INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF SOME
FOOD COLOURS, ENZYMES, FLAVOUR
ENHANCERS, THICKENING AGENTS, AND
CERTAIN FOOD ADDITIVES
WHO FOOD ADDITIVES SERIES 6
The evaluations contained in this publication were prepared by the
Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
4-13 June 19741
World Health Organization Geneva 1975
1 Eighteenth Report of the Joint FAO/WHO Expert Committee on
Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 557.
FAO Nutrition Meetings Report Series, 1974, No. 54.
INOSINIC ACID, CALCIUM AND DISODIUM SALTS
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
IMP derives from dephosphorylation of ATP (Adonosine-5-
triphosphate) to AMP and deamination. Further conversion to
hypoxanthine is slow compared with the conversion of ATP to IMP. The
greater portion of IMP in living tissue is derived from de novo
purine synthesis, less derives from ingested dietary purines or
nucleotides. IMP is the first purine formed in the complex synthesis
of purine nucleotides. Uric acid is the metabolic end-product in man
and allantoin in many mammals. Some two-thirds of uric acid appears
in urine, the rest is excreted in the gut and further degraded
(Kojima, 1973). Pharmacological studies on IMP showed no effect on the
S-A or A-V nodes in the Langendorff preparation of the rat heart
(Versporille, 1966). Intravenous administration produced hypotension
in the rabbit and dog (Flössner, 1934) but had no effect on heart rate
and ECG of the rabbit (Yabo, 1964; Hara et al., 1966). A 1% solution
decreased the mobility of isolated guinea-pig intestine (Hara et al.,
1966). Isolated guinea-pig uterus showed a biphasic response to the
application of IMP (Flössner, 1934). Topical IMP enhances the
electrical response of the chordotympani to topical MSG in rats and
cats (Adachi, 1964; Sato et al., 1965). Intravenous IMP had no
significant effect on blood electrolytes in the rabbit (Hara et al.,
1966).
Male and pregnant (day 10 or 18 of gestation) rats were given by
gavage 25 mg/kg bw of 814C labelled IMP. Radioactivity in plasma
reached maximum levels one-half to two hours after treatment and
slowly decreased to practically zero after 24 hours, the half-life
being about five hours. About 70% of total activity appeared in the
urine, 6-7% in faeces, none in expired air, between 0-2% remained in
organs, 8-17% in the organ-free carcass of males and 18 day pregnant
females 24 hours after treatment. The fetuses contained about 0.77%
activity 24 hours after treatment (Ohara et al., 1973). Groups of five
male rats (control 10) given 0%, 1% and 4% IMP for five or 10 days in
a purine-free basal diet showed levels of uric acid in serum and
urine hardly different from controls. Most of the exogenously ingested
IMP was rapidly excreted in the urine as allantoin. Only liver
hypoxanthine-guanine phosphoribosyl transferase and adenine
phosphoribosyl transferase activity were increased and also the ratio
of liver uricase/xanthine oxidase activity, suggesting metabolism by
shunt pathways for exogenously ingested IMP (Hashimoto et al., 1973).
I.v. 500 mg/kg IMP in mice caused behavioural excitement,
increased reflex response, no muscular relaxation, depressed rotating
activity during the first hour, did not modify electroshock
convulsions but decreased dose-dependently metrazol convulsive dosage.
Doses of 50-500 mg/kg i.v. prolonged loss of righting reflex.
10-50 mg/kg i.v. in cats had no effect on B.P., heart rate, ECG, blood
flow of hind limbs. 100 mg/kg s.c. IMP had no effect on intestinal
transport as measured by charcoal transportation in mice. 10-4 g/ml
IMP did not affect the contractile response of guinea-pig ileum to
acetylchlorine, histamine or barium chloride but 10-2 decreased
motility. 100 mg/kg s.c. IMP had no effect on gastric juice volume in
the rat but slightly increased pH. 100 mg/kg in mice depressed
salivary secretion. Rats given 100 mg/kg intragastric IMP showed no
diuresis. Oral 500 mg/kg IMP had no effect on analgesic response of
mice or carrageenin oedema in rats (Kojima, 1973).
