Contains Nonbinding Recommendations 
Mouse Embryo Assay for Assisted 
Reproduction Technology Devices 
______________________________________________________________________________ 
Guidance for Industry and 
Food and Drug Administration Staff 
Document issued on January 5, 2021. 
The draft of this document was issued on June 13, 2019. 
For questions about this document, contact OHT3: Office of Gastro-Renal, ObGyn, General 
Hospital, and Urology Devices/DHT3B: Division of Reproductive, Gynecology, and Urology 
Devices at (301) 796-7030. 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Devices and Radiological Health  Contains Nonbinding Recommendations 
Preface 
Public Comment 
You may submit electronic comments and suggestions at any time for Agency consideration to 
https://www.regulations.gov. Submit written comments to the Dockets Management Staff, Food 
and Drug Administration, 5630 Fishers Lane, Room 1061, (HFA-305), Rockville, MD 20852. 
Identify all comments with the docket number FDA-2019-D-2105. Comments may not be acted 
upon by the Agency until the document is next revised or updated. 
Additional Copies 
Additional copies are available from the Internet. You may also send an email request to CDRH-
Guidance@fda.hhs.gov to receive a copy of the guidance. Please include the document number 
16015 and complete title of the guidance in the request. Contains Nonbinding Recommendations 
Mouse Embryo Assay for Assisted 
Reproduction Technology Devices
 
______________________________________________________________________________ 
Guidance for Industry and 
Food and Drug Administration Staff 
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 
or Office responsible for this guidance as listed on the title page. 
I.  Introduction 
FDA is issuing this guidance to outline recommendations on conducting the mouse embryo assay 
(MEA) to support premarket submissions and lot release of assisted reproduction technology 
(ART) devices. The majority of ART devices directly or indirectly contact gametes (i.e., sperm 
and oocytes) and/or embryos during their intended use. MEA is used to assess the potential for 
embryotoxicity of devices that contact gametes and/or embryos. Several classification 
regulations under 21 CFR part 884 include special controls that require MEA testing or 
information. MEA may also be used to support premarket submissions for other devices that are 
intended to contact gametes and/or embryos during their use. However, there are no voluntary 
consensus standards that describe how to conduct the MEA. This guidance provides 
recommendations for conducting the MEA to support premarket submissions for devices that are 
intended to contact gametes and/or embryos and to comply with the special controls for those 
devices classified under 21 CFR 884 that require MEA testing or information. 
For the current edition of the FDA-recognized consensus standard(s) referenced in this 
document, see the FDA Recognized Consensus Standards Database.1 For more information 
regarding use of consensus standards in regulatory submissions, please refer to the FDA 
guidance titled Appropriate Use of Voluntary Consensus Standards in Premarket Submissions 
for Medical Devices.2
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities. Instead, guidances describe the Agencys current thinking on a topic and should 
be viewed only as recommendations, unless specific regulatory or statutory requirements are 
                                                
1 Available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfStandards/search.cfm. 
2Available at https://www.fda.gov/regulatory-information/search-fda-guidance-documents/appropriate-use-
voluntary-consensus-standards-premarket-submissions-medical-devices.
1  Contains Nonbinding Recommendations 
cited. The use of the word should in Agency guidance means that something is suggested or 
recommended, but not required. 
II.  Scope 
The scope of this document is limited to devices that are regulated under 21 CFR part 884 that 
have direct or indirect contact with gametes and/or embryos. This guidance provides 
recommendations for how to comply with the special controls for those devices classified under 
21 CFR 884 that require the MEA testing or information.3 This guidance also applies to devices 
for which MEA testing or information may be used to support a premarket submission. Devices 
that only contact sperm during use (e.g., sperm handling or storage media, separation devices, 
handling labware) and the human sperm survival assay (HSSA) are outside the scope of this 
guidance document. 
III.  Method for MEA Testing 
FDA recommends that MEA testing be used to assess the embryotoxicity of ART devices that 
have direct and/or indirect contact with gametes and/or embryos. MEA assesses blastocyst 
development from either one-cell or two-cell stage embryos (i.e., one-cell system or two-cell 
system). FDA is aware of the one-cell and two-cell system for assessing embryotoxicity and 
considers both methods acceptable. FDAs specific recommendations for MEA testing are 
described below. 
A.  Test Article 
MEA should be conducted on ART devices in their final finished form (e.g., aseptically-filtered 
media in final packaging). The number of devices evaluated for each MEA test should be 
sufficient to ensure robust evaluation. FDA recommends that a minimum of three individual 
devices be evaluated in each test to account for potential variability between devices. To support 
a premarket submission, sponsors should perform testing on devices from one lot at both time 
zero (i.e., newly manufactured devices) and the end of the proposed shelf-life. FDA believes that 
accelerated aging per the currently FDA-recognized version of ASTM F1980: Standard Guide 
for Accelerated Aging of Sterile Barrier Systems for Medical Devices can also be used to develop 
test articles at the end of the proposed shelf-life. 
Depending on the device type and intended use, the MEA test article should be prepared as 
described below: 
1.  Liquid-based devices for MEA should be tested as provided without extraction or dilution 
(i.e., neat). Liquid-based devices for MEA that are provided in a concentrated form 
should be prepared as per the instructions for use before testing 
.
                                                
