lab animalProtocol review

https://doi.org/10.1038/s41684-023-01321-y

The weights of refinement and flexibility
Check for updates

IACUCs need to ensure theapplication of the 3Rs, while allowing
researchers to conduct their workwith reasonable fexibility. We
invited experts from the Universityof Notre Dame (Eric Felde) andNorthwestern University (StefanieBittner), along with USDA and OLAW
representatives (Louis DiVincentiand Axel Wolff) to respond to achallenging situation at Great
Eastern University.

Dr. Crick, Great Eastern University(GEU)’s IACUC Chair, called upon
Dr. Jerry Silverman, GEU’s mostdistinguished Professor, to serve as
an ad-hoc consultant on a matter the IACUC
was discussing. Jerry previously served as theIACUC Chair and is a subject matter expert on
the nuances of the regulation governing animalcare and use.
The matter at hand is with regard toDr. Samantha Stevens’ triennial renewalapplication. Samantha, a neuroscientist, studies neuronal pathways under a variety ofconditions, such as stress, reward, sleep andpeer-interactions. Samantha’s IACUC protocol application, although well written, is quite
lengthy and complex. As is expected of Principal
Investigators (PIs), Samantha stays informed(e.g., new technologies, improved behavioral
assays and equipment) so she can employ theconcepts of the 3Rs in her research. For example,
the triennial renewal application now includes
the use of citric acid (CA) water as an alternative to traditional water restriction, whichis employed to help motivate the animals toperform behavioral tests. CA water permits theanimals to remain hydrated, but because of its
sour taste, still motivates animals to work foraccess to fresh water. The challenge with the CA
water is that it was only recently introduced into
Samantha’s field of expertise and has not yet been
proven to achieve the desired goals (or at thesame success rate). Samantha plans to evaluate
CA water in comparison with water restrictionthat she has performed in the past, and consequently has requested separate groups of animals
to run experiments under both paradigms.
Lab Animal | Volume 53 | February 2024 | 23–25

Crick explains to Jerry that the IACUC’sstruggle has been whether they should require
Samantha to halt all experiments using the traditional water restriction until a pilot study with
the CA water has been completed to determine
whether this less stressful (and less distressful)procedure can be employed for her research.
The results of the pilot study would then direct
the procedures that the IACUC would approvegoing forward (e.g., if CA water is just as effective in achieving the research outcomes needed,
the IACUC may not then approve any use of
the traditional water restriction). Furthermore,
Crick tells Jerry the IACUC wants to ensure thatPIs have the flexibility in their protocols to use
the tools required for their research, but cannot resolve this with their obligation to ensure
the application of the 3Rs. So, Crick asks Jerry,“What should we do?”If you were Jerry, how would you respondto Crick?

Response from EFDr. Crick described his concern that the GEU’s
IACUC needs to balance Dr. Stevens’ ability toconduct her work with reasonable flexibility
against the IACUC’s need to ensure appropriate application of the principles of reduction,refinement and replacement. I believe the GEU’sIACUC can accomplish both without considering the two goals to be in opposition.
GEU’s IACUC is completing triennial review
of Dr. Stevens’ protocol, and therefore must
consider whether the procedures it describes will
minimize distress to the animals1. The additionof a new alternative presents a challenge: canthe IACUC approve the already-approved fluid
restriction when this new alternative could resultin less distress to the animals?
In its review, the IACUC should reevaluate
information that should have been provided in
Stevens’ initial protocol, including: the necessary level of fluid regulation, potential adverse
consequences of fluid regulation and methods
for assessing the health and wellbeing of theanimals2. New information may be available
for animals already included in the study, suchas adverse event reports, records detailing fluid
consumption and hydration status and any
behavioral or clinical changes used as criteria for removal from a protocol3. If any of thisinformation suggests that potential for distressis not minimized, the IACUC may — at least —request modifications to Dr. Stevens’ protocol.
The IACUC should carefully consider the way
it approaches Dr. Stevens’ proposal to add theCA water alternative because their action may
influence researchers’ attitudes toward refinement of experimental procedures. Without
conclusive data indicating that CA water caneffectively be used in place of traditional water
restriction, suspending a study in progress
would suggest that Dr. Stevens has not followed her IACUC protocol or regulations. By
all indications, Dr. Stevens has stayed informedof advancements in practice, and she has proactively proposed refinements to the IACUC.
Unless the IACUC finds in Dr. Stevens’ data
anything that suggests animals have experienced
unexpected levels of distress to the extent that itis reportable, there does not appear to be cause
to take that action.
I would instead recommend that the IACUC
supports Dr. Stevens in her pursuit of refined
research procedures. A pilot study looking into
CA water has the potential to provide data that
could benefit the IACUC in its efforts to ensurethat researchers at GEU are applying the 3Rs
in their work. If CA water is found to result inless distress, the IACUC might encourage otherresearchers who are currently using traditional
water restriction to consider refining their own
procedures. If there is an institutional policy on
fluid restriction, it might warrant reevaluationand possible revision.
Assuming the IACUC finds no reason tosuspend Dr. Stevens’ protocol, allowing her theflexibility to include a pilot study of CA water
as an aim of her current protocol will give her
information that could support refinement of her
own work, and possibly even benefit the IACUC
and the entire animal program at the same time.

