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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION





    SAFETY EVALUATION OF CERTAIN FOOD
    ADDITIVES AND CONTAMINANTS



    WHO FOOD ADDITIVES SERIES: 44





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





    World Health Organization, Geneva, 2000
    IPCS - International Programme on Chemical Safety


    ALIPHATIC PRIMARY ALCOHOLS, ALDEHYDES, CARBOXYLIC ACIDS,
    ACETALS, AND ESTERS CONTAINING ADDITIONAL OXYGENATED FUNCTIONAL
    GROUPS

    First draft prepared by Dr P.J. Abbott

    Australia New Zealand Food Authority, Canberra, Australia

    Evaluation
       Introduction
       Estimated daily per capita intake
       Metabolism
       Application of the Procedure for the Safety Evaluation of
            Flavouring Agents
            Consideration of combined intakes
       Conclusions
    Relevant background information
       Explanation
       Additional considerations on intake
       Biological data
            Absorption, distribution, metabolism, and excretion
               Esters and diesters
               alpha-Keto-and alpha-hydroxy acids and their esters
               Acetals
               beta-Keto and beta-hydroxy acids and their esters
               gamma-Keto or gamma-hydroxy acids and their esters
               omega-Substituted derivatives
               Aliphatic di-and tricarboxylic acids and their esters
            Toxicological studies
               Acute toxicity
               Short-term and long-term studies of toxicity
               Genotoxicity
               Other relevant studies
    References

    1.  EVALUATION

    1.1  Introduction

         The Committee evaluated a group of 47 flavouring agents that
    includes aliphatic primary alcohols, aldehydes, carboxylic acids,
    acetals, and esters containing additional oxygenated functional groups
    (see Table 1) using the Procedure for the Safety Evaluation of
    Flavouring Agents (Figure 1, p. 122).

         The Committee previously evaluated eight members of this group
    for other functional uses. Fumaric acid (No. 618) was first considered
    by the Committee at its tenth meeting (Annex 1, reference 13), and at
    its thirty-fifth meeting (Annex 1, reference 88) the Committee
    established a group ADI of 'not specified'1 for fumaric acid and its
    salts. Triethyl citrate (No. 629) was first considered by the
    Committee at its twenty-third meeting (Annex 1, reference 50), and at

    its twenty-eighth meeting (Annex 1, reference 66) the Committee
    established an ADI of 0-20 mg/kg bw. Diethyl tartrate (No. 622) was
    first considered by the Committee at its twenty-third meeting (Annex
    1, reference 50), but an evaluation was not possible on the basis of
    the data available at that time. As no additional data were available
    to the Committee at its twenty-fifth meeting (Annex 1, reference 56),
    no ADI was allocated. The Committee also evaluated related terpenoid
    flavouring agents, including linalool, linalyl acetate, citronellol,
    citral, and geranyl acetate, and established a group ADI of 0-0.5
    mg/kg bw at its twenty-third meeting (Annex 1, reference 50).

    1.2  Estimated daily per capita intake

         The estimated  per capita intake of these agents, modified to
    calculate intake of flavouring agents (see p. 121), was derived from
    surveys in Europe and the United States. The total annual production
    of the 47 substances in this group is 200 tonnes in Europe and 1700
    tonnes in the United States, which is equivalent to a total estimated
    daily  per capita intake of 28 mg in Europe and 300 mg in the United
    States.

         Fumaric acid (No. 618) and (-)-malic acid (No. 619) account for
    approximately 59% of the total daily  per capita intake of these 47
    substances in Europe and 88% in the United States. The estimated total
    daily consumption of fumaric acid resulting from its use as a
    flavouring agent is approximately 0.9 mg/person in Europe and 219
    mg/person in the United States. The total daily consumption of
    (-)-malic acid is estimated to be 16 mg/person in Europe and 58
    mg/person in the United States.

         Of the 47 substances evaluated, 25 have been detected as natural
    components of traditional foods (Maarse et al., 1994). 

    1.3  Metabolism

         Studies on the absorption, metabolism, and elimination of
    aliphatic primary alcohols, aldehydes, carboxylic acids, acetals, and
    esters with additional oxygenated functional groups show that these
    substances are readily hydroly-sed and absorbed and are completely
    metabolized. Many of these substances or their metabolites are
    endogenous in humans.

                  

    1 ADI 'not specified' is a term applicable to a food component of very
    low toxicity which, on the basis of the available chemical,
    biological, toxicological, and other data, the total dietary intake of
    the substance arising from its use at the levels necessary to achieve
    the desired effect and from its acceptable background in food, does
    not, in the opinion of the Committee, represent a hazard to health.
    For this reason and for those stated in the evaluation, the
    establishment of an ADI expressed in numerical form is deemed
    unnecessary.

         Many of the substances in this group are esters or diesters and
    are expected to undergo hydrolysis to their corresponding alcohol
    (saturated linear or branched-chain aliphatic primary alcohols or
    branched-chain hydroxy or keto alcohols). The presence of a second
    oxygenated functional group has little if any effect on the hydrolysis
    of these esters. -Keto acids and derivatives such as acetoacetic acid
    easily undergo decarboxylation and, with alpha-keto and
    alpha-hydroxyacids, yield breakdown products which are incorporated
    into normal biochemical pathways. The gamma-keto acids and related
    substances may undergo complete or partial -oxidation to yield
    metabolites, which are eliminated in the urine. The omega-substituted
    derivatives are readily oxidized and/or excreted in the urine. The
    simple aliphatic di-and tricarboxylic acids either occur endogenously
    in humans or are structurally related to endogenous substances. These
    substances are metabolized through the fatty acid -oxidation pathway
    or the tricarboxylic acid cycle.

