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.
NATAMYCIN (PIMORICIN (R))
Explanation
This substance has been evaluated for acceptable daily intake for
man by the Joint FAO/WHO Expert Committee on Food Additives in 1968
(see Annex I, Ref. 17, p. 27).
Since the previous evaluation, additional data have become
available and are summarized and discussed in the following monograph.
Previously published monograph has been expanded and is reproduced in
its entirety below.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Little information is available on the absorption, distribution,
excretion or metabolism of pimaricin in the body. No pimaricin (less
than 1 µg/ml) could be detected in the blood following ingestion of
500 mg by human subjects (Anonymous, 1968) and this helps to
substantiate the statement (Raab, 1972) that pimaricin is not absorbed
from the gut in animals or man.
TOXICOLOGICAL STUDIES
Special studies on allergic effects
No allergic sensitization occurred among 111 patients being
treated with pimaricin for a variety of conditions (Gruyper, 1961,
1964). No history of allergic reactions was found in 73 workers
engaged for an average of 5 years in the manufacture of pimaricin. No
allergic reactions were obtained in the 71 who were tested by
cutaneous or intradermal challenge doses (Malten, 1967). Repeated
patch tests on 102 patients with various forms of eczema failed to
demonstrate any sensitizing potential with pimaricin (Malten, 1968).
Special studies on degradation products
(1) Acute toxicity
Recent studies indicate that similar breakdown products of
pimaricin occur in simulated gastric juice, 0.5% citric acid and urine
and it appears likely that breakdown products in stored apples
1 Natamycin is the international nonproprietary name of this
substance, hitherto referred to as pimaricin in the reports of the
Expert Committee.
resemble those produced in gastric juice. The breakdown products are
tetraenes related to pimaricin, principally the aglycone dimerized
and/or decarboxylated; whether these would be absorbed remains to be
tested (Brik, 1975). Approximately 50% pimaricin is broken down in 1
hour in simulated gastric juice and losses from the stomach of 33-43%
and 0-31% respectively occurred in fasted and non-fasted rats
(Morgenstern and Muskens, 1975).
Treatment of 1% or 5% % Mouse i.p. LD50
suspension decomposition mg/kg bw
kept at pH 2.2 with citric acid 74 200
kept at pH 6.3 in the dark 13 200-400
kept at pH 6.3 in the light 80 400-600
kept at pH 8.5 (NaOH) 0 150-250
kept at pH 8.5 (NaOH) 5 450
kept at pH 10.4 with soda 100 >800
kept at pH 6.3 with 0.1% H2O2 9 200-400
kept at pH 5.0 in UV light 0 170
kept at pH 5.0 in UV light 50 200
(Ottens, 1965)
(2) Short-term studies
Rat
Groups of 15 male and 15 female rats were fed for 98 days on
diets containing 5% water, 5% of 0.5% citric acid, 500 ppm pimaricin
or 5% of a solution of acid degraded pimaricin (pimaricin suspended in
0.5% citric acid until only 14% of the activity remained). No animals
died and weight gain was unaffected by treatment and no adverse
effects were seen in the results of haematological examination or
determination of the absolute weights of the liver and kidneys. The
minor differences in the relative organ weights were considered
coincidental and not due to treatment. Microscopic examination of a
wide range of organs failed to detect any injury due to pimaricin
degradation products (Hutchison et al., 1966).
Slices of cheese were treated with 0.05% and 5% suspensions of
pimaricin and left to dry at room temperature. The antimicrobial
activity of the two cheeses declined to less than 20% and 60-80%
during the 3 weeks storage period before they were incorporated into
rat diet, the final dietary pimaricin plus degraded pimaricin contents
being 3.6 or 360 ppm. Groups of 15 or 30 male and 15 or 30 female rats
received diet containing fresh cheese freshly dressed with 0, 0.05 and
5.0% pimaricin or diet containing cheese dressed with 0, 0.05 and 5%
suspensions and stored for 3 weeks. The test lasted 7 weeks. No
abnormalities which could be attributed to pimaricin degradation
products were found on examination of the behaviour, appearance,
morbidity, mortality, food consumption, body-weight gains,
haematological indices, liver function, organ weights and macro- and
micro-pathology of the animals (Wieriks, 1966).
