2-one (No. 1134). It was shown that the rate of reaction was extremely rapid for compounds such as 2-butenone, but that any substitution at the beta-position of the double-bond greatly reduced this reactivity. Thus, a methyl substitution at the beta-position in 4-methyl-3-penten-2-one (No. 1131) reduced the reactivity towards glutathione addition by 120 000 times as compared with 2-butenone. A similar reduction of relative reactivity was observed with 6-methyl-3,5-heptadien-2-one (No. 1134) (Portoghese et al., 1989).

Typically, two metabolic processes follow conjugation of the alpha,beta-unsaturated ketone. In one, the GSH conjugate undergoes enzyme-catalysed hydrolysis and N-acetylation, yielding the corresponding N-acetyl-L-cysteine derivative (i.e., mercapturic acid derivative) (Chasseaud, 1976). In the other, the ketone function may be reduced to the corresponding alcohol. The principal metabolite obtained from the urine of rats given 2-propenal by subcutaneous injection was N-acetyl-S-(3-hydroxypropyl)-L-cysteine (Kaye, 1973). Formation of the mercapturic acid derivative of the corresponding secondary alcohol has been reported with mesityl oxide [4-methyl-3-penten-2-one (No. 1131)] and with ethyl vinyl ketone [1-penten-3-one (No. 1147)] in vitro with rat liver homogenate (Boyland & Chasseaud, 1967; Chasseaud, 1976).

At high cellular concentrations, alpha,beta-unsaturated carbonyl compounds have the potential to react with DNA. A cyclic deoxyguanosine adduct is formed in vitro via Michael addition of the N-1 or exocyclic NH₂ of guanine to the beta position of 2-butenone, followed by condensation of the ketone carbonyl with the remaining exocyclic NH₂ or N-1 moiety (Chung et al., 1988). However, these adducts have been found to be formed at a considerably slower pace (24 h at 100 ^oC) than the glutathione reaction, suggesting that glutathione conjugation takes place in vivo before it can react with DNA (Portoghese et al., 1989).

In addition, the terminal double-bonds may undergo epoxidation, catalysed by microsomal cytochrome P450 isoenzymes (Belluci et al., 1996). In rat liver microsomes, substances such as 1-hexen-3-ol can be epoxidized to yield erythro- and threo-1,2-epoxyhexan-3-ol. These are intermediary metabolites, in that the epoxide function is rapidly hydrolysed to a diol in the presence of microsomal epoxide hydrolase or conjugated with glutathione (Chiappe et al., 1998). This results in formation of erythro- and threo-hexane-1,2,3-triol, a polar metabolite that can be rapidly excreted in the urine (Chiappe et al., 1998).

Analysis of 150 human urine samples revealed the presence of two substances used as flavouring agents. The samples were collected for 2 months on different days from the same volunteers and were analysed by gas chromatography. The urinary profiles varied among individuals but remained constant for the same person over 2 months. Dietary changes during the testing period did not change the urinary profiles. Constituents of normal urine samples were identified by mass spectrometry. Among the numerous compounds from this group of aliphatic secondary alcohols and ketones, 3-penten-2-one (No. 1124) and 4-methyl-3-penten-2-one (No. 1131) were identified as normal constituents of human urine. In a study under the same conditions for compounds present in the urine of 40 patients with diabetes mellitus, one of the several compounds identified as a normal urine constituent was again 3-penten-2-one (No. 1124) (Zlatkis et al., 1973).

A similar type of study was undertaken, in which urinary organic acid metabolites were studied in normal and diabetic C57BL/Ks male mice. The profiles of the diabetic animals showed significantly lower concentrations of certain ketones, including 6-methyl-6-hepten-2-one. These ketones were excreted in low amounts in diabetic animals with depleted fat stores, indicating that their production is related to a deficiency in fat breakdown. However, the concentrations of several other similar ketones, such as 6-methyl-5-hepten-2-one (No. 1120), 3-hepten-2-one (No. 1127) and 3-octen-2-one (No. 1128), showed no difference between normal and diabetic mice (Holland et al., 1984).

