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.
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