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.
This enzyme preparation has been evaluated for acceptable daily
intake by the Joint FAO/WHO Expert Committee on Food Additives (see
Annex 1, Ref. No. 27) in 1971.
Since the previous evaluation additional data have become
available and are summarized and discussed in the following monograph.
The previously published monograph has been expanded and is reproduced
in its entirety below.
No information available.
Special studies on aflatoxins production
Six groups of 18-20-day-old ducklings were given 0, 2 mg, 10 mg,
50 mg and 250 mg material by gavage as well as a positive control
group receiving 12.5 mg aflatoxin. Deaths occurred only in the
aflatoxin and 250 mg groups. There was some weight loss throughout the
test in the 250 mg group and less so at lower test levels. Liver
weight was increased on the eighth day at all levels. Histologically
no lesions were seen which pointed to the presence of aflatoxin (van
Logten & Kroes, 1968).
Special studies on reproduction
A four-generation study in rats was performed on the same
compound as in the long-term study by Til et al., 1972a. Four groups
of 10 males and 20 females were fed 0, 0.04, 0.2 and 1% compound in
the diet. Each generation was mated in week 12 and week 20 to get two
litters a and b. Only the b litters were raised to the next
* This enzyme preparation is prepared from the species Mucor
generation. Ten males and 10 females in each group of F3b were kept
for a five-week feeding study. The F2-generation rats showed
relatively low body weights in the 1% group in both sexes, but this
phenomenon did not occur in the F3-generation. The number of litters
born was high in all groups, and the number of youngsters per litter
was high in all groups and all generations. No consistent differences
in body weight and mortality were observed during the lactation
period. In F3b no distinct differences in body weight, food
consumption and food efficiency were found. The relative organ weight
of the spleen of males fed 1% was decreased. Gross and microscopic
examination revealed no abnormalities related to feeding the compound
(Til et al., 1972b).
Animal Route (mg/kg bw) Reference
Mouse Oral 4 500 Hara et al., 1963
i.v. 358 Ito et al., 1964
Rat Oral > 5 000 Hara et al., 1963
20% aqueous suspension was nonirritant to rabbit conjunctiva (Ito
et al., 1964).
A 10-week study in groups of male and female rats receiving 0
(0%), 2 (0.0002%), 20 (0.002%) and 1000 (0.1%) ppm in their diet
showed a dose-related weight increase compared with controls. No data
are available on organ weights or haematology but LFTs showed no
deleterious effect. Histopathology showed nothing abnormal (Ito et
A 90-day study was carried out on groups of rats using 0 (0%),
10 (0.001%), 1000 (0.1%) and 10 000 (1%) ppm in their diet. Some
reduction in growth was observed which was not dose-dependent and
statistically not significant. However, the WBC showed increases in
lymphocytes with increasing dosage and changes in monocyte and
neutrophil counts which were unrelated to dose. Organ weights and
histopathology were normal (Hara et al., 1963). Another 90-day study
was carried out in three groups of 10 male and 10 female rats
receiving 0 (0%), 1250 (0.125%) or 12 500 (1.25%) ppm in their diet.
No abnormalities were detected as regards behaviour, appearance, food
consumption, growth, haematology, organ weights, gross and
histopathology (van Logten & Kroes, 1968a).
A 90-day toxicity study was performed in three groups of rats,
each consisting of 10 male and 10 female fed 0 (0%), 1250 (0.125%) and
12 500 (1.25%) ppm enzyme preparation from Mucor pusillus Lindt in
the diet. The fungus was cultured on wheat bran, the preparation
purified by series of partial precipitation with ethanol and vacuum-
dried. Compared with controls the food intake and growth were slightly
increased. A slight, not significant dose-dependent increase in the
number of lymphocytes and increase in the relative weight of thyroid
and the prostate (P<0.05) were found. No gross or histopathological
changes were observed (van Logten et al., 1972).