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

    Step 1.   In applying the Procedure for the Safety Evaluation of
              Flavouring Agents (Figure 1, p. 122) to the above-mentioned
              aliphatic primary alcohols, aldehydes, carboxylic acids,
              acetals, and esters containing additional oxygenated
              functional groups, the Committee assigned all 47 substances
              to structural class I (Cramer et al., 1978).

    Step 2.   Metabolic data on individual members of the group are
              limited, but the common structural features and common
              pathways of metabolism allow some general conclusions to be
              drawn on the likely metabolic fate of these agents. Fourteen
              substances are found normally in human metabolism, and 28
              substances in the group are esters or diesters that would be
              expected to be metabolized to innocuous products. There was
              evidence that the other substances in the group, including
              acetals, derivatives of beta-keto and beta-hydroxy acids,
              gamma-keto and gamma-hydroxy acids, and aliphatic di-and
              tricarboxylic acids, are also metabolized to innocuous
              products. For all substances in this group, therefore, the
              evaluation should proceed via the left-hand side of the
              decision-tree.


        Table 1.  Summary of results of the safety evaluation of 47 aliphatic primary alcohols, aldehydes,
    carboxylic acids, acetals, and esters containing additional oxygenated functional groups
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            

    2-Oxobutyric acid                     589    600-18-0    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 140

    Methyl 2-hydroxy-4-methylpentanoate   590    40348-72-9  No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 141

    Methyl 2-oxo-3-methyl-pentanoate      591    3682-42-6   No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 142


    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    Citronelloxyacetaldehyde              592    7492-67-3   No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 143

    3-Oxobutanal dimethyl acetal          593    5436-21-5   No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 144

    Ethyl 3-hydroxybutyrate               594    5405-41-4   No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 145

    Ethyl acetoacetate                    595    141-97-9    Yes            Yesb           N/R            No safety concern

    CHEMICAL STRUCTURE 146


    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    Butyl acetoacetate                    596    591-60-6    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 147

    Isobutyl acetoacetate                 597    7779-75-1   No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 148

    Isoamyl acetoacetate                  598    2308-18-1   No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 149

    Geranyl acetoacetate                  599    10032-00-5  No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 150

    Methyl 3-hydroxyhexanoate             600    21188-58-9  No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 151


    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    Ethyl 3-hydroxyhexanoate              601    2305-25-1   No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 152


    Ethyl 3-oxohexanoate                  602    3249-68-1   No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 153

    Ethyl 2,4-dioxohexanoate              603    13246-52-1  No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 154

    3-(Hydroxymethyl)-2-heptanone         604    65405-68-7  No             NR             N/R            No safety concern

    CHEMICAL STRUCTURE 155


    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    1,3-Nonanediol acetate                605    1322-17-4   No             N/R            N/R            No safety concern
    (mixed esters)                               

    CHEMICAL STRUCTURE 156

    Laevulinic acid                       606    123-76-2    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 157

    Ethyl laevulinate                     607    539-88-8    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 158

    Butyl laevulinate                     608    2052-15-5   No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 159

    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    1,4-Nonanediol diacetate              609    67715-81-5  No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 160

    Hydroxycitronellol                    610    107-74-4    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 161

    Hydroxycitronellal                    611    107-75-5    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 162

    Hydroxycitronellal dimethyl           612    141-92-4    No             N/R            N/R            No safety concern
    acetal

    CHEMICAL STRUCTURE 163

    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    Hydroxycitronellal diethyl            613    7779-94-4   No             N/R            N/R            No safety concern
    acetal

    CHEMICAL STRUCTURE 164

    Diethyl malonate                      614    105-53-3    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 165

    Butyl ethyl malonate                  615    17373-84-1  No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 166

    Dimethyl succinate                    616    106-65-0    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 167

    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    Diethyl succinate                     617    123-25-1    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 168

    Fumaric acidc                         618    110-17-8    Yes            Yesd           N/R            No safety concern

    CHEMICAL STRUCTURE 169

    (-)-Malic acid                        619    97-67-6     Yes            Yesd           N/R            No safety concern

    CHEMICAL STRUCTURE 170

    Diethyl malate                        620    7554-12-3   No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 171

    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    Tartaric acid (+-, --, -, meso-)     621    87-69-4     Yes            No             Yes. NOEL      No safety concern
                                                                                           was 1200 
                                                                                           mg/kg bw
                                                                                           per day in
                                                                                           a two-year
                                                                                           study in rats  

    CHEMICAL STRUCTURE 172

    Diethyl tartrate                      622    87-91-2     No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 173

    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    Adipic acid                           623    124-04-9    Yes            No             Yes. The NOEL  
                                                                                           for the 
                                                                                           structurally 
                                                                                           related 
                                                                                           compound,
                                                                                           dibutyl
                                                                                           sebacate, was  
                                                                                           6200 mg/kg bw
                                                                                           per day in a
                                                                                           two-year
                                                                                           study in rats  

    CHEMICAL STRUCTURE 174

    Diethyl sebacate                      624    110-40-7    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 175

    Dibutyl sebacate                      625    109-43-3    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 176

    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    Ethylene brassylate                   626    105-95-3    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 177

    Aconitic acid                         627    499-12-7    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 178

    Ethyl aconitate (mixed esters)        628    -           No             N/R            N/R            No safety concern


    CHEMICAL STRUCTURE 179

    Triethyl citratec                     629    77-93-0     Yes            Yesd           N/R            No safety concern

    CHEMICAL STRUCTURE 180


    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    Tributyl acetylcitrate                630    77-90-7     No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 181

    3-Methyl-2-oxobutanoic acid           631    759-05-7    No             N/R            N/R            No safety concern
    and sodium salt                              3715-29-6   

