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    TOXICOLOGICAL EVALUATION OF CERTAIN FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES 10





    The evaluations contained in this document were prepared by the
    Joint FAO/WHO Expert Committee on Food Additives*
    Rome, 21-29 April 1976



    Food and Agriculture Organization of the United Nations

    World Health Organization




    *Twentieth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Geneva, 1976, WHO Technical Report Series No. 599, FAO Food
    and Nutrition Series No. 1.

    Butanetriols

         Butanetriols (BT) have been identified as trace contaminants of
    synthetic glycerol prepared by the hydrogenolysis of carbohydrates.
    The specific butanetriols identified were 1,2,3-butanetriol and
    1,2,4-butanetriol. The 1,2,3-butanetriol in the glycerol consists of
    erythro and threo isomers in the approximate proportion of 90% of the
    former to 10% of the latter.

    Biological Data

    Biochemical Aspects

         Young rats (approx. 100 g) were fasted and then administered
    by oral intubation 50 or 500 mg/kg uniformly labelled
    glycerine-1,2,3-C14 or 1,2,3-butanetriol-1,2,3,4-C14 (erythro rich)
    or 1,2,4-butanetriol-1,2,3,4-C14. The polyols were rapidly absorbed
    from the GI tract, less than 2% of butanetriols, and 5% of glycerine
    being found in the feces and contents of the GI tract, 12 hr post
    dosing. 57% and 50% respectively of the small and large dosage of
    glycerine, and 10% of the 1,2,3-butanetriol and 25-29% of the
    1,2,4-butanetriol was recovered in the respired air as C14O2, in a
    12 hr period. 12-24 hr post dosing 76-83% of the 1,2,3-butanetriol is
    excreted into the urine; primarily unchanged; with about 5% converted,
    possibly to a carboxylic acid derivative. The butanetriol not
    eliminated in the urine enters the general metabolic pool. About
    56-68% of this was oxidized and respired as 14CO2. The remaining 14C
    was randomly distributed throughout all the tissues in a manner
    similar to that observed with the residual 14C from glycerol.
    1,2,4-butanetriol is metabolized in a similar manner, 59-67% of the
    administered 14C being excreted in the urine in 24 hr. The major part
    being excreted in the first 12 hr post dosing. The active material in
    the urine is mainly unchanged. 1,2,4-butanetriol (ca 92%) and a
    carbosylic acid derivative. The butanetriol not eliminated enters the
    general metabolic pool. About 75-78% of this was oxidized and respired
    as 14CO2. The remaining 14C was randomly distributed throughout all
    the tissues in a manner similar to that observed with the residual
    14C from glycerol (Atlas, 1961).

         Mice (body wt 20 g) were put into hypoglycemic shock by
    injection of insulin. They were then injected intraperitoneally
    with either 2 g/kg body weight of glycine or 1,2,3-butanetriol or
    1,2,4-butanetriol or glucose (1.875 g/kg). Rapid recovery occurred
    with glycerine or glucose, but much longer periods were required for
    recovery, when the butanetriols were administered (Atlas, 1961).

        Acute Toxicity
                                                                                               

                                                           LD50
    Compound                      Animal         Route     (g/kg               Reference
                                                           body wt)
                                                                                               

    1,2,3-BT(91.5% erythro)       Rat            Oral      14.5                Atlas Chem.
    (8.4% threo)                  (female)                 (9.68-21.7)         Co., 1961

    1,2,3-BT(94.4% erythro)       Rat (male)     Oral      18.5                "
    (5.5% threo)                                           (13.6-25.3)

    1,2,3-BT(92.2% threo)         Rat            Oral      23.8                "
    (7.6% erythro)                (female)                 (19.8-28.7)

    1,2,3-BT(95% threo)           Rat (male)     Oral      24.9                "
    (4.4% erythro)                                         (21.1-29.4)

    1,2,4-BT(99.8%)               Rat            Oral      18.6                "
                                  (female)                 (16.1-21.5)

    1,2,4-BT(99.7%)               Rat (male)     Oral      24.4                "
                                                           (20.1-29.7)
                                                                                               
    
    REFERENCES

    Anderson, R. C., Harris, P. N. & Chen, K. K. (1959) J. Am. Pharm.
         Assoc., 39, 583

    Atlas Chemical Ind. (1961) Unpublished data submitted to the
         U.S.F.D.A.

