INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF CERTAIN
VETERINARY DRUG RESIDUES IN FOOD
WHO FOOD ADDITIVES SERIES: 43
Prepared by the Fifty-second meeting of the Joint FAO/WHO
Expert Committee on Food Additives (JECFA)
World Health Organization, Geneva, 2000
IPCS - International Programme on Chemical Safety
PORCINE SOMATOTROPIN
First draft prepared by Preben Olsen
Institute of Food Safety and Toxicology
Danish Veterinary and Food Administration
Copenhagen, Denmark
Explanation
Biological data
Biochemical aspects
Absorption, distribution, and excretion
Biotransformation
Effects on enzymes and other biochemical processes
Toxicological studies
Acute toxicity
Short-term studies of toxicity
Reproductive toxicity
Observations in humans
Short-term toxicity of insulin-like growth factor
Comments
Evaluation
Acknowledgement
References
1. EXPLANATION
Studies of three analogues of native porcine somatotropin (pST)
that are produced by recombinant DNA techniques1 were reviewed for
the first time by the Committee. Their use in animal production is to
increase body-weight gain and feed efficiency and to affect carcass
composition by producing pigs with more protein and less fat. The
Committee considered only the safety for consumers of foods containing
residues of recombinant porcine somatotropin (rpST) in this
toxicological monograph.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
pST, a single polypeptide chain of 190 amino acids, exists as
three variants comprising 183, 191, or 193 amino acids. Reporcin has
the amino acid methionine at the N-terminus (Henry, 1998), and in
Grolene the first seven amino acids are deleted from the N-terminus
(Sargent, 1998). Somagrepor has an additional three amino acids at the
N-terminus at position 1 and four substitutions in the amino acid
sequence at positions 6, 11, 183, and 191 (Cyanamide, 1992).
1 Grolene(R), Reporcin(R), and Somagrepor(R); common names
were not available for these products.
Although the molecular structure of recombinant products differs
from native pST, it is appropriate to consider them together in this
evaluation because they act by binding with high affinity to the pST
receptor.
pST showed cross-species bioactivity in vivo in rats
(GhiasUddin et al., 1984a) and dogs (Prahalada et al., 1998) but not
in hypophysectomized monkeys (Knobil & Greep, 1959). Although the
homology in amino acid sequence between porcine and human somatotropin
is about 66% (Wallis, 1975), differences in bioactivity are seen which
are due to the complementary molecular structures and their
non-covalent binding to highly specific receptors on the membranes of
target cells (Hughes & Friesen, 1985). Accordingly, pST did not
inhibit 125I-human somatotropin in liver tissue in vitro, indicating
that pST does not bind to human somatotropin receptors (Carr &
Friesen, 1976).
Recombinant and native pST are biologically equivalent and have
comparable activity in vitro for specific inhibition of 125I-pST in
the liver microsomal membrane fraction of pigs (Chung & Etherton,
1986) and rabbits (Martin & Yunger, 1989). In comparative studies in
vivo, growth was stimulated in hypophysectomized rats treated
subcutaneously with rpST or pST for nine days (GhiasUddin & Wilbur,
1984a; GhiasUddin et al., 1984b) or for 10 days (Biroc, 1992) and
growth performance, measured by weight gain, feed efficiency, and
carcass composition, was improved in pigs treated with daily
injections of rpST or pST for 77 days (Evock et al., 1988) or for up
to 50 days (Eager, 1992).
2.1.1 Absorption, distribution, and excretion
As pST is a protein, it is likely to be degraded by digestive
enzymes in the gastrointestinal tract (Nixon & Mawer, 1970) and has no
activity when given orally. Oral administration of excessive rpST to
rats for 15 days did not result in a detectable increase in serum
rpST, measured by radioimmuno-assay (GhiasUddin & Bailey, 1988), and
no effect was observed on key clinical and biochemical parameters. In
contrast, these parameters were affected when rpST was given
parentally (GhiasUddin & Bailey, 1988; GhiasUddin, 1989). Studies with
the related bovine somatotropin showed ready cleavage by digestive
enzymes in vitro (Annex 1, reference 105), although proteolytic
fragments had no biological activity (Hammond et al., 1990).
The serum concentrations of rpST in rats after intramuscular
injection of 10 mg/kg bw were determined by radioimmunoassay at
various times between 4 and 60 h, although the results at 4 h were
discounted because of inaccurate determination. The serum
concentration of 566 ng/ml at 6 h decreased gradually and after 60 h
reached the background level of 10 ng/ml measured in control rats. The
radioimmunoassay used showed 17% cross-reactivity with rat
somatotropin (Martin, 1989). The biological half-life of rpST in rats
could not be determined (Illi, 1989).
In a study in pigs treated with radiolabelled 125I-pST or
125I-rpST, the mean half-lives were 4 min for the fast phase for both
compounds and 38 min for the slow phase for pST and 49 min for rpST
(GhiasUddin, 1988a).
The time course of serum somatotropin concentrations in pigs
after a single intramuscular injection of pST at concentrations of
0.01 or 1 mg/kg bw showed peak values of 30 ng/ml and 290 ng/ml,
respectively, after 1 h; the concentrations had returned to baseline
by 4 and 24 h, respectively (Sillence & Etherton, 1987). The serum
concentrations of pST in untreated pigs varied from 1.6 to 7 ng/ml
(Marple & Aberle, 1972; Siers & Trenkle, 1973).