Groups of 10 male and 10 female rats were fed for 3 months on
diets containing the peel of apples which had been untreated, freshly
treated with pimaricin or treated with pimaricin and stored for 2-8
weeks to allow degradation to take place. A similar experiment was set
up in which sausage skins (untreated, freshly treated or stored with
pimaricin) were fed to rats. The dosage of pimaricin and its
degradation products cannot be calculated but the apple-skin diet
provided rats with approximately 0, 50 and 1250 times the human likely
intake and the sausage skin diet approximately 0, 1000 and 25 000
times the human intake. Some minor abnormalities were found but none
of the findings, in relation to growth rate, mortality, haematological
indices, serum enzymes, liver function, organ weights or gross and
micropathological changes, could be attributed to the intake of
pimaricin breakdown products (Wieriks, 1971).
Special studies on microbial resistance
Pimaricin is active against a wide range of mycotic organisms
such as dermatophytes and other fungi, yeasts and yeast-like organisms
(including strains pathogenic to man, animals and plants and
saprophytic varieties). Standard tests show that it has no activity on
bacteria or on actinomycetes. There is no evidence that mycotoxins-
forming species are unusually resistant to pimaricin (Raab, 1972).
No yeast or yeast-like organisms have yet been reported to show
primary resistance to pimaricin although some dermatophytes are
resistant to its activity.
Compared with bacteria and antibiotics, it is difficult to induce
resistance to pimaricin in yeasts (Khoudokmoff and Petru, 1974) and
there was some evidence that the resistance which could be obtained
was based on selection of naturally more resistant strains and not on
adaptation. Resistant cultures showed reduced pathogenicity (Athar and
Winner, 1971). No evidence of resistance in its clinical use has been
recorded.
Cross-resistance with other antimicrobials has been investigated.
Amphotericin B exhibited cross-resistance with nystatin, filipin,
endomycin and candidin but not pimaricin (Walter and Heilmeyer, 1969;
Bodenhoff, 1968; Littman et al., 1958; Stout and Pagano, 1956).
Nystatin and amphotericin B resistant organisms were susceptible to
pimaricin (Sörensen et. al., 1959) and a wide selection of nystatin
resistant yeasts also exhibited normal susceptibilities to pimaricin
(Hejzlar and Vymola, 1970). More recent in vitro studies have
established that cross-resistance between pimaricin and nystatin and
amphotericin may occur (Athar and Winner, 1971).
Special studies on mutagenicity
Groups of 10 male rats taken from the second litters of the F1
generation of a 3-generation reproduction study were fed on control
diet until sexually mature when they received, by gastric intubation,
0, 5, 15, 50 or 100 mg pimaricin/kg bw daily for 7 days. Each rat was
mated each week for 8 consecutive weeks with 2 virgin untreated
females. Each female was killed and examined 13 days after mating had
taken place. No differences between control and test animals were
found in relation to the numbers of implant sites, live and dead
foetuses or the mutagenic index (Cox et al., 1973).
Five males and 5 females were selected at random from the five
litters produced in a 3-generation study in which animals were fed on
diets providing 0, 5, 15, 50 or 100 mg pimaricin/kg bw/day. Three to 4
hours before sacrifice the animals were administered colchicine and a
bone marrow preparation was made for examination for aberrant
chromatin material. The number of abnormalities in the metaphase
chromosomal preparations of test groups did not suffer significantly
from the number occurring in sham-treated controls (Cox et al., 1973).
Special studies on reproduction
Groups of 10 females and 5 males receiving 0 or 1000 ppm
pimaricin in their diet were mated after 181 and 223 days on the test.
Other groups were mated after 48, 174 and 250 days on the diets; 4
control and 4 test female young from the second mating of this study
were fed on the same diet as their parents and mated when 107 days of
age. The pups from pimaricin-treated animals had lower mean body-
weights at weaning than control pups but examination of the results of
the 54 matings showed that their fertility, gestation, lactation and
viability indices were similar to or better than the controls. There
was a low incidence of abnormalities among pups in this study and no
abnormality could be attributed to pimaricin treatment (Levinskas,
1966; Levinskas et al., 1963).
