ILSI ® International Food Biotechnology Committee ILSI-IFBiC Task Force 10: Mammalian Toxicology Consensus Views on Protein Safety Assessment Bruce Hammond,

ILSI® International Food Biotechnology Committee

ILSI-IFBiC Task Force 10: Mammalian ToxicologyConsensus Views on Protein Safety Assessment

Bruce Hammond, PhD, DABTMonsanto Company

Society of Toxicology

Workshop Session: Risk Assessment for Proteins Introduced into Genetically Modified Crops

March 8, 2011


Task Force 10 Background

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In 2008, the International Life Sciences Institute Food Biotechnology Committee (ILSI-IFBiC) formed a new task force. Its focus was to develop international consensus recommendations when it is appropriate to undertake animal tox studies and how to design and use animal tox studies in the safety evaluation of biotech crops.

This task force also addressed issues relating to the food safety assessment of proteins introduced into biotech crops to impart desired traits.

Task Force 10 Membership

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Sue Barlow – Independent consultant in toxicology Andrew Bartholomaeus – General Manager Risk Assessment, Food Standards Australia New ZealandGenevieve Bondy – Head, Genotoxicity and Carcinogenesis Section, Food Directorate Health Products and Food Branch, Health CanadaAmechi Chukwudebe – BASFBryan Delaney – Pioneer, a Dupont CompanyBruce Hammond – Monsanto CompanyCorinne Herouet-Guicheney – Bayer CropScienceJoseph Jez – Department of Biology, Washington University, St Louis, MissouriDaland Juberg – Dow AgroSciencesHideaki Karaki – Retired (University of Tokyo)John Kough – Biopesticides and Pollution Prevention Division in the Office of Pesticide Programs, USEPASue MacIntosh – MacIntosh and Associates ConsultingWayne Parrott – Center for Applied Genetic Technologies, University of GeorgiaAlaina Sauve – Syngenta Biotechnology, Inc.Kate Walker, ILSI IFBiCFlavio Zambrone – Planitox, ILSI Brazil


Task Force 10 Progress

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The task force has met periodically and prepared a consensus document for publication. The manuscript is undergoing peer review. This presentation summarizes the consensus conclusions of the task group regarding protein safety assessment.


TF6 Consensus Views on Protein Safety

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In 2008, ILSI-IFBiC Task Force 6 published guidance onthe safety assessment of introduced proteins (Delaney et al., 2008). The major conclusions were: (1) The primary safety assessment of any protein introduced into a food/feed crop includes bioinformatics screening and testing potential digestibility when exposed in-vitro to simulated digestive fluids (2) Proteins that are not structurally or functionally related to known mammalian toxins or allergens based on bioinformatics screening and are confirmed to be digestible, are less likely to pose a hazard when consumed.


TF6 Consensus Views on Protein Safety

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(3) Where possible, the mode of action of the protein should be assessed to confirm it does not pose identifiable or anticipated safety concerns.(4) Where there are no safety issues identified, no further assessment of safety would be needed (unless required as a condition of registration - Plant Incorporated Protectants - EPA).(5) Where there are unresolved safety concerns, further toxicity testing may be indicated. The studies undertaken should be hypothesis driven.


TF10 Consensus Views on Protein Safety – New Issues*

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What is the impact of food processing on potential dietary exposure to functionally active introduced proteins?

For purposes of risk assessment, can the Threshold of Toxicological Concern model be applied for proteins?

What are the criteria for assessing “History of Safe Use” ?

How do you assess the potential for toxicological interactions between multiple proteins introduced into combined trait food crops?

* The task force did not address allergenicity since this has been well-addressed by PATC and other groups


• Protein function (e.g., enzymatic activity) is dependent on maintenance of appropriate tertiary structure. The microenvironment surrounding the protein helps to maintain protein tertiary structure.

• Many crops are processed to generate food products. Processing conditions disrupt the cell microenvironment through the use of heat, changes in pH, extraction of lipid, use of physical shear forces. Protein tertiary structure and function is often lost (denaturation).

• Food crops such as maize, soybean, wheat and rice are normally processed before they are consumed.

Impact of Food Processing onFunctional Activity of Introduced Proteins

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• In vitro heat stability tests have shown that introduced proteins exposed to temperatures similar to those used in processing lose biochemical function (e.g., enzyme activity, or ability to control insect pests - Cry proteins from Bacillus thuringiensis (Bt)).

