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Food safety and pharmacokinetic studies which supporta zero (0) meat and milk withdrawal time for use of

sometribove in dairy cowsBg Hammond, Rj Collier, Ma Miller, M Mcgrath, Dl Hartzell, C Kotts, W

Vandaele

To cite this version:Bg Hammond, Rj Collier, Ma Miller, M Mcgrath, Dl Hartzell, et al.. Food safety and pharmacokineticstudies which support a zero (0) meat and milk withdrawal time for use of sometribove in dairy cows.Annales de Recherches Vétérinaires, INRA Editions, 1990, 21 (suppl1), pp.107s-120s. �hal-00901999�

Food safety and pharmacokinetic studieswhich support a zero (0) meat and milk withdrawal

time for use of sometribove in dairy cows

BG Hammond RJ Collier MA Miller M McGrath

DL Hartzell C Kotts W Vandaele

1 Monsanto Agricultural Company, Animal Sciences Division,Boo N Lindbergh Boulevard, St Louis, MO 63167, USA;2 Monsanto Europe, Avenue de Tervuren, 220-272, Brussels, Belgium

(Pharmacokinetics of Veterinary Drugs, 11-12 October 1989, Foug6res, France)

Summary ― Sometribove (SB) is a synthetic form of bovine somatotropin (BST) whose amino acid

sequence is the same for 190 of the 191 amino acids in BST. Administration of 500 mg of SB to

dairy cows every 14 d increases the efficiency of milk production. Regulatory agencies have author-ized a zero (0) milk and meat withdrawal time for investigational use of SB. The scientific basis for

this authorization is as follows: 1) BST and other non-primate somatotropins are not active in hu-

mans, due to differences in the amino acid sequence from human somatotropin, which limits the

ability of BST to bind to receptors on human tissues. 2) SB is not orally active, as it is degraded like

other proteins when eaten. Administration of 50 000 pg/kg/d SB to rats for 90 d produced no growthresponse. 3) Residual levels of SB in meat/milk are very low (ppb) and comparable to endogenousBST levels. 4) Residual levels (ppb) of insulin-like growth factor I (IGF-I) in meat and milk are only

marginally increased by SB treatment (somatotropin stimulates local production of IGF-I in tissues to

mediate some of its biological effects. 5) IGF-I was not orally active when fed to rats at doses rang-

ing from 200 to 2 000 !g/kg for 14 d.

bovine somatotropin / milk / safety I residues / withdrawal time

Résumé ― Études d’innocuité justifiant un délai d’attente nul pour le lait et la viande prove-nant des vaches laitières supplémentées au sométribove. Le sométribove (SB) est une forme bi-

osynthétique de la somatotropine bovine (bS7), qui présente une séquence d’acides aminés iden-

tique pour 190 des 191 acides aminés constituant la bST. L’administration, tous les 14 j de 500 mg

de SB à des vaches laitières, provoque un accroissement de la production laitière. Les autorités rég-lementaires ont autorisé un délai d’attente nul (0 j) tant pour le lait que pour la viande lors de

l’utilisation du SB à des fins de recherche et de développement. Les fondements scientifiques de

cette décision sont les suivants : 1) La bST et les autres somatotropines non produites par des pri-mates n’ont aucune activité sur l’homme. En effet, les différences relevées par rapport à la somato-

tropine humaine au niveau de la séquence des acides aminés empêchent la liaison de la bST aux

récepteurs présents dans les tissus humains. 2) La SB n’est pas active en prise orale; à l’instar

d’autres protéines, elle se dégrade à l’ingestion. L’administration, durant 90 j de 50 000 ¡.1g/kglj à des

rats ne s’est traduite par aucune réponse en termes de croissance. 3) Les niveaux résiduels de SB

dans la viande et le lait sont très faibles (de l’ordre des parties par milliard), et comparables aux ni-

veaux observés pour la bST endogène. 4) Les niveaux résiduels (parties par milliard) de l’IGF-I (insu-lin-like growth factor 1) dans la viande et le lait ne s’élèvent que très marginalement en présence d’untraitement à la SB (la somatotropine stimule la production locale, dans les tissus, de l’IGF relayantcertains de ses effets biologiques. 5) L’IGF-I n’a extériorisé aucune activité biologique lorsqu’il étaitadministré par voie orale à des rats à raison de doses allant de 200 à 2 000 pglkg durant 14 j.

