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    BOVINE SOMATOTROPINS

    First draft prepared by
    Dr M.A. Miller
    Center for Veterinary Medicine
    Food and Drug Administration
    Rockville, Maryland, USA

    1.  EXPLANATION

         Four analogues of natural bovine somatotropin (bST) that are
    produced by recombinant DNA techniques (rbSTs) were reviewed by the
    Committee. These were somagrebove, sometribove, somavubove, and
    somidobove. Injection of bST or rbSt into lactating dairy cows
    stimulates milk production. In the past, commercial uses of
    endogenous bST to increase milk production in dairy cows was limited
    because there was no practical method for producing large quantities
    of this product. Recent advances in biotechnology have allowed the
    economical production of commercial quantities of the hormone. These
    products had not been previously evaluated by the Joint FAO/WHO
    Expert Committee on Food Additives.

    2.  BIOLOGICAL DATA

    2.1  Biochemical Aspects

         Bovine pituitary somatotropin exists as four variants comprised
    of 190 or 191 amino acids. The products differ in the amino acids
    which are substituted for the terminal alanine residue (Table 1)
    (Hammond  et al., 1990).

        Table 1. Products marketed as recombinant bovine somatotropins
                                                                        

    Products                      Amino Acid Substituted to
                                  Ala(191)
                                                                        

    Somagrebove                   Met-Asp-Gin

    Somidobove                    Met-Phe-Pro-Leu-Asp-Asp-
                                  Asp-Asp-Lys

    Sometribove                   Met

    Somavubove                    None
                                                                        
    

         Like other protein hormones, bST and rbSTs elicit a biological
    response by binding with high affinity to specific receptors on the
    cell membrane of target cells (OTA, 1991). Although the chemistry of
    the recombinant products varies slightly from pituitary bST, it is
    appropriate to consider them together in this human food safety
    evaluation because they all act by binding with high affinity to the
    bovine somatotropin receptor. The administered compounds are
    designated as "rbSTs" in this monograph, even though bST elicits the
    same responses in most cases.

    2.1.1  Absorption, distribution and excretion

         Like most dietary proteins, rbSTs are degraded by digestive
    enzymes in the gastrointestinal tract and are not absorbed intact.
     In vitro studies on the metabolism of rbSTs demonstrate that they
    are readily cleaved by digestive enzymes (Heiman & Harris, 1989a).
    The progressive cleavage of peptide bonds results in the loss of
    biological activity because both the C- and N-terminal and the
    appropriate tertiary structure are required for receptor binding.
     In vivo studies in laboratory animals confirm that proteolytic
    fragments of rbSTs have no biological activity (Hammond  et al.,
    1990).

    2.1.2  Biotransformation

         As discussed above, rbSTs are not absorbed intact following
    oral administration and therefore they display no biological
    activity by this route. When commercial doses of rbSTs are injected
    into dairy cows, blood levels of somatotropin increase, which does
    not translate into a biologically significant increase of
    somatotropin in muscle or liver because somatotropin is not stored
    in tissues but is rapidly degraded by cytosolic proteases and
    lysosomal enzymes (Hammond  et al., 1990; OTA, 1991).

    2.1.3  Effects on enzymes and other biochemical processes

         rbSTs increase milk production by altering the metabolism of
    several body tissues so that more nutrients are provided to the
    mammary gland for milk production, i.e., repartitioning of nutrients
    (OTA, 1991). Since rbSTs alter the metabolism of several tissues,
    e.g., adipose tissue, intestine, liver and muscle, the circulating
    levels of several nutrients and hormones are changed. Generally, the
    increase in milk production is sufficient to utilize all available
    nutrients. Milk composition data indicate that bST treatment has no
    biologically-significant effect on the major milk components (OTA,
    1991). This eliminates concern for changes in milk nutritional value
    or quality and processing characteristics.

