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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

    SUBSTANCES STRUCTURALLY RELATED TO MENTHOL 

    First draft prepared by
    M.F.A. Wouters, M.E. van Apeldoom, and G.J.A. Speijers
    National Institute of Public Health and the Environment 
    Center of Substances and Risk Assessment
    Bilthoven, The Netherlands

          Evaluation 
              Introduction
              Estimated daily  per capita intake
              Absorption, metabolism, and elimination
              Application of the Procedure for the Safety Evaluation 
                   of Flavouring Agents
              Consideration of combined intake 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
          References


    1.  EVALUATION

    1.1  Introduction

         The Committee evaluated menthol and 13 structurally related
    substances (Table 1) using the Procedure for the Safety Evaluation of
    Flavouring Agents (Figure 1, p. 222, and Annex 1, reference 131). 

         Menthol was first evaluated at the eleventh meeting of the
    Committee (Annex 1, reference 14), when it was allocated an
    unconditional ADI of 0-0.2 mg/kg bw and a conditional ADI of 0.2-2
    mg/kg bw. At the eighteenth meeting, an ADI of 0-0.2 mg/kg bw was
    established (Annex 1, reference 35). The Committee reevaluated menthol
    at its twentieth meeting (Annex 1, reference 41), when the ADI was
    maintained. At the present meeting, the Committee allocated an ADI of
    0-4 mg/kg bw to menthol (see monograph, this volume).

    1.2  Estimated daily per capita intake

         The total annual production volume of the 14 menthyl derivatives
    is approximately 140 000 kg in Europe (International Organization of
    the Flavor Industry, 1995) and 79 000 kg in the United States (US
    National Academy of Sciences, 1987). Menthol (No. 427) and menthone

    (No. 429) account for 97% of the total annual volume in Europe and 85%
    in the United States.

         Menthol and some structurally related substances occur naturally
    in a wide variety of foods, including spearmint oil, cornmint oil,
    peppermint oil, raspberries, rum, nutmeg, and cocoa (Maarse et al.,
    1994). Menthol (10-70%) and menthone (7-40%) are the principal
    constituents of peppermint oil. Eight of the substances in this group
    have been reported to occur naturally in foods (Nos 427, 428, 429,
    430, 431, 432, 434, and 435); six of the remaining substances are
    esters of menthol (Nos 433, 443, 444, and 447) or ketals of menthone
    (Nos 445 and 446). Quantitative data have been reported on the natural
    occurrence and consumption ratios of five of these substances, which
    indicate that they are consumed predominantly in traditional foods
    (i.e. consumption ratio, > 1) (Stofberg & Kirschman, 1985; Stofberg &
    Grundschober, 1987). 

    1.3  Absorption, metabolism, and elimination

         The esters in this group (Nos 427, 432, 433, and 447) would be
    expected to be readily hydrolysed to menthol and their respective
    carboxylic acids; the latter are endogenous in humans. Carbonate (-)
    esters (Nos 443 and 444) can be expected to be hydrolysed to menthol
    (No. 427) and carbonate and either ethylene glycol or propylene
    glycol. The ketals (Nos 445 and 446) are hydrolysed  in vitro to
    yield (-)- or ()-menthone and simple glycols. The ketones (Nos 429,
    430 and 435) in this group would be reduced to their corresponding
    secondary alcohols, which, like menthol, would be conjugated with
    glucuronic acid and then excreted in the urine. See also 'General
    aspects of metabolism', p. 223.

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

    Step 1.   Menthol (No. 427), (+)- neo-menthol (No. 428), menthyl
              acetate (No. 431), menthyl  iso-valerate (No. 432),
              (-)-menthyllactate (No. 433), menth-1-en-3-ol (No. 434),
              (-)-menthol ethylene glycol carbonate (No. 443), (-)-menthol
              1- and 2-propylene glycol carbonate (No. 444), and
              mono-menthyl succinate (No. 447) are classified in
              structural class I (Cramer et al., 1978). Menthone (No. 3),
              ()- iso-menthone (No. 430), (+)-piperitone (No. 435),
              (-)-menthone 1,2-glycerol ketal (No. 445), and ()-menthone
              1,2-glycerol ketal (No. 446) are classified in structural
              class II. 

