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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION





    SAFETY EVALUATION OF CERTAIN 
    FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES: 42





    Prepared by the Fifty-first meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)





    World Health Organization, Geneva, 1999
    IPCS - International Programme on Chemical Safety

    LINEAR AND BRANCHED-CHAIN ALIPHATIC, UNSATURATED, UNCONJUGATED ALCOHOLS, 
    ALDEHYDES, ACIDS, AND RELATED ESTERS

    First draft prepared by
    Dr Antonia Mattia
    Division of Product Policy, Office of Premarket Approval (HFS-206)
    Center for Food Safety and Applied Nutrition
    US Food and Drug Administration
    Washington DC, United States

          Evaluation 
              Introduction 
              Estimated daily  per capita intake 
              Absorption, metabolism, and elimination 
              Application of the Procedure for the Safety 
                   Evaluation of Flavouring Substances 
              Consideration of combined intakes from use as 
                   flavouring agents
              Conclusions 
          Relevant background information 
              Explanation 
              Intake 
              Biological data 
                   Absorption and metabolism 
                   Toxicological studies 
                        Acute toxicity 
                        Short-term and long-term studies of toxicity
                             and carcinogenicity  
                        Genotoxicity 
                        Other relevant studies 
          References 


    1.  EVALUATION

    1.1  Introduction

         The Committee evaluated 42 flavouring substances that include
    linear and branched-chain aliphatic unsaturated and unconjugated
    alcohols, aldehydes, acids, and related esters (Table 1) using the
    Procedure for the Safety Evaluation of Flavouring Agents (Figure 1, p.
    222, and Annex 1, reference 131). The Committee had evaluated one
    member of the group, oleic acid, previously at its thirty-third
    meeting (Annex 1, reference 83), when ADIs 'not specified' were
    allocated to the calcium, potassium, and sodium salts of oleic acid.
    At that meeting, the Committee noted that the safety of fatty acids,
    including oleic acid, is based on knowledge about their metabolism and
    excretion and on their occurrence in edible fats and oils that have a
    long history of use as foods or food components. 

    1.2  Estimated daily per capita intake

         The total annual volume of the 42 linear and branched-chain
    aliphatic unsaturated primary alcohols and unconjugated aldehydes,
    carboxylic acids, and related esters is approximately 39 000 kg in
    Europe (International Organization of the Flavor Industry, 1995) and
    13 000 kg in the United States (National Academy of Sciences, 1987).
    Three substances in the group,  cis-3-hexen-1-ol (No. 315), linoleic
    acid (No. 332), and oleic acid (No. 330), account for about 96% of the
    total annual volume in Europe. Four substances in the group,
     cis-3-hexen-1-ol, oleic acid, 2,6-dimethyl-5-heptenal (No. 349), and
    hexyl 2-methyl-3&4-pentenoate (No. 352) account for about 91% of the
    total annual volume in the United States. On the basis of the reported
    annual volume in Europe, the total estimated  per capita intake is
    4300 g/person of  cis-3-hexen-1-ol (71 g/kg bw per day), 130
    g/person of linoleic acid (2 g/kg bw per day), and 970 g/person of
    oleic acid (16 g/kg bw per day). On the basis of the reported annual
    volume in the United States, the total estimated  per capita intake
    is 440 g/person of oleic acid (7 g/kg bw per day), 250 g/person of
    2,6-dimethyl-5-heptenal (4 g/kg bw per day), and 520 g per person of
    hexyl-2-methyl-3&4-pentenoate (9 g/kg bw per day).

         Twenty-seven of the 42 unsaturated aliphatic primary alcohols and
    unconjugated aldehydes, acids, and related esters in this group are
    reported to occur in a wide variety of foods, including fruits and
    vegetables, dairy products, fish, and alcoholic beverages (Maarse et
    al., 1994). Oleic and linoleic acids are some of the commonest
    unsaturated fatty acids found in vegetable oils and animal fats, often
    constituting more than 50% of the total fatty acid concentration.

    1.3  Absorption, metabolism, and elimination

         The metabolism of flavouring agents in this group involves common
    pathways of intermediary metabolism, which are discussed in the
    section 'General aspects of metabolism' in "Safety Evaluations of
    Groups of Related Substances by the Procedure for the Safety
    Evaluation of Flavouring Agents".

    1.4  Application of the Procedure for the Safety Evaluation of 
    Flavouring Agents

    Step A1.  All of the linear and branched-chain aliphatic unsaturated
              primary alcohols and unconjugated aldehydes, acids, and
              related esters are classified in structural class I (Cramer
              et al., 1978).

    Step A2.  The metabolic fates of the linear and branched-chain
              unsaturated primary alcohols and unconjugated aldehydes,
              carboxylic acids, and related esters in this group of
              flavouring agents can (with one exception) be predicted
              readily because of their close structural relationship to
              endogenous substrates and the broad substrate specificities
              of the relevant enzymes. The prediction that the metabolites

              would be innocuous was supported by available data on the
              toxicity of members of this group. The Committee considered
              that inadequate data were available to predict the
              metabolism of ethyl 2-methyl-3,4-pentadienoate (No. 353),
              because of its terminal diene. 

                   At current levels of intake, the remaining 41
              substances in this group would not be expected to saturate
              the metabolic pathways, and all of them are predicted to be
              metabolized to innocuous products (see section 4.2). The
              evaluation of these 41 substances therefore proceeds via the
              left side ('A') of the evaluation scheme (see Steps A3-A5).

                   The evaluation of ethyl 2-methyl-3,4-pentadienoate (No.
              353) proceeds via the right side ('B') of the evaluation
              scheme (see Steps B3 and B4).

    Step A3.  At current levels of intake, all 41 substances were
              predicted to be metabolized to innocuous products and,
              except for  cis-3-hexen-1-ol, the intake is below the human
              intake threshold for class I substances (1800 g/person per
              day). The 40 substances were considered to be of no safety
              concern at this step on the basis of their structural class
              and low levels of estimated intake. As the daily  per 
               capita intakes of  cis-3-hexen-1-ol in Europe and the
              United States are 4300 g/person per day (71 g/kg bw per
              day) and 1100 g/person per day (18 g/kg bw per day),
              respectively, intake in Europe exceeds the human intake
              threshold for class I substances.