         The morphology of the lactating mammary gland, particularly the
    tight junction between the alveolar cells, provides an effective
    barrier against blood constituents entering the milk. However, small
    amounts of plasma proteins and circulating protein and peptide
    hormones do enter the milk (Malven, 1977). For example, trace
    amounts of insulin-like growth factor, IGF-I (0.7 to 8.1 ng/ml) are
    normally present in bovine milk (Collier  et al., 1991).

         IGF-I is a 70 amino acid polypeptide hormone of the somatomedin
    family. Human and bovine IGF-I are identical. The IGFs
    (or somatomedins) have acute metabolic effects similar (but
    considerably less potent) to insulin and long-term growth-promoting
    effects in that IGF-I mediates many of the growth effects of
    somatotropin. Plasma concentrations of IGF-I are regulated by
    somatotropin and it is the circulating hormone which changes the
    most dramatically following rbST treatment (Juskevich & Guyer,
    1990).

         Although treatment of dairy cattle with rbSTs was not expected
    to cause a biologically significant increase in milk IGF-I,
    toxicology studies were carried out to determine the oral activity
    of IGF-I and residue studies were conducted to quantitate the change
    in milk IGF-I levels following treatment with rbSTs (Juskevich &

    Guyer, 1990). The results of several studies on milk IGF-I values in
    rbST-treated and control cows demonstrate that, following treatment
    with rbSTs, milk IGF-I levels were not outside the range of values
    found naturally in milk (Miller  et al., 1991).

    2.2  Toxicological studies with rbSTs

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with rbSTs are summarized
    in Table 2.

    2.2.2  Short-term toxicity studies

    2.2.2.1  Rats

         Somidobove was administered daily to Fischer 344 rats by oral
    gavage at doses of 0.05, 0.5 or 5.0 mg/kg bw/day for two weeks.
    There were 15 rats/sex/dose including a control group.

         There were no physical or behavioural changes associated with
    treatment, and there were no toxicologically-significant changes in
    body weight, food consumption, efficiency of food utilization,
    haematology, clinical chemistry, or organ weights. There were no
    gross or microscopic lesions that were compound-related. Bovine
    somatotropin was not detected in the plasma of any of the rats
    following two weeks of oral administration. No adverse biological
    effects were observed (Fisher, 1985).

         Charles River CD (Sprague-Dawley derived) rats received
    somagrebove by gavage at concentrations of 0.1, 1 and 10 mg/kg
    bw/day for 15 consecutive days. There were 20 rats/sex/group
    including a control group. The rats were observed twice daily for
    signs of toxicity, morbidity, and mortality. Detailed observations,
    individual body weights and individual food consumption were
    recorded at 3-day intervals during the study. Haematological and
    biochemical tests were performed on 10 rats from each dose at the
    end of the study. Organ weights and microscopic examinations were
    performed at necropsy.

         There were no overt signs of toxicity observed at any treatment
    level during the course of the study. Three male rats, one each in
    the control group and at 0.1 and 1 mg/kg bw/day died during the
    first 3 days of the test. All three deaths were attributed to
    animals being misdosed in the lung. Food intake and body-weight gain
    of the orally treated animals were generally comparable to those of
    the untreated controls at all treatment levels.

        Table 2. Acute toxicity of rbSTs
                                                                                               

    Species        Route         Sex      Maximum           Effect       References
                                          Dose
                                                                                               

    Somavubove

    Rat            oral          M&F      5000 mg/kg bw     none         Marcek et al., 1988

    Rabbit         dermal        M        500 mg/site       slight       Sabaitis et al., 1987
                                                            erythema

    Rabbit         ocular        M        100 mg/eye        slight       Leong et al., 1987a
                                                            irritation

    Rat            inhalation    M&F      1.08 mg/l         none         Leong et al., 1987b

    Guinea-pig     dermal        M&F      20 mg/day         slight       Leong et al., 1988
                                                            erythema

    Sometribove

    Rat            oral          M&F      5000 mg/kg bw     none         Auletta &
                                                                         Olsen, 1986

    Somidobove

    Rat            oral          M&F      90 mg/kg bw       none         Fisher et al., 1987

    Rabbit         dermal        M&F      36 mg/kg bw       slight       Fisher et al., 1987
                                                            erythema

