INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION SAFETY EVALUATION OF CERTAIN FOOD ADDITIVES AND CONTAMINANTS WHO FOOD ADDITIVES SERIES 40 Prepared by: The forty-ninth meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) World Health Organization, Geneva 1998 ALIPHATIC LACTONES First draft prepared by Dr P.J. Abbott, Australia New Zealand Food Authority (ANZFA) Canberra, Australia 1. Evaluation 1.1 Introduction 1.2 Estimated daily per capita intake 1.3 Absorption, metabolism and elimination 1.3.1 Lactones derived from linear saturated 5-hydroxycarboxylic acid 1.3.2 Lactones derived from linear saturated 4- or 6-hydroxycarboxylic acids 1.3.3 Lactones derived from linear hydroxycarboxylic acids containing unsaturation 1.3.4 Lactones derived from branched-chain hydroxycarboxylic acids 1.3.5 Lactones containing alpha, ß-unsaturation 1.3.6 Hydroxyfuranones 1.4 Application of the procedure for the safety evaluation of flavouring agents 1.5 Consideration of combined intakes 1.6 Conclusions 2. Relevant background information 2.1 Toxicological studies 2.1.1 Acute toxicity 2.1.2 Short-term and long-term toxicity and carcinogenicity 2.1.2.1 gamma-Butyrolactone 2.1.2.2 gamma-Nonalactone and gamma-undecalactone 2.1.2.3 5-Ethyl-3-hydroxy-4-methyl-2(5H)-furanone 2.1.2.4 4,5-Dimethyl-3-hydroxy- 2,5-dihydrofuran-2-one 2.1.3 Genotoxicity 2.1.4 Other relevant study 3. References 1. EVALUATION 1.1 Introduction The Committee evaluated a group of thirty five aliphatic lactones used as flavouring substances in food using the Procedure for the Evaluation of Flavouring Agents (the 'Procedure') (see Figure 1 and Table 1). Table 1. Summary of the safety evaluation of aliphatic lactones used as flavouring agents Step 1: All of the substances in the group are in structural class I, the human intake threshold of which is 1800 µg per person per day No. Substance Estimated per capita Step 2 Step A3/B3 Step A4 Step A5/B4 Conclusion based intake USA/Europe Metabol. to Intake exceed Endogeneous? Adequate NOEL for on current levels (µg/day) innocuous threshold of substance or related of intake products? concern? substance? Structural class I 0219 4-Hydroxybutyric acid lactone 100/130 Yes No -1 -1 No safety concerns (gamma-Butyrolactone) 0220 gamma-Valerolactone 57/140 Yes No - - No safety concerns 0223 gamma-Hexalactone 19/190 Yes No - - No safety concerns 0224 delta-Hexalactone 2.5/380 Yes No - - No safety concerns 0225 gamma-Heptalactone 41/190 Yes No - - No safety concerns 0226 gamma-Octalactone 90/490 Yes No - - No safety concerns 0228 delta-Octalactone 17/270 Yes No - - No safety concerns 0229 gamma-Nonalactone 470/1200 Yes No - - No safety concerns 0230 Hydroxynonanoic acid 11/150 Yes No - - No safety concerns delta-lactone 0231 gamma-Decalactone 370/1800 Yes Yes No Yes No safety concerns 0232 delta-Decalactone 1900/8400 Yes Yes No Yes No safety concerns 0241 epsilon-Decalactone 0/0.01 Yes No - - No safety concerns 0233 gamma-Undecalactone 550/1400 Yes No - - No safety concerns 0234 5-Hydroxyundecanoic acid 180/350 Yes No - - No safety concerns delta-lactone 0235 gamma-Dodecalactone 110/220 Yes No - - No safety concerns 0236 delta-Dodecalactone 1140/6800 Yes Yes No Yes No safety concerns 0242 epsilon-Dodecalactone 0.17/0.01 Yes No - - No safety concerns 0238 delta-Tetradecalactone 2.5/120 Yes No - - No safety concerns 0239 omega-Pentadecalactone 51/84 Yes No - - No safety concerns 0221 4-Hydroxy-3-pentenoic acid 4.8/- Yes No - - No safety concerns lactone 0247 5-Hydroxy-7-decenoic acid 0.10/0.26 Yes No - - No safety concerns delta-lactone Table 1. Continued... No. Substance Estimated per capita Step 2 Step A3/B3 Step A4 Step A5/B4 Conclusion based intake USA/Europe Metabol. to Intake exceed Endogeneous? Adequate NOEL for on current levels (µg/day) innocuous threshold of substance or related of intake products? concern? substance? 0248 5-Hydroxy-8-undecenoic acid 8.6/0.01 Yes No - - No safety concerns delta-lactone 0249 1,4-Dodec-6-enolactone 8.6/0.01 Yes No - - No safety concerns 0240 omega-6-Hexadecenlactone 0.10/6 Yes No - - No safety concerns 0227 4,4-Dibutyl-œ-butyrolactone 0.10/0.14 Yes No - - No safety concerns 0244 3-Heptyldihydro-5-methyl- 0.1/0.04 Yes No - - No safety concerns 2(3H)-furanone ---- 4-Hydroxy-3-methyloctanoic 8.6/0 Yes No - - No safety concerns acid gamma-lactone 0237 6-Hydroxy-3,7-dimethyloc- 0/0.1 Yes No - - No safety concerns tanoic acid lactone 43/0 Yes No - - No safety concerns 0250 gamma-Methyldecalactone Structural class III 0246 5-Hydroxy-2-decenoic acid 0.10/12 No - - - Not evaluated2 delta-lactone 0245 5-Hydroxy-2,4-decadienoic 0.10/0.33 No - - - Not evaluated2 acid delta-lactone ---- Mixture of 5-Hydroxy-2- 2/0 No - - - Not evaluated2 decenoic acid delta-lactone, 5-Hydroxy-2-dodecenoic acid delta-lactone, and 5-Hydroxy- 2-tetradecenoic acid delta-lactone ---- 5-Hydroxy-2-dodecenoic acid 8.6/0 No - - - Not evaluated2 delta-lactone 0222 5-Ethyl-3-hydroxy-4-methyl- 6.1/13 No No No Yes No safety concerns 2(5H)-furanone 0243 4,5-Dimethyl-3-hydroxy-2,5- 0.1/2.1 No No No Yes No safety concerns dihydrofuran-2-one 1 Not applicable 2 Evaluation deferred pending consideration of other ý,œ-unsaturated compounds. Two substances in the group have been evaluated previously by the Committee, namely, gamma-nonalactone and gamma-undecalactone. At the Eleventh Meeting of the Committee, an ADI of 0-1.25 mg/kg body weight was established for each substance (Annex 1, reference 14). 