INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION TOXICOLOGICAL EVALUATION OF CERTAIN VETERINARY DRUG RESIDUES IN FOOD WHO FOOD ADDITIVES SERIES 41 Prepared by: The 50th meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) World Health Organization, Geneva 1998 RECOMBINANT BOVINE SOMATOTROPINS (addendum) First draft prepared by Professor F.R. Ungemach Institute of Pharmacology, Pharmacy and Toxicology Veterinary Faculty, University of Leipzig, Leipzig, Germany and Dr N.E. Weber Food and Drug Administration, Center of Veterinary Medicine Rockville, MD, USA 1. Explanation 2. Biological data 2.1 Use of antibiotics 2.2 Concentrations of bovine somatotropin and insulin-like growth factor in tissues and milk 2.2.1 Tissues 2.2.2 Residues of insulin-like growth factor in milk 2.2.3 Assays 2.2.4 Bioavailability and bioactivity of insulin-like growth factor residues in milk 2.3 Expression of lentiviruses and prion proteins 2.3.1 Somatotropins and the immune system 2.3.2 Effect of bovine somatotropin on expression of retroviruses 2.3.3 Effect of recombinant bovine somatotropin on prion proteins 2.4 Cow's milk and insulin-dependent type I diabetes mellitus in childhood 3. Comments 4. Evaluation 5. References 1. EXPLANATION The four analogues of bovine somatotropins, somagrebove, sometribove, somavubove, and somidobove, that are produced by recombinant DNA techniques were evaluated by the Committee at its fortieth meeting (Annex 1, reference 104). At that time, the Committee allocated an ADI 'not specified' for recombinant bovine somatotropins (rbSTs) because of the lack of activity of orally administered rbSTs and insulin-like growth factor I (IGF-I) and the low concentrations and non-toxic nature of the residues of these compounds, even after very high doses, resulting in an extremely large margin of safety for humans consuming dairy products from rbST-treated cows. The Committee concluded that use of these drugs according to good practice in veterinary medicine did not represent a dietary hazard to human health and that there was no need to specify a numerical ADI. Accordingly, MRLs 'not specified' were recommended for milk and edible tissues of cattle. The Codex Alimentarius Commission, when considering adoption of these recommended MRLs at its twenty-second session in 1997, postponed a decision pending re-evaluation of rbSTs by the Expert Committee to consider scientific information that had become available since its previous evaluation. Information was submitted by organizations and individuals relating to the following concerns: * increased use of antibiotics, with a higher rate of violative drug residues in milk due to a possible increase in the incidence of mastitis in rbST-treated cows, * the possibility that increased concentrations of IGF-I in the milk of rbST-treated cows increase cell division and the growth of tumours in humans, * the potential effect of rbST on the expression of certain viruses in cattle, particularly the retroviruses, * the possibility that the incubation period of bovine spongiform encephalopathy (BSE) is shortened by an IGF-I-induced increase in the production of pathogenic prion proteins, and * the possibility that early exposure of human newborns to milk from rbST-treated cows increases the risk for developing insulin-dependent diabetes mellitus. 2. BIOLOGICAL DATA 2.1 Use of antibiotics The induction by rbST of an increased incidence of mastitis and somatic-cell count in the milk of treated cows was not reviewed by the Committee at its fortieth meeting, as these effects on animal health were considered outside its terms of reference. At the present meeting, the Committe considered results reported in the literature and the results of a programme to monitor the effects of sometribove (Posilac(R)) on mastitis and animal health in the United States. The Committee confirmed that these effects of rbST on animal health and the resulting treatment per animal with any medication are issues outside the terms of reference of the Committee. The results of the monitoring programme with regard to the percentage of milk discarded because of drug residues above the approved limit, as a consequence of antibiotic use, were, however, considered by the Committee. The programme was initiated by the US Food and Drug Administration at the time sometribove was approved, in November 1993 and began to be commercialized, in February 1994. The objectives of this programme were to determine whether the incidence of masitits and antibiotic use were manageable under actual conditions of use and whether the labelling of rbSTs was adequate (US Food and Drug Administration, 1996). The programme was designed to address the following areas (Collier, 1996): * the incidence of mastitis and responses related to herd health (not within the terms of reference of the Committee), * treatment of 28 herds with rbST-treated cows with any medication (not within the terms of reference of the Committee), and * the percent of milk discarded because of drug residues above the allowed limit in key dairy states representing at least 50 % of US milk production. The programme was closely monitored by the US Food and Drug Administration and performed according to the sponsor's standard operating procedures for quality assurance. The US Food and Drug Administration (1996) confirmed that the integrity of the findings was acceptable and that the records and analyses showed excellent fidelity. The programme for tracking milk residues in key dairy states before and after the approval of sometribove was designed to reveal whether any increase in drug residues above the allowed limit in milk was associated with an increased frequency of use of antibiotics for mastitis and other health problems in rbST-treated herds of dairy cows (Veenhuizen et al., 1996). The results of the milk monitoring programme for the two years before commercial use of sometribove (1992-93) were compared with those for the two years after its launch on the market (1994-95). Residues in milk were traced according to the National Drug Residue Milk Monitoring Program in which the contents of all milk tanker trucks are sampled routinely. The data set represented more than 50% of the total US milk supply. The data were analysed quarterly by comparing the milk discarded before and after the launch of sometribove. As seen in Figure 1, no change was observed in 1994 after sometribove was approved. The average percent of milk discarded was 0.06% in 1992 and 1993 and 0.07% in 1994 after the launch; in 1995, the number of violations increased slightly but