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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

    ALIPHATIC ACYCLIC AND ALICYCLIC TERPENOID TERTIARY ALCOHOLS AND
    STRUCTURALLY RELATED SUBSTANCES

    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 23 flavouring agents that include
    selected tertiary alcohols 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 two members of the group previously.
    Linalool and linalyl acetate were evaluated with citral, citronellol,
    and geranyl acetate at the eleventh meeting (Annex 1, reference 14).
    The Committee recommended that at least one member of the group be
    studied for effects after long-term exposure. Linalool and linalyl
    acetate were re-evaluated at the twenty-third meeting (Annex 1,
    reference 50). A group ADI of 0-0.5 mg/kg bw was established on the
    basis of the clearly defined metabolism of these substances, their
    rapid excretion, and their low toxicity in short-term studies.

         At the forty-ninth meeting, the Committee evaluated a group of 26
    geranyl, neryl, citronellyl, and rhodinyl esters formed from
    branched-chain terpenoid primary alcohols and aliphatic acyclic linear
    and branched-chain carboxylic acids using the procedure for the safety
    evaluation of flavouring agents. The Committee concluded that these
    substances pose no safety concerns on the basis of knowledge of their
    metabolism and low levels of intake. 

    1.2  Estimated daily per capita intake

         The total annual production volume of the 23 tertiary alcohols
    and related esters is approximately 58 000 kg in Europe (International
    Organization of the Flavor Industry, 1995) and 15 000 kg in the United
    States (US National Academy of Sciences, 1987). Four substances in the
    group, linalool (No. 356), linalyl acetate (No. 359), alpha-terpineol
    (No. 366), and terpinyl acetate (No. 368) account for approximately
    96% of the total annual volume in Europe and the United States. On the
    basis of the reported annual volumes, the total estimated daily
     per capita intake of linalool from its use and that of eight of its
    esters as flavouring agents is about 4300 g/person per day (72 g/kg
    bw per day) in Europe and 1300 g/person per day (21 g/kg bw per day)
    in the United States. Similarly, the total estimated daily  per 
     capita intake of alpha-terpineol from its use of and that of six of
    its esters as flavouring agents is about 3200 g/person per day (53
    g/kg bw per day) in Europe and 1400 g/person per day (23 g/kg bw
    per day) in the United States. 

         Tertiary alcohols and related esters occur naturally in a wide
    variety of foods, including fruits, spices, and tea. Thirteen of the
    substances in this group have been reported to occur naturally in
    foods (Maarse et al., 1994).

    1.3  Absorption, metabolism, and elimination 

         The esters in this group would be readily hydrolysed to their
    component alcohols and carboxylic acids. The hydrolysis products would
    be detoxified primarily by conjugation with glucuronic acid and
    excreted in the urine. Alternatively, alcohols with unsaturation may
    be 4-oxidized at the allylic position to yield polar metabolites,
    which may be conjugated and excreted. Metabolites of acyclic alcohols
    may be further oxidized to eventually yield carbon dioxide. Ester
    hydrolysis and metabolism of the hydrolysis products are further
    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 1A.  All but one of the 23 terpenoid tertiary alcohols and
              related substances is classified in structural class I
              (Cramer et al., 1978). 2-Ethyl-1,3,3-trimethyl-2-norbornol
              (No. 440) is a non-terpene bicyclic tertiary alcohol and is
              therefore in structural class II.

    Step 2A.  Adequate data were available on the metabolism of both
              linalool and alpha-terpineol to allow prediction of the
              likely pathways of metabolism of all related compounds in
              the same group. Although the position of oxidative
              metabolism would differ between compounds, the structures of
              linalool and alpha-terpineol would cover all of the
              functional groups present in the other members of this group
              of flavouring agents. On the basis of the toxicity of
              linalool and alpha-terpineol, it was concluded that (with
              one exception) the metabolism of compounds in this group
              could be predicted to give rise to innocuous products.
              Methyl 1-acetoxycyclohexylketone (No. 442) contains a
              sterically hindered ketone group and cannot be considered
               a priori to be similar metabolically or toxicologically to
              other members of the group.

                   At current levels of intake, 22 of the substances in
              this group (21 in class I and one in class II) would not be
              expected to saturate the metabolic pathways, and these
              substances are predicted to be metabolized to innocuous
              products. The evaluation of these 22 substances thus
              proceeds via the left side ('A') of the evaluation scheme
              (steps A3-A5). Methyl 1-acetoxycyclohexyl-ketone (No. 442;
              class I) would not be predicted to be metabolized to
              innocuous products, so its evaluation proceeds via the right
              side ('B') of the scheme (steps B3-B5).

    Step A3.  Twenty-two substances were evaluated at this step. As the
              daily  per capita intakes in Europe and the United States
              of 18 of the 21 substances in class I are below the human
              intake threshold for class I (1800 g/person per day), these
              substances pose no safety concern when used as flavouring
              agents at their current levels of estimated intake. 

                   In Europe, the intakes of three substances, linalool
              (2600 g/person per day), linalyl acetate (2100 g/person
              per day), and alpha-terpineol (3000 g/person per day), are
              greater than 1800 g/person per day, and the total intakes
              of linalool (No. 356) and alpha-terpineol (No. 366) from
              their use and use of their esters (Nos 358-365 and 367-372,
              respectively) are 4300 g/person per day and 3200 g/person
              per day. Total intake in the United States of linalool and
              its esters is 1300 g/person per day, and that of
              alpha-terpineol and its esters is 1400 g/person per day.

                   Intake of the class II substance
              2-ethyl-1,3,3-trimethyl-2-norbornol is 1 g/person per day
              in Europe and 0.02 g/person per day in the United States --
              below the human intake threshold for class II (540 g/person
              per day), indicating that this substance does not pose a
              safety concern when used at current levels of estimated
              intake as a flavouring agent.

    Step A4.  Linalool, linalyl acetate, and alpha-terpineol are not
              endogenous in humans. 