    Step 2.   At current levels of intake, the 14 substances would not be
              expected to saturate the metabolic pathways, and all of the
              substances are predicted to be metabolized to innocuous
              products. Evaluation of substances in the group of
              flavouring agents that includes menthol and structurally
              related substances was based on data on the metabolic fate
              of menthol and menthone, which are metabolized by oxidation

              and conjugation. The extensive conjugation of menthol with
              glucuronic acid and its rapid elimination in urine and bile,
              combined with its simple chemical structure, gave assurance
              about the innocuous nature of the products of metabolism.
              The metabolites of (+)-piperitone (an alpha,-unsaturated
              ketone) were considered to be innocuous by comparison with
              the metabolic fate and toxicity of menthone (saturated) and
              carvone (alpha,-unsaturated). Isomers of menthone
              1,2-glycerol ketal were predicted to be converted to
              menthone and can thus be evaluated for safety with menthone
              and its metabolite menthol. This conclusion was supported by
              the available data on the toxicity of these compounds.

    Step A3.  The daily  per capita intake of all of the substances in
              this group in class I, with the exception of menthol (No.
              427), is below the human intake threshold for class I (1800
              g/person per day) in both Europe and the United States,
              indicating that they pose no safety concern at current
              levels of estimated intake as flavouring agents. Intake of
              menthol (No. 427) in Europe (18 000 g/person per day) and
              the United States (10 000 g/person per day) is greater than
              the human intake threshold for class I.

                   Total intake of menthol (No. 427) in Europe and the
              United States from its use and use of its esters (Nos 431,
              432, 433, 443, 444, and 447) as flavouring agents is above
              the human intake threshold for class I: 19 000 g/day in
              Europe and 12 000 g/day in the United States.

                   The daily  per capita intake of all of the substances
              in this group in class II, with the exception of menthone
              (No. 429), is below the human intake threshold for class II
              (540 g/person per day) in both Europe and the United
              States. Intake of menthone (No. 429) in Europe (1000
              g/person per day) and in the United States (2500 g/person
              per day) is greater than the human intake threshold for
              class II.

                   Total intake of menthone (No. 429) in Europe and the
              United States from its use and use of its ketals (Nos 445
              and 446) as flavouring agents is above the human exposure
              threshold for class II: 1000 g/day in Europe and 2900
              g/day in the United States.

    Step A4.  Menthol (No. 427) and menthone (No. 429) are not endogenous
              in humans. 

    Step A5.  An ADI of 0-4 mg/kg bw was allocated to menthol at the
              present meeting (see monograph, this volume). For menthone,
              a NOEL of 400 mg/kg bw per day was reported in a 28-day
              study of toxicity in rats (Madsen et al., 1986). There is a
              safety margin of > 1000 between this NOEL and the daily
               per capita intake of menthone itself (42 or 17 g/kg bw)

              and the total daily  per capita intake of menthone
              including its derivatives (46 or 17 g/kg bw). This
              information indicates that neither menthone nor menthol
              would be expected to be of safety concern.

         The stepwise evaluations of menthol and 13 structurally related
    substances used as flavouring agents are summarized in Table 1.

    1.5  Consideration of combined intakes from use as flavouring agents

         In the unlikely event that menthol (class I) and menthone (class
    II) were consumed concomitantly on a daily basis with the other 12
    structurally related substances, the estimated combined intake would
    exceed the human intake threshold for class II. All 14 substances are,
    however, expected to be efficiently metabolized and would not saturate
    metabolic pathways. On the basis of the evaluation of the collective
    data, the combined intake was judged by the Committee not to raise
    safety concern.
    1.6  Conclusions

         Menthol and the 13 structurally related substances evaluated do
    not pose a safety concern at current levels of intake as flavouring
    agents. The Committee noted that the available data on their toxicity
    were consistent with the results of the safety evaluations using the
    procedure. 


    2.  RELEVANT BACKGROUND INFORMATION

    2.1  Explanation

         This monograph summarizes the key data relevant to the safety
    evaluation of 13 substances structurally related to menthol (Table 1).
    A separate monograph was prepared on menthol. 

