IL-2 Luc LTT
Report of the Peer Review Panel

A JaCVAM coordinated study program addressing the validation status of the IL-2
Luc LTT for prospective identification of immunotoxic chemicals.
Report completed by the Peer Review Panel on May 2, 2023.

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Table of Contents
Executive Summary ............................................................................................ 3Peer Review Panel Composition......................................................................... 4
Background......................................................................................................... 5
Evaluation Criteria .............................................................................................. 6
Evaluation Criterion 1: A rationale for the test method should be available,including a description of the human health effect, a clear statement ofscientific need, and regulatory application. ....................................................6
Evaluation Criterion 2: The toxicological mechanisms and the relationshipbetween the test method endpoint(s) with the biological effect and the toxicity
of interest should be addressed, describing the limitations of the testmethod. .........................................................................................................6
Evaluation Criterion 3: A detailed test method protocol should beavailable………………………………………………….....................................7
Evaluation Criterion 4: The within and between laboratory reproducibility of thetest method should be demonstrated. ...........................................................7
Evaluation Criterion 5: Demonstration of the test method’s performance shouldbe based on testing a diversity of chemicals, preferably coded reference
chemicals. .....................................................................................................7
Evaluation Criterion 6: Predictive capacity should be demonstrated using
representative chemicals. .............................................................................7
Evaluation Criterion 7: All data should adequately support the assessment of thevalidity of the test method for peer review......................................................8
Evaluation Criterion 8: All data from the validation study supporting the validity ofa test method should be obtained in accordance with the principles of Good
Laboratory Practice (GLP)………………………………………………………..8Evaluation Criterion9: The applicability domain of the test method should bedefined. .........................................................................................................8
Evaluation Criterion 10: Proficiency chemicals should be set up in the proposedprotocol. ........................................................................................................9
Evaluation Criterion 11: Performance standards should be set up with theproposed protocol. …………………………………………………………….….9Evaluation Criterion 12: Advantages in terms of time, cost, and animal welfare
should be described. .....................................................................................9
Evaluation Criterion 13: Completeness of all data and documents supporting theassessment of the validity of the test method. ..............................................9
Evaluation Criterion 14: Validation Study should be managed and conductedadequately. ...................................................................................................10
Other Considerations.........................................................................................10
Conclusion ........................................................................................................10Acknowledgments……………………………………………………………………102

Executive Summary:
The interleukin-2 (IL-2) luciferase lymphocyte toxicity test (IL-2 Luc LTT) assay
has been proposed as an in vitro alternative to animal testing. The assay isrelated to the recently reviewed IL-2 luciferase assay (IL-2 Luc). While the IL-2
Luc assay provides information on adverse outcome pathways for
immunotoxicity to T cells, the IL-2 Luc LTT identifies those T cell targeting
agents that suppress IL-2 through an antimitotic mechanism, which was a gap
identified with the original IL-2 Luc assay.
The Peer Review Panel (PRP) found the Validation Management Team’s reportpresented the necessary information for an independent review.
The PRP concluded that the IL-2 Luc LTT assay was well defined and has aclear protocol and criteria for data interpretation. All necessary information,
including performance standards, was sufficiently detailed. Both within- andbetween-laboratory reproducibility were satisfactory. The PRP noted that it isimportant to clearly delineate the IL-2 Luc LTT from the IL-2 Luc assay. While
the predictive capacity was not satisfactory for a stand-alone method, the IL-2
Luc LTT assay is acceptable for use in an Integrated Approach to Testing andAssessment. In fact, the report included data showing that the combination ofthe IL-2 Luc with the IL-2 Luc LTT increased predictive capacity.