1.2 Estimated daily per capita intake Data on the estimated per capita intake were derived from surveys in the USA and Europe only (Table 2). The estimated total daily per capita intake of all aliphatic lactones from use as flavouring agents is <5.3 mg/person in the USA (NAS, 1987) or 30.3 mg/person in Europe (IOFI, 1995). In the USA, four substances, the gamma-decalactone (0.36 mg/person per day) and delta-decalactone (1.87 mg/person per day) and the gamma-dodecalactone (0.11 mg/person per day) and delta-dodecalactone (1.14 mg/person per day) account for the majority of the daily per capita intake of aliphatic lactones used as flavour ingredients (NAS, 1987). In Europe, gamma-decalactone (8.4 mg/person per day) and delta-dodecalactone (6.8 mg/person per day) account for two-thirds of the daily per capita intake of lactones in Europe (IOFI, 1987). The four lactones that contain alpha,ß-unsaturation (nos. 21, 23, 24 and 26) and the two that are hydroxyfuranones (Nos. 34 and 35) have very low estimated total daily per capita intakes in both the USA (NAS, 1987) and in Europe (IOFI, 1995). The combined estimated per capita intakes of these six substances from food use is <9 µg/person in the USA and 27 µg/person in Europe. The majority of the aliphatic lactones have been reported to occur naturally in traditional foods. The four aliphatic lactones having the highest usage as flavouring substances (gamma- and delta-decalactone and gamma- and delta-dodecalactone) are also ubiquitous in food, occurring mainly in fruits, berries, alcoholic beverages, meats, and dairy products (Engel et al., 1989; Mosandl et al., 1992; Maarse et al., 1994). 1.3 Absorption, metabolism and elimination Lactones are generally formed by acid-catalysed intramolecular cyclization of hydroxycarboxylic acids. In an aqueous environment, a pH-dependent equilibrium is established between the open-chain hydroxycarboxylate anion and the lactone ring. In basic media, such as blood, the open-chain hydroxycarboxylate anion is favoured while in acidic media, such as urine, the lactone ring is favoured. Both the aliphatic lactones and the ring-opened hydroxycarboxylic acids can be absorbed from the gastrointestinal tract. The aliphatic lactones in this group can be divided into three sub-groups on the basis of their predicted metabolism, namely, lactones derived from saturated linear and branched-chain hydroxycarboxylic acids, lactones containing alpha,ß-unsaturation, and two hydroxyfuranones. The metabolism of members of each of these sub-groups is discussed below. Table 2. Aliphatic lactones used as flavouring agents Substance CAS No. Annual production Estimated per capita Estimated dietary volumes, USA/Europe1 intake USA/Europe2 intake USA/Europe2 (kg) (µg/day) (µg/kg per day) 4-Hydroxybutyric acid lactone 96-48-0 500/880 100/130 1.60/2.10 (gamma-Butyrolactone) gamma-Valerolactone 108-29-2 300/1000 57/140 0.95/2.30 gamma-Hexalactone 695-06-7 100/1300 19/190 0.32/3.10 delta-Hexalactone 823-22-3 13/2600 2.5/380 0.04/6.20 gamma-Heptalactone 105-21-5 220/1400 41/190 0.70/3.20 gamma-Octalactone 104-50-7 480/3500 90/490 1.50/8.20 delta-Octalactone 698-76-0 90/1900 17/270 0.30/4.50 gamma-Nonalactone 104-61-0 2500/8400 470/1200 7.80/20 Hydroxynonanoic acid 3301-94-8 60/1100 11/150 0.20/2.6 delta-lactone gamma-Decalactone 706-14-9 1900/13000 370/1800 6.0/30 delta-Decalactone 705-86-2 9800/59000 1900/8400 31/140 epsilon-Decalactone 5579-78-2 0.01/0.1 0/0.01 0.0/0.0002 gamma-Undecalactone 104-67-6 2900/10000 550/1400 9.1/24 5-Hydroxyundecanoic acid 710-04-3 930/2500 180/350 2.9/5.9 delta-lactone gamma-Dodecalactone 2305-05-7 600/1600 110/220 1.9/3.7 delta-Dodecalactone 713-95-1 6000/48000 1140/6800 19/113 epsilon-Dodecalactone 16429-21-3 0.9/0.1 0.17/0.01 0.003/0.0002 delta-Tetradecalactone 2521-22-4 13/870 2.5/120 0.04/2.0 omega-Pentadecalactone 106-02-5 270/600 51/84 0.86/1.40 4-Hydroxy-3-pentenoic acid 591-12-8 25/2500 4.8/360 0.08/5.9 lactone 5-Hydroxy-2-decenoic acid 54814-64-1 0.5/80 0.10/12 0.002/0.20 delta-lactone 5-Hydroxy-7-decenoic acid 25524-95-2 0.5/1.8 0.10/0.26 0.002/0.004 delta-lactone 5-Hydroxy-2,4-decadienoic 27593-23-3 0.5/2.3 0.10/0.33 0.002/0.01 acid delta-lactone Table 2. Continued... Substance CAS No. Annual production Estimated per capita Estimated dietary volumes, USA/Europe1 intake USA/Europe2 intake USA/Europe2 (kg) (µg/day) (µg/kg per day) Mixture of 5-Hydroxy-2- 85085-23-3 