         The substances in this group are structurally related because
    they are alicyclic terpenoid ketones, secondary alcohols, related
    esters, and ketals with a 3-menthyl carbon skeleton. They therefore
    have similar metabolic and toxicological profiles. The group of
    menthyl derivatives includes three ketones (Nos 429, 430, and 435),
    one of which is a stereoisomer (No. 430), and an unsaturated analogue
    (No. 435) of menthone (No. 429); three secondary alcohols (Nos 427,
    428, and 434), which include a stereoisomer (No. 428) and an
    unsaturated analogue (No. 434) of menthol (No. 427); two carbonate
    esters (Nos 443 and 444) of (-)-menthol and either ethylene glycol or
    propylene glycol, respectively; two ketals (Nos 445 and 446) of either
    (-)-menthone or ()-menthone, respectively, and glycerol; and four
    esters (Nos 431, 432, 433, and 447) of menthol and either acetic acid,
    isovaleric acid, lactic acid, or succinic acid. The four carboxylic
    acids are endogenous. 


        Table 1.  Summary of safety evaluation of substances structurally related to menthol

    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 metabolized to innocuous products.

                                                                                                                                              

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

    Menthol              427   89-78-1   I           18 000/10 000  Yes          No           Yes.The dose of 375 mg/kg bw         No safety 
                                                                                              per day that produced no adverse     concerna
                                                                                              effects (US National Cancer 
                                                                                              Institute, 1979) is > 1000 times 
                                                                                              the daily per capita intake 
                                                                                              of 173 mg/kg bw per day and 
                                                                                              305 mg/kg bw per day from use 
                                                                                              as a flavouring agent
    CHEMICAL STRUCTURE 

    (+)-neo-Menthol      428   2216-52-6 I           3/27           No           N/R          N/R                                  No safety 
                                                                                                                                   concern
    CHEMICAL STRUCTURE 

    Table 1.  (continued)
                                                                                                                                              

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

    Menthone             429   89-80-5   II          1000/2500      Yes          No           Yes. The dose  of 400 mg/kg bw       No safety 
                                                                                              per day that produced no adverse     concern
                                                                                              effects (Madsen, 1986] is > 1000 
                                                                                              times the daily per capita intake 
                                                                                              of 42 mg/kg bw per day and 17 
                                                                                              mg /kg bw per day from use as a
                                                                                              flavouring agent
    CHEMICAL STRUCTURE 

    ()-iso-Menthone     430   491-07-6  II          200/0.1        No           N/R          N/R                                  No safety 
                                                                                                                                   concern
    CHEMICAL STRUCTURE 

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

    Menthyl acetate                 431   16409-45-3     I            420/560       No             N/R            N/R              No safety 
                                                                                                                                   concern
    CHEMICAL STRUCTURE 

    Menthyl isovalerate             432   16409-46-4     I            9/27          No             N/R            N/R              No safety 
                                                                                                                                   concern
    CHEMICAL STRUCTURE 

    (-)-Menthyl lactate             433   59259-38-0     I            26/0.1        No             N/R            N/R              No safety 
                                                                                                                                   concern
    CHEMICAL STRUCTURE 

    para-Menth-1-en-3-ol            434   491-04-3       I            0.02/0.02     No             N/R            N/R              No safety 
                                                                                                                                   concern
    CHEMICAL STRUCTURE 

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

    Piperitone                      435   6091-50-5      II           51/10         No             N/R            N/R              No safety 
                                                                                                                                   concern
    CHEMICAL STRUCTURE 

    (-)-Menthol ethylene glycol     443   156324-78-6    I            N/D/760       No             N/R            N/R              No safety 
      carbonate                                                                                                                    concern
    CHEMICAL STRUCTURE 

    (-)-Menthol 1- and 2-propylene  444   156329-82-2    I            N/D/380       No             N/R            N/R              No safety 
      glycol carbonate                                                                                                             concern
    CHEMICAL STRUCTURE 

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

    (-)-Menthone 1,2-glycerol       445   563187-91-7    II           N/D/190       No             N/R            N/R              No safety 
      ketal                                                                                                                        concern
    CHEMICAL STRUCTURE 

    ()-Menthone 1,2-glycerol       446   63187-91-7     II           N/D/190       No             N/R            N/R              No safety 
      ketal                                                                                                                        concern
    CHEMICAL STRUCTURE 

    mono-Menthyl succinate          447   77341-67-4     I            N/D/22        No             N/R            N/R              No safety 
                                                                                                                                   concern
    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 An ADI of 0-4 mg/kg bw was established for menthol at the present meeting.
    

    2.2  Intake

         The total annual production volume of the 14 menthyl derivatives
    is about 140 000 kg in Europe (International Organization of the
    Flavor Industry, 1995) and 79 000 kg in The United States (US National
    Academy of Sciences, 1987). The production volumes and intakes of each
    substance are reported in Table 2. Menthol (No. 427) and menthone (No.
    429) account for about 97% of the total annual volume in Europe and
    85% in the United States.

