Use of Whole Slide Imaging in
Nonclinical Toxicology
Studies: Questions andAnswers
Guidance for Industry
DRAFT GUIDANCEThis guidance document is being distributed for comment purposes only.
Comments and suggestions regarding this draft document should be submitted within 60 days ofpublication in the Federal Register of the notice announcing the availability of the draft
guidance. Submit electronic comments to https://www.regulations.gov. Submit writtencomments to the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630
Fishers Lane, Rm. 1061, Rockville, MD 20852. All comments should be identified with thedocket number listed in the notice of availability that publishes in the Federal Register.
For questions regarding this draft document, contact (CDER) Office of Study Integrity andSurveillance at CDER-OSIS-GLP@fda.hhs.gov or 240-402-6002, (CBER) Office ofCommunication, Outreach and Development, 800-835-4709 or 240-402-8010, (CDRH) Office ofProduct Evaluation and Quality at 301-796-5550, (CVM) Office of New Animal Drug
Evaluation at CVMONADEPolicyTeam2@cvm.fda.gov, (CFSAN) Office of Center Director at
CFSANBIMO@fda.hhs.gov, (CTP) Small Business Assistance at 1-877-287-1373, Office ofRegulatory Affairs (ORA) at ORAPolicyStaffs@fda.hhs.gov.
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
Center for Biologics Evaluation and Research (CBER)
Center for Devices and Radiological Health (CDRH)
Center for Veterinary Medicine (CVM)
Center for Food Safety and Applied Nutrition (CFSAN)
Center for Tobacco Products (CTP)
Office of Regulatory Affairs (ORA)
April 2022
Pharmacology/Toxicology

Use of Whole Slide Imaging inNonclinical Toxicology
Studies: Questions andAnswers
Guidance for Industry
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and/orPolicy and Regulations Staff, HFV-6
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U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
Center for Biologics Evaluation and Research (CBER)
Center for Devices and Radiological Health (CDRH)
Center for Veterinary Medicine (CVM)
Center for Food Safety and Applied Nutrition (CFSAN)
Center for Tobacco Products (CTP)
Office of Regulatory Affairs (ORA)
April 2022
Pharmacology/Toxicology

Contains Nonbinding Recommendations
Draft — Not for Implementation

TABLE OF CONTENTSI.

INTRODUCTION............................................................................................................. 1II.

BACKGROUND ............................................................................................................... 2

III.

QUESTIONS AND ANSWERS ....................................................................................... 2

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Use of Whole Slide Imaging in Nonclinical Toxicology Studies:
Questions and Answers
Guidance for Industry1123
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343536This draft guidance, when finalized, will represent the current thinking of the Food and Drug
Administration (FDA or Agency) on this topic. It does not establish any rights for any person and is notbinding on FDA or the public. You can use an alternative approach if it satisfies the requirements of theapplicable statutes and regulations. To discuss an alternative approach, contact the FDA staff responsible
for this guidance as listed on the title page.

I.

INTRODUCTION

This guidance provides information to sponsors and nonclinical laboratories regarding the useand management of whole slide images used during histopathology assessment and/or pathology
peer review performed for good laboratory practice (GLP)-compliant nonclinical toxicology
studies using non-human specimens. 2 When whole slide imaging is used as part of a nonclinicalstudy conducted in compliance with the GLP regulations, adequate documentation is critical. The
FDA’s expectations regarding documentation practices during generation, use, and retention ofwhole slide images have not been clearly defined and vary among nonclinical testing facilities.
This question-and-answer document is intended to clarify FDA’s recommendations concerning
the management, documentation, and use of whole slide imaging in histopathology assessment
and/or pathology peer review for nonclinical studies conducted in compliance with the GLP
regulations.
The contents of this document do not have the force and effect of law and are not meant to bindthe public in any way, unless specifically incorporated into a contract. This document is intended
only to provide clarity to the public regarding existing requirements under the law. FDA
guidance documents, including this guidance, should be viewed only as recommendations, unlessspecific regulatory or statutory requirements are cited. The use of the word should in Agency
guidances means that something is suggested or recommended, but not required.

This guidance has been prepared by the Office of Study Integrity and Surveillance in the Center for Drug
Evaluation and Research in cooperation with the Center for Biologics Evaluation and Research, Center for Devices
and Radiological Health, Center for Veterinary Medicine, Center for Food Safety and Applied Nutrition, Center for
Tobacco Products, and Office of Regulatory Affairs at the Food and Drug Administration.
2We support the principles of the “3Rs,” to reduce, refine, and replace animal use in testing when feasible. We
encourage sponsors to consult with us if it they wish to use a non-animal testing method they believe is suitable,adequate, validated, and feasible. We will consider if such an alternative method could be assessed for equivalency
to an animal test method.
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BACKGROUND

The histopathological assessment of tissue samples is one of the key activities conducted during
GLP-compliant nonclinical laboratory studies. Commonly, the histopathological assessment
includes an initial evaluation of glass histology slides 3 by the study pathologist and a subsequentreview (referred to as pathology peer review) by a second pathologist, group of pathologists, orPathology Working Group. When whole slide imaging is used as part of a nonclinical study
conducted in compliance with the GLP regulations (21 CFR Part 58), the management,
documentation, and use of whole slide images in histopathology assessment and/or pathology
peer review should be clear and follow written processes and procedures.
Use of whole slide images in casual consultations, opinion exchanges, and mentoring among
pathologists is not covered by this guidance document.
III.

