Peer Review Panel Evaluation of the Reactive Oxygen Species (ROS)
Photosafety Assay

Japanese Center for the Validation of Alternative Methods
National Institute of Health Sciences
Tokyo, Japan
16 October 2013

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16 October 2013

Table of Contents
Executive Summary
Peer Review Panel Evaluation
1. Introduction
2. Regulatory Rationale
3. Scientific Rationale And Limitations
4. Test Method Protocol
5. Inter- And Intra-Laboratory Reproducibility
6. Validation Reference Chemicals
7. Test Method Predictivity
8. Availability Of Data
9. Data Quality
10. Applicability Domain
11. Proficiency Chemicals
12. Performance Standards
13. Advantages: Cost, Time, And Animal Welfare
14. Conclusions
Appendices1. Peer Review Panel Members
2. Acknowledgements
3. Glossary
4. References

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Executive Summary
The Japanese Center for the Validation of Alternative Methods (JaCVAM) convened an
independent scientific peer review panel to evaluate the validation status of the Reactive
Oxygen Species (ROS) assay in accordance with established international criteria (OECD,2005). The ROS assay is a test method proposed as a component of an integrated photosafety
testing strategy to evaluate whether test substances such as pharmaceuticals have the potential
to cause phototoxicity.
The panel met initially in February and again in August 2013 in Tokyo, Japan. The panel
considered the reports of two international validation studies and a proposed ROS Assay
protocol at their initial meeting. The panel subsequently reviewed updated versions of the ROS
Assay protocol and the validation study reports as revised by the Validation Management Team
(VMT). This report summarizes the panel’s final evaluation and conclusions.
Overall conclusion: The panel concluded that the reproducibility and predictivity of the ROS
assay is sufficient to support its use in an integrated photosafety testing and decision strategy
for drug research and development. In this strategy, negative results in the ROS assay would
not require further testing in animals or other tests, while positive, weakly positive, andinconclusive results would proceed to the next level of testing in an in vitro test system such as
the 3T3 Phototoxicity Assay (OECD Test Guideline 432). The panel also concluded that use of
the ROS assay could potentially provide significant savings in time, cost and reduced animal
use for photosafety assessments. Furthermore, incorporating the ROS assay into a photosafety
testing strategy is expected to significantly reduce the overall number of substances that would
require additional testing in the in vitro 3T3 Phototoxicity Assay and subsequent testing inanimals.
Regulatory rationale: The panel concluded that the ROS Assay is applicable for use within the
ICH regulatory testing framework for photosafety evaluation of pharmaceutical products.
Regulatory authorities (e.g. PMDA/MHLW, U.S. FDA, EMEA) require non-clinical photosafety
testing prior to approving First-in-Human Phase I studies so that appropriate precautions andobservations can be taken during initial human studies. Such non-clinical photosafety testing
typically includes an assessment of the potential for a drug to cause phototoxic reactions, which
are characterized clinically by dermal redness, swelling, irritation, and inflammation. The panel
also agreed that the ROS assay is applicable to in-house drug research and development.
Scientific rationale: The panel recognized that ROS production is the most importantmechanism for chemically-induced phototoxicity, and is therefore a critical pathway initiating
event leading to phototoxicity. The ROS assay quantitatively measures two common reactive
oxygen species generated by photoreactive chemicals after exposure to simulated sunlight. In
this validation study, chemicals that did not produce sufficient ROS to meet the photoreactivity
threshold classification criteria for the ROS assay are uniformly non-phototoxic, while chemicals
that met or exceeded the photoreactivity classification criteria include all known phototoxicants.
Therefore if a chemical is not photoreactive in the ROS assay, it is unlikely that phototoxicity will
occur in living systems.
Limitations: The panel noted that the ROS assay assesses chemical photoreactivity in a nonbiological system, and therefore may overpredict phototoxicity potential since it does not assessthe direct interaction of chemicals with biological tissues. The assay may also overestimate the3

