Food Safety Assessment For Use Of bsT


The use of bST in dairy cows to increase milk production was considered to be controversial by some who were concerned about the safety of its use in food production. Part of this opposition, particularly in Europe, was related to their ban on the use of steroid hormone growth promotants in beef production. Steroid hormones, such as estrogen, have been safely used as growth promotants by cattle farmers in the United States for many years. They improve daily gain and feed efficiency, resulting in lower cost of meat production. Implants containing estrogen are inserted into the ear of cattle and removed prior to slaughter so that the levels of estrogen are well below limits set by the FDA, which regulates their use. Estrogen is naturally present in human and animal tissues, and estrogen activity (e.g., isoflavanoids) is present in foods derived from certain plants.35 The use of estrogen as a growth promotant in cattle is regulated and considered to be safe by the FDA, the World Health Organization (WHO), the Food and Agricultural Organization of the United Nations (FAO), the European Commission Scientific Working Group on Anabolic Agents in Animal Production, and the Codex Alimentarius Commission.35

In Europe, the use and importation of meat that had been produced from animals treated with steroid hormone growth promotants was banned in 1989. Diethylstil-bestrol (DES), a growth promotant banned in the United States in 1979 because of its link to cancer and birth defects, was used illegally in Europe for veal production. Very high levels of DES were used and some of the contaminated veal was processed into baby food consumed in Europe. This illegal use raised considerable safety concerns, leading to the total ban of all steroid growth promotants used in beef cattle production. The ban in Europe remains in effect to this day despite aforementioned scientific reviews carried out by regulatory scientists both in Europe and the United States that continue to confirm the safe use of approved growth promotants such as estrogen in beef cattle production.36 The total ban in Europe on the use of growth promotants in beef cattle occurred around the same time the safety of bST was being reviewed by European regulatory scientists [Joint FAO/WHO Expert Committee on Food Additives (JECFA)]. The public did not differentiate between protein and steroid hormone use in food production, which made the safety of bST an issue in Europe.

As shown in Figure 7.2, there are fundamental differences in the structures between protein and steroid hormones that have profound effects on the potential bioavailability of hormone residues present in meat or milk. bST is a protein hormone and, if ingested, is degraded by digestive enzymes like other dietary proteins and is not hormonally active by ingestion. Moreover, as will be discussed shortly, bST is not hormonally active in humans even following injection due to species-specific activity of somatotropins. In contrast, estrogen is a steroid hormone; it is identical in humans and farm animals. It is orally active if ingested, as a consequence of its chemical structure, which is completely different from protein hormones such as bST. Steroid hormones are much smaller than protein hormones such as bST and insulin, are not appreciably degraded in the gastrointestinal (GI) tract, and are lipid-soluble; all of these properties enhance their absorption from the GI tract. Based in part on the absence of oral activity for bST, there is no withdrawal time for its use in dairy cattle, whereas there is a withdrawal time for the use of steroid hormones in food-producing animals because they are orally active. A withdrawal time allows steroid hormone levels in tissues to return to endogenous levels found naturally in untreated cattle.

Despite these fundamental differences, bST was still caught up in the antihormone backlash in Europe, and although its food safety was ultimately confirmed following European regulatory38 and scientific review,28,39 it has not been approved for commercial use in Europe due to concerns about animal health related to bST supplementation. Concerns about long-term consequences on animal health have not been borne out since bST was approved for use in the United States in 1993. It has been estimated that more than 10 million dairy cows have been supplemented with bST during the last 12 years, and some of these cows have received bST during multiple lactations. No unexpected adverse health consequences have been observed and, as milk production increases, dairy cows continue to respond to bST.

bST also became a lightning rod for antitechnology activists who vigorously opposed its use in food production. This opposition has carried over to the subsequent use of biotechnology to develop improved agricultural crop commodities. With respect to bST, a government report acknowledged that some of the safety concerns raised regarding bST were not surprising, given the publics unfamiliarity

growth hormone (1)

o estradiol (1)

FIGURE 7.2 Structural differences between protein and steroid hormones. (a) Sometribove, MW21, 872 C978H1537 N265 O286 S9: 191 amino acids, (b) insulin, MW 5800, C256H381N65O79S6: 51 amino acids; (c) estradiol, MW 272, C18H24O2: no amino acids. (1) space-filling models shown at same scale. (Adapted from David S. Goodsell, Our Molecular Nature. New York: Springer-Verlag, 1996.)

with the FDA review process.34 Following the approval of bST by the FDA in 1993, the U.S. Government Executive Office of the President published a report34 that provided a detailed summary of all of the safety evaluations that were carried out prior to the approval of bST:

"In November 1993, bST was found safe by the Food and Drug Administration (FDA), the U.S. government's testing agency. FDA's finding was based on hundreds of formal scientific studies and tests conducted over many years around the world. FDA verified the reported data of over 120 studies and also held hearings on safety-related issues. bST has been declared safe by other respected scientific and professional organizations, including the American Dietetic Association, the National

Institutes of Health, the Congressional Office of Technology Assessment, and the HHS Office of the Inspector General. Moreover, bST has been examined, found safe, and approved for use by numerous foreign government regulatory agencies. In fact, no professionally recognized scientific group has concluded, on the basis of current knowledge, that there is doubt about the safety of bST in milk production."

A chronology of some of the key events, technical reviews, and studies that were completed on bST are summarized in Appendix 1 at the end of this chapter.34 The chronology also provides a glimpse into some of the political activities that were ongoing during the regulatory review of bST. The scientific studies that support the food safety of bST are provided below.


During the 1950s, clinicians determined that some types of human dwarfism were caused by inadequate production of human somatotropin by the pituitary. Since bovine somatotropin was readily available from extracts of cow pituitaries, it was tested as a potential source of supplemental somatotropin for therapeutic use in humans. As discussed previously, it was well known that extracts of bovine pituitaries could stimulate growth in normal and hypophysectomized rats and dogs. Since the rat responds to the growth-promoting effects of all mammalian somato-tropins and its epiphyses never close, it was possible to grow very big rats following chronic injection of pituitary extracts containing bST.40 Using highly purified pituitary preparations of bST prepared by the Armour Company, endocrinologists carried out several clinical studies but were unable to show any evidence of metabolic changes or growth-promoting activity in children with growth disorders or induced anabolic changes in normal human volunteers.41-45 Doses of bST in the aforementioned clinical studies ranged from 5 to 95 mg/person/day administered for days to weeks. There is one report in the literature of a woman receiving a cumulative dose of 674 g of bST administered over 75 days in an unsuccessful attempt to control hyperglycemia.46 When bST failed to work, some investigators tried porcine, ovine, or even whale somatotropin preparations in humans, but they were also clinically ineffective following injection.47 48

Given the lack of effectiveness of nonprimate somatotropins in man, it was proposed that an "active core" existed in the bST protein that required proteolysis to liberate its growth-promoting activity.49 There were reports that large fragments of bST produced by proteolysis had some activity in laboratory animals.50 A few clinical studies were undertaken with equivocal results.5152 There were a few reports in the literature that limited enzymatic digestion of bST produced large fragments (i.e., residues 95-134) that were biologically active when large doses (5-100 mg/day) were injected into humans.50 51 Other scientists, however, were unable to reproduce these findings so the validity of these reports was questioned.53 Further research has shown that somatotropin fragments (i.e., amino acid residues 1-134, 141-191, 95-134) possess only a small fraction (1% or less) of the biological activity of the parent molecule.54 The little biological activity that has been observed with enzymatically derived somatotropin fragments in laboratory animals has been attributed to contamination of the fragment preparation with undigested somatotropin.17

More recent work involving alanine substitution for bulky amino acids in human somatotropin indicates that the somatotropin receptor interacts with restricted regions of both the amino and carboxyl terminal ends of the somatotropin molecule.18 This is consistent with earlier work,49,53 which demonstrated that significant biologic activity with the 1-134 somatotropin fragment was possible only when it was recombined with large portions of the carboxyl terminal end of the molecule (Kostyo, personal communication). In another study, a homologous somatotropin radioreceptor assay55 was modified56 and used to compare the binding affinity of synthetic bST fragments to full-sequence bST, and none of the bST fragment peptides exhibited significant binding affinity for the bST receptor.

The species-limited activity of somatotropin in primates was subsequently confirmed as a consequence of elegantly designed studies in monkeys in which injection of primate somatotropin produced measurable anabolic responses in monkeys whereas bST did not.57 Both primate and bovine somatotropin preparations were, however, fully active in rats.