    Step A5.  NOEL values of > 50 mg/kg bw per day for linalool and
              > 24 mg/kg bw per day for linalyl acetate have been found
              in 90-day studies in rats. These NOELs provide safety
              margins of > 1000, > 500, and > 500 for intake of
              linalool (19 or 44 g/kg bw per day), linalyl acetate (3 or
              35 g/kg bw per day), and total linalool (21 or 72 g/kg bw
              per day), respectively, in both Europe and the United
              States. A NOEL of > 500 mg/kg bw per day terpinyl acetate
              was found in a 20-week study in rats. This NOEL provides
              safety margins of > 10 000 and > 500 for intake of
              alpha-terpineol (18 or 50 g/kg bw per day) and total
              alpha-terpineol (23 or 53 g/kg bw per day), respectively,
              in both Europe and the United States. A bioassay of
              carcinogenicity was conducted in rats given a mixture of the
              terpenoid esters geranyl acetate and citronellyl acetate,
              which together with linalool and linalyl acetate belong to
              the group of terpenoid substances previously reviewed by the
              Committee (Annex 1, reference 50). The NOEL of 1000 mg/kg bw
              per day (710 mg/kg bw per day geranyl acetate and 290 mg/kg
              bw per day citronellyl acetate) for the mixture provides a
              safety margin of > 10 000 for total intake of linalool and
              of alpha-terpineol.

    Step B3.  The daily  per capita intake of the class I substance
              methyl 1-acetoxy-cyclohexylketone (No. 442) in the United
              States is 4 g/person per day; no intake data were reported
              for Europe. This intake is less than the human intake
              threshold for class I (1800 g/person per day).

    Step B4.  Adequate data were not available to determine a NOEL for
              methyl 1-acetoxycyclohexylketone or structurally related
              substances.

    Step B5.  The conditions of use of methyl 1-acetoxycyclohexylketone
              result in an intake greater than 1.5 g/day; therefore,
              according to the procedure, additional information on this
              substance is required for its evaluation.

         The stepwise evaluations of the 23 aliphatic acyclic and
    alicyclic terpenoid tertiary alcohols and structurally related
    flavouring agents in this group are summarized in Table 1.

    1.5  Consideration of combined intakes from use as flavouring agents

         The data were adequate to complete an evaluation of 22 tertiary
    alcohols and related esters in this group. In the unlikely event that
    all 22 of these substances were consumed simultaneously on a daily
    basis, the estimated combined intake would exceed the human intake
    threshold for class I. These 22 substances are, however, expected to
    be efficiently metabolized and would not saturate the metabolic

    pathways. The consideration of combined intakes excludes intake of
    methyl 1-acetoxycyclohexylketone because additional data on its
    toxicity are required in order for its safety to be evaluated.

         Consumption of four additional esters of linalool (linalyl
    anthranilate, linalyl benzoate, linalyl cinnamate, and linalyl
    phenylacetate) that were not evaluated as part of this group would
    contribute an additional 10 g/person per day (Europe) or 0.2
    g/person per day (United States) to the total intake of linalool.
    Similarly, exposure to an additional ester of alpha-terpineol,
    terpinyl cinnamate, would contribute an additional 0.008 g/person per
    day (Europe) or 0.5 g/person per day (United States) to the total
    intake of alpha-terpineol. The potential consumption of linalool and
    alpha-terpineol from these additional esters is minor and would not
    lead to saturation of metabolic pathways or alter the safety
    evaluation.

    1.6  Conclusions

         The Committee concluded that 22 of the 23 terpenoid tertiary
    alcohols and related substances in this group would not present safety
    concerns when used at current levels of intake as flavouring agents.
    Knowledge of the metabolism and toxicity of these flavouring agents
    was required for application of the procedure. The Committee noted
    that these and other available data are consistent with the results of
    the safety evaluation by the procedure. For one substance, methyl
    1-acetoxycyclohexylketone, the available metabolic data were
    inadequate to predict that it would be metabolized to innocuous
    products, a relevant NOEL was lacking, and intake exceeds 1.5 g per
    day. According to the procedure, the Committee concluded that
    additional data are required for an evaluation of methyl
    1-acetoxycyclohexylketone. All of the previously established ADIs were
    maintained at the present meeting.


    2.  RELEVANT BACKGROUND INFORMATION

    2.1  Explanation

         This monograph summarizes the key data relevant to the safety
    evaluation of 23 tertiary alcohols and structurally related substances
    (see Table 1). The group comprises the aliphatic unsaturated tertiary
    alcohol linalool (No. 356) and eight esters of linalool (Nos 358-365);
    the saturated homologue of linalool, tetrahydrolinalool (No. 357); the
    alicyclic unsaturated tertiary alcohol alpha-terpineol (No. 366) and
    six esters of alpha-terpineol (Nos 367-372); three unsaturated
    alicyclic tertiary alcohols (Nos 373, 374, and 439); two saturated
    bicyclic tertiary alcohols (Nos 440 and 441); and a saturated tertiary
    ketoester (No. 442). The chemical structures of these substances are
    given in Table 1.


        Table 1. Results of safety evaluations of aliphatic acyclic and alicyclic terpenoid tertiary alcohols and structurally related substances

    Step 1: All of the substances in the group are in structural class I, except for substance 440, which is in class II. 
    The human intake threshold is 1800 g/day for class I and 540 g/day for class II substances.
    Step 2: All of the substances in this group are predicted to be metabolized to innocuous products, except methyl 1-acetoxycyclohexylketone.

                                                                                                                                              

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

    Linaloola            356   78-70-6    2600/1100       Yes         No          Yes. The dose of     Metabolized primarily      No safety
                                                                                  50 mg/kg bw per      by conjugation with        concern
                                                                                  day that had no      glucuronic acid and 
                                                                                  adverse effects in   excreted in urine. 
                                                                                  a safety evaluation  Oxidation of the allylic 
                                                                                  (Oser, 1967) is      methyl group may occur 
                                                                                  > 1000 times the     after repeated 
                                                                                  intake of linalool.  exposure.
    CHEMICAL STRUCTURE 

    Tetrahydrolinalool   357   78-69-3    55/0.1          No          N/R         N/R                  See linalool.              No safety 
                                                                                                                                  concern

    Table 1. (continued)
                                                                                                                                              
    Substance            No.   CAS No.    Estimated per   Step A3     Step A4     Step A5              Comments                   Conclusion
                                          capita intake,  Does        Is the      Adequate NOEL                                   based on
                                          Europe/USA      intake      substance   for substance                                   current 
                                          (g/day)        exceed      or its      or related                                      intake
                                          threshold?      human       metabolites
                                                          intake      substance?
                                                          endogenous?
                                                                                                                                              

    CHEMICAL STRUCTURE 

    Linalyl formate      358   115-99-1   8/13            No          N/R         N/R                  See linalyl acetate and    No safety 
                                                                                                       linalool.                  concern
    CHEMICAL STRUCTURE 

