Source Animal, Product, 
Preclinical, and Clinical 
Issues Concerning the Use of 
Xenotransplantation Products 
in Humans 
______________________________________________________________________________ 
 
Guidance for Industry
 
 
 
 
 
 
 
 
 
Additional copies of this guidance are available from the Office of Communication, Outreach 
and Development (OCOD), 10903 New Hampshire Ave., Bldg. 71, Rm. 3128, Silver Spring, 
MD 20993-0002, or by calling 1-800-835-4709 or 240-402-8010, or email ocod@fda.hhs.gov, or 
from the Internet at 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guida
nces/default.htm. 
 
For questions on the content of this guidance, contact OCOD at the phone numbers or email 
address listed above. 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and Research 
April 2003 
Updated December 2016  Contains Nonbinding Recommendations 
 
Table of Contents 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  DEFINITIONS AND ABBREVIATIONS ...................................................................... 3 
IV.  REGULATORY RESPONSIBILITY ............................................................................. 7 
V.  SOURCE ANIMAL CHARACTERIZATION .............................................................. 7 
A.  General Considerations ........................................................................................ 7 
B.  Animal Welfare Concerns .................................................................................... 8 
C.  Animal Origin........................................................................................................ 9 
D.  Animal Health and Husbandry.......................................................................... 11 
E.  Harvest of Nonhuman Live Cells, Tissues, or Organs for Use in Producing 
Xenotransplantation Products ........................................................................... 17 
F.  Source Animal History for Xenogeneic Cell Lines .......................................... 20 
G.  Disposal of Animals and Use of Byproducts ..................................................... 21 
VI.  CHARACTERIZATION OF XENOTRANSPLANTATION PRODUCTS ............. 21 
A.  General Considerations ...................................................................................... 21 
B.  Considerations for Classes of Xenotransplantation Products ........................ 22 
VII.  MICROBIOLOGICAL TESTING OF XENOTRANSPLANTATION PRODUCTS .  
  ........................................................................................................................................... 24 
A.  General Considerations ...................................................................................... 24 
B.  Considerations for Classes of Xenotransplantation Products ........................ 25 
C.  Assay Design for the Detection of Infectious Agents ....................................... 27 
VIII.  MANUFACTURING AND PROCESS-RELATED GMP CONSIDERATIONS FOR 
HARVEST AND PROCESSING OF XENOTRANSPLANTATION PRODUCTS 31 
A.  General Considerations ...................................................................................... 31 
B.  Contamination/Cross-Contamination Precautions.......................................... 31 
C.  Validation and Qualification .............................................................................. 33 
IX.  PRECLINICAL CONSIDERATIONS FOR XENOTRANSPLANTATION ........... 35 
A.  General Considerations ...................................................................................... 35 
B.  Issues Related to Infectious Agents ................................................................... 36 
C.  Xenotransplantation Product-Host Interactions ............................................. 37 
D.  Considerations for the Use of Heterogeneous Xenotransplantation Products
............................................................................................................................... 39 
E.  In Vitro and In Vivo Tumorigenicity Models for Xenotransplantation 
Products Intended for Transplantation ............................................................ 40 
F.  Combinations of Xenotransplantation Products with Devices ....................... 41 
i  Contains Nonbinding Recommendations 
 
X.  CLINICAL ISSUES IN XENOTRANSPLANTATION ............................................. 43 
A.  General Considerations ...................................................................................... 43 
B.  Clinical Protocol Review .................................................................................... 43 
C.  Xenotransplantation Site .................................................................................... 43 
D.  Criteria for Patient Selection ............................................................................. 43 
E.  Risk/Benefit Analysis .......................................................................................... 44 
F.  Screening for Infectious Agents ......................................................................... 44 
G.  Patient Follow-up ................................................................................................ 50 
H.  Archiving of Patient Plasma and Tissue Specimens ........................................ 50 
I.  Health Records and Data Management ............................................................ 52 
J.  Informed Consent ............................................................................................... 54 
K.  Responsibility of the Sponsor in Informing the Patient of New Scientific 
Information .......................................................................................................... 58 
XI.  REFERENCES ................................................................................................................ 59 
  
ii  Contains Nonbinding Recommendations 
 
Source Animal, Product, Preclinical, and Clinical Issues Concerning 
the Use of Xenotransplantation Products in Humans 
______________________________________________________________________________ 
 
Guidance for Industry 
 
 
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 
 
The Food and Drug Administration (FDA) is issuing this guidance to provide sponsors and 
applicants of xenotransplantation products with updates concerning the production, testing, and 
evaluation of products, during protocol development and during the preparation of submissions 
to FDA, e.g., Investigational New Drug Applications (INDs) and Biologics License Applications 
(BLAs).  This guidance also includes updated references and Agency practices intended to 
prevent the introduction and spread of infectious agents of animal origin into the human 
population.  This guidance amends the guidance of the same title dated April 2003 (April 2003 
guidance). 
 
For the purpose of this document, xenotransplantation refers to any procedure that involves the 
transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or 
organs from a nonhuman animal source; or (b) human body fluids, cells, tissues, or organs that 
have had ex vivo contact with live nonhuman animal cells, tissues, or organs.  Also, for the 
purpose of this document, xenotransplantation products include live cells, tissues, or organs used 
in xenotransplantation.  However, for the purpose of this guidance, xenotransplantation does not 
include transplantation, implantation, or other use of acellular animal tissues.  These acellular 
products derived from animal tissue, often referred to as xenografts, are beyond the scope of this 
guidance (See Definitions in section III of this document). 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the FDAs 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 FDAs guidances means that something is suggested or 
recommended but not required. 
1  Contains Nonbinding Recommendations 
 
II.  BACKGROUND 
 
In the mid 1990s, xenotransplantation was suggested as a mechanism for circumventing the 
shortage of human allographs for transplantation.  Proposed xenotransplantation protocols 
included implantation into humans of live organs, tissues, and cells from nonhuman animal 
sources and procedures in which human cells have had ex vivo contact with nonhuman cells, 
tissues, or organs.  Examples of procedures involving xenotransplantation products include 
transplantation of xenogeneic hearts, kidneys, or pancreatic tissue to treat organ failure, 
implantation of neural cells to ameliorate neurological degenerative diseases, administration of 
human cells previously cultured ex vivo with live nonhuman animal antigen-presenting or feeder 
cells, and extracorporeal perfusion of a patients blood or blood component perfused through an 
intact animal organ or isolated cells contained in a device to treat liver failure. 
 
The use of xenotransplantation products presents potential public health risks, such as:  
  
1.  Transmission of infectious agents that are pathogenic for humans but may not be 
pathogenic or even detectable in the source animal host; 
 
2.  Transmission of organisms that may not normally be pathogenic in humans but 
can become so in the immunosuppressed or immunocompromised individual; and, 
 
3.  Recombination or reassortment of infectious agents, particularly viruses, with 
nonpathogenic or endogenous human infectious agents, to form new pathogenic 
entities. 
 
It is difficult to predict the infectious agents that may cause disease in a recipient of a 
xenotransplantation product solely on the basis of analysis of naturally occurring zoonoses 
because there are major differences between normal contact of humans with animals and contact 
of a recipient with a xenotransplantation product.  For example, the physical barrier or distance is 
eliminated in the recipient, due to transplantation and vascularization of xenotransplantation 
products, or even due to implantation of nonvascularized cells or tissues, or ex vivo 
manipulations that cause intimate proximity or contact of xenotransplantation product materials 
with recipient cells, tissues, or fluids.  The potential for viral adaptation in immunocompromised 
or iatrogenically immunosuppressed hosts and the potential for undetected spread of previously 
latent viral infections are of particular concern.  For these reasons, during product development, 
it is important to consider the safety not only of recipients and their contacts but also of the 
public.  Public discussion of these issues previously took place in the FDA Biological Response 
Modifiers Advisory Committee (BRMAC) - Subcommittee on Xenotransplantation; the 
Department of Health and Human Services (DHHS) Secretarys Advisory Committee on 
Xenotransplantation (SACX); and other public fora.i 
 
                                                 
i The BRMAC Subcommittee on Xenotransplantation (SACX) was initiated in 2001 and was discontinued in 2005. 
Further discussions on xenotransplantation have taken place in the Cellular, Tissue, and Gene Therapy Advisory 
Committee (CTGAC).  In addition, a discussion took place in 2009 on the topic of Animal Models for Porcine 
Xenotransplantation Products Intended to Treat Type 1 Diabetes or Acute Liver Failure.  
2  Contains Nonbinding Recommendations 
 
In 1996, the Public Health Service (PHS) published a Draft Public Health Service (PHS) 
Guideline on Infectious Disease Issues in Xenotransplantation (61 FR 49920, September 23, 
1996) that was revised and issued on January 29, 2001 (66 FR 8120), based on public comments 
received and advances in fields relating to xenotransplantation[1] (hereafter referred to as PHS 
Guideline).  This FDA guidance document reiterates many of the concepts in the PHS 
Guideline but, in addition, includes specific advice, regarding all aspects of xenotransplantation 
product development and production and xenotransplantation clinical trials. 
 
Since the publication of the PHS Guideline, the World Health Organization (WHO) published 
the Second Global Consultation on Regulatory Requirements for Xenotransplantation Clinical 
Trials.[2]  
 
FDA anticipated that its approach to regulation of xenotransplantation products would evolve as 
the scientific knowledge in the area of xenotransplantation continued to accumulate.  Since the 
issuance of the final guidance in 2003, FDA has issued additional guidance that contains 
recommendation relevant to certain xenotransplantation products; see references.[3-8]  FDA 
realizes that it may not be appropriate to every aspect of the guidance to every 
xenotransplantation product.  For example, some of the recommendations for animal husbandry 
may not be needed for xenotransplantation products consisting of well-characterized, long-
established animal cell culture lines or human cells co-cultured with such lines. 
 
 
III.  DEFINITIONS AND ABBREVIATIONS 
 
Act:  The Federal Food, Drug, and Cosmetic Act (21 U.S.C. 321 et seq.). 
 
AAALAC:  Association for Assessment and Accreditation of Laboratory Animal Care, 
International.  This organization inspects and accredits biomedical animal facilities. 
 
Agents of concern:  For the purpose of this document, agents of concern are infectious agents 
that may pose a risk to the recipient and/or public, i.e., agents that can infect, potentially could 
infect, or have an inadequately defined ability to infect humans.  
 
ATCC:  American Type Culture Collection. 
 
BLA:  Biologics license application.  Approval of a biologics license application or issuance of a 
biologics license constitutes a determination that the establishment(s) and the product meet 
applicable requirements to ensure the continued safety, purity, and potency of such products. 
 
BSL:  BioSafety Level. 
 
CDC:  Centers for Disease Control and Prevention.  
 
CDRH:  Center for Devices and Radiological Health. 
 
3  Contains Nonbinding Recommendations 
 
cGMP:  Current good manufacturing practice.  For drugs, including biological drugs, cGMP 
regulations can be found at 21 CFR Parts 210 and 211.  For biological products, see  
21 CFR Part 600 Subpart B and Part 610.  For blood and blood components, additional 
regulations can be found at 21 CFR Part 606.  For devices, quality system regulations can be 
found at 21 CFR Part 820. 
 
Closed herd or colony:  Herd or colony governed by standard operating procedures (SOPs) that 
specify criteria restricting admission of new animals to assure that all introduced animals are at 
the same or higher health standard, compared to the residents of the herd or colony. 
 
CPE:  Cytopathic effects.  An effect on nucleated cells in vitro caused by some viruses that are 
observable microscopically. 
 
CVM:  Center for Veterinary Medicine. 
 
DPF:  Designated pathogen free.  This term is used to describe animals, animal herds, or animal 
facilities that have been rigorously documented to be free of specified infectious agents and that 
are maintained using well-defined routines of testing for designated pathogens and utilizing 
rigorous SOPs and practices of herd husbandry and veterinary care to assure the absence of the 
designated pathogens. 
 
EM:  Electron microscopy.  A method used to visualize very small objects, such as subcellular 
particles, or infectious agents, such as viruses. 
 
FDA:  Food and Drug Administration. 
 
GE:  Genetically engineered.  
 
Gnotobiotic:  The science of rearing laboratory animals, the microflora and microfauna of which 
are specifically known in their entirety. 
 
GVHD:  Graft versus host disease. 
 
HEPA:  High-efficiency particulate air. 
 
IACUC:  Institutional Animal Care and Use Committee.  A local institutional committee 
established to oversee the institutions animal program, facilities, and procedures.  An IACUC 
carries out semiannual program reviews and facility inspections and reviews all animal use 
protocols and any animal welfare concerns.  (See Public Health Service Policy on Humane Care 
and Use of Laboratory Animals, revised 2015.) 
 
IBC:  Institutional Biosafety Committee.  A local institutional committee, established to review 
and oversee basic and clinical research conducted at that institution.  The IBC assesses the safety 
of the research and identifies any potential risk to public health or the environment.  (See section 
IV-B-2 of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid 
Molecules.) 
4  Contains Nonbinding Recommendations 
 
 
ICH:  International Council for Harmonisation of Technical Requirements for Pharmaceuticals 
For Human Use. 
 
IDE:  Investigational device exemption application.  These are applications containing requests 
to use an unapproved device in clinical tests using human subjects.  The statutory requirement is 
at section 520(g) of the Federal Food, Drug, and Cosmetic Act (the Act) (21 U.S.C. 360(g)), and 
the implementing regulations can be found at 21 CFR Part 812. 
 
IND:  Investigational new drug application.  These applications are required for persons who 
intend to conduct clinical investigations involving unapproved drug products, including those 
subject to section 505(i) of the Act (21 U.S.C. 355(i)) or to the licensure provisions of section 
351 of the PHS Act (42 U.S.C. 262).  The statutory requirement is at section 505(i) of the Act 
(21 U.S.C. 355(i)), and the IND regulations are found at 21 CFR Part 312. 
 
INAD:  Investigational new animal drug. 
 
IRB:  Institutional Review Board.  A board, committee, or other group, designated by an 
institution established to review and approve biomedical and behavioral research involving 
human subjects in order to protect the rights and welfare of human subjects (See 21 CFR Part 56, 
Institutional Review Boards.) 
 
Lot:  Defined in 21 CFR 210.3(b)(10) as a batch or a specific, identified portion of a batch, 
having uniform character and quality within specified limits, and in 21 CFR 600.3(x) as that 
quantity of uniform material identified by the manufacturer as having been thoroughly mixed in 
a single vessel.  Each lot of final product is subjected to appropriate tests to ascertain adherence 
to specifications prior to release of the product for clinical use.  Licensed biological products 
may be subject to lot release as described in 21 CFR 610.2(a).  Often, in the case of 
xenotransplantation products, an entire lot is used for treating a single recipient. 
 
Master File:  Master Files are submitted to FDA and contain information regarding a product, 
such as product manufacture or general procedures.  Procedures and information contained in the 
Master File can be cross-referenced in INDs and IDEs on written permission from the Master 
File sponsor, but confidentiality of the information within the Master File is maintained.  
(See 21 CFR 314.420.) 
 
NADA:  New animal drug application. 
 
PBMC:  Peripheral blood mononuclear cells. 
 
PCR:  Polymerase chain reaction.  An enzymatic technique, using a thermophyllic enzyme to 
catalyze synthesis of short DNA sequences, that allows detection of nucleic acids by 
amplification of specific DNA sequences. 
 
PERV:  Porcine endogenous retrovirus(es). 
 
5  Contains Nonbinding Recommendations 
 
PHS Act:  The Public Health Service Act (42 U.S.C. 201 et seq.). 
 
PMA:  Premarket approval application.  This is a marketing application for certain devices under 
section 515 of the Act.  The regulations for PMAs can be found at 21 CFR Part 814. 
 