significantly to 0.09%. This increase coincided, however, with a change in the screening procedures in most states to include more sensitive tests. Veenhuizen et al. (1996) submitted data to the US Food and Drug Administration on 17 May 1996 showing that in New York State there had been no significant change in the rate of discard of milk in the two years after approval of Posilac in comparison with the two years before approval, but that in that State the same testing protocol had been used throughout the four-year period. The values were 0.062% before approval and 0.064% afterwards. The company that launched sometribove reported that rbST was purchased by nearly 37% of the farms in the State, and these farms represented about 50% of the State's cows. These reports indicate that: * no product-related increase in residues above the approved limit had occurred after commercialization of sometribove; * the rate of positive results was even slightly lower than those for antibiotics in grade A milk in the United States; and * use of sometribove will have no effect on the safety of milk and dairy products due to violative drug residues resulting from a slightly higher rate of medication of rbST-treated animals, as measured in the milk monitoring programme.It was concluded that use of rbST would not result in a higher risk to human health due to the use of antibotics to treat mastitis and that the increased potential for drug residues in milk could be managed by practices currently in use by the dairy industry and by following the directions for use (US Food & Drug Administration, 1996). 2.2 Concentrations of bovine somatotropin and insulin-like growth factor in tissues and milk 2.2.1 Tissues The concentrations of bST and IGF-I were measured in tissues of cattle that had been treated with a 14-day sustained-release product containing the natural variant of rbST, somavubove (Choi et al., 1997), in two experiments. In the first experiment, three groups of 12 beef cattle (except at the low dose, to which only six animals were exposed) with an average weight of 450 kg were treated by subcutaneous injection for 20 weeks. The controls received no treatment or vehicle, one group received 250 mg rbST each week, and another received 500 mg rbST at two-week intervals. The controls and animals at the high dose were further divided into groups receiving low- and high-energy feed. The stated total doses were 5 and 10 g rbST, respectively; however, the dose for both groups calculated from the stated regimens would be 5 g. Two weeks after the final treatment, the animals were slaughtered and muscle samples were obtained and stored at -20°C for analysis. In the second experiment, four groups of beef cattle were used: a control group which received no drug or vehicle, a group receiving a sustained-release low dose (0.42 mg/kg bw; 0.03 mg/kg bw per day), a group receiving an intermediate dose (0.84 mg/kg bw; 0.06 kg bw per day), and one receiving a high dose (1.26 mg/kg bw; 0.09 mg/kg bw per day). The treated groups were given the drug by subcutaneous injection every two weeks for 24 weeks, to give total doses of 2.3, 4.5, and 6.8 g, respectively. Two weeks after the final treatment, the animals were slaughtered, and samples of muscle, kidney, liver, and fat were taken and stored at -20°C. The frozen samples were assayed for bST and IGF-I residues by radioimmunoassay (RIA) in which 5 g of tissue were extracted in acidœethanol for muscle and acetic acid for kidney, liver, and fat. The RIA procedures involved standard double-antibody techniques and iodinated tracers. The detection limit for the assays (the amount that could be distinguished from zero concentration with 95% confidence) was 0.17 ng/g bST and 0.61 ng/g IGF-I. The coefficients of variation for the two assays were 6% or less, and the recoveries from liver, kidney, and fat were 64% for bST and 84% for IGF-I; similar recoveries were obtained in muscle samples. A summary of the results is given in Table 1. The authors concluded that two weeks after administration of two doses of rbST for extended times, the tissue concentrations of bST and IGF-I were not significantly different from those in untreated animals. 2.2.2 Residues of insulin-like growth factor in milk Extensive information on the residues of bST and IGF-I in the milk of rbST-treated cows was evaluated by the Committee at its fortieth meeting. IGF-I is a normal but highly variable constituent of bovine milk, the concentration depending on the state of lactation, nutritional status, and age. Over an entire lactation, the IGF-I concentrations range from 1 to 30 ng/ml, with the highest concentrations in colostrum and a constant decline thereafter. Multiparous animals have higher concentrations of IGF-I in milk than primoparous cows (Burton et al., 1994). Bulk milk from cows not given rbST had IGF-I concentrations of 1œ9 ng/ml (Juskevich & Guyer, 1990), and the fortieth Meeting cited an average value of 3.7 ng/ml for untreated cows. In milk from rbST-treated cows, the concentrations of IGF-I ranged from 1 to 13 ng/ml in most studies and were about 25-70% greater than those in untreated animals (Burton et al., 1994). The fortieth meeting reported an average IGF-I concentration of 5.9 ng/ml, and the increase was significant, even though most of the concentrations were < 10 ng/ml. Table 1. Concentrations of bovine somatotropin (bST) and insulin-like growth factor (IGF-I) in tissues after treatment with recombinant bovine somatotropin First experiment Tissue No. of Concentration (ng/g; mean ± SD) samples Controls Low dose High dose bST IGF-I bST IGF-I bST IGF-I Muscle 12 1.9 ± 1.8 88 ± 21 1.5 ± 1.6 130 ± 25 3.3 ± 2.2 110 ± 32 Second experiment Tissue No. of Concentration (ng/g; mean ± SD) samples Controls Low dose Intermediate dose High dose bST IGF-I bST IGF-I bST IGF-I bST IGF-I Muscle 5 3.4 ± 1.5 45 ± 6.5 4.9 ± 1.5 35 ± 15 3.8 ± 2.0 40 ± 5.1 1.5 ± 0.86 55 ± 19 Fat 4 5.1 ± 1.7 210 ± 85 9.3 ± 5.2 200 ± 65 4.8 ± 1.9 200 ± 53 11 ± 12 340 ± 230 Liver 4 5.2 ± 0.59 350 ± 23 3.6 ± 1.7 390 ± 130 5.4 ± 1.2 380 ± 170 4.6 ± 2.0 290 ± 88 Kidney 4 3.6 ± 1.1 910 ± 130 4.5 ± 1.6 1000 ± 140 4.5 ± 1.8 820 ± 120 3.9 ± 0.94 980 ± 220 Since the original work was reviewed, few additional data have appeared in the literature or in reports made available by the sponsors. The manufacturer of Posilac(R) - previously identified as sometribove, which is a form of rbST approved in a number of countries - submitted additional information. In a study designed to determine the concentrations of IGF-I in retail milk samples, the concentrations in milk