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Peer Review Panel Composition:
Barbara Kaplan, chair

Mississippi State University, USA

Chiyomi Kubo

Chugai Pharmaceuticals, Japan

Henk van Loveren

Maastricht University, The Netherlands

Takayuki Yoshimoto

Tokyo Medical University, Japan

Haley Neff-LaFord

Seagen Inc., USA

Support from Takao Ashikaga

JaCVAM, Japan

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Background:A healthy immune system must be in balance such that it can identifyand eradicate pathogens when needed, but not react to self-proteins. Thus, animbalance of the immune response in either direction can produce morbidity
and mortality; an insufficient immune response can cause infections or cancers,
while an overactive immune response can contribute to hypersensitivity orautoimmune diseases.
Exposure to drugs or chemicals can alter the immune response in either
direction. Therefore, there exists a need to develop and validate assays todetect immunotoxic compounds, especially those that produce immune
suppression. While an immune response is a coordinated effort among several
different cell types, the T cell is a critical initiator of adaptive immunity that can
provide help to other immune cells by producing cytokines or mediate killing of
infected cells. An important cytokine produced by T cells is interleukin-2 (IL-2),
which promotes proliferation of T cells and helps maintain specific T cell
subpopulations that contribute to immune homeostasis.
The IL-2 Luc assay was developed as a screening method to identify
chemicals that target T cells. In fact, it was the first validated in vitro assay toidentify immunosuppressive chemicals. Interestingly, the IL-2 Luc assay did notdetect well-characterized immunosuppressive drugs that acted by inhibiting
proliferation of T cells. Thus, the IL-2 Luc LTT assay was proposed to identify
those T cell targeting chemicals that acted through an anti-mitotic mechanism.
Indeed, it was demonstrated that the IL-2 Luc LTT assay accurately identified
immunosuppressive chemicals (mostly pharmaceutical drugs) that were not
identified with the IL-2 Luc assay alone. These results reinforce the idea that
immunotoxic chemicals will not likely be identified with a single assay and thatassays to identify immunotoxic chemicals will be part of integrated approaches
to testing and assessment (IATA).
The purpose of this document is to provide a constructive review of theIL-2 Luc LTT. The PRP initially met virtually in October 2022 and then in personin Shizuoka, Japan in November 2022 to continue to evaluation. Subsequentvirtual meetings occurred in January, February, March, and May 2023 toprepare this peer review report.

5Evaluation criteria and peer review panel comments to each (provided in bluetext) are detailed below:
Evaluation Criterion 1: A rationale for the test method should be available,including a description of the human health effect, a clear statement of scientificneed, and regulatory application.
The human health effect problem statement, test method, rationale for scientificneed and intent for regulatory application are stated in the document, although it
is not necessarily found in a single section (and not all sections are at thebeginning of the document). Perhaps a single section summarizing the rationale
could be included somewhere near the beginning that includes these points aspart of the rationale: 1. Environmental contaminants, food additives, and drugs
can affect the immune system, resulting in immune dysregulation; 2. Immune
dysregulation can have serious adverse health consequences, ranging fromreduced resistance to infection and neoplasia to allergic and autoimmuneconditions; 3. In vitro methods are needed due to the high cost, ethical concerns,
and questionable relevance to risk assessment for humans using animal cells
and model systems; 4. The fact that antimitotic effects of chemicals could not be
detected by the IL-2 Luc assay; and 5. That the regulatory application is eventual
generation of an OECD test method for immunotoxicity. There was also concernthat there needs to be more background description on the meaning of measuringIL-2 for determining the immune suppression and antimitotic effect would beappreciated for reader to better understand the assays as follows (maybe in thefirst paragraph of the section 3-7). IL-2 is one good marker for immune
suppression and cell proliferation. IL-2 plays a critical role in the proper activationof T cells and the lack of IL-2 production is well known to induce T cell anergy,
leading to immune tolerance. In addition, IL-2 is also a potent growth factor for T
cells and inhibition of IL-2 reduces T cell proliferation. Finally, there was someconcern that the human relevance was not entirely clear, especially regarding theadvantages of distinguishing between antimitotic and immunosuppressive effects.
Evaluation Criterion 2: The toxicological mechanisms and the relationshipbetween the test method endpoint(s) with the biological effect as well as thetoxicity of interest should be addressed, describing limitations of the test method.

