Summary of safety assessments on proteins

As discussed earlier, the oral bioavailability of digestible proteins is negligible, thus their potential to exert systemic adverse effects, if such activity were to be characteristic, is also very low. As a consequence, there is not normally the scientific case to subject proteins screened for introduction into food and feed crops to the same extensive battery of safety tests required for low-molecular-weight chemicals that end up in food or feed. As discussed in preceding chapters, no systemic toxic effects have been identified in the many dietary toxicity studies that have been carried out with proteins of variable structure and function that are used in food production.

A list of acute and subchronic oral toxicity studies conducted with these proteins is presented in Tables 11.1 and 11.2. These tables list the "no-observed-adverse-effect-levels" (NOAELs) which, for all the proteins listed, represents the highest dosages that were tested. Many of these proteins are enzymes that have been produced by microbial fermentation and are used in food processing. It has been a regulatory requirement that these enzyme preparations be tested for potential acute and sub-chronic toxicity. As discussed in Chapter 5, this testing has not been undertaken to resolve questions about safety of the enzymes themselves. Rather, testing has been

TABLE 11.1

summary of NoAELs in acute High-Dose studies with Different Proteins

TABLE 11.1

Protein

Function

NoAELab

Reference

CrytAb

Insect control

4000 mg/kg

22

Cry1A.105

Insect control

2072 mg/kg

23

CrytAc

Insect control

4200 mg/kg

22

Cry2Aa

Insect control

4011 mg/kg

22

Cry2Ab

Insect control

1450 mg/kg

22

Cry3A

Insect control

5220 mg/kg

22

Cry3Bb

Insect control

3780 mg/kg

22

CrytF

Insect control

576 mg/kg

24

Cry34Ab1

Insect control

2700 mg/kg

25

Cry35Ab1

Insect control

1850 mg/kg

25

Vip3a

Insect control

3675 mg/kg

26

ACC deaminase

Enzyme

602 mg/kg

27

Alkaline cellulase

Enzyme

10,000 mg/kg

28

Dihydrodipicolinate-synthase (cDHDPS)

Enzyme

800 mg/kg

29

ß-galactosidase

Enzyme

20,000 mg/kg

30

Enolpyruvyl-shikimate-3-phosphatesynthase

Enzyme

572 mg/kg

31

(CP4-EPSPS)

ß-glucanase

Enzyme

2000 mg/kg

32

Glutaminase

Enzyme

7500 mg/kg

33

Hexose oxidase

Enzyme

2000 mg/kg

34

Laccase

Enzyme

2700 mg/kg

35

Lactase

Enzyme

10,000 mg/kg

36

Lactose oxidase

Enzyme

900 mg/kg

37

Lipase

Enzyme

2000 mg/kg

38

Lipase

Enzyme

5000 mg/kg

39

Neomycin phosphotransferase

Enzyme

5000 mg/kg

40

Phosphinothricin acetyl transferase

Enzyme

2500 mg/kg

41

Phosphomannose isomerase

Enzyme

3030 mg/kg

42

Pullulanase

Enzyme

10,000 mg/kg

43

Xylanase

Enzyme

239 mg/kg

44

Xylanase

Enzyme

2000 mg/kg

45

a Highest dosage tested that caused no adverse effects.

b Actual delivered dosage may be lower based on the purity of the enzyme preparations tested.

TABLE 11.2

summary of NoAELs in subchronic Feeding studies with Different Proteins

TABLE 11.2

Protein

Function

study

NoAELa

Referei

Bovine somatotropin

Hormone

13 weeks

50 mg/kg

46

Dipel Bt microbial

Insect control

13 weeks

8400 mg/kg

22

Cry protein mixture

Dipel Bt microbial

Insect control

2 years

8400 mg/kg

22

Cry protein mixture

Teknar Bt microbial

Insect control

13 weeks

4000 mg/kg

22

Cry protein mixture

Bt Berliner microbial

Insect control

5 days (human)