TOXICOLOGICAL STUDIES
Special studies on reproduction
Rat
One control and three test groups of 10 male and 20 female rats
were used in a three generation study at dietary levels of 0%, 0.5%,
1% and 2% IMP. Test animals were on their diets for 60 days before
mating. No effects on mating performance, pregnancy rate or duration
of gestation were noted. Body weight gain was better than in controls
for male test animals in all generations. Litter size, pup weight, pup
mortality and incidence of abnormalities were unaffected by treatment.
Organ weight analysis, histopathology and skeletal staining of F3B
revealed no consistent pattern related to treatment (Palmer et al.,
1971).
Special studies on teratogenicity
Rat
One group of nine pregnant rats was given daily 100 mg/kg IMP
during days 9-15 of pregnancy. No significant teratogenic findings
were noted in the fetuses examined (Kaziwara, 1971).
Rabbit
Two groups of 13-18 female Japanese white rabbits received
200 mg/kg or 2000 mg/kg bw IMP in their diet during days 6-18 of
gestation. Sixteen female rabbits acted as controls. Four to five
females of each group were delivered spontaneously and pups observed
to day 30. All other dams were killed at day 29 of gestation. No
significant effects were noted on implantation sites, number of
live or dead fetuses, body weight of live fetuses and external
abnormalities. The mortality of fetuses in the 0.2 g/kg group was
lower than other groups. All groups showed some delay in ossification
but no specific skeletal abnormalities were found which appeared to be
due to IMP. Daily administration of 2000 mg/kg bw IMP by gavage had no
adverse effect on pup development (Jojima et al., 1973).
Acute toxicity
LD50
Animal Route (mg/kg bw) Reference
Mouse oral 12 000-14 000 Hara et al., 1966
oral (male) 17 600 Ichimura & Muroi, 1973
oral (female) 19 800 Ichimura & Muroi, 1973
s.c. 6 200-7 000 Hara et al., 1966
s.c. (male) 5 480 Ichimura & Muroi, 1973
s.c. (female) 5 630 Ichimura & Muroi, 1973
i.p. 5 400-5 600 Hara et al., 1966
i.p. (male) 6 300 Ichimura & Muroi, 1973
i.p. (female) 6 200 Ichimura & Muroi, 1973
i.v. 3 300-3 900 Hara et al., 1966
i.v. (male) 3 950 Ichimura & Muroi, 1973
i.v. (female) 4 600 Ichimura & Muroi, 1973
Rat oral 16 000 Usui et al., 1971
oral (male) 17 100 Ichimura & Muroi, 1973
oral (female) 15 900 Ichimura & Muroi, 1973
s.c. (male) 3 900 Ichimura & Muroi, 1973
s.c. (female) 4 340 Ichimura & Muroi, 1973
i.p. (male) 5 400 Ichimura & Muroi, 1973
i.p. (female) 4 850 Ichimura & Muroi, 1973
i.v. (male) 2 730 Ichimura & Muroi, 1973
i.v. (female) 2 870 Ichimura & Muroi, 1973
Short-term studies
Rat
Eight groups of 10 male rats received daily in their diet either
0.10, 100 or 1000 mg/kg bw of naturally derived or synthetically
prepared IMP for 90 days. No adverse effects were noted on weight
gain, organ weights, haematological parameters and histopathologically
(Hara et al., 1966).
In another experiment three groups of 10 male rats, given 0%,
0.1% or 1% of IMP in their diet for three and six months, showed no
significant abnormalities regarding their behaviour, body weight gain,
food intake, haematology, urinalysis, macroscopic and histological
findings (Usui et al., 1971).
A further six months study in one control and four treatment
groups each of eight male and eight female Sprague-Dawley rats at
levels of 0%, 0.5%, 1.0%, 2.0% and 4.0% during 12 weeks followed by
levels of 0%, 0.75%, 1.5%, 3.0% and 6.0% from week 13 to 25 showed no
significant abnormalities in any treatment group with regard to
behaviour, body weight gain, food intake, haematology and urinalysis.
Some animals in higher dosage groups showed renal medullary
calcification. Organ weights were normal but the relative mean weight
of kidney and spleen in the 6% groups were significantly raised
(Yonetani et al., 1973).