    Atlas Chemical Industry, Inc. (1969) Report No. BMRD-65, Unpublished
         data submitted to the U.S.F.D.A.

    Chambers, W. H. & Deuel, H. J. (1925) J. Biol. Chem., 65, 21

    Deichmann, W. (1941) Ind. Med. 10, Ind. Hy. Sect. 2:5

    Gidez, L. I. & Karnovsky (1954) J. Biol. Chem., 206, 229

    Guerrant, N. B., Whitlock, G. P., Wolff, M. L. & Dutcher, R. A. (1947)
         Bull. Natl. Form Comm., 15, 205

    Hine, C. H., Anderson, H. H., Moon, H. O., Dunlop, M. K. & Morse, M.
         S. (1953) Arch. Ind. Hyg. & Occ. Med., 7, 282

    Johnson, V., Carlson, A. J. & Johnson, A. (1933) Am. J. Physiol., 103,
         517

    Johnson, J. D., Hurwitz, R. & Kretchmer (1971) J. Nutr., 101, 299

    Kopf, R., Loeser, A. & Meyer, G. (1951) Arch. Exper. Path. u
         Pharmakol., 212, 405

    Smyth, H. F., Seaton, J. & Fisher, L. (1941) J. Ind. Hyg. & Tox., 23,
         259

    Spector, W. (1956) Handbook of Toxicology, Acute Toxicities, Volume 1,
         pp. 90 and 204

    Wegener, H. (1953) Arch. Exper. Path. u Pharmakol., 220, 414

    DL-, and L-menthol

    Explanation

         DL-, and L-menthol were evaluated for acceptable daily intake for 
    man in 1967 (see Ref. No. 15, p. 58) when an unconditional acceptance 
    of 0-0.2 mg/kg bw and a conditional acceptance of 0.2-2 mg/kg bw were 
    recommended. On the recommendation that allocations of conditional 
    ADIs should be abandoned (Ref. 32, p. 11), the conditional ADI for 
    menthol was eliminated and an ADI of 0-0.2 mg/kg bw was established 
    (Ref. 34, p. 14). Further data have become available and are
    summarized below. Previously published monograph has been thus 
    expanded.

    BIOLOGICAL DATA

    Biochemical aspects

         In the dog, 5% of orally administered menthol metabolizes to
    1-menthyl-5-glucuronide (Williams, 1959). For other animals, the
    reported proportions vary. In the rabbit, the larger the ingested
    dose, the less conjugation (Quick, 1924). Other workers reported
    31-34% glucuronide excretion in rats after oral or continuous i.v.
    dosing (Herken, 1961). Rabbits are said to eliminate 48% of
    1-menthol and 59% of dl-menthol as glucuronide (Williams, 1938).
    Menthol is absorbed percutaneously, and exerts a local anaesthetic
    action in mice (Macht, 1939).

         Between 40.1% and 98.7% of a 1.56 g oral dose of menthol,
    administered as an oil in water emulsion, was excreted as glucuronides
    in urine in 24 hours by 19 normal men aged 19-24 (Bolund et al.,
    1967).

         The ß-glucuronidase activity of liver, kidney and spleen was
    increased following the oral administration of 20 mg menthol/mouse 3
    times daily for 4 days and twice on the 5th day, the animals being
    killed 3 hours after the last dose (Fishman, 1940).

    TOXICOLOGICAL STUDIES

    Special studies on carcinogenicity

    Mice

         Groups of 30 female A/He strain mice received I.P. injections of
    menthol dissolved in tricaprylin at the maximum tolerated dose level
    (the maximum dose level tolerated after receiving 6 I.P. injections

    over a 2-week period), and one quarter this dose level, 3 times
    weekly, for 8 weeks. A group of 24 control female mice received a
    similar number of injections of tricaprylin. All animals which
    survived treatment were killed after 24 weeks and the numbers of
    pulmonary adenomas counted. No increase in incidence of adenomas was
    found in the test groups which had received a total of 2.0 or 0.5 g
    menthol/kg mouse. With the same test system, urethane (total dose 10
    or 20 mg) and several alkylating agents induced a marked increase in
    the incidence of pulmonary adenomas, but other substances shown to be
    carcinogenic in other test systems, e.g. safrole, produced no increase
    (Stoner et al., 1973).