somatotropine bovine / lait / sécurité / résidus / délai d’attente

INTRODUCTION

Dairy scientists first discovered the abilityof bovine somatotropin (BST) to stimulatemilk production in dairy cows approximate-ly 50 years ago. Russian scientists werethe first to report that bovine pituitary ex-tracts were galactopoietic when injectedinto dairy cows (Azimov and Krouze,1937). During World War II, attempts byBritish scientists to use BST to offset milkshortages were frustrated by the limitedamount (5-15 mg) of BST which could beextracted from a pituitary (Young, 1947).Further studies showed that administrationof BST for several weeks to dairy cowsproduced a sustained increase in milk pro-duction (Brumby and Hancock, 1955;Machlin, 1973). Commercial use of BSTwas not possible at that time, as no practi-cal method to synthesize it had beenfound.

With the advent of biotechnology, it isnow possible to clone bacteria with the

gene coding for BST and induce the bacte-ria to produce commercial quantities of theprotein. Monsanto Company has undertak-en long-term trials at various locations inthe US and Europe to investigate the safe-ty and effectiveness of biosynthetically de-rived BST to dairy cows. During these

investigations, Monsanto has been author-ized a zero (0) withdrawal time for humanconsumption of milk and meat from BST-supplemented dairy cows. This authoriza-tion has been granted by health scientists

within the US Food and Drug Administra-tion and within European regulatory agen-cies based in part on food safety data de-veloped by Monsanto. A presentation ofthe scientific data supporting the zero (0)withdrawal time authorization follows.

PURITY AND CHEMICAL IDENTITY

Pituitary BST exists as 4 variants com-prised of 190 or 191 amino acids with het-erogeneity at the amino terminus (Phe orAla-Phe) and at position 126 (Val or Leu)in the molecule (Santome et al, 1976). Thebiosynthetic form of BST developed byMonsanto (henceforth designated as

sometribove) has the same number andsequence of amino acids as one of the 191amino acid variants with the substitution ofmethionine for alanine at the amino termi-nus. E coli bacteria cloned with the some-tribove gene are grown in fermentors,killed and the sometribove is harvested.

Following precipitation and chromato-

graphic steps, a highly purified (>95%)product is obtained. The purified bulksometribove powder is formulated intofood grade oil which prolongs its releasewhen injected subcutaneously (sc) intocows. Dairy cows are injected sc with 500mg of sometribove in the oil-based pro-longed-release delivery system every 14 d,which is equivalent to 36 mg of sometri-bove/cow/d.

SPECIES LIMITED ACTIVITYOF SOMETRIBOVE

The inactivity of BST and other non-

primate somatotropins in humans was es-tablished in numerous clinical trials carried

out at various medical centers during the1950’s and 1960’s. Prior to that time, scien-tists had shown that somatotropin extract-ed from the pituitaries of cows or pigs stim-ulated growth when injected into laboratoryanimals (Knobil and Greep, 1959). Sincebovine and porcine insulin were known tobe clinically active in man, scientists postu-lated that bovine and porcine somatotropinmight promote growth in children with

growth failure due to hypopituitarism.In numerous clinical trials, the pituitary

preparations from farm animals were re-

ported to be inactive in children and adults,even when administered in large, non-

physiological doses (10 000-160 OOO,ug/d)for several weeks or months (Bennet et al,1950; Froesch et al, 1957). There was noevidence that farm animal somatotropinseither stimulated growth or consistentlycaused metabolic effects (with the possibleexception of transient hyperglycemia), in-

creased calcium excretion or nitrogen re-tention, unlike preparations of human so-matotropin which were fully active whentested in man (Beck ef al, 1957).

