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)