    Rabbit         ocular        M&F      1.8 mg            none         Fisher et al., 1987

    Somagrebove

    Rat            oral          M&F      5000 mg/kg bw     none         Fisher & Garces, 1986

    Rabbit         dermal        M&F      2000 mg/kg bw     none         Fisher & Garces, 1986

    Rabbit         dermal        M        0.5 g             none         Fisher & Garces, 1986

    Rabbit         ocular        M        100 mg            none         Fisher & Garces, 1986

    Guinea-pig     dermal        M        0.4 g/site        none         Costello, 1986
                                                                                               
    
         Haematology parameters were unaffected by the administration of
    bST. A statistically-significant decrease in alkaline phosphatase
    and a significant increase in cholesterol were observed in female
    rats which received 1 mg/kg bw/day. Although statistically
    significant, these changes were within normal limits and were not
    considered to be directly related to treatment.

         Absolute liver weights were significantly increased in female
    rats at 0.1 and 1 mg/kg bw/day, but not at 10 mg/kg bw/day. Absolute
    liver weights for treated male rats were similar to those of the
    untreated controls. Absolute spleen weights were significantly
    increased in females only at 0.1 mg/kg bw/day. Subsequent
    microscopic examinations of the liver and spleen did not show any
    pathological effects. The increase in absolute liver and spleen
    weights appeared to be related to increased body weights observed at
    these levels. The increase in relative liver weight in females at 10
    mg/kg bw/day was considered to be biologically insignificant because
    there was no corresponding increase in male rats and no untoward
    effects on liver function or histology.

         No test substance-related gross or microscopic lesions were
    seen in any tissues from any of the treatment groups. The lesions
    described among these groups were considered spontaneous in nature,
    because the incidence of occurrence was generally similar among all
    groups. The NOEL was 10 mg/kg bw/day (Fisher  et al., 1984).

         Four groups of male and female Sprague-Dawley rats were given
    daily oral doses of somavubove at 0 (vehicle), 0.5, 5 or 50 mg/kg
    bw/day for 26 days via gastric intubation. The dose groups consisted
    of 30, 25, 25 and 30 rats/sex/dose, respectively. A fifth dose group
    served as a positive control. This group consisted of 10 rats/sex
    given daily subcutaneous injections of 50 g/rat somavubove, which
    were killed on study days 5 and 22 for evaluation of serum
    somatotropin and rbST antibody production. Five rats/sex/dose from
    the 0 (vehicle control) and the 50 mg/kg bw/day groups were killed
    on study day 5; and five rats/sex/dose from all oral dose groups
    were killed on day 22 for evaluation and comparison to positive
    controls. Clinical observations were made daily. Body weight was
    measured twice weekly and food consumption was measured twice during
    for week 1 and weekly thereafter. Clinical chemistry, haematology,
    body weights, food consumption and organ weights were measured
    terminally and were analyzed for treatment-related effects of orally
    administered rbST. All orally-treated rats were necropsied. Tissues
    from rats of the 0 (vehicle control) and 50 mg/kg bw/day dose groups
    were examined histopathologically for the presence of
    treatment-related lesions.

         No treatment-related clinical observations were noted. There
    were no statistically-significant differences in the orally-treated
    groups relative to the vehicle control group in terms of
    body-weight, food consumption or haematology. There was a

    statistically-significant decrease in total protein (females only)
    values in the 5 mg/kg bw/day dose group compared to controls.
    Adrenal weights were significantly heavier in the 0.5 and 5.0 mg/kg
    bw/day dose groups (females only) compared to the control group. No
    dose-response relationship was observed for any of these
    statistically-significant parameters and no corresponding
    pathological effects were found microscopically.

         Serum somatotropin and rbST antibody production results
    indicated that orally ingested bST was not absorbed intact from the
    rat gut at the dose levels tested. Antibody titers slightly higher
    than background were present in several of the orally-treated rats,
    but rbST was not detectable by RIA. These data suggest that the
    immune system has access to an antigenic portion of the rbST (Seaman
    & Skinner, 1986). Formation of antibodies to dietary protein is a
    normal response (Bahna & Heiner, 1980; Hammond  et al., 1991).