In a classic series of studies, farm ani-mal somatotropin preparations, which wereactive in rats and dogs, were shown to becompletely inactive in normal and hypo-physectomized monkeys injected with highdoses (5000 Ji9/kg of bovine somatotro-

pin) for 20-30 d (Knobil and Greep, 1959).No changes in body weight, serum glu-cose, insulin sensitivity, non-protein nitro-

gen, amino acid nitrogen or phosphorouswere detected in monkeys treated with bo-vine somatotropin. However, when hypo-physectomized monkeys were subse-

quently injected with approximately 900xg/kg of monkey somatotropin, significantchanges in the aforementioned clinical pa-rameters were observed. Based on these

results, and the inactivity of farm animalsomatotropins in humans, it was conclud-

ed that the biological activity of somatotro-pin in primates is ’species-specific’ (Ra-ben, 1959).

The biological basis for this ’species-specific’ activity was determined some

years later, when biochemists were able to

identify the amino acid sequence of soma-totropin isolated from the pituitaries of dif-ferent species (Santome et al, 1976).While the amino acid sequence of soma-

totropin from two ruminants (bovine, ovine)is closely related (1 % difference in aminoacid sequence), the amino acid sequenceof unrelated species is more variable (35%difference between human and bovine

somatotropin) (Wallis, 1975, 1988). Thedifferences in the amino acid sequence of

somatotropins from various species hasbeen attributed to evolutionary divergencein the somatotropin gene (Wallis, 1975).

The amino acid sequence determines

the shape or conformation a protein as-sumes in the body. In order for a proteinhormone to produce a physiological re-

sponse in body tissues, it must bind to a

specific receptor on tissue cells. The re-

ceptors recognize only proteins with the

right conformation or shape; one could

consider the protein hormone a key and acell receptor the lock (Roth and Grunfeld,1981 Only the correct key (protein hor-mone) will open the lock (bind to the cellreceptor) to produce a physiological re-

sponse in the cell. Since the amino acid

sequence and, therefore, the shape of bo-vine somatotropin is different from that of

human somatotropin, bovine somatotropindoes not fit the lock or compete effectivelywith human somatotropin for binding to

somatotropin receptors on human cell sur-

faces. This has been confirmed experi-mentally in human hepatocytes and lym-phocytes (Carr and Friesen, 1976; Lesniaket al, 1977; Moore et al, 1985). The bind-ing affinity of BST for the somatotropin re-ceptor on human tissues is several ordersof magnitude lower than that of human

somatotropin. This explains the absenceof growth-promoting effects, even whenlarge non-physiological doses of BST wereinjected into humans.

The activity of bovine and porcine insu-lin in man is explainable by comparison ofthe amino acid sequence for insulin be-tween the 3 species. The amino acid se-quence of porcine insulin differs from hu-man by only 1/51 amino acid residues,bovine differs by 3 amino acid residues(Porte and Halter, 1981). Since the aminoacid sequence for insulin is well conservedin the 3 species, the conformations of therespective insulins are very similar. Thus,bovine and porcine insulins have bindingaffinities similar to that of human insulin forthe insulin receptor on human tissues andare thus pharmacologically active in man.

Prior to elucidation of the amino acidsequence of somatotropin of primates andother species, scientists hypothesized thatthere existed an active core in the soma-totropin molecule that was common to allmammalian species (Kostyo, 1974). If thisactive core could be liberated by enzymat-ic treatment of farm animal somatotropins,then an alternate source (other than hu-man pituitaries) of metabolically active

somatotropin for use in humans would beavailable. There were a few reports in theliterature that enzymatically derived frag-ments of BST were biologically activewhen large doses (5000-100 000 xg/d)were injected into humans (Forsham et al,1958; Sonenberg and Dellacha, 1967).Other scientists, however, were unable toreproduce the reported findings of meta-bolic activity of BST fragments in humans(Kostyo, personal communication), so that

the validity of these reports were ques-tioned (Kostyo, 1974). When the aminoacid sequences of bovine and human so-

matotropins were subsequently identifiedand compared, no common core of aminoacids was apparent between these pro-teins.