(iv) Summary

The studies demonstrate that these 39 aliphatic ketones, secondary alcohols and related esters are metabolized primarily by reduction of the ketone to yield the corresponding secondary alcohol, followed by conjugation with glucuronic acid and excretion in the urine. Other metabolic options include alpha-oxidation of methyl ketones and glutathione conjugation of the alpha,beta-unsaturated ketones, followed by elimination of the mercapturic acid derivative of the corresponding secondary alcohol. To a minor extent, the double-bond may undergo epoxidation and then hydrolysis or conjugation with glutathione.

Because the alpha,beta-unsaturated carbonyl group is a structural alert for toxicity, the Committee, at previous meetings, devoted considerable attention to the safety of flavouring agents containing this reactive moiety. The Committee concluded at its fifty-seventh meeting (Annex 1, reference 154) that the presence of cytoprotective processes provides adequate detoxication capacity at the low doses associated with use of such compounds as flavouring agents. With respect to alpha,beta-unsaturated ketones, these cytoprotective processes include reduction of the ketone to the corresponding alcohol (followed by conjugation of the alcohol with glucuronic acid) and conjugation of the glutathione. As reported above, these processes are operative for the aliphatic ketones used as flavouring agents.

2.3.2 Toxicological studies

(a) Acute toxicity

Oral LD₅₀ values have been reported for 15 of the 39 substances in this group. In rats, the values ranged from 320 to > 6800 mg/kg bw (Table 4).

Table 4. Studies of acute toxicity of orally administered aliphatic secondary alcohols, ketones and related esters used as flavouring agents

M, male; F, female; NR, not reported

(b) Short-term and long-term studies of toxicity

The Committee previously evaluated the structurally related aliphatic ketones 2-heptanone (No. 283), 3-heptanone (No. 285), 4-heptanone (No. 287) and 2,6-dimethyl-4-heptanone (No. 302). The NOEL for 2-heptanone and was 20 mg/kg bw per day (Gaunt et al., 1972), while those of the remaining three aliphatic ketones were > 1000 mg/kg bw per day (O’Donoghue & Krasavage, 1980; O’Donoghue et al., 1984). The Committee concluded that all four agents were of no safety concern at current levels of intake when used as flavouring agents (Annex 1, reference 138). The results of short-term studies with repeated dose of two representative aliphatic secondary alcohols and five ketones are summarized in Table 5 and described below.

Table 5. Results of short- and long-term studies of toxicity in male and female rats of orally administered aliphatic secondary alcohols, ketones and related esters used as flavouring agents

(i) 5-Methyl-5-hexen-2-one (No. 1119)

A 14-day study to screen for minimum toxicity was conducted, in which a group of five Fischer 344 rats of each sex was maintained on a diet containing 5-methyl-5-hexene-2-one (No. 1119) that provided a target intake of 10 mg/kg bw per day. Four other flavouring agents were tested, with a common control group. The animals were observed for deaths twice daily and underwent a detailed clinical examination daily. Food consumption was measured on days 7 and 14. No physical signs of toxicity, abnormal body-weight gain, abnormal food consumption or treatment-related effects were observed at necropsy. Statistically significant decreases in absolute and relative liver weights and absolute kidney weight were reported in treated males. As similar changes were found with the four other substances, the authors concluded that the decreases in organ weight were a reflection of higher than normal liver and kidney weights in the control group. This conclusion was supported by the fact that none of the five treated groups showed signs or histopathological changes in these organs (Gill & van Miller, 1987).