After repeated daily injections of rpST to pigs, serum
concentrations did not appear to increase with time, suggesting that
there it does not accumulate in serum (Chung et al., 1985; Etherton et
al., 1987; Evock et al., 1988; Campbell et al., 1988).
2.1.2 Biotransformation
As rpST is not absorbed intact after oral administration, it has
no biological activity when given by this route. When doses of 14 mg
rpST were injected twice weekly into pigs, the blood concentrations of
somatotropin increased but did not give rise to concentrations in
muscle tissues that were above physiological levels after 27 h (Schams
et al., 1989).
2.1.3 Effects on enzymes and other biochemical processes
rpST alters the metabolism of tissues such as adipose,
connective, and bone tissues, liver, skeletal muscle, kidney, thyroid,
and adrenal gland, and changes the circulating concentrations of
several nutrients and hormones (Evock et al., 1988; Molon-Noblot et
al., 1998). Somatotropin directly stimulates cell proliferation
(Paladini et al., 1984; Isaksson et al., 1985) and also has an
indirect effect mediated by insulin-like growth factor (IGF-I). The
synthesis of IGF-I in and secretion from the liver are stimulated
specifically by somatotropin (Froesch et al., 1985; Mathews et al.,
1986), and somatotropin may also stimulate local synthesis of IGF-I
within the target organ itself (Isaksson et al., 1985).
IGF-I is a 70-amino acid polypeptide hormone of the somatomedin
family (Klapper et al., 1983). Somatomedins have acute metabolic
effects similar to but considerably less potent than those of insulin;
furthermore, IGF-I mediates many of the growth-stimulating effects of
somatotropin. The serum concentrations of IGF-I are regulated by
somatotropin, and these change the most dramatically after treatment
with rpST (Chung et al., 1985; Sillence & Etherton, 1987; Evock et
al., 1988; Etherton, 1988; Puyt, 1990). The molecular structures of
porcine and human IGF-I are identical (Tavakkol et al., 1988).
2.2 Toxicological studies
2.2.1 Acute toxicity
The results of studies of the acute toxicity of rpST are
summarized in Table 1.
2.2.2 Short-term studies of toxicity
Rats
Groups of 20 male and 20 female Sprague-Dawley (Cox SD) albino
rats were given oral doses of rpST by gavage at concentrations of 0
(vehicle), 0.04, 0.4, or 4 mg/kg bw per day for 15-22 days. Clinical
observations and body weights were recorded daily. At the end of the
study, blood samples were taken and haematological parameters were
recorded, with measurement of the hormones, triiodothyronine (T3),
thyroxine (T4), glucagon, and insulin. At necropsy, the organ weights
were recorded and a thorough histopathological examination was
performed. The study was not conducted according to appropriate
standards for study protocol; no statement on observance of GLP was
provided.
No clinical signs or toxicological effects were seen during the
15-22-day test. The death of one male control rat on day 15 was due to
lung injuries associated with gavage. The body-weight gains of control
and treated rats were comparable up to day 15. A few variations in
haematological and clinical chemical parameters were seen, but they
were spontaneous, often inconsistent by sex, showed a non-linear
response to rpST dose, and were considered to be unrelated to
treatment. At necropsy, no gross pathological effect was observed. The
relative weights of the liver, adrenal, and spleen of male rats and
the thymus, kidney, brain, and pituitary gland of female rats showed
some differences from controls that were not related to dose.
Histopathological examination showed a few microscopic changes suhc as
mononuclear infiltrates in the portal region of the liver, cortical
medullary tubular mineralization in the kidneys of male rats, and
renal cortical tubular nephropathy in controls and rats at the high
dose. These changes were spontaneous and were considered to be
unrelated to treatment. The Committee noted that animals in the
various groups were sacrificed at different times, sometimes by as
much as seven days. Nevertheless, the values of none of the parameters
studied were boutside the range of historical controls. No
treatment-related toxicity was observed in rats at the high dose
necropsied on day 15. Although the control group was necropsied over
seven days, it is unlikely that the microscopic lesions seen would
have changed if all the group had been killed on day 15 (GhiasUddin &
Wilbur, 1984b).
In a pilot study, groups of five male and five female
Sprague-Dawley (Cox SD) albino rats were given oral doses by gavage of
4 mg/kg bw per day rpST (two lots, one per group) or 4 mg/kg bw per
day pST for 15 days. Groups of five rats of each sex per group served
as controls and were given either the vehicle (buffer), 4 mg/kg bw per
day of egg albumin (protein control), or no treatment. The study was
conducted in compliance with GLP.
There were no overt signs of toxicity in any group treated with
either pST, rpST, or egg albumin during the course of the study. The
mean body-weight gain on day 15 was similar in each group, and the
absolute and relative organ weights of rats given pST or rpST were
comparable and not statistically significantly different from those of
the various controls. No treatment-related effect was observed on
routine biochemical parameters at termination. At necropsy, no gross
pathologican change was noted; there was no histopathological
examination (GhiasUddin, 1988b).
