Use of Whole Slide Imaging in 
Nonclinical Toxicology 
Studies: Questions and 
Answers 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
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) 
 
May 2023 
Pharmacology/Toxicology 
   
Use of Whole Slide Imaging in 
Nonclinical Toxicology 
Studies: Questions and 
Answers 
Guidance for Industry 
 
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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) 
 
May 2023 
Pharmacology/Toxicology
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  QUESTIONS AND ANSWERS ....................................................................................... 2 
  Contains Nonbinding Recommendations 
 
Use of Whole Slide Imaging in Nonclinical Toxicology Studies: 
Questions and Answers 
Guidance for Industry1 
 
 
This guidance represents 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 not binding on FDA or the public. You can 
use an alternative approach if it satisfies the requirements of the applicable 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 use 
and 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 The guidance does not cover the use of whole slide 
imaging for clinical applications. When whole slide imaging is used as part of a nonclinical study 
conducted in compliance with the GLP regulations (21 CFR part 58), adequate documentation is 
critical. The FDAs expectations regarding documentation practices during generation, use, and 
retention of whole slide images have not been clearly defined and vary among nonclinical testing 
facilities. This question-and-answer document is intended to clarify FDAs 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. 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities. 
Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
as recommendations, unless specific 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.  
 
 
 
1 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 Devices and Radiological Health, Center for Biologics 
Evaluation and Research, Center for Veterinary Medicine, Center for Food Safety and Applied Nutrition, Center for 
Tobacco Products, and the Office of Regulatory Affairs at the U.S. Food and Drug Administration.  
 
2 We 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 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.  
  1  Contains Nonbinding Recommendations 
 
II.  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 slides3 by the study pathologist and a subsequent 
review (referred to as pathology peer review) by a second pathologist, group of pathologists, or 
Pathology Working Group. When whole slide imaging is used as part of a nonclinical study 
conducted in compliance with the GLP regulations, 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/or 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 image4 of a glass histology slide used for routine assessment in generation of the 
pathology report. The process includes four sequential parts: image acquisition (scanning), image 
processing, image file storage, and display of images. The FDA does not consider the resulting 
digital image to be an exact copy of the glass slide. A whole slide image used in a GLP-
compliant study should include all the elements from the glass slide that are needed for 
histopathological examination or pathology peer review. 
 
Q2: Should whole slide images be retained? 
 
A2: For GLP-compliant nonclinical toxicology studies, if whole slide images are assessed in lieu 
of the original glass slides and result in the generation of pathology raw data, the whole slide 
image files should be retained as study records and archived. Consideration should be given to 
ensure that archived digital images remain viewable as software/hardware updates/versions are 
implemented. 
 
Q3: If the whole slide image files are retained, should the glass slides also be retained? 
 
A3: Yes. The glass slides contain study specimens and must be retained as study specimens after 
study finalization in accordance with 21 CFR part 58. 
 
 
3 In the context of this guidance, the term histology slide refers to tissue mounted on a microscope slide, including 
organ sections and cell samples such as bone marrow and other cytological preparations. 
 
4 Digital images comprise a sequence of small images (referred to as tiles) taken from distinct locations on the glass 
slide. 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. 
  2  Contains Nonbinding Recommendations 
 
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 provided to the study pathologist for histopathological 
examination and/or provided to the peer review pathologist(s) for pathology peer review (i.e., 
files containing all image data captured by the scanner and documentation of any technical image 
processing modifications), referred to here as the original whole slide image files, should be 
retained if they are used to generate raw data. Specifically, any technical image processing 
modifications made to whole slide image files prior to being provided to the pathologist (e.g., 
smoothing, color manipulation) should be documented and retained. Technical image processing 
should not obscure elements captured from the glass slide (e.g., label, artifact). 
 
The pathologist should not permanently alter the original whole slide image files. Nonpermanent 
adjustments made by the pathologist using the image viewing software during whole slide image 
evaluation (e.g., brightness, contrast, annotations) do not need to be documented 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 because whole 
slide images may be used to generate raw data. These processes may include, for example, 
training, slide scanning, software management, and file access and exchange control. 
 
Q6: Should the whole slide imaging system be validated? 
 
A6: If the whole slide images are used to generate raw data, the whole slide imaging system 
(including software and hardware) should be validated in a manner specific to the intended use 
of the technology. 
 
Q7: Should whole slide image files be protected, including when transmitted to external 
users? 
 
A7: If the whole slide images are used to generate raw data, they should be protected to prevent 
loss or alteration of data, maintain chain of custody, control access, and secure data systems and 
data transmission. These measures should be performed following written procedures and 
processes in compliance with electronic record requirements under 21 CFR part 11 to maintain 
whole slide image file integrity. 
 
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 glass slides or whole slide images 
were used for histopathological evaluation by the study pathologist, consistent with 21 CFR 58. 
  3  Contains Nonbinding Recommendations 
 
If a pathology peer review is performed, the pathology peer review statement should indicate 
whether glass slides or whole slide images were reviewed.5 
 
5 Guidance for Industry Pathology Peer Review in Nonclinical Toxicology Studies: Questions and Answers 
(December 2021). We update guidances periodically. For the most recent version of a guidance, check the FDA 
guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
  4