    Step A4.   cis-3-Hexen-1-ol is not endogenous in humans.

    Step A5.  A NOEL of 120-150 mg/kg bw per day was reported for
               cis-3-hexen-1-ol in a 98-day study in rats (Gaunt et al.,
              1969). A safety margin of > 1000 exists between this NOEL
              and the daily  per capita intakes of 71 and 18 g/kg bw per
              day from use of  cis-3-hexen-1-ol as a flavouring agent in
              Europe and the United States, respectively. 

    Step B3.  The daily  per capita intakes of ethyl
              2-methyl-3,4-pentadienoate in Europe and the United States
              are 0.01 g/person per day (< 0.001 g/kg bw per day) and
              10 g/person per day (0.2 g/kg bw per day), respectively;
              therefore, intake does not exceed the human intake threshold
              for class I substances.

    Step B4.  Adequate data to determine a NOEL for ethyl
              2-methyl-3,4-pentadienoate were not available. The Committee
              was aware of a study in rats given ethyl
              2-methyl-3,4-pentadienoate in the diet at a concentration
              equivalent to a daily intake of  1 mg/kg bw for 92 days (Cox
              et al., 1978); however, a full report of the study was not
              available. 

         The stepwise evaluations of the selected linear and
    branched-chain aliphatic unsaturated and unconjugated alcohols,
    aldehydes, acids, and related esters in this group of flavouring
    agents, are summarized in Table 1.

    1.5  Consideration of combined intakes from use as flavouring agents

         In the unlikely event that all 41 of the linear and
    branched-chain aliphatic unsaturated and unconjugated aldehydes,
    acids, and related esters that were evaluated were consumed
    concomitantly on a daily basis, the estimated combined intake would
    exceed the human threshold for class I. All 41 substances are expected
    to be efficiently metabolized, however, and would not saturate
    metabolic pathways. On the basis of the evaluation of the collective
    data, the combined intake was judged by the Committee not to raise
    safety concerns. The consideration of combined intakes did not include
    intake of ethyl 2-methyl-3,4-pentadienoate because the evaluation of
    this substance was deferred pending review of the 90-day toxicity
    study, which was not available to the Committee.

    1.6  Conclusions

         The Committee concluded that 41 of the linear and branched-chain
    aliphatic, unsaturated, unconjugated alcohols, aldehydes, acids, and
    related esters in this group would not present safety concerns when
    used at current levels of intake as flavouring agents. Data on the
    toxicity of  cis-3-hexen-1-ol and on the metabolism of these
    flavouring agents were required for application of the procedure. The
    Committee noted that these and other available data on toxicity are
    consistent with the results of the safety evaluation with the
    procedure. The evaluation of one substance in the group, ethyl
    2-methyl-3,4-pentadienoate, was deferred pending evaluation of a
    90-day study in rats that was not available to the Committee. The ADI
    'not specified' for the calcium, potassium, and sodium salts of oleic
    acid established at the thirty-third meeting was maintained.


    2.  RELEVANT BACKGROUND INFORMATION

    2.1  Explanation

         This monograph summarizes the key data relevant to the safety
    evaluation of 42 linear and branched-chain aliphatic, unsaturated,
    unconjugated alcohols, aldehydes, acids, and related esters (see
    Table 1). This group does not include any alpha,-unsaturated (i.e.
    conjugated) aldehydes, acids, or related esters. The group of
    flavouring substances includes the following: five linear aliphatic
    unsaturated primary alcohols (Nos 315, 318, 321, 322, and 324); a
    branched-chain aliphatic unsaturated primary alcohol (No. 348); eight
    linear aliphatic unsaturated unconjugated aldehydes (Nos 316, 319,
    320, 323, 325, 326, 329, and 330); a branched-chain aliphatic
    unsaturated unconjugated aldehyde (No. 349); seven linear aliphatic
    unsaturated unconjugated carboxylic acids (Nos 314, 317, 327, 328, and
    331-333); 13 esters of linear aliphatic unsaturated unconjugated

    carboxylic acids (Nos 334-349); two branched-chain aliphatic
    unsaturated unconjugated carboxylic acids (Nos 347 and 355); and five
    esters of branched-chain aliphatic unsaturated unconjugated carboxylic
    acids (Nos 350-354). The chemical structures of the substances in this
    group are given in Table 1. 

         The substances in this group are structurally related because
    they are primary aliphatic alcohols, aldehydes, carboxylic acids, and
    related esters containing unsaturation that is not conjugated with the
    functional group. Therefore, these unsaturated unconjugated substances
    are predicted to have similar metabolic and toxicological profiles,
    with one exception, as described below. 

    2.2  Intake

         The most recent figures for the annual production volume in
    Europe and the United States are given in Table 2 for the 42
    substances in this group. Intake was estimated on the assumption that
    the production was underestimated and consumption was by 10% of the
    population, as indicated in the footnote to Table 2.

         Quantitative data on natural occurrence have been reported for 11
    of the 42 substances in the group, indicating that they are generally
    consumed predominantly from traditional foods (Maarse et al., 1994).
    Four of the 11 substa-nces (Nos 315, 317, 320, and 346) are consumed
    in greater quantities when used as flavouring substances (Stofberg &
    Kirschman, 1985; Stofberg & Grundschober, 1987). 

    2.3  Biological data

    2.3.1  Absorption and metabolism

         In general, short-chain (< C-8) linear and branched-chain
    aliphatic alcohols, aldehydes, acids, and related esters are rapidly
    absorbed from the gastrointestinal tract (Dawson et al., 1964;
    Gaillard & Derache, 1965). Long-chain carboxylic acids such as
    linoleic and oleic acid are readily absorbed as micelle aggregates,
    esterified with glycerol in chylomicrons and very low density
    lipoproteins, and transported  via the lymphatic system (Borgstrom,
    1974). Radiolabelled linoleic and oleic acids have been administered
    to a variety of amphibians, mammals, and humans by various routes.
    Fatty acid uptake occurred in all tissues, including the brain, by
    passive and/or facilitated diffusion (Dhopeshwarkar & Mead, 1973;
    Harris et al., 1980; Abumrad et al., 1984). 