11/- 2/0 0.03/0.0 decenoic acid delta-lactone, 5-Hydroxy-2- dodecenoic acid delta-lactone, and 5-Hydroxy- 2-tetradecenoic acid delta-lactone 5-Hydroxy-8-undecenoic acid 68959-28-4 45/0.1 8.6/0.01 0.14/0.0002 delta-lactone 5-Hydroxy-2-dodecenoic acid 16400-72-9 45/- 8.6/0 0.14//0.0 delta-lactone 1,4-Dodec-6-enolactone 18679-18-0 45/0.1 8.6/0.01 0.14/0.0002 omega-6-Hexadecenlactone 7779-50-2 0.5/42 0.10/6 0.0016/0.10 4,4-Dibutyl-gamma-butyrolactone 7774-47-2 0.5/1 0.10/0.14 0.002/0.002 3-Heptyldihydro-5-methyl- 40923-64-6 0.5/0.3 0.1/0.04 0.002/0.001 2(3H)-furanone 4-Hydroxy-3-methyloctanoic acid 39212-23-2 45/- 8.6/0 0.14/0.0 gamma-lactone 6-Hydroxy-3,7- dimethyloctanoic 499-54-7 0.01/0.1 0/0.1 0.0/0.0002 acid lactone gamma-Methyldecalactone 7011-83-8 220/- 43/0 0.71/0.0 5-Ethyl-3-hydroxy-4-methyl- 698-10-2 32/93 6.1/13 0.10/0.22 2(5H)-furanone 4,5-Dimethyl-3-hydroxy-2,5- 28664-35-9 0.5/15 0.1/2.1 0.002/0.04 dihydrofuran-2-one Total 27000/160000 5200/30000 86/505 1 National Academy of Science (NAS, 1987); International Organization of the Flavouring Industry (IOFI, 1995). 2 Per capita intake (µg/day) calculated as follows: [(annual usage, kg) x (1 x 109mg/kg) / (population x 0.6 x 365 days)], where population (10% "eaters only") = 24 x 106 for the USA and 32 x 106 for Europe; 0.6 represents the assumption that only 60% of the flavour annual usage was reported in the survey. Intake (µg/kg bw per day) calculated as follows: [(µg/day)/bw], where bw = 60 kg. 1.3.1 Lactones derived from linear saturated 5-hydroxycarboxylic acids Linear saturated 5-hydroxycarboxylic acids (formed from delta-lactones) are converted, via acetyl coenzyme A, to hydroxythioesters which then undergo ß-oxidation and cleavage to yield an acetyl CoA fragment and a new ß-hydroxythioester reduced by 2 carbons. Even-numbered carbon acids continue to be oxidized and cleaved to yield acetyl CoA while odd-numbered carbon acids yield acetyl CoA and propionyl CoA. Acetyl CoA enters the citric acid cycle directly while propionyl CoA is transformed into succinyl CoA, which then enters the citric acid cycle (Voet & Voet, 1990). 1.3.2 Lactones derived from linear saturated 4- or 6-hydroxycarboxylic acids Linear saturated 4- or 6-hydroxycarboxylic acids (formed from gamma- or epsilon-lactones) participate in the same pathway; however, loss of an acetyl CoA fragment produces an alpha-hydroxythioester which undergoes alpha-oxidation and alpha-decarboxylation to yield a linear carboxylic acid and eventually carbon dioxide. gamma-Butyrolactone, the only lactone in this group formed from a primary alcohol, may participate in an alternative oxidation pathway. Oxidation of gamma-butyrolactone to succinate by alcohol dehydrogenase and succinic semialdehyde dehydrogenase occurs primarily in the liver (Jakoby & Scott, 1959). Succinate then participates in the citric acid cycle (Walkenstein et al., 1964; Möhler et al., 1976; Lee, 1977; Doherty & Roth, 1978). 1.3.3 Lactones derived from linear hydroxycarboxylic acids containing unsaturation If the lactone is formed from a linear hydroxycarboxylic acid containing unsaturation, cleavage of acetyl CoA units continues along the carbon chain until the position of unsaturation is reached. If the unsaturation begins at an odd-numbered carbon, acetyl CoA fragmentation will eventually yield a 3-enoyl CoA which is converted to the trans-Delta2-enoyl CoA before entering the fatty acid pathway. If unsaturation begins at an even-numbered carbon, acetyl CoA fragmentation yields a Delta2-enoyl CoA product which is a substrate for further fatty acid oxidation. If the stereochemistry of the double bond is cis, hydration yields (R)-3-hydroxyacyl CoA which is isomerized to (S)-3-hydroxyacyl CoA by 3-hydroxyacyl CoA epimerase prior to entering into normal fatty acid metabolism (Voet & Voet, 1990). 1.3.4 Lactones derived from branched-chain hydroxycarboxylic acids For branched-chain hydroxycarboxylic acids, the principal metabolic pathways utilised for detoxication are influenced by the chain length and the position and size of alkyl substituents. Short-chain (<C6) branched aliphatic hydroxycarboxylic acids may be excreted unchanged as the glucuronic acid conjugate, or undergo alpha- or ß-oxidation followed by cleavage and complete metabolism to CO2 via the fatty acid pathway and the tricarboxylic acid cycle (Williams, 1959: Voet & Voet, 1990). Alternatively, as chain length, substitution and lipophilicity increase, the hydroxycarboxylic acid may undergo a combination of omega-, omega-1 and ß-oxidation to yield polar hydroxyacid, ketoacid and hydroxydiacid metabolites which are excreted as the glucuronic acid or sulfate conjugates in the urine and, to a lesser extent, in the faeces (Diliberto et al., 1994). 