PATULIN
First draft prepared by
Dr Margaret F.A. Wouters and Dr G.J.A. Speijers
Laboratory for Toxicology, National Institute of Public Health and
Environmental Protection, Bilthoven, Netherlands
Explanation
Biological data
Biochemical aspects
Absorption, distribution, and excretion
Effects on enzymes and other biochemical parameters
Toxicological studies
Acute toxicity studies
Short-term toxicity studies
Long-term toxicity/carcinogenicity studies
Reproductive toxicity studies
Special studies on antibiotic activity
Special studies on antitumour activity
Special studies on cytotoxicity
Special studies on genotoxicity
Special studies on immunotoxicity
Special studies on neurotoxicity
Special studies on teratogenicity and embryotoxicity
Observations in humans
Comments
Evaluation
References
1. EXPLANATION
Patulin is a mycotoxin produced by certain species of the genera
Aspergillus and Penicillium, including A. clavatus, P. expansum, P.
patulum, P. aspergillus and P. byssochlamys. P. expansum is a common
spoilage microorganism in apples, and the major potential dietary
sources of patulin are apples and apple juice made from affected fruit.
Patulin was previously evaluated by the Committee at its
thirty-fifth meeting (Annex 1, reference 88), when a PTWI of 7 µg/kg
bw was established based on a no-effect level of 0.1 mg/kg bw/day in a
combined reproductive toxicity/long-term toxicity/carcinogenicity
study in rats. Additional information has become available since the
last evaluation.
Patulin was reviewed by IARC (IARC, 1976; 1985). It was concluded
at the second of these reviews that there was inadequate evidence for
carcinogenicity of patulin in experimental animals. No evaluation
could be made of carcinogenicity of patulin in humans.
The following toxicological monograph summarizes both the
information given in the previous toxicological monograph and
information received since the previous review.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
A single oral dose of 3 mg/kg bw of 14C-patulin in citrate
buffer was given to 17 male and 12 female Sprague-Dawley rats exposed
for 41-66 weeks after birth to levels of 0 or 1.5 mg/kg bw of patulin
in 1 mol/litre citrate buffer. All animals were fasted for 24 h before
the administration of the labelled patulin. Animals were placed in
metabolic cages and faeces, urine and CO2 were collected. One or 2
animals/sex/group (untreated or pretreated with patulin) were
sacrificed at 4, 24, 48, 72 h or 7 days after blood was collected for
patulin determination. Concentrations of patulin in erythrocytes were
calculated from the difference between radioactivity of whole blood
and serum. Within 7 days, 49% and 36% of the administered radio-
activity was recovered in faeces and in urine, respectively. Most of
the excretion of label occurred within the first 24 h. Approximately
1-2% of the label was recovered as 14CO2. At the end of 7 days,
2-3% of the radioactivity was recovered in soft tissues and blood. The
major retention sites of patulin were erythrocytes and blood-rich
organs (spleen, kidney, lung and liver) (Dailey et al., 1977a).
2.1.2 Effects on enzymes and other biochemical parameters
In vivo studies
Absorption of radiolabelled glycine, alanine and lysine was
reduced in perfused intestines of rats that had received 100 mg of
patulin intraperitoneally on alternate days for 1 month (equal to
1.6 mg/kg bw/day). The authors attributed this effect to reduced total
ATPase, NaK ATPase, and alkaline phosphatase activities which were
studied in a satellite group of rats (Devaraj et al., 1982a).
A group of albino rats received 0.1 mg of patulin in 0.2 ml of
propylene glycol, injected i.p. on alternate days. A control group was
treated similarly with propylene glycol alone. The animals were
sacrificed after 15 doses. Liver, kidney and intestine were used for
the assay of various enzymes such as glycogen phosphorylase,
hexokinase, glucose-6-phosphatase, fructose-1,6-diphosphorylase,
hexokinase, glucose-6-phosphatase, fructose-l,6-diphosphatase and
aldolase.
The concentration of aldolase in the liver, kidney and intestinal
tissue was reduced during patulin toxicosis. In a follow up
experiment, groups of rats were treated similarly and the rats were
sacrificed after 20 doses. Aldolase was isolated and purified and
studies on its kinetic properties were made. These studies did not
show any significant variations in the properties of liver aldolase of
normal and patulin-treated rats. The authors concluded that the
results suggested that patulin toxicosis inhibited the biosynthesis of
liver aldolase (Sakthisekaran & Shanmugasundaram, 1990).
Forty-eight hours after i.p. injection of 5.0 or 7.5 mg/kg bw of
patulin in male ICR mice, NaKATPase and MgATPase of liver, kidney and
brain preparations were significantly inhibited. Injection of
2.5 mg/kg bw had no significant effect on enzyme activity. The same
effects were demonstrated in in vitro studies with mitochondrial and
microsomal fractions of liver, kidney and brain of ICR mice (Phillips
& Hayes, 1977).
Patulin inhibited acetylcholinesterase and NaKATPase in cerebral
hemisphere, cerebellum and medulla oblongata in rats treated for 1
month with i.p. injections of 1.6 mg/kg bw/day patulin. Concomitantly,
acetylcholine levels were raised in these brain segments (Devaraj
et al., 1982b).
A non-competitive and irreversible inhibition of the activity of
alcohol dehydrogenase derived from yeast was attributed to patulin's
ability to bind to SH-groups; the K1 was found to be 5.0 × 10-5 M
(Ashoor & Chu, 1973a).
Inhibition of yeast-derived aminoacyl-tRNA synthetase by patulin
was mainly due to modification of the enzyme's sulfhydryl groups
(Arafat, et al., 1985).
Liver lactate dehydrogenase was increased in 4 pregnant
Sprague-Dawley rats after exposure by gavage to 3 mg/kg bw/day of
patulin in tris-acetate buffer, from days 1-19 of gestation
(Fuks-Holmberg, 1980).
Malate dehydrogenase in human placental microsome- and
mitochondria-rich fractions was increased up to 15 times when
incubated with 0.5 - 3 mg/g placenta of patulin in vitro
(Fuks-Holmberg, 1980).
Placental GPT was depressed in 4 pregnant Sprague-Dawley rats
after exposure by gavage to 3 mg/kg bw/day of patulin in tris-acetate
buffer, from days 1-19 of gestation (Fuks-Holmberg, 1980).