Recipient:  An individual who receives or who undergoes ex vivo exposure to a 
xenotransplantation product (as defined in xenotransplantation). 
 
RT:  Reverse transcriptase.  An enzyme found particularly in retroviruses that catalyzes the 
synthesis of DNA from RNA. 
 
SAF:  Source animal facility. 
 
Sentinel animal:  Those animals, usually of the same species as the resident herd or colony, 
housed in direct contact with the animals being monitored and tested for the purpose of detecting 
adventitious agents in the herd or colony, including viral, fungal, and bacterial diseases as well as 
endo- and ectoparasites. 
 
SOP:  Standard operating procedure. 
 
Source animal:  An animal from which cells, tissues, and/or organs for use in xenotransplantation 
are obtained. 
 
TSE:  Transmissible spongiform encephalopathy.  TSEs are fatal, subacute degenerative diseases 
of humans and animals with characteristic neuropathology (spongiform change and deposition of 
an abnormal form of a prion protein present in all mammalian brains).  TSEs are experimentally 
transmissible by inoculation or ingestion of diseased tissue.  The abnormal prion protein is 
hypothesized to be the agent of transmission.  Alternatively, other unidentified co-factors or an 
as-yet-unidentified viral agent may be necessary for transmission. 
 
USDA:  United States Department of Agriculture. 
 
WFI:  Water for Injection. 
 
Xenograft product(s):  Xenograft products are acellular products derived from animal tissue.  
These xenografts, are beyond the scope of this guidance.  Please consult CDRH for guidance on 
xenograft products.[9] 
 
Xenotransplantation:  For the purpose of this document, any procedure that involves the 
transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or 
organs from a nonhuman animal source; or (b) human body fluids, cells, tissues, or organs that 
have had ex vivo contact with live nonhuman animal cells, tissues, or organs. 
 
Xenotransplantation product(s):  For the purpose of this document, xenotransplantation products 
include live cells, tissues, or organs used in xenotransplantation (defined above). 
 
6  Contains Nonbinding Recommendations 
 
Zoonosis:  A disease of animals (e.g., brucellosis, rabies) that may be transmitted to humans 
under natural conditions. 
 
Zoonotic:  Relating to diseases that arise from the transfer of infectious agents by normal 
contacts between animals and humans. 
 
 
IV.  REGULATORY RESPONSIBILITY 
 
FDA has regulatory oversight of xenotransplantation products, including live organs, tissues, or 
cells from a nonhuman animal source or xenotransplantation product materials used in 
encapsulated form or in which nonhuman live organs, tissues, or cells have ex vivo contact with 
human body fluids, cells, tissues, or organs that are subsequently given to a human recipient.  If 
xenotransplantation products are to be used in clinical investigation, they require an appropriate 
investigational application be in place (e.g., 21 CFR Part 312).  FDA will regulate most 
xenotransplantation products as biological products.  CBER regulates biological products, 
including cellular therapies, under authority of section 351 of the PHS Act (42 U.S.C. 262) and 
the Act (21 U.S.C. 321 et seq.).  You will find regulations for drugs, biological products, and 
devices in Title 21 of the Code of Federal Regulations (e.g., 21 CFR Part 312 for regulations 
governing INDs, 21 CFR Part 812 for regulations governing IDEs, and 21 CFR Part 601 for 
regulations governing licensing of biological products).  To assist developers of cell therapy 
products in preparing IND submissions to the Agency, FDA has published guidance on 
regulatory considerations in the development of somatic cell therapy and gene therapy and the 
content and review of chemistry, manufacturing, and control information for cell therapy product 
INDs.[3, 10]  In addition, for products derived from GE animals, please refer to 21 CFR Part 511 
and Guidance for Industry:  Regulation of Genetically Engineered Animals Containing Heritable 
Recombinant DNA Constructs[4] for recommendations regarding regulations governing 
investigational new animal drug that are regulated by FDAs Center for Veterinary Medicine 
(CVM).  The primary responsibility for designing and monitoring the conduct of clinical trials 
rests with the sponsor (e.g., 21 CFR 312.23(a)(6)(iii)(d) and 312.50).  In this document, you 
refers to the sponsor, the clinical investigator, or to any party designated by the sponsor to fulfill 
the recommended function (e.g., SAF staff, laboratory personnel, etc.). 
 
Some products may be combination products consisting of a biologic and a device, such as 
xenogeneic cells contained in a device used for extracorporeal hemoperfusion.  Refer to  
21 CFR Part 3 for issues regarding the regulation and assignment for premarket review of 
combination products.  
 
 
V.  SOURCE ANIMAL CHARACTERIZATION 
 
A.  General Considerations 
 
The cross-species infectious potential of specific animal pathogens should be a major 
consideration in the selection of source animal species.  Anatomic and physiologic 
considerations are also important.  For example, whether an organ is of appropriate size 
7  Contains Nonbinding Recommendations 
 
and will function adequately across species barriers are considerations, as are certain 
immunologic concerns, including the suitability of current regimens in prevention of 
rejection of the nonhuman animal live cells, tissues, or organs.  Please note that the 
species you use may be subject to other federal laws and regulations (see, e.g., 16 U.S.C. 
1538), such as those covering endangered or protected species.  You should consult all 
relevant PHS and FDA guidance documents on this subject prior to submitting an 
application and, specifically, should consult the document, Guidance for Industry:  
Public Health Issues Posed by the Use of Nonhuman Primate Xenografts in Humans[11] 
before submitting an application to FDA that involves the use of nonhuman primates as 
sources of a xenotransplantation product.  (The term xenograft as used in the above 
referenced document is synonymous with the term xenotransplantation product 
however, these terms are no longer synonymous in current use (see Definitions)). 
 
Due to potential infectious disease risks associated with the use of xenotransplantation 
products, you should develop appropriate source animal qualifications.  These 
qualifications should include herd management and programs for prevention and 
screening for infectious agents.  Although testing of the final xenotransplantation product 
for infectious agents is crucial, appropriate control of animal sources and husbandry 
provides important additional assurance for the safety of such products by controlling 
infections by both known and potentially even unknown agents.  Therefore, the specific 
information supplied by the sponsor, regarding animal husbandry including housing, 
feeding, veterinary care, and drug and biologic treatment of source animal herds and 
individual source animals, will be crucial for FDA evaluation of the potential for safe use 
of cells, tissues, or organs from such source animals. 
 
The SAF, production process, and records are subject to FDA inspection under section 
704 of the Act (21 U.S.C. 374) and section 351(c) of the PHS Act (42 U.S.C. 262(c)). 
 
B.  Animal Welfare Concerns 
 
Another area of consideration for SAFs and manufacturers of xenotransplantation 
products is the welfare of the source animals.  Procedures for animal husbandry, tissue 
harvesting, and termination of animals should be approved by an appropriate Institutional 
Animal Care and Use Committee, in accordance with the Animal Welfare Act (7 U.S.C. 
2131, et seq.).  In cases where funds are received from the PHS, procedures must also 
comply with the PHS Policy on Humane Care and Use of Laboratory Animals, according 
to section 495 of the PHS Act (42 U.S.C. 289(d)).  We recommend that the SAF be  
accredited by the AAALAC.  Standards for accredited facilities for when funds are 
received from the National Institutes of Health are provided in the National Research 
Councils Institute for Laboratory Animal Research, Guide for the Care and Use of 
Laboratory Animals. 
 
8  Contains Nonbinding Recommendations 
 
C.  Animal Origin 
 
1.  Animal and Herd Qualification 
 
You should derive source animals only from closed herds with documented health 
screening programs.  Individuals with expertise in infectious diseases of the 
species involved should develop a list of viruses, bacteria (including the 
rickettsiae), mycoplasma, fungi, transmissible spongiform encephalopathies 
(TSEs), and parasites for which the herd is screened and supply this information 
to FDA as part of the application to FDA (e.g., IND).  You should consider all 
infectious agents known to infect the source species.  You should justify the 
rationale for omitting agents that are found in the source animal species from the 
herd screening program in the application to FDA requesting investigational use 
(e.g., IND).  For example, the geographic location of the herd may allow 
exclusion of certain infectious agents.  You should obtain source animals from 
TSE-susceptible species only from closed herds that are documented to be free 
from TSE diseases or TSE-associated agents[12] (see also section V.C.3.c.).  You 
should not use source animals obtained from geographic areas in which TSEs are 
known to exist in the source species.  In the application to FDA requesting 
investigational use of the xenotransplantation product (e.g., IND), you should 
describe and justify the frequency of the screening, the method of assay, and the 
method of identifying which and what proportion of animals are sampled.  As 
data are accumulated that demonstrate product safety, you may modify the 
screening program in consultation with the FDA. 
 
2.  Animal History 
 
The sponsor should document the geographic origin, species, strain, and 
genealogy of the source animal(s) and herd(s).  The documentation of source 
animal history should describe factors that may pose risks to recipients, such as 
possible exposure of the predecessor animals to sources of TSEs or other 
adventitious or infectious agents of concern (see Definitions, section III.).  Source 
animals should be bred and raised in captivity and be derived from closed herds.  
You may use artificial insemination, embryo transfer, cloning, or hysterotomy 
plus foster feeding to establish animal herds with fewer endemic pathogens.  In 
particular, the PHS Guideline suggests that breeding programs use caesarean-
derived animals, whenever possible.[1]  You should document the use of these 
procedures in the animal history. 
 
3.  Source Animals from Outside the U.S. 
 
a.  You should not use animals from outside the U.S. or their first 
generation offspring as sources for the production of 
xenotransplantation products unless they are of a species or strain 
not available in the United States or have specific qualities that 
9  Contains Nonbinding Recommendations 
 
provide a unique and scientifically justified clinical advantage, 
such as transgenic animals. 
 
b.  If the use of source animals from outside the U.S. is necessary and 
justifiable, you should apply the same considerations for these 
animals as for source animals bred in the U.S. (e.g., see section 
V.D. for Animal Health and Husbandry).  You should use a 
quarantine period of sufficient length to demonstrate the absence 
of infectious agents of concern, and you should perform extensive 
screening of the animals.  In the application to FDA requesting 
investigational use of the xenotransplantation product (e.g., IND), 
you should submit thorough documentation to demonstrate that 
such source animals have been derived from closed herds, have 
been housed under appropriate conditions and subjected to 
recommended health maintenance procedures and screenings, and 
have not been fed rendered or recycled mammalian materials for at 
least two generations.  You should include in the screening agents 
that are endemic in the country of origin.  In the application to 
FDA requesting investigational use of the xenotransplantation 
product (e.g., IND), you should describe methods and conditions 
of transport of imported animals.  Descriptions should include 
means of transport and husbandry during transport, including 
isolation, caging, handling, animal treatment, and presence of other 
animals of the same or different species.  If animals from countries 
outside the U.S. are needed, you should use them as founders for a 
domestic herd that will be well-characterized for an extended 
period of time before use, using procedures sufficient to validate 
the herds acceptability as source animals. 
 
c.  You should not import source animals from any country or 
geographic region where TSEs are known to be present in the 
source species. For these purposes, only countries that are listed by 
the World Organisation for Animal Health (OIE) as having 
negligible risk of BSE in cattle should be considered 
acceptable.[13]  Use of the OIE BSE risk status for source animal 
importation is consistent with current USDA regulations  
(9 CFR Parts 92-96 and 98) with respect to importation of bovines 
and bovine products.[14] 
 
d.  You should consult the USDA and, when appropriate, CVM and 
CDC for their requirements regarding importation of animals or 
animal tissues. 
 
10  Contains Nonbinding Recommendations 
 
4.  Range and Wild Animals 
 
You should not use source animals that are raised under free-ranging conditions.  
Such animals have a higher likelihood of harboring infectious agents, due to 
potential exposure of the source animal to other animals, birds, insects, or other 
uncontrolled environmental factors.  You should not use wild-caught animals as 
source animals. 
 
5.  Animal Sources Obtained from Slaughterhouses or Abattoirs 
 
Animals from slaughterhouses or abattoirs are unsafe for use as source animals. 
Appropriate documentation and histories of animals from slaughterhouses may 
not be available because the animals are often obtained from geographically 
divergent farms or markets, and exposure to other animals or potential sources of 
infectious agents during transit or after arrival at the slaughterhouse are unknown.  
Therefore, you should not use such animals as source animals. 
 
6.  Semen Donors 
 
You should apply the same source animal considerations to semen donors, 
whether or not they are members of the herd, including, for example, screening 
for infectious agents that may be transmitted by semen. 
 
D.  Animal Health and Husbandry 
 
Production of animals as sources of live cells, tissues, or organs for use in 
xenotransplantation products involves an adequately designed facility and a program for 
the operation of the facility to minimize the animals exposure to infectious agents. 
 
You should obtain source animals exclusively from SAFs.  Your application or 
submission to FDA should include detailed plans for maintaining source animals.  These 
plans should include SOPs, detailing the containment and housing of animals, feeding 
and obtaining feed, water and bedding, performance and monitoring of the health 
screenings, removal from production and disposal of the animals and their byproducts, 
and identifying individual animals and recording their movements to, through, and out of 
the facility.  These procedures should take into consideration the source animal species 
and xenotransplantation product(s) as appropriate. 
   
1.  Facilities 
 
You should house the animals in facilities built and operated in accordance with 
recommendations described in the National Research Councils Institute for 
Laboratory Animal Research, Guide for the Care and Use of Laboratory Animals 
and accredited by the AAALAC.  You should not locate SAFs in geographic 
proximity to manufacturing or agricultural activities that could compromise the 
facilitys biosecurity by providing or enabling a source of infections. 
11  Contains Nonbinding Recommendations 
 
 
SAFs are subject to the regulations in 21 CFR Part 600, Subpart B, on 
establishment standards, including the requirements regarding animals and 
personnel in 21 CFR 600.10 and 600.11.  SAFs also are subject to the regulations 
in 21 CFR Part 600, Subpart C, regarding inspections.  These facilities are subject 
to inspection by designated representatives of the clinical protocol sponsor and 
public health agencies. 
 
You should include a detailed description of the facilities and procedures for 
housing source animals with the FDA submission (e.g., IND or Master File).  The 
information provided should include plans for the shelters, the feeding areas, the 
washing areas, the fencing, air handling systems (particularly in quarantine areas), 
lighting, temperature, and other physical attributes of the animal environment.  
Facility descriptions should also include information on physical barriers and 
operational measures intended to eliminate or minimize exposure to insects, birds, 
or other animals that may transmit disease to the source animals.  You should 
keep records of any biological or physical compromise of the animal environment 
as well as measures taken in response to this problem.   
 
These descriptions should also cover the procedures and schedules followed for 
cleaning and other routine maintenance of the animal enclosure.  You should 
include procedures for elimination of animal wastes.  Include in the description 
how qualified source animals will be housed (for example, as a batch or as 
individuals) and the methods used to decontaminate the housing after the source 
animals are used.  The SAF staff should include veterinarians with expertise in the 
infectious diseases and agents prevalent in the particular animal species being 
raised in the facility.  If a veterinarian with expertise in infectious diseases is not 
on staff, you should provide documentation in the investigational application 
(e.g., IND) that an individual with the appropriate expertise is available for 
consultation.  Staff should also include adequate numbers of caretaker personnel 
with appropriate training in the care and health of the species being housed (e.g., 
21 CFR 600.10 and 600.11).  Developers of xenotransplantation products should 
consider becoming ISO 9001:2015[15]certified. 
 
