# Presentation: 884577

## Animal Cloning:  

**Larisa Rudenko,**** PhD DABT**
**Center for Veterinary Medicine**
**FDA/DHHS**

**Developing the**
**Methodology**

**Animal Cloning:  **
**A Risk Assessment**

## Charge

- Characterization of risks
- To animal health for animals involved in cloning process
- From consumption of food from animal clones and their progeny
- Specifically exclude genetically engineered animals
- No risk management at this time

## The Risk “Triad” Simple Version

## Risk Assessment vs Risk Management

**Risk **
**   ****Communication**

**Risk **
**Assessment**

**Risk **
**Management**

- Science-based
- Identifies hazards/
- characterizes risks
- Relatively value-free
- Framework for risk management decisions

- Interactive exchange of information and opinion

-  Identification and evaluation of alternative strategies
- Selection among them based on pre-established criteria

## How Did We Approach This?

- Goals
- Considered traditional risk assessment methodologies
- Surveyed literature
- Asked fundamental questions about hazard and risk to develop framework
- Reviewed literature within risk context

## NAS Risk Analysis Steps: Two Versions

**NAS Risk Analysis Steps: Two Versions**

*2002 Animal Biotech*
- 1.	Identify potential harms
- 2.	Identify potential hazards that might produce those harms
- 3.	Define what exposure means and the likelihood of exposure
- 4.	Quantify the likelihood of harm given that exposure has occurred.

- 1983 NAS “Redbook”
- Hazard identification
- Exposure assessment
- Dose-response evaluation
- Risk characterization

- Both versions imply the presence of an etiologic agent

**Notes:**
NB:  This is non-standard language, and confusing relative to the “usual” four steps of RA...

In particular, it’s important to note that hazard in this case means only the etiologic agent, whereas in the more traditional usage it meant etiological agent and its effect (i.e., in a hazard ID, you’d determine the universe of adverse outcomes (harms) that could be caused by a hazardous substance.

Would it be helpful to list the “old” risk assessment steps???? 
Hazard ID
Exposure Assessment
Dose-Response Evaluation
Risk Characterization

## Literature Survey

- Identified approximately 500 papers on animal SCNT
- Most describe technology development
- Many address transgenic clones (not always clear)
- Emphasis is on hypothesis testing, reports of anomalies
- Very few animal health surveys; those that are surveys are of transgenic clones
- Review indicates ~ 100 relevant studies
- Model systems (mouse)

## Harms, Hazards, and Risks:

**An Overview**

## Some Definitions:Hazards, Harms, and Risks

- Hazard **≡** an act or phenomenon that has the potential to produce a harm.
- Harm **≡** an adverse outcome, injury, or some sort of loss or detriment.
- Risk **≡**** **conditional probability that estimates the probability of harm given that exposure has occurred.
- from 2002 NAS/NRC *Animal Biotechnology*

**Notes:**
Part of the general overview of hazards and risks.  

Based on the discussion of hazard and risk in the 2002 NAS report on tx/cloned animals.

General idea of the whole section is to clearly set up what we’re doing, because THERE IS NO PRECEDENT for looking at cloned animals.  Everything must be derived de novo, based on our previous ability/knowledge base in dealing with conventionally derived animals.  In other words, we could be looking at everything, or we could be looking at nothing.

Is this slide unnecessary if we use the next one?

## Challenges

**Animal Health and Food Consumption Risk Assessments**

## Challenges:  General

- RA method suitable for animal health/food consumption
- Identification of Potential Harms Difficult (AH/FC)
- Reliance on publicly available information
- Lack of theoretical framework
- No Etiologic Agent  Technology Change
- No inserted genes, gene product(s), insertional mutagenesis
- Risk Questions Difficult to Formulate; Absence of Risk (Safety) Difficult to Prove
- How to prove a negative? (absence of risk) (FC more than AH)
- How to limit uncertainty? (decisions in absence of large data sets) (AH/FC)
- Risk Metric
- How to measure risk?
- Comparator groups