Groups of 10 male and 20 female rats were fed on diet providing 0
(two groups), 5, 15, 50 or 100 mg pimaricin/kg bw/day for 11 weeks.
These formed generation F0 of a 3-generation reproduction study, with
2 litters being produced in each generation. In the 100 mg/kg group
there was an increased number of foetuses born dead, a decrease in the
number born alive and a decrease in the number surviving at 21 days.
Pup weight was depressed in the second litters of the F0 and F1
generations and both litters of the F2 generation. However, the
fertility, gestation, viability and lactation indices were within
normal limits for both litters of all 3 generations. The 5, 15 and
50 mg/kg dosage levels had no detectable effect on growth or
reproduction (Cox et al., 1973).
Special studies on teratogenicity
Groups of 20 female rats from the second litters of generation F1
of a 3-generation study on pimaricin were reared to maturity on
control diet and mated with untreated males. The females were given by
gastric intubation the same dose level of pimaricin as their parents
(0, 5, 15, 50 or 100 mg pimaricin/kg bw/day) during the 6-15 days of
pregnancy. They were killed and examined on the 20th day of pregnancy.
No differences between control and test animals were seen in relation
to the number of pregnancies, live letters, implant sites, resorption
sites, live and dead foetuses or skeletal and soft tissue
abnormalities (Cox et al. 1973).
Groups of 10-12 female rabbits were administered 0, 5, 15 or
50 mg pimaricin/kg bw/day by gavage on the 6-18th days of pregnancy.
They were examined on the 29th day and the numbers of corpora lutea,
implantation sites, resorption sites and live and dead foetuses
recorded. No adverse effects of pimaricin on nidation or maternal or
foetal survival were found. The number of abnormalities seen in the
soft or skeletal tissues did not differ from the number occurring
spontaneously in controls (Bailey and Morgareidge, 1974).
Acute toxicity
LD50
Animal Sex Route mg/kg bw Reference
Mouse - oral 1 500 Anonymous, 1965
- oral 2 500 Anonymous, 1965
Rat male oral 2 730 Levinskas et al., 1966
female oral 4 670 Levinskas et al., 1966
Guinea-pig female oral 450 Struyk et al., 1958
Rabbit male oral 1 420 Levinskas et al., 1966
Dog - oral 1 000 Anonymous, 1965
In rabbits a dose of 500 mg/kg and above caused diarrhoea and
animals which died had a haemorrhagic gastric mucosa. Raab (1972)
reports that pimaricin complexed with one-third its weight of a
modified polysaccharide increases its toxicity sixfold and that when
fed to rats pimaricin could be detected in blood.
Short-term studies
Rat
Oral administration of 50-70 mg pimaricin/kg bw daily for 5-10
weeks had no effect on the growth, blood or tissues of rats. A daily
oral dose of 150 mg/kg for 9 weeks caused some growth inhibition and a
daily dose of 500 mg/kg caused 30% of the rats to die within 2 weeks
(Struyk, 1958).
Groups of 20 male and 20 female rats were fed on diets containing
0, 125, 500, 2000 or 8000 ppm pimaricin for 94-96 days. None of the 5
deaths could be attributed to treatment. Growth was retarded and food
intake was diminished at the 2 highest dosage levels. The results of
haematological examinations and organ weights were within normal
limits and no gross or microscopic lesions could be attributed to
pimaricin intake (Levinskas et al., 1966).
Dog
Pimaricin was fed to groups of 3 male and 3 female beagle dogs at
dietary levels of 0, 125, 250 or 500 ppm for 2 years. All but one dog,
receiving 250 ppm diet, survived for 2 years; the death was unrelated
to exposure to pimaricin. No effect was seen on food intake but males
receiving the 500 ppm diet did not grow as rapidly as controls
initially and after 15 months when the dietary intake was reduced some
animals were unable to maintain a satisfactory body-weight. Results of
haematological examinations and clinical chemical studies revealed no
abnormalities. No effects of significance were found on determination
of organ weights or on gross and microscopic examination for
pathological changes (Levinskas et al., 1966).
Long-term studies
Rat
Groups of 35-40 male and 35-40 female rats received diet
containing 0, 125, 250, 500 and 1000 ppm pimaricin for 2 years.