2.  Maintenance of Source Animals  
 
a.  General 
 
You should maintain source animals in accordance with standard 
operating procedures appropriate to the species, xenotransplantation 
product, and the intended clinical application.  SOPs should provide for 
admission of new animals to the SAF and source animal pool, for 
quarantine, and for removal, isolation, or elimination of diseased animals.  
You should provide this information to FDA in the application for 
investigational use (e.g., IND).  You should not reintroduce animals that 
have been removed from the source animal pool due to illness or infection. 
12  Contains Nonbinding Recommendations 
 
 
You should develop procedures to identify incidents that negatively affect 
the health of the herd or colony.  This information is relevant to the safety 
review of every xenotransplantation product application.  You should 
report such information to FDA as well as procedures to collect the 
information as part of the application for investigational use (e.g., IND). 
 
b.  Health Screening 
 
i.  You should maintain source animals in barrier facilities that 
are considered free of designated pathogens.  For the 
purpose of this document, such facilities are termed 
Designated Pathogen Free (DPF), and animals derived 
from them are termed DPF animals.  Initial screening and 
routine monitoring are important to validate that such 
facilities maintain DPF status.  Protocols for monitoring the 
herd for disease and infectious agents should exist, and you 
should include a copy or a summary of the SOPs in the 
FDA submission requesting investigational use (e.g., IND).  
You may modify the frequency of testing as the reliability 
of the production system is established using data from 
earlier screens.  You should consult appropriate experts, 
such as infectious disease consultants, virologists, 
microbiologists, accredited microbiological laboratories, 
and veterinarians, to generate a list of agents for which all 
source animals should be screened and a list of appropriate 
diagnostic tests.  In addition to screening for specific 
infectious agents, you should use more general assays for 
detection of classes of agents.  For example, you should 
examine feces from source animal herds on a regular basis 
for evidence of parasitic infections.  If infectious agents, 
including normal flora that could potentially be infectious 
in an immunosuppressed recipient, have been identified in 
source animals, the use of such animals should be avoided.  
You should consult with CBER if the use of such animals 
is contemplated (see, for example, section VII.C.4.d.).  
Techniques for introducing new animals, such as artificial 
insemination, caesarean section, cloning, or novel 
gnotobiotic techniques, should be fully described. 
 
ii.  Subclinical infections of source animals may not be 
apparent at the time of harvest of the nonhuman live cells, 
tissues, or organs and may be identified only 
retrospectively.  Sampling of individual animals from the 
herd of origin for screening and the use of sentinel animals 
should help minimize this problem and may help identify 
13  Contains Nonbinding Recommendations 
 
infections in recipients, post transplantation.  You should 
consider establishing a sentinel animal program that 
includes periodic necropsy and extensive histology and 
pathology evaluations.  The screening procedures should be 
appropriate for the animal species, the xenotransplantation 
product, and the clinical application.  Specific screening 
procedures should include appropriate physical 
examination and laboratory tests, and should be able to 
detect zoonoses known to exist in the species or 
geographical regions in which the source animals originate 
and are maintained.  For example, a sentinel animal 
program for ruminants might specify that the program 
maintain some animals into old age and test all diseased 
animals eliminated from the herd as well as all sentinels at 
necropsy for evidence of BSE using USDA-approved 
postmortem diagnostic tests for BSE with brainstem tissue 
collected as specified by USDA.[16] 
 
iii.  You should quarantine and screen individual source 
animals before harvest of cells, tissues, or organs, as 
discussed in section V.D.4.b., below, and in the PHS 
Guideline[1]. 
 
c.  Health Care 
 
The herd health surveillance system should include comprehensive 
documentation of all veterinary care received by source animals.  This 
includes documentation of all illnesses, medical care, procedures, drugs 
administered, vaccinations, routine physical exams, and any treatments 
received by each animal.  You should carefully document use of 
antimicrobial agents due to potential risk to allergic recipients receiving 
unprocessed nonhuman animal live cells, tissues or organs.  You should 
validate residual drug levels to be insignificant in cells, tissues, or organs 
taken from source animals that previously have received medications.  
Exclusive use of killed vaccines generally is warranted both in the source 
animal and in the herd with which it is associated.  You should use live 
vaccinations only when alternative immunogens for vaccinations are not 
available and only if scientific evidence exists to support that the live 
cells, tissues, or organs from the vaccine-treated animal will not pose a 
risk of infection for the human recipient.  You should describe the 
procedures to deal with illnesses or other incidents that affect the health of 
the herd in your application to FDA for investigational use.  You should 
not use as source animals those animals requiring treatment with blood, 
blood products, or tissues obtained from animals outside the closed herd, 
and you should remove these animals from the herd.  You should use 
aseptic techniques and sterile equipment for all parenteral interventions, 
14  Contains Nonbinding Recommendations 
 
including vaccinations, treatment with drugs or biologics, phlebotomy, 
and biopsies.  If animals within the closed herd have been treated with a 
biological product (e.g., vaccine, monoclonal antibody), you should 
document the treatment in the application to FDA requesting 
investigational use (e.g., IND), and you should provide copies of package 
inserts or labeling.  You should document and maintain records showing 
the treatment of animals with drugs for any reason, including the 
withdrawal period following drug treatment.  You should develop 
procedures for disposal of dead animals (see section V.G.). 
 
d.  Feed 
 
You should describe the storage and delivery of feed, water, and other 
consumables in the application to FDA for investigational use (e.g., IND).  
Records should include manufacturer, batch numbers, and other pertinent 
information, and you should describe recordkeeping procedures in an 
SOP.  You should record in the individual source animals records the 
vendor and contents of feed given to a source animal for at least two 
generations before use as a source for live cells, tissues, or organs used in 
xenotransplantation.  You should not use feeds containing animal proteins 
or other cattle materials that are prohibited by the FDA feed ban as 
expanded in 2008 as source animals (21 CFR 589.2000).[17]  You should 
not use feeds containing significant drug contamination or pesticide or 
herbicide residues for source animals (21 CFR 589.2001).[9]  You should 
not use natural, non-sterile foods, such as hay, to minimize potential risks 
of exposure to pests or infectious agents.  Water should be of sufficient 
quality to prevent unnecessary exposure of animals to infectious or 
adventitious agents, drugs, pesticides, herbicides, and fertilizers.  You may 
include pasteurized milk products in feeds.  You should feed newborn 
animals colostrum or milk from dams only if the dams have been 
specifically qualified by the same procedures used for herd qualification. 
 
e.  Caretakers 
 
You should provide SOPs for animal caretakers in the FDA submission 
requesting investigational use (e.g., IND), and you should include entry 
and exit procedures, clothing requirements, and all interactions with the 
animals, e.g., feeding, watering, exercising, delivery of immunizations and 
medications, etc. (e.g., 21 CFR 600.11).  There should be a documented 
training program for personnel as described in the cGMP regulations  
(21 CFR 211.25). 
 
You should monitor the health of humans in contact with animals on a 
routine basis.[18]  You should predetermine and customize the program for 
screening and monitoring of caretaker and other staff to maximize 
screening information, and you should describe the program in an SOP.  
15  Contains Nonbinding Recommendations 
 
Health monitoring of humans who come into contact with the animals 
should include physical exams with baseline and, if indicated, either 
periodic sampling and storage of serum or plasma for individuals having 
frequent and close contact with source animals or less rigorous monitoring 
for those with occasional contact.  You should obtain baseline samples 
from all caretakers. 
 
3.  Animal and Personnel Traffic through the Source Animal Facility 
 
You should develop SOPs for entry and exit of animals, and they should include 
transportation of animals to and from the facility.  You should subject all animals 
entering the facility to a defined quarantine period, allowing for completion of 
any screening procedures.  The minimum quarantine period for animals used in 
manufacture is seven days (21 CFR 600.11(f)(2)).  However, you should use 
longer quarantine periods that extend beyond the incubation period for infectious 
agents in the source animal species for animals entering a SAF.  You should 
devise a tracking system that allows unique identification of each individual 
animal in the facility.  You should minimize entry and exit of animals and human 
staff to avoid exposures to transmissible infectious agents.  We encourage the use 
of an all in/all out or batch approach for moving qualified source animals as a 
method of minimizing the potential for infectious agent transmission. 
 
You should describe personnel traffic patterns in the FDA submission requesting 
investigational use (e.g., IND) and should minimize transmission of infectious 
agents. Caretakers should not work in more than one animal facility or with more 
than one species of animal.  Caretakers should not work with more than one 
isolated group of animals or more than one herd within any given day unless 
validated SOPs for caretaker decontamination and disinfection are used.  
 
4.  Individual Source Animal Qualification  
 
a.  Testing for infectious agents 
 
You should screen all individual source animals for presence of the same 
infectious agents used for herd qualification.  In addition, you should 
perform further laboratory tests for infectious agents as described in 
section V. for testing of the xenotransplantation product (e.g., viral co-
cultivation assays) on appropriate samples of source animal blood or 
tissue.  When fetal or neonatal animals will be used as source animals, you 
should conduct testing of the mothers, which may supplant testing of the 
fetus or neonate if technical and temporal difficulties render such testing 
unfeasible. 
 
When feasible, you should examine a biopsy of the live animal cells, 
tissue, or organ or other relevant tissue (e.g., contra-lateral kidney as a 
surrogate for a kidney graft or thoracic tissue as a surrogate for a heart) by 
16  Contains Nonbinding Recommendations 
 
histopathology and tested for infectious agents by appropriate assays.  You 
should take care in performing biopsies to use fastidious sterile technique 
so as not to introduce new microbial contaminants.  You should archive 
remaining biopsy tissue as described in section V.E.3. 
 
You should perform all tests at a time as close as possible to the date of 
harvest of the live cells, tissues, or organs but which allows the results to 
be obtained before their use.  If more than 3 months have elapsed since the 
initial testing or biopsy of the source animal, you should repeat tests 
before harvest. 
 
You should consider the nature, timing, and results of surveillance of the 
herd from which the individual animal is procured in designing 
appropriate screening of individual animals. 
 
b.  Quarantine 
 
You should generally quarantine individual source animals for a minimum 
of three weeks before harvest of their live cells, tissues, or organs.  It may 
be appropriate to modify individual quarantine periods, depending on the 
characterization and surveillance of the source animal herd, the design of 
the facility, and the clinical indication.  If the quarantine is shorter, you 
should provide justification in the application to FDA for investigational 
use (e.g., IND).   During the quarantine period, in addition to tests for 
infectious agents, source animals should undergo physical examination by 
a veterinarian, including complete blood count, peripheral blood smear, 
and fecal exam for parasites. 
 
E.  Harvest of Nonhuman Live Cells, Tissues, or Organs for Use in Producing 
Xenotransplantation Products 
 
1.  Harvest and Documentation 
 
You should describe the procedures and physical facilities used for harvesting of 
live cells, tissues, or organs from source animals in detail in the application to 
FDA requesting investigational use (e.g., IND).  Qualified and controlled 
procedures for avoiding the introduction of infectious agents during harvesting 
should be in place.  Qualification of the procurement and screening procedures 
should include documented performance of the processes, with documented 
results supporting successful harvest of live cells, tissues, or organs from source 
animals that meet lot release criteria including identity, potency (or activity), and 
safety (e.g., microbiological sterility).  Source animal anesthetic agents should not 
be harmful to the human recipient.  A summary of the health records (e.g., health 
status and microbiological screening reports, results of lot release tests, and  
17  Contains Nonbinding Recommendations 
 
anesthetic used, if relevant) regarding the source animal should accompany the 
xenotransplantation product, and you should incorporate them into the recipients 
records. 
 
SOPs should permit rapid, accurate, and facile tracking of tissue from an 
individual source animal to the recipient. 
 
2.  Transportation 
 
Transportation of source animals may expose them to risks not encountered in 
closed herds and should be avoided if possible.  Therefore, we recommend that, 
when feasible, and particularly in cases where source animal tissues or cells are 
going to be processed further prior to use, live cells, tissues, or organs should be 
procured at the animal facility prior to shipping.  In some cases, particularly when 
the xenotransplantation product is a whole organ intended for immediate 
transplantation, it may be necessary to ship live animals.  In those cases where 
transportation is necessary, you should maintain barriers, equivalent to or better 
than those in place at the SAF during transit, to ensure that source animal 
contamination does not occur en route.  Transportation should occur in dedicated 
vehicles in which source animals are not exposed to any other animals, and you 
should document the method in the submission to FDA (e.g., in the IND).  If there 
is any question regarding the effectiveness of the transportation and containment 
procedures, you should quarantine and rescreen animals in a fashion comparable 
to that used for entry of new animals into a closed herd. 
 
You should develop and implement procedures for avoiding shipping errors, 
avoiding contamination, and documenting transfer of animal materials to the 
correct patient.  You should describe in detail the method of transporting the live 
animal cells, tissues, or organ from harvest site to the clinical xenotransplantation 
site in the application to FDA requesting investigational use (e.g., IND). 
 
3.  Source Animal Sample Archive 
 
a.  Timing of Sample Acquisition 
 
If the source animal is sacrificed at the time its live cells, tissues, or organs 
are harvested, you should conduct a full necropsy, including gross, 
histopathological, and microbiological evaluation, and you should obtain 
archival samples, including portions of the product for storage as 
described in section V.E.3.b. 
 
If the source animal is not sacrificed at the time its cells, tissues, or organs 
are harvested, you should archive portions of the harvested material and 
plasma and leukocytes from the source animal, and you should monitor 
the health of the source animal for life. 
 
18  Contains Nonbinding Recommendations 
 
When source animals die or are euthanatized, you should perform a full 
necropsy, and you should obtain archival samples for storage as described 
in section V.E.3.b. 
 
b.  Samples to be Archived and Storage Conditions 
 
You should store archived samples of source animal tissues and body 
fluids at -70 degrees Celsius or lower temperatures, as appropriate for 
preserving the sample, or you should maintain fixed samples at room 
temperature.  Section 3.7.1 of the PHS Guideline[1] recommends that you 
cryopreserve at least ten 0.5 cc aliquots of citrated- or EDTA-
anticoagulated plasma and at least five aliquots of viable leukocytes 
(1x107/aliquot, for subsequent isolation of nucleic acids and proteins or for 
use as a source of viable cells for co-culture or other tissue culture assays).  
You should select the conditions of cryopreservation and storage for 
viable samples to maintain cell viability for the period of storage (see 
section V.E.3.c.).  You should collect appropriate tissue samples for 
formalin fixation and paraffin-embedding and for cryopreservation from 
source animals at the time the live cells, tissues, or organs are procured.  
You should collect and cryopreserve tissue samples representative of 
major organ systems of source animals (e.g., spleen, liver, bone marrow, 
central nervous system, lung,) at necropsy.  As appropriate to the 
xenotransplantation product, you should archive other body fluids, such as 
cerebrospinal fluid, at the time of procurement of the product and/or 
necropsy.  If sentinel animals are used, you should also archive tissue 
samples and body fluids obtained at necropsy. 
 
c.  Archive Rationale, Duration, and Responsibility 
 
The PHS Guideline[1] recommends that a sufficient quantity of materials 
be harvested and cryopreserved for three different uses: 
 
i.  dedicated sample(s) for use by the PHS, 
 
ii.  for use if needed for recipient diagnosis and care, and 
 
iii.  for use by the sponsor, as appropriate. 
 
You should include detailed plans for obtaining and storing the archive 
samples in the application to FDA requesting investigational use (e.g., 
IND).  The PHS Guideline[1] recommends that samples should be stored 
for 50 years from the time of sample acquisition.  You should clearly 
designate responsibility for the archives and access to the specimens. 
 
19  Contains Nonbinding Recommendations 
 
4.  Herd Records 
 
You should keep records pertaining to the source animals and facilities.  These 
records are subject to inspection, and you should maintain the records for 50 years 
beyond the date of procurement of the nonhuman animal live cells, tissues, or 
organs for use in xenotransplantation. 
 
5.  Disposition of Records on Closing of a Source Animal Facility 
 
If a SAF ceases operation, all records and archived samples should be transferred 
to the respective sponsors, or the sponsors should be notified of a new archive 
site.  You should make provisions for all records to be maintained for the 
requested period in the event that the establishment ceases operation.  If you are a 
sponsor who plans to cease doing business, you should consult FDA regarding the 
disposition of records and archive samples. 
 