     Hydrolysis of linear and branched-chain esters

         The esters are hydrolysed in digestive fluids and  in vivo to
    yield the corresponding alcohols and unsaturated carboxylic acids, as
    discussed in the section, 'General aspects of metabolism'. The results
    of studies of hydrolysis  in vitro indicate that the rate of
    hydrolysis of straight-chain esters is approximately 100 times greater
    than that of branched-chain esters (Drake et al., 1975). 


        Table 1. Summary of results of safety evaluations of linear and branched-chain aliphatic, unsaturated, unconjugated alcohols, aldehydes, 
    acids, and related esters

    Step 1: All of the substances in this table are in structural class I.
    The human intake threshold is 1800 g/day for class I.
    Step 2: All of the substances in this group are predicted to be metabolized to innocuous products, except ethyl 2-methyl-3,4-pentadienoate.

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    4-Pentenoic acid       314     5/3             No          N/R          N/R                Converted to coenzyme            No safety concern
                                                                                               A thioester which 
                                                                                               undergoes dehydrogenation and
                                                                                               selective hydrogenation 
                                                                                               and then participates in 
                                                                                               the fatty acid and 
                                                                                               tricarboxylic acid pathways
    CAS No. 591-80-0
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    cis-3-Hexen-1-ol       315    4300/1100        Yes         No           Yes. The NOAEL     Expected to be oxidized          No safety concern
                                                                            of 120-150 mg/kg   to the corresponding 
                                                                            bw per day [Gaunt  aldehyde and carboxylic 
                                                                            et al., 1969] is   acid which is completely 
                                                                            >1000 times the    metabolized in the
                                                                            daily per capita   fatty acid and tricarboxylic 
                                                                            intake.            acid pathways
    CAS No. 928-96-1
    CHEMICAL STRUCTURE

    cis-3-Hexenal          316    5/3              No          N/R          N/R                See cis-3-hexenol.               No safety concern
    CAS No. 6789-80-6
    CHEMICAL STRUCTURE

    3-Hexenoic acid        317    11/1             No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                                                                concern
    CAS No. 4219-24-3
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    4-Hexen-1-ol           318    3/0.04           No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                                                                concern
    CAS No. 6126-50-7
    CHEMICAL STRUCTURE

    cis-4-Hexenal          319    0.03/0.1         No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                                                                concern
    CAS No. 4634-89-3
    CHEMICAL STRUCTURE

    4-Heptenal             320    2/0.1            No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                                                                concern
    CAS No. 6728-31-0
    CHEMICAL STRUCTURE

    cis-3-Octen-1-ol       321    6/0.01           No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                                                                concern
    CAS No. 20125-84-2
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    cis-5-Octen-1-ol       322    0.5/0.04         No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                                                                concern
    CAS No. 64275-73-6
    CHEMICAL STRUCTURE

    cis-5-Octenal          323    0.001/29         No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                                                                concern
    CAS No. 41547-22-2
    CHEMICAL STRUCTURE

    cis-6-Nonen-1-ol       324    3/0.1            No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                                                                concern
    CAS No. 35854-86-5
    CHEMICAL STRUCTURE

    cis-6-Nonenal          325    2/0.1            No          N/R          N/R                See cis-3-hexenol.               No safety
                                                                                                                                concern
    CAS No. 2277-19-2
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    4-Decenal              326    1/0.01           No          N/R          N/R                See cis-3-hexenol.               No safety
                                                                                                                                concern
    CAS No. 30390-50-2
    CHEMICAL STRUCTURE

    5&6-Decenoic acid      327    4/0.2            No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                                                                concern
    CAS No. 72881-27-7
    CHEMICAL STRUCTURE
    CHEMICAL STRUCTURE

    9-Decenoic acid        328    0.1/0.2          No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                               concern
    CAS No. 14436-32-9
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    9-Undecenal            329    1/0.2            No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                                                                concern
    CAS No. 143-14-6
    CHEMICAL STRUCTURE

    10-Undecenal           330    0.4/0.04         No          N/R          N/R                See cis-3-hexenol.               No safety 
                                                                                                                                concern
    CAS No. 112-45-8
    CHEMICAL STRUCTURE

    10-Undecenoic acid     331    30/0.8           No          N/R          N/R                See cis-3-hexenol.               No safety
                                                                                                                                concern
    CAS No. 112-38-9
    CHEMICAL STRUCTURE

    Linoleic acid          332    133/6            No          N/R          N/R                Readily metabolized in           No safety 
                                                                                               the fatty acid oxidation         concern
                                                                                                                                pathway
    CAS No. 60-33-3
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    Oleic acida            333    970/440          No          N/R          N/R                Readily metabolized in           No safety 
                                                                                               the fatty acid oxidation         concern
                                                                                                                                pathway
    CAS No. 112-80-1
    CHEMICAL STRUCTURE

    Methyl 3-hexenoate     334    0.7/0.01         No          N/R          N/R                Expected to be hydrolysed        No safety 
                                                                                               to its component                 concern
                                                                                               alcohol and acid
                                                                                               See also cis-3-hexenol.
    CAS No. 2396-78-3
    CHEMICAL STRUCTURE

    Ethyl 3-hexenoate      335    4/1              No          N/R          N/R                See methyl 3-hexenoate.          No safety 
                                                                                                                                concern
    CAS No. 2396-83-0
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    cis-3-Hexenyl          336    4/0.8            No          N/R          N/R                See methyl 3-hexenoate.          No safety 
     cis-3-hexenoate                                                                                                            concern
    CAS No. 61444-38-0
    CHEMICAL STRUCTURE

    Methyl                 337    0.4/0.04         No          N/R          N/R                See methyl 3-hexenoate.          No safety 
     cis-4-octenoate                                                                                                            concern
    CAS No. 21063-71-8
    CHEMICAL STRUCTURE

    Ethyl                  338    1/0.04           No          N/R          N/R                See methyl 3-hexenoate.          No safety 
     cis-4-octenoate                                                                                                            concern
    CAS No. 34495-71-1
    CHEMICAL STRUCTURE