Animals remained in good health and their survival was unaffected by
pimaricin. Inhibition of growth rate and a diminished food intake
occurred in both sexes receiving the 1000 ppm diet but lower dosage
levels had no adverse effects. The results of haematological
investigations and determination of organ weights and the gross and
microscopic pathology showed no differences between treated and
control groups. The number and types of tumours found in pimaricin-
treated rats were not significantly different from untreated animals
(Levinskas et al., 1966).
OBSERVATIONS IN MAN
In man nausea, vomiting and diarrhoea have occasionally been
caused by oral doses of 300-400 mg pimaricin daily; no changes in
peripheral blood have been observed (Royal Netherlands Fermentation
Industries, 1966). In a group of 10 patients with systemic mycoses who
received oral doses of 50-1000 mg/day for 13-180 days, nausea,
vomiting and diarrhoea occurred in those receiving 600-1000 mg/day
(Newcomer et al., 1960).
Comments
Information available on the metabolism of pimaricin suggests it
is not absorbed to a significant extent from the gastrointestinal
tract. The only adverse effects found in animal studies were a
decrease in food intake with a decrease in the rate of body-weight
gain. The dog appeared to be more sensitive than the rat, the response
appearing in dogs with doses of the order of 10 mg/kg/day. In man mild
gastrointestinal symptoms begin to appear at daily dosage levels of
about 5 mg/kg, although much higher dosage levels have been taken
without ill-effects being observed. Adequate studies have demonstrated
no adverse effects on reproduction nor any carcinogenic, mutagenic or
teratogenic potential. Work on the composition of breakdown products
suggests that those formed in food are likely to be the same as those
formed in acid conditions in the stomach; the feeding studies carried
out on pimaricin are therefore relevant to breakdown products. Studies
in rats on food containing breakdown products or citric acid degraded
pimaricin also suggest no adverse effects are likely from the
breakdown of pimaricin. Although widely used there are no reports of
allergic reactions. Despite general reservations concerning the use as
food additives of therapeutically useful antimicrobial substances, the
Committee agreed that data demonstrated that problems related to the
development of clinically significant microbial resistance or cross-
resistance were unlikely to occur with pimaricin (see Appendix 4 of
the 20th Report).
EVALUATION
Level causing no toxicological effects
Rat - 500 ppm in diet, equivalent to 25 mg/kg bw
Dog - 250 ppm in diet, equivalent to 6 mg/kg bw
Man - approximately 200 mg/man/day, equivalent to 3 mg/kg bw
Estimate of acceptable daily intake for man
0-0.3 mg/kg bw
REFERENCES
Anonymous (1965) Data on the safety of the use of pimaricin as
preservative against mold growth on cheese. Summary of the
results of acute and chronic toxicity tests. Unpublished report
from Royal Netherlands Fermentation Industries Ltd, Delft, The
Netherlands, submitted to the World Health Organization
Anonymous (1968) Absorption of pimaricin following oral
administration. Unpublished report from Royal Netherlands
Fermentation Industries Ltd, submitted to the World Health
Organization
Athar, M. A. and Winner, H. I. (1971) The development of resistance by
Candida species to polyene antibiotics in vitro, J. Med.
Microbiol., 4, 505-517
Bailey, D. E. and Morgareidge, K. (1974) Teratogenicity test with
pimaricin. Unpublished report (No. 1-1052) from Food and Drug
Research Laboratories Inc., submitted to the World Health
Organization
Bodenhoff, J. (1968) Resistenzuntersuchungen von Candida albicans, mit
besonderer Berücksichtigung von zwei während einer längeren Zeit
mittels antibiotica behandelten Patienten, Scand. Dent. J.,
76, 279
Brik, H. (1975) Natamycin (pimaricin). New high-molecular
decomposition products with intact lactone-ring. Unpublished
report submitted to the World Health Organization by Gist-
Brocades NV, Delft, The Netherlands
Cox, G. E., Bailey, D. E. and Morgareidge, K. (1973) Unpublished
report (No. 1-1052) from Food and Drug Research Laboratories
Inc., submitted to the World Health Organization
Grupper, Ch. (1961) Personal communication from the Hōpital Saint-
Louis, Paris
Grupper, Ch. (1964) Pimaricin in the treatment of superficial
mucocutaneous monoliasis. Intern. Congr. Trop. Dermat. Naples,
June 1964
Hejzlar, M. and Vymola, F. (1970) Comparative study of pimaricin and
fungicidin activity in vitro, J. Hyg. Epidem. (Praha), 14,
211
Hutchison, E. B., Ribelin, W. E. and Levinskas, G. J. (1966) 98-day
study in the rat. Unpublished report by American Cyanamid Co.,
submitted to the World Health Organization
Khandokormoff, B. and Petru, M. (1974) On the possible development of
antibiotic resistance amongst fungi with special reference to the
use of pimaricin as a preservative in the food industry.