Response from SB

As an experienced IACUC Chair and knowingthat regulations around IACUC processes are
not always black and white, Jerry understands
that this can lead to great deliberation and disagreement amongst the Committee and sympathizes with the Committee’s current struggle.
Jerry discusses with Crick that Dr. Stevens
should be applauded for not only doing what is
expected by continuously researching alternatives, but also proactively including the possible
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lab animalProtocol review

Box 1 | A Word from USDA and OLAWA Word from USDA
In 2021, APHIS amended the Animal Welfare
Regulations (AWR) to replace the requirementfor annual continuing reviews with acomplete resubmission and review at least
every 3 years. The triennial review should be
conducted as if it were a new activity. This“de novo review” is meant to prevent a study
from continuing “indefnitely without everbeing fully revisited to ensure its underlying
design or foundational assumptions are instep with current science and regulatory
policy relating to animal welfare6.” Dr. Crick’s
approach to the triennial review ofDr. Stevens’ protocol is appropriate.
If the animals are USDA-covered
species, the AWR also require the IACUC,
as part of its review, to determine thatprocedures involving animals will avoid
or minimize discomfort, distress, andpain to the animals and that the principal
investigator (PI) has considered alternatives
to procedures that may cause more thanmomentary or slight pain or distress7.
Importantly, the AWR do not require theIACUC to determine that there are noalternatives, only that the PI has consideredalternatives based on a written narrativedescription of the methods and sources
used. In this case, the IACUC has met its
obligation in determining that Dr. Stevens
has considered a possible alternative totraditional water restriction, a procedurethat can cause distress. However, thealternative is unproven. Allowing Dr. Stevens
to compare the alternative to the traditional
method is appropriate. If Dr. Stevens
determines that the alternative is equivalentor superior to traditional water restriction,
the IACUC is required to carefully consider
Dr. Stevens’ justifcation for not adopting thealternative should she propose to continue
using the traditional method.
A Word from OLAW
This scenario involves IACUC considerations regarding IACUC approval of

alternative in her protocol to see if it would bea viable alternative to her current studies. The
PI indicated that CA water treatment is not yet
proven to work in her field; so performing a setof pilot studies to ensure that the CA water treatment not only works, but also doesn’t have any
adverse effects on her specific research makes
Lab Animal | Volume 53 | February 2024 | 23–25

