Preclinical ToxPathology is often considered as an hidden scientific field in Pathology and it offers a lot of complexity. When implementing Digital Pathology strategies within translational workflows it is crucial to understand the basics of ToxPathology. So, let's start with the introduction !

1. PRECLINICAL DEVELOPMENT
Processes & Timelines & Acceptance
Global R&D: Pharma & CRO
By: Dr. Neyens Elizabeth, DVM, DABT, Pathologist
Certified in Digital Pathology by NSH/DPA

2. OBJECTIVES:
UPON COMPLETION OF THIS SESSION
PARTICIPANTS WILL BE RESPONSIBLE TO:
1. Identify the main regulatory authorities responsible for new
drug developments
2. Understanding the importance of a GLP environment
3. Concept of preclinical research and its stakeholders
4. Able to discuss with pathologist about SEND and
INHAND
5. Understanding the number of slides and tissues in safety
studies
6. Histology & Histopathology
7. Image analysis versus Histopathology
8. What are Animal Models

3. Food & Drug Administration
- 21 CFR Part 11 section of the Code of
Federal Regulations
- Introduced in 1997
- Apply to records in electronic form that are
created, modified, maintained, archived,
retrieved, or transmitted, under any records
requirements set forth in agency regulations
- Provide criteria under which the agency
determines the reliability of electronic
records, electronic signatures, and
handwritten signatures that are executed to
electronic records

4. ü Thalidomide scandal
ü Early’s ‘70, FDA discovered poor
laboratory practices whole over the
US
ü Invalid equipment, results, reports
Applies to Non-Clinical Studies
TRACIBILITY

5. FDA
Efficacy & Safety
ü Efficacy is tested in Proof of Concept
studies
ü Safety of the drug or medical device is
tested in safety & toxicity studies
ü Small molecules & Biologics
ü Extra category: Medical Devices

6. DRUG
DEVELOPMENT
PIPELINE

7. Pre-Clinical RESEARCH
Locations & Species:
1. Contract research organizations
2. Pharmaceutical Industry
3. Academia – Research Centers
Macroscopic Changes:
1. Necropsy procedures
2. Repetitive tasks
3. Technicians & Pathologists
Microscopic Changes:
1. Protocol-driven tissues
2. Repetitive tasks
3. Normal vs. Pathology
4. Peer-Reviews !

8. Preclinical non-GLP
• Although it’s non-GLP, often studies are conducted within the GLP-manner
• Try not to focus, we need to do it GLP
• Non-GLP studies are as important as the GLP ones
• Many advantages in the non-GLP studies:
• Pathologists may be too expensive for non-GLP
• Pathologists prefer to read within GLP context
• Pathologists in pharmaceutical industry read (often) non-GLP
• non-GLP needs to be accurate and performant
• GLP studies will rely on the non-GLP data

9. Types of
Toxicity-
Safety Studies
• Animal Species:
• Rodents: rats & mice
• Rabbits (guinea pigs)
• Dogs, Minipigs & NHP
• Oral or Gavage studies
• Inhalation Studies: focus
respiratory tract
• Infusion studies: single or
multiple site
• Neurological Studies
• Dermal Studies & topical
applications
• Ocular Studies &
specialized pathology
• Reproduction &
developmental studies

10. STUDY
PROTOCOL
LIST OF ORGANS & TISSUES

11. STUDY PROTOCOL
Pathology Section: experimental design, macroscopic, microscopic Changes

12. INHAND & SEND TERMINOLOGIES
• The INHAND Proposal (International Harmonization of Nomenclature and Diagnostic Criteria for
Lesions in Rats and Mice) has been operational since 2005.
• Organ Working Groups (OWG), drawing upon experts from North America, Europe and Japan
• Great progress has been made with 9 systems published to date
• www.goreni.org
• INHAND nomenclature guides offer diagnostic criteria and guidelines for recording lesions observed in
rodent toxicity and carcinogenicity studies.
• During 2012, incorporation of INHAND terminology as preferred terminology for SEND (Standard for
Exchange of Nonclinical Data) submissions to the FDA.
• The interest in utilizing the INHAND nomenclature, based on input from industry and government
toxicologists as well as information technology specialists, suggests that there will be wide acceptance of
this nomenclature.