    Ethyl                  339    2/0.01           No          N/R          N/R                See methyl 3-hexenoate.          No safety 
     cis-4,7-octadienoate                                                                                                       concern
    CAS No. 69925-33-3
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    Methyl 3-nonenoate     340    2/76             No          N/R          N/R                See methyl 3-hexenoate.          No safety 
                                                                                                                                concern
    CAS No. 13481-87-3
    CHEMICAL STRUCTURE

    Ethyl                  341    2/0.04           No          N/R          N/R                See methyl 3-hexenoate.          No safety 
     trans-4-decenoate                                                                                                          concern
    CAS No. 76649-16-6
    CHEMICAL STRUCTURE

    Methyl 9-un-decenoate  342    39/0.4           No          N/R          N/R                See methyl 3-hexenoate.          No safety 
                                                                                                                                concern
    CAS No. 5760-50-9
    CHEMICAL STRUCTURE

    Ethyl 10-undecenoate   343    2/44             No          N/R          N/R                See methyl 3-hexenoate.          No safety 
                                                                                                                                concern
    CAS No. 692-86-4
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    Butyl 10-undecenoate   344    0.04/5           No          N/R          N/R                See methyl 3-hexenoate.          No safety 
                                                                                                                                concern
    CAS No. 109-42-2
    CHEMICAL STRUCTURE

    Ethyl oleate           345    69/3             No          N/R          N/R                See methyl 3-hexenoate           No safety 
                                                                                               and oleic acid.                  concern
    CAS No. 111-62-6
    CHEMICAL STRUCTURE

    Methyl linoleate       346     N/D/23          No          N/R          N/R                See methyl 3-hexenoate           No safety 
     & Methyl linolenate                                                                       and linoleic acid.               concern
     (mixture)
    CAS No. 301-00-8
    CHEMICAL STRUCTURE
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    2-Methyl-3-pentenoic   347    1/2              No          N/R          N/R                Undergoes  oxidation to         No safety
     acid                                                                                      yield products that are          concern
                                                                                               completely metabolized in
                                                                                               the tricarboxylic acid cycle
    CAS No. 37674-63-8
    CHEMICAL STRUCTURE

    2,6-Dimethyl-          348    N/D/0.1          No          N/R          N/R                See 2-methyl-3-pentenoic         No safety
     6-hepten-1-ol                                                                             acid.                            concern
    CAS No. 36806-46-9
    CHEMICAL STRUCTURE

    2,6-Dimethyl-          349    31/251           No          N/R          N/R                See 2-methyl-3-pentenoic         No safety
     5-heptenal                                                                                acid.                            concern
    CAS No. 106-72-9
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    Ethyl 2-methyl-        350    6/6              No          N/R          N/R                Expected to be hydrolysed        No safety
     3-pentenoate                                                                              to its component                 concern
                                                                                               alcohol and carboxylic
                                                                                               acid.  See also 
                                                                                               2-methyl-3-pentenoic acid.
    CAS No. 1617-23-8
    CHEMICAL STRUCTURE

    Ethyl 2-methyl-        351    0.3/0.02         No          N/R          N/R                Expected to be hydrolysed        No safety
     4-pentenoate                                                                              to its component                 concern
                                                                                               alcohol and carboxylic
                                                                                               acid.  See also 
                                                                                               2-methyl-3-pentenoic acid.
    CAS No. 53399-81-8
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    Hexyl 2-methyl-        352    0.03/525         No          N/R          N/R                Expected to be hydrolysed        No safety 
     3&4-pentenoate                                                                            to its component                 concern
                                                                                               alcohol and carboxylic 
                                                                                               acid.  See also 
                                                                                               2-methyl-3-pentenoic acid.
    CAS No. 58625-95-9
    CHEMICAL STRUCTURE
    CHEMICAL STRUCTURE

    Ethyl 2-methyl-        353    0.01/10          -           -            -                  Cannot be predicted to           Evaluation of this 
     3,4-pentadienoate                                                                         be metabolized to innocuous      substance was 
                                                                                               products; therefore, , steps     deferred pending 
                                                                                               A3-5 are not applicable          review of a 90-day 
                                                                                               to this substance, which         study not available 
                                                                                               must be evaluated on             at this meeting.
                                                                                               the right side ('B') of 
                                                                                               the evaluation scheme.
    CAS No. 60523-21-9
    CHEMICAL STRUCTURE

    Table 1. (continued)

                                                                                                                                                 
    Substance              No.    Estimated        Step A3     Step A4      Step A5            Comments                         Conclusion
                                  per capita       Does        Is the       Adequate                                            based on current
                                  intake ('eaters  intake      substance    NOEL for                                            intake
                                  only'),          exceed      or its       substance 
                                  Europe/USA       intake      metabolites  or related 
                                  (g/day)         threshold?  endogenous?  substance?
                                                                                                                                                 

    Methyl 3,7-dimethyl-   354    0.2/0.1          No          N/R          N/R                Expected to be hydrolysed        No safety concern
     6-octenoate                                                                               to its component 
                                                                                               alcohol and carboxylic 
                                                                                               acid.  See also 
                                                                                               2-methyl-3-pentenoic acid.
    CAS No. 2270-60-2
    CHEMICAL STRUCTURE

    2-Methyl-4-pentenoic   355    N/D/0.04         No          N/R          N/R                See 2-methyl-3-pentenoic         No safety concern
     acid                                                                                      acid. 
    CAS No. 1575-74-2
    CHEMICAL STRUCTURE

                                                                                                                                                 

    N/R, Not required for evaluation because consumption was determined to be of no safety concern at Step 3A of the procedure
    N/D, No intake data reported
    a  The ADI 'not specified' for the calcium, potassium, and sodium salts established at the thrity-third meeting of the Committee (Annex 1, 
       reference 83) was maintained.
    