    CHEMICAL STRUCTURE 182

    3-Methyl-2-oxopentanoic acid          632    1460-34-0   No             N/R            N/R            No safety concern
    and sodium salt                              3715-31-9

    CHEMICAL STRUCTURE 183

    4-Methyl-2-oxopentanoic acid          633    816-66-0    No             N/R            N/R            No safety concern
    and sodium salt                              4502-00-5   

    CHEMICAL STRUCTURE 184


    Table 1. (continued)
                                                                                                                            

    Substance                             JECFA  CAS No.     Step A3a       Step A4        Step A5        Conclusion based
    and structure                         No.                Does intake    Is the         Adequate NOEL  on current intake
                                                             exceed the     substance or   for substance 
                                                             threshold for  are its        or related 
                                                             human intake?  metabolites    substance?
                                                                            endogenous?
                                                                                                                            
    2-Oxopentandioic acid                 634    328-50-7    No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 185

    3-Hydroxy-2-oxopropionic acid         635    1113-60-6   No             N/R            N/R            No safety concern

    CHEMICAL STRUCTURE 186
                                                                                                                            

    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, and all of the substances in the group are metabolized to innocuous products.

    a The threshold for human inatke of substances in class I is 1800 g per day.
    b Ethyl acetoacetate is expected to be hydrolysed to acetoacetic acid, which is endogenous in humans.
    c The ADI for this substance was maintained.
    d Fumaric acid, (-)-malic acid, and triethyl citrate are components of the tricarboxylic acid cycle.  
    

    Step A3.  The estimated daily  per capita intakes in Europe and the
              United States of 41 of the substances in this group are
              below the threshold of concern for substances in class I
              (1800 g), indicating that they would not raise concern for
              safety. The intakes of six substances, namely, ethyl
              acetoacetate (No. 595; 1900 g/person per day in Europe and
              3900 g/person per day in the United States), fumaric acid
              (No. 618; 220 000 g/person per day in the United States);
              (-)-malic acid (No. 619; 16 000 g/person per day in Europe
              and 58 000 g/person per day in the United States), tartaric
              acid (No. 621; 4400 g/person per day in Europe and 14 000
              g/person per day in the United States), adipic acid (No.
              623; 18 000 g/person per day in the United States), and
              triethyl citrate (No. 629; 3400 g/person per day in Europe
              and 2400 g/person per day in the United States), are
              greater than the threshold for human intake for class I
              (1800 g). The evaluation of the safety of these six
              substances therefore proceeds to step A4.

    Step A4.  Four of the six substances for which the intake exceeds the
              threshold of concern for class I are endogenous in humans.
              Three of these four substances, namely, fumaric acid (No.
              618), (-)-malic acid (No. 619), and triethyl citrate (No.
              629), are components of the tricarboxylic acid cycle. The
              fourth substance, ethyl acetoacetate (No. 595), is expected
              to be hydrolysed to acetoacetic acid, which is endogenous in
              humans and is formed from the condensation of two acetyl
              coenzyme A units in the fatty acid pathway. For tartaric
              acid and adipic acid, the evaluation should proceed to
              step A5.

    Step A5.  The NOEL for tartaric acid in a two-year study of toxicity
              in rats was 1200 mg/kg bw per day, the highest dose tested,
              which provides adequate margins of safety (> 10 000 and
              > 1000) for the known levels of intake (74 and 230 g/kg bw
              per day in Europe and the United States, respectively). No
              NOEL was available for adipic acid, but the NOEL for the
              structurally related material, dibutyl sebacate, in a
              two-year study in rats was 6200 mg/kg bw per day, which
              provides adequate margins of safety (> 100 000 000 and >
              10 000 times) for the known levels of intake of adipic acid
              (0.2 and 300 g/kg bw per day in Europe and the United
              States, respectively). These substances would not therefore
              be expected to raise concern.

         Table 1 summarizes the stepwise evaluation of the 47 aliphatic
    primary alcohols, aldehydes, carboxylic acids, acetals, and esters
    containing additional oxygenated functional groups used as flavouring
    agents.

    1.5  Consideration of combined intake

         All of the 47 aliphatic primary alcohols, aldehydes, carboxylic
    acids, acetals, and esters containing additional oxygenated functional
    groups that were evaluated would be efficiently metabolized by common
    biochemical pathways to innocuous substances.

         In the unlikely event that foods containing all 47 substances
    were consumed simultaneously on a daily basis, the total estimated
    daily per capita intake of these substances in Europe and the United
    States would exceed the threshold for human intake of substances in
    class I. The Committee considered that such intake would not give rise
    to perturbations outside the physiological range.

    1.6  Conclusions

         The Committee concluded that the safety of flavouring agents in
    this group would not raise concern when they were used at he current
    levels of estimated intake.

         No data on toxicity were available for application of the
    Procedure to 45 of the 47 substances in this group. For the remaining
    two substances, tartaric acid (No. 621) and adipic acid (No. 623), the
    data on toxicity were consistent with the results of the safety
    evaluation made with the Procedure.

         The ADIs for fumaric acid and its salts and for triethyl citrate
    were maintained at the present meeting.

    2.  RELEVANT BACKGROUND INFORMATION

    2.1  Explanation

         Forty-seven aliphatic primary alcohols, aldehydes, carboxylic
    acids, acetals, and esters containing additional oxygenated functional
    groups are included in this group of flavouring agents (see Table 1).
    The substances were selected on the basis of the criteria that all
    members of the group are simple aliphatic primary alcohols, aldehydes,
    carboxylic acids, acetals, and esters and contain additional
    oxygenated functional groups. Eight substances in this group (Nos 589,
    591, 603, 631-635) are alpha-keto acids, esters, or related
    substances; five substances (Nos 590, 619-622) are alpha-hydroxy
    acids, esters, or related substances; 12 substances (Nos 593-602, 614,
    615) are beta-keto or beta-hydroxy alcohols, aldehydes, carboxylic
    acids, and related acetals and esters; five substances (Nos 605-609)
    are gamma-keto acids, esters, or related substances; four substances
    (Nos 610-613) are omega-substituted alcohols, aldehydes, or acetals;
    and 22 substances (Nos 614-631) are simple, aliphatic di-and
    tricarboxylic acids or their esters.

