# Presentation: 522398

## CVM VMACExperience with 558.15 Studies

**Thomas R. Shryock, Ph.D.**
**Elanco Animal Health**
**January 23, 2002**

## Discussion Outline

**558.15 Study Protocol Review**
**Actual experience with 558.15 salmonella shedding studies**
**Limitations of salmonella shedding studies**
**Lessons learned **
**Relevance to Proposed Pathogen Load studies**
**Conclusions**

## 21 CFR 558.15 Studies1970  FDA Task Force report rationale to test test in-feed antimicrobials for effects on salmonella 

## 21 CFR 558.15 StudiesObjectives

**For salmonella shedding studies a sponsor must show:**
**drug does not adversely impact quantities, prevalence or duration of salmonella shed (over baseline non-med control).**
**Drug does not increase salmonella or coliform resistance (over baseline non-med control) to drugs used in either human or animal medicine**
***558.15 regulation gives no specifics in study design or expectations for the type of data to be submitted for CVM review. Sponsors were to consult with CVM***

## Industry Experience with Conducting 558.15 Studies

**FDA required for feed additive for **_**>**_** 14 days**
**Salmonella shedding (Quantities, Prevalence, Duration) + tissues at study end**
**Antibiotic resistance of salmonellae and coliforms**
**Most studies done by C.A.R.E. (Dr. Fagerberg)**
**Initially a single study to assess all parameters**
**Evolved to salmonella study + separate coliform study**
**Currently 3 studies****:**
**Salmonella Quantitation (high dose challenge)**
**Salmonella Prevalence & Duration (lower challenge dose)**
**Coliform Resistance Study**

## General Study Timeline

_**Day   -14     -7      -1    0     1     4      7     14     21     28     35     42      49     56**_
- Culture X     X              X     X    X      X      X      X       X       X      X       X      X
- Weigh X								      X
- Intake X	      X	               X    X      X      X	    X      X       X      X        X      X
- Clin ObX      X       X    X     X    X      X      X       X      X       X      X        X      X
- Necropsy							      X
- 	liver, spleen, MLN, cecal
**Note: Quant-4 week study, Prev/Dur 8 week study**

**Fast 12-24h ****Challenge 2X ****Feed (Med or Non-med)**

**High Challenge 10****11**** cfu**
**Low Challenge 10****6**** cfu**

## General Salmonella Study Design

_**Animals**_**-SPF or Salmonella-free, **_**<**_**20% baseline resistance**
**	**** ****in *****E. coli***
_**Salmonella**_**-Nal****R**** strain specific to animal species on test**
_**Antibiotics**_**-12 (AG, b-lactams, phenicol, quinolone, sulfa, tet)**
**	****MICs determined **
_**Feed**_**- Requires validated analytical feed assay**
**	****Requires dose from efficacy studies**
**	****Requires formulated premix product ****	**
_**Study Groups**_
**	****10-12 animals/group, housed individually**
**	****Med, Env. Control, Non-med. **
**	****Separate caretakers, strict biosecurity**
**	****Isolation cage - minimal coprophagia**
_**GLP Study**_

## Industry Experience with 558.15 Studies

_**Discovery **_**               **_**Development**_**              **_**Product Support**_

**Approval**

**Registration**
**Activities**

**Sales**

**Find New **
**Technology**
- A.	Target I.D. Screen Dev
- B. Screen Opt Early SAR
- C. New Screens

**Lead **
**Optimization**
- A.	Select Final Chemistry
- B.	Formulation
- C.	Candidate Selection
- D. Toxicology

**Product **
**Development**
- Non Clinical
- Clinical
- Data for CM&C

**Submission**

**558.15 studies**

## General Coliform Resistance Study Design

**Animals-SPF or Salmonella-free, **_**<**_**20% baseline resistance**
**	****	*****in E. coli***
**No salmonella challenge**
**Antibiotics-12 (AG, b-lactams, phenicol, quinolone, sulfa, tet)**
**Feed-same ration lot for medicated and non-medicated**
_**Study Groups**_
**	****10-12 animals/group, housed individually**
**	****Med, Non-med. **
**	****Separate caretakers, strict biosecurity**
**	****Isolation cage - minimal coprophagia**

## Interpretation Issues	Pass-Fail for Candidate

**Statistical significance vs. biological significance interpretation evolved from Larry Rollins, CVM**
**Pass if med. Vs. non-med. Groups had:**
**Salm Quantitation ****	****< 1-2 logs difference**
**Salm Prevalence****	****<20-30% difference**
**Salm Duration ****	****	****<7-10 days difference**
**Antibiotic Resistance Moot Point (no differences ever observed)**
**Unclear if all 3 salmonella parameters, or just 2, had to be within "bounds" to Pass the molecule**
***No scientific evidence that these criteria actually relate to farms, meat, or illness***