F.  Source Animal History for Xenogeneic Cell Lines 
 
Cell lines from animals may be established and used in the production of 
xenotransplantation products.  You should include the history of the cell line in the 
application to FDA requesting investigational use.  Especially for long-term cultures, it 
need not always include all the detailed information about the source animal and source 
animal facility mentioned above.  However, it should include, at a minimum, the species 
and tissue of derivation.  You should include, whenever possible, information such as age 
and sex of the source animal, laboratory of derivation, date of derivation, and the 
immediate provider of the cell line.  For short-term cultures, (e.g., derived less than one 
year previously), you should also include a description of the husbandry and health status 
of the particular source animal and herd or colony used to derive the cells.  The history of 
the cell line should also include the above information on feeder cells or animals used for 
passage in vivo if such techniques were used to develop the cell line. Information 
regarding the commercial source and country of origin of any animal products used in 
deriving or maintaining the cell line should also be included in the history.  You should 
characterize and test the final product as described in section VII.  You may also consult 
the Points to Consider in the Characterization of Cell Lines Used to Produce Biological 
Products[19] for pertinent recommendations regarding the production, identification, and 
characterization of cell lines used in manufacture.  Additional information may be found 
in relevant ICH Guideline.[20] 
 
FDA has considered how this guidance relates to human embryonic stem (hES) cell lines 
that were in existence prior to August 9, 2001.  These hES cell lines had used murine 
feeder layer cells and, thus, fit the definition of xenotransplantation used in this guidance 
and the PHS Guideline.  FDA does not intend that the Agencys regulation of 
xenotransplantation will preclude the use of these hES cell lines.  It may be necessary for 
a sponsor who wishes to investigate a stem cell product derived from existing hES cell 
lines in a clinical trial to demonstrate to FDA that the hES cell line is free from infectious 
agents, including murine infectious agents.  Given current technology, a sponsor should 
20  Contains Nonbinding Recommendations 
 
be able to do this without undue burden.  For example, it will not be necessary for a 
sponsor to provide FDA with complete animal husbandry information for mice from 
which the mouse feeder layers were derived.  If continued expansion on murine feeder 
cell layers is planned, FDA may request that a sponsor demonstrate that the mouse 
colonies, from which such murine feeder layers are to be obtained, are maintained 
appropriately to ensure safety.  (See section V. of this guidance and section 3 of the PHS 
Guideline[1]).  The same recommendations apply to other xenotransplantation products 
that contain human cells with a history of co-culture with nonhuman animal cells. 
 
G.  Disposal of Animals and Use of Byproducts 
 
There is a need for advance planning for the ultimate disposition of source animals, 
including those animals in which the insertion of genetic information failed (no-takes) 
and sentinel animals bred for use in producing xenotransplantation products, especially 
animals of species ordinarily used to produce food.  Food or feed derived from such 
animals may be adulterated under the Act.  Generally, you should not use such animals as 
sources of human food via milk or meat or as ingredients of feed for other animals.  You 
should not use such animals as pets or breeding animals because of the potential for them 
to enter the food/feed chain, either directly or through the rendering process.  You should 
dispose of source animals in a manner consistent with the disposal of medical waste in 
compliance with federal, state, and local requirements. 
 
There may be infrequent situations where animals from xenotransplantation facilities can 
be considered safe for human food use or as feed ingredients when disposed of through 
rendering.  Persons wishing to offer animals into the human food or animal feed supply 
or who have food safety questions should first consult with FDAs Center for Veterinary 
Medicine.  CBER will refer food safety issues from sponsors to CVM, or you may 
contact CVM directly through the Division of Compliance, HFV-235, FDA, Center for 
Veterinary Medicine, 7500 Standish Place, Rockville, MD 20855, 240-276-9227.  
 
 
VI.  CHARACTERIZATION OF XENOTRANSPLANTATION PRODUCTS 
 
A.  General Considerations 
 
In general, you should test the xenotransplantation product for safety, identity, purity, and 
potency.  Part 610 (21 CFR Part 610) describes types of assays that are required for 
licensed biologics.  You should use similar tests during investigational stages of product 
development.  We discuss in more detail assays for safety testing, including infectious 
agent tests and tests for endotoxin, in section VII. of this document.  Assays for testing 
identity and potency will depend on the product, itself.  Assays for purity should include 
tests for endotoxin or pyrogen and, for certain xenotransplantation products, should 
include measurements of cell populations in the xenotransplantation product.  For further 
guidance in this section, see references.[20-22]  
 
21  Contains Nonbinding Recommendations 
 
Additional recommendations and comments regarding microbiologic tests are found in 
section VII. of this document. 
 
B.  Considerations for Classes of Xenotransplantation Products 
 
1.  Xenotransplantation Products Used Immediately after Procurement from 
the Source Animal 
 
When xenotransplantation products are transplanted directly after removal from a 
source animal, it may not be possible to perform all tests on the final product and 
have the results available before use.  However, you should use a biopsy of the 
organ or a relevant surrogate sample (e.g., adjacent tissues or contra-lateral 
organs) for assay of the xenotransplantation product.  Safety analyses should 
include fungal and bacterial sterility, mycoplasma, and virus testing.  You should 
also perform tests for endotoxin or pyrogen.  Although we realize that results of 
these tests will not be available before transplantation, you should still complete 
assay or culture periods and record the results.  As appropriate, you may use 
histology, performed on a retention sample or biopsy of the xenotransplantation 
product, to document identity of the product. 
 
2.  Stored or Processed Xenotransplantation Products 
 
For live xenogeneic cells or tissues that are stored, processed, or expanded ex 
vivo, in addition to safety testing, you should perform additional product 
characterization to measure identity, purity, and potency.  As much as possible, 
results of these assays should be available before xenotransplantation and used for 
lot release.  You should also apply these same product characterization steps to 
xenotransplantation products comprised of human cells that have had ex vivo 
contact, for example, by co-culture, with cells or tissues of nonhuman origin. 
 
a.  Safety 
 
We discuss tests for bacterial and fungal sterility, mycoplasma, and viral 
agents, generally considered safety tests, in detail in section VII. of this 
document. 
 
b.  Identity 
 
You should develop a means to assess identity of the active component of 
the xenotransplantation product.  This may include identification of 
relevant cell or tissue types, using immunological, immunohistological, or 
biochemical cell markers.  In some cases, it may be possible to use 
histological evaluation.  Depending on the manufacturing process, 
verification of the species or strain identity of the final product may be 
necessary, such as when the SAF handles more than one strain or species 
of animal. 
22  Contains Nonbinding Recommendations 
 
  
c.  Purity 
 
If the final product is a heterogeneous xenotransplantation product, i.e., a 
tissue, possessing several types of cells, or a cellular implant, containing 
extraneous tissues or cells which may be incompletely removed during 
tissue dissection, cell processing, or ex vivo culture, the determination of 
the purity of the cell population is especially important.  You should 
develop a quantitative method to assess the presence of the putatively 
active cell type as well as contaminating cell types in the final product.  
You may achieve this, for example, using morphologic, histologic, 
molecular genetics, biochemical and/or immunocytochemical techniques 
to identify contaminating cells and/or their products.  For 
xenotransplantation products comprised of human cells that have had ex 
vivo contact with cells or tissues of nonhuman origin, you should perform 
quantitative assays to assess the presence of nonhuman cells in the final 
product.  You should validate purity assays.  Purity assays are important 
for production of a consistent product.  You should use results of such 
tests as a lot release specification if possible.  In those cases where the 
final product is a purified population of cells of a single or few types, such 
as an established cell line, you should still test the product for purity, and 
you should develop tests for identity of the cells. 
 
You should measure endotoxin levels on the final product, and results 
should be available for use as a lot release specification.  We also discuss 
tests for endotoxin in the context of tests for infectious agents (section 
VII.C.3.). 
 
d.  Potency 
 
You should perform potency assays that measure and reflect the intended 
biological activity of the final product.  For example, potency assays may 
measure biologically active molecules secreted/produced by the 
xenotransplantation product, such as cytokines, hormones, or 
neurotransmitters.  If necessary, development of appropriate potency 
assays may proceed along with product development.  In addition, you 
should assay cell viability and use the results for lot release.[5] 
 
 
23  Contains Nonbinding Recommendations 
 
VII.  MICROBIOLOGICAL TESTING OF XENOTRANSPLANTATION PRODUCTS 
 
A.  General Considerations 
 
1.  Framework 
 
This section of the guidance document provides a general framework for the 
microbiological testing of xenotransplantation products.  We suggest some 
specific examples of tests and infectious agents.  However, we encourage 
sponsors to consider all available up-to-date information, regarding potential 
pathogens and testing strategies, to evaluate their own systems, to perform 
experiments to identify potentially infectious agents, and to propose and validate 
appropriate tests in consultation with CBER.  During the initial stages of 
investigations, complete validation for all assays may not be necessary, with the 
exception of standard sterility tests.  However, you should establish the 
specificity, sensitivity, and reproducibility for all procedures used to detect 
infectious agents to the extent possible. 
 
2.  General Biological Products Standards 
 
For general standards on testing of biologics for infectious agents, refer to  
21 CFR Part 610 (see references).[3, 10, 20-22] 
 
You can also find additional guidance on these issues as they relate to 
xenotransplantation in section 3.3 of the PHS Guideline.[1] 
 
3.  Inactivation or Removal of Infectious Agents 
 
Whenever possible, without compromising the integrity and effectiveness of the 
xenotransplantation product, we encourage you to develop and incorporate 
validated procedures for inactivation or removal of adventitious agents, infectious 
agents, or other microbiological contaminants into the manufacture of the 
xenotransplantation product. 
 
4.  Archiving 
 
You should cryopreserve and archive for further testing, as needed, samples of all 
final xenotransplantation products (i.e., cells or tissues or biopsies of organs), 
whether fresh or from culture ex vivo.  In some cases, for example if the 
xenotransplantation product is a whole intact organ, it may be acceptable to 
archive a relevant surrogate sample (e.g., adjacent tissues or contra-lateral organ).  
If the final product consists of human cells, you should archive tissues or organs 
that have been in contact ex vivo with live nonhuman animal cells, tissues, or 
organs and samples of the final product and the nonhuman animal cells, tissues, or  
24  Contains Nonbinding Recommendations 
 
organs.  As in the case of the animal source samples (see section V.E.3.c.), you 
should harvest and cryopreserve sufficient quantities and numbers of replicates of 
the xenotransplantation product for three different uses: 
 
a.  dedicated sample(s) for use only by PHS[1]; 
 
b.  for use if needed for recipient diagnosis and care; and 
 
c.  for use by the sponsor, as appropriate. 
 
You should include detailed plans for obtaining and storing archive samples in the 
application to FDA requesting investigational use (e.g., IND).  You should store 
samples for 50 years from the time of manufacture of the xenotransplantation 
product.  You should clearly describe responsibility for the archives and access to 
the specimens. 
 
If you are the sponsor, you should provide for all samples and attendant records to 
be maintained for the requested period of time in the event that an establishment 
ceases operation. 
 
B.  Considerations for Classes of Xenotransplantation Products 
 
1.  Xenotransplantation Products Used Immediately after Procurement from 
the Source Animal 
 
In procedures in which the xenotransplantation product is transplanted 
immediately after removal from the source animal, such as xenotransplantation of 
whole organs, results of testing of the xenotransplantation product may not be 
available before its clinical use.  In such cases, testing of the source animal, itself, 
may be all the testing that is possible before the procedure.  Testing of samples 
taken from such xenotransplantation products or appropriate relevant biological 
surrogates, e.g., adjacent tissues or contra-lateral organs, is also warranted, even 
though the results will not be available before use of the xenotransplantation 
product because results may contribute to patient management.  (See also section 
VI.B.1.) 
 
2.  Stored or Processed Xenotransplantation Products 
 
For xenogeneic cells or tissues that are stored, processed, or expanded ex vivo, 
you should accomplish or, at a minimum, initiate testing for infectious agents 
before xenotransplantation.  If cells or tissues are maintained in culture, you 
should validate cell culture procedures and reagents for maintenance of microbial 
sterility, including both xenogeneic infectious agents and other cell culture 
adventitious agents.  You should perform testing periodically during the culture 
period.  Performance of all tests at every time point may not be necessary, but you  
25  Contains Nonbinding Recommendations 
 
should provide FDA, as part of the application for investigational use (e.g., IND), 
with a scientific rationale to support the selection of tests performed at each given 
time.  As an example, you may test samples: 
 
a.  at the initiation of culture ex vivo; 
 
b.  before cryopreservation if performed as a step in manufacture; 
 
c.  as late as possible during culture, such that final results (or useful 
preliminary results) will be available before the release and use of 
the product; 
 
d.  two to three days before clinical use (e.g., for microbiological 
cultures used in lot release); and 
 
e.  at the time of final product harvest, though results may not be 
available before clinical use. 
 
3.  Xenotransplantation Product/Device Combination Products 
 
In certain biologic/device combination products, physical barriers may separate 
the xenogeneic component from human fluids or tissues and might prevent or 
reduce transmission of certain classes of infectious agents.  If such claims are to 
be made or are implicit, or if the existence of the physical barrier is to be used in 
lieu of certain other precautions to lower the risk of transmission, you should 
provide to FDA, as part of the application for investigational use (e.g., IND), the 
results of validation studies that demonstrate the inhibition of transmission of 
specific infectious agents and the maintenance of device/barrier integrity.  For 
specific guidance on the design of these types of studies, see reference.[23]  For 
example, if such claims are to be made in the patient informed consent document, 
you should provide the results of these studies in the application to FDA 
requesting investigational use; if such claims are to be made during marketing, 
you should provide the data in the marketing application.  The design of these 
studies should take into consideration the following parameters: 
 
a.  Conditions of normal physiologic use of the xenotransplantation 
product/device combination product and conditions under which 
the combination is subjected to physical and biological stress; 
 
b.  Use of infectious agents that are representative of those potentially 
present in the xenotransplantation product.  Note that the size and 
plasticity of selected agents should be considered; and 
 
c.  Use of agents that would demonstrate the physical properties of the 
barrier (i.e., permeability to viruses or other particles with differing 
properties, such as size, charge, hydrophobicity, shape, etc.). 
26  Contains Nonbinding Recommendations 
 
 
Without supporting data obtained from such studies, you should not assume that 
xenotransplantation products contained within a barrier present lesser risk of 
infection to humans than xenotransplantation products not contained within a 
barrier. 
 
C.  Assay Design for the Detection of Infectious Agents 
 
1.  General 
 
The choice of tests will vary, depending on the animal source, including the 
species, strain, and geographic origin, the type of tissue, the processing of tissue 
before use, and the proposed use or clinical indication.  You should give special 
consideration to infectious agents known to infect the source animal and those 
known to cause zoonoses.  You should base the list of infectious agents for which 
you will test on that used for individual source animal qualification.  We 
encourage discussions with CBER.  You should include data in the FDA 
application for investigational use to document the specificity, sensitivity, and 
reproducibility of novel assays used to detect infectious agent(s). 
 
2.  Tests for Bacteria, Fungi, and Mycoplasma 
 
You can find standards concerning the types of methods used for detection of 
bacteria, fungi, and mycoplasma in licensed biologics in 21 CFR Part 610.  You 
may use alternative methods during product development, but you should support 
use of such methods by data on the sensitivity, specificity, and reproducibility of 
the method.  For xenotransplantation products, you should obtain such data using 
infectious agents appropriate to the source animal species, geographic origin of 
the source animal, and the cells, tissues, or organ(s) to be used.  You should 
submit these data to FDA in the application for investigational use. 
 
In addition to testing the final product for viable organisms, you should perform 
Gram stains on appropriate samples of all final xenotransplantation products.  The 
results of these stains should be available before use of the product in humans, 
and you should set a negative Gram stain as a lot release criterion. 
 