    Linalyl acetatea     359   115-95-7   2100/180        Yes         No          Yes. The dose of     Hydrolysed to linalool     No safety 
                                                                                  24 mg/kg bw per      and acetic acid. See       concern
                                                                                  day that had no      also linalool.
                                                                                  adverse effects in 
                                                                                  a safety evaluation
                                                                                  (Oser, 1967) is 
                                                                                  > 500 times the 
                                                                                  intake of linalyl 
                                                                                  acetate.
    CHEMICAL STRUCTURE 

    Table 1. (continued)
                                                                                                                                              
    Substance            No.   CAS No.    Estimated per   Step A3     Step A4     Step A5              Comments                   Conclusion
                                          capita intake,  Does        Is the      Adequate NOEL                                   based on
                                          Europe/USA      intake      substance   for substance                                   current 
                                          (g/day)        exceed      or its      or related                                      intake
                                          threshold?      human       metabolites
                                                          intake      substance?
                                                          endogenous?
                                                                                                                                              

    Linalyl propionate   360   144-39-8   16/2            No          N/R         N/R                  See linalyl acetate and    No safety 
                                                                                                       linalool.                  concern
    CHEMICAL STRUCTURE 

    Linalyl butyrate     361   78-36-4    10/4            No          N/R         N/R                  See linalyl acetate and    No safety 
                                                                                                       linalool.                  concern

    CHEMICAL STRUCTURE 

    Linalyl isobutyrate  362   78-35-3    36/1            No          N/R         N/R                  See linalyl acetate and    No safety
                                                                                                       linalool.                  concern
    CHEMICAL STRUCTURE 

    Table 1. (continued)
                                                                                                                                              
    Substance            No.   CAS No.    Estimated per   Step A3     Step A4     Step A5              Comments                   Conclusion
                                          capita intake,  Does        Is the      Adequate NOEL                                   based on
                                          Europe/USA      intake      substance   for substance                                   current 
                                          (g/day)        exceed      or its      or related                                      intake
                                          threshold?      human       metabolites
                                                          intake      substance?
                                                          endogenous?
                                                                                                                                              

    Linalyl isovalerate  363   1118-27-0  5/6             No          N/R         N/R                  See linalyl acetate and    No safety
                                                                                                       linalool.                  concern
    CHEMICAL STRUCTURE 

    Linalyl hexanoate    364   7779-23-9  1/0.4           No          N/R         N/R                  See linalyl acetate and    No safety
                                                                                                       linalool.                  concern
    CHEMICAL STRUCTURE 

    Linalyl octanoate    365   10024-64-3 0.1/1           No          N/R         N/R                  See linalyl acetate and    No safety 
                                                                                                       linalool.                  concern
    CHEMICAL STRUCTURE 

    Table 1. (continued)
                                                                                                                                              
    Substance            No.   CAS No.    Estimated per   Step A3     Step A4     Step A5              Comments                   Conclusion
                                          capita intake,  Does        Is the      Adequate NOEL                                   based on
                                          Europe/USA      intake      substance   for substance                                   current 
                                          (g/day)        exceed      or its      or related                                      intake
                                          threshold?      human       metabolites
                                                          intake      substance?
                                                          endogenous?
                                                                                                                                              

    alpha-Terpineol      366   98-55-5    3000/1100       Yes         No          Yes. The dose of     Metabolized primarily      No safety 
                                                                                  500 mg/kg bw per     by conjugation with        concern
                                                                                  day that had no      glucuronic acid and 
                                                                                  adverse effects      excreted in urine. 
                                                                                  (Hagan et al.,       Oxidation of the allylic 
                                                                                  1967) is > 5000      methyl group followed by 
                                                                                  times the intake     hydrogenation to yield the 
                                                                                  of the component     corresponding saturated 
                                                                                  alpha-terpineol.     acid may occur 
    CHEMICAL STRUCTURE 

    Terpinyl formate     367   2153-26-6  0.1/2           No          N/R         N/R                  See linalyl acetate and    No safety 
                                                                                                       alpha-terpineol.           concern
    CHEMICAL STRUCTURE 

    Terpinyl acetate     368   8007-35-0  260/390         No          N/R         N/R                  See linalyl acetate and    No safety 
                                                                                                       alpha-terpineol.           concern
    CHEMICAL STRUCTURE 


    Table 1. (continued)
                                                                                                                                              
    Substance            No.   CAS No.    Estimated per   Step A3     Step A4     Step A5              Comments                   Conclusion
                                          capita intake,  Does        Is the      Adequate NOEL                                   based on
                                          Europe/USA      intake      substance   for substance                                   current 
                                          (g/day)        exceed      or its      or related                                      intake
                                          threshold?      human       metabolites
                                                          intake      substance?
                                                          endogenous?
                                                                                                                                              

    Terpinyl propionate  369   80-27-3    0.03/1          No          N/R         N/R                  See linalyl acetate and    No safety 
                                                                                                       alpha-terpineol.           concern
    CHEMICAL STRUCTURE 

    Terpinyl butyrate    370   80-26-6    6/6             No          N/R         N/R                  See linalyl acetate and    No safety 
                                                                                                       alpha-terpineol.           concern
    CHEMICAL STRUCTURE 

    Terpinyl             371   7774-65-4  0.7/0.02        No          N/R         N/R                  See linalyl acetate and    No safety 
     isobutyrate                                                                                       alpha-terpineol.           concern
    CHEMICAL STRUCTURE 

    Table 1. (continued)
                                                                                                                                              
    Substance            No.   CAS No.    Estimated per   Step A3     Step A4     Step A5              Comments                   Conclusion
                                          capita intake,  Does        Is the      Adequate NOEL                                   based on
                                          Europe/USA      intake      substance   for substance                                   current 
                                          (g/day)        exceed      or its      or related                                      intake
                                          threshold?      human       metabolites
                                                          intake      substance?
                                                          endogenous?
                                                                                                                                              

    Terpinyl             372   1142-85-4  0.1/1           No          N/R         N/R                  See linalyl acetate and    No safety 
     isovalerate                                                                                       alpha-terpineol.           concern
    CHEMICAL STRUCTURE 

    para-Menth-3-en-1-ol 373   586-82-3   41/0.4          No          N/R         N/R                  See alpha-terpineol.       No safety 
                                                                                                                                  concern
    CHEMICAL STRUCTURE 