        Table 2. Most recent annual usage of linear and branched-chain aliphatic unsaturated 
    unconjugated alcohols, aldehydes, acids, and related esters  as flavouring substances in Europe 
    and the United States

                                                                                                  

    Substance (No.)                     Most recent      Per capita intakea    Per capita intakeb,
                                        annual volume                          alcohol equivalents
                                        (kg)             g/day    g/kg bw    (g/kg bw per day)
                                                                   per day
                                                                                                  

    4-Pentenoic acid (314)
      Europe                            32               5         0.08        NA
      United States                     15               3         0.05

    cis-3-Hexen-1-ol (315)
      Europe                            30 000           4 300     71          NA
      United States                     5 600            1 100     18

    cis-3-Hexenal (316) 
      Europe                            34               5         0.08        NA
      United States                     18               3         0.06

    3-Hexenoic acid (317) 
      Europe                            77               11        0.2         NA
      United States                     5                1         0.02

    4-Hexen-1-ol (318) 
      Europe                            20               3         0.05        NA
      United States                     0.2              0.04      0.001

    cis-4-Hexenal (319) 
      Europe                            0.2              0.03      0.0005      NA
      United States                     0.5              0.1       0.002

    4-Heptenal (320) 
      Europe                            13               2         0.03        NA
      United States                     0.5              0.1       0.002

    cis-3-Octen-1-ol (321)
      Europe                            39               6         0.1         NA
      United States                     0.05             0.01      0.0002

    cis-5-Octen-1-ol (322) 
      Europe                            3.3              0.5       0.01        NA
      United States                     0.2              0.04      0.001

    cis-5-Octenal (323)
      Europe                            0.01             0.001     0.00002     NA
      United States                     150              29        0.5

    Table 2. (continued)

                                                                                                  

    Substance (No.)                     Most recent      Per capita intakea    Per capita intakeb,
                                        annual volume                          alcohol equivalents
                                        (kg)             g/day    g/kg bw    (g/kg bw per day)
                                                                   per day
                                                                                                  

    cis-6-Nonen-1-ol (324 )
      Europe                            18               3         0.04        NA
      United States                     0.5              0.1       0.002

    cis-6-Nonenal (325) 
      Europe                            14               2         0.03        NA
      United States                     0.5              0.1       0.002

    4-Decenal (326)
      Europe                            8                1         0.02        NA
      United States                     0.05             0.01      0.0002

    5&6-Decenoic acid (327)
      Europe                            28               4         0.07        NA
      United States                     0.9              0.2       0.003

    9-Decenoic acid (328)
      Europe                            0.8              0.1       0.002       NA
      United States                     0.9              0.2       0.003

    9-Undecenal (329)
      Europe                            8                1         0.02        NA
      United States                     0.9              0.2       0.003

    10-Undecenal (330)
      Europe                            2.6              0.4       0.01        NA
      United States                     0.2              0.04      0.001

    10-Undecenoic acid (331)
      Europe                            210              30        0.5         NA
      United States                     4                0.8       0.01

    Linoleic acid (332)
      Europe                            940              130       2           NA
      United States                     31               6         0.1

    Oleic acid (333)
      Europe                            6 800            970       16          NA
      United States                     2 300            440       7

    Table 2. (continued)

                                                                                                  

    Substance (No.)                     Most recent      Per capita intakea    Per capita intakeb,
                                        annual volume                          alcohol equivalents
                                        (kg)             g/day    g/kg bw    (g/kg bw per day)
                                                                   per day
                                                                                                  

    Methyl 3-hexenoate (334)
      Europe                            4.6              0.7       0.01        0.003
      United States                     0.05             0.01      0.0002

    Ethyl 3-hexenoate (335)
      Europe                            26               4         0.1         0.03
      United States                     6                1         0.02

    cis-3-Hexenyl cis-3-hexenoate (336)
      Europe                            26               4         0.1         0.05
      United States                     4                0.8       0.01

    Methyl cis-4-octenoate (337)
      Europe                            3                0.4       0.01        0.002
      United States                     0.2              0.04      0.001

    Ethyl cis-4-octenoate (338)
      Europe                            10               1         0.02        0.005
      United States                     0.2              0.04      0.001

    Ethyl cis-4,7-octadienoate (339)
      Europe                            15               2         0.04        0.01
      United States                     0.04             0.01      0.0001

    Methyl 3-nonenoate (340)
      Europe                            13               2         0.03        0.006
      United States                     400              76        1

    Ethyl trans-4-octenoate (341)
      Europe                            15               2         0.04        0.009
      United States                     0.2              0.04      0.001

    Methyl 9-undecenoate (342)
      Europe                            280              39        1           0.20
      United States                     2                0.4       0.01

    Ethyl 10-undecenoate (343)
      Europe                            12               2         0.03        0.006
      United States                     230              44        1

    Table 2. (continued)

                                                                                                  

    Substance (No.)                     Most recent      Per capita intakea    Per capita intakeb,
                                        annual volume                          alcohol equivalents
                                        (kg)             g/day    g/kg bw    (g/kg bw per day)
                                                                   per day
                                                                                                  

    Butyl 10-undecenoate (344)
      Europe                            0.3              0.04      0.0007      0.0002
      United States                     25               5         0.08

    Ethyl oleate (345)
      Europe                            490              69        1           0.10
      United States                     17               3         0.1

    Methyl linoleate & methyl 
      linolenate (mixture) (346)
      Europe                            NR               N/D
      United States                     120              23        0.4         0.046

    2-Methyl-3-pentenoic acid (347)
      Europe                            10               1         0.02        NA
      United States                     9                2         0.03

    2,6-Dimethyl-6-hepten-1-ol (348)
      Europe                            NR               N/D                   NA
      United States                     0.5              0.1       0.002

    2,6-Dimethyl-5-heptenal (349)
      Europe                            220              31        1           NA
      United States                     1 300            250       4

    Ethyl 2-methyl-3-pentenoate (350)
      Europe                            40               6         0.1         0.03
      United States                     31               6         0.1

    Ethyl 2-methyl-4-pentenoate (351)
      Europe                            0.2              0.03      0.0005      0.0002
      United States                     0.9              0.2       0.003

    Hexyl 2-methyl-3&4-pentenoate (352)
      Europe                            0.2              0.03      0.0005      0.0003
      United States                     2 760            520       9

    Ethyl 2-methyl-3,4-pentadienoate (353)
      Europe                            0.1              0.01      0.0002      0.00007
      United States                     50               10        0.2