• Analysis of processed food fractions has confirmed loss of function for introduced proteins.

• Consequently, dietary exposure to functionally active introduced proteins in processed food is likely negligible.

Impact of Food Processing on Functional Activity of Introduced Proteins

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• TTC principle has been recommended for ranking and prioritizing risk from exposure to substances present at low levels in food where toxicology data is limited.

• Proteins were previously excluded from establishing a TTC since a safe threshold for dietary exposure to allergenic proteins had not been established.

• In regard to thresholds for allergens , a recent review of human studies with 286 subjects in France suggest a population threshold of approximately 1 mg/person for highly sensitive peanut allergic patients. (Taylor et al., 2010)

Threshold of Toxicological Concern - TTC

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• The introduced protein could be considered for TTC if it does not fit the profile of known allergens:

– Digestible– Low (ppm) levels in food – Not structurally related to known allergens

• TTC levels for consumption of proteins were estimated based on published literature (Hammond and Cockburn, 2008)

– The highest dietary levels tested in all studies produced no test article related effects

– The NOAELs were corrected for enzyme purity (default to 10%) and divided by 100 and were averaged

– ~18 mg/kg/day (acute)– 2.5 mg/kg/day (chronic)


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• Case study - CP4 EPSPS enzyme– An enzyme in the aromatic amino acid synthesis pathway,

from the bacterial strain CP4, that imparts tolerance to glyphosate herbicide

– Prior to its introduction into food crops, there was no history of consumption in food; however, homologous EPSPS enzymes are commonly found in food crops.

• Potential chronic intake of CP4 EPSPS from consumption of herbicide tolerant corn was estimated to be 4 µg/kg/day.

• This is 600× lower than TTC chronic limit, assuming no denaturation during processing of corn.

• More realistic exposure – processing reduces CP4 EPSPS ~ 2 orders of magnitude – 60,000× lower than TTC


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• EFSA has recommended all introduced proteins with no history of safe use (HOSU) in foods be subjected to at least 28-day repeat dose toxicity testing unless there is reliable information to demonstrate their safety.

• Proteins without a HOSU have been labeled as “novel”. According to Websters’ dictionary, novel means “new and not resembling something formerly known or used”.

• Does changing one, several, or many amino acids in a protein with a HOSU make it truly novel?

History of Safe Use Considerations

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• ~74% of the known proteins assigned to ~ 9,000 protein families based on structural/functional relatedness. Within these families, evolutionary divergence accounts for considerable variability in amino acid content of proteins of the same family

Yet the residues forming the active sites of the proteins are conserved

Accordingly, functional activity is preserved

• The amino acid content of EPSPS in soy, corn, and Baker’s yeast varies considerably from CP4 EPSPS amino acid content

23 to 41% identity (invariant amino acid content at a given residue)

49 to 59% similarity (conservative substitutions of amino acids)

• Functionally related proteins can vary considerably in amino acid content yet maintain similar functions through homologous active sites and tertiary structures.

• Consequently, the HOSU of related EPSPS found in foods could be considered as evidence for the safety of CP4 EPSPS.

Structures of E. coli and CP4 EPSPS

The X-ray structures are represented as ribbon diagrams with the E. coli enzyme shown in violet and the CP4 enzyme in red.

Although E. coli and CP4 EPSPS enzymes share only 27.6% identity, X-ray crystal structure analysis indicate that both proteins have evolved to fold similarly and are superimposable.

Structures of E. coli and CP4 EPSPS

The X-ray structures are represented as ribbon diagrams with the E. coli enzyme shown in violet and the CP4 enzyme in red.

Although E. coli and CP4 EPSPS enzymes share only 27.6% identity, X-ray crystal structure analysis indicate that both proteins have evolved to fold similarly and are superimposable.

Monsanto Scientific Literature #17600

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Cytochrome c oxidase structural homology

Cytochrome c oxidases from bacteria, fungi and animals share less than 30% sequence identity yet have a similar tertiary structure – catalytic site has been conserved

Modified from Voet and Voet (1995) Biochemistry, 2nd Ed


• There is a HOSU for tuna cytochrome c oxidase.