Further research has shown that soma-totropin fragments (eg, amino acid resi-dues 1-134, 141-191, 95-134) possessonly a small fraction (1% or less) of the bi-ological activity of the parent molecule(Reagan et al, 1981 The little biologicalactivity that has been observed with soma-totropin fragments in laboratory animalshas been attributed to contamination of thefragment preparation with intact somatotro-pin (Aubert et al, 1986). Biosynthetic orchemically synthesized somatotropin frag-ments that are free of intact somatotropinare largely devoid of biological activitywhen tested in vitro (Krivi et al, 1987; Au-bert et al, 1986). More recent work indi-cates that somatotropin receptors interactwith an extensive domain consisting of re-gions of both the N- and C-terminal endsof the somatotropin molecule (Cunning-ham et al, 1989). This explains earlier workwhich demonstrated that significant biologi-cal activity with the 1-134 fragment waspossible only when it was recombined with

large portions of the C-terminal region ofthe somatotropin molecule (Kostyo, per-sonal communication). Therefore, there isno reason to believe that smaller peptidesderived from BST would possess somato-

tropin activity and their consumption innanomolar amounts in meat/milk would

pose no health risks to man.

ABSENCE OF ORAL ACTIVITYOF SOMETRIBOVE

Dietary proteins are degraded in the diges-tive tract by the combined action of stom-

ach acids and enzymes, such as trypsin,chymotrypsin, carboxypeptidases, etc to

individual amino acids and oligopeptides(Lehninger, 1971 ). Sometribove would beexpected to be similarly degraded if ingest-ed, as it is digested in a manner similar toBST when incubated in vitro with trypsin,carboxypeptidase and elastase (Monsanto,unpublished data).When sometribove was administered

orally to rats for 90 d at dosages varyingfrom 100 to 50 000 ,ug/d, there was no evi-dence of a growth response, whereas in-

jection of 1 000 yg/kg/d produced a clearincrease in body weight (table I) (Monsan-to, unpublished data). While there was evi-dence of slight anemia and increased or-gan weights in the injected positive controlanimals, no such changes were observedin the gavage-treated rats at any dose. Nodose-related gross or microscopic histolog-ical changes were evident in gavage-dosed animals either. Thus, oral adminis-tration of up to 50 000 !g/kg/d of sometri-bove to rats for 90 d produced no growthresponse or deleterious effects.

SOMETRIBOVE RESIDUESIN MILK AND TISSUES

Following administration of 500 mg of

sometribove every 14 d to a dairy cow, the

equivalent daily exposure is around 36 mg/cow/d. This level of exposure is approxi-mately 4-6 times the daily output of BSTfrom the pituitary, which is reflected in the2-10-fold increase in endogenous BSTblood levels (0-2 ng/ml) observed in dairycows administered 500 mg of sometribove

every 2 weeks (Monsanto, unpublisheddata). The increase in blood levels does

not translate into a significant increase in

sometribove levels in muscle and liver.

Studies carried out in laboratory animalsindicate that somatotropin is not stored in

tissues following exogenous administra-

tion, because it is readily degraded by cy-tosolic proteases and lysozomal enzymes(Postel-Vinay et al, 1982; Johnson and

Maack, 1977). As shown in tables II and

III, sometribove administration to cows

leads, at most, to only a 2-fold increase insomatotropin levels in muscle and liver

when blood levels are at their highest in

the mid-point of the injection cycle. Thus,the exposure potential to sometribove in

muscle and liver is not significantly higherthan endogenous exposure to BST.

Bovine milk has been reported to con-tain trace (ng/ml) levels of BST (Malven,1977), although it is not clear how it enters

the milk, since no BST receptors havebeen identified on the surface of the mam-

mary gland (Akers, 1985). Exogenous ad-ministration of BST (15-100 mg/d) or

sometribove (500 mg/14 d) to dairy cowshas not been reported to increase the en-dogenous levels (0-10 ng/ml) of BST in

milk (Mohammed and Johnson, 1985; Hartet al, 1985; Torkelson et al, 1987; Schams,1988). Taken in context with all the other

proteins in milk, a level of 10 ng/ml of BSTrepresents only 0.00002% of the total pro-tein (3.5 g/100 ml) in milk.