Groups of 20 male and 20 female Sprague-Dawley (Crl:CDBR) rats
were given doses by gavage of 0 (vehicle), 0.44, 4.4, 44, or 130 mg/kg
bw per day of rpST for 15 days, and groups of 10 males and 10 females
received daily intramuscular injections of 0.44, 4.4, or 44 mg/kg bw
per day of rpST; a further 10 males and 10 females given the vehicle
by intramuscular injection served as controls. Clinical observations
were made daily, and body weight was recorded before treatment and on
days 1, 3, 7, 10, and 13 and at the end of the study. Food consumption
was measured on days 7 and 14. Clinical chemical parameters, including
determination by radioimmunoassay of the concentrations of circulating
T3, T4, insulin, and glucagon, and haematological and urinary
measurements were examined in all animals at the end of the study. All
rats were necropsied and their organs weighed; the tissues were not
examined microscopically. Apart from the lack of histopathological
examination, the study was conducted according to appropriate
standards for study protocol and conduct.
There were no adverse clinical signs attributable to treatment.
One female rat receiving 44 mg/kg bw per day rpST orally died on day
11 with renal hydropelvis and calculi at necropsy. No statistically
significant differences were found between the groups treated orally
and intramuscularly in comparison with the vehicle controls in terms
of body-weight gain, food consumption, or urinary parameters. No
treatment-related effects on clinical chemical parameters, organ
weights, or gross pathological appearance were found in groups treated
orally when compared with the controls. The mean group values for
haematological parameters were within the normal reference range for
this strain of rats but were statistically significantly different
( p < 0.05) from those of their respective vehicle controls, as
follows: decreased haematocrit in males at 44 and 130 mg/kg bw per day
orally, decreased haemoglobin in males at 130 mg/kg bw per day orally,
and increased platelet count in females at 4.4 mg/kg bw per day
intramuscularly. The clinical chemical changes in rats given rpST
orally did not alter the blood concentrations of glucagon, insulin,
T3, T4, total cholesterol, albumin, or aspartate aminotransferase,
but when rpST was given by intramuscular injection, statistically
significant ( p < 0.05) changes were seen, as follows: decreased T3
and increased total cholesterol in males at 4.4 mg/kg bw per day,
decreased alkaline phosphatase activity, albumin, T3, and T4 in
males at 44 mg/kg bw per day, and decreased aspartate aminotransferase
activity in females at 4.4 mg/kg bw per day.
The mean serum concentrations of rpST in rats 24 h after oral
treatment were no higher than the mean background levels of vehicle
control rats treated orally or by intramuscular injection. Some of the
control animals had concentrations of immunoreactive rpST above the
detection limit (5 ng/ml, by radioimmunoassay), possibly because of
non-specific binding of labelled immunoreactive rpST with rat serum
proteins. Rats given intramuscular injections of 4.4 or 44 mg/kg bw
per day rpST had mean serum values of 22 and 73 ng/ml of rpST for
males and 27 and 56 ng/ml for females, respectively.
At necropsy, no treatment-related gross pathological changes were
seen in any of the animals, apart from dark foci in the skeletal
muscle at the injection site. Rats given rpST by intramuscular
injection had statistically significantly ( p < 0.05) increased
weights of the adrenals (absolute and relative) in males at 4.4 and 44
mg/kg bw per day, of the testes (relative) in males at 44 mg/kg bw per
day, and of the liver (relative) in females at 44 mg/kg bw per day
when compared with vehicle controls.
These results indicate that oral administration of rpST to male
and female rats at doses of 0.44-130 mg/kg bw per day for 15 days did
not result in detectable blood concentrations of rpST and did not
induce any significant physiological responses. In addition, rpST was
not absorbed from the gastrointestinal tract in a hormonally active or
immunologically recognizable form and has no significant physiological
activity when administered orally to rats (GhiasUddin & Bailey, 1988).
The effects of rpST containing a high level of endotoxin carried
over from the production technique were investigated in groups of five
male and five female Sprague-Dawley albino rats given oral doses of 0,
44, or 130 mg/kg bw per day of rpST or intramuscular injections of 0
or 44 mg/kg bw per day for 15 days. Apart from the lack of
histopathological examination, the study was conducted according to
appropriate standards for study protocol and conduct. No
treatment-related effects were observed on clinical signs, body-weight
gain, haematological parameters, creatine kinase, glucagon, glucose,
insulin, T3, or T4 concentrations, or gross pathological appearance
(GhiasUddin, 1989).
Table 1. Acute toxicity of recombinant porcine somatotropin
Species Route Sex Maximum Effect Reference
dose
Somagrepor
Rat Oral M, F 5000 mg None Fischer (1991a)
Rabbit Dermal M 500 mg/site None Fischer (1991b)
Rabbit Eye M 100 mg Slight Fischer (1991c)
irritation
Reporcin
Guinea-pig Dermal F 12.5 mg/site None Bolt (1991a)
Rabbit Dermal F 12.5 mg/site Mild dermal Bolt (1991b,
irritation 1992a,b)
Rat Dermal M,F 1000 mg/kg bw None Bolt (1991c)
Grolene
Rabbit Dermal M,F 2000 mg/kg bw None Frank (1989a)
Rabbit Dermal F 500 mg/site None Frank (1989b);
Sullivan (1985)
Rabbit Eye F 100 mg None Franck (1988a)
Guinea-pig Dermal M,F 10 mg/day None Gorman & Sullivan
(1986);
GhiasUddin & Smith
(1989);
Frank (1988b)
Guinea-pig Intradermal M 0.2 mg/day Skin GhiasUddin et
sensitization al. (1984a)
Guinea-pig Intratracheal F 10 mg/ml Sensitization Campbell et al.