    Table 2. (continued)

                                                                                                  

    Substance (No.)                     Most recent      Per capita intakea    Per capita intakeb,
                                        annual volume                          alcohol equivalents
                                        (kg)             g/day    g/kg bw    (g/kg bw per day)
                                                                   per day
                                                                                                  

    Methyl 3,7-dimethyl-6-octenoate (354)
      Europe                            1.1              0.2       0.003       0.0005
      United States                     0.3              0.1       0.001

    2-Methyl-4-pentenoic acid (355)
      Europe                            NR               N/D                   NA
      United States                     0.2              0.04      0.001

    Total
      Europe                            39 168           NA        NA          NA
      United States                     13 130           NA        NA          NA
                                                                                                  

    NR, not reported;  NA, not applicable

    a  Intake (g/day) calculated as follows: 
       [(annual volume, kg)  (1  109 g/kg)]/[population  0.6  365 days], where population 
       (10% 'eaters only') = 32  106 for Europe (International Organization of the Flavor Industry,
       1995) and 24  106 for the United States (US National Academy of Sciences, 1989); 
       0.6 represents the assumption that only 60% of the flavour volume was reported in the survey.
       Intake (g/kg bw per day) calculated as follows: [(g/day)/body weight], where body 
       weight = 60 kg. Slight variations may occur due to rounding off.
    b  Calculated as follows: (molecular mass alcohol/molecular mass ester)  daily per capita 
       intake ester
    
     Linear unsaturated primary alcohols, aldehydes, and acids

         The alcohols (including those formed from ester hydrolysis) and
    aldehydes in this group are efficiently oxidized to the corresponding
    unsaturated carboxylic acids by high-capacity enzyme pathways, as
    discussed in the section 'General aspects of metabolism'. The
    resulting linear unsaturated carboxylic acids participate in normal
    fatty acid metabolism. Short-chain acids containing terminal
    unsaturation, such as 4-pentenoic acid (No. 314), may be metabolized
    by desaturation to yield a substrate that may participate in the fatty
    acid pathway.

         Alternative minor metabolic pathways have been characterized for
    linear long-chain fatty acids (linoleic acid and oleic acid) and
    short-chain acids containing unsaturation (i.e. 4-pentenoic acid).
    While linoleic and oleic acids participate in -oxidation and normal
    fatty acid metabolism in most tissues (Masoro, 1977), they may undergo
    omega-oxidation in the liver and alpha-oxidation in the brain (Wakil &
    Barnes, 1971; Gibson et al., 1982). In another minor pathway for the
    oxidative metabolism of 4-pentenoic acid,  trans-2,4-pentadienoyl
    coenzyme A is produced, which is further metabolized via -oxidation
    to yield 3-keto-4-pentenoyl coenzyme A (Schulz, 1983). 

     Branched-chain unsaturated primary alcohols, aldehydes, and acids

         Generally, branched-chain aliphatic alcohols are oxidized to the
    corresponding aldehydes, which, in turn, are oxidized to the
    corresponding carboxylic acids (Bosron & Li, 1980; Levi & Hodgson,
    1989). The aldehyde may also be reduced to the corresponding alcohol,
    which is probably a short-lived intermediate  in vivo. Like their
    saturated analogues, unsaturated branched-chain aliphatic alcohols and
    aldehydes are converted by the pathways cited above to the
    corresponding acids. The acids may undergo -oxidative cleavage and
    complete metabolism to carbon dioxide (Williams, 1959; Voet & Voet,
    1990) in amino acid pathways, the fatty acid pathway, and the
    tricarboxylic acid cycle. Alternatively, they may undergo a
    combination of omega-, (omega-1)-, and -oxidation and selective
    dehydrogenation and hydration to yield polar 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., 1990). The
    principal metabolic pathways for detoxification of these
    branched-chain substances is determined primarily by four structural
    characteristics: carbon chain length and the position, number, and
    size of the alkyl substituents.

         These multiple pathways are illustrated by 2-ethylbutyric acid
    and 2-ethyl-1-butanol. An oral dose of 2-ethyl-1-butanol was excreted
    unchanged in the urine, principally as the glucuronic acid conjugate
    (Kamil et al., 1953). 2-Ethylbutyric acid is metabolized by dogs to
    2-pentanone, which is presumably derived from -oxidation and
    decarboxylation (Deuel, 1957). Urinary metabolites of 2-ethylbutyric
    acid reported in diabetic patients include the glucuronide conjugates
    of 3-keto-2-ethylbutyric acid and 3,3'-diketo-2-ethylbutyric acid

    formed by -oxidation of one or both ethyl chains (Granger et
    al.,1954).

         Higher homologues of 2-ethyl-1-butanol have a more complex
    pattern of metabolism in which omega-oxidation competes with excretion
    of the glucuronide conjugate of the corresponding acid. Female Fischer
    344 rats were given a single oral dose of 50 or 500 mg/kg bw
    [1-14C-ethyl]-2-ethylhexanol by gavage or repeated daily oral doses
    of 50 mg/kg bw 2-ethyl-hexanol for 14 days. The major urinary
    metabolites of the structurally related saturated branched-chain
    alcohol 2-ethyl-1-hexanol collected over 0-24 h included the
    glucuronic acid conjugate of 2-ethyl-hexanoic acid, omega- and
    (omega-1)-oxidation metabolites, 2-ethyl-1,6-dihexanoic acid,
    5-hydroxy-2-ethylhexanoic acid and its corresponding -lactone, and
    6-hydroxy-2-ethylhexanoic acid. Minute quantities of the
    omega-desaturation metabolite, 2-ethyl-5-hexenoic acid, and
    metabolites formed from  oxidation and oxidative decarboxylation, 2-
    and 4-heptanone, were also detected (Albro, 1975; Deisinger  et al., 
    1994). After single or repeated doses of 50 mg/kg bw, the
    omega-oxidation pathway predominated, as the principal urinary
    metabolites excreted were 2-ethyl-1,6-hexane-dioic acid (30%) and its
    precursors (8.5%). Only 6.8% of the dose was excreted in the urine as
    the glucuronic acid conjugate of 2-ethylhexanoic acid. With 500 mg/kg
    bw, 2-ethylhexanoic acid (25%) was the principal urinary metabolite,
    indicating that saturation of the omega-desaturation pathway occurs at
    this dose (Deisinger et al., 1994; Figure 1). 

