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        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, -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 g 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
                                     (g/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
                                     (g/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--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 ,-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,-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 g/person
    in the USA and 27 g/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,-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)                    (g/day)                (g/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)                    (g/day)                (g/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 (g/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 (g/kg bw per day) calculated as follows: [(g/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 -oxidation and cleavage to yield
    an acetyl CoA fragment and a new -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; Mhler  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 -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 -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,-unsaturation

        For the four substances containing alpha,-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,-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,-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,-unsaturation may
    conjugate with glutathione and be excreted as the cysteine or
    mercapturic acid. Data for two structurally related
    alpha,-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,-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,-unsaturation, metabolism
    may occur either via hydrolysis followed by -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,-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 g/person per day), delta-decalactone (8400
    mg/person per day) and delta-dodecalactone (6800 g/person per day)
    have intakes equal to or greater than the threshold of concern for
    Class I (1800 g/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 g/person
    per day, respectively) are well below the threshold of concern for
    structural class III (90 g/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 g/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,-unsaturation, the evaluation should be
    deferred pending the general consideration of substances containing
    alpha,-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            Br & Griepentrog, 1967
    gamma-Undecalactone               rat (M/F)          diet        18 - 24             >250            Br & 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 (Br & 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 moles/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 g/plate1   negative      NTP, 1992
                                                        TA100, TA1535, TA1537
                                      Gene mutation     S. typhimurium TA98,      0-10 000 g/plate1     negative      Haworth et al., 1983
                                                        TA100, TA1535, TA1537
                                      Gene mutation     S. typhimurium            5000 g/plate1         negative      MacDonald, 1981
                                                        TA98, TA100, TA1537
                                      Gene mutation     S. typhimurium TA98,      500 g/plate1          negative      Garner et al., 1981
                                                        TA100, TA1535, TA1537
                                      Gene mutation     S. typhimurium TA100,     4-2500 g/plate1       negative      Trueman, 1981
                                                        TA1535, TA1537, TA1538
                                      Gene mutation     S. typhimurium TA92,      0.2-2000 g/plate1     negative      Brooks & Dean, 1981
                                                        TA98, TA100, TA1537,
                                                        TA1538, TA1535
                                      Gene mutation     S. typhimurium            1000 g/plate          negative      Baker & Bonin, 1981
                                                        TA98, TA100, TA1535,
                                                        TA1537, TA1538

    4-Hydroxybutyric acid lactone
    (gamma-Butyrolactone)             Gene mutation     S. typhimurium            500 g/plate           negative      Rowland & Severn, 1981
                                                        TA98, TA100, TA1535,
                                                        TA1537, TA1538
                                      Gene mutation     S. typhimurium            500 g/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 g/plate1    negative      Richold & Jones, 1981
                                                        TA1537, TA1538

    Table 6.  Continued...

                                                                                                                                               
    Substance                         Test system       Test object               Dose                   Result        Reference
                                                                                                                                               

                                      Gene mutation     S. typhimurium            500 - 1000 g/ml       negative      Ichinotsubo et al., 1981
                                                        TA98, TA100
                                      Fluctuation       S. typhimurium            500 g/ml1             negative      Hubbard et al., 1981
                                      Test              TA98, TA100
                                      Forward mutation  S. typhimurium TM677      1000 g/ml2            negative      Skopeck et al., 1981
                                      Microtiter        S. typhimurium TA98,      10-1000 g/ml1         negative      Gatehouse, 1981
                                      fluctuation       TA1535, TA1537            
                                      Gene mutation     E. coli                   500 g/plate1          negative      Venitt & Crofton-Sleigh,
                                                                                                                       1981
                                      Gene mutation     E. coli SA500             250 g/plate           lethal        Dambly et al., 1981
                                      Differential      E. coli WP2, WP67 & M871  2500 g/plate1         negative      Green, 1981
                                      killing test
                                      Differential      E. coli P2,WP67 & CM871   1000 g/ml1            negative      Tweats, 1981
                                      killing assay