animal activities including assessment of
the new, but unproven, use of citric acidwater as an alternate to water restriction
for a neuroscience research protocol. Inorder to approve proposed animal activities
or proposed signifcant changes, thePHS Policy on Humane Care and Use ofLaboratory Animals (PHS Policy) requires
the IACUC to conduct a review and confrm
compliance with the Policy, the institution’s
Animal Welfare Assurance, and the AnimalWelfare Act and regulations, as applicable1(and see Related links). Among theserequirements, and of importance to this
scenario, IACUCs are required to determinethat procedures involving animals will
avoid or minimize discomfort, distress,
and pain to the animals1,8. “Refnement
and reduction goals should be balanced
on a case-by-case basis4,” and “limitedpilot studies, designed to assess boththe procedure’s effects on the animals
and the skills of the research team andconducted under IACUC oversight, areappropriate4.” For the pilot studies(see Related links), the IACUC shouldengage with Dr. Stevens and evaluate
the lab’s current practices involving
traditional water restriction and consider
the proposed procedures, monitoring,and documentation with careful attention
to the Guide’s Food and Fluid Regulation
section4 (and see Related links). Monitoring
frequency may need to be increased
with pilot studies that evaluate new
methods, especially when procedures
involve water restriction. Goodcommunication and a collaborative effort
between the IACUC and the researcher
are recommended as both parties sharethe obligation of humane animal careand use. The IACUC is responsible forthe approval of animal activities that areconsistent with PHS Policy requirements
and institutional policies, and all animal
activities must be conducted under anapproved IACUC protocol.

sense. Jerry reminds the IACUC that per theGuide4, “the IACUC is obliged to weigh theobjectives of the study against potential animal
welfare concerns. By considering opportunities
for refinement, the use of appropriate nonanimalalternatives, and the use of fewer animals, boththe institution and the principal investigator (PI)can begin to address their shared obligations forhumane animal care and use.” While PHS Policy
IV C.1.a indicates1: “Procedures with animals will
avoid or minimize discomfort, distress, and pain
to the animals, consistent with sound research
design,” Jerry asks the Committee to consider:• Does the PI have adequate justifcation to haveboth paradigms to remain in the protocol?
• Would requiring the PI to remove a provenmethod of motivation to attempt an unprovenmethod of motivation inhibit the PI’s researchplans?
• Has the PI considered if there will be strainor species variances in the acceptance of CA
water treatment as an alternative to water
restriction if working with various models?
• Could the IACUC require a modifcation
to secure approval that the PI conduct theunproven CA water pilot study alongside theirproven methods, and that they would only
continue to use the water restriction procedure in cases where the CA water paradigm
demonstrates an inability to meet the samescientifc goals at the same success rate as theirwater restriction procedure?
∘ Could the Committee request a review of thepilot study results be submitted for review at
the next meeting once conducted?
∘ Could the Committee request a postapproval monitoring visit to review the procedure and records with a report back to theCommittee?
If the pilot study cannot achieve the goals at
the same or similar success rate, then although
the animals may be undergoing a less stressfulprocedure, more animals may be required which
would be trading refinement for reduction to gain
statistical signifcance in Dr. Stevens’ research. If
asked how Jerry would cast his vote on the matter,
since the protocol is well written, he would be infavor that the PI be allowed to conduct the pilot
study alongside their current proven paradigm.

Compliance considerations

The Protocol Review coordinators offer the following compliance considerations:
Refinement refers to modifications of husbandry or experimental procedures to enhance
animal well-being and minimize or eliminate
pain and distress4. During protocol review,
the IACUC must consider 4, 5 :• Te “availability or appropriateness of the useof less invasive procedures, other species, isolated organ preparation, cell or tissue culture,or computer simulation”;• That “unnecessary duplication of experiments” is not occurring; and,• Te “impact of the proposed procedures on
the animals’ well-being.”
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lab animal

The Guide further states that “At times, protocols include procedures that have not beenpreviously encountered…If little is known
about a specific procedure, limited pilot studies,designed to assess both the procedure’s effects
on the animals and the skills of the researchteam and conducted under IACUC
oversight, are appropriate 4 .”Consequently, GEU’s IACUC has a regulatory
and ethical obligation to consider CA water as arefinement to a stressful/distressful procedure(i.e., water restriction).
Recall that “The IACUC may invite consultants to assist with the review of complex issues4,”
which may help the IACUC determine how toproceed. Ultimately, however, the IACUC must
determine, for example, whether:• CA water is a refnement that must be implemented (i.e., can Samantha continue withwater restriction whilst investigating theimpact of CA water on her research, or must
she halt all activities until CA water has been
established as a refnement); and,• A pilot study or some other criteria arerequired to establish CA water as a viable
refnement (i.e., it will not introduce a newvariable that could skew the data outcome(s)and/or prohibit comparison to animals
already studied).