         Eight of the substances in this group (Nos 427-432, 434, and 435)
    have been reported to occur naturally in foods (Maarse & Visscher,
    1994). Six of the remaining substances are esters of menthol (Nos 433,
    443, 444, and 447) or ketals of menthone (Nos 445 and 446), which are
    hydrolysed to menthol or menthone  in vivo. Menthol (10-70%) and
    menthone (7-40%) are primary constituents of peppermint oil.
    Quantitative data have been reported on the natural occurrence and
    consumption ratios of five of these substances (see Table 2);
    according to the authors, these data indicate that these substances
    are consumed predominantly from traditional foods (Stofberg &
    Kirschman, 1985; Stofberg & Grundschober, 1987).

    2.3  Biological data

    2.3.1  Absorption and metabolism

         In general, menthyl esters are hydrolysed to menthol and their
    respective carboxylic acids. The carbonate esters in this group are
    hydrolysed to menthol and carbonate and either ethylene glycol or
    propylene glycol. The ketals in this group are hydrolysed to menthone
    and their respective alcohols. Ester hydrolysis is catalysed by
    classes of enzymes recognized as carboxylesterases or esterases
    (Heymann, 1980), the most important of which are the B-esterases
    (Heymann, 1980; Anders, 1989). Acetyl esterases are the preferred
    substrates of C-esterases (Heymann, 1980). These enzymes occur in most
    mammalian tissues (Heymann, 1980; Anders, 1989) but predominate in
    hepatocytes (Heymann, 1980). Esters (Nos 431-433 and 447) and
    carbonate esters (Nos 443 and 444) of menthol are expected to be
    hydrolysed in humans to yield menthol and their respective saturated
    aliphatic carboxylic acids or alcohols. 

         In two studies, (-)-menthol ethylene glycol carbonate (No. 443)
    and (-)-menthol propylene glycol carbonate (No. 444) were hydrolysed
    after incubation with rat liver homogenate  in vitro, the mean values
    for hydrolysis to menthol being 85% for the ethylene glycol carbonate
    and 75% for the propylene glycol carbonate (Emberger, 1994a,b). More
    than 80% of radiolabelled cyclandelate (Figure 1), a structurally
    related cyclohexyl ester, was hydrolysed after 20 min of incubation
    with rat hepatic microsomes (White et al., 1990).

    Table 2. Most recent annual usage volume of menthol and related 
    substances as flavouring agents in Europe and the United States 

                                                                        

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

    Menthol (427)
      Europe                     128 000           18 000     300
      United States              54 000            10 000     170

    (+)-neo-Menthol (428)
      Europe                     15                3          0.05
      United States              140               27         0.4

    Menthone (429)
      Europe                     7 300             1 000      17
      United States              13 300            2 500      42

    ()-iso-Menthone (430)
      Europe                     1 100             200        3
      United States              0.5               0.1        0.002

    Menthyl acetate (431)
      Europe                     2 200             420        7
      United States              2 900             560        9

    Menthyl isovalerate (432)
      Europe                     48                9          0.2
      United States              140               27         17

    (-)-Menthyl lactate (433)
      Europe                     140               26         0.4
      United States              0.5               0.1        0.002

    para-Menth-1-en-3-ol (434)
      Europe                     0.1               0.02       0.0003
      United States              0.1               0.02       0.0003

    Piperitone (435)
      Europe                     270               51         0
      United States              50                10         0.2

    (-)-Menthol ethylene gylcol carbonate (443)
      Europe                     NR                ND         ND
      United States              4 000             760        13

    Table 2. (continued)
                                                                        

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

    (-)-Menthol 1- and 2-propylene gylcol carbonate (444)
      Europe                     NR                ND         ND
      United States              2 000             381        6

    (-)-Menthone1,2-glycerol ketal (445)
      Europe                     NR                ND         ND
      United States              1 000             190        3

    ()-Menthone1,2-glycerol ketal (446)
      Europe                     NR                ND         ND
      United States              1 000             190        3

    mono-Menthyl succinate (447)
      Europe                     NR                ND         ND
      United States              110               22         0.4

    Total
      Europe                     140 000
      United States              79 000

    Total menthol
      Europe                     NA                12 000     190
      United States              NA                19 000     310

    Total menthone
      Europe                     NA                2 800      46
      United States              NA                1 000      17
                                                                        

    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 and 32  106 for the United States; 0.6 
        represents the assumption that only 60% of the flavour volume was 
        reported in the surveys 
        (US National Academy of Sciences, 1970, 1982, 1987; International 
        Organization of the Flavor Industry, 1995). 
        Intake (g/kg bw per day) calculated as follows: 
        [(g/day)/body weight], where body weight = 60 kg. Slight variation 
        may occur from rounding off.