(ii) 2,6,10-Trimethyl-2,6,10-pentadecatrien-14-one (No. 1123)

Groups of five weanling rats of each sex were given 2,6,10-trimethylpenta-decatrien-14-one in corn oil by gavage at a dose of 0, 0.35 or 3.5 mg/kg bw per day on 6 days per week for 2 weeks. Individual body weights and food intake were recorded on days 1, 7 and 14, and haemoglobin concentration was measured on day 14. All animals survived and were killed at the end of the study. Kidney and liver weights were recorded, and all organs were examined macro-scopically and microscopically. No differences in body-weight gain, food intake or food use efficiency were reported between treated and control groups. Histopathological examination revealed no evidence of alterations to the liver or kidneys. Liver weights were increased in male rats at the lower dose, while kidney weights were increased in male rats at the higher dose. In the absence of histopathological effects, these changes were considered to be of no toxicological significance (deGroot et al., 1974).

(iii) (E,R)-3,7-Dimethyl-1,5,7-octatrien-3-ol (No. 1154)

Groups of seven Sprague-Dawley rats of each sex were given (E,R)-3,7-dimethyl-1,5,7-octatrien-3-ol (No. 1154) in 1 ml of corn oil by gavage at a dose of 10 mg/kg bw per day for 14 consecutive days. Seven vehicle controls of each sex were given 1 ml of corn oil by gavage daily for 14 days. Individual doses were adjusted for body weight on days 1 and 8. All animals were observed daily for signs of gross toxicity and deaths. Weekly measurement of body weights and food consumption revealed no differences between test and control groups. All animals survived to completion of the study. At termination, gross and histopathological examination of the liver and kidneys from all treated animals revealed no lesions that could be associated with administration of the test substance (Wnorowski, 1997).

(iv) 4-Hexen-3-one (No. 1125)

Groups of 23 weanling Sprague-Dawley rats of each sex were maintained on a diet containing 4-hexene-3-one (No. 1125) at a concentration calculated to provide an average daily intake of 6.7 mg/kg bw for males and 6.6 mg/kg bw for females. Weekly measurements of body weight revealed no differences between treated and control groups. Biochemical, haematological and urinary analyses conducted after weeks 6 and 13 also showed no significant differences between treated and control rats. After 90 days, the animals were killed, and a complete necropsy was performed. A number of incidental lesions were reported, the incidences of which were low and randomly distributed among control and treated animals. The authors did not attribute the gross pathological lesions seen in the tissues to treatment with 4-hexen-3-one (Shellenberger, 1970a). In an addendum to the final report, which included the results of histopathological evaluation of tissues obtained from the rats, Shellenberger (1970b) concluded that administration of 4-hexen-3-one had no toxicologically significant effects.

(v) 2-Octen-4-one (No. 1129) and 1-octen-3-one (No. 1148)

In two studies with the same protocol, groups of 15 Wistar rats of each sex were maintained on a diet containing 2-octen-4-one (No. 1129) (Cox, 1974a) or 1-octen-3-one (No. 1148) (Cox, 1974b) at a concentration providing an average target intake of 7.5 mg/kg bw per day, for 90 days. Groups of 15 control rats of each sex were maintained on basal diet. The rats were housed individually and giventheir respective diet and tap water ad libitum. Measurements of food consumption throughout the study indicated that the treated groups received 6.7 mg/kg bw per day of 2-octen-4-one or 1-octen-3-one. All rats were monitored daily for survival, behaviour and physical appearance. Weekly measurement of body weight and food consumption revealed no significant differences between treated and control groups. Haematological, blood biochemical and urine analyses performed during weeks 6 and 12 on eight males and eight females from each group showed normal values. At 90 days, complete necropsies were performed. A detailed histopathological examination was undertaken on 23 major organs from eight male and eight female rats, and the livers and kidneys of the remaining seven animals in each group were examined for histopathological changes. No difference was found between treated and control animals in absolute or relative organ weights, and there was no evidence of gross or microscopic alterations with either substance (Cox, 1974a). A sporadic incidence of slight vacuolization of liver cells was observed in both control and treated animals (Cox, 1974b).