When white male albino mice were injected with 10 doses of
0.1 mg of patulin in propylene glycol on alternate days, glycogen
phosphorylase in the liver was activated, and blood glucose levels
increased by 60%. These results were confirmed by in vitro studies
(Madiyalakan & Shanmugasundaram, 1978).
Groups of 10 rats were fed regular diet, diet infected with
Penicillium patulum, or received i.p. injections of purified patulin
(1 mg/kg bw on alternate days) for 3 months. Fasting blood glucose
levels were elevated and a glucose tolerance test revealed an elevated
glucose curve and reduced insulin production. The authors concluded
that patulin was diabetogenic (Devaraj et al., 1986).
Male F344 rats received a single i.p. injection of 0, 0.5, 5 or
10 mg/kg bw patulin. Liver mixed function oxidase and cytochrome P-450
activity were determined 4 days after treatment. Oxidative cleavage of
phosphonothioate and aryl hydrocarbon hydroxylase were elevated at
10 mg/kg bw. No effect was observed on p-nitroanisole O-demethylase or
on cytochrome P-450 (Kangsadalampai et al., 1981).
Patulin was reported to induce mixed function oxidase in male ICR
mice treated with 0.5, 1.0 or 2.0 mg/kg bw of patulin intraperitoneally
(Siraj & Hayes, 1978).
Patulin inhibited protein prenylation in mouse FM3A cells.
An inhibition of 50% and 80% was observed at 7 µM and 100 µM,
respectively. Protein synthesis, as measured by the incorporation of
14C-leucine, was also inhibited by patulin. The inhibition was 50%
at 3 µM and >90% at 30 µM. In a cell-free assay, patulin inhibited
rat brain farnesyl protein transferase, one of the enzymes responsible
for protein prenylation. The inhibition was 50% at a concentration of
290/µM (Miura, et al., 1992).
The concentration of glycogen in liver, kidney and intestinal
tissues was reduced during patulin toxicosis. The decrease in hepatic
glycogen indicated glucose intolerance which may be due to insulin
insufficiency. This may be reflected in decreased concentration of
insulin-dependent enzymes. Glycogen phosphorylase was markedly
increased, while glycolytic enzymes such as hexokinase and aldolase
were significantly lowered. Gluconeogenesis was stimulated as
evidenced by increased glucose-6-phosphatase and fructose-1,6-
diphosphatase activity (Sakthisekaran et al., 1989).
In vitro studies
Oxygen uptake stimulated by Krebs-cycle intermediates was
reported to be inhibited in tissue extracts from mice, rats and golden
hamsters. Inhibition of oxygen uptake in liver homogenates was
observed at levels of patulin as low as 0.033 mM. Inhibition of oxygen
uptake in heart and muscle homogenates was greater than in liver
homogenates. Patulin competitively inhibited succinate dehydrogenase
in mouse liver homogenates. The P/O ratio was not affected by
the toxin. In comparative studies, the golden hamster was more
susceptible, and the rat less susceptible to patulin inhibition than
the mouse (Hayes, 1977).
Kidney explants from male Osborne-Mendel rats, when incubated for
18 h in media containing 0.5, 0.75, or 1.0 mM patulin in vitro, lost
their respiratory ability as measured by conversion of 14C-glucose
to 14CO2. During measurement of respiration, patulin was not
present in the reaction mixture. At 1.0 mM patulin, respiration was
increased. Leakage of protein into the medium at a concentration of
1.0 mM patulin may indicate increased cell membrane permeability
(Braunberg et al., 1982).
Patulin inhibited the in vitro activity of NaKATPase in
microsomes prepared from mouse brain. Activity was partially restored
by washing. Preincubation of patulin with dithiothreitol or
glutathione prevented the inhibition (Phillips & Hayes, 1978).
Non-competitive inhibition was demonstrated when patulin was
incubated with rabbit-muscle aldolase; the Ki was 1.3 × 10-5 M.
The cysteine adduct of patulin was a less effective inhibitor (Ashoor
& Chu, 1973b).
Patulin, at a level of 4.35 µmol/ml, was reported to inhibit by
29% and 84% the activity of DNA-dependent RNA polymerase I and II
prepared from rat liver nuclei (Tashiro et al., 1979).
Patulin at a level of 200 µg/ml inhibited in vitro the chain
initiation stage of RNA synthesis in rat liver nuclei (Moule & Hatey,
1977).
Ribonuclease H, prepared from rat liver nuclei, was inhibited by
patulin in vitro by 62% at a concentration of 0.32 µmol/mol, and by
47% at a concentration of 1.07 µmol/ml (Tashiro et al., 1979).
Acid RNAse in human placental microsome and mitochondria-rich
fractions was increased up to 1.5 times when incubated with
0.5-3 mg patulin/g of placenta in vitro (Fuks-Holmberg, 1980).
Patulin caused a competitive inhibition of lactate dehydrogenase
from rabbit muscle (K1 = 7.2 × 10-5 M). The presence of cysteine
reversed the inhibitory effect of patulin on lactate dehydrogenase
(Ashoor & Chu, 1973a).
2.2 Toxicological studies
2.2.1 Acute toxicity studies
The acute toxicity of patulin is summarized in Table 1. Toxic
signs consistently reported in all studies were agitation, in some
cases convulsions, dyspnea, pulmonary congestion and edema, and
ulcerations, hyperemia and distension of the GI tract.
Acute toxicity of i.p. administered patulin was reported to be
reduced by simultaneous administration of another mycotoxin,
rubratoxin B (Kangsadalampai et al. 1981).
When a patulin/cysteine adduct was administered to mice
intraperitoneally, no acute toxicity was observed at levels up to
150 mg of patulin/mouse (Ciegler et al., 1976).
2.2.2 Short-term toxicity studies
2.2.2.1 Mice
When patulin was administered by gavage in citrate buffer to
groups of 10 male Swiss ICR mice at doses of 0, 24 or 36 mg/kg bw,
daily or on alternate days for 14 days, body weight was depressed and
mortality was increased in a dose-dependent manner. Histopathological
lesions were found in the GI tract, which included epithelial
degeneration, haemorrhage, ulceration of gastric mucosa, and exudation
and epithelial desquamation in the duodenum (McKinley & Carlton,
1980a).