         Eighty Sprague-Dawley Crl CD(SD)BR rats were divided into 4
    groups of 20 rats/sex/group. Animals were gavaged daily for 28 days
    with 0 (vehicle), 0.06, 0.6 or 6 mg of sometribove/kg bw/day.
    Survival, clinical observations, body weight, food consumption,
    clinical pathology, gross pathology, organ weights and
    histopathology were assessed.

         No animals were found dead or were sacrificed during the study.
    No compound-related trends were noted in clinical observations of
    either sex. Mean growth rate and mean total food consumption values
    were not significantly different between control and treated groups.
    Clinical and gross pathology findings were unremarkable. There were
    no compound-related trends in the mean terminal body-weight of
    treated animals. Mean ovary weight and mean ovary/body-weight ratios
    were significantly lower in the 0.06 and 6 mg/kg bw/day group
    females when compared to controls. Mean kidney/brain weight ratios
    in the 0.06 and 6 mg/kg bw/day groups and the mean adrenal/brain
    weight ratios in the 6 mg/kg bw/day group females were significantly
    higher than in the control animals. These effects were not
    considered related to treatment because there was no dose-dependent
    relationship. No treatment-related microscopic changes were
    observed. The NOEL was 6 mg/kg bw/day (Serota, 1984).

         Groups of 15 male and 15 female young adult Fischer 344 rats
    were given daily gavage doses of 0 (vehicle), 10, 30, or 100 mg/kg
    bw/day of somidobove for three months. Clinical observations were
    made daily. Body weight and food consumption were determined weekly.
    Clinical chemistry, haematology, urinalysis, organ weights and
    histopathology were performed at the end of the study.

         All rats survived the three-month study period. One mid-dose
    female had chromodacryorrhea of the right eye for one week; another
    female in this group had a small growth at the right corner of the
    mouth two days prior to necropsy. Because of the low incidence and

    the lack of a dose-response relationship, these observations were
    not considered to be related to treatment. There were no
    compound-related effects on body weight, weight gain, food
    consumption or efficiency of food utilization. Sporadic but
    statistically-significant changes in haematological parameters,
    including decreased nucleated erythrocytes in the high-dose females
    and increased monocytes in the high-dose males, were small and not
    considered treatment-related. Also, small but
    statistically-significant increases in alanine transaminase,
    aspartate transaminase and cholesterol occurred in the low and
    high-dose males. These changes were not dose-related and therefore
    were probably not related to somidobove treatment. There were no
    compound-related changes in urinalysis parameters or absolute and
    relative organ weights. There were no gross or microscopic lesions
    associated with somidobove treatment. In conclusion, no adverse
    effects due to the administration of somidobove were observed in
    this study. Therefore, the NOEL was 100 mg/kg bw/day (Fisher, 1987).

         Sometribove was administered daily by gavage at doses of 0.1,
    0.5, 5 or 50 mg/kg bw/day or subcutaneously at 1 mg/kg bw/day
    (positive control) to Charles River CD VAF rats (30 rats/sex/group)
    for 13 consecutive weeks. The negative control animals were gavaged
    with vehicle only. After 13 weeks, 5 animals/sex/group were
    maintained without dosing for a 14-week recovery phase.

         No mortality was observed in the groups treated orally except
    for one male in the mid-dose group which died due to a dosing
    accident. After the eighth week of treatment, three deaths occurred
    in the positive control group; one animal of each sex was found dead
    and one female was killed  in extremis because the animal displayed
    thin appearance, reduced motor activity and blood discharge around
    the nose.

         No treatment-related ocular changes were observed. Oral
    treatment did not increase body weight or feed consumption.
    Concomitant marked increases in body-weight gain and feed
    consumption were observed from week 2 and throughout the treatment
    phase in the positive control group animals. During the recovery
    phase, body-weights of the male rats in the positive control group
    remained higher than in those of the negative control animals, but
    feed consumption rapidly returned to normal.

