The Committee evaluated a group of 10 flavouring agents consisting of aliphatic acyclic acetals (see Table 1) using the Procedure for the Safety Evaluation of Flavouring Agents (see Figure 1, Introduction). None of these flavouring agents had been evaluated previously by the Committee.

Aliphatic acetals are geminal diethers in which two molar equivalents of alcohol are condensed with an aldehyde. Three of the 10 acetals are formed from acetaldehyde and simple aliphatic alcohols; the remaining seven acetals are formed from methanol or ethanol and aldehydes of carbon chain-length C₇–C₁₀. Acetals are known to hydrolyse in vivo to yield the corresponding alcohols and aldehydes. Of the component alcohols (methanol, ethanol, and cis-3-hexen-1-ol) and aldehydes, acetaldehyde (No. 80), heptanal (No. 95), 4-heptenal (No. 320), octanal (No. 98), and decanal (No. 104) were considered previously, at the forty-ninth and fifty-first meetings of the Committee (Annex 1, references 131 and 137), which concluded that they were of no safety concern under current levels of intake when used as flavour agents.

Three of the 10 flavouring agents in this group (No. 940), acetal (No. 941), and acetaldehyde ethyl (cis)-3-hexenyl acetal (No. 943 ) have been reported to occur as natural components of foods (Maarse et al., 1999). They have been detected in foods such as orange juice, strawberry, cider, peas, coffee, and cognac. A consumption ratio of 66 has been reported for acetal (No. 941) (Stofberg & Grundschober, 1987).

The total annual production of the 10 aliphatic acyclic acetals is approximately 2200 kg in Europe (International Organization of the Flavor Industry, 1995) and 4900 kg in the USA (Lucas et al., 1999). Approximately 97% of the total annual production in Europe and 99% of that in the USA is accounted for by two flavouring agents: the acetals formed from acetaldehyde and methanol or acetaldehyde and ethanol (Nos 940 and 941).

In general, aliphatic acetals undergo acid-catalysed hydrolysis to their component aldehydes and alcohols (Knoefel, 1934; Morgareidge, 1962). They are hydrolysed within 1–5 h in simulated gastric fluid in vitro and to a lesser extent in simulated intestinal fluid. Indirect evidence from a study in which rabbits were given aliphatic acetals in aqueous suspension by stomach tube indicated that rapid hydrolysis occurs in the stomach (Knoefel, 1934). The acetals formed from the reaction of alkyl-substituted pentanal and methanol, ethanol, and isopropyl alcohol are metabolized to the corresponding alcohols and acids in rat liver homogenate by an oxidative mechanism involving cytochrome P450 enzymes (Vicchio & Callery, 1989). It is anticipated that aliphatic acetals would undergo similar metabolism in humans to the corresponding alcohols and acids. There are insufficient data to exclude the possibility that significant amounts of the parent acetals reach the general circulation; however, the parent compounds are all in structural class I (Cramer et al., 1978). The low intake resulting from use as flavours would not be expected to saturate metabolic enzymes, and the acetals are metabolized to innocuous compounds by hydrolysis or oxidation.

On the basis of their recognized or presumed metabolic fate, the component alcohols and aldehydes fall into one of three structural classes: (1) linear, aliphatic, primary, saturated and unsaturated alcohols, and aldehydes; (2) alpha,beta-unsaturated aldehydes; and (3) branched-chain aliphatic aldehydes. The metabolic detoxication of linear, aliphatic, primary alcohols and aldehydes in vivo occurs primarily by oxidation of the alcohol to the corresponding aldehyde, with subsequent oxidation of the aldehyde to the corresponding carboxylic acid. The acid can serve as a substrate for fatty acid oxidation pathways and the citric acid cycle (Bosron & Li, 1980; Brabec, 1981). In general, alpha,beta-unsaturated aldehydes are metabolized by oxidation to the corresponding carboxylic acid, which may then participate in the fatty acid pathway. The aldehyde may be conjugated with glutathione in a Michael-type addition (Lamé & Segall, 1986; Mitchell & Petersen, 1987). Branched-chain aliphatic aldehydes have been reported to be oxidized primarily to more polar metabolites, which are excreted mainly in the urine. A mixture of diacids and hydroxyacids resulting from omega-oxidation, reduction, and hydration of the alkene function and oxidation of the aldehyde function are the principal urinary metabolites of branched aldehydes. It is anticipated that the alcohol and aldehyde products of aliphatic acetal hydrolysis would undergo similar metabolism in humans.