Other scientists had also been at work isolating and characterizing somatotropin from human pituitaries.58,59 Human somatotropin was found to be highly potent in stimulating the growth of patients with pituitary dwarfism.60 The sequence of human somatotropin has diverged considerably from bovine and other nonprimate somato-tropins.61 Primate and nonprimate somatotropins differ by approximately 59-63 amino acids (~33%), whereas nonprimate somatotropins differ by only 0-4 amino acids from one another.62 These changes in the primate somatotropin molecule, as great as they are, are not the only critical factor in the species-limited action of somatotropins in man. Primate somatotropin retains its potency in rats and most mammalian species.52 Once primate somatotropin was isolated and purified, it was soon shown that primate somatotropin bound to the somatotropin receptor on human liver membranes, but bST did not.63 Years later, when biotechnological techniques became available, cloning of the human and rabbit somatotropin receptors64 led to the subsequent elucidation of the amino acid sequences of bovine, ovine, rat, and mouse somatotropin receptors.65

The human somatotropin receptor differs from other nonprimate somatotropin receptors by having an arginine residue at position 43 of the receptor, whereas nonprimate receptors have the neutral amino acid leucine at this location. Arginine bears a strong, positive charge at physiological pH. Primate somatotropin has an aspartate residue (position 171), which bears a negative charge and forms a slat bridge with arginine (position 43) of the somatotropin receptor.62 Somatotropin molecules from nonprimate species all have a histidine residue instead of aspartate at position 171, and histidine has a slight positive charge at physiological pH. The interaction of histidine with arginine (position 43) in the human receptor would lead to an unfavorable charge repulsion/steric hindrance that would inhibit the binding of nonprimate somatotropins to the human somatotropin receptor.62 The substitution of arginine for leucine on the human somatotropin receptor, and aspartate for histidine in the human somatotropin molecule, are major factors contributing to the species-limited activity of somatotropin in primates.65

7.3.2 Digestibility of bST and Lack of Oral Bioavailability

Degradation of orally consumed proteins begins in the stomach. The acidic pH of the stomach can cause loss of tertiary structure and denaturation of most ingested proteins. Denaturation exposes inner hydrophobic portions of the protein molecule to attack by digestive enzymes. Pepsin, an endopeptidase that is active at the low-pH environment of the stomach, contributes to protein degradation by breaking a variety of peptide bonds between different amino acids in the protein. Degradation of ingested proteins continues in the small intestine where proteins and their peptide degradation fragments are subjected to further proteolysis by digestive enzymes that attack other pep-tide bonds that pepsin does not break. The ultimate degradation products of ingested proteins are very small peptides and individual amino acids that can be absorbed from the gastrointestinal tract and used to make new proteins by body tissues.

Protein hormones such as bST are degraded in like manner as other ingested proteins present in the diet. Due to their susceptibility to digestion if consumed, therapeutic protein hormones such as insulin, somatotropin, and gonadotropins cannot be given by mouth but must be administered parenterally (by injection) to humans.66,67 When bST has been incubated in vitro with enzymes such as trypsin, the hydrolysis of peptide bonds results in the loss of biological activity as measured in vivo in laboratory animals.59,68 There are 24 tryptic sites on bST that can yield 25 peptide fragments.69

Unlike steroid hormones, which are lipophilic and can traverse cell membranes, protein hormones must first bind to receptors on the surface of the target cell before they can be translocated into the cell to exert their pharmacologic effect. The affinity of the protein hormone for its receptor is determined by the shape or tertiary structure of the protein.70 Loss of tertiary structure due to degradation or denaturation can reduce the binding affinity of a protein hormone for its receptor, limiting its phar-macologic effects. Biotechnology-derived or chemically synthesized somatotropin fragments that are not contaminated with intact somatotropin are essentially devoid of biologic activity when tested in vitro.11,11

When an investigational veterinary drug is being evaluated for safety and efficacy as required by FDA/CVM regulations, food such as meat and milk derived from the treated animals cannot enter the human food chain until CVM scientists have affirmed the safety of the food products for human consumption. Sometimes a withdrawal time will be specified that requires investigators to wait a required number of days or weeks before a farm animal can be used for human food, to minimize the potential for residues to be left in meat or milk. In some cases, the animals cannot be used for food and must never enter the food chain. There are similar requirements for investigational animal drugs being tested in Europe.

Based on the previous research that has been discussed, it was recognized that bST was not hormonally active in man. Although it was presumed that Monsanto's bST (sometribove) would be digested and destroyed when eaten like other dietary proteins, CVM required data to confirm its absence of oral activity. To establish the absence of oral activity would require an animal model that would be sensitive to the effects of sometribove should it be absorbed when eaten. The rat responds to somatotropins of general mammalian origin60 and has been used in bioassays to measure the potency of somatotropin preparations.12,13 Since the epiphyses of the rat never close, it is possible to produce very large rats if large doses of somatotropin are administered throughout most of their lives.40

Therefore, CVM required that rats be fed exaggerated doses of sometribove to confirm its absence of oral activity, and a similar request was made by European regulatory scientists. The results of the 4- and 13-week repeat-dose oral gavage studies with bST (sometribove) are summarized below.

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