2.2.2.2 Rats
When patulin was administered by gavage to groups of 10 male
Sprague-Dawley rats at doses of 28 or 41 mg/kg bw, daily or on
alternate days for 14 days, initial loss of body weight was observed;
animals recovered after day 4. Mortality was increased in all treated
groups, but no dose dependency was observed. Gross lesions were found
in the stomach and small intestine; the gastric mucosa was reddened
and the stomach was distended. The duodenum and jejunum were distended
by fluid. Histopathological lesions were found in the stomach which
consisted of ulceration of the mucosa, epithelial degeneration,
haemorrhage, and neutrophil and mononuclear cell infiltration
(McKinley et al., 1982).
Table 1. Acute toxicity of patulin
Species Sex Route LD50 References
(mg/kg bw)
Mouse M oral 29-48 Escoula, et al., 1977
Lindroth & von Wright, 1978
F oral 46.31 McKinley & Carlton, 1980a
M&F " 17 Hayes et al., 1979
? " 25 Katzman et al., 1944
M i.p. 5.7-8.17 Ciegler et al., 1976
Escoula et al., 1977
McKinley & Carlton, 1980a
F i.p. 10.85 Escoula et al., 1977
M&F " 7.6 Hayes et al., 1979
? " 4-5.7 Katzman et al., 1944
Ciegler et al., 1976
M&F i.v. 8.57 Escoula et al., 1977
? s.c. 8-10 Katzman et al., 1944
M s.c. 10 McKinley & Carlton, 1980a
Rat M oral 30.53-55.0 Escoula et al., 1977
McKinley et al., 1982
F oral 27.79 Escoula et al., 1977
? " 32.5 Dailey et al., 1977b
M&F " 108-118 Hayes et al., 1979
M i.p. 4.59-100 Escoula et al., 1977
McKinley et al., 1982
neonatal F oral 5.70 Escoula et al., 1977
rats M&F " 6.8 Hayes et al., 1979
weanling M&F i.p. 5.9 Hayes et al., 1979
rats M i.v. 8.57 Escoula et al., 1977
M s.c. 11.0 McKinley et al., 1982
? s.c. 25 Katzman et al., 1944
Hamster M oral 31.5 McKinley & Carlton, 1980b
i.p. 10 McKinley & Carlton, 1980b
s.c. 23 McKinley & Carlton, 1980b
Drinking-water containing 0, 25, 85, or 295 mg/litre of patulin
in 1 mM citrate buffer was given to groups of 6 SPF RIVM:Tox
(Wistar-derived) rats for 4 weeks. Food and liquid intake were
recorded three times per week. Body weights were determined at the
start of the experiment and at termination. Urinalysis, including
urine volume, bilirubin, and urinary protein were determined in the
last week. Creatinine clearance was calculated from serum and urine
levels of creatinine. At termination, the animals were examined
macroscopically, and the liver, spleen, thyroid glands, brain,
kidneys, heart, mesenteric lymph nodes, adrenal glands, thymus, testes
and ovaries were weighed. Histopathological examination was carried
out on all organs and tissues of the high-dose and the control groups.
Food and liquid intake were reduced in the mid- and high-dose animals.
Body weights at the high-dose level were decreased. Creatinine
clearance was lower in the high-dose animals, but no morphological
glomerular damage was observed. In the high-dose group, fundic ulcers
in the stomach were observed in combination with enlarged and active
pancreatico-duodenal lymph nodes, while villous hyperemia of the
duodenum was observed at the mid- and high-dose levels. The authors
suggested, based on normal appearance of the adrenal glands, that the
observed effects in the GI tract were a direct effect of patulin on
the tissue, which was not mediated through adrenal gland stimulation
(stress). The NOEL in this study was 25 mg/litre (Speijers et al.,
1985, 1988).
Albino rats were given 0.1 mg patulin in 0.1 ml propylene glycol
injected intraperitoneally on alternate days. Control rats were
treated with propylene glycol only. The animals were sacrificed after
15 doses. Blood was collected and liver, kidney and intestine were
used for the estimation of DNA and RNA. In plasma, the total protein
level, albumin concentration and A/G ratio were significantly
decreased in the dosed animals. The levels of DNA and RNA in liver,
kidney and intestine were significantly reduced (Gopalakrishnan &
Sakthisekaran, 1991).
Twenty rats/sex received by gavage 0 or 0.1 mg/kg bw patulin
dissolved in water on alternate days for 30 days. At the end of the
30th day, rats were killed and the intestine was removed and used for
the estimation of lipids and Na+ -K+ dependent ATPase.
Total lipids (25.8%), phospholipid (20.6%) and triglycerides
(59.8%) decreased significantly whereas total cholesterol levels
showed a slight increase (12.6%) in the experimental rats. A marked
inhibition of Na+-K+ dependent ATPase (32.4%) was observed in the
intestines of experimental rats (Devaraj & Devaraj, 1987).
Groups of Wistar rats (10/sex/group) were given drinking-water
containing patulin at concentrations of 0, 6, 30 or 150 mg/litre in
1 mM citrate buffer for 13 weeks. Food and water intake were decreased
in the mid- and high-dose groups. Body-weight gain was decreased only
in animals of the high-dose group. Haematological parameters were
slightly altered in the high-dose group. No effect of patulin on the
intestinal microflora was observed. A slight impairment of the kidney
function and a villous hyperaemia in the duodenum in the mid- and
high-dose groups were observed. The NOAEL in this study was 6 mg/litre
drinking-water, equivalent to 0.8 mg patulin/kg bw/day (Speijers,
et al., 1986).
2.2.2.3 Hamsters
When patulin was administered by gavage to groups of 10 male
Syrian golden hamsters at doses of 0, 16 or 24 mg/kg bw, daily or on
alternate days for 14 days, loss of body weight was observed and
mortality was increased in all treated groups, but no dose dependency
was observed. Gross lesions were found in the stomach and duodenum.
Histopathological lesions were found in the GI tract and included
epithelial degeneration, haemorrhage and ulceration (McKinley &
Carlton, 1980b).