1.3.5 Lactones containing alpha,ß-unsaturation For the four substances containing alpha,ß-unsaturation (nos. 246, 245, 5-hydroxy-2-dodecenoic acid delta-lactone, and a mixture of 5- hydroxy-2-decenoic acid delta-lactone, 5-hydroxy-2-dodecenoic acid delta-lactone, and 5-hydroxy-2-tetradecenoic acid delta-lactone) there is no direct evidence of hydrolysis available for these substances. While hydrolysis to the corresponding ring-opened alpha,ß-unsaturated hydroxycarboxylic acids may occur, there is no information available on the four substances considered to predict that this is the major route of metabolism (Koppel & Tenczer, 1991). If hydrolysed to the corresponding ring-opened form, condensation of the alpha,ß-unsaturated hydroxycarboxylic acid with acetyl CoA would yield a Delta2-enoyl CoA product which is a substrate in the fatty acid pathway. Since the stereochemistry of the double bond in a lactone is cis, hydration would yield (R)-3-hydroxyacyl CoA which is then isomerized to (S)-3-hydroxyacyl CoA by 3-hydroxyacyl CoA epimerase prior to entering into normal fatty acid metabolism (Voet & Voet, 1990). Alternatively, the lactones containing alpha,ß-unsaturation may conjugate with glutathione and be excreted as the cysteine or mercapturic acid. Data for two structurally related alpha,ß-unsaturated lactones provide evidence for the direct conjugation pathway. Liver glutathione (GSH) levels were significantly reduced (11% of control values after 30 minutes; 26% after 2 hours) following an intraperitoneal injection of 5-hydroxy-2-hexenoic acid delta-lactone (parasorbic acid lactone) to rats (Boyland & Chasseaud, 1970). In vitro, the reaction is catalysed by glutathione-S-transferase (Chasseaud, 1979). The relative rate of reaction with GSH is faster for 5-hydroxy-2-hexenoic acid delta-lactone compared to a series of aromatic (coumarin and dihydrocoumarin) and saturated (e.g., (-valerolactone) lactones. The reaction slowed in the presence of liver microsomes obtained from male Wistar rats or humans which suggests that cytochrome P-450 monooxygenase is not required for the GSH conjugation. Conversely, 5-hydroxy-2,4-pentadienoic acid delta-lactone did not react directly with GSH, but did deplete GSH in the presence of liver microsomes, which suggests that an oxidized metabolite of the lactone may be conjugated with GSH (Fry et al., 1993). 1.3.6 Hydroxyfuranones For the two hydroxyfuranones (Nos. 222 & 243), there is no direct evidence of hydrolysis to the corresponding ring-open compound available. Alternative metabolic pathways are likely and no prediction of metabolic route is possible for these substances. 1.4 Application of the Procedure for the Safety Evaluation of Flavouring Agents Step 1. According to the decision tree structural class classification, twenty-nine members of this group are in Class I, while the four members of the group which contain alpha,ß-unsaturation and the two hydroxyfuranones are in structural Class III. Step 2. The available data indicate that for the 29 lactones in Class I derived from linear and branched-chain hydroxycarboxylic acids, the corresponding aliphatic hydroxycarboxylic acids are metabolized via the fatty acid pathway. For these substances, the evaluation should proceed via the A side of the scheme. For the four lactones in Class III that contain alpha,ß-unsaturation, metabolism may occur either via hydrolysis followed by ß-oxidation or via conjugation with glutathione. There was insufficient information from consideration of these four substances alone to predict the route of metabolism with confidence. The Committee considered that further information on the metabolism of these four substances was required and that they should be evaluated together with other substances containing alpha,ß-unsaturation and that, therefore, their evaluation should be deferred. For the two lactones in Class III that are hydroxyfuranones, no information was available to indicate the route of metabolism and, therefore, the evaluation for these two substances should proceed via the B side of the scheme. Step A3/B3. For the 29 lactones derived from saturated linear and branched-chain hydroxycarboxylic acids in Class I, three lactones, gamma-decalactone (1800 µg/person per day), delta-decalactone (8400 mg/person per day) and delta-dodecalactone (6800 µg/person per day) have intakes equal to or greater than the threshold of concern for Class I (1800 µg/person per day). These substances, therefore, proceed to step A4. For the other 26 lactones of similar structure, the intake levels are below the threshold of concern for Class I and these would not be expected to be of safety concern. For the two hydroxyfuranones in Class III , the estimated levels of intake (13 and 2.4 µg/person per day, respectively) are well below the threshold of concern for structural class III (90 µg/person per day). These substances, therefore, proceed to step B4. Step A4. For the three lactones derived from saturated linear hydroxycarboxylic acids, namely, gamma-decalactone, delta-decalactone, and delta-dodecalactone, none are known to be endogeneous