    2.2  Additonal considerations on intake

         The total annual production of each of the 47 substances in this
    group is shown in Table 2. 

    2.3  Biological data

    2.3.1  Absorption, metabolism, and elimination

    2.3.1.1  Ester and diesters

         Twenty-eight substances in this group (Nos 590, 591, 594-603,
    605, 607-609, 614-617, 620, 622, 624-626, and 628-630) are esters or
    diesters, including one cyclic diester, which are expected to undergo
    hydrolysis to their corresponding alcohol (saturated linear or
    branched-chain aliphatic primary alcohols or branched-chain hydroxy or
    keto alcohols) and acid components (alpha, beta-, or gamma-keto or
    hydroxy acids or simple aliphatic acids, diacids, or triacids), which
    would be further metabolized. Hydrolysis occurs in the intestinal
    tract, blood, and liver and in most tissues and is catalysed by
    carboxylesterases or esterases, the most important of which are the
    B-esterases (Anders, 1989; Heymann, 1980). Acetyl esters are the
    preferred substrates of C-esterases (Heymann, 1980). The presence of a
    second oxygenated functional group has little if any effect on
    hydrolysis of these esters. 

         Evidence for hydrolysis of these esters has come from various
    experiments. Incubation of aqueous methyl 2-oxo-3-methylpentanoate
    (No. 591) with a 2% pancreatin solution (pH 7.5) resulted in virtually
    complete hydrolysis (> 98%) within 80 min (Leegwater & Van Straten,
    1979). Dibutyl sebacate (No. 625) in 10% acacia solution was also
    hydrolysed  in vitro in a 10% crude pancreatic lipase solution
    (Smith, 1953). 14C-Tributylacetyl citrate (No. 630) administered to
    male Sprague-Dawley rats by gavage at a dose of 70 mg/kg bw was
    rapidly absorbed (half-life, 1 h) and partially hydrolysed. More than
    87% of the radiolabel was eliminated within 24 h of dosing. At least
    nine urinary metabolites representing 59-70% of the dose were
    detected. Five were identified as the partially hydrolysed mono-, di-,
    and trialkylesters of citric acid. Three metabolites representing
    25-26% of the dose were identified in the faeces. Approximately 2% was
    eliminated as 14CO2 (Hiser et al., 1992). Hydrolysis of the cyclic
    diester ethylene brassylate (No. 626) would be expected to occur on
    the basis of the hydrolysis of structurally related lactones like
    omega-6-hexadecenlactone. In simulated intestinal fluid,
    omega-6-hexadecenlactone underwent nearly complete hydrolysis (92%) to
    its open-chain form within 15 min (Morgareidge, 1962a).

         The alcohol, aldehyde, and acid components of these esters,
    diesters, and cyclic diester are completely metabolized. At higher
    concentrations, they may be conjugated with glucuronic acid and
    excreted. 

    2.3.1.2  alpha-Keto-and alpha-hydroxy acids and their esters

         alpha-Keto-and alpha-hydroxyacids and their esters (Nos 589-591,
    603, 631-635) would be expected to be metabolized in the same way as
    endogenous alpha-ketoacids formed from oxidative deamination of amino
    acids, such as isoleucine, methionine, and valine,  in vivo.
    2-Oxobutyric acid (alpha-ketobutyric acid, No. 589) is produced
    endogenously in humans as a product of methionine degradation and
    undergoes alpha-decarboxylation to yield propionyl-coenzyme A, which

    ultimately enters the tricarboxylic acid cycle as succinyl-coenzyme A.
    Nos 631-635 are intermediates formed endogenously from the oxidative
    deamination of valine, isoleucine, leucine, glutamic acid, and serine,
    respectively (Voet & Voet, 1990).

    2.3.1.3  Acetals

         Three substances in this group are acetals (Nos 593, 612, and
    613), which are likely to undergo uncatalysed hydrolysis  in vivo to
    yield their component aldehydes and alcohols. 3-Oxobutanal dimethyl
    acetal (No. 593) would be expected to undergo hydrolysis to yield
    methanol and acetoacetaldehyde, which may be oxidized to acetoacetic
    acid. More than 99% of hydroxycitronellal dimethyl acetal (No. 612)
    was hydrolysed to the terpenoid hydroxycitronellal and methanol in
    simulated gastric juice (pH 2.1) after 1 h, and > 6% was hydrolysed
    in intestinal fluid (pH 7.5) after 2 h (Morgareidge, 1962b).
    Hydroxy-citronellal diethyl acetal (No. 613) would be expected to
    undergo similar metabolism.

    2.3.1.5  beta-Keto-and beta-hydroxy acids and their esters

         Esters of beta-keto or beta-hydroxy acids (Nos 594-603, 605) are
    hydrolysed to acetoacetic acid or its beta-hydroxy or aldehyde
    precursor. The last two can be oxidized  in vivo to acetoacetic acid,
    which is endogenous in humans and is formed from the condensation of
    two acetyl coenzyme A units in the fatty acid pathway. It is released
    from the liver into the bloodstream and transported to peripheral
    tissues, where it is converted to acetyl coenzyme A and is completely
    metabolized. When the endogenous levels are high, beta-ketoacids may
    undergo non-enzymatic decarboxylation, which for acetoacetic acid
    yields acetone and carbon ioxide (Voet & Voet, 1990).