3 These classification regulations include 21 CFR 884.6100, 884.6110, 884.6130, 884.6160, 884.6165, 884.6170, 
884.6180, and 884.6195. 
2 Contains Nonbinding Recommendations 
2.  Oil indicated for oocyte/embryo culture should be evaluated under the intended use 
conditions. Embryos should be cultured in a drop of embryo culture medium overlaid 
with the culture oil. The type of medium to be used in this study is recommended in 
Section III.C below. 
3.  Plates and dishes that are indicated for embryo culture should be tested directly without 
extraction. 
4.  Solid devices that are not indicated for embryo culture (e.g., embryo transfer catheters, 
cryopreservation devices) should be extracted before use. Only the portions of devices 
that will come in direct and/or indirect contact with oocytes or embryos should be used to 
prepare test article extracts. 
B.  Preparation of Test Article Extract 
When preparing test article extracts for solid devices that are not indicated for embryo culture, 
the devices should be extracted in a standard embryo culture medium at 37 C. For devices 
where the clinical useis less than 30 minutes, the devices should be extracted for at least 30 
minutes to represent worst-case exposure conditions. For devices wherethe clinical use is more 
than 30 minutes, the devices should be extracted for at least twice the clinical use time to 
represent worst-case exposure conditions. FDA recommends that the volume of extraction media 
is consistent with the FDA guidance Use of International Standard ISO 10993-1, 'Biological 
evaluation of medical devices Part 1: Evaluation and testing within a risk management 
process'4 and ISO 10993-12: Biological evaluation of medical devices Part 12: Sample 
preparation and reference materials.
C. Test Procedure
(1)  Culture media and labware 
Culture media and labware used in the study, except for the test article(s), should be intended for 
use in ART procedures. FDA recommends that a medium indicated for embryo culture during 
ART procedures be used in MEA. Manufacturers can check the marketing status of culture 
media and labware using a publicly available FDA database5 and provide this information to 
FDA in the premarket submission. 
                                                