or to be metabolized to endogeneous substances. The safety evaluation of these substances, therefore, proceeds to step A5. Step A5. While adequate studies were not available on which to base a NOEL for the three lactones derived from saturated linear hydroxycarboxylic acids, the following NOELs in 2-year rat studies have been reported for structurally-related lactones: gamma-nonalactone (250 mg/kg bw per day), gamma-undecalactone (250 mg/kg bw per day) and gamma-butyrolactone (112 mg/kg bw per day). A 2-year study in mice with gamma-butyrolactone indicated a NOEL of 262 mg/kg bw per day. The studies on gamma-nonalactone and gamma-undecalactone were considered previously by the Committee and ADIs were established at the eleventh meeting (Annex 1, reference 14). While these studies have not been conducted according to modern standards, the results do not provide cause for concern. These NOELs provide an adequate margin of safety (>1000) for gamma-decalactone, delta-decalactone and delta-dodecalactone and, therefore, these substances would not be expected to be of safety concern. Step B4. In considering the two lactones in Class III, for 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone a 90-day dietary study in rats has been performed in which the NOEL was >1.3 mg/kg bw per day, and for 4,5-dimethyl-3-hydroxy-2,5-dihydrofuran-2-one a 1-year dietary study in rats has been performed in which the NOEL was >46 mg/kg bw per day. These NOELs provide an adequate margin of safety (>1000) for these substances and, therefore, they would not be expected to be of safety concern. Table 1 summarizes the evaluation of the 35 aliphatic lactones using the procedure. 1.5 Consideration of combined intakes Of the 35 aliphatic lactones considered in this evaluation, the 29 lactones derived from linear and branched-chain hydroxycarboxylic acids would be expected to be efficiently metabolized via commonly known biochemical pathways to innocuous substances. In the unlikely event that all of these substances were consumed similtaneously on a daily basis, the estimated daily per capita consumption in Europe and the USA would exceed the human intake threshold for substances in class I, but, in the option of the Committee this would not give rise to perturbations outside the physiological range. For the two hydroxyfuranones whose metabolic route is unknown, their combined intake is very low (15 µg/person per day) compared to the known NOELs for each of these substances, and, in the opinion of the Committee, would not present a safety concern. 1.6 Conclusions In applying Procedure, the Committee concluded that for the four substances that contain alpha,ß-unsaturation, the evaluation should be deferred pending the general consideration of substances containing alpha,ß-unsaturation. The safety evaluation of the two hydroxyfuranones proceeded because of the existence of supporting toxicity studies. The results of the evaluation of the 29 lactones derived from linear and branched-chain hydroxycarboxylic acids and the substances, 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone and 4,5-dimethyl-3-hydroxy- 2,5-dihydrofuran-2-one concluded that the use of these lactones as flavouring substances would not present safety concerns at the current estimated intake levels. In using the Procedure, the Committee noted that all available toxicity data were consistent with the results of the safety evaluation. The ADIs for gamma-nonalactone and gamma-undecalactone were maintained at the present meeting. 2. RELEVANT BACKGROUND INFORMATION 2.1 Toxicological studies 2.1.1 Acute toxicty The results of acute toxicity studies on aliphatic lactones are shown in Table 3. 2.1.2 Short-term and long-term toxicity and carcinogenicity The results of all short-term toxicity studies are shown in Table 4. The results of all long-term toxicity studies are shown in Table 5. Details of the studies which were critical to the safety evaluation of aliphatic lactones are given below. 2.1.2.1 gamma-Butyrolactone Groups of B6C3F1 mice (10/sex/dose level) were administered gamma-butyrolactone in corn oil by gavage at dose levels of 0, 65, 131, 262, 535 or 1050 mg/kg bw per day for 13 weeks. The mean body weight gain and final mean body weight of high dose male mice were lower than those of the controls. For females, the body weight gain and mean body weight were similar to those of the controls. The NOEL was 525 mg/kg bw per day. Table 3. Acute toxicity studies on aliphatic lactones Substance Species Sex1 Route LD50 Reference (mg/kg bw) 4-Hydroxybutyric acid lactone mouse nr1 gavage 1245 Schafer & Bowles, 1985 gamma-Valerolactone rat nr gavage >5000 Moreno, 1978 rabbit nr gavage 2640 Deichmann et al., 1945 gamma -Hexalactone rat nr gavage >5000 Moreno, 1977c delta-Hexalactone rat nr gavage 13,030 Smyth et al., 1969 gamma-Heptalactone rat nr gavage >5000 Moreno, 1977b gamma -Octalactone rat nr gavage >5000 Moreno, 