(1989)
Four groups of 20 male and 20 female Sprague-Dawley (CD) rats
were given rpST at concentrations of 0.088, 0.88, 8.8, or 26 mg/kg bw
per day by gavage for 15 days. Three groups of 20 rats of each sex per
group served as controls: one was untreated, a second was given the
vehicle (buffer), and the third received 8.8 mg/kg bw per day of pST
and served as positive controls. Clinical observations were made
daily; body weight and food consumption were measured before treatment
and weekly throughout the study. Haematological, clinical chemical,
and urinary analyses were performed on 10 rats of each sex from each
groups at study termination; the measurements included determination
of the concentrations of circulating T3, T4, insulin, and glucagon.
All rats were necropsied and their organs were weighed. Only gross
lesions were examined microscopically. Apart from the limited
histopathology, the study was conducted according to appropriate
standards for study protocol and conduct.
No treatment-related clinical changes were noted, and no
statistically significant differences from controls were aeen in body
weight, food consumption, haematological, clinical chemical, or
urinary parameters, or in absolute or relative organ weights. At
necropsy, red discolouration of the thymus was seen in some rats of
each sex, at somewhat higher incidence in treated animals than in
controls. No concurrent histopathological changes were seen in the
thymus, and the gross observations were judged to be artefacts induced
by the exsanguination technique. Increased concentrations of serum T3
were noted in male rats given 8.8 mg/kg bw per day pST or 26 mg/kg bw
per day rpST when compared with vehicle controls, but they were
comparable to those of untreated controls. The plasma glucagon
concentrations were statistically significantly decreased in males
given 8.8 or 26 mg/kg bw per day rpST or 8.8 mg/kg bw per day pST when
compared with vehicle controls (Sullivan & Atkinson, 1986).
In order to corroborate the increased serum concentrations of T3
and the decreased glucagon concentrations, two additional 15-day
studies in rats were carried out in two independent laboratories. The
study design was similar to that of Sullivan & Atkinson, except that
organs were not weighed; particular attention was paid to the
collection, handling, and analysis of blood samples. The studies were
conducted in compliance with GLP.
Neither study reproduced the effects on circulating hormone
concentrations after daily oral administration of rpST or pST, and
there were no treatment-related effects on clinical appearance,
body-weight gain, blood concentrations of glucagon, insulin, T3, T4,
or glucose, or gross pathological appearance (Sullivan, 1987;
GhiasUddin & Waterhouse, 1987).
Groups of 10 male and 10 female Charles River CDR rats were given
rpST by gavage at 0 (vehicle), 0.25, 2.5, or 25 mg/kg bw per day for
21 days. A fifth group of 10 male and 10 female rats received rpST by
subcutaneous injection at a dose of 2.5 mg/kg bw, also for 21 days.
Clinical observations were made daily, and body-weight gain and food
consumption were recorded weekly. At the end of the study, blood
samples were taken and haematological and clinical chemical parameters
were measured. At necropsy, organ weights were recorded and a thorough
histopathological examination was performed. The study was conducted
according to appropriate standards for study protocol and conduct.
Apart from the death of two female rats given 25 mg/kg bw per day
of rpST orally, which were due to accidents during gavage, no clinical
signs of toxicity were observed. There were no treatment-related
differences between orally-treated animals and controls in terms of
body-weight gain, food consumption, haematological or clinical
chemical parameters, gross or histopathological appearance, or
absolute or relative organ weights. The group treated subcutaneously
with 2.5 mg/kg bw per day of rpST had increased body-weight gain,
which was not significant for males and significant ( p < 0.05) for
females. No other effects were observed in the group treated
subcutaneously (Fischer, 1992).
Dogs
Thirty-two beagle dogs, 54-69 weeks old and weighing 6.9-16 kg,
were randomized to groups of four males and four females and given
subcutaneous injections of pST (native or recombinant not specified)
at doses of 0 (vehicle), 0.014, 0.05, or 0.55 mg/kg bw per day for 14
weeks. Clinical observations were made daily, food consumption was
determined four times per week, and body weights were recorded weekly.
Haematological, and clinical chemical parameters were determined after
3, 7, 11, and 14 weeks of treatment. Necropsy, organ weighing, and
histopathology were performed at the end of the study.
During the study, all treated dogs developed increased skin
thickness, especially on the head, which was histologically correlated
to thickening of dermal collagen. All dogs survived the study, and
those at the intermediate and high doses had a dose-related decrease
in body-weight gain. Persistent, statistically significant
( p < 0.05) normochromic normocytic anaemia developed in dogs at the
high dose after three weeks of treatment and was seen in those at the
low dose after seven weeks. From week 3 onwards, a statistically
significant ( p < 0.05) decrease in serum urea nitrogen and creatinine
was seen in dogs at the high dose and increases in serum alkaline
phosphatase activity, calcium, phosphorus, potassium, cholesterol, and
triglyceride concentrations were found in dogs at the intermediate and
high doses. The serum concentrations of T3, T4, and cortisol were
unaffected by treatment. Dogs at the high dose showed polyuria
associated with decreased specific gravity of the urine.