    4-Carvomenthenol     439   562-74-3   170/51          No          N/R         N/R                  See alpha-terpineol.       No safety
                                                                                                                                  concern
    CHEMICAL STRUCTURE 

    Table 1. (continued)
                                                                                                                                              
    Substance            No.   CAS No.    Estimated per   Step A3     Step A4     Step A5              Comments                   Conclusion
                                          capita intake,  Does        Is the      Adequate NOEL                                   based on
                                          Europe/USA      intake      substance   for substance                                   current 
                                          (g/day)        exceed      or its      or related                                      intake
                                          threshold?      human       metabolites
                                                          intake      substance?
                                                          endogenous?
                                                                                                                                              

    para-Menth-8-en-1-ol 374   138-87-4   2/21            No          N/R         N/R                  See alpha-terpineol.       No safety 
     (-Terpineol)                                                                                                                concern
    CHEMICAL STRUCTURE 

    2-Ethyl-1,3,3-       440   18368-91-7 1/0.02          No          N/R         N/R                  The structurally related   No safety 
     trimethyl-2-                                                                                      bicyclic tertiary          concern
     norbornanol                                                                                       alcohols thujyl alcohol 
                                                                                                       and -sante-nol 
                                                                                                       are conjugated with 
                                                                                                       glucuronic acid.
    CHEMICAL STRUCTURE 

    4-Thujanol           441   546-79-2   1/0.04          No          N/R         N/R                  The structurally related   No safety 
                                                                                                       bicyclic tertiary          concern
                                                                                                       alcohols thujyl alcohol 
                                                                                                       and -sante-nol are 
                                                                                                       conjugated with 
                                                                                                       glucuronic acid.
    CHEMICAL STRUCTURE 

    Table 1. (continued)
                                                                                                                                              
    Substance            No.   CAS No.    Estimated per   Step A3     Step A4     Step A5              Comments                   Conclusion
                                          capita intake,  Does        Is the      Adequate NOEL                                   based on
                                          Europe/USA      intake      substance   for substance                                   current 
                                          (g/day)        exceed      or its      or related                                      intake
                                          threshold?      human       metabolites
                                                          intake      substance?
                                                          endogenous?
                                                                                                                                              

    Methyl 1-acetoxy-    442   52789-73-8 N/D/4           -           -           -                    Cannot be predicted        Additional 
     cyclohexyl                                                                                        to be metabolized          data on 
     ketone                                                                                            to innocuous               toxicity
                                                                                                       products. Therefore,       are required
                                                                                                       Steps A3-A5 are not        because a
                                                                                                       applicable to this         NOEL is not
                                                                                                       substance, which must      available 
                                                                                                       be evaluated on the        for this or
                                                                                                       right side ('B') of the    a related
                                                                                                       evaluation scheme.         substance 
                                                                                                                                  (Step B4)
                                                                                                                                  and intake
                                                                                                                                  exceeds 1.5
                                                                                                                                  g/person 
                                                                                                                                  per day 
                                                                                                                                  (Step B5).
    CHEMICAL STRUCTURE 

                                                                                                                                              

    N/R, not required for evaluation because consumption of the substance was determined to be of no safety concern at Step A3 of the procedure;
    N/D, no intake data reported
    a   The group ADI of 0-0.05 mg/kg bw established at the twenty-third meeting for citral, citronellol, linalool, and linalyl acetate as citral
        was maintained.
    

         The substances in this group are structurally related because
    they each contain a tertiary alcohol function (without aryl
    substitution); they therefore have similar metabolic and toxicological
    profiles. Four esters of the tertiary alcohol linalool (linalyl
    anthranilate, linalyl benzoate, linalyl cinnamate, and linalyl
    phenylacetate) and one ester of alpha-terpineol (terpinyl cinnamate)
    used as flavouring substances in Europe and the United States are not
    included in this group because they may have different toxicological
    profiles, owing to their carboxylic acid components. It would be
    appropriate to evaluate these esters in subsequent groups, with
    anthranilic acid, benzoic acid, cinnamic acid, phenylacetic acid, and
    related substances used as flavouring substances. Any additional
    intake of linalool and alpha-terpineol from these four esters is
    minimal and would not have a significant impact on the total combined
    intake of linalool, alpha-terpineol, and related tertiary alcohols and
    esters (see section 1.5).

    2.2  Intake

         The total annual volume of the 23 tertiary alcohols and related
    esters is approximately 58 000 kg in Europe (International
    Organization of the Flavor Industry, 1995) and 15 000 kg in the United
    States (US National Academy of Sciences, 1987). Production volumes and
    intake levels of each flavouring substance are reported in Table 2. 

         Thirteen tertiary alcohols and related esters have been reported
    to occur naturally in foods (Maarse et al., 1994); quantitative data
    reported for eight of these substances (see Table 2) indicate that
    they are consumed predominantly from traditional foods (Stofberg &
    Kirschman, 1985; Stofberg & Grundschober, 1987).

    2.3  Biological data

    2.3.1  Absorption and metabolism

         In general, esters are hydrolysed to their corresponding alcohol
    and carboxylic acid. Hydrolysis is catalysed by classes of enzymes
    recognized as carboxylesterases or esterases (Heymann, 1980), the most
    important of which are the B-esterases. In mammals, these enzymes
    occur in most tissues throughout the body (Anders, 1989; Heymann,
    1980) but predominate in hepatocytes (Heymann, 1980). 

         Esters of linalool (Nos 358-365) and alpha-terpineol (Nos
    367-372) are expected to be hydrolysed in humans to yield linalool and
    alpha-terpineol, respectively, and the corresponding saturated
    aliphatic carboxylic acid. Methyl 1-acetoxycyclohexyl ketone (No. 442)
    is expected to be hydrolysed to acetic acid and methyl
    1-hydroxycyclohexyl ketone. 