## Baseline Assumptions: Animal Safety and Food Consumption RAs

- Compliance with existing regulatory requirements for
- Conventional food animals
- Food derived from conventional animals
- 	e.g., inspection, adulteration

## Nature of Food Consumption Hazards and Risks from SCNT Animals

- Assuming that
- Domestic animals consumed for food do not naturally produce toxins in edible tissues, and
- Frankly diseased or defective animals do not enter the food supply
- Then....
- Postulate changes due to gene dysregulation (epigenetic changes)
- Characterized these as “subtle hazards”

## Subtle Hazards

- Outside “conventional” range of food hazards
- Alterations in gene expression (phenotypic variability such as coat color, behavior, longevity)
- Disruption of immune function (increased sensitivity to pathogens)
- Alterations in metabolism (changes in physiological “set-points” with physiological compensation but potential alterations in milk/meat)
- How would these differ from “subtle hazards” arising in conventional animals?
- How would one detect them?
- Would subtle hazards pose risks?  How to measure risks if they arise??

## Food-Related Hazards and Risks: Hypothetical Examples

| Subtle Hazard | General Risk to Animal | Hypothetical Food
Consumption Risk |
| --- | --- | --- |
| Δ in gene expression Δ protein structure or function | Toxicity due to aberrant protein expression | ↑allergy to milk
Δ nutrient content of meat/milk |
| Δ in immune function | Δ susceptibility to pathogens | ↑ potential for food-borne disease (Salmonellosis) |
| Δ in metabolic “set-points” | Δ in reproductive hormone levels  Δ in fertility  | Δ in sexual maturation due to ↑ estrogen levels in milk |

## Developing the Methodology

**First Iteration**

## Two-Pronged Approach to Evaluating Food Safety

**Two-Pronged Approach to Evaluating Food Safety**

**Critical Biological Systems Approach**
**a healthy animal is likely to produce safe food**

**Compositional Analysis**
**food products from healthy AC/P are not **
**materially different from those derived from **
**conventional animals**

**Entry into **
**food supply**

- +

## Critical Biological Systems Approach (CBSA)

- Mechanistically derived
- HACCP-like Systems Approach
- Accepts SCNT as biologically imprecise and inefficient
- Allows for biological repair or correction
- Intrinsic or following human intervention
- Cumulative nature allows incorporation of favorable and unfavorable outcomes
- Suitable framework for both animal health and food consumption risk characterization

## Critical Biological Systems Approach

**Critical Biological Systems Approach**

**Perinatal**
**Development **
**and Function**

**Reproductive Development**
**And Function**

**Post-Pubertal**
**Maturation**

**Selection of donor and recipient cells, oöcyte maturation/activation,**
**nuclear transfer, embryo culture and transfer  to surrogate dam, **
**implantation, placentation, pregnancy maintenance**

**Cell Fusion Through **
**Fetal Development**

**Induction of labor, parturition, post-natal survival**

**Gametogenesis, secondary sex characteristics, **
**gender appropriate behavior; estrus, fertility, ease of delivery**

**Growth and development measures**

**Physical and physiological functions **
**associated with normal maturation**

**Offspring **

**Juvenile Development**
**And Function**

## Critical Biological Systems Approach

**Critical Biological Systems Approach**

**Perinatal**
**Development **
**and Function**

**Reproductive Development**
**And Function**

**Post-Pubertal**
**Maturation**

- Selection of donor and recipient cells, oöcyte maturation/activation,
- nuclear transfer, embryo culture and transfer  to surrogate dam,
- implantation, placentation, pregnancy maintenance

**Cell Fusion Through **
**Fetal Development**

- Induction of labor, parturition, immediate post-natal survival

- Spermatogenesis, oögenesis, secondary sex characteristics,
- gender appropriate behavior; estrus, fertility, ease of delivery

- Growth and development measures

- Physical and physiological functions
- associated with normal maturation

**Progeny **

**Milk**

**Juvenile Development**
**And Function**

**Milk**

**Meat**

**Meat**

## Interim Conclusions Regarding CBSA Based on Literature Review

- With a few notable exceptions,
- Most information on 1st node in cattle
- Several studies often cited address transgenic clones
- Intermingling of transgenic and “just” clones without individual animal identification (Pace et al., Forsburg et al)
- Very few animals
- multiple reports on one cohort
- Cursory information on health status of non-neonatal animals