    FIGURE 1

    2.3.2  Toxicological studies

    2.3.2.1  Acute toxicity

         Oral LD50 values have been reported for a number of the 42
    substances in this group and are in the range 470-19 000 mg/kg bw,
    demonstrating that the acute toxicity of linear and branched-chain
    aliphatic unsaturated and unconjugated alcohols, aldehydes, acids, and
    related esters is low when they are given orally (Jenner et al., 1964;
    Briggs et al., 1976).

    2.3.2.2  Short-term and long-term studies of toxicity and
             carcinogenicity

     cis-3-Hexen-1-ol (No. 315)

         Groups of 15 male and 15 female weanling rats were given
    drinking-water containing 0, 310, 1250, or 5000 ppm  cis-3 hexen-1-ol
    for 98 days. Weekly measurements of body weight, food intake, and
    water consumption revealed no differences in body-weight gain or food
    intake between treated and control animals, but a significant
     (p < 0.01) increase in water intake was recorded for males at the
    high dose. A significant  (p < 0.001), transitory anaemia was seen
    at six weeks in females given 5000 ppm; however, this effect was not
    observed at 14 weeks or in the males and therefore was considered not
    to be related to treatment. Kidney and liver function in animals at
    the low and intermediate doses was comparable to that of controls.
    After 98 days, males at the high dose had a statistically significant
    increase in relative kidney  (p < 0.05) and adrenal weights
     (p < 0.01) and a slight increase in urine specific gravity during
    the first 2 h after a standard water load; however, there were no
    histological signs of renal damage and no indications of abnormal
    kidney function associated with treatment. Histopathological
    examination revealed lymphocyte infiltration in the lung, suggesting a
    mild pulmonary infection, and foci of tubular degeneration and
    dilatation in the kidney. The incidence of these findings was similar
    in test and control animals and could not be attributed to treatment.
    The NOEL of 1250 ppm, reported by the authors to be equivalent to an
    intake of 120-150 mg/kg bw per day (Gaunt et al., 1969), is > 1000
    times the current daily  per capita intake of 71 and 18 g/kg bw per
    day from use of  cis-3-hexen-1-ol as a flavouring substance in Europe
    and the United States, respectively.

     10-Undecenoic acid (No. 331)

         In an abstract describing the results of a study in which 100,
    200, or 400 mg/kg bw per day of 10-undecenoic acid were administered
    to Sprague-Dawley rats by gavage for six months, no effects were
    reported (Tislow et al., 1950). Because a full report of this study
    was not available, it was considered inadequate for deriving a NOEL.

         In another study of 10-undecenoic acid, Sprague-Dawley rats were
    maintained on diets containing 0.5, 1, or 2.5% (approximately 500,
    1000, and 2500 mg/kg bw per day (US Food & Drug Administration, 1993))
    for eight weeks. An inhibitory effect on growth, possibly related to
    decreased food consumption, was reported during the first five weeks
    (Newell et al., 1949). These data, described by the authors as
    preliminary, were not used to derive a NOEL. 

     Oleic acid (No. 333)

         The Committee previously allocated an ADI 'not specified' to the
    aluminum, ammonium, calcium, magnesium, potassium, and sodium salts of
    oleic acid (Annex 1, reference 70). Recently, the results of a 13-week
    and a two-year study of oleic acid diethanolamine condensate
    administered dermally to Fischer 344/N rats and B6C3F1 mice were
    reported (US National Toxicology Program, 1997). These studies were
    not considered relevant to the safety evaluation of oleic acid used as
    a flavouring substance.

         Numerous other studies are available of the role of high
    concentrations of oleic acid and other fatty acids in the diet. As the
    effects observed are probably the result of the extremely high intake
    of total dietary fat and fatty acids, these studies were not
    considered relevant to the safety evaluation of oleic acid at its
    current level of intake as a flavouring substance.

     2,6-Dimethyl-5-heptenal (No. 349)

         Groups of 15 male and 15 female rats were fed diets providing
    2,6-dimethyl-5-heptenal at average intakes of 0, 9, 37, or 150 mg/kg
    bw per day for at least 90 days. Statistically significant
     (p < 0.05) increases in haemoglobin concentrations were found in
    males at the high dose at weeks 6 and 14 and in males at the
    intermediate dose at week 14; however, these effects were not
    biologically significant and were not considered by the authors to be
    adverse. Most of the absolute and relative weights of organs were
    similar in treated and control animals, but statistically significant
     (p < 0.05) increases in the relative and absolute spleen weights
    were observed in males at the intermediate dose. A statistically
    significant increase in serum glucose concentration was observed in
    male and female rats at the high dose, which the authors noted was of
    unknown origin. Also at the high dose, the renal concentrating ability
    was reduced in males at week 6 and in females at week 14, resulting in
    proteinuria. Slight, statistically significant increases  (p < 0.05
    by Student's  t test) in relative kidney and liver weights were also
    observed in females at this dose. Histological examination of rats at
    the high dose showed no statistically significant difference between
    the treated group and controls, other than alveolar thickening in
    males  (p < 0.05). Since the histopathological appearance was normal
    in animals of each sex and the kidney weights were normal in males,
    the proteinuria could not be associated with treatment. Additionally,
    no structural kidney damage was seen by light microscopy. A functional
    effect on the kidney might have been caused by treatment at the

    highest level, however, because males and females at the high dose
    failed to concentrate urine after a period of dehydration during weeks
    6 and 13, respectively, and the kidney weights of females were
    marginally increased. The NOEL for 2,6-dimethyl-5-heptenal was 37
    mg/kg bw per day (Gaunt et al., 1983) which is > 1000 times the daily
     per capita intakes of 1 and 4 g/kg bw per day from use of
    2,6-dimethyl-5-heptenal as a flavouring substance in Europe and the
    United States, respectively. 