Unpublished report from Gist-Brocades NV, Research and
Development Division, Delft, The Netherlands, submitted to the
World Health Organization
Levinskas, G. J., Shaffer, C. B., Bushey, C., Kinde, M. L.,
Stackhouse, D. W. and Vidone, L. B. (1963) Two-year feeding to
rats. Unpublished report from the Central Medical Dept., American
Cyanamid Co., submitted to the World Health Organization
Levinskas, G. J., Ribelin, W. E. and Shaffer, C. B. (1966) Acute and
chronic toxicity of pimaricin, Tox. and Appl. Pharmacol., 8,
97-109
Littman, M. L., Pisano, M. A. and Lancaster, R. M. (1958) Induced
resistance of Candida species to nystatin and amphoteracin B. In:
Antibiotics Ann.: 981. Medical Encyclopedia, New York, NY
Malten, K. E. (1967) Report of an investigation concerning possible
allergic side effects of pimaricin in humans. Unpublished report
from the Instituut voor Geneeskunde en Maatschappij, Nijmegen,
The Netherlands, submitted to the World Health Organization by
Gist-Brocades NV, Delft, The Netherlands
Malten, K. E. (1968) Report on investigation into possible sensitising
side effects of pimaricin in human beings. Unpublished report
from the Instituut voor Geneeskunde en Maatschappij, Nijmegen,
submitted to the World Health Organization by Gist-Brocades NV,
Delft, The Netherlands
Morgenstern, A. P. and Muskens, G. J. A. M. (1975) Further data on the
toxicity of the decomposition products of pimaricin. Unpublished
report submitted to the World Health Organization by Gist-
Brocades NV, Delft, The Netherlands
Newcomer, V. D., Sternberg, T. H., Wright, E. T., Reisner, R. M.,
McNall, E.G. and Sorensin, L. J. (1960) The treatment of systemic
diseases with orally administered pimaricin: Preliminary report,
Ann. N.Y. Acad Sci., 89, 240-246
Ottens, H. (1965) Unpublished report submitted to the World Health
Organization by Royal Netherlands Fermentation Industries, Delft,
The Netherlands
Raab, W. P. (1972) Natamycin (pimaricin). Its properties and
possibilities in medicine. Georg Thieme Publishers, Stuttgart
Sörensen, L. J., McNall, E.G. and Sternberg, T. H. (1959) The
development of strains of Candida albicans and Coccidioides
immitis which are resistant to amphotericin B. In: Antibiotics
Ann.: 920-923. Medical Encyclopedia, New York, NY
Stout, H. A. and Pagano, J. F. (1956) Resistance studies with
nystatin. In: Antibiotics Ann.: 704. New York, NY
Struyk, A. P., Hoette, I., Drost, G., Waisvisz, J. M., van Eek, T. and
Hoogerheide, J. C. (1958) Pimaricin, a new antifungal antibiotic.
In: Antibiotics Annual 1957-1958 (H. Welch and F. Marti-Ibanez,
eds.), pp. 878-885. Medical Encylopedia, Inc., New York
Walter, A.M. and Heilmeyer, L. (1969) Antibiotika Fibel. Thieme
Verlag, Stuttgart
Wieriks, J. (1966) Pimaricin in cheese: a toxicity test of seven weeks
in rats. Unpublished report from the KNGSF-Research of the Royal
Netherlands Fermentation Industries Ltd, submitted to the World
Health Organization
Wieriks, J. (1971) Pimaricin in apples: a toxicity test of three
months in rats. Unpublished report from the Royal Netherlands
Fermentation Industries Ltd, submitted to the World Health
Organization