         No toxicologically-meaningful changes were observed in clinical
    chemistry or haematology parameters in the orally-treated animals.
    In animals treated subcutaneously, cholesterol and calcium values
    were increased in male rats. In female rats, albumin was slightly
    decreased which caused a decrease in total protein and
    albumin/globulin ratio. Phosphorus and alkaline phosphatase were
    elevated in these females, suggesting an alteration in bone
    metabolism. Moderate increases in the mean corpuscular volume and
    mean corpuscular haemoglobin were observed in both sexes. In males,

    this was accompanied by decreases in red blood cell count,
    haematocrit, haemoglobin concentration and mean corpuscular
    haemoglobin concentration. In females, white blood cell counts were
    increased which correlated with an increased number of lymphocytes.
    No changes of toxicological significance were noted in the
    urinalysis.

         No drug-related changes in organ weights were observed in
    animals treated orally. In animals treated subcutaneously, almost
    all organs had increased absolute weights or organ/brain weight
    ratios. In addition, spleen and liver (both sexes) and adrenals
    (males) presented increased organ/body weight ratios.

         No gross findings were observed in animals treated orally.
    Enlarged submaxillary lymph nodes were noted in a few animals
    treated subcutaneously. Renal cortical tubular basophilia was
    observed microscopically in some males of the high-dose group and
    positive control groups, but only in one male of the highest-dose
    group. Fibrosis of the pancreatic islets was observed in some males
    of the positive control group, and in a few animals treated orally.
    Since these latter two findings were not dose-related, and since
    these lesions are often found in this rat strain, a causal
    relationship with product administration was doubtful.
    As a general conclusion, there were no systemic effects following
    oral administration of rbST at doses up to 50 mg/kg bw/day (Richard
     et al., 1989).

    2.2.2.2  Dogs

         Groups of four male and four female young adult beagle dogs
    were given daily oral doses of 0 (vehicle), 1, 3, or 10 mg/kg bw/day
    of somidobove by capsule for three months. Groups of two male and
    two female beagle dogs were given a single subcutaneous dose of 0 or
    1 mg/kg bw/day of somidobove to serve as a positive control group.

         All dogs survived the three-month study period. The only
    treatment-related clinical sign was soft or mucoid stools. Abnormal
    stools were observed in the control dogs and the frequency of
    occurrence increased in a dose-dependent manner. The effect was
    probably related to a combination of phosphoric acid diluent and
    somidobove. The stool changes were considered to be of minor
    toxicological significance. There were no adverse ophthalmic effects
    attributable to somidobove administration. There were no
    compound-related effects upon body weight, food consumption,
    haematology, clinical chemistry and urinalysis parameters or
    absolute and relative organ weights. There were no gross or
    microscopic lesions associated with somidobove treatment.

         Somidobove was not detectable in the serum of dogs treated
    orally except for one dog treated with 10 mg/kg bw/day orally, which
    had a single positive response 2 h after treatment on day 30. The

    serum level returned to below detectable limits by four hours. This
    one positive serum sample out of 144 was believed to represent a
    false positive. In the serum of dogs treated subcutaneously,
    somidobove was detectable at maximum concentrations of 195 to
    551 ng/ml. The NOEL was 10 mg/kg bw/day (Fisher & Russell, 1988).

    2.2.3  Special studies with rbSTs in hypophysectomized rats

         A study was conducted to determine if orally administered rbST
    can be absorbed in the hypophysectomized rat in sufficient
    quantities to result in weight gain when dosed at greatly
    exaggerated doses with respect to amounts in the human diet. rbST
    was given orally or subcutaneously to groups of female
    hypophysectomized rats daily for 9 days. Ten rats per dose were
    given oral dosages of 0 (vehicle), 0.04, 0.4, 2 or 4 mg rbST/kg
    bw/day. One replicate each of 10 rats per dose group received
    subcutaneous doses of 0 (vehicle), 15, 30 or 60 g rbST per day.
    Rats were weighed daily to observe body-weight gain, a measure of
    absorption and activity of somatotropin in the hypophysectomized
    rat.