At the end of the study, the absolute and relative weights of the
liver and kidneys of animals of each sex and of the adrenals in males
were statistically significantly ( p < 0.05) increased. Dose-related
histopathological changes were seen in all groups, comprising
cytoplasmic rarefaction of pancreatic islet cells, renal mesangial
thickening, lateral widening of the cartilage-bone junction of the
ribs and focal osteoblast proliferation, physis thickening of the
bones, erythroid depletion of the bone marrow, extramedullary
haematopoiesis of the spleen, rarefaction of hepatocytes (at the
intermediate and high doses), and mucous cell hyperplasia of the
glandular stomach (at the intermediate and high doses) (Prahalada et
al., 1998).
Pigs
A number of studies in pigs of the effects of pST or rpST on
growth and other biological parameters after parenteral administration
were reported which were nor designed to comply with accepted
standards for the protocol and conduct of studies of toxicity.
Groups of 12 Yorkshire barrows weighing 32 kg received
intramuscular injections of 0 or 0.022 mg/kg bw per day of pST for 30
days. The pigs were individually penned and fed a corn-soyabean-based
diet formulated to contain 16% protein ad libitum. One animal treated
with pST was withdrawn during the study because it developed
proliferative ileitis. At the end of the study, the treated pigs
showed increased ( p < 0.05) body-weight gain and feed efficiency;
the serum concentrations of glucose, triglycerides, urea nitrogen,
pST, and IGF-I were comparable to those of controls. The serum insulin
concentration of pST-treated pigs was increased during the study but
not at termination. No pST antibodies were detected in plasma. The
absolute weights of the liver, kidney, and heart of treated pigs was
increased ( p < 0.05), while the weight of the pancreas was
comparable to that of controls. No histopathological changes were
observed in lung, thyroid, adrenal, liver, kidney, spleen, myocardium,
skeletal muscle, or intestine. The weight of the pituitary gland was
not affected by pST treatment, but the concentration of pST per mg
pituitary gland was reduced ( p < 0.001) more than 40%. No effect on
carcass adipose tissue mass was seen after treatment, but the carcass
muscle mass was increased ( p < 0.01) in comparison with controls
(Chung et al., 1985).
In a subsequent study, the results of Chung et al. (1985) were
confirmed in pigs that received pST by intramuscular injection at
doses of 0, 0.01, 0.03, or 0.07 mg/kg bw per day for 35 days. The
dose-related effect of pST on increasing feed efficiency and carcass
muscle mass and decreasing carcass lipid mass indicate that metabolic
effects and stimulation of growth can be expected at doses below 0.01
mg/kg bw per day and do not have an upper limit at 0.07 mg/kg bw per
day (Etherton et al., 1987).
Groups of 12 Yorkshire-Duroc barrows weighing 27 kg received
daily intramuscular injections of pST at doses of 0, 0.035, or 0.07
mg/kg bw or rpST at 0.035, 0.07, or 0.14 mg/kg bw for 77 days. The
growth rate was increased and feed efficiency was improved similarly
by pST and rpST. The improved feed efficiency was associated with a
decrease in feed intake. Both pST and rpST increased carcass muscle
mass, while rpST was less effective in decreasing carcass lipid mass.
All of the other parameters measured, including serum concentrations
of glucose, insulin, blood urea nitrogen, pST, rpST, and IGF-I,
indicated that rpST mimics the biological effects of pST, including
binding to pig liver membranes and induction of IGF-I production
(Evock et al., 1988).
As use of pST in pigs may affect their mobility, investigations
have been conducted on leg soundness focusing on the major part of the
bones, the growth centres, and the joints. Daily treatment with rpST
progressively affected the physical mobility of pigs (initial body
weight, 27 kg) over a period of 11 weeks, and doses of 0.07 and 0.14
mg/kg bw, but not 0.035 mg/kg bw, caused an increased ( p < 0.05)
incidence of osteochondrosis in the growth centres of the femur and
tibia characterized by focal deep zones of hypertrophied chondrocytes
protruding into the metaphysis. No concurrent differences in the
calcium or phosphorus concentrations of bones or serum were observed
(Evock et al., 1988).
Pigs weighing 40 kg and treated daily with pST at 0.1 mg/kg bw
for eight weeks had an increased ( p < 0.05) incidence and increased
severity of osteochondrotic lesions of the articular-epiphyseal
complex of the humerus and ulna when compared with controls (Carlson
et al., 1988).
The weight and length of the humerus and ulna and the cartilage
thickness of their distal joints were increased ( p < 0.05) in pigs
treated daily with 4 mg pST for six weeks when compared with controls
(Joergensen & Tang Sorensen, 1994).
When osteochondrotic lesions were scored on a three-point scale,
the forelimbs of control pigs had an average score of 1.06 while those
of pigs treated with 4 mg rpST per day from a weight of 55 to 109 kg
had a score of 1.43; the differences in soundness scores on a
nine-point scale approached significance (6.5 versus 5.3; p < 0.10)
(Skaggs et al., 1989a). The reported prevalence and severity of
osteochondrotic lesions were associated with increased body-weight
gain (Skaggs et al., 1989a) and with increased somatotropin serum
concentrations in pigs selected for rapid growth (Arbona et al.,
1988).