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potential for phototoxicity because some chemicals may not achieve sufficient concentration inskin for phototoxic reactions to occur, or photodegradation may occur. Accordingly, positive
results in the ROS assay are generally recommended for further evaluation in a photosafety
testing strategy.
Validation study reference chemicals: The panel agreed that the reference chemicals
selected for the validation studies were appropriate and sufficiently representative of thechemicals likely to be evaluated in the assay. The 42 reference chemicals incorporated most
known human phototoxicants and included 23 known positives and 19 negatives. The chemicals
were backed by data from human patch testing and in vitro 3T3 phototoxicity assay results. Alldata from the validation studies were made available in the validation study reports.
Assay Reproducibility: The panel concluded that the assay had excellent reproducibility bothwithin and between laboratories for the 42 reference chemicals evaluated in the validation
studies. Additionally, the positive and negative control chemicals had 100% reproducibility within
and between laboratories based on classification outcome, which further supports the
reproducibility of the ROS assay.
Test method predictivity: After reviewing analyses provided in the validation study reports, thepanel agreed that conducting a single assay per chemical provided optimal predictivity. The
panel concluded that the classification criteria for test outcomes have been appropriately
optimized to avoid false negatives while minimizing false positives. The panel also noted thatchemicals positive for both reactive oxygen species were uniformly phototoxic.
Data quality: The panel agreed that the high level of within and between laboratory
reproducibility suggested a consistently high level of quality of the validation studies. While thestudies were not conducted in strict accordance with GLPs, most of the labs were GLP certified.
The validation management team also confirmed that quality control audits found that validation
report data accurately reflected the raw data results.
Test method protocols: The panel considered the test method protocols used for the twovalidation studies and key aspects of a proposed standardized ROS assay protocol. The panel
recommended that the solar simulator should be equipped with an appropriate temperature
control unit or fan since ROS production can be influenced by temperature. The panel
concluded that the list of proficiency chemicals provided in the test method protocol forlaboratories to use to demonstrate ability to perform the assay was appropriate. The panel
recommended that each lab should develop historical positive and negative control value
acceptance ranges that can be used to determine the acceptability of an individual test. The
panel also agreed with the appropriateness of the reference chemicals identified for qualificationof solar simulators other than the two used in the validation studies.
Applicability domain: The applicability domain of the ROS assay is currently restricted to only
those chemicals that meet the solubility criteria outlined in the protocol. The panel
recommended that as experience is gained from use of the ROS assay, the applicability domain
could be more fully described in terms of physicochemical properties and/or chemical classes.
This will contribute to increased efficiency by providing criteria that can be used to identify
whether a chemical may be satisfactorily tested in the ROS assay, or whether an alternate
assay should be used initially.

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Peer Review Panel Evaluation of the ROS Assay
Introduction
The Japanese Center for the Validation of Alternative Methods (JaCVAM) convened an
independent scientific peer review panel to evaluate the validation status of the Reactive
Oxygen Species (ROS) Assay in accordance with established international validation andacceptance criteria (OECD, 2005). The ROS Assay is a test method proposed to evaluate
whether test substances such as pharmaceuticals may have the potential to cause
phototoxicity.
The panel met initially in February and again in August 2013 in Tokyo. The panel considered thereports of two international validation studies and a proposed outline for a ROS assay protocol
at their initial meeting. Following provision of a complete ROS assay protocol by the Validation
Management Team (VMT) and updating of the validation study reports, the panel met a second
time to complete its evaluation. In conducting its evaluation, the panel addressed each of theevaluation criteria that correspond to internationally harmonized validation and acceptancecriteria. This report summarizes the panel’s final evaluation and conclusions.
Evaluation Criterion 1: A rationale for the test method should be available, including
description of toxicological mechanisms, a clear statement of scientific need, andregulatory application.
The panel concluded that the ROS assay is applicable for use within the ICH regulatory testing
framework for photosafety evaluation of pharmaceutical products. Regulatory authorities (e.g.
PMDA/MHLW, U.S. FDA, EMEA, KFDA) require non-clinical photosafety testing prior toapproving First-in-Human Phase I studies so that appropriate precautions and observations canbe taken during initial human studies. Such non-clinical photosafety testing typically includes anassessment of the potential for a drug to cause phototoxic reactions, which are characterized bydermal redness, swelling, irritation, and inflammation. The panel also recognized that the ROS
assay is applicable to in-house drug research and development. A proposed integrated
photosafety testing strategy incorporating the ROS assay is provided below as Figure 1.
Chemicals that exhibit the potential for phototoxicity should be identified and if appropriate,
eliminated in the early stages of drug discovery and development. Ideally, drugs should not be
phototoxic. However, some beneficial drugs that have phototoxicity potential may be
unavoidable, in which case it is important to ensure that there are appropriate precautions on
drug labels so that patients can avoid exposures to sunlight that could lead to adverse reactions.
ROS production is the most important mechanism for inducing chemical phototoxicity.
Physicochemical tests such as the ROS Assay enable the identification of ROS production bychemicals after exposure to UV and/or visible light.
.