• Paracoccus and Rhodospirillum cytochrome c oxidases may have no HOSU, but if they are:

1. not related to known allergens or toxins

2. capable of being digested

• They should also be safe to consume based on their similar mode of action to related cytochrome c oxidases that have a HOSU.

• This could be considered “reliable” information indicating no need for further toxicity testing for consumption of Paracoccus and Rhodospirillum cytochrome c oxidases.


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• Through conventional breeding, it is possible to cross parent lines containing new traits (introduced proteins) to generate progeny with combined traits.

• Combined trait products have been generated to reduce potential for resistance development and/or to combine traits in the same plant that reduce environmental stress factors (drought, weed competition for soil nutrients, protection against insect pests, etc.)

• Mix and match traits depending on the needs of the grower

Assessment of Potential Protein Interactions

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Combining Traits to Reduce Plant Stress


• Interactions are unlikely when the mode of action of introduced proteins are fundamentally different. – Enzymes that exhibit substrate specificity and/or carry out

different catalytic reactions (CP4 EPSPS or mEPSPS and PAT enzymes that impart tolerance to structurally different herbicides)

• Interactions that could cause adverse effects on non- target organisms are unlikely when different insect control proteins with the same mode of action are combined in the same crop. – If the mode of action does not pose a risk for non-target

organisms, combinations of such proteins are also unlikely to pose a risk – Bt Cry proteins.

• Cry proteins whether tested individually or in combination are not toxic to non-target organisms.

Impact of Potential Protein-Protein Interactions

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Cry Protein Mechanism of Toxicity

Environmental Health Criteria 217, Microbial Pest Control Agent - Bacillus Thuringiensis (WHO IPCS, 1999)


Mammalian toxicology testing may be needed if there is uncertainty about protein safety.

Toxicological evaluation of a functionally active introduced protein may be appropriate if it is:– structurally or functionally related to known mammalian toxins,– stable in simulated gastric fluids and to processing conditions,– has a mode of action that raises a toxicological concern.

When toxicology testing is considered necessary, it should be driven by specific endpoint-related hypotheses and employ relevant and appropriate techniques to address the hypotheses.

Consensus Conclusions

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Mammalian toxicology testing would not be needed if the following information is available:

– An introduced protein without a HOSU is structurally and functionally similar to proteins that do have a HOSU (e.g., CP4 EPSPS and mEPSPS); its mode of action is likely to be demonstrably similar and non-toxic.

– Modifications in the primary structure of a non-toxic protein are unlikely to make it toxic when stability is not significantly changed. As evidenced by:• Evolutionary divergence in protein families across species • Engineering of proteins to improve biological function (e.g.,

enzymes).


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Most proteins denature and lose biological function when heated or exposed to food/feed processing conditions. Thus, human dietary exposure to functionally active introduced proteins in such foods is likely to be negligible, and far below an estimated TTC for proteins.

Acute and repeated-dose toxicology testing of proteins introduced into food crops to date has found no evidence they are toxic to non-target organisms (includes homologous proteins with variant structures, e.g., amino acid sequence or content)


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ILSI® International Food Biotechnology Committee 25

Dankie

Backup Slides

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EFSA Recommendation (2008)

“Unless reliable information is provided demonstrating the safety of the newly expressed protein, the safety assessment of proteins with no history of safe use (for consumption as food) should normally include a repeated-dose toxicity test (normally 28 days) and not rely on acute toxicity testing. Depending on the results of this test, further testing may be necessary.”

EFSA 2009 View on Safety Testing

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• For enzymes, it has been estimated that 50-70% of random modifications in amino acid sequence are approximately neutral in regard to enzymatic function, 30-50% are strongly deleterious to function and only 0.01 to 0.5% are beneficial to function. It is much easier to disrupt function that to enhance it.

Learnings from Protein Engineering

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• It is highly unlikely that modifications to a non-toxic protein will make it toxic. It has been estimated that the likelihood that 9 substitutions in the amino acid sequence of the enzyme phytase being an exact match in sequence to a toxic protein are just 1 in 2 x 1011 (Pariza and Cook, 2010).

• Putting this into perspective, the probability of winning the Powerball lottery has been estimated at 1 in 1.95 × 108.

Learnings from Protein Engineering

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Documents

ILSI ® International Food Biotechnology Committee ILSI-IFBiC Task Force 10: Mammalian Toxicology Consensus Views on Protein Safety Assessment Bruce Hammond,