A Word from USDA and OLAW

Comments from USDA and OLAW can befound in Box 1.

Lab Animal | Volume 53 | February 2024 | 23–25

Protocol review

This protocol review was coordinated by Lauren
Danridge and Bill Greer.
Lauren Danridge1 , Bill Greer2, Eric Felde3,
Stefanie Bittner4, Louis DiVincenti5 &Axel Wolff6
1
Associate Director - IACUC/IBC/PAM,Princeton University, Princeton, NJ, USA.
2Animal Care & Use Offce, University ofMichigan, Ann Arbor, MI, USA. 3Director of
Research Compliance, University of Notre
Dame, Notre Dame, IN, USA. 4IACUC Offce,Northwestern University, Chicago, IL, USA.
5Senior Veterinary Medical Offcer – Research,Animal Care National Policy Staff, APHIS,USDA, Riverdale, MD, USA. 6Acting Director,
Offce of Laboratory Animal Welfare, OER, OD,NIH, HHS, Bethesda, MD, USA.
e-mail: laurendanridge@princeton.edu

5. USDA. Animal Welfare Act and Animal Welfare
Regulations. https://www.aphis.usda.gov/animal_welfare/downloads/AC_BlueBook_AWA_508_comp_version.pdf
6. AWA Research Facility Registration Updates, Reviews,and Reports. 86 FR 66919 (November 24, 2021;
to be codifed at 9 C.F.R. pts. 2, 3, 4). https://www.
federalregister.gov/documents/2021/11/24/202125614/awa-research-facility-registration-updatesreviews-and-reports
7. Animal Welfare Regulations. 9 CFR § 2.31(d)(1).
8. Offce of Science and Technology Policy. U.S. Government
Principles for the Utilization and Care of Vertebrate Animals
Used in Testing, Research, and Training. Federal Register,May 20, 1985, Vol. 50, No. 97, [FR Doc. 85-12059].
Principle IV. https://olaw.nih.gov/policies-laws/phs-policy.
htm#USGovPrinciples

Competing interests
The authors declare no competing interests.

Disclaimer

Published online: 2 February 2024

The views expressed in “A word from OLAW andUSDA” represent the positions of OLAW/NIH andUSDA, respectively, but the remainder of thecontributions in this article do not represent offcial
support or an endorsement by NIH, USDA, or any
government agency.

References

Related links:

1. Public Health Service. PHS Policy on Humane
Care and Use of Laboratory Animals (U.S. Department
of Health and Human Services, National Institutesof Health, Bethesda, MD, 2015). Section IV.C.1.
Review of PHS-Conducted or Supported ResearchProjects. https://olaw.nih.gov/policies-laws/phs-policy.ht
m#ReviewofPHS-ConductedorSupportedResearchProjects
2. National Research Council. Guidelines for the careand use of mammals in neuroscience and behavioral
research (National Academies Press, Washington
DC, 2003).
3. Morton, D. B. ILAR J. 41, 80–86 (2000).
4. Institute for Laboratory Animal Research. Guide for the Careand Use of Laboratory Animals 8th edn. (National Academies
Press, Washington DC, 2011).

PHS Policy on Humane Care and Use of Laboratory
Animals – FAQs. Protocol Review, Question No. D.6.
https://olaw.nih.gov/faqs#/guidance/faqs?anchor=50318.
PHS Policy on Humane Care and Use of Laboratory
Animals – FAQs. Protocol Review, Question No. D.7.
https://olaw.nih.gov/faqs#/guidance/faqs?anchor=50319.
PHS Policy on Humane Care and Use of Laboratory
Animals – FAQs. Protocol Review, Question No. D.11.
https://olaw.nih.gov/faqs#/guidance/faqs?anchor=50323.
PHS Policy on Humane Care and Use of Laboratory
Animals – FAQs. Animal Use and Management,
Question No. F.19.
https://olaw.nih.gov/faqs#/guidance/faqs?anchor=52964

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