4 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/use-international-standard-iso-10993-
1-biological-evaluation-medical-devices-part-1-evaluation-and. 
5 The 510(k) database is available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMN/pmn.cfm. 
3 Contains Nonbinding Recommendations 
(2)  Mouse embryos 
One-cell or two-cell embryos should be obtained from hybrid mouse strains (e.g., CBA/B6 
hybrid6) that are sufficiently sensitive to detect embryotoxicity.7 FDA recommends that the 
mouse strain(s) used in MEA are scientifically justified. A minimum of 21 embryos should be 
exposed to the test article, while a minimum of 15 embryos should be exposed to the control 
medium in each MEA. FDA believes that this is a sufficient sample size to evaluate potential 
embryotoxicity and control for biological variability based on previous submissions for legally 
marketed devices. FDA recommends that the number of embryos cultured together be justified 
because the sensitivity of the assay may be reduced when embryos are cultured in large groups. 
(3)  Control 
Each MEA should include a control group that uses a medium indicated for embryo culture 
during ART procedures, and the same test procedure as the test article group. 
(4)  Duration of exposure of mouse embryos to test articles 
For MEA test exposure to represent intended use, FDA recommends: 
  In a standard MEA, embryos should be incubated in the test article extract under 
normal culture conditions (i.e., 37 C and 5% CO ) for 96 hours if a one-cell system 
2
is used or 72 hours for a two-cell system. FDA recommends that modifications to the 
culture conditions be justified.
  If the device is a plate or dish intended for embryo culture, the embryos should be 
directly incubated in the device in a standard embryo culture medium under normal 
culture conditions until the end of MEA culture time.
  If the device is a one-step medium (e.g., a single mediumused for culture of embryos 
until Day 5/6 of development), MEA should be conducted using the test articles until 
the end of intended incubation period under normal culture conditions. For example, 
MEA should be assessed after six days, inclusive of the beginning and ending days,
of incubation (120 hours) for a medium intended for use until Day 6 of development. 
  If the device is a media product with a shorter intended contact duration than a 
standard MEA(e.g., less than 72 ina two-cell system or 96 hours in a one-cell 
system), MEA should be conducted by exposing the embryos to the device for the 
total intended use duration provided in the labeling of the device.The embryos should
then be transferred to standard culture medium for the remaining culture time. 
 If the device is a culture oil, embryos should be cultured in standard culture medium 
overlaid with the device under normal culture conditions for 96 hours if a one-cell 
system is used or 72 hours for a two-cell system. 
                                                
6 For more information about embryo strains, see (1) Khan, Zaraq, et al. Mouse strain and quality control testing: 
improved sensitivity of the mouse embryo assay with embryos from outbred mice. Fertility and sterility 99.3 
(2013): 847-854; and (2) Puntvan der Zalm, J. P. E. M., et al. Toxicity testing of human assisted reproduction 
devices using the mouse embryo assay. Reproductive biomedicine online 18.4 (2009): 529-535. 
7 FDA supports the principles of the 3Rs, to reduce, refine, and replace animal use in testing when feasible. We 
encourage sponsors to consult with us if it they wish to use a non-animal testing method they believe is suitable, 
adequate, validated, and feasible. We will consider if such an alternative method could be assessed for equivalency 
to an animal test method. 
4 Contains Nonbinding Recommendations 
  If the device includes a series of solutions used sequentially (e.g., vitrification and 
warming of oocytes or embryos), testing should be conducted using a step-in/step-out 
approach. This approach sequentially exposes the embryos to all vitrification and 
warming solutions. Exposure to each solution should match the maximum exposure 
conditions described in the labeling. After step-in/step-out exposure, embryos should 
then be cultured in a standard culture medium under normal culture conditions until 
the end of MEA culture time. 
(5)  Evaluation of embryo development 
At the completion of embryo culture, embryos should be evaluated for blastocyst development 
using the acceptance criteria described in Section III.D. 
D.  Acceptance Criteria 
Depending on the system used, FDA recommends using the following MEA acceptance criteria 
for test and control articles: 
1.  One-cell system: 80% embryos developed to expanded blastocyst at 96 hours; or 
2.  Two-cell system: 80% embryos developed to expanded blastocyst at 72 hours. 
When test articles are incubated beyond 96 hours, the test report should include the actual 
incubation period and provide a justification for the incubation period. The sponsor should also 
propose an acceptance criterion based on the test procedure. 
E.  Test report 
For information on the recommended content and format of test reports for the testing described 
in this section, refer to FDAs guidance, Recommended Content and Format of Complete Test 
Reports for Non-Clinical Bench Performance Testing in Premarket Submissions.8 To facilitate 
FDAs review, we recommend that your MEA complete test report also include the MEA test 
method used, including mouse strain, number of embryos for test and control groups, culture 
conditions (e.g., gas, temperature, size of medium drop, number of embryos in each drop), and 
procedures. The name of the embryo culture medium used in the control group and test article 
group (as applicable) should be provided. 
IV.  MEA Information in Device Labeling 
When MEA testing is conducted, FDA recommends that the package and vial labels and 
instructions for use state the MEA acceptance criterion used. 
V.  Certificate of Analysis (COA) 
                                                
8 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/recommended-content-and-format-
non-clinical-bench-performance-testing-information-premarket. 
5 Contains Nonbinding Recommendations 
When MEA testing is conducted, FDA recommends that any COA state the MEA acceptance 
criterion used and include the lot-specific pass/fail results. 
6