1000 mg/adult

22

Cry protein mixture

Cry1Ab

Insect control

28 days

0.45 mg/kg/day

22

Amylase

Enzyme

90 days

17.5 mg/kg/day

47

Amylase

Enzyme

90 days

890 mg/kg

48

Amyloglucosidase

Enzyme

14 days

1640 mg/kg

49

Amino peptidase

Enzyme

90 days

2000 mg/kg

50

Arabinofuranosidase

Enzyme

14 days

103 mg/kg

49

Chymosin

Enzyme

90 days

1000 mg/kg

51

Chymosin

Enzyme

90 days

11.9 mg/kg

51

ß-galactosidase

Enzyme

6 months (rat)

4000 mg/kg

30

30 days (dog)

1000 mg/kg

Glucanase

Enzyme

90 days

1258 mg/kg

52

Glutaminase

Enzyme

90 days

9000 mg/kg/day (yeast

33

CK)1200 mg/kg/day

(yeast CKD10)10,000 mg/

kg/day (yeast TK)

365 days

13,000 mg/kg(yeast CK)

Hexose oxidase

Enzyme

90 days

5000 HOX units/kg

34

Laccase

Enzyme

90 days

1720 mg/kg

35

Lactase

Enzyme

28 days

1540 mg/kg

36

Lactose oxidase

Enzyme

90 days

900 mg/kg

37

Lipase

Enzyme

90 days

658 mg/kg

39

Lipase

Enzyme

90 days

1680 mg/kg

38

Lipase G

Enzyme

90 days

1516 mg/kg

53

Lipase AY

Enzyme

90 days

2500 mg/kg

54

Pectin methylesterase

Enzyme

14 days

133 mg/kg

49

Phosphodiesterase

Enzyme

28 days

165 mg/kg

55

Phospholipase-A

Enzyme

90 days

1350 mg/kg

49

Phytase

Enzyme

90 days

1260 mg/kg

49

Pullulanase

Enzyme

28 days

5000 mg/kg

56

Tannase

Enzyme

91 days

660 mg/kg

57

Xylanase

Enzyme

90 days

1850 mg/kg

49

TABLE 11.2 (CoNTINuED)

summary of NoAELs in subchronic Feeding studies with different Proteins

TABLE 11.2 (CoNTINuED)

summary of NoAELs in subchronic Feeding studies with different Proteins

Protein

Function

study

NoAELa

Reference

Xylanase

Enzyme

90 days

4095 mg/kg

49

Lactoferrin (human)

Iron transport

90 days

2000 mg/kg/d

58

Lactoferrin (bovine)

Iron transport

90 days

2000 mg/kg/d

59

Silkworm pupae

Not defined

30 days

1500 mg/kg/d

60

protein

Thaumatins

Sweetner

90 days

2696 mg/kg/d

61

Ice-structuring

Cryo

90 days

580 mg/kg/d

62

protein

preservation

a In all cases, the NOAELs were the highest dose tested.

a In all cases, the NOAELs were the highest dose tested.

considered necessary to confirm the absence of possible toxic contaminants (myco-toxins, bacterial toxins) from the fermentation medium that might be present in the enzyme preparation. Such testing, also applied to protein based vaccines, is also known as "freedom from abnormal toxicity" (FAT) testing.

These studies confirm the absence of oral toxicity even when the protein preparations were administered at very high dosage levels. The studies listed in Tables 11.1 and 11.2 have been published, but there are many others that have been completed and have not been published. According to a recent review,63 as of 2001 almost 800 toxicity tests have been conducted on approximately 180 enzymes by member companies of the European Association of Manufacturers and Formulators of Enzyme Products (AMFEP). According to AMFEP, these studies raised no issues of toxicological concern.63 Given the history of safe use for certain microorganisms to make enzyme preparations, it has been proposed that routine toxicology testing of highly characterized specific enzyme preparations prepared from these microorganisms is no longer scientifically justified and is inhumane because of its unnecessary use of laboratory animals for toxicology testing.63