The effects of rpST on quantitative and qualitative carcass
traits were investigated in genetically selected normal and
stress-susceptible pigstreated with 4 mg rpST per day from 55 to 109
kg body weight. The meat of genetically selected stress-susceptible
pigs was improved with regard to muscle pH and colour by rpST
treatment (Skaggs et al., 1989b). In another study, treatment with pST
at 2 or 4 mg/day for 75 days improved the carcass composition without
increasing the incidence of pale, soft, exudative meat (Ender et al.,
1989).
The immunotoxicity of rpST was studied in groups of growing pigs
which received intramuscular injections of doses of 5, 15, or 25
mg/head for 57 days. One group received the vehicle and served as
controls. No consistent, significant affect of rpST treatment was
observed on the gross or histopathological appearance of the organs of
the immune system, total and differential leukocyte counts, antibody
titres to Actinobacillus pleuropneumonia isolates of
B. bronchiseptica, and P. multocida from nasal swabs, the
lymphocytic blastogenic response to mitogens, or the neutrophil
functions of chemotaxis, ingestion, reduction of cytochrome c, and
antibody-dependent cell-mediated cytotoxicity. Significantly effects
of rpST-treatment included decreased haemoglobin and packed cell
volume, increased random neutrophil migration, and a temporary
decrease in the IgG antibody response to tetanus toxoid. There was no
observed effect on the overall clinical health of the treated pigs
(Goff et al., 1991).
2.2.3 Reproductive toxicity
Information was available only on pigs. A number of studies have
been carried out in pigs to investigate the effects of pST on
reproduction, including maternal sexual maturation, fetal development,
and post-natal performance. Parenteral administration was used in all
of the studies.
Cultured ovarian granulosa cells derived from prepubertal gilts
treated with pST at a dose of 0.07 mg/kg bw for 25 days produced more
progesterone in the presence of luteinizing hormone or
follicle-stimulating hormone than cells from control gilts (Bryan et
al., 1988). In another study, however, a reduced response of granulosa
cells to gonadotropin was found in vitro (Bryan et al., 1989).
In young boars, treatment with rpST at a dose of 3.5 or 7 mg/day
from 70 to 118 kg body weight did not affect the weights of the
reproductive organs and glands or serum testosterone concentrations
(Hagen et al., 1989)
The age at puberty, the length of estrus, the length of the
estrus cycle, and the ovulation rates at second oestrus of gilts
treated with pST at a dose of 6 mg/day from 50 to 110 kg body weight
were similar to those of controls (Terlouw et al., 1991). Similar
results were found in prepubertal gilts treated with 5 mg/day for 30
days (Bryan et al., 1990).
In gilts treated with rpST at a dose of 5 mg/day on days 30-43
of gestation, fetal and implantation length were not affected, but
fetal and placental weights and maternal serum IGF-I concentrations
were increased ( p < 0.05) when compared with controls (Sterle et
al., 1995).
Sows were given rpST by injection at a dose of 6 mg/day from day
108 of gestation to day 28 of lactation. The litter size was
standardized at 8-10 piglets per litter on day 3 of lactation. The
milk yield and average piglet weaning weights were not affected by
treatment. In a second study, similar results were found in sows
receiving 70 mg rpST on days 3, 10, 17, and 24 of lactation (Cromwell
et al., 1992). Daily injection of 10 mg/day rpST of sows with their
first litter on days 8 and 39 of lactation did not change the milk
yield or composition with respect to fat, protein, and lactose content
in comparison with controls (Toner et al., 1996). In another study, an
increased fat content was found in colostrum and in milk at day 13 but
not at day 20, and increased milk production was found by three weeks
in sows treated with 5.3 mg/day pST during the last 13 days of
gestation and the first 21 days of lactation.
In sows and gilts treated with pST at 10 mg/day during the last
21 days of gestation, no effects were found on birth weight, the
number born alive, or 21-day survival; however, one gilt and three
sows out of 20 treated pigs died before or at delivery (Kveragas et
al., 1986). Sows treated with 5 or 15 mg/day rpST during the last 14
days of gestation had litters with increasing birth weight ( p <
0.10), but this result was not confirmed in a subsequent study which
showed no effect on birth weight, weaning weight, or survival at day
21 among the offspring of sows treated with 4 or 8 mg/day pST from day
94 of gestation through weaning (Baile et al., 1989).
2.3 Observations in humans
During the 1950s several clinical trials were conducted of the
injection of pituitary preparations derived from farm animals,
including pigs, into humans for treatment of dwarfism. Although some
of the initial studies were thought to show an effect, the porcine
pituitary preparations did not stimulate growth in humans (Raben,
1959; Kaplan, 1965) or in hypophysectomized monkeys (Knobil & Greep,
1959). Serum triglycerides were not affected in humans (Raben, 1959).
Mills et al. (1976) also failed to detect any metabolic activity for
the retention of nitrogen, phosphorus, potassium, sodium, and
chloride; they found no increase in plasma free fatty acids, no
decrease in plasma alpha-amino nitrogen, no impaired glucose
tolerance, and no hyperinsulinaemia in growth hormone-deficient
children after injection of either native pST or serum
plasmin-digested pST. It was concluded that somato-tropins were
species-limited with somatotropins from lower species having no
activity in humans. The biological basis for this species specificity
was discovered years later when it was determined that the binding of
pST to the human somatotropin receptor is several orders of magnitude
lower than that of human somatotropin (Carr & Friesen, 1976).