         Results showed that rbST administered subcutaneously increased
    average daily body-weight gain. No daily body-weight gain occurred
    in the rats administered rbST orally and little or no gain occurred
    in rats receiving subcutaneous or oral vehicle control. This study
    showed that rbST displays no growth-promoting activity following
    oral administration (Seaman  et al., 1986).

         Fifty-nine female hypophysectomized Sprague-Dawley rats were
    randomly assigned to one of eight treatments. Rats were administered
    0 (vehicle), 40, 80 or 120 g/day rbST daily by gavage or by
    subcutaneous injection for seven days. All groups consisted of 7
    animals except for the control group, which had 10 animals. The
    objective of the study was to utilize a somatotropin-sensitive
    model, the hypophysectomized rat, to determine whether ingested rbST
    could be absorbed in a biologically-active form. Body weight was
    measured on day 0 and on day 7 for determination of body-weight gain
    and relative body-weight gain.

         Hypophysectomized rats injected with rbST had increased weight
    as a log function of the dose of somatotropin administered. Maximal
    weight gain, approximately 16 grams above basal level, was achieved
    with the 80 and 120 g/day doses. No growth response was observed
    among the hypophysectomized rats administered somatotropin orally.
    The results support the hypothesis that ingested somatotropin is
    degraded and is not absorbed in a biologically-active form (Lanza &
    Hoffman, 1986).

    2.2.4  Special studies on genotoxicity

         The results of genotoxicity studies on rbSTs are summarized in
    Table 3.

        Table 3. Results of genotoxicity assays on rbSTs
                                                                                               

    Test system          Test object        Concentration     Result      Reference
                                                                                               

    Micronucleus assay1  Mouse bone         250 mg/kg         negative    Li et al., 1989
                         marrow cells       subcutaneous

    Forward mutation     Chinese hamster    200-1000 g/ml    negative    Vanrell et al., 1989
    assay                ovary cells
                         HGPRT locus
                                                                                               

    1    Both with and without metabolic activation using rat liver S9 fraction.
    


    2.3  Observations in humans

         Humans are naturally exposed to trace levels of bST in beef,
    milk and other dairy products. bST, like other dietary proteins, is
    degraded and shows no growth-promoting activity. bST also displays
    no growth-promoting activity when injected into humans. During the
    1950s, clinical trials were conducted injecting large quantities of
    farm animal pituitary preparations (10 000 to 160 000 g/day) into
    children and adults for several weeks or months. These pituitary
    preparations did not stimulate growth or cause consistent metabolic
    effects (Bennett  et al., 1950, Froesch  et al., 1957). It was
    concluded that somatotropins 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 bST to the human somatotropin
    receptor is several orders of magnitude lower than that of human
    somatotropin (Carr & Friesen, 1976, Moore  et al., 1985). Since
    receptor binding is governed by the laws of mass action, the low
    affinity of bST for the human somatotropin receptor renders it
    biologically inactive even at grossly pharmacological doses.

    2.4  Toxicology studies with IGF-I

    2.4.1  Short-term toxicity studies

    2.4.1.1  Rats

         This study was designed to evaluate the oral activity of IGF-I
    when administered daily by gavage for two weeks at dose levels of
    20, 200, or 2000 g/kg bw/day to three groups of 20 male and 20
    female Sprague-Dawley (Crl:CDBR) rats. To examine the efficiency of
    uptake and its growth promoting potential, IGF-I was also
    administered using an osmotic pump at a release rate of 50 or
    200 g/day to 20 male and 20 female rats. In addition, 20 rats per
    sex per group were given vehicle at the same dosing volume by gavage
    or at the same release rate via osmotic pumps (negative controls).
    As a positive control, 20 rats per sex received somatotropin, a
    proven growth promoter, using an osmotic pump at a release rate of
    4000 g/day. Criteria evaluated for compound effect included
    survival, clinical pathology, plasma hormone assay, gross pathology,
    organ weights, tibia length and epiphyseal width.