(vi) 1-Octen-3-ol (No. 1152)

A group of 14 male and 14 female Charles River rats was maintained on a diet containing 1-octen-3-ol at a concentration calculated to provide an average intake of 12 mg/kg bw per day for males and 14 mg/kg bw per day for females. The rats were given their respective diet and tap water ad libitum. All rats were monitored daily for survival, behaviour and physical appearance. Weekly measurements of body weight and food consumption revealed no significant differences between treated and control animals. In general, haematological, blood biochemical and urine analyses performed during weeks 7 and 13 revealed normal values, with the exception of increased haemoglobin and corpuscular values. At necropsy, gross and histopathological examination revealed no evidence of effects attributable to intake of the substance (Posternak, 1964).

(c) Genotoxicity

Seven representative substances in this group have been tested for genotoxicity in vitro. The results of these tests are summarized in Table 6 and described below.

Table 6. Studies of genotoxicity in vitro with aliphatic secondary alcohols, ketones and related esters used as flavouring agents

^a With and without metabolic activation

^d Toxic in some species at 300 µg/plate

(i) Reverse mutation

Assays for reverse mutation were performed with 6-methyl-5-hepten-2-one (No. 1120), 3-penten-2-one (No.1124), 4-methyl-3-penten-2-one (No. 1131), 6-methyl-3,5-heptadien-2-one (No. 1134), (E)-7-methyl-3-octen-2-one (No. 1135) and 1-decen-3-ol (No. 1153). There was no evidence of mutagenicity at concentrations up to 1500 µg/ml in TA98, TA100, TA1535, TA1537 or TA1538 strains of Salmonella typhimurium (Florin et al. 1980; Cheh, 1986; Durward, 1995; Thompson, 1996).

1-Penten-3-one (No. 1147) caused reverse mutation in an assay with preincubation in a single strain of S. typhimurium (TA100) in the presence and absence of metabolic activation (Deininger et al., 1990). No increase over spontaneous background reversion rates was reported with 1-penten-3-one at a concentration of 42 µg/plate in the presence of metabolic activation; however, at a concentration of 84 µg/plate, it induced a threefold increase in the rate of reversions. Bacterial toxicity was reduced in the presence of a metabolic activation system, probably due to detoxication by reaction with nucleophilic components in the system, such as glutathione and other SH-group-containing compounds. Similarly, the toxicity was reduced by addition of glutathione, with no effect on mutagenicity (Deininger et al., 1990; Eder et al., 1993).

In order to evaluate the effect of epoxidation of the double-bond on potential mutagenicity, 1-penten-3-one was further evaluated in the presence of SKF 525A, an inhibitor of microsomal monooxygenases, and 1,1,1-trichloropropene-2,3-oxide, an inhibitor of epoxide hydrolase. No mutagenic activity was observed in the presence of 100 µg/ml of SKF 525A, but addition of 1,1,1-trichloropropene-2,3-oxide resulted in an increase in the frequency of reverse mutations in a concentration-dependent manner (Deininger et al., 1990).

(ii) DNA damage

In a non-standardized assay for SOS DNA repair, Escherichia coli strain PQ37, with rfA and uvrA mutations, was incubated with 1-penten-3-one (No. 1147), with and without metabolic activation. sfiA gene-linked beta-galactosidase activity was used as a measure of the induction of SOS repair. In the absence of metabolic activation, 1-penten-3-one was a weak SOS-inducing agent, with a potential of 0.027 calculated from the linear part of the dose–response curve. Adding metabolic activation did not increase the genotoxicity. The authors concluded that the genotoxicity of 1-penten-3-one is probably a result of its toxicity (Deininger, 1990). As has been pointed out by other authors, it is difficult to detect genotoxic effects of alpha,beta-unsaturated substances in vitro because of their general toxicity to bacteria (Eder et al., 1991).

(iii) Conclusion

Positive results in vitro were obtained only with 1-penten-3-one, for which sufficient metabolic processes are available for detoxication at low levels of intake. These include known conjugation of alpha,beta-unsaturated ketones with glutathione and metabolic conversion of metabolically formed epoxides to dihydrodiols by epoxide hydrolase.

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       Toxicological Abbreviations