1974 delta-Octalactone rat nr gavage >5000 Levenstein, 1977 gamma -Nonalactone rat M/F gavage 9780 Jenner et al., 1964 rat nr gavage 6600 Moreno, 1972 guinea pig M/F gavage 3440 Jenner et al., 1964 gamma -Decalactone rat nr gavage >5000 Moreno, 1975a delta-Decalactone rat nr gavage >5000 Levenstein, 1975 epsilon-Decalactone mouse M/F gavage 5252 Moran et al., 1980 gamma -Undecalactone rat M/F gavage 18 500 Jenner et al., 1964; Moreno, 1975b 5-Hydroxyundecanoic acid lactone rat nr gavage >5000 Moreno, 1975b gamma -Dodecalactone rat nr gavage >5000 Moreno, 1974 delta-Dodecalactone rat nr gavage >5000 Moreno, 1977a epsilon-Dodecalactone mouse M/F gavage 7898 Moran et al., 1980 omega-Pentadecalactone rat nr gavage >5000 Levenstein, 1974 4-Hydroxy-3-pentenoic acid mouse M/F gavage 2800 Moran et al., 1980 lactone 5-Hydroxy-2,4-decadienoic acid rat M/F gavage 1600-5000 Piccirillo & Hartman, 1980 delta-lactone Mixture of 5-hydroxy-2-decenoic rat M/F gavage 2952 Reagan & Becci, 1984 acid, 5-hydroxy-2-dodecenoic acid, and 5-hydroxy-2- tetradecenoic acid delta-lactones 5-Hydroxy-8-undecenoic acid delta-lactone rat nr gavage 5000 Moreno, 1980 1,4-Dodec-6-enolactone rat M/F gavage >5000 Watanabe & Kinosaki, 1990 omega-6-Hexadecenlactone rat nr gavage >5000 Wohl, 1974 Table 3. Continued... Substance Species Sex1 Route LD50 Reference (mg/kg bw) 6-Hydroxy-3,7-dimethyloctanoic rat M/F gavage 5000 Palanker, 1979 acid lactone gamma -Methyldecalactone rat nr gavage >5000 Moreno, 1976 1 M = male; F = female; nr = not reported Table 4. Short-term toxicity studies on aliphatic lactones Substance Species Route Duration NOEL2 Reference (sex1) (days) (mg/kg bw per day) 4-Hydroxybutyric acid lactone (gamma-Butyrolactone) mouse (M/F) gavage 16 175 NTP, 1992 rat (M/F) gavage 16 300 rat (M/F) gavage 90 225 mouse (M/F) gavage 90 525 gamma-Valerolactone rat diet 90 >50 Hagan et al., 1967 rat diet 90 >49(M) >51.1(F) Oser et al., 1965 gamma-Nonalactone rat diet 90 >62.8(M) >72.5(F) Oser et al., 1965 gamma-Undecalactone rat (M) diet 90 >14.6 Oser et al., 1965 rat (F) 4-Hydroxy-3-pentenoic acid lactone rat (M/F) diet 90 >17.4 Shellenberger, 1971 5-Hydroxy-2,4-decadienoic acid rat (M/F) diet 90 >12.1 Cox et al., 1974 delta-lactone 5-Ethyl-3-hydroxy-4-methyl- rat diet 90 >1.294 Posternak et al., 1969 2(5H)-furanone 4,5-Dimethyl-3-hydroxy-2,5- rat diet 52 weeks >46 Munday & Kirkby, 1973 dihydrofuran-2-one 1 M=male; F=female. 2 A NOEL (no-observed-effect level) given in this table as "greater than" (>) indicates that no adverse effects were observed at the highest dose level in the study. Table 5. Long-term studies on aliphatic lactones Substance Species Route Duration NOEL1 Reference (sex) (months) (mg/kg bw per day) 4-Hydroxybutyric acid lactone rat (M/F) gavage 24 112 NTP, 1992 (gamma-Butyrolactone) mice (M/F) gavage 24 >525 gamma-Nonalatone rat diet 18 - 24 >250 Bär & Griepentrog, 1967 gamma-Undecalactone rat (M/F) diet 18 - 24 >250 Bär & Greipentrog, 1967 1 A NOEL (no-observed-effect level) given in this table as "greater than" (>) indicates that no adverse effects were observed at the highest dose level in the study. Groups of F344/N rats (10/sex/dose level) were administered gamma- butyrolactone (97% pure) in corn oil by gavage at dose levels of 0, 56, 112, 225, 450 or 900 mg/kg bw per day for 13 weeks. The final mean body weight and mean body weight gain were significantly lower than those of the controls at 450 mg/kg bw per day, while the body weights and body weight gains of females at all dose levels were similar to those of the controls. There was an increased incidence of focal inflamation of the nasal mucosa in rats administered gamma-butyrolactone. The NOEL was 225 mg/kg bw per day (NTP, 1992). Groups of B6C3F1 mice (50/sex/dose level) were administered gamma-butyro-lactone in corn oil by gavage at dose levels of 0, 262 or 525 mg/kg bw per day for 2 years. The final mean body weight was, for male mice, 6% lower than that of controls and, for female mice, 14-17% lower than controls. The incidence of proliferative lesions was higher in male mice at the low dose level only, and the incidence of hepatocellular lesions was lower in male mice at both dose levels. The conclusion of this study was that there was equivocal evidence of carcinogenic activity of gamma-butyrolactone in male B6C3F1 mice, based on marginally increased incidences of adrenal medulla pheochromocytomas and hyperplasia in the low-dose group. There was no evidence of carcinogenic activity of gamma-butyrolactone in female B6C3F1 mice given 262 or 525 mg/kg bw per day in corn oil. The NOEL was >525 mg/kg bw per day (NTP, 1992). Groups of F344/N rats (50/sex/dose level) were administered gamma- butyrolactone in corn oil by gavage at dose levels of 0, 112 or 225 mg/kg bw per day for 2 years. The mean body weights of male rats were similar to those of controls throughout the study. The mean body weights of females were 10-20% lower than controls throughout the second year. There was no increased incidence of neoplasms in male or female rats in relation to treatment. The NOEL was 112 mg/kg bw per day (NTP, 1992). 