Groups each comprising four male and four female dogs were fed
dietary levels of 0, 0.5, 1.0 or 2.0% of fungal rennet (from the same
batch used in the rat study described below) for two years. There was
only one death unrelated to compound administration. General behaviour
and appearance, body weight gain and food intake were all comparable
between groups. Blood pressures and heart rates were generally within
normal limits and electrocardiograms revealed no gross abnormalities.
Slight ophthalmological abnormalities observed in two control dogs,
three dogs fed 0.5% and five fed 2.0% were considered not related to
compound administration. Haemograms, clinical chemistry and
qualitative urinary analyses were comparable for all groups. There
were no gross or histopathological abnormalities that could be related
to feeding fungal rennet (Hollingsworth & Woodard, 1968b).
Groups each comprising 20 male and 20 female rats were fed
dietary levels of 0, 0.5, 1.0 or 2.0% of fungal rennet (identified as
microbial rennet from Meito Sangyo Co. Ltd, Lot No. R2G7801). General
appearance and behaviour were comparable in test and control groups.
Incidence of mortality appeared unrelated to feeding the test
compound. Body weight gain and food intake were normal between the
groups. Periodic haemograms including haemoglobin, microhaematocrit,
coagulation time and general clinical chemistry determination as well
as absolute and relative organ weights and gross and histopathology
revealed no abnormalities related to feeding the rennet (Hollingsworth
& Woodard, 1968a).
In a two-year study four groups of Wistar rats, each group 30
males and 30 females, received in the diet respectively, 0, 0.04, 0.2,
and 1.0% rennet from Mucor pusillus Lindt (strain: University of
Tokyo, No. F-27). The diets were prepared once a fortnight and stored
at room temperature. Mean body weight and food consumption showed no
significant differences. Biochemical blood parameters and urine
analyses showed no dose-related differences. The relative weights of
testes were significantly increased in all the test groups. Gross and
histopathological findings were related to aging and infectious
diseases. At autopsy of about 90% of the animals, the tumour rate in
the control group was 80% and 82% in the 1% group (Til et al., 1972a).
This preparation has been tested by short-term studies in rats
and dogs. A duckling study was done and the results of a further
90-day study and two-year study in rats and a four generation
reproduction study showed no significant abnormalities at the 1%
level, the highest level tested. This meets the requirements laid down
by the Committee.
Acceptable daily intake not specified.*
Hara, S. et al. (1963) Report of the Department of Pharmacology, Tokyo
Medical College submitted to WHO
Hollingsworth, R. C. & Woodard, G. (1968a) Unpublished Report dated
7 November 1968 from Woodard Research Corporation submitted by
Noury & van der Lande, N. V.
Hollingsworth, R. C. & Woodard, G. (1968b) Unpublished report dated
6 November 1968 from Woodard Research Corporation submitted by
Noury & van der Lande, N. W.
* 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 any acceptable
daily intake (ADI) in mg per kg of body weight is not deemed
Ito, H., Kond, H. & Tokunaga, Y. (1964) Fd. Hyg. Mag., Japan,
Nakamura, K. (1966) Report submitted by the National Institute of
Hygienic Sciences, Tokyo to WHO
Til, H. P., van der Meulen, H. C. & de Groot, A. P. (1972a)
Unpublished report dated December 1972 from CIVO/TNO submitted By
Noury & van der Lande, N. V.
Til, H. P., Spanjers, M. T. & Willems, M. I. (1972b) Unpublished
report dated December 1972 from CIVO/TNO submitted by Noury & van
der Lande, N. V.
van Logten, M. J. & Kroes, R. (1968) Report 82/68 Tox submitted by
van Logten, M. J. & Kroes, R. (1968a) Report 88/68 Tox submitted by
van Logten, M. J. et al. (1972) Fd. Cosmet Toxicol., 10, 649
Woodard Research Co. (1968) Unpublished report dated 6 November 1968
submitted to WHO
Woodard Research Co. (1968a) Unpublished report dated 7 November 1968
submitted to WHO