2.2.2.4 Chickens
Fifteen one-day old white leghorn chicks were given by intubation
0 or 100 µg patulin, every 48 h. At the end of the 15th dose, the
birds were fasted overnight and killed. Kidney and intestine were
removed and assayed. The experimental chicks showed reduced enzyme
activity. The reduction in the activity of total ATPase in the kidney
was 40% and in the intestine 52% while the reduction in the Na+-K+
dependent ATPase activity in the kidney was 46% and in the intestine
55% (Devaraj et al., 1986).
2.2.2.5 Monkeys
Groups of 1 male and 1 female pigtail monkeys (Macaca
nemestrina) received doses of 0, 0.005, 0.05, or 0.5 mg/kg bw/day of
patulin for 4 weeks. Monkeys of the highest dose group received
5 mg/kg bw/day patulin for 2 additional weeks. Weekly determinations
were made of SGOT, SAP, BUN, cholesterol, sodium and potassium as well
as haematological parameters. Plasma protein electrophoresis was
performed and glucose and lipoprotein levels were determined. No signs
of toxicity were noted, except that the monkeys receiving 5 mg/kg
bw/day of patulin started to reject their food during the last 3 days
of the experiment. No statistically significant differences were
observed in any of the parameters studied (Garza et al., 1977).
2.2.3 Long-term toxicity/carcinogenicity studies
2.2.3.1 Rats
Subcutaneous injections of 0.2 mg of patulin in 0.5 ml of arachis
oil were administered biweekly for 61 or 64 weeks to 2 groups of 5
male Wistar rats, weighing 100 g at the start of the experiment. Local
(fibro)sarcomas was produced at the injection site in 4/4 and 2/4 rats
surviving at the time when the first tumour was observed. No
metastases were observed, and of 3 tumours tested only one was
transplantable in 3 of 12 recipient rats. Control animals receiving
arachis oil did not develop local tumours (Dickens & Jones, 1961).
Patulin in water containing 0.05% lactic acid was administered by
gavage twice weekly to 50 female SPF Sprague-Dawley rats at a dose of
1 mg/ kg bw for 4 weeks, and 2.5 mg/kg bw for the following 70 weeks
(total dose: 358 mg/kg bw of patulin). No effects were observed on
weight gain or survival. No significant differences were observed in
tumour incidence. The occurrence of 4 forestomach papillomas and 2
glandular stomach adenomas, as compared to none in the control
animals, is noteworthy. The Committee noted a discrepancy between the
reported duration of the study (64 weeks) and the reported duration of
administration (74 weeks) (Osswald et al., 1978).
Groups of 70 FDRL Wistar rats of each sex were exposed to
0, 0.1, 0.5, or 1.5 mg/kg bw/day of patulin in citrate buffer by
gavage 3 times/week for 24 months. The rats were derived from the F1
generation of a 1-generation reproductive toxicity study. Mortality
was increased in both sexes at the highest dose: all males had died by
19 months; 19% of females survived until termination at 24 months.
Body weights of males were reduced at the mid and high dose, but for
females body weights were comparable in all groups. No difference in
tumour incidence was observed. The NOEL in this study was 0.1 mg/kg
bw, administered 3 times weekly (Becci et al., 1981).
2.2.4 Reproductive toxicity studies
2.2.4.1 Rats
Groups of Sprague-Dawley rats (30/sex/group) received doses of
0, 1.5, 7.5, or 15.0 mg/kg bw/day of patulin in citrate buffer by
gavage 5 times per week for 7 weeks before mating. The pregnant dams
were gavaged daily at the same levels during gestation. Half the
dams were sacrificed on day 20 of gestation, and used for teratological
evaluation. The remaining dams were allowed to produce the F1
generation. Some of the F0 and F1 males were used for a dominant
lethal experiment. Twenty three controls and 15 low-dose animals per
sex were continued to produce an F2 generation. One-half of the
latter generation were again used for teratological evaluation.
Haematological and blood chemistry examinations were performed on
10 males and 10 females of the F2 generation 23 days after
weaning. The only lesion found at necropsy of parent animals was
gaseous distension of the GI tract. All treated males of the F0
generation had a dose-related reduction in weight gain. High mortality
occurred at 7.5 and 15.0 mg/kg bw/day in both males and dams. Although
Jitter size at 7.5 mg/kg bw/day was comparable to controls, survival
of male progeny was severely impaired. At the 1.5 mg/kg bw/day level,
pup growth of both sexes was reduced, and there was increased
mortality among the F2 females. No significant alterations were
found in the haematology and blood chemistry levels in selected
animals of the F2 generation (Dailey et al., 1977b).
Groups of FDRL Wistar rats (50/sex/group) were exposed to
0, 0.1, 0.5, or 1.5 mg/kg bw/day of patulin in citrate buffer by
gavage for 4 weeks before mating, and pregnant females were dosed
through gestation and lactation. The parent generation was sacrificed
after weaning. Body-weight gain was comparable among groups. Ten
females died in the high-dose group. Reproductive parameters such as
mating success, litter size, fertility, gestation, viability, and
lactation indices, and pup weight at birth, at 4 days and at weaning,
were not statistically different among experimental groups.
Histopathological evaluation of grossly abnormal tissues of the F0
generation did not show any effects of patulin treatment. The F1
generation was used for a 2-year toxicity/carcinogenicity study (see
section 2.2.3.1) (Becci et al., 1981).
2.2.5 Special studies on antibiotic activity
Twelve species of bacteria and two species of yeast were tested
for sensitivity against 11 different mycotoxins, including patulin,
using a disc diffusion assay. Bacillus brevis appeared to be the
most sensitive microorganism. The lowest amount that could be detected
under optimal conditions was 1 µg/disc for patulin (Madhyastha
et al., 1994).
Clear synergy was shown with patulin plus rifampin and patulin
plus bottromycin. Synergy of patulin with efrotomycin was weak and
there was no synergy of patulin plus kasugamycin (Dulaney & Jacobsen,
1987).
2.2.6 Special studies on antitumour activity
A comparison was made between the cytotoxicity and antitumour
activity of patulin and five structural analogs (isopatulin,
dehydroisopatulin, dimethylisopatulin, trimethylisopatulin and
isopropylisopatulin). In vitro assays using L1210 and P 388 cells
showed that the structure of the pyranic ring as well as the nature of
the substituents influenced the observed activities. Among the five
structural analogs of patulin assayed in vivo against Ehrlich
carcinoma, L1210 and P388 leukemias, dehydroisopatulin was the only
one being active on all 3 types of tumours at a dose of 100 mg/kg
bw/day. The ratios between the LD50 in mice and the active dose was
5, while with patulin it was 10 (Seigle-Murandi et al., 1992).