     Ethyl 2-methyl-3,4-pentadienoate (No. 353)

         The Committee was aware of a study on the toxicity of
    ethyl-2-methyl-3,4-pentadienoate (Cox et al., 1978); however, a full
    report of the study was not available. 

    2.3.2.3  Genotoxicity

         Three representative linear and branched-chain aliphatic
    unsaturated primary alcohols and unconjugated aldehydes, carboxylic
    acids, and related esters in this group have been tested for
    genotoxicity. The results of these tests are summarized in Table 3 and
    described below.

         No evidence of mutagenicity was reported for oleic acid, methyl
    linoleate, methyl linolenate, or 2,6-dimethyl-5-heptenal in standard
    and preincubation assays in  Salmonella typhimurium strain TA98,
    TA100, TA1535, TA1537, or TA1538 with or without the addition of
    metabolic activation (Wild et al., 1983; Shimizu et al., 1985;
    Mortelmans et al., 1986; Heck et al., 1989). The maximum
    concentrations used in these studies ranged from 333 to 50 000
    g/plate. In further bacterial assays, such as the  rec assay in
     Bacillus subtilis incubated with oleic acid (Osawa & Namiki, 1982),
    the  his+ reversion assay in  S. typhimurium incubated with methyl
    linoleate or methyl linolenate (MacGregor et al., 1985), and a
    modified Ames test in  Escherichia coli WP2 uvrA incubated with
    oleic acid (Shimizu et al., 1985), aliphatic unsaturated unconjugated
    acids and esters were also shown to be non-mutagenic.

         No unscheduled DNA synthesis was seen after exposure of rat
    hepatocytes to 2,6-dimethyl-5-heptenal at concentrations up to 1 mg/ml
    (Heck et al., 1989). 

         In  S. typhimurium strain TA98, a concentration of 0.1 mmol/L
    oleic acid was reported to inhibit (by approximately 50%) the
    mutagenicity of known mutagens, including
    3-amino-1,4-dimethyl-5 H-pyrido[4,3- b]indole,
    2-amino-9 H-pyridol[2,3- b]indole,
    2-amino-3-methylimidazo[4,5- d]quinoline, benzo[ a]-pyrene, and
    aflatoxin B1, when tested in the presence or absence of metabolic
    activation (Hayatsu et al., 1981). 


        Table 3. Results of assays for the genotoxicity studies for linear and branched-chain aliphatic unsaturated unconjugated alcohols, aldehydes, 
    acids and related esters 

                                                                                                                                                 

    Substance                 No.       End-point            Test object                     Maximum          Result     Reference
                                                                                             concentration
                                                                                                                                                 

    Oleic acid                333       Reverse mutationa    S. typhimurium TA1535,          5000 mg/plate    Negativeb  Shimizu et al. (1985)
                                                             TA1537, TA98, TA100, TA1538
                                        Reverse mutationa    S. typhimurium TA1535,          333 mg/plate     Negativeb  Mortelmans et al. (1986)
                                                             TA98, TA100, TA1537 
                                        Reverse mutationa    E. coli                         5000 mg/plate    Negativeb  Shimizu et al. (1985)
                                        Rec assay            B. subtilis                     1 mg/plate       Negativeb  Osawa & Namiki (1982)
    Methyl linoleate          Part of   His+ reversion       S. typhimurium TA100,           1 mg/plate       Negativeb  MacGregor et  al. (1985)
                              346                            TA98, TA102, TA97, TA1537
                                        His+ reversion       S. typhimurium TA100,           1 mg/plate       Negativeb  MacGregor et  al. (1985)
                                                             TA98, TA102, TA97, TA1537
    2,6-Dimethyl-5-heptenal   349       Reverse mutation     S. typhimurium TA1535,          3.6 mg/plate     Negativeb  Wild et al. (1983)
                                                             TA100, TA1537, TA1538, TA98
                                        Reverse mutation     S. typhimurium TA1535,          50 mg/plate      Negativeb  Heck et al. (1989)
                                                             TA100, TA1537, TA1538,  TA98
                                        Unscheduled DNA      Rat hepatocytes                 1 mg/ml          Negativeb  Heck et al. (1989)
                                        synthesis
                                        Micronucleus         Mouse                           1540 mg/kg bw    Negative   Wild et al. (1983)
                                        formation
                                        Basc test            Drosophila melanogaster         25 mmol/L        Negative   Wild et al. (1983
                                                                                                                                                 

    a  Modified Ames' assay, with preincubation
    b  With and without metabolic activation
    

         2,6-Dimethyl-5-heptenal at a concentration of 25 mmol/L did not
    cause  Basc reversion in  Drosophila melanogaster (Wild et al.,
    1983).

         In mice given a maximum single dose of 1540 mg/kg bw
    2,6-dimethyl-5-heptenal, all of which survived treatment, the
    incidence of polychromatic erythrocytes was not statistically
    significantly increased (Wild et al., 1983).

         Oleic acid did not induce oxidative damage in isolated DNA (de
    Kok et al., 1994). In a three-week study in volunteers, ingestion of
    oleic acid did not alter the frequency of micronucleated lymphocytes
    in peripheral blood (Record et al., 1992). 

         These negative results indicate that the substances in this group
    of linear and branched-chain aliphatic unsaturated and unconjugated
    alcohols, aldehydes, acids, and related esters that are used as
    flavouring substances are neither mutagenic nor genotoxic.

    2.3.2.4  Other relevant studies

         In a study of teratogenicity, two groups of 15 female NMRI mice
    were mated with males for 2 h, and 4-pentenoic acid (No. 314) was
    administered as the sodium salt in water by subcutaneous injection to
    pregnant mice on day 8 of gestation as a single 600 mg/kg bw dose. A
    group of control mice was included. Implantation sites were counted,
    and each live fetus was weighed and inspected for neural tube defects.
    Effects on the visceral or skeletal system were not examined.
    4-Pentenoic acid had no effect on fetal weight, the number of live
    fetuses, embryolethality, or exencephaly in comparison with control
    animals (Nau & Loscher, 1986). These results could not be used to
    derive a NOEL for oral intake of 4-pentenoic acid.


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    See Also:
       Toxicological Abbreviations