Although few studies on the absorption, distribution, and elimination of aliphatic acyclic acetals have been reported, the metabolism of the component alcohols and aldehydes has been investigated. These studies are considered relevant to the safety evaluation of orally administered acetals that are expected to be hydrolysed in the acid environment of the stomach.

Citral is predicted to be a metabolite of citral dimethyl acetal (No. 944) and citral diethyl acetal (No. 948). The absorption, distribution, and excretion of citral have been studied extensively in rats and mice. Citral was reported to undergo rapid absorption from the gastrointestinal tract and to be distributed uniformly throughout the body (Phillips et al., 1976). Rapid elimination of citral and its metabolites was reported to occur primarily in the urine and to a minor extent in exhaled air and faeces (Phillips et al., 1976; Diliberto et al., 1988).

Step 1 All 10 of the compounds in this group are aliphatic acetals. They have acyclic structures that vary only in the length of their hydrocarbon chains and the number and placement of double bonds. All of the substances were classified in structural class I.

Step 2 At current levels of intake, none of the 10 substances would be expected to saturate their metabolic pathways. They are all predicted to be metabolized to their component aldehydes and alcohols, which will then be metabolized to innocuous products¹. The parent acetals are all in structural class I.

Step A3 The daily per capita intakes of all the substances in this group in Europe and the USA are below the threshold of human intake for class I (1800 µg), indicating that they pose no safety concern at current levels of estimated intake when used as flavouring agents.

The considerations on intake and other information used to evaluate the aliphatic acetals according to the Procedure are summarized in Table 1.

In the unlikely event that all 10 substances were to be consumed concurrently on a daily basis, the estimated combined intake would not exceed the human intake threshold for class I (1800 µg/person per day). All flavouring agents in this group are expected to be efficiently metabolized and would not saturate their metabolic pathways. On this basis of the evaluation of all the data, there is no safety concern about combined intake.

The Committee concluded that none of the flavouring agents in the group of aliphatic acetals would present a safety concern at the current levels of estimated intake. Other data on the toxicity of aliphatic acetals were consistent with the results of the safety evaluation.

This monograph summarizes the key data relevant to the safety evaluation of 10 aliphatic acetals used as flavouring agents (see Table 1).

The daily per capita intake of each agent is reported in Table 2.

2.3.1 Biochemical data

In general, aliphatic acetals undergo hydrolysis to their component aldehydes and alcohols (Knoefel, 1934; Morgareidge, 1962). 1,1-Dimethoxyethane (No. 940), acetal (No. 941), and related acetals were hydrolysed within 1–5 h in simulated gastric fluid and to a lesser extent in simulated intestinal fluid (Morgareidge, 1962). In a study in which rabbits were given 1,1-dimethoxyethane (No. 940), acetal (No. 941), and other aliphatic acetals in aqueous suspension by stomach tube, rapid hydrolysis occurred in the stomach (see Figure 1). A correlation was reported between decreased narcotic effects, which are observed at high doses of acetals, and resistance to acid hydrolysis (Knoefel, 1934).

A study was conducted on the feasibility of using acetals of 2-propylpentanal as pro-drugs in treatment with valproic acid (2-propylpentanoic acid). The acid and alcohol of 2-propylpentanal were identified in the supernatant and microsomal fractions of rat liver incubated with the dimethyl, diethyl, and di-isopropyl acetals of 2-propylpentanal, indicating that dimethoxy-, diethoxy- and diisopropyl-2-propyl-pentane hydrolyse to yield the corresponding alcohols and parent aldehyde 2-propylpentanal. The aldehyde is subsequently oxidized to the corresponding acid or reduced to the corresponding alcohol (Vicchio & Callery, 1989). It is anticipated that aliphatic acetals would undergo similar metabolism in humans to the corresponding alcohols and acids. There are insufficient data to exclude the possibility that significant amounts of the parent acetals may reach the general circulation; however, all the substances are in structural class I, and the available data indicate that they have little toxicity.

Hydrolysis of paraldehyde, the cyclic acetal formed from three molecules of acetaldehyde, occurs in the human liver to yield acetaldehyde, which is subsequently oxidized to acetic acid. The acetaldehyde produced in this pathway is completely metabolized, as no trace of the substance is found in the serum of treated animals (Levine & Bodansky, 1940; Hitchcock & Nelson, 1943; Thurston et al., 1968).