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The rationale for developing the test method and the toxicity of interest wereclearly stated. The limitations of the test method were also laid out with discussionof the applicability domain and the combination of the IL-2 Luc LTT with theoriginal IL-2 Luc assay to strengthen predictivity.
Evaluation Criterion 3: A detailed test method protocol should be available.
The detailed test method protocol was provided. There were 2 points that wereinconsistent: there was a difference between total amount and sum of required
amount in Table 6 of 8-2-2 (page 56), and the centrifuge speed for thawing 2H4cells was wide range (120~350xg) in 8-2-4 (page 58). These points were properly
corrected in the final version of the validation report.
Evaluation Criterion 4: The within and between laboratory reproducibility of thetest method should be demonstrated.
The initial pre-validation trial (phase 0) was conducted with 3 non-codedchemicals (bleomycin sulfate, dexamethasone and 6-thioguanine). Within- andbetween-laboratory reproducibility of this assay (phase I) was checked using fivecoded chemicals in three test facilities (mycophenolic acid, indomethacin,cyclosporin A 5-FU, and mannitol). Between-laboratory reproducibility (phase II)
was determined using 20 coded chemicals in three test facilities. Importantly,
there were different chemicals tested in each of the 3 phases (except for use ofa negative control chemical). Reproducibility was demonstrated. Withinlaboratory reproducibility in the Phase I trial in Lab A, Lab B, and Lab C
demonstrated 100%, 100%, and 100% reproducibility, respectively. Betweenlaboratory reproducibility result for Lab A, Lab B, and Lab C was 92.0 % for thecombined data of the Phase I and Phase II trials. These results satisfied theacceptance criteria for the validation study of a within-laboratory reproducibility ofat least 80% and a between-laboratory reproducibility of at least 80%.
Evaluation Criterion 5: Demonstration of the test method’s performance should
be based on testing of representative, preferably coded reference chemicals.
There was a Chemical Selection Committee (CSC) that determined the chemicals
that should be used in all three phases, including use of positive and negative

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control chemicals. Chemicals were selected based in part on those chemicals
determined by the literature (i.e., Luster et al, 1998) and other sources (e.g.,National Toxicology Program) to be immunotoxic. Moreover, there was purposefulselection of immunotoxic chemicals that are known to act through an antimitotic
mechanism as well as others that do not depend on antimitotic action to exhibit
immunotoxicity. All chemicals were coded.
Evaluation Criterion 6: Predictive capacity should be demonstrated using
representative chemicals.
Predictivity was provided for several endpoints. First, predictivity was provided
based on the IL-2 Luc LTT for phases I and II, considering whether the known
mechanism of the chemical had been determined to involve an antimitotic
mechanism. Predictivity for the chemicals evaluated in phases I and II for the IL2 Luc LTT was 76%. Second, predictivity was provided considering use of the IL2 Luc LTT in combination with the IL-2 Luc, which was also 76% for the chemicals
evaluated in phases I and II. Third, the predictivity was provided based onassessment of 85 test compounds (46 pharmaceutical drugs and 39 nonpharmaceutical chemicals). Predictivity for IL-2 Luc LTT for antimitotic effects was80.8%. When the applicability domain was applied, the predictivity was slightly
reduced to 79.5%. Finally, when combined with the IL-2 Luc, the predictivity was77.2%. Predictivity is also provided for the pharmaceutical drugs for the IL-2 Luc
compared to the IL-2 Luc in combination with the IL-2 Luc LTT and for the nonpharmaceutical chemicals for the IL-2 Luc compared to the IL-2 Luc in
combination with the IL-2 Luc LTT (i.e., the 85 test compounds were separated
into drugs versus chemicals and the predictivity calculated). Overall, there isextensive information provided on the predictivity of the IL-2 Luc LTT and howthat might change when used in combination with the IL-2 Luc.
Evaluation Criterion 7: All data should adequately support the assessment of thevalidity of the test method for peer review.
Data were appropriately included and described in a way that allowed for peer
review.
Evaluation Criterion 8: All data from the validation study supporting the validity of8a test method should be obtained in accordance with the principles of Good
Laboratory Practice (GLP).
Assays and quality assurance were carried out in the spirit of GLP, as not all theparticipating laboratories routinely worked under GLP certification. Overall, theprinciples of GLP were followed for this validation.
Evaluation Criterion 9: Applicability domain of the test method should be defined.
Applicability domain was described clearly in section 10-5, although it isdiscussed before this section so perhaps this could be moved further up in thedocument. Overall, the PRP agrees with the applicability domain as defined: 1.)
chemicals that interfere with luciferase or luminescence that confound its
activity/measurement are out of the applicability domain; 2) the use of PMA/Io asa stimulant bypasses signaling through the T cell receptor and the subsequent
intracellular signaling events that precede activation of phospholipase C, andtherefore precludes detection of chemicals that act on those upstream signaling
molecules; and 3) the Jurkat T cell line (from which 2H4 cells are derived) mightlack several key proteins involved in the activation of normal T cells in response
to TCR stimulation, and therefore may not be able to detect effects of chemicals
that act on those key proteins.
Evaluation Criterion 10: Proficiency chemicals should be set up in the proposed
protocol.
Bleomycin sulfate and cyclosporin are used as positive and negative controls for
antimitotic effects, respectively. There were used to demonstrate proficiency
across laboratories. NOTE: page 36 states that dexamethasone was negative
control for antimitotic effects, but all other tables and verbiage state cyclosporin;needs to be edited.
Evaluation Criterion 11: Performance standards should be set up with theproposed protocol.
Various criteria to establish the success of the assay were stated several times,
although they were not called “performance standards”. Moreover, some criteria