2.2.7 Special studies on cytotoxicity
The ID50 (inhibitory dose) of patulin tested on the protozoan
Tetrahymena pyriformis was 0.32 µg/ml (Nishie, et al., 1989).
Patulin at a concentration of 3.2 µg/ml inhibited by 50% the
growth rate of the ciliate Tetrahymena (Bürger et al., 1988).
To evaluate its inhibitory effect on cells, hepatoma tissue
culture cells in suspension were incubated in the presence of 30 µM of
patulin for 7 h and investigated by transmission and scanning electron
microscopy. The most significant difference observed between treated
and control cells was the disorganization of the cytoplasmic
microfilaments in the treated cells (Rihn, et al., 1986).
In an immortalized rat granulosa cell line, effects of patulin on
GSH levels and alterations in the partitioning of rhodamine 123 were
detected at 0.1 µM within 1 h. Alterations in Ca2+ homeostasis,
intracellular pH and gap junction mediated intercellular communication
were detected between 1 and 2 h with 1.0/µM patulin (Burghardt et al.,
1992).
A study in cultural renal cells on the effect of patulin on ion
influx, and the influence of dithiothreitol and glutathione on patulin
effects was performed. It was hypothesized that patulin altered
intracellular ion content via Na+-K+ ATPase and non-Na+-K+ ATPase
mechanism (Hinton et al., 1989).
In cultured renal cells LLC-PK1, concentrations of
patulin above 10/µM caused a transient increase in intracellular
electronegativity (< 1 h) followed by a sustained depolarization
(> 1 h), which was correlated with complete Na+ influx, K+
efflux, total LDH release, and bleb formation. However, patulin
concentrations of 5-10 µM caused a sustained increased intracellular
electronegativity (4-8 h) which was associated with partial Na+
influx and K+ efflux, no significant LDH release, and relatively few
blebs. The hyperpolarizing effect may be a result of increased
intracellular electronegativity. The toxic effects of patulin are
irreversible in LLC-PK1 cells, even after short pretreatment with
patulin (Riley et al., 1990).
In LLC-PK1, cells exposed to 50 µM patulin lipid peroxidation,
abrupt calcium influx, extensive blebbing and total LDH release
appeared to be serially connected events with each representing a step
in the loss of structural integrity of the plasma membrane. Patulin
also caused depletion of nonprotein sulfhydryls, increased 86Rb+
efflux, dome collapse and eventually the loss of cell viability (Riley
& Showker, 1991).
2.2.8 Special studies on genotoxicity
The results of in vitro and in vivo genotoxicity studies with
patulin are summarized in Tables 2 and 3, respectively.
Patulin was negative in mutagenicity tests with S. typhimurium
but was clearly positive in the initiator tRNA acceptance assay for
carcinogens (Hradec & Vesely, 1989).
The mutagenic effect of patulin was studied with a mutant of
bacteriophage M13am6H1. A 50% decrease in liberation of M13 phage per
cell (ED50) was observed at a concentration of 0.85 µg patulin/ml,
and a 50% decrease in growth rate of E. coli host cells was observed
at a much higher concentration of the mycotoxin (6.3 µg/ml). The
reversion frequency of M13am6H1 to the wild-type phenotype in the
presence of patulin compared to the spontaneous reversion increased by
a factor of 7-19.5 depending on the addition of patulin to bacteria or
phages only or simultaneously.
The same authors studied the effects of patulin on protein and
DNA synthesis. At a concentration of 3.2 µg/ml, the protein synthesis
of the ciliate Tetrahymena was inhibited by 85% and the RNA
synthesis by 86% compared with the control. Four hours after addition
of patulin, DNA synthesis was reduced to 20%; it rose to the value of
the control after an additional 2 h. An in vitro system could be
developed consisting of permeabilized cells of Tetrahymena. This
system allowed the separation of regulatory and secondary effects
induced by patulin. Patulin reduced DNA synthesis by 50%, whereas RNA
and protein synthesis were less inhibited than in the in vivo system
(Burger et al., 1988).
Table 2. Results of in vitro genotoxicity assays on Patulin
Test system Test object Concentration Result Reference
Ames test E. coli 1 µg/ml (to phage) Positive Burger et al., 1988
M 13am6H1 &/or 5 µg/ml
(to bacteria)
Ames test(3,4) S. typhimurium 0.01, 0.1, 1, Negative Ueno et al., 1978
TA98, TA100 10, 100, & 500
µg/plate
Ames test(3) S typhimurium 0.25 23, 25 & Negative Wehner et al., 1978
TA98, TA100, 250 µg/plate
TA1535, TA1537
Ames test(3) S, typhimurium 0.1, 1, 10 & 100 Negative Kuczek et al,, 1978
TA1535, TA1537, µg/plate
TA1538
Ames test(5) S. typhimurium 5, 10, 20 & 30 Negative Von Wright & Lindroth,
TA98, TA100 µg/plate 1978
Ames test(3,5) S. typhimurium <0.0065 Negative Bartsch et al., 1980
TA100, TA1538 µmoles/plate
Ames test(5) S. typhimurium 12-960 µg/plate Negative Wurgler et al., 1991
TA102 Conc. >35
µg/plate
were toxic;
Table 2 (cont'd)
Test system Test object Concentration Result Reference
Chromotest(3) E. coli 0.01, 0.02 & 0.05 Positive Auffray &
K12 PQ37 µ/ml (No S-9) Boutibonnes, 1987
Negative
(with S-9)
SOS Chromotest E. coli K12 0.01, 0.02 & 0.05 Weakly Auffray &
µg/l positive Boutibonnes, 1988