Although few studies have been reported on the absorption, distribution, and elimination of aliphatic acetals per se, studies have been conducted on the component alcohols and aldehydes. These were considered to be relevant to the safety evaluation of orally administered acetals, as acetals are readily hydrolysed in the acidic environment of the stomach, intestinal fluid, or in the liver to yield the component alcohol and aldehyde. The absorption, distribution, and excretion of the aliphatic acetal metabolites, ethanol and citral, have been studied in humans and rodents, respectively.

Within 1 h of ingestion of the acetal, ethanol was reported to be absorbed from the stomach and upper intestine by passive diffusion (Wallgren & Barry, 1970; Halsted et al., 1973). As discussed below, the metabolic detoxication of linear, aliphatic, primary alcohols in vivo occurs primarily by oxidation of the alcohol to the corresponding aldehyde (Bosron & Li, 1980).

After administration of a single dose of up to 960 mg/kg bw to rats and 100 mg/kg bw to mice by gavage, citral underwent rapid absorption from the gastrointestinal tract and was distributed uniformly throughout the body (Phillips et al., 1976). An oral dose of citral was reported to be eliminated primarily in the urine and also in exhaled air and faeces (Phillips et al., 1976; Diliberto et al., 1988). Excretion in the faeces was not a primary route of elimination, but a significant quantity of citral was present in the bile, suggesting that it readily enters the enterohepatic circulation (Diliberto et al., 1988), consistent with the observation that citral induces mitochondrial oxidation and hepatic cytochrome P450, glucuronyl transferase, and alcohol dehydrogenase activity (Parke & Rahman, 1969; Boyer & Petersen, 1990).

On the basis of their recognized or presumed metabolic fate, the component alcohols and aldehydes fall into one of three structural classes: (i) linear, aliphatic, primary, saturated and unsaturated alcohols (i.e. methanol, ethanol, and cis-3-hexen-1-ol) and aldehydes (i.e., acetaldehyde, heptanal, octanal, decanal, and 4-heptenal); (ii) alpha,beta-unsaturated aldehydes (i.e., 2,6-nonadienal); and (iii) branched-chain aliphatic aldehydes (i.e., citral). The metabolism of the component alcohols and aldehydes in humans can be reasonably predicted by analogy with the known metabolic fate of the substance or structurally related substances in animals. The alcohol and aldehyde products of acetal hydrolysis would undergo similar metabolism in humans.

Linear, aliphatic, primary, saturated and unsaturated alcohols and aldehydes are detoxicated in vivo primarily by oxidation of the alcohol, first to the corresponding aldehyde and subsequently to the corresponding carboxylic acid (Bosron & Li, 1980). Oxidation of alcohols is catalysed by an NAD⁺/NADH-dependent enzyme, alcohol dehydrogenase (Pietruszko et al., 1973). Oxidation of aldehydes is catalysed by the NAD⁺/NADH-dependent enzyme, aldehyde dehydrogenase (see Figure 2). Direct conjugation of the alcohol with glucuronic acid has been reported to occur as a minor metabolic pathway (Bosron & Li, 1980).

Aldehydes are oxidized to carboxylic acids, which, in turn, participate in the fatty acid oxidation pathway and the citric acid cycle (Brabec, 1981). The resulting carboxylic acid metabolites become labile substrates for beta-oxidation and cleavage to yield acetyl coenzyme A (CoA) or propionyl CoA, which eventually enter the citric acid cycle. Unsaturated carboxylic acids also participate in the fatty acid pathway. If the stereochemistry of the double bond is cis (e.g., cis-3-hexen-1-ol), the acid is unable to participate in fatty acid oxidation until it is enzymatically converted to the trans 2-isomer, which ultimately forms the acyl CoA derivative (Feldman & Weiner, 1972; Lehninger, 1975; Voet & Voet, 1990).

Ethanol is a primary aliphatic alcohol which is reported to be rapidly oxidized in vivo by alcohol dehydrogenase to form acetaldehyde. Acetaldehyde may be further oxidized by aldehyde dehydrogenase to form acetic acid, with further oxidation to form CO₂ and water (Timbrell, 1982). Alcohol and acetaldehyde dehydrogenases have been reported in numerous tissues, with the greatest activity in the liver (Sipes & Gandolfi, 1986); however, these enzymes are also found in the gastrointestinal tract, suggesting that ethanol can undergo first-pass metabolism at that site subsequent to absorption (Sato & Kitamura, 1996). Small amounts of ethanol were also reported to undergo conjugation with glucuronic acid (Williams, 1959).