    FIGURE 1

         Menthyl derivatives are metabolized like other alicyclic ketones
    and secondary alcohols. Ketones are reduced to their corresponding
    secondary alcohols and conjugated mainly with glucuronic acid (Quick,
    1928; Williams, 1940; Atzl et al., 1972). The metabolites of menthol
    are eliminated in the urine or faeces either unchanged or conjugated
    with glucuronic acid (Yamaguchi et al., 1994). The parent ketone,
    menthone, is primarily reduced to the correspon-ding secondary
    alcohol,  neo-menthol, which is metabolized and eliminated by
    pathways similar to those of its stereoisomer, menthol (Williams,
    1940). 

         (-)-Menthone given to rabbits at a dose of 1000 mg/kg bw was
    stereoselectively reduced to (+)- neo-menthol (Figure 2). Similarly,
    the stereoisomer (+)- iso-menthone was reduced to (+)- iso-menthol.
    About 67% of a 1000-mg/kg bw dose of (+)- neo-menthol given to
    rabbits by stomach tube was eliminated in the urine as the glucuronic
    acid conjugate (Williams, 1940). Like other alicyclic terpenoids such
    as menthol, ()- iso-menthone may also undergo omega-oxidation of the
    alkyl ring substituents, to yield the corresponding hydroxyketones.
    Data on structurally related alicyclic terpenoids suggest that
    oxidation occurs preferentially on the isopropyl or methyl substituent
    and not on the cyclohexane ring (Yamaguchi et al., 1994).

    FIGURE 2

    2.3.2  Toxicological studies

    2.3.2.1  Acute toxicity

         Oral LD50 values have been reported for 11 of the 14 substances
    (Table 3). The values are in the range 940-7300 mg/kg bw, indicating
    that the acute toxicity of orally administered menthol and related
    substances is low. The lowest reported value was not, however,
    supported by other studies in the same species.

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

         The results of short-term studies and of long-term studies of
    toxicity and carcinogenicity on substances related to menthol are
    summarized in Table 4 and described below. Studies on menthol are
    summarized in the separate monograph on that substance.

     Menthone (No. 429)

         Three short-term studies on peppermint oil and peppermint oil
    components, i.e. menthone, pulegone, and menthol, from the same
    laboratory showed 'cyst-like spaces' in the white matter of the
    cerebellum of rats. Groups of male and female rats were given menthone
    at doses of 0, 200, 400, or 800 mg/kg bw per day (Madsen et al.,
    1986); peppermint oil at 0, 10, 40, or 100 mg/kg bw per day (Thorup et
    al., 1983a); pulegone at 0, 20, 80, or 160 mg/kg bw per day (Thorup et
    al., 1983b); or menthol at 0, 200, 400, or 800 mg/kg bw per day
    (Thorup et al., 1983b) by gavage daily for 28 days. Cyst-like spaces
    in the white cerebellar matter were reported at the two highest doses
    of peppermint oil and pulegone and at all doses of menthone. A similar
    effect was not observed with menthol. 

         The slides of the brains of the animals in these studies were
    subsequently reviewed independently (Smith et al., 1996). Three
    conclusions were reached:

    (1)  No cellular reaction was seen in tissue adjacent to the cyst-like
         spaces in the white matter of the cerbellum, either in the three
         studies or in a subsequent 90-day study in rats given peppermint
         oil at a dose of 0, 10, 40, or 100 mg/kg bw per day (Spindler &
         Madsen, 1992). The appearance and extent of the cyst-like spaces
         were no different in the last, longer study than in the first
         three.

    (2)  The cyst-like spaces in cerebellar tissue were not seen in
         five-week studies in which rats of the same strain were given
         peppermint oil at doses of 150 or 500 mg/kg bw per day and dogs
         were given peppermint oil in gelatin capsules daily at a dose of
         25 or 125 mg/kg bw per day (Mengs & Stotzem, 1989). 