2.1.2.2 gamma-Nonalactone and gamma-undecalactone Groups of rats (10/sex/dose level) were given a diet containing either gamma-nonalactone or gamma-undecalactone at a level of 0, 1000 or 5000 mg/kg diet for 2 years. Animals were observed throughout the study period. No adverse effects were reported. It was noted, however, that the study protocol and the presentation of the results was less than the standard required of current studies. However, the results of the study did not give rise to any safety concerns (Bär & Greipentrog, 1967). 2.1.2.3 5-Ethyl-3-hydroxy-4-methyl-2(5H)-furanone In a 90-day dietary study in rats, groups of Charles River CD rats (10-16/sex) were fed a diet containing 5-ethyl-3-hydroxy-4-methyl- 2(5H)-furanone at dose levels of 1.29 mg/kg bw per day (males) and 1.47 mg/kg bw per day (females) for 90 days. Body weights and food consumption were recorded weekly. Haematological examinations and blood urea were recorded at weeks 7 and 13. At the end of the study period, liver and kidney weights were recorded and a histological examination was performed on a wide range of organs. There were no treatment-related effects on growth, food consumption or efficiency of food utilisation. Haematological parameters were unremarkable. There were no treatment-related effects on organ weights or on organ pathology. The NOEL was >1.29 mg/kg bw per day (males) and >1.47 mg/kg bw per day (females) (Posternak et al., 1969). 2.1.2.4 4,5-Dimethyl-3-hydroxy-2,5-dihydrofuran-2-one In a 52-week dietary study in rats, groups of Wistar rats (24/sex) were fed a flavour cocktail containing 4,5-dimethyl-3-hydroxy-2,5- dihydrofuran-2-one at a dose level of 46 mg/kg bw per day for one year. The animals were examined throughout the study period and body weights measured at regular intervals. Haematological parameters were also examined. At the end of the study period, the animals were sacrificed, organs weights recorded and tissues taken for histological examination. There were no treatment-related effects on body weight, haematological parameters or organ weights. Histological examination of a variety of tissues did not reveal any treatment-related effects. The NOEL was >46 mg/kg bw per day (Munday & Kirkby, 1973). 2.1.3 Genotoxicity The results of genotoxicity studies are shown in Table 6. 2.1.4 Other relevant study In a teratogenicity study, Sprague-Dawley rats were given gamma-butyrolactone at dose levels up to 500 mg/kg per day on days 6-15 of gestation. There was no evidence of embryotoxicity or malformations in the fetuses (Kronevi et al., 1988). Table 6. Genotoxicity studies on aliphatic lactones Substance Test system Test object Dose Result Reference 4-Hydroxybutyric acid lactone (gamma-Butyrolactone) Gene mutation S. typhimurium 0.1-50 µmoles/plate1 negative Loquet et al., 1981 TA1535, TA98, TA100 Gene mutation S. typhimurium 0.013-1.3 mmol1 negative Aeschbacher et al., 1989 TA98, TA100, TA102 Gene mutation S. typhimurium TA98, 100-10 000 µg/plate1 negative NTP, 1992 TA100, TA1535, TA1537 Gene mutation S. typhimurium TA98, 0-10 000 µg/plate1 negative Haworth et al., 1983 TA100, TA1535, TA1537 Gene mutation S. typhimurium 5000 µg/plate1 negative MacDonald, 1981 TA98, TA100, TA1537 Gene mutation S. typhimurium TA98, 500 µg/plate1 negative Garner et al., 1981 TA100, TA1535, TA1537 Gene mutation S. typhimurium TA100, 4-2500 µg/plate1 negative Trueman, 1981 TA1535, TA1537, TA1538 Gene mutation S. typhimurium TA92, 0.2-2000 µg/plate1 negative Brooks & Dean, 1981 TA98, TA100, TA1537, TA1538, TA1535 Gene mutation S. typhimurium 1000 µg/plate negative Baker & Bonin, 1981 TA98, TA100, TA1535, TA1537, TA1538 4-Hydroxybutyric acid lactone (gamma-Butyrolactone) Gene mutation S. typhimurium 500 µg/plate negative Rowland & Severn, 1981 TA98, TA100, TA1535, TA1537, TA1538 Gene mutation S. typhimurium 500 µg/plate1 negative Simmon & Shepard, 1981 TA98, TA100, TA1535, TA1537, TA1538 Gene mutation S. typhimurium not reported1 negative Nagao & Takahashi, 1981 TA98, TA100, TA1537 Gene mutation S. typhimurium TA98, TA100, TA1535, 10-10 000 µg/plate1 negative Richold & Jones, 1981 TA1537, TA1538 Table 6. Continued... Substance Test system Test object Dose Result Reference Gene mutation S. typhimurium 500 - 1000 µg/ml negative Ichinotsubo et al., 1981 TA98, TA100 Fluctuation S. typhimurium 500 µg/ml1 negative Hubbard et al., 1981 Test TA98, TA100 Forward mutation S. typhimurium TM677 1000 µg/ml2 negative Skopeck et al., 1981 Microtiter S. typhimurium TA98, 10-1000 µg/ml1 negative Gatehouse, 1981 fluctuation TA1535, TA1537 Gene mutation E. coli 500 µg/plate1 negative Venitt & Crofton-Sleigh, 1981 Gene mutation E. coli SA500 250 µg/plate lethal Dambly et al., 1981 Differential E. coli WP2, WP67 & M871 2500 