        Table 2. Most recent annual usage of aliphatic acyclic and alicyclic terpenoid tertiary 
    alcohols and structurally related substances 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
                                                                                                

    Linalool (356)
      Europe                        18 000          2600      44           NA
      United States                 5 800           1100      19           NA

    Tetrahydrolinalool (357)
      Europe                        390             55        0            NA
      United States                 0.5             0.1       0.002        NA

    Linalyl formate (358)
      Europe                        57              8         0.14         0.1
      United States                 70              13        0.22         0.2

    Linalyl acetate (359)
      Europe                        14 000          2100      35           27
      United States                 970             180       3            2

    Linalyl propionate (360)
      Europe                        110             16        0.3          0.2
      United States                 8               2         0.03         0.02

    Linalyl butyrate (361)
      Europe                        69              10        0.2          0.1
      United States                 22              4         0.07         0.05

    Linalyl isobutyrate (362)
      Europe                        250             36        0.6          0.4
      United States                 3               0         0.00         0.00

    Linalyl isovalerate (363)
      Europe                        38              5         0.0          0.06
      United States                 34              6         0.11         0.07

    Linalyl hexanoate (364)
      Europe                        7               0         0.02         0.01
      United States                 2               0.4       0.00         0.004

    Linalyl octanoate (365)
      Europe                        1               0.1       0.002        0.001
      United States                 5               0         0.02         0.00

    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
                                                                                                

    alpha-Terpineol (366)
      Europe                        21 000          3000      50           NA
      United States                 5 500           1100      18           NA

    Terpinyl formate (367)
      Europe                        1               0.1       0.002        0.002
      United States                 10              2         0.03         0.03

    Terpinyl acetate (368)
      Europe                        1 800           260       4            3
      United States                 2 000           390       6            5

    Terpinyl propionate (369)
      Europe                        0.2             0.03      0.0005       0.0004
      United States                 5               0         0.02         0.01

    Terpinyl butyrate (370)
      Europe                        42              6         0.10         0.07
      United States                 33              6         0.10         0.07

    Terpinyl isobutyrate (371)
      Europe                        5               0.7       0.01         0.00
      United States                 0.1             0.02      0.0003       0.0002

    Terpinyl isovalerate (372)
      Europe                        1               0.1       0.002        0.002
      United States                 5               0         0.02         0.01

    para-Menth-3-en-1-ol (373)
      Europe                        290             41        0            NA
      United States                 2               0.4       0.00         NA

    4-Carvomenthenol (439)
      Europe                        1200            170       3            NA
      United States                 270             51        0            NA

    para-Menth-8-en-1-ol (374)
      Europe                        11              2         0.03         NA
      United States                 110             21        0.3          NA

    2-Ethyl-1,3,3-trimethyl-2-norbornol (440)
      Europe                        6.1             1         0.02         NA
      United States                 0.1             0.02      0.0003       NA

    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
                                                                                                

    4-Thujanol (441)
      Europe                        7.5             1         0.02         NA
      United States                 0.2             0.04      0.0006       NA

    Methyl 1-acetoxycyclohexyl-ketone (442)
      Europe                        NR
      United States                 21              4         0.07         NA

    Total
      Europe                        58 000          NA        NA           NA
      United States                 15 000          NA        NA           NA

    Total linalool
      Europe                        NA              4300      72           28
      United States                 NA              1300      21           3

    Total alpha-terpineol
      Europe                        NA              3200      53           4
      United States                 NA              1400      23           5
                                                                                                

    NR, not reported; NA, not applicable; +, reported to occur naturally in foods (Maase et al., 
    1994), but no quantitative data available; -, not reported to occur naturally in foods

    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
    
         In a study of hydrolysis  in vitro, linalyl acetate was easily
    hydrolysed in water and simulated gastric and pancreatic fluids, with
    mean half-lives of 5.5 min in gastric fluid and 53 min in pancreatic
    fluid (Hall, 1979). Terpenoid alcohols formed in the gastrointestinal
    tract are rapidly absorbed (Phillips et al., 1976; Diliberto et al.,
    1988).

         In humans and animals, terpenoid tertiary alcohols are conjugated
    primarily with glucuronic acid and are excreted in the urine and
    faeces (Williams, 1959; Parke et al., 1974a,b; Horning et al., 1976;
    Ventura et al., 1985). Unsaturated terpenoid alcohols may undergo
    allylic oxidation to form polar diol metabolites, which may be
    excreted either free or conjugated. If the diol contains a primary
    alcohol function, it may under-go further oxidation to the
    corresponding carboxylic acid (Madyastha & Srivatsan, 1988; Horning et
    al., 1976; Ventura et al., 1985).  

         The metabolic fate of the aliphatic tertiary alcohol linalool
    (No. 356) has been studied in mammals (Figure 1). Linalool labelled
    with 14C in positions 1 and 2 was administered orally to rats at a
    single dose of 500 mg/kg bw. The majority (55%) of the radioactivity
    was excreted in the urine as the glucuronic acid conjugate, while 23%
    was excreted as carbon dioxide in expired air, and 15% was excreted in
    the faeces within 72 h of administration; only 3% of the radiolabel
    was detected in tissues after 72 h, with 0.5% in the liver, 0.6% in
    the gut, 0.8% in the skin, and 1.2% in skeletal muscle (Parke et al.,
    1974a). Reduction metabolites such as dihydro- and tetrahydrolinalool
    were identified in the urine after administration of a single dose of
    linalool to rats (Rahman, 1974).

    FIGURE 1

         In a separate study, male IISc strain rats were given daily oral
    doses of 800 mg/kg bw linalool for 20 days. Urinary metabolites formed
    by cytochrome P450 (CYP450)-induced allylic oxidation of linalool
    included 8-hydroxylinalool and 8-carboxylinalool. CYP450 activity in
    liver microsomes was increased by about 50% after three days, but the
    activity had decreased to control values after six days (Chadha &
    Madyastha, 1984). Linalool administered to four-week-old male Wistar
    rats by gavage at a dose of 500 mg/kg bw per day for 64 days did not
    induce CYP450 until the 30th day of treatment (Parke et al., 1974b).

         These results suggest that glucuronic acid conjugation and
    excretion are the primary route of metabolism of linalool and allylic
    oxidation becomes an important pathway only after repeated dosing. It
    has been suggested that biotransformation of the diol metabolite of
    linalool to the corresponding aldehyde by the action of the
    NAD+-dependent enzyme alcohol dehydrogenase is inhibited because of
    the bulky nature of the neighbouring alkyl substituents and the
    substrate specificity of the enzyme (Eder et al., 1982). 

         When male albino IISc strain rats were given the alicyclic
    tertiary alcohol alpha-terpineol (No. 366) orally at a daily dose of
    600 mg/kg bw for 20 days, oxidation of the allylic methyl group was
    observed to yield the corresponding carboxylic acid which was
    hydrogenated to a small extent to yield the corresponding saturated
    carboxylic acid (Madyastha & Srivatsan, 1988; Figure 2). When
    alpha-terpineol was administered orally to rats, it increased the
    liver microsomal P450 content and the activity of NADPH-cytochrome c
    reductase (Madyastha & Srivatsan, 1988), suggesting that the oxidation
    is mediated by CYP450.