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Figure 1
Proposed integrated photosafety testing strategy incorporating the ROS
Photosafety Assay (courtesy of Dr. Hosoi)

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Evaluation Criterion 2: The relationship between the test method endpoint(s) and thebiological effect and to the toxicity of interest should be addressed, describing
limitations of the test methods.
Scientific rationale The ROS assay is based on identifying reactive oxygen species producedby photoreactive chemicals after exposure to UV and/or visible light. This mechanism is thebasis for phototoxic reactions in the skin of humans, e.g., redness, swelling, irritation, andinflammation. The panel recognized that ROS production is the most important mechanism forchemically-induced phototoxicity, and is therefore a critical pathway initiating event leading tophototoxicity. The ROS assay quantitatively measures two common reactive oxygen species
generated by photoreactive chemicals after exposure to simulated sunlight. In this validation
study, chemicals that did not produce sufficient ROS to meet the positive photoreactivity
threshold classification criteria for this assay are uniformly non-phototoxic, while chemicals that
met or exceeded the positive classification criteria include all known phototoxicants. Therefore if
a chemical is negative in the ROS assay it is unlikely that phototoxicity will occur in living
systems.
Limitations: The panel noted that the ROS assay assesses chemical photoreactivity in a nonbiological system, and therefore may overpredict phototoxicity potential since it does not assessthe direct interaction of chemicals with biological tissues. Additionally, the initiation of phototoxic
reaction in humans depends on pharmacokinetics and sufficient concentration in the target
tissue, which cannot be assessed in this assay. The assay may also overestimate the potential
for phototoxicity because some chemicals may not achieve sufficient concentration in skin forphototoxic reactions to occur, or photodegradation may occur. Accordingly, positive results in
the ROS assay are generally recommended for further evaluation in a photosafety testing
strategy.
Evaluation Criterion 3: A detailed test method protocol should be available.
The panel considered the test method protocols used for the two validation studies and key
aspects of a proposed standardized ROS assay protocol. The panel concluded that the
proposed ROS assay protocol was sufficiently detailed to allow for users to successfully performthe procedure. The panel also concluded that the protocol included adequate and appropriate
analysis and classification criteria. The panel recommended that the solar simulator should beequipped with an appropriate temperature control unit or fan since ROS production can be
influenced by temperature. The panel concluded that the list of proficiency chemicals provided
in the test method protocol for laboratories to use to demonstrate ability to perform the assay
was appropriate. The panel recommended that each lab should develop historical positive andnegative control value acceptance ranges that can be used to determine the acceptability of anindividual test.