## Considerations of Next Steps

- Develop “wish list” of information that would be useful in assessing animal health
- Several presentations to professional societies and other public fora
- Conversations with clone producers

## “Wish list” for Assessing Animal Health

**Species- and Life Stage-Appropriate Comprehensive Veterinary Examinations (CVE) and Clinical Measurements of Blood and Urine**
**Life Stages at which CVE and Clinical Measurements would be desirable:**
**Perinatally**
**Juvenile**
**Sexual Maturity**
**Maturity** (prior to food use)
- Necropsies of animal clones that die prior to use, and whose deaths are not immediately attributable to “normal” events (e.g., accidents, herd-wide disease)

## Standard Menu of ClinChem/Hemogram Panels

**Blood**
**Albumin**
**Alkaline Phosphatase**
**Amylase**
**ALT (SGPT)**
**AST (SGOT)**
**Bile acids**
**BUN**
**CIAP**
**Creatine phosphokinase**
**Gamma glutamyl transferase**
**Lipase**
**Sorbitol Dehydrogenase**
**Cholesterol**
**Creatinine**
**Glucose**
**Serum Protein**
**Calcium**
**Chloride**
**Potassium**
**Sodium**
**PLUS**
**Serum IGF-1 (?)  reserve sample**
**Estrogen (?)  reserve sample**

**Hemo/Leukograms**
**Reticulocyte**
**Differential WBC**
**Eosinophil**
**Platelet**
**Urinalysis**
**Specific gravity**
**Glucose**
**Ketones**
**Bilirubin**
**pH**
**Cells**
**Protein**
**Bacteria**
**Blood (including leukocytes)**
**Nitrate**

## Two-Pronged Approach to Evaluating Food Safety

**Two-Pronged Approach to Evaluating Food Safety**

**Critical Biological Systems Approach**
**a healthy animal is likely to produce safe food**

**Compositional Analysis**
**food products from healthy AC/P are not **
**materially different from those derived from **
**conventional animals**

**Entry into **
**food supply**

- +

## Compositional Analysis Scheme: Goals

**Regulatory Requirements:**
- Must meet requirements as for products from conventionally-bred animals
- PMO
- USDA inspection criteria
- CFSAN nutrition labeling
- CVM drug residues

**Identity Standard Analyses:**
- Constituents within contemporary normal ranges of variability for that food product
- Reflect the genetics of the animal that is being propagated

- Outcome criterion:  “Not materially different from conventional food products”

## Compositional Analysis Scheme:  Summary of Published Data

- Until this fall, no (0) peer-reviewed publications relevant to SCNT animal-derived meat or milk composition
- Very few clones have been bred
- Little impetus to publish compositions
- Meat composition requires sacrificing animal
- Devised “wish list” for milk and meat composition

## Compositional Analyses Showing “No Material Differences” for Milk and Meat from Clones

**MILK**
**Proximates plus**
**Vitamins and minerals for which milk is a moderate to major source**
**Vit A, C, B-1,B-2, B-12, niacin, pantothenic acid**
**Ca, Fe, P**
**Fatty Acid Profiles**
**Saturated:  4:0, 6:0, 8:0, 10:0, 12:0, 14:0, 16:0, 18:0**
**Unsaturated:  18:1, 18:2, 18:3**
**Protein characterization (c****aseins,whey)**
**Amino acid profile**
**Somatic cell count**

**MEAT**
**Proximates plus**
**Nutritionally important vitamins and minerals (for which meat is a moderate to major source)**
**Vit A, C, B-6, B-12, Niacin**
**Ca, Fe, P, Zn**
**Fatty Acid Profiles**
**Saturated: 10:0, 12:0, 14:0, 16:0, 18:0**
**Unsaturated:  18:1, 18:2, 18:3, 20:4**
**Cholesterol **
**Amino acid profile**