µg/plate1 negative Green, 1981 killing test Differential E. coli P2,WP67 & CM871 1000 µg/ml1 negative Tweats, 1981 killing assay 4-Hydroxybutyric acid lactone (gamma-Butyrolactone) Microtiter E. coli WP2 uvrA 10-1000 µg/ml1 negative Gatehouse, 1981 fluctuation Gene mutation E. coli WP2 uvrA not reported1 negative Matsushima et al., 1981 pKM102 Sister chromatid Chinese hamster 148-1480 µg/ml3 negative NTP, 1992 exchange ovary cells 494-4940 µg/ml2 positive 3010-5010 µg/ml2 (weak)6 positive6 Chromosome Chinese hamster 500-4990 µg/ml3 negative NTP, 1992 aberration ovary cells 400-3990 µg/ml2 positive6 2210-2950 µg/ml2 positive6 ADP-ribosyl Human FL cells 10-3 to 10-7 mol/l negative Yingnian et al., 1990 transf. act. Polyploidy Human leucocyte 0.7 mmol/litre negative Withers, 1966 Gene Mutation Schizosaccha- 20 µg/ml1 negative Loprieno, 1981 romyces pombe Mitotic crossing- Saccharomyces 1000 µg/ml negative Kassinova et al., 1981 over cervevisiae Table 6. Continued... Substance Test system Test object Dose Result Reference Rec assay Bacillus subtilis 20 µl/disc4 positive6 Kada, 1981 H17, M45 Unscheduled DNA Human HeLa 0.1-100 µg/ml1 negative Martin & McDermid, 1981 synthesis S3 cells Mitotic gene Saccharomyces cerevisiae 750 µg/ml1 negative Sharp & Parry, 1981 conversion 4-Hydroxybutyric acid lactone (gamma-Butyrolactone) Clastogenic Rat liver cell line RL1 250 µg/ml negative Dean, 1981 activity Mammalian cell BHK21C1B/HRC1 cells 2500 µg/ml1 ?5 Daniel & Dehnel, 1981 transformation Mammalian cell BHK-21 hamster 250 µg/ml2 positive Styles, 1981 transformation kidney cells Degranulation Rat 25 mg/ml positive Fey et al., 1981 assay Cell growth Saccharomyces cerevisiae 750 µg/ml1 negative Sharp & Parry, 1981 inhibition Haploid yeast Saccharomyces cerevisiae 222 µg/ml1 ?5 Mehta & von Borstel, 1981 reversion DNA pol I E. coli W3110 & P3478 330 µg/plate positive3 Rosenkranz et al., 1981 inhibition negative2 Sperm head (CBA x Balb/c)F1 mice 0.1-1.0 mg/kg/ negative Topham, 1981 abnormality day ip (5 days) Sex-linked Drosophila melanogaster 20 000 or 28 000 mg/ negative Foureman et al., 1994 recessive test kg (diet) or 15 000 mg/kg (injection) Micronucleus B6C3F1 mice 0.7 mg/kg/day ip negative Katz et al., 1981 test (2 days) Micronucleus B6C3F1 mice 80% LD50 ip negative Salamone et al., 1981 test (2 days) Micronucleus CD-1 mice 0.11-0.44 mg/kg/day negative Tsuchimoto & Matter, test ip (2 days) 1981 Table 6. Continued... Substance Test system Test object Dose Result Reference gamma-Heptalactone Gene mutation S. typhimurium TA98, 100 000 µg/plate1 negative Heck et al., 1989 TA100, TA1535, TA1537, TA1538 UDS Rat hepatocytes 3000 µg/ml negative Heck et al., 1989 gamma-Nonalactone Gene mutation S. typhimurium TA98, 37 500 µg/plate1 negative Heck et al., 1989 TA100, TA1535, TA1537, TA1538 Gene mutation Human leucocytes 0.7 mM positive Withers, 1966 Gene mutation Mouse lymphoma L5178y 1000 µg/ml negative3 Heck et al., 1989 TK+/- 400 µg/ml positive2 UDS Rat hepatocytes 500 µg/ml negative Heck et al., 1989 Gene mutation E.coli WP2 uvrA 0.2-1.6 mg/plate negative Yoo, 1986 Rec-assay Bacillus subtilis 20 µl/disk positive Yoo, 1986 gamma-Undecalactone Gene mutation S. typhimurium TA92, 5000 µg/plate1 negative Ishidate et al., 1984 TA94, TA98, TA100, TA1535, TA1537 Gene mutation S. typhimurium TA97, 100 µg/plate negative Fujita & Sasaki, 1987 TA102 Chromosome Chinese hamster 500 µg/ml negative Ishidate et al., 1984 aberration fibroblast Rec-assay Bacillus subtilis H17 19 µg/disc negative Oda et al., 1978 & M45 Rec-assay Bacillus subtilis H17 10 µl/disc positive Yoo, 1986 & M45 Rec-assay Bacillus subtilis H17 10 µl/disc positive2 Kuroda et al., 1984 & M45 negative1 Mouse 2-6 ddY male mice 250-2000 mg/kg/day negative Hayashi et al., 1988 micronucleus ip (2 days) 5-Hydroxyundecanoic acid delta-lactone Rec-assay Bacillus subtilis H17 19 µg/disc negative Oda et al., 1978 & M45 Table 6. Continued... Substance Test system Test object Dose Result Reference omega-Pentadecalactone Gene mutation S. typhimurium TA98, 50 µmol/plate1 negative Aeschbacher et al., 1989 TA100, TA102 Chromosome Human leukocytes 70 µmole/ml negative Withers, 1966 aberration 1,4-Dodec-6-enolactone Gene mutation S. typhimurium TA98, 500 µg/plate1 negative Watanabe & Kinosaki, 1990 TA100, TA1535, TA1537 Rec-assay E. coli WP2 uvrA 500 µg/plate1 negative Watanabe & Kinosaki, 1990 1 With and without rat liver S-9 metablic activation 2 With rat liver S-9 metabolic activation 3 Without rat liver S-9 metabolic activation 4 With yellowtail S-9 metabolic activation 5 Ambiguous result 6 These positive results with gamma-butyrolacone were only seen at relatively high dose levels and may be artifactual. There was no evidence of positive genotoxicity in in vivo studies. Overall, the genotoxicity of gamma-butyrolactone was considered to be negative. 3. 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See Also: Toxicological Abbreviations