3.  Endotoxin Test 
 
During the product development phase, you may perform a bacterial endotoxin 
test in lieu of the rabbit pyrogen test as described for licensed products  
(21 CFR 610.13(b)).  You should describe the type of endotoxin assay and its 
specificity and sensitivity in the application for investigational use submitted to 
FDA.  If you intend to use an endotoxin assay in lieu of the rabbit pyrogen test 
after licensure, you should demonstrate equivalency with the pyrogen test for the 
specific xenotransplantation product at or before the time of license application 
(21 CFR 610.9). 
27  Contains Nonbinding Recommendations 
 
 
Since it is possible to perform an endotoxin assay within a few hours, you should 
select and perform an appropriate assay on the final product.  Results should be 
available before use for any xenotransplantation product that has been cultured, 
stored, or processed for more than the few hours required to perform the assay.   
You should use these results as a lot release specification.  Consult the USP <85> 
and the FDA Guidance for Industry:  Pyrogen and Endotoxins Testing:  
Questions and Answers[6] for information on bacterial endotoxins testing.  
 
4.  Viruses 
 
a.  Culture Assays 
 
You should test xenogeneic cells used for xenotransplantation (fresh or 
cultured) by co-culture with a panel of appropriate indicator cells to 
amplify potential viral contaminants. The panel of cells used in this 
analysis should include a cell line representative of the source animal 
species, a cell line representative of the animal tissue(s) type used in the 
manufacture of the xenotransplantation product, and a human cell line.  
For additional guidance, see the Points to Consider in the 
Characterization of Cell Lines Used to Produce Biological Products.[19]  
This reference provides especially useful information for testing rodent 
xenotransplantation products for viral adventitious agents.  You may find 
additional information in Viral Safety Evaluation of Biotechnology 
Products Derived From Cell Lines of Human or Animal Origin 
Q5A(R1).[23]  When possible, you should also co-cultivate manipulated 
and/or unmanipulated source animal cells with recipient cells, such as 
peripheral blood cells.  You should routinely observe co-cultivation 
cultures for CPE, focus formation, RT activity, and changes in cell growth 
or other unexpected changes.  We recommend visualization of co-cultures 
by EM to identify morphologic changes or to recognize certain viruses.  
You should try to identify any viruses detected using immunoassay, PCR, 
or other assays using virus-specific probes.  At the end of the culture 
period, you should test cultures for hemagglutination and hemadsorption 
with erythrocytes of three different species[19].  Additional efforts may be 
necessary to characterize viruses that are detected that may be novel or for 
which specific probes may not yet be available. 
 
You should base lot release specifications on available data.  You should 
use these specifications for release of xenotransplantation products for 
which results can be available before administration of the product to 
humans, such as for products that can be cryopreserved.  For cells that are 
manipulated ex vivo, if time allows, you should perform viral tests during 
the period of culture or manipulation so that the results are available 
before delivery of the product to the recipient.  If it is not possible to 
obtain the results prior to use, you should still test samples of each product 
28  Contains Nonbinding Recommendations 
 
lot.  In these instances, you should qualify assay procedures and obtain 
data on a number of representative final product lots before beginning 
human trials. 
 
b.  Activation of Latent Viruses 
 
You should give special consideration to testing for viruses known to 
occur in the latent state.  Transmission of viruses with long clinical 
latencies is of concern, due to the possibility of transmission of these 
viruses from the recipient to the recipients contacts in the absence of 
symptoms or signs of disease.  Immunosuppression and transplantation, 
either alone or in combination, may activate latent viruses.[24]  
Manipulation or culture of cells ex vivo may also activate latent viruses 
(see references).[25, 26]  Which experiments might be appropriate to detect 
latent viruses in animal cells, tissues, or organs would depend upon the 
tissue type and the virus in question.  Examples of experiments that have 
been used to detect viral activation and may be useful in the 
xenotransplantation product setting include the following:  
 
i.  the expression of endogenous retroviruses is induced by 
culturing in vitro; or  
 
ii.  by treatment with iododeoxyuridine or demethylating 
agents, such as 5-aza-cytidine[27]; and  
 
iii.  cultivation in vitro of ganglia latently infected with Herpes 
simplex virus results in the production of infectious 
virus[28]. 
 
In certain cases, positive results may not necessarily preclude use of such 
tissue (see section VII.C.4.d. for information regarding 
xenotransplantation products containing porcine endogenous retrovirus(es) 
(PERV)), but the identification and characterization of the resulting virus 
may provide useful information and materials for monitoring the recipient 
of the xenotransplantation product for the presence of the activated virus 
(see section X.F.3.). 
 
You should attempt to evaluate the potential of the processing or clinical 
use of the xenotransplantation product to activate latent viruses (e.g., 
PERV) before use. 
 
c.  In Vivo Assays for the Detection of Viruses 
 
You should test xenotransplantation products by assay in vivo for 
detection of certain viruses that may not be found by culture methods in 
vitro.  For example, many serotypes of Coxsackie A virus are only 
29  Contains Nonbinding Recommendations 
 
detected upon inoculation of newborn mice.[28]  Therefore, we recommend 
that if there are no reliable in vitro assays, appropriate in vivo assays 
should be applied (see Points to Consider in the Characterization of Cell 
Lines Used to Produce Biological Products[19] and Viral Safety 
Evaluation of Biotechnology Products Derived From Cell Lines of Human 
or Animal Origin Q5A(R1).[23]  Certain in vivo assays, such as antibody 
production assays, may be particularly useful for testing for rodent 
viruses.[19, 23]  
 
d.  Assays Suitable for the Detection of Porcine Endogenous 
Retrovirus(es) (PERV) 
 
All live cells, tissues, or organs derived from pigs contain sequences for 
PERV in their genome[28].  It has been demonstrated in some primary 
porcine cells these sequences are expressed, resulting in production of 
infectious retroviruses.  In light of data demonstrating that PERV can 
infect human cell lines in vitro[19, 23] we recommend that all porcine-
derived xenotransplantation products be evaluated using appropriate 
assays for the production of infectious retrovirus.  You should assess 
several lots of each xenotransplantation product.  Each time major 
manufacturing changes occur, such as use of a new source or herd or 
method of product procurement, you should repeat testing for infectious 
PERV.[29] 
 
You should test xenotransplantation products (e.g., a fresh sample of the 
xenotransplantation product or relevant surrogate tissue, e.g., adjacent 
tissues or contra-lateral organs or the cultured xenogeneic cells) by co-
culture with appropriate indicator cells to amplify any infectious 
retrovirus(es).  Indicator cells that have been demonstrated to be 
permissive for PERV replication include the human embryonic kidney cell 
line 293 (American Type Culture Collection (ATCC CRL-1573)), mink 
lung fibroblasts (ATCC CCL-64), certain feline cell lines (such as PG-4, 
ATCC CRL-2032), and a swine testis (ST) cell line (ATCC CRL-1746).  
You should choose one or more of these cell lines for initial analysis of the 
porcine xenotransplantation product or appropriate relevant surrogate 
tissue.  After co-culture for a period of at least 30 days or 10 cell passages, 
you should analyze the cells for the transfer of PERV from the porcine 
cells to the indicator cell by either an optimized RT assay or use of PERV-
specific primers to amplify, by PCR, reverse-transcribed viral RNA or 
cellular RNA.  Evidence for virus production will not necessarily result in 
the xenotransplantation products being considered unsuitable for clinical 
use.  Rather, you should pursue additional characterization of the virus in 
consultation with Center for Biologics Evaluation and Research (CBER) 
in order to ensure appropriate reagents are available for recipient follow-
up (section X.F.).  Additional characterization may include analysis for the 
cell substrate most sensitive to infection by the particular strain of PERV 
30  Contains Nonbinding Recommendations 
 
present in the xenotransplantation product and sequence analysis of the 
infectious virus produced by the xenotransplantation product.  These steps 
will provide important information and development of diagnostic tools to 
optimize the protocols for follow-up of recipients for evidence of infection 
(section X.F.). 
 
 
VIII.  MANUFACTURING AND PROCESS-RELATED GMP CONSIDERATIONS FOR 
HARVEST AND PROCESSING OF XENOTRANSPLANTATION PRODUCTS 
 
A.  General Considerations 
 
You should use facilities for the harvest and/or processing of xenotransplantation 
products that have been designed to minimize the potential for contamination of the 
harvested and/or processed xenogeneic cells, tissue, or organs and cross-contamination 
between lots of these cells, tissues, or organs. 
 
For sponsors of investigational trials, you should phase in the validation activities 
described in this section, during the investigational phase, as the clinical studies progress 
toward submission of an application for approval to market (e.g., BLA).  The exception 
to this is sterility assurance validation, which you should complete before initiating 
clinical trials.  Manufacturing process controls should be in adherence to cGMP 
regulations (21 CFR Parts 210 and 211).  The IND regulations (21 CFR 312.23(a)(7)) 
allow that some controls may be introduced as appropriate for the phase of development. 
 
B.  Contamination/Cross-Contamination Precautions 
 
You should take precautions to prevent contamination/cross-contamination during 
harvest and manipulation of xenogeneic cells or tissues.  You should give consideration 
to:  
 
  personnel, animal, material and waste flow into and out of the facility;  
  proposed air cleanliness classifications;  
  cleaning/sanitizing agents used and demonstration of their efficacy in relation to 
facility isolates, viruses, and other potential adventitious agents; and  
  environmental monitoring and gowning procedures.  
 
1.  Flows 
 
Your facilities should be designed such that personnel, animal, material, product, 
and waste flows into and out of the facility exclude mixing of clean and dirty 
activities.  Ideally, flows should be one way so that personnel, animals, materials, 
and product enter and exit separately.  Using this design, waste would only exit 
through designated airlocks, pass-throughs, and/or autoclaves.  Alternatively, you 
may accomplish segregation of activities procedurally and/or temporally.  In this 
case, you should take special care to avoid contamination or cross-contamination.  
31  Contains Nonbinding Recommendations 
 
For example, more stringent cleaning and sanitization schedules should be in 
place. 
 
Of special concern is the transfer of animals to the harvesting area (i.e., operating 
room).  You should ensure that animals are prepared in such a way as to exclude 
potential surface contaminants, which may be carried from the animal facility.  
 
2.  Cleaning and Sanitizing Agents 
 
You should demonstrate that agents used for cleaning and sanitizing work 
surfaces and equipment as well as other surfaces in the harvesting and processing 
areas (e.g., floors, walls), are effective against facility isolates, viruses, and other 
potential adventitious agents. You should establish cleaning schedules which 
maintain acceptable control in relation to the activities performed in the specified 
area.  We expect that validation studies, demonstrating the efficacy of the agents 
used, will be performed as the trial progresses toward submission of a marketing 
application  
 
3.  Environmental Monitoring 
 
You should establish a program for monitoring the environment in the harvesting 
and processing areas, based on the criticality of the manufacturing process 
involved. 
 
You should perform nonviable particulate monitoring to verify air cleanliness 
classifications in the harvesting and processing areas (see section VIII.C.1. for 
recommended air cleanliness classifications).  This verification should include 
laminar flow areas in the harvesting area and biological safety cabinets in the 
processing area.  After initial verification, you should perform nonviable 
particulate monitoring at established intervals to demonstrate maintenance of the 
assigned air cleanliness classification.[30] 
 
You may monitor viable particulates, i.e., microbes, using a variety of techniques.  
The use of settling plates during harvesting and processing activities, while not 
quantitative, provides some assurance that the quality of the environment has not 
been compromised.  You should establish quantitative methods as clinical trials 
progress toward submission of a marketing application.  You should monitor 
surfaces, including those of personnel performing production activities (e.g., 
gloved hands), using contact plates or swabs to demonstrate the continued 
efficacy of the cleaning regimen, and maintenance of asepsis for personnel.  We 
recommend that personnel engaged directly in harvesting and processing 
activities be monitored at the conclusion of each critical activity (e.g., surgery, 
aseptic surgery).  Additionally, you may randomly sample operators performing 
cell expansion activities. 
 
32  Contains Nonbinding Recommendations 
 
4.  Changeover Procedures 
 
You should establish, follow, and document changeover procedures designed to 
prevent contamination between harvests of xenotransplantation products.  These 
procedures should include clearance of all materials and waste from the operating 
room or cell processing cabinet and cleaning/sanitization of surfaces.  In addition, 
you should address segregation procedures if multiple lots of xenogeneic cells or 
tissues are processed at the same time.  Adequate labeling of processing vessels 
(e.g., tissue culture flasks) and dedication of equipment or portions of equipment 
(e.g., shelves within incubators) are examples of such segregation procedures.  
Centrifuges used for processing are of particular concern in terms of cross-
contamination.  We recommend that only one lot of xenogeneic cells or tissues be 
centrifuged at a time.  You should demonstrate integrity of centrifuge tubes or 
closed systems employed, when possible.  You should adequately clean 
centrifuges between each lot operation. 
 
C.  Validation and Qualification 
 
As noted previously, validation and qualification efforts should be ongoing as clinical 
trials progress.  Minimally, we expect assurance that systems and equipment are 
functioning as needed.  You should submit validation protocols and data summaries to 
FDA for review as part of the ongoing investigational file. 
 
1.  Air Handling Systems 
 
HVAC systems should be designed to provide adequate air quality for harvesting 
and processing of xenotransplantation products.  You may use laminar flow units 
above the operating table to provide high quality air during harvesting operations.  
You may use biological safety cabinets to maintain aseptic conditions during 
processing.  We expect that this equipment will be capable of producing Class 
100 conditions for the most critical of processes although we understand that 
maintenance of these conditions may be difficult, during harvesting.  Minimally, 
the environment surrounding the Class 100 laminar flow units and/or biological 
safety cabinets should be Class 100,000.  Proceeding toward licensure, areas 
surrounding critical Class 100 processes should meet Class 10,000 conditions. 
 
Validation of these systems and units should include verification of air changes 
and pressure differentials and should achieve the desired cleanliness level (see 
section VIII.B.3.).  Testing of the HEPA filters contained in the system should 
address integrity and efficiency. 
 
Routine environmental monitoring (see section VIII.B.3.), pressure differential 
checks, and recertification of HEPA filters should demonstrate maintenance of the 
desired conditions. 
 
33  Contains Nonbinding Recommendations 
 
2.  Water 
 
We expect that water used to formulate necessary reagents or for critical cleaning 
purposes (i.e., equipment and surfaces in the harvesting and processing areas) will 
meet the United States Pharmacopoeia USP<1231> monograph on Water for 
Pharmaceutical Purposes[31].  If WFI is purchased, you should perform lot specific 
testing and validate hold times for open containers.  If WFI is generated at the 
facility, you should properly validate and routinely monitor the system to ensure 
continued quality. 
 
3.  Equipment 
 
You should adequately calibrate and qualify equipment used for harvesting and/or 
processing of xenogeneic cells and tissues.  You should then routinely monitor 
temperature-controlled equipment, such as refrigerators/freezers and incubators, 
to assure proper conditions.  Carbon dioxide supplied to incubators used for cell 
expansion should be 0.2 micron filtered to minimize the potential for 
contamination.  If water baths are used, you should use maintenance procedures 
for water quality.  This may include the addition of agents to control 
contamination. 
 
4.  Aseptic Processing 
 
Generally, manipulation or expansion of xenogeneic cells or tissues is an entirely 
aseptic process, i.e., there is no final sterilizing filtration of the product.  In order 
to validate this process, you should perform media fills (substitution of media for 
product) to demonstrate that sterility may be maintained consistently.  Assurance 
of sterility of the final product is necessary from the very beginning of the clinical 
studies[32].  You should adequately train and monitor personnel performing these 
functions to assure consistent performance during normal production.  
 