Acetaldehyde is an important intermediate in the cellular metabolism of mammals, including humans, and undergoes rapid enzymatic oxidation in the liver, with approximately 80% conversion to acetate. Acetate produces energy via the citric acid cycle (Asmussen et al., 1948; Lundquist et al., 1962; Lehninger, 1975). Hald & Larsen (1949) reported that an average-sized rabbit could metabolize 7–10 mg of acetaldehyde per minute. The oxidation rate in mammals was 0.75 µmol/min per g of liver (Lundquist et al., 1962). An alternative minor pathway that has been reported is reduction of acetaldehyde to ethanol, catalysed by aldehyde dehydrogenase.

In general, alpha,beta-unsaturated aldehydes (e.g., 2,6-nonadienal) are oxidized to the corresponding carboxylic acids (Lamé & Segall, 1986; Mitchell & Petersen, 1987. Results reported for the structurally related alpha,beta-unsaturated aldehydes trans-2-hexenal and 2-nonenal indicate that secondary pathways may involve conjugation with glutathione. The metabolites would ultimately be excreted as the mercapturic acid derivatives (Esterbauer et al., 1982; Cadenas et al., 1983).

The metabolism of the branched-chain aliphatic aldehyde citral has been studied in laboratory animals. In rats, citral is metabolized to a mixture of diacids and hydroxyacids resulting from omega-oxidation, reduction, and hydration of the unsaturation at C-2, and oxidation of the aldehyde function. Hepatic reduction of the aldehyde may precede oxidation pathways. Citral was reported to be rapidly reduced to the corresponding alcohol by alcohol dehydrogenase in rat hepatic mitochondrial and cytosolic fractions (Boyer & Petersen, 1990; Diliberto et al., 1990).

2.3.2 Toxicological studies

Aliphatic acetals have been reported to have little acute toxicity after oral administration, with LD₅₀ values > 4300 mg/kg bw. LD₅₀ values have been reported for seven of the 10 aliphatic acetals used as flavouring agents and for their corresponding metabolites. These values are presented in Table 3.

In a study designed to evaluate the narcotic effects of acetals, no effects were reported in rabbits given a single oral dose of 1800 mg/kg bw (Knoefel, 1934). In the same study, 1,1-dimethoxyethane at a single oral dose of 2700 mg/kg bw was reported to have no effect in three of four rabbits; the fourth showed semi-erectness or staggering.

One short-term toxicological study has been reported with citral diethyl acetal (No. 948). Although no studies on the other nine aliphatic acetals have been identified, studies have been conducted with the component alcohols and aldehydes, and these are considered relevant to the safety evaluation of orally administered acetals, which are presumed to be readily hydrolysed in gastric juice, intestinal fluid, or the liver. The short-term study with citral diethyl acetal and various metabolites of aliphatic acetals is summarized in Table 4 and described in detail below.

A blend of equal parts by weight of citral diethyl acetal and citral was administered to a group of 21 rats in the diet for 12 weeks, providing an average intake of the combination of 110 mg/kg bw per day and an average daily intake of citral diethyl acetal of 56 mg/kg bw. A control group of 21 rats received an unsupplemented diet. The treated animals had normal behaviour and appearance during the study. Growth, food intake, and efficiency of food use were reported not to be affected, and gross examination revealed no changes in organ weights or haemoglobin concentration. The NOEL of 56 mg/kg bw per day for citral diethyl acetal is > 100 000 times the estimated daily intake (‘eaters only") of 0.07 µg/kg bw from its use as a flavouring agent in Europe (Trubek Laboratories Inc., 1958a).

cis-3-Hexen-1-ol: Groups of 15 male and 15 female weanling rats were given drinking-water containing cis-3-hexen-1-ol at a concentration of 0, 310, 1250, or 5000 mg/L for 98 days, reported to provide average daily intakes of 0, 30, 130, and 410 mg/kg bw per day for males and 0, 42, 170, and 720 mg/kg bw per day for females. Slightly increased relative weights of the kidney and adrenal gland were reported in males at 5000 mg/L. The authors reported a NOEL of 1250 mg/L, equivalent to average intakes of 130 and 170 mg/kg bw per day for male and female rats, respectively (Gaunt et al., 1969). These doses are > 10 000 times the combined total daily per capita intake (‘eaters only’) of 11 µg/kg bw per day for the 10 aliphatic acetals used as flavouring agents in the USA. This large margin of safety would accommodate any anticipated difference in toxicity between the aliphatic acetals and their component alcohols.