## Slide 11

## Slide 12

## Industry Experience with 558.15 Studies

**Failure of 558.15 studies kills the candidate molecule in late Development Phase**

**Discovery****	****Development****	****Product Support**

** ****Phase I****  ****	****Phase II ****	****                 **** Phase III ****   ****           Phase lV****   **

**Approval**

**Registration**
**Activities**

**Sales**

**Find New **
**Technology**
- A.	Target I.D. Screen Dev
- B. Screen Opt Early SAR
- C. New Screens

**Lead **
**Optimization**
- A.	Select Final Chemistry
- B.	Formulation
- C.	Candidate Selection
- D. Toxicology

**Product **
**Development**
- Non Clinical
- Clinical
- Data for CM&C

**Submission**

_**558.15 studies (GLP)**_
**Formulated Premix (GMP)**
**Validated analytical feed assay method**
**Dose selected **

## Industry Experience with Conducting 558.15 Studies

**Most drugs tested were G+ active, all in-feed **
**Baseline resistance (<20% rule) in coliforms in test animals is difficult to achieve **
**73% Pass rate; variety of antibiotic classes**
**Failure of studies may be due more to interpretation of "borderline" data than a biologically significant "shedding"**
**A failed study could be repeated and be passed**
**If Pass in one species, usually pass in others**
**"Study Creep"**
**One initial study became 3 separate studies**
**Separate series of studies for each species of use**

## Study Limitations

**Relationship of test model results to commercial farms has not been established**
**Relationship of test model results to carcass contamination levels was not established**
**Relationship of test model results to public health was not established**
**Only one strain of salmonella tested for "shedding"**
**Salmonella given prior to medication would sequester intracellularly, bypassing normal flora (minimized by fasting). **
**High-challenge dose is unrealistic exposure**
**Overcomes protective flora, causes disease**
**Sick animals don't eat as much**

**Notes:**
G+ drugs tested so far are poorly absorbed or extracellular.
"Drug-bug" interaction would be minimal. 
Salmonella are not susceptible to G+ antibiotics. 
Normal flora re-establishes extracellularly; does not prevent salmonella shedding

## Model Study vs. Field Situation

**The field situation is considerably different:**
**Animals previously exposed (immune) or no salmonella present **
**Natural, continuous exposure, often at young age**
**Small proportion of herd/flock culture positive, many undetected carriers**
**Antibiotic superimposed in presence of low levels of shedding**
**Antibiotic administered to diseased and stressed animals**
**Housing, biosecurity, environment are not "research grade" status**
***Model study cannot "factor in" these real world situations, nor predict what will happen there.***

**Notes:**
The challenge model would seem to be something of unrealistic, worst-case scenario because of:
(1) The lack of prior exposure and development of some level of immunity,
(2) The huge challenge inoculum.

## 558.15 Lessons to apply to Proposed Pathogen Load Studies

**Only database so far is for in-feed medication for multi-week studies with salmonella challenge**
**No experience with higher doses, other routes, shorter durations or post-medication withdrawal effects**
**Therapeutic drugs usually given weeks to months before slaughter. When to sample? Relevance?**
**No experience with other foodborne pathogens-will multiple studies be needed ("study creep")?**
**Very little experience with broad-spectrum drugs**
**G- spectrum likely to decrease shedding!**
**Biological variation occurs in individual animals; results may not always be clear-cut for interpretation.**

## Other Lessons to apply to Proposed Pathogen Load Studies

**CVM Pre-approval Workshop groups found no value to conducting Pathogen Load studies**
**Exponent Report found no consistent evidence to indicate an association of salmonella shedding with antibiotics**
**FDA Task Force recommendations did not include therapeutic drugs**
**A standardized, validated protocol, with clear interpretation of results does not exist for Pathogen Load studies. A modify-as-you-go approach, as was done for 558.15 studies, is unacceptable. **
**De facto implementation of Pathogen Load studies has already begun as CVM is asking sponsors to conduct these studies for therapeutic drugs****  ****	**

## Pathogen Load Study Relevance

**8% of US Swine salmonella positive, HACCP pork carcass baseline is 8%. Feed additive use is 90%.**
**The magnitude of salmonella for these food animal species is already quite low.**
**How would pre-approval pathogen load studies on therapeutic uses of new antibiotics be relevant?**
**Six of top ten human salmonella serotypes differ from those recovered from food animals**** **
**How can Pathogen Load studies be designed to be predictive of public health impact?**

**Notes:**
Human enteric disease rates and feed additive usage:
Human salmonella infection rate
US 14/100,000 vs. Denmark 43/100,000
US human campylobacter infection rate:
 US 16/100,000 vs. 82/100,000 in Denmark. 
DK no longer uses feed additives for performance; US does.
 Is there a relationship? What other factors must be important?

## Pathogen Load Study Relevance

**Salmonella "load" in intestinal tract increases from farm to slaughter plant; different serotypes emerge**
**Pathogen load is more affected near slaughter by transport, stress, feed, environment than prior antibiotic use.**
**Contamination post-slaughter can occur but on-farm origin assumed**
**Effective interventions occur at/after slaughter plant (HACCP, cooking, etc.) to minimize foodborne pathogen contamination of carcasses**
**Muscle is sterile upon arrival- meat is contaminated upon cut-up so containment is especially important**

## Conclusions

**558.15 remains in place for in-feed antibiotic products**
**Pathogen Load studies for therapeutic antibiotics cannot be designed to have relevance to on-farm practices, meat contamination and subsequent human illness**
**Pathogen Load study requirements are  an unnecessary impediment to the development of new food animal therapeutic antibiotics.**