IMPACT OF SOMETRIBOVEON ENDOGENOUS SOMATOMEDINLEVELS IN MILK AND MEAT

Somatomedins are thought to mediate, in

part, some of the biological effects of so-matotropin on tissues. Somatomedin activi-ty in blood and tissues has been attributedto two different peptides known as insulin-like growth factor-I (IGF-I) and insulin-likegrowth factor-11 (IGF-11) (Daughaday et al,1987). IGF-I is a 70 amino acid proteinwhose production is under the control of

somatotropin; IGF-II is a 67 amino acid

protein whose production is less depen-dent upon somatotropin. Both of these in-sulin-like growth factors are structurally re-lated to proinsulin and, like insulin, their

amino acid sequences have been highlyconserved across species (Porte and Hal-ter, 1981 ).

For example, the amino acid sequencesof bovine and human IGF-I are identical,while the amino acid sequence of bovine

IGF-11 differs by 3 out of 67 amino acidsfrom the human form (Zapf and Froesch,1986). IGF-I appears to be more importantfor post-natal growth and development,while IGF-11 may play a role as a fetal

growth factor (Herington et al, 1983).IGFs are not stored in granules, but are

continually synthesized and secreted by avariety of tissues, such as liver, kidney andlung, among others (Zapf and Froesch,1986). IGFs circulate in blood bound to

specific carrier proteins which prolongstheir half-life in serum (Nissley and

Rechler, 1984).

Exogenous administration of BST or

sometribove to dairy cows has been re-

ported to produce a 2-4-fold increase in

endogenous levels (29-372 ng/ml) of IGF-Iin blood (Collier et al, 1988; Schams et al,1989). Levels of IGF-I in tissues of dairycows increased at most only 2-fold as

shown in tables 11 and 111. Elevated levels

of IGF-I in tissues would not be sustained,since tissues contain proteases and lyso-zomal enzymes that degrade IGF-I in a

manner similar to insulin (Roth et al, 1984;Bhaumick and Bala, 1987; D’Ercole and

Underwood, 1987).While the lactating mammary glands of

dairy cows do not appear to have recep-tors for somatotropin, receptors for IGF-Iand -II have been identified in the mam-

mary tissue of pregnant cows (Malven etal, 1987). Since somatotropin does not ap-pear to have a direct effect on mammary

secretory tissue, it is conceivable that the

galactopoietic effect of somatotropin maybe mediated, at least in part, through IGF-1, since this somatomedin has a stimulato-

ry effect on mammary growth (Malven etal, 1987).

High levels of IGF-I (150 ng/ml) andIGF-II (600 ng/ml) are found naturally in

bovine colostrum (Malven et al, 1987;

Ronge and Blum, 1988). These concentra-tions fall off rapidly during the first few

days following parturition, so that betweenpostpartum d 4-6, the levels of IGF-I de-cline to 24 ng/ml and of IGF-11 to 117 ng/ml.

Studies carried out by Monsanto (Tor-kelson et al, 1988) found that endogenousIGF-I levels in milk varied from 0 to 30 ng/ml, depending upon the age and stage oflactation of the cow.

Daily administration of 30 mg of sometri-bove to Jersey cows for 7 d was reportedto increase milk IGF-I from 3.7 ng/ml (con-trol) to 13.6 ng/ml (treated) (Prosser et al,1989). Further studies during which 500

mg of sometribove were administered to

dairy cows every 14 d for 10 cycles oftreatment found only a modest increase inmilk IGF-I levels and no increase in milkIGF-11 levels (tables IV and V) (Monsanto,unpublished data). The modest increase inmilk IGF-I following sometribove treatmentis less than the natural variation in levelsobserved during lactation and less thanthe fluctuation observed between the treat-ed and control groups prior to treatment in-itiation (table IV). Moreover, endogenouslevels of IGF-I in human breast milk are re-ported to be higher (7-31 ng/ml) than thelevels in milk from sometribove-treatedcows (Baxter et al, 1984; Corps et al,1988).