No adverse effects were reported in rats given a diet containing 0.5% 1-hexanol for 13 weeks, calculated to provide an average daily intake of 420 mg/kg bw. In the same study, 1-hexanol in the diet at increasing concentrations from 1 to 6% for 13 weeks (calculated to provide an average daily intake of 1200 mg/kg bw) also produced no adverse effects in rats (Eibert, 1992). No adverse effects were reported in dogs given 1-hexanol at a concentration of 0.5% in the diet for 13 weeks, calculated to provide an average daily intake of 200 mg/kg bw (Gaunt et al., 1969).

Acetaldehyde, heptanal, octanal, and decanal: Acetaldehyde was added to the drinking-water of rats at concentrations providing a daily intake of 0, 25, 120, or 680 mg/kg bw for 4 weeks. The only treatment-related effect reported was hyper-keratosis of the forestomach in animals at the high dose. The NOEL of 120 mg/kg bw per day for acetaldehyde is > 10 000 times the combined total daily per capita intake ("eaters only") of 11 µg/kg bw per day for the 10 aliphatic acetals used as flavouring agents in the USA. This large margin of safety would accommodate any anticipated difference in toxicity between the aliphatic acetals and their component aldehydes (Til et al., 1988).

Groups of 12 male and 12 female rats were maintained for 86 days on diets containing a mixture of aldehydes: C-8, octanal (4 ppm); C-9, nonanal (9 ppm); C-10, decanal (2.2 ppm); C-11, (6 ppm); C-12, (6 ppm); C-12 (6 ppm); and methyl nonyl acetaldehyde (8 ppm). The diet was calculated to provide an average daily intake of 112 mg/kg bw of the aldehyde mixture for 12 weeks. Controls were maintained on an unsupplemented diet. After 12 weeks, urine samples were examined for the presence of glucose and albumin, and blood was analysed for haemoglobin. At necropsy, the liver and kidney were weighed and examined histologically. Measurements of growth, food intake, efficiency of food use, haematological examinations, urine analyses, liver and kidney weights, and histological examination of liver and kidney provided no evidence of toxicity (Trubek Laboratories Inc., 1958b).

No adverse effects were reported in rabbits given 1-heptanol (a metabolite of heptanal) at a dose of 1.4 mg/kg bw per day by gavage for 6 months. In the same study, a mixture of 1-hexanol, heptanol, octanol (a metabolite of octanal), and IM-68 (a mixture of these three alcohols) was administered by gavage to mice for 1 month, providing doses of 200, 150, 180, and 230 mg/kg bw per day, respectively. No cumulative effects were seen (Voskoboinikova, 1966).

Ten albino male and female rats of mixed strain, weighing 50–80 g, were fed a rice diet containing decanoic acid (a metabolite of decanal) at a concentration of 10%, calculated to provide an average daily intake of 5000 mg/kg bw, for 150 days. After treatment, the animals were killed and their stomachs were examined for gross lesions. The author reported no remarkable changes either in the forestomach or glandular stomach (Mori, 1953).

2,6-Nonadienal: Although no short-term studies of toxicity have been identified for the acetal metabolite 2,6-nonadienal, results have been reported for the structurally similar compounds, trans,trans-2,4-decadienal, trans-2-cis-6-dodeca-dienal, and trans-2-cis-4-cis-7-tridecatrienal. No adverse effects were reported when rats were maintained for 13 weeks on diets containing trans,trans-2,4-decadienal at 3.4, 11, or 34 mg/kg bw per day (Damske et al., 1980). The dose of 34 mg/kg bw per day is > 1000 times the combined total daily per capita intake (‘eaters only’) of 11 µg/kg bw per day for the 10 aliphatic acetals used as flavouring agents in the USA. The large margin of safety would accommodate any anticipated difference in toxicity between the aliphatic acetals and their component aldehydes.