Chromotest(2) E. coli PQ37 0.001-30 µg/ml Negative Krivobok et al., 1987
Recombinogenesis B. subtilis 20 & 100 µg/disc Positive Ueno & Kubota, 1976
Prophage induction E. coli 5, 10, 25 & 50 Positive Lee & Roschenthaler,
X8011 (lambda) µg/ml 1986
Spot test E. coli K12 1-10 µg/assay(2) Positive Auffray &
Boutibonnes 1986
Reverse S. cerevisiae 50 /No S-9) & Positive Kuczuk et al., 1978
mutagenesis(3) D-3 100 (with S-9)
µg/plate
Forward S. cerevisiae 10, 25, 50 & 75 Positive Mayer & Legator
mutagenesis (haploid) µg/ml 1969
Table 2 (cont'd)
Test system Test object Concentration Result Reference
Forward FM3A mouse 0.032, 0.1, & Positive Umeda et al., 1977
mutagenesis mammary 0.32 µg/ml
(8-azoquanine carcinoma cells
resistance)
SOS microplate E. coli PQ37 2, 8, 24 µg/ml Negative Sakai et al.. 1992
assay(3)
Somatic mutations Drosophila 3.2×10-2, Weakly Belitsky et al., 1985
melanogaster 3.2×10-3, M positive
4×10-4
Chromosome FM3A mouse 0.032, 0.1 & 0.32 Positive Mori et al., 1984
aberration mammary µg/ml
induction carcinoma
cells
Chromosome Chinese hamster 1, 2.5. 5 & 10 µM Positive Thust et al., 1982
aberration V79-E cells (No S-9)
induction(3) Negative
Chromosome Human 3.5 µM Positive Withers, 1966
aberration leucocytes
induction
Cell cycle Prim. Chinese 0.5, 1 & 2 µg/ml Positive Kubiak &
retardation hamster cells Kosz-Vnenchak, 1983
Table 2 (cont'd)
Test system Test object Concentration Result Reference
Cell cycle Human 0.075 & 0.30 Positive Cooray et al., 1982
retardation peripheral blood mg/ml
lymphocytes
Cell cycle Human 0.075 & 0.30 Positive Cooray et al., 1982
retardation peripheral blood µg/ml
lymphocytes
Sister chromatid Chinese hamster 1, 2.5, 5 & 10 µM Negative Thust et al., 1982
exchange V79-E cells
induction(3)
Sister chromatid Prim. Chinese 0.5, 1 & 2 mg/ml Positive Kubiak & Vnencha, 1983
exchange induction hamster cells
Sister chromatid Human 0.075, 0.10, 0.20 Weakly Cooray et al., 1982
exchange induction peripheral blood & 0.30 mg/ml positive
lymphocytes
DNA synthesis T. pyriformis 3.2 µg/ml Positive Burger et al., 1988
retardation
DNA synthesis AWRF cells 1, 2, 4 & 8 µg/ml Positive Stetina & Votova, 1986
retardation CHO cells 0.24, 0.5, 1, 2, 4
µg/ml
Table 2 (cont'd)
Test system Test object Concentration Result Reference
DNA breakage ColE1 plasmid 0.25, 0.5, 1.0 & Negative Lee & Roschenthaler,
5.0 mM(1) 1986
Lambda DNA 0.5. 1, 5, 10 &
14 mM
Unscheduled DNA Primary ACI rat 60 & 600 µM Negative Mori et al., 1984
synthesis induction hepatocytes
Primary C3H 65 & 650 µM Negative Mori et al., 1984
mouse
hepatocytes
DNA breakage E. coli 10, 20, 25 & 50 Positive Lee & Roschenthaler,
D110 polA µg/ml 1986
DNA-repair human or rat 1.6×10-3 - Negative Belitsky et al., 1985
liver cells 1.6×10-3
DNA breakage FM3A mouse 1.0, 3.2, 10 µg/ml Positive Umeda et al., 1977
mammary
carcinoma cells
DNA breakage AWRF cells 2 & 10 µg/ml Positive Stetina & Votava, 1986
CHO cells 2, 8 & 10 µg/ml
DNA synthesis human 1.0×105 Negative Yanagisawa et al., 1987
inhibition test fibroblasts
Table 3. Results of in vivo genotoxicity assays on Patulin
Test system Test object Concentration Result Reference
Chromosome Chinese hamster oral 2 × 10 and 20 Positive Roll et al., 1990
aberrations bone marrow mg/kg bw
Chromosome Chinese hamster 2 × 20 mg/kg bw Positive Korted et al., 1979
aberration bone marrow cells by gavage(6)
induction
Chromosome Chinese hamster 2 × 1, 10 & 20 Positive Korte, 1980
aberration bone marrow cells mg/kg bw by
induction gavage
Chromosome Chinese hamster 2 × 10 & 20 mg/kg Positive Korte & Ruckert, 1980
aberration bone marrow cells bw
induction
Host mediated S. typhimurium i.m. 3 × <500 µg Negative Gabridge & Legator, 1969
assay in Swiss G46
albino mice
Host mediated S. typhimurium 10 × 20 mg/kg bw Negative Von Wright & Lindroth,
assay in male TA1950, TA1951 gavage 1978
NMRI mice
Dominant lethal ICR/Ha Swiss 0.1 & 0.3 mg/kg Negative Epstein et al., 1972
assay
Dominant lethal Sprague-Dawley 1.5 mg/kg bw Negative Dailey et al., 1977b
assay rats 5x/wk × 10-11 wk,
by gavage
Table 3. (cont'd)
Test system Test object Concentration Result Reference
Dominant lethal Texas ICR x 3.0 mg/kg bw i.p. Negative Reddy et al., 1978
assay Sprague-Dawley
Sch:Ars(CFI)f
Sister chromatid Chinese hamster 2 × 1, 10 & 20 Negative Korte, 1980
exchange bone marrow cells mg/kg bw by
induction gavage
Dominant lethal NMRI mice (m) i.p 2.5 and 5 Negative Roll et al., 1990
assay mg/kg bw
(1) Positive when CuCl2 & NADPH were added
(2) Both with and without S-9 fraction (source not specified)
(3) Both with and without rat liver S-9 fraction
(4) Both with regular plate and preincubation methods
(5) Both with and without mouse liver S-9 fraction
(6) Effect negative if animals first given ethanol as only liquid for 9 wk prior to exposure
2.2.9 Special studies on immunotoxicity
2.2.9.1 In vitro studies
Peritoneal exudate cells of mice (C57BL/6J) collected by washing
the peritoneal cavity, were preincubated for 2 h with 0.01-2 µg
patulin/ml. Phagocytosis and phagosome-lysosome fusion were diminished
above 0.1 µg/ ml, and lysosomal enzymes and microbiological activity
above 0.5 µg/ml, whereas O2 production was inhibited only above
2 µg/ml (Bourdiol et al., 1990).