    2.3.1.6  gamma-Keto and gamma-hydroxy acids and their esters

         Small amounts of gamma-hydroxy and gamma-keto acids and related
    substances (Nos 606-609) are expected to be completely metabolized to
    carbon dioxide. With greater exposure, the ketone function may be
    reduced to the corresponding secondary alcohol (Bosron & Ting-Kai,
    1980) and excreted as the glucuronic acid conjugate (Williams, 1959).
    Products of partial beta-oxidation or glucuronic acid conjugation have
    been identified in the urine. For example, a 1-g dose of the
    structurally related substance gamma-hydroxybutyrate was excreted in
    human urine unchanged and as S-3,4-dihydroxybutyrate and glycolate
    (Lee, 1977).

    2.3.1.7  omega-Substituted derivatives

         omega-Substituted derivatives (Nos 610-613) may undergo complete
    oxidation or conjugation with glucuronic acid and are then excreted
    primarily in the urine. Products of incomplete oxidation and reduction
    have also been observed. In rabbits, orally administered
    hydroxycitronellal (No. 611) is reduced to hydroxy-citronellol (No.
    610) and oxidized to hydroxycitronellic acid, both of which are
    excreted in the urine (Ishida et al., 1989).

    2.3.1.8  Aliphatic di- and tricarboxylic acids and their esters

         The simple aliphatic di- and tricarboxylic acids either occur
    endogenously in humans (Nos 618, 619, 627, and 634) or are
    structurally related to endogenous substances (Nos 621-626, and 630).
    The esters of these acids (616, 617, 620, 628, and 629) are
    hydrolysed, as discussed above. Succinic acid, derived from the esters
    (Nos 616 and 617), fumaric acid (No. 618), (-)-malic acid (No. 619),
    aconitic acid (No. 627), citric acid derived from triethyl citrate
    (No. 629), and 2-oxopentandioic acid (No. 634) are components of the
    tricarboxylic acid cycle (Voet & Voet, 1990). Fumaric acid is present
    in the blood, brain, liver, muscle, and kidney of normal rats
    (Marshall et al., 1949), and citric, tartaric, malic, aconitic,
    fumaric, and adipic acids are present in adult human urine (Osteux &
    Laturaze, 1954). alpha-Ketoglutaric acid is an intermediate metabolite
    of citric acid, fumaric acid, and succinic acid and is formed by
    alpha-oxidation (Krebs et al., 1938; Simola & Krusius, 1938).

         Simple aliphatic di-and tricarboxylic acids and their esters (Nos
    614-635) are metabolized (after hydrolysis in the case of esters) in
    the fatty acid beta-oxidation pathway or tricarboxylic acid cycle.
    When 14C-labelled (-)-malic acid (No. 619) was administered to male
    albino Wistar rats by gavage at a dose of 2.5 mg/kg bw, 93% of the
    radiolabel was recovered in expired air, urine, and faeces (Daniel,
    1969). Radiolabelled adipic acid fed to rats by stomach tube at a dose
    of 200-300 mg/kg bw was partially or completely metabolized, and the
    radiolabelled products identified in the urine included glutamic acid,
    lactic acid, beta-ketoadipic acid, and citric acid. The presence of
    the beta-oxidation metabolite beta-ketoadipic acid indicates that
    adipic acid participates in beta-oxidation in the fatty acid pathway
    (Rusoff et al., 1960).

         The linear and branched-chain aliphatic primary alcohol
    components would be oxidized in the presence of alcohol dehydrogenase
    to their corresponding aldehydes which, in turn, would be oxidized to
    their corresponding carboxylic acids (Bosron & Ting-Kai, 1980; Levi &
    Hodgson, 1989; Feldman & Weiner, 1972). The resulting carboxylic acids
    would be metabolized in the fatty acid pathway and tricarboxylic acid
    cycle (Voet & Voet, 1990). Branched-chain diols or keto alcohols may
    undergo oxidation to their corresponding aldehydes and carboxylic
    acid, which would be further metabolized or excreted.

    2.4  Toxicological studies

    2.4.1  Acute toxicity

         The available data on this group of aliphatic primary alcohols,
    aldehydes, carboxylic acids, acetals, and esters which contain
    additional oxygenated functional groups demonstrate that they have
    little acute toxicity when given orally. Oral LD50 values have been
    reported for 29 of the 47 substances in the group; these range from
    1628 to > 34 000 mg/kg bw in male and female rats and from 1900 to >
    31 000 mg/kg bw in male and female mice (Smyth et al., 1949, 1951;
    Smith, 1953; Smyth et al., 1954; Horn et al., 1957; Finkelstein &
    Gold, 1959; Wolven & Leverstein, 1962; Jenner et al., 1964;

    Levenstein, 1969; Smyth et al., 1969; Hart & Wong, 1971; Levenstein,
    1973; Moreno, 1973; Pellmont, 1973; Shelanski & Moldovan, 1973;
    Lawrence et al., 1974; Moreno, 1976, 1977; Vernot et al., 1977;
    Moreno, 1978; Pellmont, 1978; Moreno et al., 1979; Moreno, 1980;
    Levenstein, 1981; Hoechst, 1995).

    2.4.2  Short-term and long-term studies of toxicity

         The results of short-term and long-term studies of the toxicity
    of the substances in this group are shown in Table 3. Details of the
    studies which were critical to the evaluation of the safety of
    tartaric acid and adipic acid are given below.