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Evaluation Criterion 4: Within- and between-laboratory reproducibility of the test methodshould be demonstrated.
The panel concluded that the assay demonstrated excellent reproducibility both within andbetween laboratories for the 42 reference chemicals evaluated in the validation studies.
Additionally, the positive and negative control chemicals had 100% reproducibility within andbetween laboratories based on classification outcome, which further supports the reproducibilityof the ROS assay.
Evaluation Criterion 5: Demonstration of the test method’s performance should be basedon testing of representative, preferably coded reference chemicals.
The panel agreed that the reference chemicals selected for the validation studies wereappropriate, and sufficiently representative of the chemicals likely to be evaluated in the assay.
The 42 reference chemicals incorporated most known human phototoxicants and included 23
known positives and 19 negatives. The chemicals were backed by data from human patch
testing and in vitro 3T3 phototoxicity assay results. The validation reference chemicals wereappropriately coded to minimize bias by performing labs. All data from the validation studies
were made available in the validation study reports.
The panel noted the potential importance of chemical structure, and acknowledged the VMT for
incorporating chemical structures for all chemicals in the validation report. In addition, the panel
noted that the VMT also assessed and described whether the current drug label information forJapan and U.S. included precautionary language for phototoxicity.
Evaluation Criterion 6: Accuracy or predictive capacity should be demonstrated using
representative chemicals. The performance of test methods should have been evaluatedin relation to existing relevant toxicity data as well as information from the relevant target
species.
After reviewing analyses provided in the study reports, the panel agreed with the VMT that asingle assay per chemical provided optimal predictivity. The panel concluded that theclassification criteria for test outcomes had been appropriately optimized to avoid false
negatives while minimizing false positives (see ROS assay protocol judgment criteria).
Appropriate criteria are provided for photoreactive, weakly photoreactive, non-photoreactive,
and inconclusive classifications. In the first validation study (Atlas solar simulator), twophototoxic and one non-phototoxic reference chemicals were classified as inconclusive due tosolubility issues, and were not included in the integrated accuracy calculations. In the second
validation study (Seric solar simulator), three phototoxic and four non-phototoxic reference
chemicals were classified as inconclusive due to solubility issues, and were not included in the
integrated accuracy calculations.
All of the phototoxic reference chemicals that produced conclusive results were identified as
photoreactive in both validation studies, resulting in a sensitivity of 100% and a false negative
rate of 0%. In the first validation study, of the 18 non-phototoxic reference chemicals thatprovided conclusive results, 15 were identified as non-photoreactive and three were classified
as weakly photoreactive, resulting in a specificity of 83.3 %(15/18), and a false positive rate of
16.7% (3/18). In the second study, of the 15 non-phototoxic chemicals for which there wereconclusive results, 12 were identified as non-phototoxic, two were classified as weakly
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photoreactive, and one was classified as phototoxic, resulting in a specificity of 80% (12/15),
and a false positive rate of 20% (3/15). However, it is important to note that of the nonphototoxic chemicals producing photoreactive results, all three responses were categorized as
weakly photoreactive in the first study, and 2 of the 3 responses were categorized as weakly
photoreactive in the second study.
Evaluation Criterion 7: All data supporting the assessment of the validity of the test
method should be available for expert review.
All raw data for the two validation studies was provided in the validation study reports, which arereadily available electronically from the Japanese Center for the Validation of Alternative
Methods at the National Institute of Health Sciences, Tokyo, Japan.
Evaluation Criterion 8: Ideally, all data supporting the validity of a test method shouldhave been obtained in accordance with the principles of Good Laboratory Practice (GLP).
The panel concluded that there was a high level of within and between laboratory
reproducibility, which suggested a consistently high level of quality of the validation studies.
While the studies were not conducted in strict accordance with GLPs, six of the sevenlaboratories participating in the validation studies were GLP certified. This included two of threeof the labs in Study #1 (Atlas), and all four of the labs participating in Study #2 (Seric). There
was no significant variability between laboratories, which suggested a consistent level of quality.
The validation management team also confirmed that quality control audits found that validation
report data accurately reflected the raw data results.
.
Evaluation Criterion 9: The applicability domain of the validity of the test method should
be defined for expert review.
The applicability domain of the ROS Assay is currently restricted to only those chemicals thatmeet the solubility criteria outlined in the protocol. The panel recommended that as experienceis gained from use of the ROS assay, the applicability domain could be more fully described interms of physicochemical properties and/or chemical classes. This would contribute toincreased efficiency by providing criteria that can be used to identify whether a chemical may besatisfactorily tested in the ROS assay, or whether an alternate assay should be used initially.
Chemicals that are insoluble in the recommended vehicles and therefore are not suitable fortesting with this assay may be able to be tested in other vehicles, such as BSA, alcohol, andacetone. However, further characterization and standardization of procedures using thesealternative vehicles should be performed before incorporation into routine use.
Evaluation Criterion 10: Proficiency chemicals should be provided in the proposed
protocol.
The panel concluded that the list of 9 proficiency chemicals provided in the test method protocol
for laboratories to use to demonstrate ability to perform the assay was appropriate. These 9chemicals were selected from the validation study reference chemicals and represent a wide
range of responses in the assay as well as a wide range of solubilities.
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Evaluation Criterion 11: Performance standards should be developed for the proposed
protocol.
The panel agreed with the appropriateness of the 17 reference chemicals identified for
qualification of proposed solar simulators other than the two solar simulators used in thevalidation studies. The reference chemicals were appropriately selected from the reference
chemicals used for the validation studies. While performance standards were not specifically
proposed, the panel considered that these reference chemicals would be appropriate forincorporation in future performance standards for the ROS assay.