Although the vast majority of subchronic feeding studies with food enzymes have consistently found no evidence of treatment-related adverse effects in test animals, a couple of studies reported local irritation to the stomach caused by feeding high levels of protease enzymes to rats. Such effects might be anticipated due to proteolytic effects of the enzymes on the stomach mucosa at high exposures.64 A few other subchronic feeding studies reported adverse effects usually limited to the highest dosages tested, and at lower dosages no adverse effects were reported. Since lower dosages were still many times higher than potential human dietary exposures, a very large safety margin existed for the use of these enzymes in food production. The adverse effects were not attributed to the enzymes themselves, but rather to other constituents in the enzyme preparation. For example, enzyme preparations with high levels of ash (salts and minerals) from the fermentation medium produced nephrocalcinosis43 or increased water consumption in rats.64 Other effects, such as slight anemia32 or reduced urine pH, found in other studies were either not correlated with any microscopic evidence of pathologic changes or were not reproducible

(salivary gland enlargement when rats were fed the enzyme in the diet but not by stomach tube).65 At a recent (2005) European Toxicology Forum conference on the safety assessment of food enzymes, a European regulator was asked whether he had ever seen evidence of adverse effects in submitted subchronic toxicology studies that were directly attributable to the enzyme fed to rats.66 He responded that in his many years of experience, he had not.

No evidence of pre-neoplastic microscopic changes have been reported in the tissues of laboratory animals fed proteins (enzymes, etc.) in subchronic feeding studies. As discussed in Chapters 5 and 6, proteins are not considered to be capable of mutagenic interactions with DNA, and this would be even less likely for proteins consumed in the diet. Mutagenicity studies have been carried out with many enzyme preparations to confirm they did not contain genotoxic contaminants (e.g., mycotox-ins) from the fermentation medium. Members of the United States Enzyme Technical Association (ETA) reported that, as of 1999, 102 bacterial mutagenesis tests and 63 mammalian chromosomal aberration mutagenesis tests had been carried out with enzyme preparations that were from conventional and genetically modified microor-ganisms.67 The vast majority of these tests found no evidence of mutagenic activity; the few tests that had positive results were considered to be largely attributable to artifacts in the test system (e.g., presence of free histidine in the enzyme preparation gave false positive results in the histidine reversion bacterial mutagenicity tests).67 It was concluded that testing enzymes for potential genotoxicity was not necessary for safety evaluation.67

Similar conclusions were stated in Chapter 6 regarding International Conference on Harmonization (ICH) guidelines for safety testing of protein pharmaceuticals. The ICH guidelines for genotoxicity testing comment that biologicals (which include protein therapeutics) are not expected to interact directly with DNA. They are degraded to peptides and amino acids which are not considered to have genotoxic potential. Routine genotoxicity testing of protein pharmaceuticals is not considered necessary to confirm safety.

There are a few published examples of enzyme preparations being tested in rat teratology and/or one generation rat reproduction studies to confirm the absence of fermentation contaminants that might exert adverse effects. No evidence of adverse effects attributable to the enzymes on progeny development or reproductive performance were reported in these studies.28,30,64,68

A few chronic feeding studies have been carried out with protein preparations produced by fermentation.22,69 This was done to determine whether there were any chronic adverse effects attributable to potential contaminants from the microorganisms used in the fermentation production. These studies did not report that protein preparations caused cancer in laboratory animals. There is no evidence to that proteins directly induced cancer, birth defects, or mutagenic effects when fed in the diet of laboratory animals.67

In the 1980s there was some controversy regarding the chronic effects of trypsin inhibitor proteins on the rat pancreas and the relevance of these findings to humans. Trypsin inhibitors are considered to be antinutrients and members of a larger family of protease inhibitors found naturally in a variety of food crops such as legumes, cereals, and potatoes.70 As the name implies, trypsin inhibitors block the protease activity of trypsin in the gut, interfering with protein digestion. Protease inhibitors may play a role in plant defense by interfering with insect digestion and reducing insect feeding on the crop. The safety controversy began in the UK when rats that had been fed a diet containing raw (unprocessed) soybean meal were dosed with azaserine, a low-molecular-weight chemical that induces pancreatic cancer.71 Soybean meal must be subjected to thermal processing to inactivate trypsin inhibitors before the meal is used as food/feed or the trypsin inhibitors will interfere with protein digestion. The aforementioned study found that trypsin inhibitors in soybeans promoted the development of pancreatic cancer induced by azaserine. In addition, control animals that had not been treated with azaserine, but maintained chronically on unprocessed soybean meal also developed hypertrophic and hyperplastic changes in the pancreas.