         All animals survived to termination of the study. There were no
    significant treatment-related clinical observations. Mean body
    weights were slightly but significantly increased throughout the
    study for males receiving 2000 g IGF-I/kg bw/day by gavage. This
    increase in body weight actually occurred in only one of the two
    replicates of high-dose males receiving 2000 g of IGF-I/kg bw/day
    and was not considered biologically significant. For animals that
    were implanted with an osmotic pump, the 50 g IGF-I/day males and
    both sexes receiving 200 g IGF-I/day exhibited significant
    increases in body weight throughout most of the study. In addition,
    both sexes of the positive control animals exhibited a significant
    increase in body weight throughout the study. These findings were
    considered treatment-related.

         Food consumption values were increased for both sexes of rats
    receiving 200 g IGF-I/day by osmotic pump and for both sexes of the
    positive control animals at all intervals. A
    statistically-significant increase in food consumption was also seen
    in the male animals receiving 50 ug IGF-I/day by osmotic pump on
    days 1 to 7. These effects were probably related to treatment. Food
    consumption was decreased in females in the 20 g IGF-I/kg/day
    gavage group but this result was considered sporadic.

         In animals receiving somatotropin by subcutaneous infusion,
    both epiphyseal width and tibia length were increased. Epiphyseal
    widths were increased in females receiving the high-dose of IGF-I by
    osmotic pump. Tibia lengths were increased in the males receiving
    IGF-I by osmotic pump. In rats receiving IGF-I by gavage, epiphyseal

    widths were decreased in both sexes of the high-dose group and tibia
    lengths were increased in the low- and high-dose males. These
    contradictory findings in tibia length and epiphyseal width for
    gavage-dosed animals suggest this effect is sporadic rather than
    treatment-related.

         The organ weights of animals receiving IGF-I by osmotic pump
    were significantly increased compared to control animals in many
    instances. For animals receiving IGF-I by osmotic pump, the males
    showed a decrease in relative brain weights and in relative
    testis/epididymis weights. The females showed decreased relative
    brain weights. Significant increases for the males were seen in
    final body weights, absolute heart weights, absolute and relative
    kidney weights and absolute liver weights. Significant increases in
    final body weights, absolute adrenal weights, absolute heart
    weights, absolute and relative spleen weights and absolute and
    relative kidney weights were observed in females.

         No increases in circulating IGF-I levels in the blood of rats
    receiving IGF-I by gavage were observed indicating that significant
    absorption of IGF-I did not occur. The rats which received IGF-I by
    osmotic pump had a significant increase in circulating IGF-I levels.

         In conclusion, no biologically-significant effects were
    produced in male rats at gavage doses up to 2000 g/kg bw/day. The
    results suggest that no biologically-significant absorption occurs
    following gavage administration. Subcutaneous administration of
    IGF-I by osmotic pump produced an increase in body weight, food
    consumption, blood levels of IGF-I, tibia length, epiphyseal width
    and numerous changes in organ weights. The effects seen with IGF-I
    administered by osmotic pump were similar to those produced by
    somatotropin administered by a similar route, although somatotropin
    was more effective (Terrill, 1989).

    2.4.2  Special study with IGF-I on hypophysectomized rats

         Hypophysectomized Sprague-Dawley rats (20 sex/group) were given
    daily oral doses of 0 (vehicle), 0.01, 0.1 or 1 mg/kg bw/day IGF-I
    by gavage for two weeks. An additional group of 20 males and 20
    females were given 1 mg/kg bw/day of bovine serum albumin by gavage
    as a positive oral protein control and a group of 10 males and 10
    females was given 1 mg/kg bw/day IGF-I via subcutaneously implanted
    osmotic pumps to serve as a positive parenteral control. Animals
    were examined daily for physical condition. Body weight and body
    length were measured twice weekly. Haematology, clinical chemistry,
    organ weights and pathology were performed at the termination of the
    test period.