Evaluation Criterion 12: Are there advantages in terms of time, cost and animal welfare?
The ROS assay can potentially provide significant savings in time, cost and reduced animal use
when used in an integrated photosafety testing strategy by allowing decisions to be made earlier
and with fewer overall tests for many chemicals. These advantages are illustrated in Figure 1,
which shows that chemicals that are non-photoreactive in the ROS assay need not be tested inanimals or other tests. The ROS assay also reduces the number of chemicals which progress totesting in the 3T3 Phototoxicity Assay, with a subsequent reduction in the number of positive
results in the 3T3 assay that may progress to in vivo tests for confirmation.
Conclusion
The panel concluded that the reproducibility and predictivity of the ROS assay is sufficient tosupport its use in an integrated photosafety testing and decision strategy for drug research anddevelopment. In this integrated strategy, negative results in the ROS assay would not require
further testing in animals or other tests, while positive, weakly positive, and inconclusive results
would proceed to the next level of testing in an in vitro test system such as the 3T3 Phototoxicity
Assay (OECD Test Guideline 432). The panel also concluded that use of the ROS assay will
provide significant potential savings in time, cost and reduced animal use for photosafetyassessments. Furthermore, incorporating the ROS assay into a photosafety testing strategy will
significantly reduce the overall number of substances that require additional testing in the invitro 3T3 Phototoxicity Assay, and substantially reduce the number of substances that require
subsequent testing in animals.

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Appendix 1
Independent Peer Review Panel Members
Horst Spielmann, M.D., Panel Chairman
Dr. Spielmann is a Professor for Regulatory Toxicology in the Institute for Pharmacy, Faculty ofBiology, Chemistry, and Pharmacy at the Freie Universität Berlin in Berlin, Germany. He is theformer and first head of the National German Center for the Validation of Alternative Methods(ZEBET) at the Federal Institute for Risk Assessment (BfR) in Berlin, Germany, where hedirected the standardization and international validation of several in vitro methods and also
served as the head of the Department of Scientific Services. He served as Germany’s firstnational representative on the Scientific Advisory Committee for the European Centre for the
Validation of Alternative Methods at the European Commission’s Joint Research Centre in
Ispra, Italy, and has served as an expert for many years for the European Commission’s
Framework Programs on alternatives to animal testing and in vitro toxicology. In December
2012, he was appointed as the Animal Welfare Officer for the State of Berlin.
William S. Stokes, D.V.M., Rapporteur
Dr. Stokes is an Adjunct Professor in the Department of Molecular Biomedical Sciences,
College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, U.S.A.
He recently retired from the United States Public Health Service where he was an Assistant U.S.
Surgeon General. He served as the first Director of the U.S. National Toxicology Program’s
Interagency Center for the Evaluation of Alternative Toxicological Methods at the National
Institute of Environmental Health Sciences from 1997-2012, where he directed the international
validation and scientific peer review of numerous new test methods for regulatory safetyassessments. He also served as the first Executive Director of the U.S. Interagency
Coordinating Committee on the Validation of Alternative Methods from 1997-2012. He is a
Fellow of the Academy of Toxicological Sciences and a board certified environmental scientist,and is also board certified in laboratory animal medicine and animal welfare.
Ikuo Horii, Ph.D.
Dr. Horii is a global consultant for Pfizer and former Executive Director of Pfizer Drug Safety
Research and Development in Nagoya, Japan. He is a Certified Toxicologist in the JapaneseSociety of Toxicology with a background in biochemistry, pharmacology, pathology, andmolecular toxicology. He is currently a Visiting Professor at several institutions, including the
School of Pharmacy at Showa University in Japan, Dalian Medical University in China, andCambridge University in the United Kingdom. He is a Visiting Research Fellow at the National
Institute of Health Sciences in Tokyo, Japan and Lecturer at Kyoto University, Tokyo University,
and Chiba Institute of Science. He is President of Horii Science Associates and a board
member of the Japanese Society of Toxicology.
Bae-Hwan Kim, D.V.M., Ph.D.
Dr. Kim is a Professor in the Department of Public Health at Keimyung University in Daegu,
Republic of Korea, where he leads a biomedical research program and lectures in the Collegeof Natural Sciences. He previously worked in the pharmaceutical and cosmetic industry for 15
years as a Team Leader in the Preclinical Department. His research includes investigation of
the oxidative stress and oxidative photodamage induced by UV radiation and interventional
strategies for avoidance of UV irradiation damage. His focus is on the safety evaluation ofsubstances applied to the skin and the development of alternative methods to animal
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experiments. Dr. Kim serves on the Editorial Board of the Journal of Biomedical Research andis a Council Member of the Korean Association for Laboratory Animal Science and the Korean
Society for Alternatives to Animal Experiments.