It was subsequently shown that this response was not due to a direct effect of trypsin inhibitors on the pancreas but, rather, to negative hormone feedback by cholecystokinin (CCK), a hormone produced in the stomach. CCK is released in response to undigested protein and feeds back on the pancreas to increase production of proteases for release into the digestive tract to increase protein digestion. The continued presence of trypsin inhibitor prevented protein digestion; more CCK was released to stimulate the pancreas and the cycle continued. Rats chronically fed unprocessed soybean meal had very high levels of blood CCK levels due to impaired protein digestion, resulting in chronic stimulation of pancreatic growth which eventually led indirectly to the development of tumors.72

Questions were raised about the relevance to human food safety72-74 since it was reported that the average adult intake of trypsin inhibitors from consumption of normal foods in the UK diet was approximately 330 mg/person/day.74 Feeding studies with raw soybean meal in other species (dog, pig, calf) did not demonstrate hyper-trophic or hyperplastic changes in the pancreas,74 suggesting that rats were more sensitive than other species and may not be a relevant model for humans. It was recognized that trypsin inhibitors mediated their effects on the rat pancreas through the endocrine system. Moreover, according to Gumbmann et al. in 1986, "[T]here is no evidence of absorption from the gastrointestinal tract, direct neoplastic action or tumor induction, genotoxicity, interaction with cellular genetic material or epi-demiological indication of a potential risk in man."75 It was ultimately concluded that "humans are not at increased risk for pancreatic neoplasia for foods containing natural trypsin inhibitor activity."72 Thus, the earlier observation of lack of evidence for direct carcinogenic effects of proteins fed in the diet remains true.

As discussed in Chapter 2, certain proteins are known to be toxic to humans.76 Some of these toxins are produced by pathogenic bacteria that elaborate the toxins in the GI tract when ingested. Some pathogenic bacteria are present in food and form protein toxins in food. Understanding each step in the life cycle of protein toxins can help to define their mode of action and explain why some are toxic when ingested and others are not (Chapter 2). There are also protein antinutrients, such as protease inhibitors and lectins, that are naturally present in a number of foods that are traditionally consumed (legumes, grain, potatoes, etc.).70,77 Although there is a history of safe consumption to many of these proteins, a few of them are toxic, particularly when the food is not properly cooked to inactivate the toxin (e.g., kidney bean lectin).78 The are other examples, such as the castor bean plant, which is not consumed for food but its oil has been used as a cathartic. Castor plants produces ricin, a highly toxic lectin that causes poisoning in humans and animals that accidentally consume the bean.79

Lastly, there is the example of a unique class of proteins known as prions that are components of mammalian neurons. Prion structure can be modified by spontaneous mutations in the prion gene to form stable, pathogenic forms that cause neuro-degenerative diseases. The modified prions cause unmodified prions in neurons to assume the altered structural configuration that induces neuropathologic changes. Modified prions can contaminate surgical equipment or blood and be transmitted to others. Ruminants with bovine spongioform encephalopathy (BSE) caused by modified prions may "infect" those who consume meat from these animals.80 Modified prion proteins are unusually stable as they are resistant to proteases, standard sterilization, and disinfection agents.

As will be discussed below, developers of improved crop varieties initially screen the proteins that are being considered for introduction into agricultural crops for a range of attributes. In particular, the efficacy of the trait to be conferred (e.g., insecticidal activity), and they do not have properties that would pose a risk to consumers or farm animals. Subsequently, following selection and first proof of concept, they undergo systematic bioinformatics, in vitro and in vivo testing on a case-by-case basis. To date, none of the proteins introduced into agricultural crops has shown any evidence of adverse effects, confirming the rigorousness of the screening system that has been developed.

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