2.4 Short-term toxicity of insulin-like growth factor
IGF-I, given as recombinant human somatomedin-C, was administered
by gavage at doses of 0.1, 0.25, or 0.5 mg/kg bw per day to three
groups of 25 male and 25 female hypophysectomized Sprague-Dawley
(Crl:CDBR) rats for 15 days. Three control groups, each consisting of
25 male and 25 female rats, were given the vehicle (buffer) orally by
gavage, a single daily intramuscular injection of IGF-I at 0.5 mg/kg
bw, or a single daily intramuscular injection of the vehicle. Clinical
observations, body weights, food consumption, haematological, urinary,
and clinical chemical parameters (including adrenocorticotropic
hormone, glucagon, cortisol, insulin, T3, and T4), gross
pathological appearance, and organ weights were assessed. Tissues were
not examined microscopically. Apart from the limited histopathological
examination, the study was conducted according to appropriate
standards for study protocol and conduct.
The treatment with IGF-I did not cause any clinical signs of
toxicity during the study. The mean values for food consumption, body
weight (days 1-13), and body-weight gain of treated male and female
rats were comparable to those of the controls, and no
treatment-related abnormalities were seen at necropsy. The only
difference from controls in absolute and relative organ weights was a
statistically significant ( p < 0.05) increase in relative kidney
weight and an inverse dose-related decrease in terminal body weight of
female rats given 0.25 or 0.5 mg/kg bw per day. Haematological,
urinary, and clinical chemical parameters were not affected by
treatment, except for a statistically significant ( p < 0.05) decrease
in serum glucose in female rats, which was also observed in control
females receiving vehicle by intramuscular injection. The authors
considered the observed decrease in serum glucose in female rats to be
of doubtful biological significance as the changes were small and not
related to dose. The serum concentrations of IGF-I were below the
limit of detection (7 ng/ml) 2.5-3.5 h after the last treatment in all
animals treated orally. IGF-I was found by a validated
radioimmunoassay in the serum of rats given IGF-I by intramuscular
injection at mean concentrations of 72 and 90 ng/ml in male and female
rats, respectively. Intramuscular injection increased the platelet
counts in females, decreased the serum glucose concentration in
females, decreased alanine aminotransferase activity in males,
decreased the total cholesterol concentration in females, decreased
the albumin concentration in males, and decreased the relative weight
of the liver in males. Two female vehicle controls died on days 5 and
9 of the study from unidentified causes. Thus, no toxicological or
physiological effects were seen in hypophysectomized rats treated
orally for two weeks with IGF-I at doses up to 0.5 mg/kg bw per day
(Howard & Bailey, 1989; Edwards et al., 1989).
Similar results were reported from two other studies of
hypophysectomized rats given IGF-I orally at doses up to 1 or 2 mg/kg
bw per day for two weeks (Juskevich & Guyer, 1990).
The half-lives of 125I-labelled IGF-I in fasted adult rats were 8
min in the stomach, 2 min in ligated segments of the duodenum, 2 min
in the ileum, and 38 min in the colon. Results obtained in vitro
were comparable except that IGF-I was degraded more rapidly in the
colon in vivo (Xian et al., 1995).
3. COMMENTS
The Committee considered the results of studies on the acute and
short-term toxicity and reproductive toxicity of pST and rpST and the
results of studies on IGF-I, the production of which is stimulated by
somatotropin. All of the pivotal studies of toxicity were performed
according to appropriate standards for study protocol and conduct.
Most of the short-term studies focused on effects on biological
parameters such as body-weight gain, ahematology, and clinical
chemistry after oral or parenteral administration of rpST, and only a
few included comprehensive histopathological examination.
Somatotropin and IGF-I are found in all mammalian species. The
structural homology between porcine and human somatotropin is
approximately 66%. The differences in amino acid sequences result in
the 'species specificity' of somatotropins. Pituitary-derived pST is
biologically inactive when injected into hypphysectomized rhesus
monkeys and in humans who have received pST or plasmin-digested pST by
injection. In addition, it has been shown that pST does not bind to
human somatotropin receptors in liver in vivo. Rats displayed a
physiological response to parenterally administered pST and rpST,
although the hormones are about 250 times less potent in this species
than in pigs. The physiological effects of rpST were indistinguishable
from those of native pST in pigs or hypophysectomized rats after
parenteral administration. pST and rpST showed similar inhibition of
binding of 125I-labelled pST to liver microsomal membrane fractions
from pigs or rabbits in vitro.
Peak serum concentrations of pST in rats occurred 6 h after an
intramuscular injection of 125I-labelled rpST, while pST was no longer
measurable 60 h after treatment. The biological serum half-life was
not established in rats. The half-lives of 125I-pST and 125I-rpST in
pigs are short and were determined to be 4 min for the fast phase and
up to 49 min for the slow phase. Biodegradation appears to be rapid,
and parenterally administered rpST at the doses used for growth
promotion in pigs did not lead to concentrations of pST in blood or
muscle that were greater than the physiological levels 27 h after
treatment.
A study in rats given rpST as single doses of up to 5 g/kg bw
showed no biological or toxicological effect. No adverse biological
effects were observed in a study in which rats were given pST or rpST
orally at a dose of 4 mg/kg bw per day for 15 days, and in two 15-day
studies in which rats were given rpST at doses up to 130 mg/kg bw per
day, no adverse effects were seen and no pST could be found in serum.