    2.4.2.1  Tartaric acid (No. 621)

         Rats

         The toxicity of fumaric, tartaric, oxalic, and maleic acids was
    compared in groups of 12 weanling Osborne-Mendel rats of each sex,
    with 24 of each sex in the control group. The animals were given diets
    containing tartaric or fumaric acid at concentrations of 0, 0.1, 0.5,
    0.8, or 1.2%, equivalent to 100, 500, 800, or 1200 mg/kg bw per day.
    The mortality rates in treated groups were not different from those of
    controls, and there was no statistically significant difference in
    body-weight gain or weekly food consumption. Necropsy performed on
    most animals at two years did not reveal any macroscopic changes.
    Histopathological examination of a wide range of tissues revealed no
    treatment-related changes. The NOEL was 1200 mg/kg bw per day
    (Fitzhugh & Nelson, 1947).

         Rabbits

         In a study of the toxicity of citric, fumaric, and tartaric
    acids, 15 New Zealand rabbits (sex not specified) weighing 1-3 kg were
    given the sodium salt of tartaric acid in the diet at a concentration
    of 7.7% for 150 days, equivalent to 2300 mg/kg bw per day. A control
    group was fed ground diet alone. Each animal was examined daily, and
    food intake and body weights were determined weekly. Haematological
    and urinary analyses were performed after 60 days of treatment on five
    treated and six control rabbits. Two animals were examined grossly 30
    days after treatment, and one animal was examined after 60 days. The
    testis was examined histologically. At 100 days, half of the surviving
    rabbits were examined grossly, and the liver, kidney, and testis were
    examined microscopically. At the end of the study at 150 days, all
    animals were killed and examined grossly and histologically.
    Haematological and urinary analyses showed no changes. No significant
    gross or histopathological changes attributable to tartaric acid were
    observed (Packman et al., 1963).


        Table 3.  Results of short-term and long-term studies of the toxicity of aliphatic
    primary alcohols, aldehydes, carboxylic acids, acetals, and esters with additional
    oxygenated functional groups
                                                                                                                          

    No.   Substance             Species     Sex   No. test      Route     Duration     NOEL         Reference
                                                  groupsa/no.                          (mg/kg bw
                                                  per test                             per day)
                                                  groupb
                                                                                                                          

    595   Ethyl acetoacetate    Rat         M/F   3/32          Diet      28-29 days   300          Cook et al. (1992)

    606   Laevulinic acid       Rat         NR    2/3           Diet      16 days      1000         Tischer et al. (1942)

    611   Hydroxycitronellal    Rat         M/F   2/20, 2/60    Diet      2 years      250          Bar & Griepentrog 
                                                                                                    (1967)

    614   Diethyl malonate      Rat         M/F   2/20          Diet      13 weeks     < 500c,d     Posternak (1964)

    614   Diethyl malonate      Rat         M/F   2/20-32       Diet      90 days      406          Posternak et al. 
                                                                                                    (1969)

    618   Fumaric acid          Rat         M/F   8/12          Diet      2 years      1200         Fitzhugh & Nelson 
                                                                                                    (1947)

    618   Fumaric acid          Rat         NR    2/14, 14/20   Diet      2 years      1380         Levey et al. (1946)

    618   Fumaric acid          Guinea-pig  M/F   NR            Diet      1 year       400          Levey et al. (1946)

    618   Fumaric acide         Rabbit      NR    3/15          Diet      150 days     2070         Packman et al. (1963)

    621   Tartaric acid         Rat         M/F   8/12          Diet      2 years      1200         Fitzhugh & Nelson 
                                                                                                    (1947)

    621   Tartaric acide        Rabbit      NR    3/15          Diet      150 days     2300c        Packman et al. 
                                                                                                    (1963)

    621   Tartaric acid         Dog         NR    1/4           Oral      90-114 days  < 990c       Krop et al. (1945)

    Table 3. (continued)
                                                                                                                          

    No.   Substance             Species     Sex   No. test      Route     Duration     NOEL         Reference
                                                  groupsa/no.                          (mg/kg bw
                                                  per test                             per day)
                                                  groupb
                                                                                                                          
    624   Diethyl sebacate      Rat         M/F   2/10          Diet      17-18 or     1000         Hagan et al. (1967)
                                                                          27-28 
                                                                          weeks

    625   Dibutyl sebacate      Rat         M     4/10          Diet      1 year       1250         Smith (1953)

    625   Dibutyl sebacate      Rat         M     5/16          Diet      2 years      6250         Smith (1953)

    629   Triethyl citrate      Rat         M/F   3/7           Diet      2 months     4000         Finkelstein 
                                                                                                    & Gold (1959)

    629   Triethyl citrate      Cat         NR    1/6           Gavage    2 months     < 285        Finkelstein 
                                                                                                    & Gold (1959)

    630   Tributyl              Rat         M/F   2/4           Diet      2 months     5000         Finkelstein 
          acetylcitrate                                                                             & Gold (1959)

    630   Tributyl              Cat         NR    1/2           Gavage    2 months     < 5700c      Finkelstein 
          acetylcitrate                                                                             & Gold (1959)
                                                                                                                          

    M, male; F, female; NR, not reported
    a  Number of test groups does not include controls.
    b  Number per test group comprises male and female animals.
    c  Only one dose tested
    d  Changes in relative liver weight and glomerular and renal tubular histological appearance observed
    e  Administered as the sodium salt
    

         Dogs

         As part of a comparison of the toxicity of hydroxyacetic acid,
    citric acid, and tartaric acid, four dogs (sex not specified) received
    tartaric acid daily in a gelatin capsule at a dose of 990 mg/kg bw per
    day for periods of 90 to 114 days. The changes in body weight varied
    from a 30% gain to a 32% loss. Haematological and urinary parameters
    were examined. Urinary casts (gelled protein) were observed in all
    dogs and were graded as hyaline (clear) in three dogs. Blood chemical
    parameters remained normal except in one dog which showed azotaemia
    (increased concentrations of urea in the blood) and died at 90 days,
    according to the authors due to nephrotoxicity. There was no NOEL
    (Krop et al., 1945).