         On test day 3 female rats from the negative control group and
    one male rat from the 0.01 mg/kg bw/day treatment group died. On
    test day 6, one female died and one male from the 0.01 mg/kg bw/day
    group was killed in moribund condition. Animals which died appeared
    dehydrated. Therefore, deaths were attributed to failure to
    acclimate and to the delicate physical condition of
    hypophysectomized rats, and not to treatment. Subcutaneous treatment
    with IGF-I caused physiologic effects of increased body weight,
    increased neutrophil count, decreased BUN, creatinine and albumin
    and increased kidney and spleen weights of both males and females.
    Oral treatment caused no effects upon body weight, body length,
    haematology or clinical chemistry parameters. The only change in
    clinical chemistry of the orally treated rats was a 5.7% decrease in
    total protein in the males of the 1 mg/kg bw/day group. This finding
    was believed to be incidental and not associated with treatment.
    There were no statistically-significant changes in organ weights of
    the orally treated animals that were compound-related. There were no
    treatment-associated gross or microscopic lesions.

         In conclusion, no toxicological or physiological effects
    occurred in hypophysectomized rats treated orally for two weeks with
    IGF-I at doses up to 1 mg/kg bw/day (Fisher & Russell, 1989).

    3.   COMMENTS

         Somatotropins and insulin-like growth factors which mediate
    many of the physiological effects of somatotropins are found in all
    mammalian species. Pharmacological studies on the four compounds
    show that their physiological effects are indistinguishable from
    those of naturally occurring bST in dairy cows. The somatotropins
    display species specificity and pituitary-derived bST is inactive
    even when administered parenterally to humans. However, rats display
    a physiological response to parenterally administered bST and rbSTs.

         Somatotropins, including rbSTs, are degraded by enzymes of the
    gastrointestinal tract and should therefore be inactive when
    administered orally. Acute oral toxicity studies in rats with rbST
    doses up to 5 g/kg bw failed to show any biological or toxicological
    effect. No adverse biological effects were observed in two 2-week
    oral feeding studies in rats with doses of rbSTs up to 10 mg/kg
    bw/day, and in two 4-week oral feeding studies in rats with doses up
    to 50 mg/kg bw/day, rbSTs caused no dose-dependent effects. In two
    90-day oral feeding studies in rats with rbSTs at doses up to 100
    mg/kg bw/day, no drug-related changes were observed, and in a 90-day
    oral feeding study in dogs at doses up to 10 mg/kg bw/day, rbST
    treatment caused no adverse toxicological effects. Two 9-day oral
    feeding studies in hypophysectomized rats with rbST doses up to 4
    mg/kg bw/day demonstrated that ingested rbSTs are degraded and are
    not absorbed in a biologically active form. These studies confirm
    that rbSTs have no biological activity when administered orally.

         The genotoxic potential of the rbSTs was evaluated in two
    assays, both of which were negative.

         Many of the physiological effects of rbSTs are mediated by
    bovine insulin-like growth factor-I (IGF-I), which is structurally
    identical to human IGF-I. The liver is the major site of IGF-I
    synthesis, but it is also present in human milk, saliva, and
    pancreatic secretions. Two feeding studies, one in intact and one in
    hypophysectomized adult rats, confirmed that IGF-I at doses up to 2
    mg/kg bw/day has no biological activity when administered orally.
    Because human and bovine IGF-I are structurally identical, the role
    of dietary IGF-I on the gastrointestinal tract was evaluated in
    several studies which showed that IGF-I is degraded by digestive
    enzymes, is present in human saliva and digestive juices, and is not
    active in the upper gastrointestinal tract.

    4.  EVALUATION

         The lack of oral activity of rbSTs and IGF-I and the low levels
    and non-toxic nature of the residues of these compounds, even at
    exaggerated doses, results in an extremely large margin of safety
    for humans consuming dairy products from rbST-treated cows. In view
    of the lack of impact on human food safety, the Committee
    established an ADI "not specified" for rbSTs which applied to
    somagrebove, sometribove, somavubove, and somidobove.

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    See Also:
       Toxicological Abbreviations