In a comprehensive study in rats given rpST at doses up to 26 mg/kg bw
per day or pST at 8.8 mg/kg bw per day, no clinical signs of toxicity
or treatment-related changes in body-weight gain, haematological,
clinical chemical, or urinary parameters, organ weights, or gross
pathological appearance were observed. A statistically significant
decrease in the serum concentration of glucagon was observed in male
rats at 8.8 and 26 mg of rpST and 8.8 mg of pST per kg bw per day;
however, the observed effect on serum glucagon concentration was not
reproduced in two additional studies of identical design which were
conducted in two independent laboratories. In a further study, oral
administration of rpST to rats at 25 mg/kg bw per day for 21 days did
not cause treatment-related histopathological changes. These studies
demonstrated that pST and rpST have no biological activity when
administered orally.
When rpST was given intramuscularly to rats for 15 days,
decreased concentrations of the thyroid hormones triiodothyronine and
thyroxine, decreased activities of serum alkaline phosphatase and
aspartate aminotransferase, decreased albumin concentrations, and
increased serum cholesterol concentrations, platelet counts, and
weights of the liver, testis, and adrenal were observed at doses of
4.4 mg/kg bw per day and above.
In several studies, pigs weighing 27-40 kg were treated
parenterally with pST or rpST at doses of 0.01-0.07 mg/kg bw per day
for up to 77 days. The observed effects included increased body-weight
gain, feed efficiency, serum glucose, triglycerides, blood urea
nitrogen, and IGF-I concentrations, and increased weights of the
liver, kidneys, and heart. These effects were considered to be due to
specific binding of pST and rpST to pST receptors. Doses of 0.035 mg
of pST or rpST per kg bw or above affected the physical mobility of
the pigs by causing lesions of the bone and cartilage of the major leg
joints. Since pST and rpST do not bind to the human somatotropin
receptor, the Committee considered that the effects seen in pigs are
unlikely to occur in humans. No effect on key immune functional
parameters was observed in pigs receiving daily intramuscular
injections of rpST at doses of up to 25 mg/animal for 57 days.
No information was available on the reproductive toxicity of pST
or rpST in laboratory animals. Reproductive effects in pigs given
intramuscular injections of approximately 5 mg of pST or rpST per
animal per day were investigated in a number of studies. No effects
were observed on the age at onset of puberty, length of estrus or
estrus cycle, or ovulation rate in nulliparous sows, while treatment
of pregnant nulliparous sows increased placental and fetal weights.
The lactation performance of sows and the composition of their milk
were not affected. Treatment of sows late in gestation and during
lactation had no effect on the birth weight of their offspring, the
number of live births, or survival up to 21 days.
No information was available on the genotoxic potential of rpST,
but the Committee noted that the structurally related compound
recombinant bovine somatotropin did not show evidence of genotoxicity
in two assays (Annex 1, reference 135).
Many of the physiological effects of somatotropin are mediated by
IGF-I. The chemical structures of human, porcine, and bovine IGF-I are
idential. The bioactivity of IGF-I residues in tissues and milk was
considered at the fortieth meeting in relation to use of bovine
somatotropin (Annex 1, reference 104). At that time, the Committee
concluded that, although the liver is the major site of IGF-I
synthesis, it is also present in human milk, saliva, and pancreatic
secreations. The Committee further concluded that IGF-I is not
biologically active when administered orally to hypophysectomized
rats, as dietary IGF-I is almost completely degraded by digestive
enzymes and is not expected to contribute significantly to the high
endogenous concentration of IGF-I in the intestine.
4. EVALUATION
Using data considered at the fiftieth meeting, when recombinant
bovine somatotropins were last evaluated (Annex 1, reference 134),
the Committee concluded at its present meeting that when rpST is used
in pigs, the excess levels of IGF-I residues in edible tissues are
orders of magnitude lower than the amount produced endogenously in
humans and are therefore extremely unlikely to represent any health
risk for consumers.
The Committee noted that recombinant proteins may cause allergy;
however, because there was no evidence that pork meat, which contains
pST, is allergenic in humans and because rpSTs are antigenically
similar to native pST, residues of rpST in food are not likely to
cause an allergic response in humans after consumption.
In reaching a conclusion on the safety of rpST, the Committee
noted the following:
* the lack of biological activity of rpST in rats after oral
administration;
* the lack of biological activity of rpST in humans, as evidenced
by the substantial difference in the amino acid sequence of
somatotropin from pigs and humans, the absence of binding of rpST
to human somatotropin receptors, and the lack of effect in humans
injected with either pST or serum plasmin-digested pST; and
* the lack of biological activity of ingested IGF-I.
From the above, the Committee concluded that rpST can be used in
pigs without any appreciable health risk for consumers from the
administered rpST or from IGF-I residues in rpST-treated pigs. It
established an ADI 'not specified'1 for rpST, which applies to the
three products that were evaluated at the present meeting.
5. ACKNOWLEDGEMENTS
Dr K. Sejrsen and Dr T. Tang-Sorensen, Danish Institute of Animal
Science, Denmark, are acknowledged for their assistance with the
preparation of the first draft of this monograph.
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See Also:
Toxicological Abbreviations