    2.4.2.2  Diethyl sebacate (No. 624)

         Rats

         In a study of the toxicity of about 50 flavouring agents, groups
    of five weanling Osborne-Mendel rats of each sex were fed diethyl
    sebacate (referred to in the paper as ethyl sebacate) at a dietary
    concentration of 1000 mg/kg for 27-28 weeks or 10 000 mg/kg for 17-18
    weeks, equivalent to 100 and 1000 mg/kg bw per day. A group of 10
    males and 10 females served as controls. Body weights, food intake,
    and general condition were recorded weekly, and haematological
    examinations were performed at the end of the study. All tissues were
    examined grossly at necropsy. The livers, kidneys, spleens, hearts,
    and testes from six controls and eight animals at the high dose,
    evenly divided by sex, were weighed and examined microscopically.
    There was no difference in growth rate or food consumption between
    test and control animals, and haematological examination revealed
    normal values. No macroscopic or microscopic changes were observed in
    the tissues. The NOEL was 1000 mg/kg bw per day (Hagan et al., 1967).

    2.4.2.3  Dibutyl sebacate (No. 625)

         Rats

         Groups of 10 male Sprague-Dawley rats, five weeks old, were fed
    dibutyl sebacate at dietary concentrations of 0, 0.01, 0.05, 0.25, or
    1.25%, equivalent to 0, 10, 50, 250, and 1250 mg/kg bw per day, for
    one year. Body weight and food intake were measured periodically
    throughout the study. Measurement of haematological parameters and
    microscopic examination at necropsy revealed no adverse effects
    (Smith, 1953).

         Groups of 16 five-to six-week-old male Sprague-Dawley rats were
    given dibutyl sebacate in the diet at concentrations of 0 (two control
    groups), 0.01, 0.05, 0.25, 1.25, or 6.25%, equivalent to 0, 10, 50,
    250, 1250, and 6250 mg/kg bw per day, for two years. Administration of
    dibutyl sebacate did not adversely affect the growth or survival of
    the animals. Body weight and food intake were measured periodically
    throughout the study. Measurement of haematological parameters and
    microscopic examination at necropsy revealed no adverse effects. The
    lesions observed in older control and treated rats at necropsy
    included inflammatory changes in the lungs, enlarged and discoloured

    kidneys, and fatty changes in the liver. The incidence of these gross
    lesions was not considered to be associated with the administration of
    dibutyl sebacate. The NOEL was 6250 mg/kg bw per day (Smith, 1953). 

    2.4.4  Genotoxicity

         The results of tests for the genotoxicity of substances in this
    group are shown in Table 4. 

    2.4.5  Other relevant studies

    2.4.5.1  Adipic acid (No. 623)

         In a study of teratogenicity, groups of 20-24 pregnant rats were
    given adipic acid by oral intubation on days 6-15 of gestation at
    doses of 0, 3, 13, 62, or 288 mg/kg bw per day. A sixth group of 24
    pregnant females was given aspirin at a dose of 250 mg/kg bw per day
    as a positive control. The maternal parameters evaluated included
    clinical signs of toxicity, body weight, and food consumption. The
    fetuses were removed surgically from all dams on day 20. The numbers
    of implantation sites, resorption sites, and live births were counted,
    and the body weights of live pups and external, visceral, and skeletal
    abnormalities were evaluated. Administration of adipic acid had no
    adverse effect on the maternal parameters evaluated, nor did it
    adversely affect fetal survival or the number of abnormalities in soft
    or skeletal tissues (Morgareidge, 1973).

         In a study of potential peroxisome proliferation, male Fischer
    344 rats were fed adipic acid at a dietary concentration of 2%,
    equivalent to about 2000 mg/kg bw per day, for three weeks. Control
    animals received powdered Purina rat chow alone. No effect on hepatic
    peroxisomes or their associated enzymes was observed in treated
    animals (Moody & Reddy, 1978). 

    2.4.5.2  Tartaric acid (No. 621)

         The potential immunotoxicity of tartaric acid was evaluated in a
    rapid screening protocol in which groups of 10-20 female CD1 or
    B6C3F1 mice were given the material orally at doses up to 3000 mg/kg
    bw per day (doses not specified) for five days. A group of control
    animals was also evaluated. The animals received an infectious
    challenge on day 3 of dosing and immunization on day 5, and the
    antibody plaque-forming cell response was measured four days later.
    Deaths and survival were monitored for 10 days after infection. There
    were no statistically significant differences in spleen weight, thymus
    weight, spleen cellularity, anti-sheep red blood cell or
    plaque-forming cell response, or death due to  Listeria infection
    between test and control animals (Vollmuth et al., 1989).