    FIGURE 2

         In a minor pathway, the endocyclic alkene of alpha-terpineol is
    epoxidized and then hydrolysed to yield a triol metabolite
    1,2,8-trihydroxy- para-menthane, which was also reported in humans
    after inadvertent oral ingestion of a pine-oil disinfectant containing
    alpha-terpineol (Horning et al., 1976). It is expected that after
    single doses, alpha-terpineol would be metabolized like linalool
    (Chadha & Madyastha, 1984), primarily by glucuronic acid conjugation
    and excretion in the urine.

         Bicyclic tertiary alcohols (Nos 440 and 441) are relatively
    stable  in vivo, but are eventually conjugated with glucuronic acid
    and excreted. In rabbits, the structurally related bicyclic tertiary
    alcohols thujyl alcohol and -santenol
    (2,3,7-trimethylbicyclo(2.2.1)heptan-2-ol) are conjugated with
    glucuronic acid (Williams, 1959). 

         In a study of the metabolism of the structurally related
    terpenoid tertiary alcohol  trans-sobrerol in humans, dogs, and rats,
    10 metabolites were isolated in urine, eight of which were found in
    humans. Two principal modes of metabolism were observed: allylic
    oxidation of the ring positions and alkyl substituents and conjugation
    of the tertiary alcohol functions with glucuronic acid. These
    metabolic patterns are common modes of converting tertiary (Ventura et
    al., 1985) and secondary (Yamaguchi et al., 1994) terpenoid alcohols
    to polar metabolites, which are easily excreted in the urine and
    faeces (see Figure 3). Menthol forms similar oxidation and conjugation
    products in rats (Yamaguchi et al., 1994). 

    FIGURE 3

         After hydrolysis of linalyl and terpinyl esters, the component
    aliphatic carboxylic acids undergo -oxidation and cleavage to
    eventually yield carbon dioxide and water. Successive two-carbon units
    are removed from the carbonyl end, which cleaves acids with even
    numbers of carbons to acetyl coenzyme A and those with odd numbers of
    carbons to acetyl and propionyl coenzyme A. Acetyl coenzyme A enters
    the citric acid cycle directly or reacts with propionyl coenzyme A to
    form succinyl coenzyme A, which also enters the citric acid cycle
    (Voet & Voet, 1990).

    2.3.2  Toxicological studies

    2.3.2.1  Acute toxicity

         Oral LD50 values have been reported for 16 of the 23 substances
    in this group. They range from 1300 to > 36 000 mg/kg bw, indicating
    that the acute toxicity of terpenoid tertiary alcohols and related
    esters after oral exposure is low (Jenner et al., 1964; Moreno, 1977).

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

         The results of studies with representative terpenoid tertiary
    alcohols, esters, and related substances are summarized in Table 3 and
    described below.

     Linalool

         In a safety evaluation, a 50:50 mixture of linalool and
    citronellol was fed to male and female rats (numbers and strain
    unspecified) in the diet. The daily intake was calculated to be 50
    mg/kg bw of each substance. Haematological, clinical chemical, and
    urinary analyses at weeks 6 and 12 showed no statistically significant
    difference between treated and control groups. Histopathology revealed
    no dose-related lesions. A slight retardation of growth was observed
    in males, but was considered by the authors to be biologically
    insignificant (Oser, 1967). The dose of linalool that had no adverse
    effect (50 mg/kg bw) is > 1000 times the daily  per capita intake of
    44 or 19 g/kg bw from use of linalool as a flavouring substance in
    Europe and the United States, respectively, and > 500 times the total
    daily  per capita intake of 72 or 21 g/kg bw from use of linalool
    and its esters (i.e. alcohol equivalents) as flavouring substances in
    Europe and the United States, respectively.

     Linalyl esters

         A mixture of linalyl acetate, linalyl isobutyrate, and geranyl
    acetate was added to the diet of male and female rats (strain not
    specified) at concentrations calculated to result in average daily
    intakes of 24, 27, or 48 mg/kg bw, respectively, for 12 weeks.
    Haematological, clinical chemical, and urinary analyses at weeks 6 and
    12 showed normal values. Histopathological examination revealed no
    dose-related lesions. Slight retardation of growth was observed in

    females but was considered by the authors to be biologically
    insignificant (Oser, 1967). The dose of linalyl acetate that had no
    adverse effect (24 mg/kg bw per day) is > 500 times the daily
     per capita intake of 34 g/kg bw from use of linalyl acetate as a
    flavouring substance in Europe and > 1000 times the daily  per 
     capita intake of 3 g/kg bw in the United States. Similarly, the
    dose of linalyl isobutyrate that had no adverse effect (27 mg/kg bw
    per day) is > 10 000 times the daily  per capita intake of 1 g/kg
    bw from use of linalyl isobutyrate as a flavouring substance in Europe
    and > 1 000 000 times the daily  per capita intake of 0.01 g/kg bw
    in the United States.

         Four groups of 10 male and 10 female Osborne-Mendel rats were fed
    the structurally related ester of linalool, linalyl cinnamate, at
    dietary concentrations of 0, 1000, 2500, or 10 000 mg/kg for 17 weeks.
    These concentrations were calculated (US Food and Drug Administration,
    1993) to provide intakes of 0, 50, 125, and 500 mg/kg bw per day,
    respectively. Body weights were measured and haematological and gross
    examinations were performed on all treated animals; only those at the
    high dose were examined microscopically. No differences were found
    between treated and control animals (Hagan et al., 1967). 

     Terpinyl acetate

         Groups of 10 male and 10 female weanling Osborne-Mendel rats were
    fed terpinyl acetate in the diet for 20 weeks at concentrations of 0,
    1000, 2500, or 10 000 mg/kg (Hagan et al., 1967). These dietary levels
    were calculated (US Food and Drug Administration, 1993) to result in
    intakes of 0, 50, 125, and 500 mg/kg bw per day, respectively. All
    animals were examined for changes in growth, haematological
    parameters, and gross histological appearance. Microscopic examination
    was performed on six to eight male and female animals at the high dose
    and in the control groups. No statistically significant adverse
    effects were reported (Hagan et al., 1967). The dose of terpinyl
    acetate that had no adverse effects (500 mg/kg bw per day) is
    > 50 000 times the daily  per capita intake of 6 g/kg bw from its
    use as a flavouring substance in Europe and the United States. This
    dose is > 5000 times the total daily  per capita intakes of 53 or 23
    g/kg bw from use of alpha-terpineol and its esters (i.e. alcohol
    equivalents) as flavouring substances in Europe and the United States.