specifically labelled as coming from cows that had not been treated with bST were compared with those in unlabelled milk. While the sponsor assumed that all of the unlabelled milk was from cows that had been treated with bST, the extent to which this was true was not ascertained. The study was conducted under US Food and Drug Administration GLP regulations (21CFR 58). The labelled and unlabelled status was determined of 127 of 129 retail milk samples collected as 2% fat cow's milk in 51 retail outlets in 34 cities in Wisconsin, Minnesota, and Iowa, USA, from four goats and 125 cows, and the samples were analysed by RIA for IGF-I. For 78 samples, the farmer had certified that rbST had not been used. As rbST is not approved for use in goats in the United States, it is assumed that the goat-milk samples were from untreated animals. The results are shown in Table 2 (Eppard et al., 1994). The values for IGF-I are not unlike those reported in the monograph prepared at the fortieth meeting of the Committee. Although the contribution of milk from rbST-treated cows to the unlabelled milk is unknown, the results indicate that the IGF-I concentrations in retail milk did not increase in the first year after the launch of rbST. Table 2. Concentrations of insulin-like growth factor in milk certified by farmers as coming from cows not treated with recombinant bovine somatotropin (labelled) and from unlabelled milk Insulin-like growth factor (ng/ml) Labelled Unlabelled (n = 78) (n = 45) Raw mean 4.3 ± 0.09 4.5 ± 0.12 Logea 1.47 ± 0.044 1.55 ± 0.031 p = 0.01769 Antilog (95% 4.4 (4.0-4.7) 4.7 (4.4-5.0) confidence interval) a Least square means adjusted for state where purchased and dairy 2.2.3 Assays Because of variations in the IGF-I values reported in different studies, questions have been raised in submissions to the Committee about their accuracy. Lower values were found in some studies because the assay used involved acid-ethanol extraction; an assay involving acidic gel filtration has been reported to allow better recovery. In a comparison of the two assays for determining IGF-I in pre- and post-partum mammary secretions, the acid-ethanol assay was found to result in a 24 ± 6.6% underestimate in comparison with acid gel filtration (Vega et al., 1991). Use of the acidœethanol assay to recover 125I-IGF-I in bovine serum and colostrum, which also contain binding proteins, gave values of 86 ± 6% and 88 ± 7% recovery by the antibody, respectively (Hadsell, 1991). The difference is due to the fact that the binding protein with affinity for IGF-I competes with the antibody used in the RIA procedure; however, gel filtration removes the hormone that is extensively dissociated by the acid common to both assays. The studies suggest that the acidœethanol assay probably results in 15-25% underestimates of the concentrations in milk and plasma. The relative difference in milk will probably not affect a decision, as the concentrations are low, although higher control values may affect the inferences. Although incomplete removal of IGF-binding proteins or differences in the source of standards might affect the reported results, the Committee considered these factors immaterial. 2.2.4 Bioavailability and bioactivity of insulin-like growth factor residues in milk At its fortieth meeting, the Committee concluded that many of the physiological effects of rbSTs are mediated by bovine IGF-I, which is structurally identical to human IGF-I. It noted that there is substantial endogenous synthesis of IGF-I, mainly in the liver but also in human milk, saliva, and pancreatic secretions. It further concluded that IGF-I has no bioactivity when administered orally to normal and hypophysectomized rats at doses up to 2 mg/kg bw per day, as dietary IGF-I is degraded by digestive enzymes and is not active in the upper gastrointestinal tract. Concern has been expressed that widespread use of rbSTs in dairy production would lead to a sustained increase in the concentrations of IGF-I in bulk cow's milk and that, if IGF-I survives digestion, the greater exposure of consumers would cause adverse health effects (Hansen et al., 1997). For a quantitative assessment of risk, the slight increases in the concentration of IGF-I in milk from rbST-treated cows should be compared with the physiological variations in the concentrations of this growth factor during lactation (see section 2.2.2) and with the concentrations in human breast milk, in the secretions of the gastrointestinal tract, and in serum. The concentrations of IGF-I are 8-28 ng/ml in colostrum and 5-10 ng/ml thereafter (Zumkeller, 1992; Burton et al., 1994), indicating that breast-fed newborns are usually exposed to IGF-I at concentrations equal to or higher than those in milk from rbST-treated cows. Assuming a daily intake of 1.5 L of milk from rbST-treated cows with an average IGF-I concentration of 6 ng/ml, the amount of IGF-I ingested would be 9000 ng/day. The additional daily ingestion of IGF-I over that of people drinking milk from untreated animals, with an average IGF-I concentration of 4 ng/ml or 6000 ng/1.5 L, would be 3000 ng. The slightly increased IGF-I concentrations would contribute to the endogenous concentrations of IGF-I in the gastrointestinal tract of consumers; however, the main site of IGF-I production in animals and humans is the liver. It is also produced in the human gastrointestinal mucosa and is found in saliva, bile, and pancreatic juice (Olanrewaju et al., 1992). The average concentrations of IGF-I in the five human gastrointestinal secretions were 0.9 nmol/L in saliva, 3.5 nmol/L in gastric juice, 24.6 nmol/L in jejunal chyme, 3.6 nmol/L in pancreatic juice, and 0.9 nmol/L in bile (Chaurasia et al., 1994). On the basis of a molecular mass of 7.5 kDa for IGF-I (Zumkeller, 1992) and the volume of each of the fluids produced (Vander et al., 1990), the total volume of IGF-I emptying into the gastrointestinal tract is 383 000 ng/day. Table 3 shows that the amount of endogenous IGF-I emptied into the gastrointestinal tract daily is more than (383 000/9000) 42 times greater than the amount present in 1.5 L of milk from rbST-treated cows. The 9000 ng value is 2.3% of the estimated daily gastrointestinal secretion of IGF-I in adults. The additional daily ingestion of 3000 ng IGF-I over that in milk from untreated animals thus represents 0.78% of the gastrointestinal secretion. Table 3. Concentration of insulin-like growth factor (IGF-I) in gastrointestinal tract secretions Secretion Volume Concentration Total IGF-I (ml/day)a (average; ng/ml) secreted (ng) Jejunal chyme 1500 184.5 