    4-Hydroxybutyric acid lactone
    (gamma-Butyrolactone)             Microtiter        E. coli WP2 uvrA          10-1000 g/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 g/ml3        negative      NTP, 1992
                                      exchange          ovary cells               494-4940 g/ml2        positive
                                                                                  3010-5010 g/ml2       (weak)6
                                                                                                         positive6
                                      Chromosome        Chinese hamster           500-4990 g/ml3        negative      NTP, 1992
                                      aberration        ovary cells               400-3990 g/ml2        positive6
                                                                                  2210-2950 g/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 g/ml1              negative      Loprieno, 1981
                                                        romyces pombe             
                                      Mitotic crossing- Saccharomyces             1000 g/ml             negative      Kassinova et al., 1981
                                      over              cervevisiae

    Table 6.  Continued...

                                                                                                                                               
    Substance                         Test system       Test object               Dose                   Result        Reference
                                                                                                                                               

                                      Rec assay         Bacillus subtilis         20 l/disc4            positive6     Kada, 1981
                                                         H17, M45
                                      Unscheduled DNA   Human HeLa                0.1-100 g/ml1         negative      Martin & McDermid, 1981
                                      synthesis         S3 cells                  
                                      Mitotic gene      Saccharomyces cerevisiae  750 g/ml1             negative      Sharp & Parry, 1981
                                      conversion

    4-Hydroxybutyric acid lactone
    (gamma-Butyrolactone)             Clastogenic       Rat liver cell line RL1   250 g/ml              negative      Dean, 1981
                                      activity
                                      Mammalian cell    BHK21C1B/HRC1 cells       2500 g/ml1               ?5         Daniel & Dehnel, 1981
                                      transformation
                                      Mammalian cell    BHK-21 hamster            250 g/ml2             positive      Styles, 1981
                                      transformation    kidney cells
                                      Degranulation     Rat                       25 mg/ml               positive      Fey et al., 1981
                                      assay
                                      Cell growth       Saccharomyces cerevisiae  750 g/ml1             negative      Sharp & Parry, 1981
                                      inhibition
                                      Haploid yeast     Saccharomyces cerevisiae  222 g/ml1                ?5         Mehta & von Borstel, 1981
                                      reversion
                                      DNA pol I         E. coli W3110 & P3478     330 g/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 g/plate1      negative      Heck et al., 1989
                                                        TA100, TA1535, TA1537,
                                                        TA1538
                                      UDS               Rat hepatocytes           3000 g/ml             negative      Heck et al., 1989

    gamma-Nonalactone                 Gene mutation     S. typhimurium TA98,      37 500 g/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 g/ml             negative3     Heck et al., 1989
                                                        TK+/-                     400 g/ml              positive2
                                      UDS               Rat hepatocytes           500 g/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 l/disk             positive      Yoo, 1986

    gamma-Undecalactone               Gene mutation     S. typhimurium TA92,      5000 g/plate1         negative      Ishidate et al., 1984
                                                        TA94, TA98, TA100,
                                                        TA1535, TA1537
                                      Gene mutation     S. typhimurium TA97,      100 g/plate           negative      Fujita & Sasaki, 1987
                                                        TA102
                                      Chromosome        Chinese hamster           500 g/ml              negative      Ishidate et al., 1984
                                      aberration        fibroblast
                                      Rec-assay         Bacillus subtilis H17     19 g/disc             negative      Oda et al., 1978
                                                        & M45
                                      Rec-assay         Bacillus subtilis H17     10 l/disc             positive      Yoo, 1986
                                                        & M45
                                      Rec-assay         Bacillus subtilis H17     10 l/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 g/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 mol/plate1         negative      Aeschbacher et al., 1989
                                                        TA100, TA102
                                      Chromosome        Human leukocytes          70 mole/ml            negative      Withers, 1966
                                      aberration

    1,4-Dodec-6-enolactone            Gene mutation     S. typhimurium TA98,      500 g/plate1          negative      Watanabe & Kinosaki, 1990
                                                        TA100, TA1535, TA1537
                                      Rec-assay         E. coli WP2 uvrA          500 g/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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    See Also:
       Toxicological Abbreviations