     Geranyl acetate and citronellyl acetate 

         The Committee previously evaluated linalool and linalyl acetate,
    at the twenty-third meeting in 1979 (Annex 1, reference 50) as part of
    a group of terpenoid flavouring substances including citral,
    citronellol, and geranyl acetate. Although the Committee established a
    group ADI of 0-0.5 mg/kg bw, they maintained that a long-term study
    was required for at least one member of the group. Since that time,
    long-term studies have been performed in rats and mice of a mixture of
    the structurally related terpenoid ester geranyl acetate (71%) and
    citronellyl acetate (29%) (US National Toxicology Program, 1987).
    These studies were considered by the Committee at its forty-ninth

    meeting (Annex 1, reference 131) in a safety evaluation of the group
    of flavouring substances that includes 26 geranyl, neryl, citronellyl,
    and rhodinyl esters formed from branched-chain terpenoid primary
    alcohols and branched-chain carboxylic acids. The Committee determined
    that the study of carcinogenicity in mice was inadequate owing to
    dosing errors and the low survival associated with infections. The
    NOEL of the mixture in rats was 1000 mg/kg bw per day, which
    corresponds to estimated doses of 710 mg/kg bw per day geranyl acetate
    and 290 mg/kg bw per day citronellyl acetate. This NOEL is > 10 000
    times the estimated daily  per capita intakes of 44 and 19 g/kg bw
    per day from use of the structurally related substance linalool as a
    flavouring substance in Europe and the United States, respectively. 

    2.3.2.3  Genotoxicity

         Five representative alcohols and esters in this group have been
    tested for genotoxicity, including a complete battery of tests with
    linalool  in vitro. The results of these tests are summarized in
    Table 4 and described below.

         Linalool (No. 356) was inactive in  Salmonella typhimurium 
    strains TA92, TA94, TA98, TA100, TA1535, TA1537, and TA1538 with and
    without metabolic activation (Rockwell & Raw, 1979; Eder et al., 1980;
    Florin et al., 1980; Ishidate et al., 1984; Heck et al., 1989).
    Linalool did not induce chromosomal aberrations when incubated with
    Chinese hamster fibroblasts at a maximum concentration of 0.25 mg/ml
    (Ishidate et al., 1984), nor did it induce unscheduled DNA synthesis
    in rat hepatocytes at concentrations up to 50 g/ml (Heck et al.,
    1989). 

         Linalool did not induce mutation in  E. coli WP2 uvrA at
    concentrations of 0.125-1 mg/plate (Yoo, 1986). When incubated with
     Bacillus subtilis H17  (rec+) and M45  (rec-), linalool was
    not mutagenic at 17 g/plate (Oda et al., 1978) but was mutagenic at
    10 l/disc (Yoo, 1986). Linalool was inactive in mouse lymphoma L5178Y
     tk+/- cells with metabolic activation at 200 g/ml but gave a
    weakly positive response without activation at 150 g/ml (Heck et al.,
    1989).

         The 24-h urine of Sprague-Dawley rats given 0.5 ml of linalool or
    -terpineol (No. 374) was incubated with  S. typhimurium strains TA98
    and TA100 to observe mutagenicity. The following assays were
    conducted: linalool or -terpineol (with metabolic activation),
    aliquot of 24-h urine (with and without metabolic activation), ether
    extract of 24-h urine (with and without metabolic activation), and
    aqueous phase of 24-h urine ether extract (with and without metabolic
    activation). The urine samples were diluted to 60 ml and incubated in
    the presence of chloroform and -glu-curonidase before ether
    extraction. The only positive response was found with the ether
    extract of -terpineol.


        Table 3. Short-term and long-term studies of toxicity and carcinogenicity and special studies of aliphatic acyclic and alicyclic 
    terpenoid tertiary alcohols and structurally related substances 

                                                                                                                                            

    Substance                No.     Species    Sex     No. groups/     Route      Time          NOEL                Reference
                                                        no. per group                            (mg/kg bw 
                                                                                                 per day)
                                                                                                                                            

    Linalool                 356     Rat        M/F     1/--            Dieta      84 days       50b                 Oser (1967)
    Linalyl acetate          357     Rat        M/F     1/NR            Dietc      84 days       24b                 Oser (1967)
    Linalyl isobutyrate      362     Rat        M/F     1/NR            Dietd      84 days       27b                 Oser (1967)
    Terpinyl acetate         368     Rat        M/F     3/20            Diete      140 days      500b                Hagan et al. (1967)
    Linalyl cinnamatee       None    Rat        M/F     4/20            Dietf      17 weeks      500b                Hagan et al. (1967)
    Geranyl acetate/         None    Mouse      M/F     2/50            Gavage     103 weeks     -g                  US National Toxicology
      Citronellyl acetate                                                                                            Program(1987)
    Geranyl acetate/         None    Rat        M/F     2/5             Gavage     103 weeks     1000                US National Toxicology
      Citronellyl acetate                                                                        (710 geranyl        Program (1987)
                                                                                                 acetate; 
                                                                                                 290 citronellyl 
                                                                                                 acetate)

    Special study of immunotoxicity
    Linalool                 356     Mouse      F       3/30            Gavage     5             375b                Gaworski et al. (1994)
                                                                                                                                            

    M, male; F, female; NR, not reported
    a   Administered with citronellol as part of a 50/50 mixture
    b   The study was performed at a single dose or multiple doses that had no adverse effects; therefore, no NOEL was determined. The NOEL 
        is probaby higher than the dose reported here, which is the highest dose that had no adverse effect.
    c   Administered with linalyl isobutyrate and geranyl acetate as part of a mixture
    d   Administered with linalyl acetate and geranyl acetate as part of a mixture
    e   Structurally related ester of linalool, not evaluated as part of this group
    f   Structurally related terpenoid esters administered as a mixture: geranyl acetate, 71%; citronellyl acetate, 29%
    g   There was no NOEL owing to a high incidence of gavage errors and low survival associated with pneumonia.