ABSENCE OF ORAL ACTIVITY OF IGF-I

As part of the food safety package devel-oped for sometribove, a study was carriedout in rats to assess the potential oral ac-tivity of IGF-I (Hammond et al, 1989).Young male and female rats were dosedorally with 20, 200 or 2000 !g/kg/d of IGF-Ifor 14 d. Positive controls included in the

study were administered either 50 or 200xg/d IGF-I or 4000 ug/d porcine somato-tropin (PST) via implanted osmotic pumpsfor 14 d. Blood levels of IGF-I increased inall positive control groups but no increaseswere observed in orally treated rats.

PST systemically treated rats exhibitedthe largest increase in body and organweights. Statistically significant increasesin average daily gain (g/d) were evident inmale rats and less so in females adminis-tered IGF-I systemically via implanted os-motic pumps (table VI). No statistically sig-nificant change in average daily gain wasobserved in orally treated rats with the ex-ception of high dose males (table VI). Oneof the two replicates in the high dose malegavage group exhibited an increased aver-

age daily gain which remained significantwhen the groups were combined statisti-

cally (table VII). Since this increase wasnot consistently observed in both repli-cates, and there was not an accompanyingincrease in blood IGF-I, the increased

body weight gain may not be treatment re-lated. For practical purposes, IGF-I, like

other protein hormones (eg, insulin, go-

nadotropins) is not biologically active fol-

lowing ingestion (Astwood, 1970; Gallowayand Root, 1972).

ESTIMATION OF SAFETY FACTORSFOR CONSUMPTION OF SOMETRIBOVEAND IGF-I IN MEAT AND MILK

Based on the residue studies for determi-nation of sometribove and IGF-I in un-

cooked meat and milk and the ’no-effect’

levels determined in rat gavage studies, it

is possible to approximate safety factorsfor ingestion of these two proteins. As apractical matter, however, estimation of

safety factors for sometribove consumptionis not necessary, since it would not be hor-

monally active in humans even if it could

be absorbed.

Meat

Consumption by a 60 kg adult of 500 g ofuncooked meat containing 2.1 ng/g of BST(untreated cow) or 3.1 ng/g sometribove(treated cow) results in a potential expo-sure of 0.018 /ig/kg/d BST (untreated cow)versus 0.025 !g/kg/d sometribove (treat-ed) cow. Compared to the ’no-hormonaleffect level’ of 50 000 !g/kg/d in the 90 drat study, this translates into a safety mar-gin of 2 000 000.

Consumption by a 60 kg adult of thesame amount of uncooked meat (IGF-I isdenatured by heat treatment (Miller et al,

1989)) containing 160 ng/g of IGF-I resultsin a daily exposure of 1.3 !g/kg, which is

approximately 2 times the endogenous ex-posure to IGF-I. This level of exposure is150-1500 times lower than the 200 or

2000 xg/kg/d doses of IGF-I administeredorally to rats.

Milk

No increase in BST levels has been de-tected in milk from sometribove-treatedcows (at ng/ml levels, the antibody in ourradioimmunoassay cannot differentiate be-

tween sometribove and pituitary BST).However, assuming that all of the BST

found in milk was sometribove, consump-tion by a 10 kg infant of 1 I containing 10 0nc!’ml of sometribove would represent anexposure of 1 !g/kg/d, which is 50 000

times lower than the dose administered torats. If the same assumptions are used tocalculate potential oral exposure to IGF-I inmilk, a safety factor of 200-2000 can beapproximated.

CONCLUSION

A substantial body of food safety data is

available to support the authorization of a

zero (0) withdrawal time for human con-sumption of meat and milk from sometri-bove-treated cows. This is based on the

following scientific conclusions: 1) by anal-ogy to BST, sometribove is not hormonallyactive in humans; 2) sometribove adminis-tered to dairy cows produces, at best, onlya marginal increase in sometribove and

IGF-I residual levels in meat and milk; 3)sometribove and IGF-I are not orally activein laboratory animals supporting a largemargin of safety for their consumption.

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