## Milk Composition Study (Walsh et al. 2003)

- Clones described by Forsburg et al. (2002)
- 17 clones derived from 5 cell lines bred by AI
- 2 Holsteins, 2 Holstein x Jersey,
- 1 female progeny of bull clone (unsp. breed)
- Comparators
- Approximately age and lactation-stage matched
- Not housed at same farm
- 5 Holsteins (Farm 1), 1 Brown Swiss (Farm 2)
- Different rations

## Milk Composition Study:  Analytes (Walsh et al. 2003)

- Milk components analyzed
- Total fat
- Nitrogen
- Solids
- Lactose
- pH
- Somatic Cell Count
- Acid Degree Value

- Elements
- Na, **Ca**, S, K, Zn, **Fe**, Sr, **P**
- Fatty Acids
**4:0**, **6:0**, **8:0**, **10:0**, **12:0**,**14:0**, 14:1, **16:0**,**18:0**, **18:1**,**18:2**,**18:3**, 20:0
- Protein
- Total protein, caseins (, ,), -lactoglobulin, -lactalbumin, Igs, “other”

## Walsh et al. (2003): Results

- Within Comparator or Published Ranges
- Somatic cell counts (comparator)
- pH (comparator)
- Acid degree values (comparator)
- Gross chemical composition
- Gross levels of milk proteins over entire lactation period (comparator)
- Levels of -casein, -casein, -lactalbumin (comparator)
- 13/14 fatty acids not significantly different (comparator)
- 16:0 (palmitic) 18:3 (linoleic acid) (published)
- FA over lactation cycle (published)
- K, P, Zn,(published), except Sr (? only approximate value published)

## Walsh et al. (2003):Conclusions and Reflections

- “Composition of milk from somatic cell cloned cattle was similar to that from non-cloned animals”
- Differences between clones and comparators likely attributed to differences in breeds, diets, housing
- Small number of animals,
- BUT
- Results consistent with health data

## Model Systems

- Relevance of Mouse Data
- to Livestock Cloning

## What Does the Mouse Literature Tell Us?

- Similar to livestock literature:  tends to focus on interesting outcomes rather than overall health surveys
- May provide insights into underlying biology
- Short generation time may provide clues to reproduction, longevity, and ageing
- Some anomalies similar to those noted in livestock species; others different
- Need to interpret with care

## Anomalies Noted in Mice

- Similar to Livestock
- Large, abnormal placentae reported (Wakayama et al. 1998, Wakayama and Yanagamichi 1999, Ogura et al. 2000, Oho et al. 2001, Yanagamichi 2002).
- Perinatal respiratory difficulties (Wakayama et al. 1999, Wakayama and Yanagamichi 2000, Eggan et al. 2001, Frank et al. 2002)
- Distinctive Phenomena
- Shorter lifespans; liver pathology: limited to one laboratory and strain (Ogonuki et al. 2002)
- Unique obese phenotype in one laboratory
- (Tamashiro et al. 2000, 2002)

## Implications for Domestic Livestock Cloning

- Insights into physiological mechanisms that may be perturbed in animal clones
- Placentation difficulties’ contribution to overgrowth phenomena
- Epigenetic changes appear responsible for clone anomalies
- Rapid resolution of perinatal fragility
- Anomalies noted in clones are not transmitted to progeny
- Obese phenotype not inherited
- Predictive interspecies extrapolation for specific outcomes should be attempted with caution
- Obese phenotype
- Lifespan

## Influx of New Data

**Cyagra, Inc.**
**Supplies Detailed Information on Clone Health Status**

## Cyagra Data Set

- Made available early summer of 2003
- “Snapshot in time” survey, not longitudinal study
- Individual animal data on 134 clones and 85 comparators
- Comparators ~ age-/breed-matched; same farms
- Data collected at 3 developmental stages
- Health records, veterinary exams, laboratory work
- Decided to include these data, even though we knew that the RA would be delayed

## Assessing Risk

**Starting points, biases, and uncertainties**

## Restate Initial Assumptions

- The same criteria that apply to conventional food animals and their edible products also apply to clones and their food products.
- Animals appear normal and healthy; those exhibiting gross abnormalities or disease are culled, and do not enter food supply.
- Milk and meat appear normal, and meet or exceed USDA/FDA/PMO standards.