        Table 3.   Studies of the acute toxicity of substances structurally related to menthol used as flavouring agents

                                                                                                                                

    Substance                                          No.    Species     Sex    Route     LD50          Reference
                                                                                           (mg/kg bw)
                                                                                                                                

    (+)-neo-Menthol                                    428    Mouse       NR     Gavage    4000          Wokes (1932)
    Menthone                                           429    Rat         M/F    Oral      1600-1950     Levenstein (1973); 
                                                                                                         Igmi & Ide (1974)
    Menthyl acetate                                    431    Rat         M/F    Gavage    > 7000        Levenstein (1973)
                                                              Rat         M/F    Oral      > 5000        Shelanski &
                                                                                                         Moldovan (1972)
    Menthyl iso-valerate                               432    Rat         NR     Oral      > 5000        Moreno (1976)
    (-)-Menthyl lactate                                433    Rat         M/F    Oral      7257          Reagan & Becci (1984)
    (-)-Menthol ethylene glycol carbonate              443    Rat         M/F    Oral      > 2000        Tuffnell (1992)
    (-)-Menthol 1- and 2-propylene glycol carbonate    444    Rat         M/F    Oral      > 2000        Driscoll (1993)
    ()-Menthone 1,2-glycerol ketal                    445    Rat         M/F    Oral      5716          Reijnders (1991a)
                                                              Rat         M/F    Gavage    > 2000        Skydsgaard (1991)
    Monomenthyl succinate                              447    Rat         M/F    Oral      > 5000        Mercier (1994)
                                                                                                                                

    NR, not reported; M, male; F, female

    Table 4. Short-term and long-term studies of the toxicity of substances structurally related to menthol used as flavouring agents

                                                                                                                                           

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

    Menthone                         429   Rats      M/F    3/20             Gavage            28 weeks    400        Madsen (1986)
                                           Mice      F      2/NR             Intraperitoneal   24 weeks    --a        Stoner et al. (1973)
    ()-Menthone 1,2-glycerol ketal  446   Rats      M/F    3/10             Gavage            28 days     50         Reijnders (1991b)
                                                                                                                                           

    M, male; F, female; NR, not reported
    a No NOEL was determined.
    

    (3)  No cyst-like spaces were seen in cerebellar tissue when the
         brains of rats were perfused with peppermint oil (Olsen, 1994).

         The authors of the independent review concluded that the
    cyst-like spaces found after treatment of rats with peppermint oil,
    pulegone, or menthone were artefacts arising from inadequate
    preparation and fixation of the cerebellar tissue (Adams et al., 1996;
    Smith et al., 1996).

         Other reported effects in the groups of 10 male and 10 female
    rats receiving menthone at doses of 0, 200, 400, or 800 mg/kg bw per
    day by gavage for 28 days included a signficant reduction in food
    consumption among males receiving the high dose, among females at all
    doses during the first two weeks, and among females at the high dose
    during week 3 (Madsen et al., 1986). The reduction was attributed to
    the unpalatibility of the diet and is similar to that observed in
    other studies with menthone (Mengs & Stotzem, 1989). It was further
    reported that the relative weights of the kidney, spleen, liver, and
    brain in females and of the spleen, liver, and brain in males were
    statistically significantly increased at all doses. Furthermore, the
    bilirubin concentration and alkaline phosphatase activity in plasma
    were increased in all treated animals. A further statistical review
    showed, however, that the increases in organ weights and clinical
    chemical parameters occurred only at the high dose. The NOEL for
    menthone was therefore 400 mg/kg bw per day (Madsen et al., 1986),
    which is more than 10 000 times the daily  per capita intake ('eaters
    only') of 17 and 42 g/kg bw from its use as a flavouring agent in
    Europe and the United States, respectively (see Table 2).

         Groups of female A/He mice were given intraperitoneal injections
    of menthone at doses of 1900 or 4750 mg/kg bw three times weekly for
    eight weeks. The animals were observed for an additional 16 weeks;
    20-45% of the treated animals and 15% of the controls died before the
    end of the study. No increases were seen in the incidences of
    non-neoplastic or neoplastic lesions in the lung, liver, kidney,
    spleen, thymus, intestine, or salivary or endocrine glands of treated
    animals. The authors reported that the vehicle used, tricaprylin, had
    caused a 3-4 g loss of weight in control animals during the first week
    of the study, high mortality rates, and higher mean incidences of
    tumours (Stoner et al., 1973).