All product contact equipment should be sterile and free of pyrogens when 
aseptically processing cells or tissues.  You may use disposable labware where 
possible.  The sterility and depyrogenicity of the containers and closures used for 
the final product are of particular importance.  For equipment and components 
that are to be sterilized, there should be evidence that the autoclave cycle(s) is 
validated to provide an acceptable level of sterility assurance.  Minimally, you 
should establish and follow basic load configurations, and you should place 
biological indicators within each load to verify lethality.  As studies progress, we 
expect that formal validation of all sterilizing/depyrogenating processes will be 
performed. 
 
34  Contains Nonbinding Recommendations 
 
5.  Process Validation 
 
Ultimately, before licensure or product approval, you should validate all critical 
processes used to manufacture the product.  We have previously defined process 
validation.[7, 32] We expect that process validation, when performed, will be 
prospective and at full scale, with the exception of studies performed to 
demonstrate viral clearance (removal/inactivation).  Laboratory studies may also 
help to establish appropriate operating and process parameters and may be used in 
support of the formal study.  We expect that information on the validation 
protocol(s) and summaries of data resulting from its execution will be included in 
the license application. 
 
 
IX.  PRECLINICAL CONSIDERATIONS FOR XENOTRANSPLANTATION 
 
A.  General Considerations 
 
This section provides a general framework for the preclinical testing of 
xenotransplantation products before use in clinical trials.  You may also apply the general 
principles as set forth in the document generated by the ICH on the safety of 
biotechnology-derived pharmaceuticals to these products (see reference[33]).  In general, 
studies to support the safety characterization of therapeutic agents should focus on the 
intended alteration to the human pathophysiologic state (i.e., activity) as well as 
unintended effects (i.e., toxicity) to the host system.  Such studies serve to assess the 
potential for clinical risks and constitute an important component of an FDA regulatory 
application.  Preclinical studies are particularly valuable for gaining insight into safety 
issues which cannot be evaluated in human recipients for ethical or practical reasons.  
Consequently, you should design strong preclinical safety programs and also consult the 
FDA Guidance for Industry:  Preclinical Assessment of Investigational Cellular and 
Gene Therapy Products.[34] 
 
Specific considerations in the design of preclinical studies that are intended to support the 
safety of xenotransplantation products should include: 
 
1.  the animal source for the xenotransplantation product; 
 
2.  the tissues anatomic and physiologic similarity to its human homologue; 
  
3.  the determination of function of the xenotransplantation product; 
 
4.  the animal model system; 
 
5.  the integrity of the device components (if a device is used); 
 
6.  the dose levels (based on tissue mass as well as pharmacologic/metabolic 
activity or release kinetics of bioactive molecules); 
35  Contains Nonbinding Recommendations 
 
 
7.  the route of administration (site of implantation/injection, extracorporeal 
or ex vivo use); 
 
8.  the study duration (as related to potential human exposure); 
  
9.  reactions between source animal and host immune systems; 
 
10.  interspecies extrapolation (i.e., cross-species activity of secreted 
proteins/hormones at receptors); and 
 
11.  device biocompatibility. 
 
Since a primary intent of the preclinical animal and in vitro studies is to identify potential 
clinical risk factors, these evaluations should focus on maximizing the similarity between 
animal and human testing strategies in test substance, route of administration, and dosing 
regimen.  Animal models of xenotransplantation should utilize a xenotransplantation 
system evaluating the cell, tissue, or organ type being examined for use in humans and 
should utilize clinically relevant immunosuppressive therapy.  Rigorous preclinical 
program design is needed to ensure comparability of preclinical to clinical study design 
and is important for planning the clinical testing program, including selecting an 
appropriate clinical indication, inclusion/exclusion criteria, recipient monitoring scheme, 
dose, and concomitant therapies, as well as for advising potential recipients of risks 
(informed consent). 
 
B.  Issues Related to Infectious Agents 
 
Since the transfer of infectious agents that are pathogenic, latent, or even non-pathogenic 
in their natural animal host may cause serious disease in an immunosuppressed patient, 
you should consider the microbiologic burden carried by the xenotransplantation product 
as well as the immune status of the recipient in preclinical study designs.  Additionally, 
designs of preclinical studies should incorporate: 
 
1.  careful veterinary monitoring of animals, taking note of any early signs of 
infection; and 
 
2.  procedures needed to assign a cause of mortality (using appropriate 
serologic or immunohistochemical identification of pathogens). 
 
To prevent the spread of demonstrable or potential infectious agents, you should care for 
animals with appropriate precautions, including isolation if necessary.  Deaths from 
infections in animal models may occur, due to immunosuppressive regimens that may be 
intentionally more extreme than expected for use in humans in order to avoid rejection of 
the xenogeneic live cells, tissues, or organs, and to obtain proof-of-concept data.  
Therefore, data identifying cause of death (e.g., xenogeneic infectious agents or 
activation of latent host infection) could assist in interpreting human risk, may be helpful 
36  Contains Nonbinding Recommendations 
 
in refining animal experimental models, and may identify pathogenic infectious agents in 
the source animal.  Animal models of xenotransplantation, while exploring these issues, 
are limited by uncertainties in extrapolation of cross-species infectivity information; e.g., 
data indicating no infections in animal, even primate species, are not adequate to assure 
that humans will not be susceptible to infections transmitted by the xenotransplantation 
product. 
 
Additional insight into refinements of animal immunosuppressant regimens may come 
from evaluation of host resistance.  You may evaluate host immunocompetence by 
measuring resistance to infection by various pathogens, including those that may be 
contained within the xenotransplantation product. 
 
C.  Xenotransplantation Product-Host Interactions 
 
1.  Immunologic Rejection 
 
You should assess survival of the xenogeneic cells, tissues, or organs in animal 
models, with attention given to: 
 
a.  identifying infiltration of immune or inflammatory cells into the 
xenotransplantation product or alteration of such cells in other 
relevant compartments, such as the blood and cerebrospinal fluid; 
 
b.  fibrotic encapsulation of the xenotransplantation product, e.g., 
resulting in impaired function or xenotransplantation product loss; 
 
c.  xenotransplantation product necrosis; 
 
d.  any evidence of graft versus host disease (GVHD); 
 
e.  in vivo function and durability of encapsulation or barriers 
intended to diminish rejection or inflammatory responses; 
 
f.  any special concerns, regarding the site and nature of the 
xenotransplantation product; and 
 
g.  if relevant to the particular xenotransplantation product, the 
possibility that rejection of that product might predispose the 
recipient to rejection of subsequent xenotransplantation products or 
allotransplants. 
 
2.  Immunosuppression 
 
Preclinical animal studies in which xenotransplantation is used in an 
immunosuppressed host may raise questions, regarding the relevance of the model 
to clinical pharmacology, toxicology, or immunology.  Consideration of how both 
37  Contains Nonbinding Recommendations 
 
the host and source species handle the immunosuppressive drugs may be 
necessary (for instance, where a nephrotoxic drug is metabolized by hepatic 
enzymes), but intra-species differences in metabolism exist.  Immunosuppressive 
drugs often have very restricted therapeutic indices so that pharmacokinetics and 
metabolism may markedly affect the activity and/or toxicity of the agents in the 
host or xenotransplantation products.  You should attempt to delineate toxicities 
due to immunosuppressive drugs from toxicities due to the xenotransplantation 
product. 
 
You should consider and study, where appropriate, relative activity of 
immunosuppression on the source species of the live xenogeneic cells, tissues, or 
organs since immunosuppressive treatment that selectively suppresses immunity 
in the host species may be permissive to GVHD.  This might occur in cases where 
immunologically active cells are contained, either intentionally or inadvertently, 
within the xenotransplantation product. 
 
3.  Tumorigenicity in the Immunosuppressed Host 
 
In addition, the tumorigenic potential of the xenotransplantation product, perhaps 
due to altered cell growth regulation or to immunosuppression of the host, is an 
important concern (refer to section IX.E.). 
 
4.  Cross-Species Compatibility of Bioactive Molecules 
 
For xenotransplantation products for which you intend that the product synthesize 
and provide bioactive molecules, such as cytokines or hormones, you should 
provide to FDA, as part of the investigational application , data from preclinical 
experiments that support that the molecules produced will be active in humans.  
Experiments to address this issue should evaluate concentration-response issues.  
You should perform the experiments in vitro and/or in appropriate preclinical 
models in vivo. 
 
Even when the xenotransplantation product is composed of a single cell type, the 
product may secrete unintended molecules that could alter normal host 
physiology.  Moreover, host substances might affect product function.  Therefore, 
preclinical models should evaluate the overall health of the recipient (i.e., clinical 
signs, gross pathology, and histopathology) as well as markers of activity of the 
xenotransplantation product.  You may use combination toxicity and activity 
studies to evaluate both potential therapeutic and constitutive functions of the 
xenotransplantation product.  In some instances, the ability to biopsy 
xenotransplantation products periodically is potentially an available tool for 
evaluating the histopathologic status of the product and host immune response, 
especially when evaluated in conjunction with clinical chemistries.  You might 
also perform control experiments to test the in vivo effects of live xenogeneic 
cells, tissues, or organs taken from anatomic sites other than those used for 
therapeutic procurement of the xenotransplantation product but lacking the 
38  Contains Nonbinding Recommendations 
 
therapeutic cell or tissue type and its anticipated pharmacologic activity.  (Please 
note that this advice also pertains to heterogeneous xenotransplantation products 
as discussed in section IX.D.2.). 
 
5.  Migration of Xenogeneic Cells 
 
Cells from xenotransplantation products may migrate within the host, thus 
presenting clinical concerns regarding adverse reactions deriving from displaced, 
bioactive cells or unexpected anatomical impediments.  This may be especially 
true for incompletely differentiated cells (see section IX.D.3.) and may be 
evaluated in animals using histopathology, possibly coupled with enhancing 
techniques, such as fluorescent dye loading and/or species-specific antibodies, or 
more sensitive techniques, such as PCR. 
 
D.  Considerations for the Use of Heterogeneous Xenotransplantation Products 
 
You should apply the following principles to the development of appropriate preclinical 
testing of heterogeneous xenotransplantation products in order to assess potential adverse 
effects.  A xenotransplantation product may be considered heterogeneous if it is, for 
example, a tissue or solid organ, possessing many varieties of cells; or a cellular implant, 
containing extraneous tissues or cells which may be incompletely removed during tissue 
dissection or present in short term cultures ex vivo. 
 
1.  Characterization of Constituent Cell Types in a Heterogeneous 
Xenotransplantation Product 
 
The procedures used in preclinical studies for the collection, isolation, and, where 
relevant, activation or expansion of the xenotransplantation product, should 
mimic the procedures intended for use in clinical trials, and you should 
characterize cell types in the product being tested in an analogous fashion to the 
proposed clinical xenotransplantation product.  See section VI.B.2.c., regarding 
recommendations for the evaluation of purity of heterogeneous 
xenotransplantation products. 
 
2.  Secretion of Biologically Active Molecules by Xenotransplantation 
Products 
 
Uncharacterized cells or tissues present in the xenotransplantation product may 
produce biologically active molecules with unintended activities.  You should 
perform experiments to identify released, bioactive substances of potential 
biologic significance and appropriate to the particular xenotransplantation product 
(e.g., neurotransmitters, hormones, cytokines), whether by intended or extraneous 
cell types in the xenotransplantation product.  For example, you may maintain or 
culture in vitro samples of tissues being prepared for transplantation and test the  
39  Contains Nonbinding Recommendations 
 
supernatants for activities or relevant bioactive substances.  You should consult 
the ICH guidance document on safety preclinical evaluations in biotechnology for 
additional guidance on these studies.[33] 
 
In addition to assessment in vitro, you should evaluate heterogeneous 
xenotransplantation products in appropriate animal models.  (See section IX.C.4. 
for additional discussion of this topic). 
 
3.  Differentiation in Heterogeneous Xenotransplantation Products 
 
Xenotransplantation products derived from fetal animal sources, dedifferentiated 
cells, or tissues or cells, expanded ex vivo, may comprise a heterogeneous 
population with regard to cell maturity.  The degree of heterogeneity may depend 
on the cell or tissue type from which the xenotransplantation product is obtained, 
the period of fetal development during which the tissue is procured, and/or the 
time in culture.  For such products, preclinical studies should compare the viable 
cell types initially transplanted with those that exist subsequently in the 
xenotransplantation product.  This comparison may warrant preclinical studies 
with sequential sacrifice groups or biopsies.  Techniques, such as 
immunohistochemical staining, trypan blue exclusion, bioassays, or PCR assays, 
may be useful in identifying heterogeneous cell differentiation.  You should 
develop models to evaluate the effects of differentiation on the function of the 
xenotransplantation product, using, for example, measurements of release or 
secretion of biologically active molecules, including those that may not be 
intended for efficacy of the xenotransplantation product but that it may produce.  
Viable products may change over time as they respond to, adapt to, and 
functionally integrate with the host environment.  Therefore, you may monitor 
cell viability, morphology, and functional endpoints (e.g., endocrine, behavioral, 
or immunological) over time to guide development of clinical monitoring 
regimens. 
 
E.  In Vitro and In Vivo Tumorigenicity Models for Xenotransplantation 
Products Intended for Transplantation  
 
Tumorigenicity is an important part of preclinical testing for certain xenotransplantation 
products, such as those manipulated ex vivo.  For further guidance, applicable to this 
topic, see references.[3, 10, 33, 34] 
 
Xenotransplantation products may be tumorigenic in a new species because of various 
factors, such as transgenic manipulations, endogenous viruses, ex vivo culture, and 
immunosuppression of the host.  Therefore, for xenotransplantation products intended for 
implantation, you should consider evaluation of tumorigenicity in vivo and in vitro. 
 
1.  Multiple models exist for testing tumorigenesis in vivo.  The role of 
immune challenge, immunosuppressive drugs, and exposure to certain 
infectious agents comprise an important set of safety concerns that may be 
40  Contains Nonbinding Recommendations 
 
addressed preclinically.  Preclinical experiments should include careful 
evaluation of controls, background tumor growth rates, tumor incidence 
and type, location, and time of appearance of tumors over an extended 
period.  These should make use of histopathologic evaluation as a primary 
endpoint. 
 
2.  Colony formation in soft agar (clonogenic assays) and growth in organ 
culture may be useful in vitro assays of the tumorigenic potential, 
particularly for cell lines.  These tests may provide information on stability 
or abnormal characteristics of cell lines and may substitute for testing in 
animals if you demonstrate that the tests have equivalent sensitivity in 
your hands. 
 
For xenotransplantation products consisting of cells that have been 
expanded ex vivo, a change in cellular growth pattern, morphology, or 
growth factor dependence may suggest transformation and a need for more 
rigorous investigation. 
 
F.  Combinations of Xenotransplantation Products with Devices 
 
A number of products for therapeutic use are combinations of xenotransplantation 
products and device components, either for use as implants or extracorporeally.  All of 
the preceding recommendations in section IX. apply to such products.  These products 
also warrant further preclinical characterization for bioreactivity and biocompatibility of 
the device components.  Preclinical testing often will include characterization of the 
device intended for human use, rather than a homologous product that has been made in 
scale with a small laboratory species.  This, in turn, may dictate that the device be studied 
in an animal species with blood volume and size and, possibly, anatomic structures, 
similar to those of humans. 
 
Device elements may be reviewed jointly by staff in CBER and the Center for Devices 
and Radiological Health (CDRH).  Failure of device components (e.g., membranes and 
filters) that serve to isolate animal tissue from the recipient is an important aspect of 
safety assessment addressed by review staff in CDRH.  Additional device toxicity issues, 
also considered by staff at CDRH, are covered in the biocompatibility guidance published 
by the International Organization for Standardization. 
 