276 750 Pancreatic juice 1500 27.0 40 500 Gastric juice 2000 26.2 52 400 Bile 500 6.8 3 400 Saliva 1500 6.8 10 200 Adapted from Baumann (1995) a From Vander et al. (1990) In contrast to the conclusion of the Committee at its fortieth meeting, that IGF-I is completely and rapidly degraded in the gastrointestinal tract, milk-borne IGF-I may escape digestion by proteases and therefore be bioactive in the intestine (Hansen et al., 1997) or even be absorbed as intact peptide into the systemic circulation (Epstein, 1996). In a study designed to investigate the potential therapeutic use of IGF-I and to determine whether oral formulations were feasible, the degradation of 125I-IGF-I was determined in various segments of the gastrointestinal tract of rats in vivo and in vitro (Xian et al., 1995). The extent of degradation was measured by one of three methods: receptor binding, immunoprecipitation, and trichloracetic acid precipitation. Two segments of the duodenum, ileum, or whole stomach and part of the colon were ligated in an anaesthetized male Sprague-Dawley rat that had been fasted for 24 h. A bolus of labelled IGF-I (8.6 ng/ml in 0.2% bovine serum albumin in saline) was injected into each segment and incubated for various times up to 1 h. The reactions were stopped, and the flushed luminal contents were examined for intact IGF-I. In a parallel set of experiments, the flushed luminal contents from each of the four gut segments were used as a source of degredation enzymes in vitro. The results are shown in Table 4. The most rapid degradation occurred in the duodenum and ileum and in their contents in vitro, followed by the stomach and then by the colon. In all cases the values seen in vitro were equal to or greater than those seen in vivo. The authors also examined the effectiveness of slowing the degradation rate of IGF-I in the gut by protecting the molecule in several ways. Casein at a concentration of 10 mg/ml conferred > 90% protection on stomach contents in both the trichloracetic acid and receptor assays; in duodenal flushings, casein conferred 80% protection against IGF-I degradation in the trichloracetic acid assay but only 36% protection in the receptor assay at a maximal casein concentration of 40 mg/ml. The half-life of IGF-I in the upper gastrointestinal tract in the receptor assay increased from 2-3 min in the absence of casein to 35 min in its presence. While these results appear to demonstrate significant protection by casein at a concentration similar to those in milk, the authors acknowledge that the observed effects could be explained by simple competition by the additional proteins for degradation by proteases in the segments. The results demonstrate that biological receptor-stimulating activity, which is the best indicator of biological activity, is dramatically reduced even with good protection from protease activity. The authors noted with regard to milk residues of IGF-I that 'the protective effect of casein makes irrelevant the argument that human saliva contains IGF-I at concentrations greater than the quantities that would be consumed in milk. As the IGF-I produced by salivary glands is free IGF-I, without protective effect of casein, it is unlikely to survive digestion.' (Hansen et al., 1997). That argumentation neglects the following facts: Table 4. Half-lives of intact 125I-insulin-like growth factor (IGF-I) in ligated Sprague-Dawley rat gut segments in vivo and in the flushed contents in vitro Test for Half-life (min) intact IGF-I Duodenum/ileum Stomach Colon In vivo In vitroa In vivo In vitro In vivo In vitro Trichloracetic 2 2 8 50 38 > 60 Antibody binding 2 5 33 Receptor binding 2 2 2.5 3 16 ND ND, not determined a Antibody and membrane receptor values reported as receptor binding * Saliva is not the only source of IGF-I in the gastrointestinal tract: most is secreted into the gut, and the high concentration in intestinal chyme indicates that IGF-I is secreted in substantial amounts by the mucosa throughout the gastrointestinal tract (Olanrewaju et al., 1992; Chaurasia et al., 1994). * Casein has a flexible structure and is readily degraded in the stomach and small bowel (Xian et al., 1995). Thus, the protective effect is present only in the upper gastrointestinal tract. * The half-life of IGF-I in the intestine in the presence of casein is only 35 min (Xian et al., 1995). Therefore, less than 5% of an initial dose of IGF-I will survive for more than 2 h during passage through the upper gastrointestinal tract. * In the presence of casein ingested with milk, endogenous IGF-I in the gastrointestinal tract will also be protected. Therefore, even if casein has a limited protective effect, the amount of bioactive IGF-I ingested with milk from rbST-treated cows would still be negligible. Because of the protective effect of casein, some IGF-I might escape digestive degradation and be absorbed intact. The absorption of large (1 mg/kg) oral doses of 125I-labelled recombinant human IGF-I was studied in fasted adult rats, with trichloracetic acid precipitation of plasma proteins. The baseline bioavailability of the administered IGF-I was 9.3% of the dose, but this was was increased by co-administration of 4 mg/kg aprotinin (47%) and 10 mg/kg casein (67%). RIA of the plasma confirmed the bioavailability of IGF-I in this model, and the administered radiolabel was found in the form of high-molecular-mass complexes (Kimura et al., 1997). It should be noted, however, that the receptor assay, which is the most accurate, was not used. The relatively good bioavailability of intact IGF-I in this adult rat model is in contrast to the lack of bioactivity of orally administered IGF-I in adult animals (Annex 1, reference 104) and to the results of studies with neonatal animals which have an incomplete mucosal barrier and reduced intestinal proteolytic activity (Burrin, 1997). Studies in neonatal rats and piglets indicated that although 30% of an orally administered dose of 125I-IGF-I can be recovered in the intestinal mucosa there is limited absorption into the peripheral circulation (Phillips et al., 1995; Donovan et al., 1997). When suckling transgenic rats ingested 1000-fold higher concentrations of des(1,3) human IGF-I, no des(1,3)-IGF-I was detected in the plasma of their pups (Burrin, 1997). Furthermore, in newborn calves and piglets given large doses of IGF-I in milk replacers, no substantial increase in the plasma concentration of this growth factor was found (Donovan et al., 1997; Hammon & Blum, 1997; Houle et al., 1997). In one study with newborn calves fed milk replacer, a small amount