     ()-Menthone 1,2-glycerol ketal (No. 446)

         Four groups of five male and five female Wistar rat were given
    ()-menthone 1,2-glycerol ketal at doses of 0, 50, 200, or 800 mg/kg
    bw per day by gavage for 28 days and were observed daily throughout
    the study. The body weights and food consumption of the animals were
    measured weekly and on the day before necropsy, when macroscopic
    observations and organ weights were recorded. The adrenal glands,
    heart, kidney, liver, and stomach were examined histologically.
    Decreased serum glucose concentrations and increased kidney weights
    were noted in males at the two highest doses. Periportal
    hepatocellular hypertrophy was reported in these animals and in

    females at the highest dose. The hypertrophy in the males was
    accompanied by fine hepatocellular vacuolation. Increased liver
    weights were reported in males and females at the highest dose. The
    NOEL was 50 mg/kg bw per day (Reijnders, 1991b), which is more than
    10 000 times the daily  per capita intake ('eaters only') of 3 g/kg
    bw from its use as a flavouring agent in the United States (see Table
    2).

    2.3.2.3   Genotoxicity

         The results of tests for genotoxicity with five representative
    menthyl derivatives are shown in Table 5.

         Menthone (No. 429) was not mutagenic in the standard Ames test
    with  Salmonella typhimurium strains TA98, TA100, or TA1535 when
    tested at concentrations of up to 800 g/plate, either with or without
    metabolic activation; however, it induced reverse mutation in
     S. typhimurium TA97 at concentrations up to 160 g/plate in the
    presence of metabolic activation and at concentrations up to 800
    g/plate in the absence of activation. It was also mutagenic in strain
    TA1537 at concentrations of 32 and 6.4 g/plate (Andersen & Jensen,
    1984). 

         (-)-Menthol ethylene glycol carbonate (No. 443), (-)-menthol
    1,2-propylene glycol carbonate (No. 444), and ()-menthone
    1,2-glycerol ketal (No. 446) were not mutagenic in the standard Ames
    test or in the preincubation protocol with  S. typhimurium strains
    TA98, TA100, TA1535, TA1537, and TA1538 at concentrations up to 5000
    g/plate, either with or without metabolic activation (King, 1991;
    Poth, 1991; King, 1992, 1993). (-)-Menthol ethylene glycol carbonate
    (No. 443) and (-)-menthol 1,2-propylene glycol carbonate (No. 444) did
    not induce chromosomal aberrations in human peripheral blood
    lymphocytes when tested at concentrations up to 300 g/ml (King,
    1994a,b). ()-Menthone 1,2-glycerol ketal (No. 446) given orally to
    NWRI mice at doses up to 2500 mg/kg bw did not induce micronuclei in
    bone-marrow cells (Vlkner, 1991).


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        Table 5. Results of assays for the genotoxicity of substances structurally related to menthol

                                                                                                                                          

    Substance                     No.   End-point           Test object                  Dose                Result       Reference
                                                                                                                                          

    In vitro
    Menthone                      429   Reverse mutation    S. typhimurium TA 1535,      800 g/plate        Negativea    Anderson &
                                                            TA 100, TA 98                                                 Jensen (1984)
                                        Reverse mutation    S. typhimurium TA 1537,      6.4-800 g/plate    Positivea    Andersen &
                                                            TA 97                                                         Jensen (1984)

    (-)-Menthol ethylene          443   Reverse mutation    S. typhimurium TA 1535,      5000 g/plate       Negativea    King (1992)
    glycol                                                  TA 1537, TA 98, TA 100,
                                                            TA 1538
                                        Chromosomal         Human peripheral blood       300 g/ml           Negativea    King (1994a)
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    (-)-Menthol 1- and            444   Reverse mutation    S. typhimurium TA 1535,      5000 g/plate       Negativea    King (1993)
    2-propylene glycol                                      TA 1537, TA 1538, TA 98,
    carbonate                                               TA 100

                                        Chromosomal         Human peripheral blood       300 g/plate        Negativea    King (1994b)
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    ()-Menthone 1,2-glycerol     446   Reverse mutation    S. typhimurium TA 1535,      1500 g/plate       Negativea    King (1991)
    ketal                                                   TA 1537, TA 98, TA 100,
                                                            TA 1538
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                                                            TA 1537, TA 98, TA 100,
                                                            TA 1538

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    ketal                               formation
                                                                                                                                          
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    See Also:
       Toxicological Abbreviations