    Special studies on mutagenicity

         No evidence was obtained that natural menthol was mutagenic when
    examined extensively in bacteria, in host mediated assays using
    bacteria and saccharomyces as indicators, in in vitro and in vivo
    cytogenic studies and by the host mediated assay (Weir and Brusick,
    1975).

    Special studies on pharmacological effects

         Menthol stimulated the ciliary activity of frogs oesophagus; its
    action was not blocked by atropine treatment (Das et al., 1970). No
    effect on the activity of isolated rabbit intestine was found when the
    concentration of menthol in bath fluid was 0.01%. A 0.03% solution
    depressed contractions and at a 1.5% concentration the intestinal
    motility was completely arrested (Chiu, 1972). A depressive action
    directly on the muscle of isolated frog and rabbit heart was noted
    with menthol (Heathcote, 1922). A dose of 0.25 mg/kg of menthol in
    propylene glycol given intravenously produced a fall of blood pressure
    in anaesthetized cats and rabbits (Rakieten and Rakieten, 1957). A
    concentration of 0.4-400 µg/ml menthol reduced the taste threshold of
    human subjects by 20-63% (Skouby and Zilstorff-Pedersen, 1955).
    Stimulation of bile flow following oral administration of menthol is
    well documented (Möersdorf, 1966; Förster and Oettel, 1954).

    Acute toxicity

                                                                        

                                        LD50           References
    Animal           Route       (mg/kg body-weight)
                                                                        

    (a) l-menthol

    Mouse            s.c.        5000-6000             Flury, 1920

                     i.p.        2000 (LD)             Macht, 1939

    Rat              oral        3300                  Herken, 1961

                     s.c.        1000-2500             Flury, 1920

                     i.p.        710                   Herken, 1961

                                 1500 (LD)             Macht, 1939

    Guinea-pig       i.p.        4000 (LD)             Macht, 1939

    Cat              oral        800-1000              Flury, 1920

                     i.p.        800-1000              Flury, 1920

                     i.v.        34 (LD)               Macht, 1939

    Rabbit           i.p.        approx. 2000          Herken, 1961

    (b) dl-menthol

    Mouse            s.c.        1400-1600             Flury & Seel, 1926

    Rat              oral        2900                  Herken, 1961

                                 3180                  Jenner et al., 1964

                     i.p.        750                   Herken, 1961

    Cat              oral        1500-1600             Flnry & Seel, i926

                     i.p.        1500-1600             Flury & Seel, 1926

    Rabbit           i.p.        approx. 2000          Herken, 1961
                                                                        

    Short-term studies

    Rat

         Groups of 40 male and 40 female rats received 0, 100 and
    200 mg/kg body-weight of either l- or dl-menthol in their diet for
    5-1/2 weeks.  There was no adverse effect on weight gain, excretion of
    glucuronide, water and electrolytes, nor interference with CNS
    reactions to cardrazol or electric shock, or on i.v. hexobarbital
    sleeping time as compared with controls (Herken, 1961).

    Long-term studies

         None available.

    Observations in man

         Smoking 80 mentholated cigarettes resulted in irritability, 
    gastrointestinal upsets, tremors, ataxia, bradycardia and toxic 
    psychosis. Taking 64 mg of menthol three times a day produced
    tiredness and apathy within 3 days and nausea, exhaustion and
    bradycardia in 7 days (Luke, 1962). Chronic urticaria with basophil
    leucopenia on challenge has been reported after contact with menthol
    in toothpaste, mentholated cigarettes, peppermint sweets, etc. (Papa &
    Shelley, 1964; McGowan, 1966).

         Of 877 persons with primary contact, atopic, nummular and stasis
    dermatitis and exzema, 1% developed a positive reaction (erythema and
    infiltration) to patches of 5% menthol in yellow paraffin within 96
    hours. This reaction rate was approximately one-fifteenth, one-fifth
    and two-fifths the reaction rate respectively to paraphenylene
    diamine, nickel sulfate and lanolin with salicylic acid (Rudzki and
    Kleniewska, 1970).

         The usual human oral dose is 60-120 mg. The probable lethal dose
    for man is 50-500 mg/kg B.W. (Gleason et al., 1969).