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Appendix 2
Acknowledgements
The Peer Review Panel members gratefully acknowledge Steven Venti from BHK Limited for his
invaluable assistance with translation and editing during the peer review process. The Panelalso acknowledges the members of the Validation Management Team for the completeness ofthe validation study reports, their cooperation and responsiveness to requests for additional
information and analyses, and their responsive consideration of suggestions and updating of thevalidation study reports and test method protocol. Finally, the Panel expresses its appreciation
to Dr. Hajime Kojima and his staff at the National Institute of Health Sciences for their excellent
support and arrangements for the peer review panel meetings.

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Appendix 3

Glossary1

3T3 NRU-PT: In vitro 3T3 neutral red uptake phototoxicity test.
Dose of light: The quantity [= intensity Å~ time (seconds)] of UV or visible light incident on asurface, expressed in J/m2 or J/cm2.
Irradiance: The intensity of UV or visible light incident on a surface, measured in W/m2 or
mW/cm2.
MEC: Molar Extinction Coefficient (also called molar absorptivity) is a constant for any given
molecule under a specific set of conditions (e.g., solvent, temperature, and wavelength) andreflects the efficiency with which a molecule can absorb a photon (typically expressed as L mol1 cm-1).
Photoreactivity: the property of a chemical to react with another molecule as a consequence ofphoton absorption. Excitation of molecules by light can lead to generation of reactive oxygen
species (ROS) such as superoxide anion (SA) and singlet oxygen (SO) through energy transfer
mechanisms.
Phototoxicity: acute toxic response that is elicited after the first exposure of skin to certain
chemicals and subsequent exposure to light, or that is induced similarly by skin irradiation after
systemic administration of a chemical.
ROS: Reactive Oxygen Species, including superoxide anion (SA) and singlet oxygen (SO).
UVA: Ultraviolet light A (wavelengths between 320 and 400 nm).
UVB: Ultraviolet light B (wavelengths between 290 and 320 nm).
UVC: Ultraviolet light C (wavelengths between 190 and 290 nm).

1

Note: definitions derived from OECD TG 432 and the ROS assay protocol
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Appendix 4
References
1. OECD. Guidance Document No. 34: Guidance Document on the Validation andInternational Acceptance of New or Updated Test Methods for Hazard Assessment.
Organization for Economic Cooperation and Development, Paris, France. 2005.
Available at:
http://search.oecd.org/officialdocuments/displaydocumentpdf/?doclanguage=en&c
ote=env/jm/mono(2005)14

2. OECD. Test Guideline 432: In Vitro 3T3 NRU Phototoxicity Assay. OECD
Guidelines for the Testing of Chemicals, Section 4: Health Effects. Organization for
Economic Cooperation and Development, Paris, France. 2005. Available at:
http://www.oecd-ilibrary.org/environment/test-no-432-in-vitro-3t3-nru-phototoxicitytest_9789264071162-en

3. ROS Assay Validation Management Team. Validation report for the international
validation study on ROS (Reactive Oxygen Species) assay as a test evaluating
phototoxic potential of chemicals (Atlas Suntest version), 20 September 2013.

4. ROS Assay Validation Management Team. Validation report for the international
validation study on ROS (Reactive Oxygen Species) assay as a test evaluating
phototoxic potential of chemicals (Seric version), 9 October 2013.

5. ROS Assay Validation Management Team. Reactive Oxygen Species (ROS)
Assay to Examine Photoreactivity of Chemicals: ROS Assay Protocol, version 3.1,
20 September 2013.

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