of orally administered 125I-IGF-I was detected in plasma (Baumrucker et al., 1992); however, the increase was observed only three days after administration and in only three of six animals. Even in newborns, therefore, IGF-I is absorbed to only a small extent, and absorption is unlikely in adults. Furthermore, the amount absorbed should be compared with the normal concentrations of IGF-I in human serum, which show considerable variation with age. The values are lowest in infants under two years of age, then increase steadily to reach a maximum in late puberty, and afterwards decrease to the adult values (Table 5). Assuming a blood volume that is 5% of the body weight (Ganong, 1971), the serum load of IGF-I can be calculated to be 50 000 ng in a 15-kg child, 714 000 ng in a 60-kg adult, and 1 220 000 ng in a 50-kg teenager. The total IGF-I production in adults has been estimated at 107 ng per day (Guler et al., 1989). These amounts should be compared with the 9000 ng IGF-I in 1.5 L of milk, which constitutes only 0.09% of the daily IGF-I production. Since only one-third of the concentrations in milk can be attributed to IGF-I due to rbST treatment and only a small amount if any will be absorbed, the milk-borne IGF-I that reaches the systemic circulation is negligible and this small amount is immediately sequestered by unsaturated binding proteins. Table 5. Concentrations (ng/ml) of insulin-like growth factor (IGF-I) in human plasma Age Males Females Mean Range Mean Range 0-2 years 42 14-98 56 14-238 3-5 years 56 59-210 84 21-322 6-10 years 98 28-308 182 56-364 Before puberty > 10 years 126 84-182 182 70-280 Early puberty 210 140-240 224 84-392 Late puberty 364 224-462 434 224-686 Adult > 23 yeras 112 42-266 140 56-308 From Schaff-Blass et al. (1984) Concern has been expressed about the possible adverse effects on the health of consumers exposed to increased concentrations of IGF-I in milk from rbST-treated cows (Epstein, 1996; Hansen et al., 1997). The most important potential adverse effects of IGF-I arise from the fact that it is a mitogen for a number of cell types and has been associated with the growth of tumours including those of the colon, breast, and lung and osteosarcoma (Pines et al., 1985; Macaulay, 1992; National Institute of Health, 1995). The mitogenic effect could also result in proliferative reactions locally in the gut. Thus, orally administered IGF-I increased the cellularity of the intestinal mucosa of rats in vivo (Olanrewaju et al., 1992) and increased the rate of proliferation in cultures of human duodenal epithelial crypt cells (Challacombe & Wheeler, 1994). Since IGF-I receptors can be detected throughout the epithelium of the intestine, with a high density in the colon (Laburthe et al., 1988), and the incidence of colorectal cancer is increased in acromegalic patients who have excessively high concentrations of free IGF-I in their plasma (Ezzat & Melmed, 1991), concern has been expressed that increased concentrations of milk-borne IGF-I may increase the risk for colon cancer. Although the normal biological effects of IGF-I mean that it could promote the growth of tumours, this hazard would become a risk only if there were adequate exposure of consumers to IGF-I. Since exposure to IGF-I in milk from rbST-treated cows is negligible when compared with endogenous IGF-I production, it is extremely unlikely that IGF-I residues cause any systemic or local mitogenic reaction. 2.3 Expression of lentiviruses and prion proteins 2.3.1 Somatotropins and the immune system Somatotropin enhances the immune system in many species, including cattle (Comens-Keller et al., 1995). The primary effect appears to be altered responsiveness of the immune system, although substantive evidence of this effect is lacking (Burton et al., 1994), and information on changes in cytokine concentrations or secretion and on their binding sites are needed in order to define the nature of the immune-enhancing effects of somatotropins. The studies reported in the literature differ with regard to the source of somatotropin, the treatment schedule, and the age of the animals. The differences between findings in vitro and in vivo may be due to the release in vivo of mediators such as IGFs and cytokines, which are not present in vitro (Kelley, 1989). A better understanding of somatotropin-mediated immune enhancement as a homeorhetic regulator of the overall health and disease resistance of animals is needed. Lymphocytes from rbST-treated cows have a greater average maximum lymphoblastogenic response to rbST than to other mitogens around the time of parturition (Comens-Keller et al., 1995), and this effect might prevent mastitis and the other infectious diseases that occur during this period of immunosuppression. 2.3.2 Effect of bovine somatotropin on the expression of retroviruses Concern has been expressed that the immunomodulatory effect of bST might affect retroviral expression in treated animals and thus cause resurgence of latent retroviral and lentiviral infections in the ruminant population and the presence of these viruses in somatic cells in milk. The concern is based largely on a review by Lerondelle et al. (1994), who discussed the evidence for induction of these viruses in small ruminants by steroid hormones and the evidence for induction of lentiviruses in other species by hormones including growth hormone and IGF-I, and an unpublished study by Lerondelle et al. (1996), who investigated the effects of rbST on the expression of caprine arthritis encephalitis virus (CAEV) in goats. This virus belongs to the group of lentiviruses which, like maediœvisna, can infect small ruminants. Ruminant lentiviruses are of interest for at least three reasons. First, they may cause disease in persons who drink milk. Second, if use of rbST increases the prevalence of ruminant viruses, by the presence of rbST itself or by the action of IGF-I, the small additional amounts of growth hormone in the milk of treated cows could also affect the retroviruses that attack the human immune system, HIV-1 and HIV-2. Finally, the severity or kinetics of expression of the disease in ruminants might be increased. The study of Lerondelle and coworkers (1996) attempted to address the question of whether rbST increases the expression of CAEV. Viral expression was measured by assaying reverse transcriptase activity in milk cells, clinical examination of the udders and joints of the animals at the beginning and end of the study, and evidence of infection in an immunodiffusion assay. Twelve pregnant Saanen goats that were seronegative for CAEV were