In another experiment, six male and six female Sprague-Dawley
rats were given 0%, 1%, 4% and 8% IMP in the diet for 52 weeks. The
only adverse effects noted were slight depression of body weight gain
of the 8% groups. More renal calcifications were found in the 4% and
8% females compared with others, probably related to urine osmolarity.
The 8% males and 2% and 8% females showed more severe nephrosis than
the others (Yonetani et al., 1973).
Dog
Two groups of one male and one female beagle were given 3.6-3.9%
or 8% of IMP for four to six weeks without any adverse effects (Noel
et al., 1971).
Long-term studies
Rat
Five groups of 14 male and 14 female Sprague-Dawley rats were
given for 95 weeks 0%, 1%, 2%, 4% and 8% IMP in their diet. No
significant changes were seen in behaviour, body weight gain, food
intake, haematology, blood chemistry, urinalysis, histopathology or
mortality (Yonetani et al., 1973).
Dog
One control and three test groups each of four male and four
female beagles were given 0, 0.5, 1 and 2 g/kg IMP in their diet for
two years. No significant abnormalities were found clinically in body
weight gain, food consumption or ophthalmoscopy. Haematology,
biochemistry and urinalysis were normal. Dogs on 2 g/kg/day had
significantly raised allantoin levels in the serum but these were not
dose related. Exhaustive histopathology showed no significant
abnormalities (Rivett et al., 1973).
OBSERVATIONS IN MAN
Three healthy volunteers were given 1, 1.5, 2 and 2.5 g IMP for
seven consecutive days on a constant purine diet. Serum uric acid and
urinary uric acid excretion doubled without signs of ill effects
(Kojima, 1973).
Comments:
Inosinates, guanylates and ribonuoleotides are substances
normally present in all tissues and their role in purine metabolism as
well as their breakdown in the majority of mammals, but not man, to
uric acid and allantoin is well known. The various products have been
studied adequately in long-term, reproduction and teratology tests.
Ingestion of large amounts of these compounds by man can increase the
serum uric acid level and urinary uric acid excretion and this needs
to be considered in relation to people with gouty diathesis and those
taking uric-acid retaining diuretics. Hence, specific mention of the
addition of these substances on the label may be indicated. The
changes in dietary purine intake from the use of flavour enhancers are
no greater than those likely to be occasioned by changes in
consumption of those dietary items which are the main contributors of
purine.
EVALUATION
Acceptable daily intake not specified.*
* The statement "ADI not specified" means that, on the basis of the
available data (toxicological, biochemical, and other), the total
daily intake of the substance, arising from its use or uses at the
levels necessary to achieve the desired effect and from its acceptable
background in food, does not in the opinion of the Committee,
represent a hazard to health. For this reason, and for the reasons
stated in individual evaluations, the establishment of an acceptable
daily intake (ADI) in mg/kg bw is not deemed necessary.
REFERENCES
Adachi, A. (1964) J. Physiol. Soc. Jap., 26, 347
Flössner, O. (1934) Arch. Exptl. Path. Pharmakol., 174, 245
Hara, S. et al. (1966) J. Tokyo Med. Coll., 24, 553
Hashimoto, S. et al. (1973) Report from Life Sciences Laboratories,
Ajinomoto Co., Japan
Ichimura, M. & Muroi, K. (1973) Report from Life Sciences
Laboratories, Ajinomoto Co., Japan
Jojima, M. et al. (1973) Report from Life Sciences Laboratories,
Ajinomoto Co., Japan
Kaziwara, K., Mizutani, M. & Ihara, T. (1971) J. Takeda Res. Lab., 30
(2), 314
Kojima, K. (1973) Toxicology, 2 (In press)
Noel, P. R. B. et al. (1971) Personal communication, HRC
Ohara, V., Matsuzawa, Y. & Takeda, J. (1973) Report from Life Sciences
Laboratories, Ajinomoto Co., Japan
Rivett, K. F. et al. (1973) Unpublished data of HRC
Sato, M. et al. (1965) Jap. J. Physiol., 15, 53
Usui, T. et al. (1971) J. Takeda Res. Lab., 30 (3), 614
Versprille, A. (1966) Arch. Ges. Physiol., 291, 261
Yabo, S. (1964) Folia Pharmacol. Japon., 60, 194
Yonetani, S. et al. (1973) Unpublished data
See Also:
Toxicological Abbreviations