The effects of patulin were investigated on immunological
responses of Balb/c mice. In vitro, patulin had a stimulatory effect
on splenocytes at lower concentration (1 nM to 10 nM) and strongly
inhibited lymphocyte proliferation at higher concentrations (ID50
from 0.02 to 0.24/uM depending on mitogens) (Pancod et al., 1990).
At concentrations from 0.25-1 µg/ml, patulin decreased the
chemotactic index of dog neutrophilic granulocytes stimulated by
opsonized zymosan. At the same concentrations patulin favoured the
migration of the cells. At 1 µg/ml it inhibited the liberation of
superoxide ions by neutrophils, but did not modify their ability to
phagocyte Saccharomyces cerevisiae even at concentrations up to 10
µg/ml. The immunosuppressive actions may be explained by a fixation of
patulin on sulfhydric groups present on the neutrophil membrane
(Dubech, et al., 1993).
In alveolar macrophage harvested from male Long-Evans hooded
rats, patulin caused a significant increase in mean cell volume after
2 h exposure at 10-3 M. Chromium release from alveolar macrophage
following exposure to patulin was both time- and concentration-
dependent. Treatment with > 1.5 × 10-4 M caused significant
chromium release within 30 minutes. ATP concentrations in alveolar
macrophage monolayer cultures were markedly inhibited within 1 h at
concentrations > 5 × 10-5 M patulin. Incorporation of [3H]-precursors
into protein and RNA was also strongly inhibited by patulin. Inhibition
was both time- and concentration-dependent for both classes of molecules
but protein synthesis was sensitive to 10- to 100- fold lower
concentrations of patulin than RNA synthesis at the same time interval.
The dose producing 50% inhibition at 1 h (ED50) was estimated at
ca. 1.6 × 10-6 M and 1.0 × 10-5 M for [3H]-leucine and [3H]-uridine
incorporation, i.e. protein and RNA synthesis, respectively. Patulin
strongly inhibited phagocytosis of [51Cr]-sheep erythrocytes and
there was significant inhibition of phagocytosis at >5 × 10-7 M
patulin (Sorenson et al., 1986).
2.2.9.2 In vivo studies
The effects of patulin were investigated on immunological
responses of Balb/c mice. In mice, patulin at dose levels of 2 and
4 mg/kg bw significantly reduced delayed type hypersensitivity to
Bordetella pertussis antigen and did not reduced anti-KLH antibody
production (Paucod et al., 1990).
Mice (Swiss female IFA CREDO) receiving 10 mg/kg bw/day patulin
for 4 days showed enhanced resistance to i.p. challenge with 108
viable Candida albicans at day 2. Immunoglobulin levels (IgA, IgM
and IgG) were markedly depressed (10-75%) (Escoula et al., 1988a).
Mice (Swiss female IFA CREDO) were given by gavage 10 mg/kg
bw patulin daily from day 0 to day 4, and rabbits received
intraperitoneally 2.5 mg/kg bw. The mice were lymphopenic on days
5 and 10, but not on day 20.
There was no effect on neutrophil count on day 5. A significant
suppression of the chemilumminescene response of peritoneal leucocytes
was observed in both species. Mitogenic responses of mice splenic
lymphocytes and rabbit peripheral cells were slightly suppressed
(ConA) by treatment with 0.05 µg/ml and markedly inhibited with
0.5 µg/ml. The inhibition was more pronounced on B-cell mitogen
compared with T-cell mitogen. In mice and rabbits IgG, IgA and IgM
levels obtained on day 5 were lower when treated with patulin (Escoula,
et al., 1988b).
Patulin inhibited DNA synthesis in peripheral lymphocytes. These
effects were mitigated by cysteine which suggested that sulfhydryl
binding was involved in patulin induced toxicity. In mice, an
increased resistance to Candida albicans was observed and decreased
concentrations of circulating immunoglobulin. In rabbits decreased
serum immunoglobulin, reduced blasto-genesis of lymphocytes and
reduced chemiluminescence of peritoneal leucocytes were observed. No
details about concentrations were given (Sharma, 1993).
2.2.10 Special studies on embryotoxicity
Groups of 25 albino rats (sex not specified) weighing 25-30 g
received 0 or 100 mg of patulin in propylene glycol intraperitoneally
on alternate days (dose approximately 1.6 mg/kg bw/day) for 1 month.
The patulin-treated animals showed convulsions, tremors, impaired
locomotion, stiffness of hindlimbs, and wagging of the head. Patulin
inhibited acetylcholinesterase and NaKATPase in the cerebral
hemisphere, cerebellum and medulla oblongata. Concomitantly,
acetylcholine levels were raised in these brain segments (Devaraj
et al., 1982a).
2.2.11 Special studies on teratogenicity and embryotoxicity
2.2.11.1 Mice
Twelve pregnant Swiss mice received 0 or 2 mg/kg bw/day of
patulin in water containing 0.05% lactic acid twice daily by gavage
for 6 days starting 14 days after mating. The control mice received
0.05% lactic acid by gavage.
Mean survival time was significantly reduced in the patulin
treated dams, while 2/12 control animals and 5/12 experimental animals
developed tumours. Of the offspring, 8/43 male and 11/52 female
suckling mice died in the first 6 days of life, with hyperemia and
bleeding in the brain, lungs and skin. When these early deaths were
excluded from the calculations, patulin did not affect survival time
in the animals exposed in utero. No evidence of carcinogenicity was
observed in the offspring exposed only to patulin in utero (Osswald
et al., 1978).
Groups of 22-31 mice (NMRI) received orally during days 12
and 13 of gestation 0 or 3.8 mg/kg bw/day, or i.p. 0, 1.3, 2.5 or
3.8 mg/kg bw/day. Higher dose levels were maternally toxic. Oral
administration caused no effects on the number of implantation,
delivered fetuses, number of resorption, dead fetuses, fetal weight,
or malformations of the skeleton and organs. Intraperitoneal
administration showed at 3.8 mg/kg bw/day a slight increase in early
resorption, compared to controls. An increase in cleft palate was seen
(10.6% compared to 1.5% in controls), and an increase in malformations
of the kidney (2.8% compared to none in the control group) were also
seen at this dose level (Roll et al., 1990).