    Table 4. Results of assays for the genotoxicity of aliphatic acyclic and alicyclic terpenoid tertiary alcohols and structurally 
    related substances

                                                                                                                                            

    Substance          No.   End-point           Test object                       Concentration        Result       Reference
                                                                                                                                            

    Linalool           356   Reverse mutation    S. typhimurium TA100              0.01-3 l/2-ml       Negativea    Eder et al. (1980)
                             (modified test)                                       incubation volume

    Linalool           356   Reverse mutation    S. typhimurium  TA92, TA100,      1 mg/plateb          Negativea    Ishidate et al. (1984)
                                                 TA1535, TA1537, TA94, TA98

    Linalool           356   Reverse mutation    S. typhimurium  TA98, TA100       0.05-100 l/plate    Negativea    Rockwell & Raw  (1979)

    Linalool           356   Reverse mutation    S. typhimurium  TA100, TA1535,    10 000 g/plateb     Negativea    Heck et al. (1989)
                                                 TA1537, TA1538, TA98

    Linalool           356   Gene mutation       Mouse lymphoma L5178Y tk+/-       200 g/ml            Negativec    Heck et al. (1989)
                                                                                   150 g/ml            Weakly 
                                                                                                        positived

    Linalool           356   Unscheduled DNA     Rat hepatocytes synthesis         50 g/mlb            Negativee    Heck et al. (1989)

    Linalool           356   Chromosomal         Chinese hamster fibroblasts       0.25 mg/mlb          Negatived    Ishidate et al. (1984)
                             aberration

    Linalool           356   Gene mutation       Bacillus subtilis H17 (rec+)      17 g                Negative     Oda et al. (1978)
                                                 & M45 (rec-)

    Linalool           356   Gene mutation       Bacillus subtilis H17 (rec+)      10 l/discb          Positive     Yoo (1986)
                                                 & M45 (rec-)

    Linalool           356   Gene mutation       E. coli WP2 uvrA                  0.125-1 mg/plate     Negative     Yoo (1986)

    Linalyl acetate    359   Reverse mutation    S. typhimurium  TA1535, TA1537,   25 000 g/plateb     Negativea    Heck et al. (1989)
                                                 TA1538, TA98, TA100

    Linalyl acetate    359   Unscheduled DNA     Fischer or Sprague-Dawley rat     300 g/mlb           Negativee    Heck et al. (1989)
                             synthesis           hepatocytes

    Table 4. (continued)

                                                                                                                                            

    Substance          No.   End-point           Test object                       Concentration        Result       Reference
                                                                                                                                            

    Linalyl acetate    359   Gene mutation       Bacillus subtilis H17 (rec+)      18 g/plate          Negative     Oda et al. (1978)
                                                 & M45 (rec-)

    alpha-Terpineol    366   Reverse mutation    S. typhimurium  TA1535, TA1537,   10 000 g/plateb     Negativea    Heck et al. (1989)
                                                 TA1538, TA98, TA100

    alpha-Terpineol    366   Spot test           S. typhimurium  TA1535, TA1537,   3 mol/plate         Negativea    Florin et al. (1980)
                                                 TA98, TA100

    alpha-Terpineol    366   Gene mutation       Mouse lymphoma L5178Y tk+/-       250 g/mlb           Negativec    Heck et al. (1989)
                                                                                   300 g/mlb           Negatived

    Terpinyl acetate   368   Gene mutation       Bacillus subtilis H17 (rec+)      19 g/plate          Negative     Oda et al. (1978)
                                                 & M45 (rec-)

    -Terpineol        374   Reverse mutation    S. typhimurium  TA100, TA98       0.05-100 l/plate    Negativec    Rockwell & Raw  (1979)

    Terpineolf         366   Gene mutation       S. cerevisiae                     Not reported         Negativec    Oda et al. (1978)
                       and/or
                       374
                                                                                                                                            

    a   With and without metabolic activation
    b   The highest inactive or lowest active concentration; range not specified
    c   With metabolic activation
    d   Without metabolic activation
    e   75 or 150 cells analysed per dose
    f   Not specified whether alpha- or -terpineol
    

         Linalyl acetate (No. 359) and alpha-terpineol (No. 366) were
    inactive in  S. typhimurium strains TA98, TA100, TA1535, TA1537, and
    TA1538 with and without metabolic activation (Eder et al., 1980;
    Florin et al., 1980; Ishidate et al., 1984; Heck et al., 1989).
    -Terpineol was inactive in  S. typhimurium strains TA98 and TA100
    with metabolic activation (Rockwell & Raw, 1979). When incubated with
     B. subtilis H17  (rec+) and M45  (rec-), linalyl acetate and
    terpinyl acetate were not mutagenic at 18 or 19 g, respectively (Oda
    et al., 1978).

         Thus, all but two of 19 tests of the genotoxicity of terpenoid
    tertiary alcohols and related esters  in vitro gave negative results.
    Linalool was active in a  rec assay, in which differences in the
    growth rates of two strains of  B.subtilis are used as a measure of
    DNA damage (Yoo, 1986). In contrast, no evidence of DNA damage was
    observed in an assay for unscheduled DNA synthesis in rat hepatocytes
    (Heck et al., 1989). The authors of a study in which linalool gave a
    weak positive result in the mouse lymphoma assay (Heck et al., 1989)
    emphasized that positive results are commonly observed for polar
    substances in this assay in the presence of metabolic activation and
    may be associated with changes in physiological culture conditions (pH
    and osmolality). The weight of the evidence for terpenoid tertiary
    alcohols and related esters indicates that this group of flavouring
    substances would not be expected to have genotoxic potential
     in vivo.

    2.3.2.4  Other relevant studies

         No adverse effects were reported at doses of linalool up to
    375 mg/kg bw in a screening test in mice for humoral and cell-mediated
    immune responses (see Table 4). Linalool in 1% methylcellulose was
    administered intragastrically to 10-20 six- to eight-week-old female
    B6C3F1 mice for five days at a dose of 94, 188, or 375 mg/kg bw per
    day. Cell-mediated immunity was assessed in a host resistance assay
    with a  Listeria monocytogenes bacterial challenge. Humoral immunity
    was measured by the antibody plaque-forming cell response to sheep
    erythrocytes. Body weights, lymphoid organ weights, and spleen
    cellularity were also measured. Cyclophosphamide was used as a
    positive control. Linalool did not adversely modulate the immune
    response in either assay (Gaworski et al., 1994).


    4.  REFERENCES

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    See Also:
       Toxicological Abbreviations