Implanted devices may be intended for permanent or extended residence in the human 
body.  Since these are considered chronic therapies, long-term risks (such as chronic 
inflammation, carcinogenicity, consequences of re-implantation, and local/systemic 
toxicities) of implanted xenotransplantation product/device combinations may necessitate 
evaluation before product premarket approval and, to some extent, before initiation of 
investigational studies in humans.  Studies before initiation of clinical investigational 
studies would usually last a minimum of 3 months.  
 
41  Contains Nonbinding Recommendations 
 
Membranes with pores may partially isolate xenogeneic tissue housed in devices from 
attack by host immune cells, but proteins and pathogens from the xenogeneic tissue may 
still be released into the host, along with desired pharmacologically active molecules.  
Such devices may reduce but may not eliminate a risk of xenogeneic infections.  They 
also may act as a stimulus, leading to local inflammation and fibrin deposition.  
Adhesions and granulomas may form in host tissue, and deposits on implants may 
interfere with activity and implanted cell viability.  You should evaluate encapsulated 
xenotransplantation products intended to be permeable to bioactive substances (such as 
encapsulated islets) for preimplantation activity, and you should retrieve and assess the 
products for activity, capsule integrity, and tissue viability after various periods of time in 
the animals. 
 
You should conduct extended animal studies (e.g., 12 to 24 months), using the clinical 
route of administration (e.g., implant site) and clinical grade materials.  You should 
design studies to include groups that elucidate reactions to the biomaterials, alone, as well 
as groups exposed to clinical and supraclinical doses of the complete product.  
Toxicology studies for implanted biomaterials which have previously been utilized in 
non-cellular devices may be relevant to safety determinations of the 
xenotransplant/device combination products but cannot completely satisfy the need for 
toxicity evaluation of the new product in its complete clinical form.  You should be aware 
that later changes in manufacture of the xenotransplantation product may necessitate new 
toxicology studies. 
 
For devices used for extracorporeal hemoperfusion, you should conduct studies to 
evaluate the hemodynamic effects of establishing and discontinuing the extracorporeal 
circuit, the products released from the tissues housed in the device (e.g., proteins that 
could cause anaphylactic responses or stimulate unintended autoimmunity), the 
deposition of blood cells (such as platelets) on device tubing or other components, 
coagulation or complement activation, and removal of drugs from the recipients 
circulation through filtration or device-localized cellular metabolism.  Assessment of the 
biologic activity of the combination product is often a component of preclinical safety 
evaluations.  For instance, studies should evaluate the duration and predictability of 
cellular (e.g., cell cartridge) activity so that you may replace the biologic component of 
the device at appropriate intervals to maintain life-supporting pharmacologic or metabolic 
activity. 
 
In summary, you should design animal studies of xenotransplantation product/device 
combinations, as with other preclinical experiments, taking into consideration all aspects 
of the clinical trial and proposed patient population, the need to study both desired and 
undesired activities of the xenotransplantation product, and toxicities at the local and 
systemic levels. 
42  Contains Nonbinding Recommendations 
 
X.  CLINICAL ISSUES IN XENOTRANSPLANTATION  
 
A.  General Considerations 
 
This section provides general principles rather than specific guidance.  Since the 
available basic knowledge and clinical experience with xenotransplantation are limited, 
current issues may be resolved as new knowledge is acquired and new concerns may 
emerge. 
 
B.  Clinical Protocol Review 
 
Sponsors are responsible for ensuring reviews, as appropriate, by local review bodies, 
including Institutional Review Boards (IRBs), Institutional Animal Care and Use 
Committees (IACUCs), and Institutional Biosafety Committees (IBCs).[1] 
 
In addition to the human subject protection issues traditionally addressed by local IRBs, 
institutional review of xenotransplantation clinical trial protocols should also address: 
 
1.  the potential risks of infection for the contact populations (including health 
care providers, family members, friends, and the community at large); 
 
2.  source animal husbandry (e.g., screening program, animal quarantine); and 
 
3.  adequacy of the proposal to address issues related to human and veterinary 
infectious diseases (including virology, laboratory diagnostics, 
epidemiology, and risk assessment). 
 
C.  Xenotransplantation Site 
 
The PHS Guideline recommends that all clinical xenotransplantation procedures be 
performed in transplantation centers with appropriate experience and expertise for 
comparable allotransplantation procedures and with the capability to culture and to 
identify viral agents, using in vitro and in vivo methods, either on-site or through active 
and documented collaborations.[1] 
 
D.  Criteria for Patient Selection 
 
Due to the potentially serious public health risks of possible zoonotic infections, you 
should limit xenotransplantation to patients with serious or life-threatening diseases for 
whom adequately safe and effective alternative therapies are not available, except when 
very high assurance of safety can be demonstrated.  You should limit candidates to those 
patients who have potential for a clinically significant improvement with increased 
quality of life, following the procedure.  You should also consider the patients ability to 
comply with public health measures as stated in the protocol, including long-term 
monitoring. 
 
43  Contains Nonbinding Recommendations 
 
E.  Risk/Benefit Analysis 
 
We understand that the lack of other therapeutic options and the severity of disease may 
raise the benefit-to-risk ratio for some individuals.  However, consideration and 
evaluation of risks and benefits of xenotransplantation should address both recipient and 
public health concerns.  You should consider the following in providing a benefit-to-risk 
analysis. Infectious disease is among the potential risks both to the recipient and to the 
public posed by the use of xenotransplantation products.  You should describe and 
examine the likelihood and uncertainties associated with any potential recipient and 
public health consequences of each of the following events.  Transmission of microbial 
agents from xenotransplantation products could lead to systemic disease (for example, 
infection or neoplasia) or failure of the xenotransplantation product in the recipient.  In 
addition, transmission of infectious agents could result in outbreaks of zoonotic disease, 
silent transmission of latent viruses, or emergence of new strains of pathogens.  
Experience has shown that widespread horizontal or vertical transmission of new 
pathogens is possible before the pathogens are recognized (e.g., Human 
Immunodeficiency Virus).  You should describe and examine the possibilities and 
uncertainties associated with any other types of adverse events that could have potential, 
significant recipient or public health impact, arising from the xenotransplantation 
product.  Similarly, you should describe any immunological risks, including rejection of 
the live xenogeneic cells, tissues, or organs, and, in some cases, GVHD. 
 
F.  Screening for Infectious Agents 
 
Consult the PHS Guideline[1] for additional guidance and information on testing 
recipients of xenotransplantation products. 
 
1.  Infectious Agents of Concern 
 
Infectious agents of concern will differ among source animal species and among 
cell or tissue types within each species.  Therefore, you should individualize 
clinical tests for the specific xenotransplantation product in question.  The 
categories of infectious agents of concern include bacteria (such as the 
rickettsiae), fungi, mycoplasma, viruses, and the agent(s) causative for TSEs.  
Tests should be available for agents known potentially to be present, including 
those that are pathogenic in the source animal species and agents that are known 
to infect human cells in vivo or in vitro.  You should have the capability to test for 
latent viruses or pathogens, and you should be prepared to develop and validate 
clinical tests for new pathogens that may not be recognized at the time of 
xenotransplantation.  You should identify specific infectious agents for which 
tests will be performed.  (See section X.F.3. for additional information on testing.) 
 
44  Contains Nonbinding Recommendations 
 
2.  Collection and Analysis of Clinical Samples 
 
You should only perform xenotransplantation at clinical centers with available 
state-of-the-art virology and microbiology laboratories that include a staff with 
knowledge and experience in the isolation and identification of unusual 
pathogens.  In addition, there should be access to laboratory facilities where viral 
cultures can be done in vivo, such as in embryonated eggs and suckling mice.  
You should place specimens into viral transport medium at the bedside, store at 4 
degrees Celsius, and inoculate into cell cultures as soon as possible and always 
within 24 hours of collection.  The sample(s) selected for culture will depend 
upon clinical evaluation of the recipient.  In your investigational application to 
FDA (e.g., IND), you should describe tissue cell culture systems.  These may 
include primary monkey, primary human embryonic kidney, semicontinuous 
human diploid, and continuous human heterodiploid cells.  If isolation remains 
difficult, then inoculation in vivo, e.g., into embryonated hens eggs and/or 
suckling mice, may be necessary.  In addition to culture, you may examine tissue 
by EM.  Immunohistopathology, immunofluorescent antibody, 
radioimmunoassay, Enzyme-Linked Immunosorbent Assay, and PCR may be 
helpful when appropriate antibodies and probes are available. 
 
3.  Testing and Scheduling of Testing of Recipients for Infectious Agents 
 
In your investigational application to FDA, you should describe tests of clinical 
specimens from recipients for specific agents of concern.  The tests may include, 
for example, serological and culture assays.  You should also describe tests for 
latent agents known to be in the source animal species (e.g., retroviruses, 
herpesviruses). Assays should be able to distinguish between an infectious agent 
derived from the source species and a related infectious agent present in humans 
(i.e., porcine versus human Cytomegalovirus (CMV)).  Data should be available 
to demonstrate specificity, sensitivity, and reproducibility for all tests that are not 
in widespread use or for newly-developed tests.  In some cases, completion of 
development of new tests, which have already demonstrated some level of utility, 
specificity, and reproducibility, may proceed concurrently with the clinical trial. 
 
You should also describe the schedule for screening of recipients for infections in 
the application for investigational use. 
 
a.  Acute Infections 
 
Recipients are at risk for the same infections that are common among 
individuals who have received allografts.  In general, these infections will 
be related to the use of immunosuppression and will arise from the 
recipients endogenous flora, reactivation of latent infections, and 
environmental sources.  The detection methods useful for these diseases 
will not differ from the methods used to detect infections after 
allotransplantation.  
45  Contains Nonbinding Recommendations 
 
 
In addition to being at risk for these infections, a recipient may be at risk 
for infection by agents contained in the xenotransplantation product.  
Little clinical experience exists with xenogeneic infectious agents 
infecting humans from xenotransplantation products.  We anticipate that a 
recipient will be at greatest risk for infection during the first few months 
after the procedure.  However, there may be significant delay in the 
clinical manifestations of infection in some cases.  The timing of 
occurrence of infectious episodes may vary, depending on 
immunosuppression.  It is important that you collect relevant data, during 
the clinical trial and for the lifetime of the recipient, and that investigation 
of acute infectious episodes includes appropriate tests.  It is difficult to 
predict the diagnostic symptoms and signs of such infections in the 
immunosuppressed patient.  When the source of a recipients post-
transplant illness remains obscure, you should perform testing on 
appropriate fluid and tissue samples. Such testing should include the use 
of serology as well as various cell and microbial culture systems and in 
vivo systems.  Culturing may detect infections that serologic testing has 
missed (for example, when immunosuppressed transplant recipients are 
unable to mount the usual immunological response to a pathogen). 
 
Patient care workers who work with acutely ill recipients should follow 
recommended procedures for handling and disinfection/sterilization of 
medical instruments and disposal of infectious waste[35].  When there is a 
suspicion of a possible xenogeneic infection, you should notify FDA 
promptly if a non-xenogeneic causative infectious agent is not readily 
identified and notify FDA immediately if a potentially xenogeneic, 
causative infectious agent is identified. 
 
b.  Chronic Infections 
 
An immunosuppressed recipient will be at risk for infection by the 
pathogens most commonly associated with allotransplantation.  In 
addition, you should consider pathogens potentially derived from the 
source animal.  With adequate preclinical and xenotransplantation product 
testing before the procedure, the most likely chronic pathogens from the 
animal may be endogenous or exogenous viruses although parasites, such 
as Toxoplasma, should be considered. 
 
c.  Routine Screening for Clinically Inapparent Infections and 
Seroconversions 
 
In addition to diagnostic testing when a recipient appears ill, it is 
important to establish ongoing recipient screening programs.  Sponsors 
should describe and validate their screening programs, taking into 
46  Contains Nonbinding Recommendations 
 
consideration the source animal species and type of cell, tissue, or organ 
used. 
 
i.  Passive Screening Program 
 
In passive screening programs, appropriate clinical samples, such 
as blood, plasma, urine, etc., are obtained periodically and archived 
for possible future testing.  In the event of a diagnosed infection or 
the onset of symptoms that may represent infection in one 
recipient, these samples are then available for retrospective 
screening of persons, regardless of the presence or absence of 
symptoms, who shared a common or similar exposure to a 
xenotransplantation product.  We recommend that a passive 
screening program be accomplished through an established 
schedule for routine sample collection and storage of samples from 
asymptomatic recipients.  Such a passive screening program would 
be in addition to the collection and archiving of biologic specimens 
designated for PHS use as described in the PHS Guideline[1].  
However, the time points identified by the PHS Guideline as 
appropriate for archiving specimens designated for PHS use also 
provide guidance on the minimal frequency with which specimens 
should be obtained and stored as part of a passive screening 
program.  These time points include: 
 
  before xenotransplantation (two samples, one month apart);  
  at the time of transplantation; 
  in the immediate post-transplant period; 
  at one month and six months after transplant; 
  annually, for the first two years; and 
  every five years, subsequently. 
 
In certain cases, more frequent acquisition of samples may be 
appropriate. The sponsor should consider the animal source and 
type of product in proposing the schedule and tests to be used in 
the passive screening program. 
 
See section X.H. for recommendations regarding the number, size, 
use, and duration of storage of collected samples. 
 
47  Contains Nonbinding Recommendations 
 
ii.  Active Screening 
 
In addition to a passive screening program, you should consider an 
active screening program.  Possible mechanisms of active 
screening range from testing of samples immediately after 
collection from recipients to periodic performance of specific 
additional laboratory tests on a subset of the samples collected in 
the passive screening program.  In addition, you should consider 
implementation of centralized review of routinely collected clinical 
data to detect trends suggestive of emerging diseases.  Section 
4.1.1.2 of the PHS Guideline[1] suggests an active screening 
program for agents known to be in the xenotransplantation product 
with testing of samples acquired at 2, 4, and 6 weeks after the 
patient receives the xenotransplantation product. 
 
A significant advantage of such a program is screening 
prospectively for evidence of infection in the absence of symptoms 
provides for a prospective understanding of the patterns of 
infection and disease that may be occurring in recipients.  Active 
screening could allow potential detection of a novel infection in the 
asymptomatic recipient and enable implementation of infection 
control practices to contain it before secondary human to human 
transmission or widespread dissemination in the general public, 
even in the absence of manifestation of associated disease (which 
may be absent altogether or simply delayed in onset).  If a 
xenotransplantation product known to harbor an infectious agent is 
used for xenotransplantation, you should implement active 
screening for that infectious agent.  For example, you should assess 
all recipients of xenotransplantation products involving the use of 
porcine cells, tissues, or organs for evidence of infection by 
PERV.[29]  Recipient screening for PERV should include analysis 
by multiple methods.  Ideally, you should use all of the following 
detection methods: 
 
  PCR of recipients PBMC for PERV DNA sequence,  
  serologic analysis for PERV-specific antibodies, and 
  assays capable of detecting plasma virions, such as RT-
PCR for detection of viral RNA or highly sensitive 
methods for detection of RT activity.[29] 
 
You should collect a sufficient quantity of each sample in the 
active screening program to permit archiving for future use, should 
the need arise.  See section X.H. for recommendations regarding 
the number, size, use, and duration of storage of collected samples. 
 