given an intramammary injection of monocytes infected in vitro with the Cork strain of CAEV at the time of drying off. Seven weeks after giving birth, four goats were treated daily with 5 mg/goat rbST (sometribove), a second group was treated daily with 10 mg/goat thyroxine, and the control group was untreated. The compounds were administered in suspension in sterile water for 30 days, followed by a 45-day observation period. Milk samples were taken to measure reverse transcriptase activity on days 7, 14, 21, and 28 of treatment and three times during the observation period. Examination of the udders and joints and immunodiffusion tests were carried out at the beginning and end of the study. Milk production and milk-cell counts were evaluated every two days. The occurrence of CAEV and the onset of effects are shown in Table 6. More positive cultured cells were seen in the controls than after treatment with either hormone. Perhaps the most striking effect is the lack of increase in the rate of infectivity and even a suggestion of a decrease after treatment with rbST, particularly after the first milk sampling. When the results were expressed as a ratio of transcriptase activity to the number of cells considered to contain virus (Table 7), there was no correlation between the effect of rbST or thyroxine and the activity of reverse transcriptase in the milk samples. In fact, there was no evidence of increased transcriptase activity in any group. In particular, even though the group receiving rbST had a lower initial rate of infection than the other two groups, including the controls, the rate of infection was not increased, as measured by the number of positive cultures or increased transcriptase activity. The authors interpreted their results as showing a tendency to increased viral expression with increased milk production. The results for rbST were, however, biased by the fact that only two animals were included in the final evaluation. The authors concluded that, owing to the Table 6. Onset of appearance of cytopathic effect (in days) for each of 10 milk samplings from groups of four goats given no treatment or thyroxine or recombinant bovine somatotropin (rbST), as a function of the period of hormonal treatment Treatment Milk sampling Before treatment During treatment After treatment 1 2 3 4 5 6 7 8 9 Control 4 8 6 10 6 6 6 8 10 4 6 6 6 8 6 6 6 - 4 6 4 6 4 4 6 4 4 6 8 10 6 10 6 - - - Mean ± SD (n) 6 ± 1.91 (12) 6.4 ± 1.71 (15) 7 ± 2.39 (8) Thyroxine 8 6 10 4 6 6 - - 6 6 4 4 4 4 4 4 4 ND 4 4 4 4 4 4 6 4 ND 4 8 6 4 4 4 6 4 ND Mean ± SD (n) 5.67 ± 2.06 (12) 4.53 ± 0.92 (15) 4.25 ± 0.71 (8) rbST 8 - - - - - - - - 4 8 8 - 8 - - - 4 ND - - - - - - - - 4 4 4 4 4 4 4 4 4 Mean ± SD (n) 5.71 ± 2.41 (7) 4.8 ± 1.79 (5) 4 (4) ND, not determined Table 7. Number of samples found to contain virus by the reverse transcriptase-positive culture ratio in groups of four goats given no treatment, thyroxine, or recombinant bovine somatotropin (rbST) Treatment Before During After Total treatment treatment treatment Control 3/6 4/7 3/4 10/17 3/6 3/5 1/3 7/14 6/6 4/5 5/6 15/17 4/4 2/8 3/6 9/18 Total 16/22 13/25 12/19 41/66 Thyroxine 0/3 2/4 0/3 2/10 4/4 5/7 6/6 15/17 6/6 8/8 6/6 20/20 6/6 8/8 4/6 18/20 Total 16/19 23/27 16/21 55/67 rbST 2/4 0/6 0/1 2/11 6/6 7/8 2/5 15/19 0/2 0/4 0/3 0/8 4/4 4/5 5/6 12/14 Total 12/16 11/23 7/15 29/52 heterogeneity of the effects on milk production and the small number of animals tested, the evidence for a promoting effect of rbST on viral expression was insufficient. These results provide no evidence that treatment of cows infected with lentiviruses with rbST will cause resurgence of viral infections or pose any risk to human health. Lentiviruses are retroviruses that replicate only in activated cells of the immune system. They may stay dormant for months or years before they gradually wear down the immune system to the point of collapse. The phylogenetic tree of lentiviruses includes the subfamily of bovine immunodeficiency virus (also called bovine leukaemia virus, BLV) and the subfamily of HIV-1 and HIV-2 which are the causative agents of AIDS. BLV and HIV are widely separated phylogenetically (Robertson, 1997). BLV is not known to cause disease in humans although it can infect human cells in vitro, where host defence mechanisms are not present (Georgiades et al., 1978; van der Maaten & Miller, 1990); infection of human cells in vivo could not be demonstrated, and all attempts to obtain direct evidence of infection in exposed human populations have yielded negative results (Straub, 1981; van der Maaten & Miller, 1990). Failure to infect humans has also been shown for other ruminant lentiviruses, such as CAEV and maediœvisna (Straub, 1981). Excretion of virus with milk somatic cells can infect the offspring of infected cows. Such transmission can be effectively blocked by procedures similar to pasteurization, which destroy the virus at 60°C within 30 s (Abramova et al., 1974; Baumgartner et al., 1976; van der Maaten & Miller, 1990). BLV therefore cannot induce disease in humans and is completely inactivated by routine pasteurization. Furthermore, the company that sells sometribove has reported that there is no indication that the incidence of BLV infection has increased in cattle after eight years of continuous use of rbST in Mexico and Brazil and four years of use in the United States (Collier & Kowalczyk, 1998), although this statement is not further qualified. An increase in the expression of HIV in humans who ingest milk from rbST-treated cows is extermely unlikely because of the negligible residues of rbST and IGF-I. Treatment of AIDS patients for six weeks with recombinant human growth hormone and IGF-I had no effect on the HIV titres in peripheral blood mononuclear cells, on the CD3, CD4, or CD8 counts in peripheral blood, or on serum HIV p24 antigen concentrations (Waters et al., 1996). 