QUESTIONS AND ANSWERS

Q1: What is whole slide imaging?
A1: Whole slide imaging includes the software and hardware used to generate a two-dimensional
digital image 4 of a glass histology slide used for routine assessment in generation of thepathology report. The process includes four sequential parts: image acquisition (scanning), image
processing, image file storage, and display of images. Due to inherent limitations of the current
technologies used in the process that digitalizes the spatial and color information from thescanned histology slides, FDA does not consider the resulting digital image to be an exact copyof the glass slide. For example, the scanning systems have limited spatial and color resolution
and loss of depth of field. 5
Q2: Should whole slide image files be retained?
A2: If whole slide images are assessed in lieu of the original glass slides during histopathology
assessment and/or pathology peer review performed for GLP-compliant nonclinical toxicology
studies, the whole slide image files should be retained as study records and archived after study
finalization. Consideration should be given to ensure that archived digital images remain
viewable as software/hardware updates/versions are implemented.

In the context of this guidance, the term histology slide refers to tissue mounted on a microscope slide, includingorgan sections and cell samples such as bone marrow and other cytological preparations.
4Digital images comprise a sequence of small images (referred to as tiles) taken from distinct locations on the glassslide. Whole slide imaging systems typically determine the optimal focal plane at a limited, discrete set of locations
on the glass slide and interpolate the optimal focal plane to generate all of the tiles. The individual tiles are then
combined to create the “whole slide” image.
5
Guidance for Industry and Food and Drug Administration Staff Technical Performance Assessment of Digital
Pathology Whole Slide Imaging Devices (April 2016), section IV (A)(6) Image Processing Software and section IV(A)(7) Image Composition. We update guidances periodically. For the most recent version of a guidance, check theFDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.
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Q3: If the whole slide image files are retained, should the glass slides also be retained?
A3: Yes. The glass slides are study specimens and must be retained as study specimens after
study finalization in accordance with 21 CFR Part 58.
Q4: What should be retained with respect to the whole slide image file? Should modified
whole slide image files be retained?
A4: The whole slide image files assessed by the pathologist for histopathology assessment and/orpathology peer review (i.e., files containing all image data captured by the sensor anddocumentation of any modifications), referred to here as the original whole slide image files,
should be retained. Specifically, any technical image processing modifications made to wholeslide image files prior to being provided to the pathologist (e.g., smoothing, color manipulation)
should be documented and retained. Viewing software should not allow the original whole slide
image files to be changed. Simple adjustments made by the pathologist using the image viewing
software during whole slide image evaluation (e.g., brightness, contrast) do not need to bedocumented or retained.
Q5: Should written procedures for whole slide imaging processes be in place?
A5: Yes, written procedures for whole slide imaging processes should be in place. These may
include slide scanning, validation, training, maintenance, software version control,
backup/disaster recovery, virus protection, archival, secure access controls, and chain of custody
processes.
Q6: Should the whole slide imaging system be validated?
A6: If the whole slide images are assessed in lieu of the original glass slides duringhistopathology assessment and/or pathology peer review performed for GLP-compliant
nonclinical toxicology studies, the whole slide imaging system (including software andhardware) should be validated and maintained in a manner specific to the intended use of thetechnology, consistent with 21 CFR Part 58.
Q7: How should whole slide image files be protected, including when transmitted toexternal users?
A7: If the whole slide images are assessed in lieu of the original glass slides duringhistopathology assessment and/or pathology peer review performed for GLP-compliant
nonclinical toxicology studies, generation of a backup file, chain of custody, access controls, andsecuring data systems and data transmission should be performed following written proceduresand processes in compliance with an electronic record under 21 CFR Part 11 to maintain wholeslide image file integrity.

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Q8: Should the signed pathology report/peer review statement state that whole slide images
were evaluated in lieu of glass slides?
A8: Yes, the signed pathology report should state whether the glass slides or whole slide images
were used for histopathological evaluation by the study pathologist, consistent with 21 CFR
58.185(a). If a pathology peer review is performed, the pathology peer review statement shouldindicate whether whole slide images or glass slides were reviewed. 6Guidance for Industry Pathology Peer Review in Nonclinical Toxicology Studies: Questions and Answers(December 2021).

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