48  Contains Nonbinding Recommendations 
 
d.  Identification of Xenogeneic Retroviruses in Recipients 
 
One particular concern is the potential transmission of xenogeneic 
retroviruses, such as PERV in the case of recipients of porcine products.  
If you are a sponsor of porcine xenotransplantation product clinical trials, 
you should develop a plan to address the possibility that a recipient tests 
positive for the presence of PERV or other similar xenogeneic infectious 
agents.  The plan should include the following: 
 
i.  Strategies to identify the source of a positive signal in the 
screening test (e.g., infection versus false positive).  For 
example, in the case of porcine xenotransplantation 
products, we recommend PCR of DNA isolated from 
recipient PBMC for detection of PERV genetic sequences.  
However, if a positive result is obtained from this analysis, 
one possible explanation would be the presence of porcine 
cells.  Therefore, you should perform additional DNA PCR 
for a repetitive porcine genetic element to determine 
whether the source of the positive result may be from 
microchimerism for pig cells rather than from human cells 
infected with a pig retrovirus.  If this analysis suggests the 
latter possibility, additional analysis should include an 
attempt to isolate the virus from relevant recipient 
specimens in an appropriate co-culture assay;  
 
ii.  Determination of infectivity of the agent using appropriate 
assays (e.g., co-cultivation) and additional characterization 
of the agent as necessary; 
 
iii.  A plan to notify FDA and relevant sponsors and 
investigators and appropriate IRBs; 
 
iv.  A contingency plan to modify the clinical trial (including 
suspension or termination of enrollment); 
 
v.  Provisions for acute and follow-up medical care and 
counseling of the patients in the study; and 
 
vi.  Additional actions, if required, for the safety of the 
recipient and intimate contacts and to address possible 
public health risks. 
 
49  Contains Nonbinding Recommendations 
 
e.  Post-mortem Detection of Agents and Archiving of Autopsy 
Samples 
 
You should request a complete postmortem examination, including 
histopathology and cultures of all recipients.  At post-mortem, you should 
fix and embed samples of body tissue for examination by light and 
electron microscopy.  You should obtain samples from the 
xenotransplantation product and, as appropriate, all major organs related 
to the product or to clinical syndromes that resulted in the recipients 
death, were deemed to have been serious, or were of unexplained etiology.  
You should archive tissue and fluid samples at -70 degrees Celsius or 
lower, as appropriate for preserving the sample, for 50 years beyond the 
recipients death as discussed in section X.H.1. 
 
4.  Infections in Recipient Contacts 
 
We recommend that you develop a program to educate and monitor health care 
providers and to monitor other intimate contacts of recipients (e.g., persons with 
whom recipients repeatedly engage in activities that could result in intimate 
exchange of body fluids).  In these groups, passive screening (see section 
X.F.3.c.i.) may be appropriate. You should obtain baseline samples of plasma and 
archive them at -70 degrees Celsius, and you should obtain leukocytes and 
archive them in liquid nitrogen for health care personnel, for example, when they 
join the clinical teams.  You should also ensure that such contacts are advised and 
counseled, regarding potential risks. 
 
G.  Patient Follow-up 
 
The sponsor should propose and submit a plan for clinical follow-up of recipients in a 
xenotransplantation protocol in the FDA application, requesting investigational use.  This 
plan should take into account the timetable for collection and storage of specimens for the 
passive screening program and should extend for the life of the recipient (see section 
X.F.3.c.i.).  The frequency of follow-up will decrease with time, post-procedure.  It is 
reasonable to plan for a tapering frequency of clinical monitoring and follow-up, with the 
flexibility to increase the frequency for individual recipients or trial participants as a 
whole, if events occur to make this appropriate. 
 
H.  Archiving of Patient Plasma and Tissue Specimens 
 
1.  Before patients are treated, you should create protocols or SOPs for 
archiving all samples of patient tissue and fluids, including samples 
archived as part of recipient screening, post-mortem samples, and samples 
for PHS use. 
 
a.  You should follow appropriate biosafety precautions in collection 
of clinical samples from recipients.  You should follow standard 
50  Contains Nonbinding Recommendations 
 
precautions in obtaining blood from recipients.[35]  The PHS 
Guideline[1] recommends the use of at least a BioSafety Level 
(BSL)-2 containment facility with BSL-3 practices for any 
manipulation of clinical samples. 
 
b.  For the schedule for archiving biological specimens recommended 
by PHS, see reference 1 and section X.F.3.c.i. of this document.  
The specific protocol or the recipients medical course may 
indicate more frequent archiving. 
 
c.  Plans should exist to maintain all archived samples, according to 
the procedures recommended in the PHS Guideline[1], including 
those obtained from patients during acute infectious episodes and 
from health care workers. 
 
d.  You should store patient blood and plasma samples in volumes and 
quantities, according to the recommendations for animal plasma 
and blood cell samples (see section V.E.3.b.). 
 
In addition to recipient samples collected during screening programs or 
post mortem, when xenotransplantation recipient tissues are collected for 
any medical use, such as a biopsy for diagnostic purposes, you should also 
archive samples of such tissues.  You should store samples at -70 degrees 
Celsius or lower as appropriate for preserving the sample.  
 
2.  Archive Samples 
 
a.  You should follow the recommendations in the PHS Guideline[1] 
and also section X.F.3.c.i. of this document, regarding archiving of 
plasma, blood, and other specimens.  You should collect, archive, 
and reserve samples for use by PHS in the event that the need for a 
PHS-led investigation arises.  The PHS Guideline[1] recommends 
that biologic specimens for PHS use be maintained for 50 years 
beyond the date of xenotransplantation, based on the latency 
periods of known human pathogenic persistent viruses and the 
precedents established by the U.S. Occupational Safety and Health 
Administration with respect to recordkeeping requirements. 
 
b.  In addition to the designated PHS samples, the sponsor should 
archive separate samples of patient plasma, blood cells, 
xenotransplantation product, or other tissues for clinical follow-up 
and for storage as part of a passive screening program, as detailed 
above.  (See section X.H.1.). 
 
51  Contains Nonbinding Recommendations 
 
c.  You should not use samples archived for use by PHS (see section 
X.H.2.a.) or for monitoring of the recipient through a passive 
screening program (see section X.H.2.b.) for other purposes, such 
as research. 
 
I.  Health Records and Data Management 
 
1.  You should ensure that the recipients medical record contains information 
on the recipients health and all xenotransplantation-related information, 
including procedures, a description of the xenotransplantation product, and 
any xenotransplantation product-related adverse events.  In addition, you 
should develop an appropriate tracking system for all recipients of their 
xenotransplantation products and use this tracking information to facilitate 
notification in the case of a serious adverse event related to a 
xenotransplantation product.  You should collect information when events 
occur, such as a xenotransplantation procedure or an adverse event, and at 
the time of clinical follow-up examinations. 
 
Reporting forms should be uniform and include information relevant to the 
recipient. We recommend that the information to be collected and tracked 
include, at a minimum, the following: 
 
a.  Facility information - Sponsors should record information 
regarding their animal facilities, manufacturing facilities, and 
clinical centers associated with each source animal, 
xenotransplantation product, and recipient. 
 
b.  Recipient information - You should identify recipients by code 
number or other identifier to link the recipient to relevant 
information in the tracking system. 
 
c.  Procedure information - You should record information about each 
xenotransplantation procedure.  This information should include 
but is not limited to: 
 
i.  recipient identifiers; 
 
ii.  the date of the procedure; 
 
iii.  the clinical center where the procedure was performed; 
 
iv.  the physician or investigator who performed the procedure; 
 
v.  the clinical indication for the xenotransplantation 
procedure; 
 
52  Contains Nonbinding Recommendations 
 
vi.  medications and therapies administered at the time of the 
procedure; 
 
vii.  a description of the xenotransplantation product(s); 
 
viii.  identification of the animal source(s); 
 
ix.  animal facilities for each animal source; 
 
x.  xenotransplantation product manufacturing facilities; and 
 
xi.  other pertinent clinical information. 
 
d.  Adverse Event Reports - A sponsor must record adverse events and 
report the events to FDA, pursuant to existing regulation  
(21 CFR 312.32).  Sponsors should keep records of each event. 
 
e.  Recipient clinical follow-up examinations - You should 
periodically collect clinical status information for recipients of 
xenotransplantation products (see section X.F.).  This information 
should include but is not limited to: 
 
i.  the date of the clinical follow-up examination; 
 
ii.  the location of the clinical follow-up examination; 
 
iii.  the status of the xenotransplantation product in the 
recipient; 
 
iv.  any new significant co-morbidities; 
 
v.  any hospitalizations since the recipients last clinical 
follow-up examination. 
 
f.  Animal Health Events - Animal facilities should record animal 
health events.  These events include but are not limited to: 
 
i.  breaks in environmental barriers of the secured animal 
facility; 
 
ii.  disease outbreaks; and 
 
iii.  sudden, unexplained or unexpected animal deaths.  
 
53  Contains Nonbinding Recommendations 
 
The animal facility should report animal health events to the IND 
sponsor.  The sponsor should include this information in its 
tracking system for recipients and in reports to the FDA. 
 
g.  Recipient Death Reports - Sponsors should maintain death reports 
on recipients.  This information should include recipient 
identifying information, the date of death, and the cause of death.  
You should record death certificate and autopsy information, if 
available.  You should also report deaths to FDA. 
 
2.  You should maintain health records for at least 50 years beyond the date 
of transplantation. 
 
3.  The sponsor should make provisions for all records and samples 
(including post-mortem samples) to be maintained for the requested period 
of time in the event that the establishment ceases operation.  
  
J.  Informed Consent 
 
1.  General Comments 
 
The informed consent document must include the standard contents (see  
21 CFR Part 50). 
 
2.  Specific Issues 
 
Within the general outline of the informed consent document, you should address 
certain specific issues regarding recipients. 
 
a.  Participation in the Study 
 
i.  Since the zoonotic, opportunistic, and xenogeneic 
infectious risks to the recipient may extend to the 
recipients family or intimate contacts, the patient should 
consent to inform his or her current and future intimate 
contacts of their potential risks from the source animal 
species and of their deferral from blood donation.  Intimate 
contacts of xenotransplantation product recipients include 
persons who have engaged repeatedly in activities that 
could result in intimate exchange of body fluids, including 
blood or saliva, with a xenotransplantation product 
recipient.  Examples of intimate contacts include but are 
not limited to sexual partners, household members who 
share razors or toothbrushes, and health care workers or 
laboratory personnel with repeated percutaneous, mucosal 
or other direct exposures.  Mere sharing of housing or 
54  Contains Nonbinding Recommendations 
 
casual contact, such as hugging or kissing without the 
exchange of saliva, would not be interpreted as intimate 
contact.  You should offer the recipient assistance with this 
education process, if desired.  This discussion should 
include the recipients potential to transmit zoonotic or 
opportunistic infections if such an infection were to occur 
and the possibly increased risk of such transmission to 
individuals who may be at increased risk for zoonotic or 
opportunistic pathogens, such as infants, pregnant women, 
the elderly, and chronically ill or immunosuppressed 
individuals. 
 
ii.  Pending further information, clarifying risks, and further 
public consultation and discussion, xenotransplantation 
product recipients and their intimate contacts (see section 
X.J.2.a.i.) should be deferred from donation of Whole 
Blood; blood components, including Source Plasma and 
Source Leukocytes; tissues; breast milk; ova; sperm; or any 
other body parts for use in humans.  For more detailed 
information, please consult the FDA Guidance for 
Industry:  Eligibility Determination for Donors of Human 
Cells, Tissues, and Cellular and Tissue-Based Products 
(HCT/Ps).[8] 
 
iii.  You should counsel the recipient, regarding other 
behavioral modifications.  You should provide, as 
appropriate, advice on the use of barriers to transmission of 
infectious agents during sexual activity and the use of 
appropriate precautions for nonsexual contacts. 
 
iv.  The informed consent document should contain 
information about the proposed life-long surveillance for 
all recipients and the need for clinical and laboratory 
monitoring throughout.  You should explain, to the extent 
possible, the schedule for such clinical and laboratory 
monitoring. 
 
v.  The document should address the need for archiving 
plasma and tissue specimens from the source animal and 
the recipient for analysis in the case of xenogeneic disease 
concerns.  The document should explain that such 
specimens may be tested in the future by the sponsor or 
PHS agencies as needed to evaluate concerns regarding 
xenogeneic infections. 
 
55  Contains Nonbinding Recommendations 
 
vi.  The document should inform the recipient of the 
responsibility to inform the investigator or his/her designee 
of any change in address or telephone number for the 
purpose of enabling life-long health surveillance. 
 
vii.  The document should inform the recipient of the long term 
need for access by the appropriate public health agencies 
(e.g., FDA, CDC) to the recipients medical records.  To 
the extent permitted by applicable laws and/or regulations, 
you should maintain the confidentiality of medical records. 
 
viii.  You should include a request for autopsy in the informed 
consent document signed by the intended recipient or 
his/her appropriate representative. 
 
b.  Risks to the Recipient and his/her Intimate Contacts 
 
i.  The informed consent document should address the specific 
and known risks of all protocol-related activities not 
directly associated with source animal issues as well as the 
known and unknown zoonoses that may be associated with 
the source species.  It should mention the uncertainty of the 
risks of infection or its transmission and of the risk of 
tumorigenesis.  It should mention the possibility of a long 
latency period before detection of possible adverse effects.  
It should specify the need for and risks from prophylactic 
antimicrobial, antiviral, or other chemo- or immunotherapy.  
It should provide in an attachment for the recipient and the 
recipients family the reasoning behind the use of any 
prophylactic treatments. 
 
ii.  In addition, it should describe the possible need for 
confinement, reverse isolation, or other specialized medical 
housing, including the estimated duration of such 
confinement.  It should describe any specialized dietary, 
travel, or other precautions in as much detail as possible. 
 
iii.  It should include any known time course for the risks of 
disease development and transmission.  It should discuss 
infectious diseases with protracted incubation periods, 
including TSEs and other unusual pathogens. 
 
iv.  In the specific case of xenotransplantation products from 
porcine sources, the informed consent document should 
include the following information: 
 
56  Contains Nonbinding Recommendations 
 
  PERV can be transmitted from pig cells to human 
cells in culture, and this virus can be transmitted 
from a human cell line to other human cell lines in 
culture. 
  The clinical significance, if any, of this observation 
is unknown and is an area of active research; 
however, it is known that infection by certain type 
C retroviruses, similar in structure to PERV, can 
cause neurological disorders and diseases, such as 
lymphomas and other malignancies, in certain 
animal models. 
 
c.  Potential Benefits 
 
It should be clear in the informed consent whether xenotransplantation is 
being studied as a first-line, second-line, or salvage therapy of the 
condition for which it is being proposed for the individual recipient.  It 
should clearly convey the specific desired benefits, e.g., limited 
prolongation of survival, improved specific organ function, 
xenotransplantation product support until allograft becomes available, or 
experimental use without known or anticipated benefit. 
 
d.  Alternative Treatments 
 
The informed consent document should also explain in detail the 
anticipated therapeutic options available to participants in the event of 
failure of the xenotransplantation product. 
 
e.  Possible Consequences and Subsequent Treatment Options 
 
The informed consent document, to the extent possible, should explain the 
consequences to the patient, should the product fail or undergo irreversible 
rejection, including clear and unambiguous statements about the options 
that may not be possible after rejection of the xenotransplantation product, 
e.g., allotransplantation. 
 
f.  Confidentiality Issues 
 
The informed consent should inform the patient that all data, including 
data collected during the follow-up period, could be made available to 
PHS agencies. 
 
57  Contains Nonbinding Recommendations 
 
K.  Responsibility of the Sponsor in Informing the Patient of New Scientific 
Information 
 
You should commit to providing recipients with updated information as soon as possible, 
in the event that new data on risks, benefits, or the need for additional treatments relevant 
to the recipients clinical course become available or necessary.  You should be willing to 
make a long-term commitment to provide information to the recipients families, in the 
event that a recipient has died and new safety information of relevance to their potential 
exposures becomes known.  If you are the sponsor, you should ensure that the 
investigators are also willing to commit to providing new information to recipients and 
their families. 
58  Contains Nonbinding Recommendations 
 
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2.  Second WHO Global Consultation on Regulatory Requirements for Xenotransplantation 
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59  Contains Nonbinding Recommendations 
 
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60  Contains Nonbinding Recommendations 
 
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