2.3.3 Effect of recombinant bovine somatotropin on prion proteins Concern has been expressed that rbST treatment could increase the risk for BSE in dairy cows (Hansen et al., 1997). Little evidence is available to support this concern, and that which has been provided is indirect. It is currently thought that the infectious agent of BSE is a prion protein (Prusiner, 1982). Prion proteins are found normally in all animals and are encoded by a prion-protein gene. BSE is associated with a post-translationally modified protease-resistant prion protein, which differs in its three-dimensional structure from the normal protease-sensitive prion protein. Normal prion proteins are bound to membranes on the surface of all nerve cells, some lymphocytes, and other tissues (Prusiner, 1991). To date, no function has been ascribed to normal prion protein. The most widely accepted theory of BSE is the conversion of normal prion protein to the abnormal protease-resistant form, which in turn causes more normal prion protein to convert to protease resistance. The mechanisms of the conversion to the disease-causing protease-resistant prion protein are not clearly understood. In contrast to the normal form, the protease-resistant form cannot be turned over and builds up in cells, forming large oligomers which are observed as plaques (amyloids) in the brains of affected individuals (Gajdusek, 1993). IGF-I increased the production of prion protein mRNA in vitro in a rat phaeochromocytoma cell line (PC12 cells), with a rather flat dose-response curve showing a 40% increase at 10 ng/ml and a doubling at 100 ng/ml (Lasmézas et al., 1993). In transgenic mice that harbour multiple copies of the prion protein gene, the progession of scrapie was accelerated (Prusiner, 1991). It has been speculated that the increased IGF-I concentrations in rbST-treated cows could increase prion protein production and possibly speed up the progression of BSE; however, no studies are available that directly address the question of whether rbST or IGF-I increases the formation of normal prion protein or its pathogenic protease-resistant mutant in the brains of the cattle, and the possibility of a link between rbST treatment and BSE is highly speculative. 2.4 Cow's milk and insulin-dependent type I diabetes mellitus in childhood Epidemiological studies have shown that environmental factors such as chemicals, viral infections, a short duration of breastfeeding, and early dietary exposure of newborns to cow's milk increase the risk for insulin-dependent type I diabetes mellitus (IDDM) by about 1.5 times (Scott, 1990; Dahlquist et al., 1991; Jorgensen et al., 1991; Virtanen et al., 1993; Gerstein, 1994; Verge et al., 1994; Virtanen et al., 1994). IDDM develops as a consequence of autoimmune destruction of the insulin-producing b cells of the pancreatic islets. The precise trigger of the autoimmune reaction is unknown, but it is assumed to be a genetically acquired immune defect in susceptible individuals (Gerstein, 1994). The epidemiological evidence indicates that IDDM is geographically and temporally related to neonatal feeding with cow's milk and that avoidance of cow's milk during the first few month of life can protect genetically predisposed individuals (Gerstein, 1994; Verge et al., 1994). Serological evidence supports the theory that the immune defect is triggered by exposure to proteins in cow's milk (Gerstein, 1994). It has been postulated that, in newborns, milk proteins cross the immature gut wall, initiating an immune response that cross-reacts with a beta-cell surface antigen (Verge et al., 1994). Children aged five to nine, who have an intact intestinal barrier, are not at risk of acquiring IDDM by drinking cow's milk (Dahlquist et al., 1991). The possible triggering factors in cow's milk have not been identified. Casein is unlikely to be involved, since replacement of milk proteins by casein in the diets of rats susceptible to diabetes completely prevented the disease (Jorgensen et al., 1991). Increased concentrations of immunoglobulin A antibodies to cow's milk and b-lactoglobulin appear to be associated with an increased risk for IDDM (Dahlquist et al., 1991; Virtanen et al., 1994). It is unlikely that exposure of human newborns to milk from rbST-treated cows would increase the risk for IDDM for the following reasons: * The composition of milk from rbST-treated cows is well within the normal variations observed during lactation, * The slightly increased IGF-I concentrations can be excluded as a triggering factor because bovine and human IGF-I are identical and because the concentrations of IGF-I in breast milk are equal to or initially higher than those found in cow's milk. 3. COMMENTS Use of antibiotics After reviewing the available information, the Committee considered the risk for mastitis associated with use of rbST as an issue of animal health that is not within its terms of reference; however, the possibly increased use of antibiotics was considered. A post-approval monitoring programme was established in the United States to address the following issues: * the incidence of mastitis and responses related to herd health (not within the terms of reference of the Committee), * treatment with any medications of 28 herds of rbST-treated cows (not within the terms of reference of the Committee), and * the percent of milk discarded because of violative drug residues in key dairy states representing at least 50% of United States milk production. In New York State, the percentage of milk discarded because of the presence of antibiotic residues was not significantly changed after the introduction of rbST. In other states, however, a small but statistically significant, increase was observed in 1995, which coincided with a change to a more sensitive testing method in those states. The Committee concluded that the use of rbST would not result in a higher risk to human health due to the use of antibiotics to treat mastitis and that the increased potential for drug residues in milk could be managed by practices currently in use within the dairy industry and by following the directions for use. Concentrations of insulin-like growth factor in milk and tissues Insulin-like growth factor (IGF-I) is a normal component of milk and is found in abundance in a variety of body fluids (Table 8). The presence and concentrations of IGF-I were the source of much of the scientific discussion during the original review of bST undertaken at the fortieth meeting of the Committee and in submissions to the present one. The information that was reviewed is summarized in FAO Food and Nutrition Paper No. 41/5 (Annex 1, reference 106). The concentrations of IGF-I in milk are variable and have been shown to depend on the state of lactation, nutrition, and age. Methods for assaying IGF-I were considered by the Committee. Although incomplete removal of IGF-binding proteins or differences in the source of standards and extraction methods might affect the reported values, these factors were considered not to alter any conclusions materially. The relatively high values previously reported in milk were considered to reflect inadequate extraction. Table 8. Insulin-like growth factor in milk and body fluids Medium Concentration (ng/ml) Milk Human 5-10 Colostrum 8-28 Bovine (bulk