13. Maximum
Tolerated
Dose
Escalating
Dose
When the first dose level needs
to be established NOAEL,
dosing regimes are increased in a
MTD study. Animals will be
monitored & necropsied in order
to find the maximum tolerated
dose level of the new drug
Cause of death & necropsies are
prioritized
Small species & large species are
used
Histopathology is limited on
target organs such as liver, brain,
spleen, lungs, heart
Often these studies are non-GLP
The number of animals are
limited to approximately
10/Group
Total tissue to be examined by
pathologist: 50 tissues of target
organs to be screen for
abnormalities
Often these studies are lacking
normal animals

14. Single dose
vs. Repeat
dose
This is the second step
in the establishment of a
safety profile of the new
drug
Necropsies are
prioritized BUT with
advanced
histopathological
evaluations
Cause of Death are not
welcome !
Small species & large
species are used
Histopathology is has an
extensive organ and
tissue list according to
the protocol (guidelines)
Often these studies are
moving to GLP-status
The number of animal
groups is often up to 4
(control, low – mid –
high dose)
The number of animals
= group varies from 10
females + 10 males
Control animals are
mandatory for these
studies
Total tissue to be
examined by pathologist:
xx tissues of target
organs to be screen for
abnormalities
Each tissue can go up till
10 different diagnosis
(sometimes nihil)
Recovery animals are
often included in this
type of study

15. Pre-Clinical GLP
Organization needs
to be controlled by
authorities & GLP
approved
Needs to follow 21
CFR Part 11
Needs to have
Standard Operating
Procedures in place
Study Protocol
needs to mention
GLP Compliance
Quality Assurance
Team
Internal Audit &
External FDA –
OECD inspections

16. Sub-
chronic &
Chronic
Duration from 3-months up
to 6-months Small & large species
Often conducted GLP-status
Main animals from at least 3
dose levels and control +
Recovery animals from high
doses (all dose levels)
Extensive tissue list with at
least 25 organs/tissues to
evaluate (trimming protocols)
Number of animals/group
varies from 10
animals/group/sex up to 20
animals
Control animals are
mandatory
Tissue list can be extended
within special
administrations studies
•Inhalation studies with respiratory tract
•Infusion studies with injection sites
•Implant studies

17. Carcinogenicity
studies
• This is the one of the final safety studies in the establishment of a safety
profile of the new drug
• Complex histopathological evaluations because there is the risk of drift of
the pathologist
• Only Small species are used, which are treated during 2-years
• The purpose of the carcinogenicity study is the identify an increased
amount of a type of tumor following the administration of the test item
• Cause of Death are requested in order to validate such a long term study
• Histopathology is has an extensive organ and tissue list according to the
protocol (guidelines) + a lot of abnormalities !
• These studies are always GLP-status
• The number of animal groups is often up to 4 (control, low – mid –high
dose)
• The number of animals = group varies from 40 females + 40 males
• Control animals are mandatory for these studies
• Total tissues (min) to be examined by pathologist: xx tissues of target
organs to be screen for abnormalities
• Each tissue can go up till 10 different diagnosis (sometimes nihil)

18. Special Safety Studies
• Route of administration needs
to mimic the route of
administration in human for its
intended use
• INHALATION STUDIES
• Specific protocol driven
• Extensive respiratory tract
organs & tissues to be
trimmed & evaluated
• Serial sections
• Nasal cavity
• Larynx
• Pharynx
• Trachea & Lungs

19. Inhalation Studies

20. Reproduction studies (1)
• Male Reproductive tract
• Mandatory to confirm absence of infertility
• Mature laboratory animals: Rats
• Immature & Mature laboratory animals: Non
Human Primates & Dogs
• Screening exercise for Normal spermatogenic
stages
• Diagnostic exercise for Infertility & Pathology
(reversible – irreversible)

21. Reproduction studies (2)
• Female Reproductive tract
• Mandatory to confirm absence of infertility
• Mature laboratory animals: Rats & Rabbits
• Immature & Mature laboratory animals: Non
Human Primates & Dogs
• Screening exercise for Normal Cycle (vagina &
ovaria)
• Diagnostic exercise for Infertility & Pathology
(reversible – irreversible)

22. IND
• Pharmacology – Pharmacokinetics
• Toxicology – Pathology
• Small & Large animal species
• Single Dose, Maximum Tolerated Dose
• Repeat Dose Toxicity
• Genotoxicity
• Reproductive & Developmental Toxicity
• Carcinogenicity Toxicity
• Special Toxicology & Pathology

23. HISTOPATHOLOGY
VS. IMAGE ANALYSIS

24. PART 2 : IMAGE
ANALYSIS
• IMAGE ANALYSIS via microscope =performed as
early as 17th century
• DIGITAL IMAGE ANALYSIS = extracting
meaningful information from a digital image via
processing techniques
• Extracting quantitative information from a
digitized histology slide via computer processing
• FDA-approved algorithms for IHC analysis
(Oncology) as aids to pathologists

25. Applications: Image
Analysis

26. ü Study protocols
ü Objectives
ü Material & Methods
ü Sampling & Tissues
ü Histopathology
ü Primary readings
ü Peer-Review
ü Reporting
ü Archiving
ü GLP /non-GLP
ü Conclusions

27. PART 3 : Proof of
Concept
• Non-GLP environment
• Proof of efficacy – Toxicity is not evaluated
• Typical Animal Models
• Research Institutes, Commercial Biotechnologies, CRO’s – Less
in Pharmaceutical Industy
• Mimicking Human Pathologies
• Typical Jackson Laboratory Catalogue