    Comments

         Evidence from human studies suggest that menthol is well absorbed
    from the gut. A large proportion is excreted in urine as glucuronides
    but the metabolic fate of the remainder has not been elucidated. No
    long-term studies have been carried out but a 24 week lung adenoma
    study and extensive mutagenicity studies gave negative results. The
    results of one study suggested that adverse effects may occur in man
    ingesting about 2 mg menthol/kg/day. Other evidence from human
    exposure shows that adverse effects are unlikely to occur when
    0.2 mg menthol/kg/day is ingested. The Committee agreed, however, that

    further information on human menthol intake from food and medicines
    and, if possible, observations on a group of people with a higher than
    average intake would need to be carried out if any increase in the ADI
    is to be contemplated.

    Evaluation

    Level causing no toxicological effect:

         Rat: 200 mg/kg/body weight of dl- or l-menthol

    Estimate of acceptable daily intake for man:

         0-0.2 mg/kg BW

    Work desirable

    1.   Long-term toxicity and carcinogenicity study in rats.

    2.   Information on the average and maximum likely intakes of menthol.

    3.   Clinical observation of subjects with a higher than average
         intake of menthol.

    4.   Metabolic studies.

    REFERENCES

    Bolund, S., Falus, F. and Jorgensen, K. (1967) Scand. J. clin. Lab.
         Invest., 11, 288

    Chiu, Y. Y. (1972) Shikoku Acta Med., 6, 35

    Das, P. K., Rathor, R. S., Sinha, P.S. and Sanyal, A. K. (1970)
         Indian J. Physiol. Pharmacol., 14, 257

    Fishman, W. H. (1940) J. Biol. Chem., 136, 229

    Flury, F. (1920) Abderhalden's Handbuch der Biologischen Arbeits-
         methoden, 39, 1365

    Flury, F. and Seel, H. (1926) Synthetishes Menthol Muench. Mediz.
         Wochenschr., 48, 2011-2012

    Förster, W. and Oettel, H. (1954) Naunyn-Schmiedebergs Arch. Exptl.
         Pathol. Pharmakol., 222, 244

    Gleason, M. N., Gosselin, R. E., Hodge, H. C. and Smith, R. P. (1969)
         Clinical toxicology of commercial products, 3rd ed., The
         Williams and Wilkins Co., Philadelphia

    Heathcote, R. St. A. (1922) J. Pharm. and Exp. Therapeutics, 21, 177

    Herken, H. (1961) Pharmakologisches Gutachten über die Vertraglichkeit
         von natürlichem (l-) und synthetischem (d,l-) Menthol.
         Unpublished report from the Director, Pharmakologischen Institute
         der Freien Universität, Berlin-Duhlem, submitted to the World
         Health Organization by Schering, A. G.

    Jenner, P.M., Hagan, E. C., Taylor, J. M., Cook, E. L. and Fitzhugh,
         O. G. (1964) Fd. Cosmed. Toxicol., 2, 327

    Luke, E. (1962) Lancet, i, 110

    Macht, D. I. (1939) Arch. Int. Pharmacodyn., 63, 43

    McGowan, E. M. (1966) Arch. Derm., 94, 62

    Möerdorf, K. (1966) Chim. Ther., 1966(7), 442

    Papa, M. and Shelley, W. B. (1964) J. Amer. med. Ass., 189, 546

    Quick, A. J. (1924) J. Biol. Chem., 61, 679

    Rakieten, N. and Rakieten, M. (1957) J. Am. Pharm. Assoc., 46, 82

    Rudzki, E. and Kleniewska, D. (1970) Br. J. Derm., 83, 543

    Skouby, A. P. and Zilstorff-Pedersen, K. (1955) Acta Physiol. Scand.,
         34, 250

    Stoner, G. D., Shimkin, M. B., Kniazeff, A. J., Weisburger, J. H.,
         Weisburger, E. K. and Gori, G. B. (1973) Cancer Research.,
         33, 3069

    Weir, R. J. and Brusick, D. (1975) Mutagenic evaluation of compound
         FDA 71-57, Menthol. Unpublished report from Litton Bionetics,
         Inc., Kensington, Md., USA, submitted to the World Health
         Organization by the U.S. Food and Drug Administration

    Williams, R. T. (1938) Biochem. J., 32, 1849-185

    Williams, R. T. (1959) Detoxication Mechanisms, Second Edition,
         Chapman Hall, London


    See Also:
       Toxicological Abbreviations
       Menthol (WHO Food Additives Series 42)
       MENTHOL (JECFA Evaluation)
       Menthol  (SIDS)