2.2.11.2 Rats
In a 2-generation reproductive toxicity study (see section
2.2.4.1), offspring of 15 Sprague-Dawley dams of the F1 and F2
generation exposed by gavage to 0 or 1.5 mg/kg bw/day of patulin in
citrate buffer were evaluated for teratological abnormalities. Patulin
caused an increase in resorption in the F1 litters, but this effect
was not observed in the F2 generation. The average weight of male
fetuses of the F2 generation was significantly less than controls.
No increase in skeletal or soft tissue abnormalities was observed
(Dailey et al., 1977b).
However, when patulin was administered i.p. to groups of 10-17
pregnant Charles River CDI rats at doses of 1.5 or 2.0 mg/kg bw/day, a
significant decrease in average fetal body weight was observed at the
lower dose, and at 2.0 mg/kg bw/day all implanted embryos were
resorbed (Reddy, et al., 1978).
2.2.11.3 Chickens
Patulin was injected into the air cell of chick eggs. It was
reported to be embryotoxic at levels of 2.4-69 µg/egg depending on the
age of the embryo, and teratogenic at levels of 1-2 µg/egg. Patulin/
cysteine adducts exhibited the same toxic effects, but at much higher
doses: 15-150 µg of patulin equivalents (Ciegler et al., 1976).
2.2.11.4 In vitro studies
Whole rat embryo culture was used to determine the teratogenic
potential of patulin in vitro. Embryos were exposed to untreated or
patulin-treated (0 - 62 µM) rat serum for 45 h. The embryos exposed to
62 µM patulin were not evaluated because they did not survive the 40 h
incubation time. The results indicated that patulin induced a
concentration-dependent reduction in protein and DNA content, yolk sac
diameter, crown rump length, and somite number count. Patulin
treatment also resulted in an increase in the frequency of defective
embryos. Anomalies included growth retardation, hypoplasia of the
mesencephalon and telencephalon, hyperplasia and/or blisters of the
mandibular precess (Small et al., 1992, summary only).
2.3 Observations in humans
Patulin was tested as an antibiotic for treatment of the common
cold in humans. Application was through the nasal route (1:10 000 or
1:20 000 solutions, every 4 h). Most of the information is anecdotal
(Gye, 1943).
A report on a controlled trial failed to identify the number of
patients tested, and was unclear as to which clinical tests were
performed to support the authors assertion that no ill effects were
observed (Hopkins, 1943).
3. COMMENTS
In rats, most of the administered dose was eliminated within 48 h
in faeces and urine, less than 2% being expired as carbon dioxide. No
other metabolites have been identified. About 2% of the administered
dose was still present after 7 days, located mainly in erythrocytes.
Patulin has a strong affinity for sulfhydryl groups, which
explains why it inhibits the activity of many enzymes. Patulin adducts
formed with cysteine were less toxic than the unmodified compound in
acute toxicity, teratogenicity, and mutagenicity studies.
In acute and short-term studies, patulin caused gastrointestinal
hyperaemia, distension, haemorrhage and ulceration. Pigtail monkeys
(Macaca nemestrina) tolerated patulin consumption of up to 0.5 mg/kg
bw/day for 4 weeks without adverse effects.
The NOEL in a 13-week toxicity study performed in rats was
0.8 mg/kg bw/day, based on a slight impairment of kidney function and
a villous hyperaemia in the duodenum in the mid- and high-dose groups.
Two reproductive toxicity studies in rats and teratogenicity
studies in mice and rats were available. No reproductive or
teratogenic effects were noted in mice or rats at dose levels of up
to 1.5 mg/kg bw/day. However, maternal toxicity and an increase in
the frequency of fetal resorptions were observed at higher levels,
which indicated that patulin was embryotoxic.
Both in vitro and in vivo experiments indicated that patulin
had immuno-suppressive properties. However, the dose levels at which
these effects occurred were higher than the NOEL in both the
short-term toxicity study and a combined reproductive toxicity/
long-term toxicity/carcinogenicity study.
Although the data on genotoxicity were variable, most assays
carried out with mammalian cells were positive while assays with
bacteria were mainly negative. In addition, some studies indicated
that patulin impaired DNA synthesis. These genotoxic effects might be
related to its ability to react with sulfhydryl groups and thereby
inhibit enzymes involved in the replication of genetic material.
Nevertheless, it was concluded from the available data that patulin is
genotoxic.
The mortality seen in short-term toxicity, reproductive toxicity
and long-term toxicity studies with conventional rats, due to
dilatation of the gut and/or pneumonia, was most probably secondary to
the fact that patulin acts like an antibiotic on Gram-positive
bacteria, thereby giving a selective advantage to pathogenic
Gram-negative bacteria. This conclusion was supported by the fact
that, in 13-week studies at similar dose levels with specific
pathogen-free (SPF) rats, no such mortality was seen.
In combined reproductive toxicity, long-term toxicity/
carcinogenicity study in rats, a dose level of 0.1 mg/kg bw/day of
patulin produced no effect in terms of decreased weight gain in males.
However, as patulin was administered only three times per week during
24 months, the NOEL derived from this study was 43 µg/kg bw/day.
An additional long-term carcinogenicity study in a rodent species
other than the rat, which was recommended at the previous meeting for
the further evaluation of the toxicity of patulin, was not available.
4. EVALUATION
Since in the most sensitive experiment, patulin was administered
only three times per week, the existing PTWI was changed. As it does
not accumulate in the body and in the light of the consumption
pattern, the PTWI was changed to a provisional maximum tolerable daily
intake (PMTDI). Based on a NOEL of 43 µg/kg bw/day and a safety factor
of 100, a PMTDI of 0.4 µg/kg bw was established.
Submission of the results of a long-term toxicity/carcinogenicity
study in a rodent species other than the rat is desirable.
Patulin levels in apple juice are generally below 50 µg/litre and
maximum intakes have been estimated to be 0.2 µg/kg bw/day for
children and 0.1 µg/kg bw/day for adults, i.e. well below the
tolerable intake established by the Committee. However, apple juice
can occasionally be heavily contaminated and continuing efforts are
therefore needed to minimize exposure to this mycotoxin by avoiding
the use of rotten or mouldy fruit.
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