Citral: No adverse effects were reported when citral was added to the diet of rats at concentrations up to 10 000 mg/kg for 13 weeks (Hagan et al., 1967). The concentration was calculated to result in an average daily intake of 500 mg/kg bw (Food & Drug Administration, 1993). The NOEL of 500 mg/kg bw per day is > 10 000 times the combined total daily per capita intake ("eaters only") of 11 µg/kg bw for the 10 aliphatic acetals used as flavouring agents in the USA. The large margin of safety would accommodate any anticipated difference in toxicity between the aliphatic acetals and their component aldehydes.

No long-term studies of toxicity and carcinogenicity have been reported for the 10 aliphatic acetals in this group of flavouring agents. However, a 2-year bioassay was performed on a substance structurally related to citral, an aldehyde formed by hydrolysis of its corresponding acetal. Citral is a mixture of geranial and a minor amount of neral. As geraniol and its acetate ester are metabolic precursors of geranial, they are structurally related to citral. The acetate ester of genaniol is expected to be hydrolysed rapidly to geraniol (Grundschober, 1977; Longland et al., 1977) and then be oxidized to geranial in vivo. A mixture of geranyl acetate (71%) and citronellyl acetate (29%) was administered to groups of mice and rats by gavage at multiple doses, 5 days per week for 103 weeks. No significant toxic or carcinogenic effects were reported when the mixture was administered at a dose of 1000 or 2000 mg/kg bw per day to mice and rats, respectively, corresponding to calculated doses of 710 and 1400 mg/kg bw per day for geranyl acetate, respectively, representing 71% of the administered dose, which is the fraction of geranyl acetate contained in the mixture (National Toxicology Program, 1987).

Although no studies of genotoxicity have been reported with aliphatic acetals, several studies were conducted with their component alcohols and aldehydes. The results of these tests are summarized in Table 5 and described below.

Acetaldehyde: Acetaldehyde did not cause reverse mutation in the Salmonella/mammalian microsome assay with S. typhmiurium strains TA97, TA98, TA100, TA1535, and TA1537 with and without metabolic activation (Mortelmans et al., 1986). Acetaldehyde was reported to be mutagenic in mouse lymphoma cells with and without metabolic activation (Wangenheim & Bolcsfoldi, 1988). It did not cause chromosomal aberrations in normal human lymphocytes, but positive results were found in lymphocytes from a patient with Fanconi anaemia (Obe et al., 1979). Acetaldehyde increased the frequency of sister chromatid exchange in adult human lymphocytes and peripheral lymphocytes (He & Lambert, 1985; Norppa et al., 1985); however, aldehydes are rapidly oxidized to the corresponding acids and have a short plasma-life, and these important conditions that hold in vivo are difficult to establish in vitro.

Ethanol: Ethanol was not mutagenic in L5178Y mouse lymphoma cells with or without metabolic activation (Wangenheim & Bolcsfoldi, 1988).

Heptanal, octanal, and nonanal: The homologous series of aliphatic aldehydes did not induce reverse mutation in S. typhimurium strains (e.g., TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538) with or without metabolic activation (Florin et al., 1980; Marnett et al., 1985; Mortelmans et al., 1986; Zeiger et al., 1992) when concentrations of up to 3333 µg/plate were used in standard (Florin et al., 1980) and preincubation (Marnett et al., 1985; Mortelmans et al., 1986; Zeiger et al., 1992) protocols. No gene mutation was induced in a variation on the standard assay, with preincubation and metabolic activation (Mortelmans et al., 1986).

There was no evidence of unscheduled DNA synthesis when rat or human hepatocytes were incubated with nonanal at concentrations up to 100 mmol/L (Martelli et al., 1994). In standard assays, no significant increase in the frequency of chromosomal aberrations was reported when concentrations of nonanal up to 100 µmol/L (16 200 ug/plate) were incubated with primary hepatocytes from Fischer 344 rats. No increase in the mitotic index or the frequency of micronuclei was seen when nonanal at 16 200 µg/plate was incubated with freshly prepared rat hepatocytes (Esterbauer et al., 1990; Eckl et al., 1993). Nonanal induced a significant increase in the incidence of sister chromatid exchange in rat hepatocytes, but there was no dose–response relationship (Eckl et al., 1993).

Decanal and octanal metabolites: Decanoic acid and octanoic acid (metabolites of decanal and octanal, respectively) did not induce reverse mutation in S. typhimurium strains TA97, TA98, TA100, TA1535, and TA1537 in the presence or absence of an exogenous metabolic activation system from the livers of Aroclor-induced male Sprague-Dawley rats and Syrian hamsters (Zeiger et al., 1988).