## Hypothesis 1 (Assumes Clones “Same”)
Animal clones are biological copies of the donor animal, and data confirming overall findings of health and food product comparability are sufficient to indicate that no additional risk is posed by the consumption of such food products relative to food from conventional animals.

Hypothesis 2 (Assumes Clones “Different”)
Animal clones may appear to be biological copies of the donor animal, but subtle hazards may have been introduced by the SCNT process. To avoid additional risks above those posed by consumption of foods from conventional animals, comprehensive health and compositional data are required to demonstrate that the animals are healthy, and that food products derived from them do not differ materially from those derived from conventional animals.

- Animal clones *are* biological copies of the donor animal, and data confirming overall findings of health and food product comparability are sufficient to indicate that no additional risk is posed by the consumption of such food products relative to food from conventional animals.
**Hypothesis 2 (Assumes Clones “Different”)**
- Animal clones* may appear to be* biological copies of the donor animal, but subtle hazards may have been introduced by the SCNT process. To avoid additional risks above those posed by consumption of foods from conventional animals, comprehensive health and compositional data are required to demonstrate that the animals are healthy, and that food products derived from them do not differ materially from those derived from conventional animals.

**Step 1:  Bound Risk Space**

## Step 2: Evaluate Weight of the Evidence for Animal Health and Food Safety

**Literature**
**Submitted data**

## Step 3: State Conclusions Regarding Risk

**Biological assumptions**
**Empirical evidence**
**Consistency of observations **
**Applicability of model systems**
**Uncertainties**
**Confidence in estimates of risk**

## Limitations on Conclusions

**Qualitative, comparative risk assessment**
- Comparators of known or inferred safety
**Strongest conclusion “likely to be as safe as”**** **
- For animal health, a finding of “as safe as” means that the cloning process is likely to be as safe as other ARTs
- For food derived from clones, the finding means that food derived from animal clones is likely to be as safe as foods we eat every day

## Step 4: Recommendations to Decrease Uncertainties

- Risk assessment is an iterative process
- Can always call for more data
- “Paralysis by analysis”
- Determine relative merit(s) of additional data
- Make clear recommendations for risk managers so that risk tolerance can be transparent

## Risk Assessment Document: Overall Structure 

**I.****	****Executive Summary**
**II.****	****Technology Overview**
**III.****	****Hazard, Risk, and Cloning**
**IV. ****Animal Health Risks**
**V.****	****Food Consumption Risks**
**VI. ****Overall Conclusions**

**Appendices**
- Overall Reproductive Efficiency and Health Statistics for US Animal Agriculture
- Comparisons of Success Rates Among Various Assisted Reproductive Technologies
- Cyagra Data
- The Comprehensive Veterinary Exam and its Interpretation
**Bibliography**
**Glossary**

## The Value of This Risk Assessment

**Logical framework**
- tailored for specific questions
**Systematic analysis of available data for multiple uses**
- animal health and food consumption risks
**Identification of data gaps**
- To characterize relative merit of additional data
**Weight of evidence evaluations accommodate **
- Data
- Assumptions
- Biases
- Uncertainties
- Degree of confidence in conclusions
**Stakeholders see transparent decision-making process**
- Scientific deliberations clear
- Relative science/policy contributions to risk management explicit

## Acknowledgments

- CVM Primary Team
- 	Amey Adams
- 	Eric Dubbin
- 	Kevin Greenlees
- 	Barry Hooberman
- 	Wendelyn Jones
- 	John Matheson
- 	Christina Musgrave

- CVM Reviewers
- Laura Hungerford
- Gail Schmerfeld
- Gary Sherman
- Marty Schoenemann
- Jodie Fleming (Rutgers)
- And many others for support in important ways.

## Acknowledgments

- For data:
- Ray Page, Cyagra.
- And other producers whose data we have not yet included.
- For photos:
- Cyagra
- Nexia
- TransOva
- ViaGen
- Jorge Piedrahita