milk) Untreated 1-9 rbST-treated 1-13 Plasma Child 17-250 Adolescent 180-780 Adult 120-460 Gastrointestinal secretions (human) Saliva 6.8 Gastric juice 26 Pancreatic juice 27 Bile 6.8 Jejunal chyme 180 Daily production of adult humans 107 ng/d Since the previous evaluation, few additional data on residues have appeared in the literature or in reports provided by interested parties; however, the manufacturer of sometribove submitted additional information on the concentrations of IGF-I in retail milk after the approval of rbST in the United States. The results showed no difference in the IGF-I concentrations of milk certified as derived from cows not treated with rbST and of unlabelled milk; however, the percentage of the unlabelled milk that was derived from cows receiving rbST was not specified. Concern has been expressed that any rbST-induced increase in the concentration of IGF-I in milk would contribute to the endogenous levels of IGF-I in the gastrointestinal tract and serum if it were not biodegraded and were absorbed. In rats, IGF-I is rapidly degraded in the gastrointestinal tract; however, a protective effect of casein on IGF-I was demonstrated in these studies. It was postulated that the retarded degradation leads to increased serum levels of IGF-I (as shown in one study in rats) and to prolonged exposure of the gut. The Committee noted that seven days' oral administration of high doses of IGF-I in milk replacer did not increase the circulating concentrations of IGF-I in newborn calves and piglets, indicating that significant absorption of IGF-I is unlikely to occur under physiological conditions. In view of the decreased rate of degradation in the small intestine of rats in the presence of casein, the concentrations of the growth factor would probably decrease to less than 5% of their initial values within 2 h, so that milk-borne IGF-I would not be expected to contribute to the concentrations of IGF-I in the large intestine. If 1.5 L of milk are ingested per day, the average intake of IGF-I will be 6000 ng in milk from untreated cows containing an assumed IGF-I concentration of 4 ng/ml and 9000 ng in milk from rbST- treated cows with an approximate average concentration of 6 ng/ml. It has been calculated that IGF-I in gastrointestinal secretions amounts to about 380 000 ng/day. Therefore, the additional amount of IGF-I in 1.5 L of milk from rbST-treated cows would represent only about 0.8% of the IGF-I secreted in the gastrointestinal tract. The total amount of IGF-I in serum has been calculated to range from 50 000 to 1 220 000 ng, depending on age. The total daily IGF-I production in adult humans has been estimated to be 107 ng. Therefore, the daily amount of IGF-I ingested with milk from rbST-treated cows would represent less than 0.09% of the daily production of adults. Even if all of the milk-borne IGF-I were absorbed, the additional amount would be negligible. When sustained-release rbST was administered to cattle once every two weeks for a total of 20 weeks, the tissue concentrations of rbST and IGF-I two weeks after the final dose were not significantly increased. The Committee concluded that any increase in the concentration of IGF-I in milk from rbST-treated cows is orders of magnitude lower than the physiological amounts produced in the gastrointestinal tract and in other parts of the body. Thus, the concentration of IGF-I would not increase either locally in the gut or systemically, and the potential for IGF-I to promote tumour growth would not increase when milk from rbST-treated cows was consumed; there is thus no appreciable risk for consumers. Expression of retroviruses Concern that treatment of cattle with rbST would increase the expression of retroviruses, including BLV, were addressed in experiments with caprine arthritis encephalitis virus in goats. Infectivity was not increased when measured as numbers of infected cells, and there was no evidence of increased reverse transcriptase activity. These studies provide no evidence that rbST affects the expression of BLV, a lentivirus in cattle. Furthermore it has been shown that BLV is destroyed in simulated pasteurization conditions by heating milk to 60 .C for 30 s. In addition, there is no evidence of human susceptibility or response to ruminant retroviruses. Expression of prion proteins Concern has been expressed that treatment with rbST could shorten the incubation period for BSE. This hypothesis is based on results obtained with a neuronal cell line in vitro indicating increased formation of prion protein mRNA in response to IGF-I. Furthermore, increased formation of prion protein shortened the incubation period for scrapie in transgenic mice harbouring multiple copies of the gene that codes for prion proteins. No data were available, however, to address directly the question of whether rbST or IGF-I increases the formation of normal prion protein or its pathogenic protease-resistant mutant in the brains of cattle. The Committee considered that the possibility of a link between rbST treatment and BSE was highly speculative. Risk for insulin-dependent diabetes mellitus Exposure of human newborns to cow's milk increases the risk for insulin-dependent diabetes mellitus by about 1.5-fold. The Committee considered whether exposure of newborns to milk from rbST-treated cows further increases this risk. It concluded that, because of its unchanged composition, the milk of rbST-treated cows would not pose an additional risk to the development of insulin-dependent diabetes mellitus. 4. EVALUATION On the basis of the following: * insignificant changes in the quantities of milk discarded after testing for antibiotic residues following the introduction of rbST into commercial use; * low concentrations of rbST and IGF-I residues in milk; * degradation of IGF-I in the gut and its abundance in gut secretions; * the extremely low concentrations of ingested IGF-I in comparison with endogenous production; * lack of evidence that rbST stimulates the expression of retroviruses; * lack of a direct link between rbST treatment and BSE; and * the absence of significant changes in the composition of milk from rbST-treated cows that could contribute to an additional risk for the development of insulin-dependent diabetes mellitus, the Committee concluded that rbST can be used without any appreciable risk to the health of consumers. The Committee reaffirmed its previous ADIs1 and MRLs2 'not specified' for somagrebove, sometribove, somavubove, and somidobove. 5. 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See Also: Toxicological Abbreviations