        Table 4. Results of studies of the genotoxicity of aliphatic primary alcohols, aldehydes, carboxylic acids,
    acetals, and esters with additional oxygenated functional groups
                                                                                                                                 

    No.  Substance             End-point         Test system               Concentration        Results       Reference
                                                                                                                                 

    595  Ethyl acetoacetate    Gene mutation     B. subtilis               20 mg/disc           Negative      Oda et al. 
                                                 H17, M45 rec+/-                                              (1978)

    595  Ethyl acetoacetate    Gene mutation     B. subtilis               20 ml/disc           Positive      Yoo (1986)
                                                 H17, M45 rec+/-

    595  Ethyl acetoacetate    Gene mutation     E. coli WP2 uvrA          25-320 mg/plate      Positive      Yoo (1986)

    595  Ethyl acetoacetate    Gene mutation     B. subtilis               10-20 ml/ml          Weakly        Kuroda et al. 
                                                 H17, M45 rec+/-                                positive      (1984)
                                                 (test tube)               

    595  Ethyl acetoacetate    Chromosomal       Chinese hamster           2 mg/ml              Negative      Ishidate et al. 
                               aberration        cells                                                        (1984)

    595  Ethyl acetoacetate    Gene mutation     S. typhimurium            25 mg/plate          Negativea     Ishidate et al. 
                                                 TA92, TA1535, TA100,                                         (1984)
                                                 TA1537, TA94, TA98 
                                                 (preincubation
                                                 protocol)

    595  Ethyl acetoacetate    Gene mutation     S. typhimurium TA97,      0.01-10 mg/plate     Negativea     Fujita & Sasaki 
                                                 TA102 (preincubation                                         (1987)
                                                 protocol)

    610  Hydroxycitronellol    Gene mutation     S. typhimurium TA1535,    3.6 mg/plate         Negativea     Wild et al. 
                                                 TA100, TA1537, TA1538,                                       (1983)
                                                 TA98

    610  Hydroxycitronellol    Micronucleus      Mouse                     1204 mg/kg bw        Negative      Wild et al. 
                               formation                                                                      (1983)

    610  Hydroxycitronellol    Gene mutation     D. melanogaster           10 mmol/L            Negative      Wild et al. 
                                                                                                              (1983)

    611  Hydroxycitronellal    Gene mutation     S. typhimurium TA1535,    3.6 mg/plate         Negativea     Wild et al. 
                                                 TA100, TA1537, TA1538,                                       (1983)
                                                 TA98

    Table 4. (continued)
                                                                                                                                 

    No.  Substance             End-point         Test system               Concentration        Results       Reference
                                                                                                                                 

    611  Hydoxycitronellal     Micronucleus      Mouse                     861 mg/kg bw         Negative      Wild et al.
                               formation                                                                      (1983)

    611  Hydoxycitronellal     Gene mutation     D. melanogaster           37 mmol/L            Negative      Wild et al. 
                                                                                                              (1983)

    612  Hydroxycitronellal    Gene mutation     S. typhimurium TA1535,    3.6 mg/plate         Negativea     Wild et al. 
         dimethyl acetal                         TA100, TA1537, TA1538,                                       (1983)
                                                 TA98

    612  Hydroxycitronellal    Micronucleus      Mouse                     763 mg/kg bw         Negative      Wild et al. 
         dimethyl acetal       formation                                                                      (1983)

    612  Hydroxycitronellal    Gene mutation     D. melanogaster           25 mmol/L            Negative      Wild et al. 
         dimethyl acetal                                                                                      (1983)

    614  Diethyl malonate      Gene mutation     S. typhimurium TA98,      3 mmol/plate         Negativea     Florin et al. 
                                                 TA100, TA1535, TA1537     (480 mg/plate)b                    (1980)

    616  Dimethyl succinate    Gene mutation     S. typhimurium TA100,     20 000 mg/plate      Negativea     Andersen & Jensen 
                                                 TA1535, TA1537, TA98                                         (1984)

    616  Dimethyl succinate    Gene mutation     S. typhimurium TA97,      10 mg/plate          Negativea     Zeiger et al.
                                                 TA98, TA102, TA104,                                          (1992)
                                                 TA1535, TA1538

    618  Fumaric acid          Gene mutation     S. typhimurium TA100      1000 mg/plate        Negativea     Rapson et al. 
                                                                                                              (1980)

    618  Fumaric acid          Gene mutation     S. typhimurium TA98,      2000 mg/plate        Negative      Zeiger et al. 
                                                 TA100, TA1535, TA97                                          (1988)
                                                 (preincubation
                                                 protocol)

    619  (-)-Malic acid        Gene mutation     S. typhimurium TA97,      2000 mg/plate        Negativea     Al-Ani & Al-Lami 
                                                 TA98, TA100, TA104                                           (1988)

    Table 4. (continued)
                                                                                                                                 

    No.  Substance             End-point         Test system               Concentration        Results       Reference
                                                                                                                                 

    623  Adipic acid           Gene mutation     E. coli WP2 uvrA          5000 mg/plate        Negativea     Shimizu et al. 
                                                                                                              (1985)

    623  Adipic acid           Gene mutation     S. typhimurium TA100,     5000 mg/plate        Negativea     Shimizu et al. 
                                                 TA98,                                                        (1985)

    623  Adipic acid           Gene mutation     D. melanogaster           4000 ppm             Negative      Ramel & Magnusson 
                                                                                                              (1979)

    625  Dibutyl sebacate      Gene mutation     S. typhimurium TA1535,    3.6 mg/plate         Negativea     Wild et al. 
                                                 TA100, TA1537, TA1538,                                       (1983)
                                                 TA98

    625  Dibutyl sebacate      Micronucleus      Mouse                     2829 mg/kg bw        Negative      Wild et al. 
                               formation                                                                      (1983)

    625  Dibutyl sebacate      Gene mutation     D. melanogaster           19 mmol/L            Negative      Wild et al. 
                                                                                                              (1983)

    626  Ethylene brassylate   Gene mutation     S. typhimurium TA1535,    3.6 mg/plate         Negativea     Wild et al. 
                                                 TA100, TA1537, TA1538,                                       (1983)
                                                 TA98

    627  Aconitic acid         Gene mutation     S. typhimurium TA100,     20 000 mg/plate      Negativea     Andersen & Jensen
                                                 TA1535, TA1537, TA98                                         (1984)
                                                                                                                                 

    a  With and without metabolic activation
    b  Calculation based on relative molecular mass of 160.17
    

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    See Also:
       Toxicological Abbreviations