Citral: Citral induced mutation in Bacillus subtilis strains M45 and H17 at a concentration of 2.5 µL (Yoo, 1986); but no effect was seen with a concentration of 17 µg/disc (Oda et al., 1979). Citral was not mutagenic in Escherichia coli when tested at concentrations of 0.013–0.1 mg/plate (Yoo et al., 1986). Furthermore, it did not induce chromosomal aberrations in a Chinese hamster fibroblast cell line or reverse mutations in S. typhimurium strains TA92, TA94, TA98, TA100, TA1535, and TA1537, with and without metabolic activation (Eder et al., 1982; Lutz et al., 1982; Ishidate et al., 1984; Zeiger et al., 1987; Ishidate, 1988). It was also inactive in S. typhimurium (strains not specified) with metabolic activation (no further details provided) (National Toxicology Program, 1983).

Acetaldehyde: Acetaldehyde did not cause reciprocal translocations or sex-linked recessive lethal mutation in germ cells of Drosophila melanogaster after oral administration; however, it induced sex-linked recessive lethal mutation when administered by injection (Woodruff et al., 1985).

Acetaldehyde administered by intraperitoneal injection to mice and hamsters induced sister chromatid exchange in bone-marrow cells (Obe et al., 1979; Korte & Obe, 1981).

Ethanol: Ethanol provided in the drinking-water of Chinese hamsters for 46 weeks at a concentration of 10% (v/v) did not induce chromosomal aberrations or sister chromatid exchange in peripheral lymphocytes or bone-marrow cells, respectively (Korte & Obe, 1981); however, sister chromatid exchange was induced in a study in which 1.0 ml ethanol was administered by intraperitoneal injection at a concentration of 10^–4% (v/v) (Obe et al., 1979).

On the basis of the results of the studies of genotoxicity, the Committee concluded that this group of aliphatic acetals is not genotoxic in vivo.

Groups of 30 rats were given citral at a dose of 0, 50, 160, or 500 mg/kg bw per day for 2 weeks before mating through to day 20 of gestation. The fetuses were removed surgically from half of the rats on day 20 of gestation, while the other half remained on the citral diet until 21 days after parturition, the offspring thus being exposed to citral during lactation. Dose-related increases in maternal mortality rates, adverse clinical signs, and reductions in body-weight gain and feed consumption were reported at the two higher doses. No effects on estrous cycling, mating, fertility, or length of gestation were reported at any dose. A statistically significant decrease in pup body weight at birth was found at the highest dose. The NOEL for dams was 50 mg/kg bw per day, and that for the offspring was 160 mg/kg bw per day (Hoberman et al., 1989).

In a screening study, groups of 10 female Sprague-Dawley rats were given the acetal orally by gavage at a dose of 120, 250, or 500 mg/kg bw per day for 7 days before cohabitation and then throughout cohabitation, gestation, parturition, and a 4-day lactation post partum, for a total of 39 days. Measurements of body weight and food consumption, clinical observations, and gross examination of dams and pups killed at the end of the study gave NOELs of 120 mg/kg bw per day for maternal toxicity and 250 mg/kg bw per day for reproductive and developmental toxicity (Vollmuth et al., 1990).

Four groups of 10 virgin Crl CD rats were given an acetal formed from ethanol and a mixture of geranial (> 90%) and neral (> 10%) at a dose of 0, 125, 250, or 500 mg/kg bw per day by gavage once daily, 7 days before cohabitation and throughout cohabitation (maximum of 7 days), gestation, parturition, and a 4-day post-partum period, for a total of 39 days. The dams were monitored twice daily, and body weights, food consumption, duration of gestation, and fertility parameters (mating, fertility, and gestation indexes, number of offspring per litter) were measured. The offspring were observed daily for clinical signs, examined for gross external malformations, and weighed. The NOEL for both maternal and developmental toxicity was 500 mg/kg bw per day (Vollmuth et al., 1990).

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¹ Some aldehydes, including acetaldehyde, are genotoxic in vitro in a number of test systems, and acetaldehyde has been reported to produce tumours of the respiratory tract in rats and hamsters exposed to high doses by inhalation. The relevance of this observation to oral administration is questionable, as various metabolic processes in the intestinal wall and liver (i.e. oxidation and conjugation) are predicted to result in extensive first-pass metabolic inactivation, especially at the low concentrations expected from use as flavouring agents.

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       Toxicological Abbreviations