9or performance standards were not clear (i.e., a concrete attainment goal of thepredictivity was not mentioned). Specific success criteria are not mentioned
because this assay is not a stand-alone method but should be combined with IL2 Luc assay. The predictivity was improved when IL-2 Luc LTT was used in
combination with the IL-2 Luc assay plus the applicability domain. For instancethe predictivity was highest when examining the combined results from the IL-2
Luc and IL-2 Luc LTT for those drugs or chemicals within the applicability domain.
Evaluation Criterion 12: Advantages in terms of time, cost, and animal welfare.
These advantages were described, although the cost advantage wasn’t
completely evident. This assay was considered to be suitable for addressing 3Rsof animal research. However, the advantage was not as high when compared tosimilar in vitro assays. It might be better to compare the cost with other in vitro
assays after the cost was calculated including human resources, availability of
the cells, time for the cell maintenance, and number of samples that can beconducted in one experiment.
Evaluation Criterion 13: Completeness of all data and documents supporting theassessment of the validity of the test method.
Quality assurance issues were clear, and 3 areas were identified (temperature ofluciferase activity measurement; time of P/I stimulation not consistent in one lab;
and that positive and negative chemical controls need to be done at the sametime as experiment). A detailed summary of the data was provided. Although we
did not have access to raw data or paperwork, there were extensive analyses
provided.
Evaluation Criterion 14: Validation Study Management (VSM) and Conduct.
The activity of the VSM and conduct of the study was described. A timeline forthe assay development was also provided. A list of the various participants in thestudy was also included.
Other considerations

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There is still some concern that there will continue to be confusion with IL-2 Luc
versus IL-2 Luc LTT as it is currently presented. Even in the introduction and as
part of the rationale, there are data shown from the IL-2 Luc, which might be
confusing if people are not reading the document carefully. Perhaps consider
presenting only the IL-2 Luc LTT and associated data first then dedicate aseparate section at the end to an “IATA effort” using the combination of the IL-2
Luc and IL-2 Luc LTT.
Conclusions
In summary, the IL-2 Luc LTT assay was well described, well defined, and has aclear protocol and criteria for data interpretation. All necessary information,
including performance standards, was sufficiently detailed. Both within- andbetween-laboratory reproducibility were satisfactory. While the predictive
capacity was not satisfactory for a stand-alone method, the IL-2 Luc LTT assay
is acceptable for use in an Integrated Approach to Testing and Assessment for
immunotoxicity.
Acknowledgments
The peer review panel would like to acknowledge the hard work provided Dr. Aibaand Dr. Kimura in leading the laboratory effort, and Dr. Kojima and Dr. Ashikaga
in coordinating the trials through JaCVAM. The peer review panel alsoacknowledges the validation management team, the chemical selection
committee, data management team, and members of the laboratories in theparticipating test facilities. The report is extensive and there has been a strong
effort in not only conducting the necessary studies to get the IL-2 Luc LTT
submitted as a test guideline but presenting additional data and analyses to use
it in combination with IL-2 Luc as part of an IATA for immunotoxicity. The peer
review panel also thanks Dr. Aiba for being very responsive to our concerns
throughout the review process.

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