ROS Assay Protocol
Version 3.2
28November 2014

REACTIVE OXYGEN SPECIES (ROS) ASSAY TO EXAMINEPHOTOREACTIVITY OF CHEMICALS

Issued by: ROS assay Validation Management Team
Date: 28 November 2014.

1.

INTRODUCTION

The purpose of this document is to recommend a protocol for assaying the photoreactivity ofchemicals based on reactive oxygen species (ROS).

Photoreactivity is defined as the property of achemical to react with another molecule as a consequence of photon absorption.

Excitation ofmolecules by light can lead to generation of ROS such as superoxide anion (SA) and singlet oxygen(SO) through energy transfer mechanisms.

The ROS assay does not measure phototoxicity directly,

but rather is a physicochemical test that can be applied to that purpose, similar to measurement of
UV absorbance.

2.

INITIAL CONSIDERATION

Validation studies conducted by JaCVAM showed that the ROS assay has 100% sensitivity forpredicting phototoxicants but can result in some false positives [1-3].

Based on the results of thevalidation studies, conducting this assay would classify a test chemical into one of the following:
photoreactive, weakly photoreactive, non-photoreactive, or inconclusive.

Photoreactive, weakly

photoreactive, or inconclusive results would be a flag for follow-up phototoxicity assessment.
Non-photoreactive results indicate a very low probability of phototoxicity, and no further
phototoxicity testing would be suggested.

In the ROS assay, measurement is first made at aconcentration of 200 µM (final concentration).

If interference such as precipitation or coloration(exhibiting peak absorbance at 440 or 560 nm) is observed at 200 µM, measurements are made at 20µM.

When precipitation or coloration is found at 20 µM, the substance is considered incompatible

with the ROS assay.

Since the ROS assay is designed to evaluate directly the photoreactivity ofchemicals, it is not suitable for detecting chemicals that induce in vivo phototoxicity by indirect
mechanisms such as porphyria and pseudoporphyria.

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PRINCIPLE OF THE TEST METHOD

3.

Drug-induced photoirritation can be defined as an inflammatory reaction of the skin after topical orsystemic administration of pharmaceutical substances.

There are several classes of drugs(including antibacterials, thiazide diuretics, non-steroidal anti-inflammatory drugs, quinolones, andtricyclic antidepressants) which are nontoxic by themselves but could become reactive when
exposed to environmental light and thereby result in undesired side effects.

The primary event inany photosensitization process is the absorption of photons of a wavelength that induces excitationof the chromophore.

The excitation energy is often transferred to oxygen molecules, followed bygeneration of ROS, including SA through type I reaction and SO through type II reaction byphoto-excited drug molecules.
phototoxic response.

These appear to be the principal intermediate species in the

From the standpoint of risk assessment, previous research has demonstrated

that determination of ROS from pharmaceutical substances irradiated with UVA/B and visible lightwould be of help in recognizing their phototoxic potential.
In the ROS assay, SO generation was detected by spectrophotometric measurement of
p-nitrosodimethyl aniline (RNO) bleaching, followed by decreased absorbance of RNO at 440 nm[4].

Although SO does not react chemically with RNO, the RNO bleaching is a consequence of SO

capture by the imidazole ring, which results in the formation of a trans-annular peroxideintermediate capable of inducing the bleaching of RNO, as follows:
SO+ Imidazole → [Peroxide intermediate] → Oxidized imidazole[Peroxide intermediate] + RNO → RNO + Products
SA generation was detected by the observing the reduction of nitroblue tetrazolium (NBT) as
indicated below; NBT can be reduced by SA via a one-electron transfer reaction, yielding partiallyreduced (2 e-) monoformazan (NBT+) as a stable intermediate [5].

Thus, SA can reduce NBT to+

NBT , the formation of which can be monitored spectrophotometrically at 560 nm.
SA + NBT → O2 + NBT+4.

DESCRIPTION OF THE TEST METHODTechnical equipment
Solar simulators:
Suntest CP series (Atlas Material Technology, Chicago, IL, USA) or SXL-2500V2 (Seric, Tokyo,
Japan) with a fan and UVC cut filter (Spectrum shown in Appendix 1)
UVA detector: e.g. #0037 (Dr. Hönle, München, German) or UD series (Topcon, Tokyo, Japan)
Quartz reaction container (Ozawa Science, Aichi, Japan, Appendix 2) or equivalent
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Microplate spectrophotometer, equipped with 440 and 560 nm filters
Microscope
Thermometer
Vortex mixer
Plate shaker
Sonicator
Pipettes
Polypropylene tubes
Plastic 96-well plates (clear, non-treated, flat-bottom)
Plastic and glass laboratory ware

Solar simulator
An appropriate solar simulator is to be used for irradiation of UV and visible light.

The irradiation

power distribution is to be kept as close to that of outdoor daylight as possible by using anappropriate UVC cut filter.

Recommended solar simulators and UVA intensity on the plate position

measured by UVA detector #0037 (Dr. Hönle) are as follows:
Suntest CPS+ or CPS (Atlas) with UV cut filter (<290 nm),
1.8 to 2.2 mW/cm2 (e.g. the indicator setting value of 250 W/m2 for CPS+) for 1 hour,
6.5 to 7.9 J/cm2 of UVA intensity (Appendix 1)
SXL-2500V2 (Seric) with UV cut filter (<300 nm),
3.0 to 5.0 mW/cm2 for 1 hour,
11 to 18 J/cm2 of UVA intensity (Appendix 1)
The solar simulator is to be equipped with an appropriate temperature control or fan to stabilize thetemperature during irradiation, because ROS production is affected by temperature.
temperature for a solar simulator with temperature control is 25°C.
range during irradiation is 20° to 29°C.

Standard

The acceptable temperature

If a solar simulator other than the two recommended

models is used, the reference chemical set listed in section 6 is to be tested prior to performing the
ROS assay to ensure that measured values of SO and SA are close to those mentioned in section 6.

Quartz reaction container
A quartz reaction container is used to avoid loss of UV due to passing through a plastic lid andvaporization of the reaction mixture [6].

A made-to-order container (See Appendix 2.) or its

equivalent is recommended.

If a different container is used, a lid or seal with high UVtransmittance must be used.

In this case, a feasibility study using the reference chemicals is to be

conducted to determine an appropriate level of exposure to UV and visible light.

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Reagents
The following reagents are to be used and stored according to the instructions of manufacturers.
NaH2PO4・2H2O (CAS No. 13472-35-0)
Na2HPO4・12H2O (CAS No. 10039-32-4)
p-Nitrosodimethylaniline (RNO, CAS No. 138-89-6)
Imidazole (CAS No. 288-32-4)
Nitroblue tetrazolium chloride (NBT, CAS No. 298-83-9)
Purified waterPreparation of reagents
All reagents are to be sonicated and used within 1 month after preparation.

Representative

preparation methods are shown as follows.
20 mM sodium phosphate buffer (NaPB), pH 7.4
Weigh 593 mg of NaH2PO4・2H2O and 5.8 g of Na2HPO4・12H2O, add 900 mL of purified
water, adjust with HCl to a pH of 7.4, dilute with purified water up to 1 L, and mix.
Store in a refrigerator or at room temperature.
0.2 mM p-Nitrosodimethylaniline (RNO)
Dissolve 3 mg of RNO in 100 mL of 20 mM NaPB.
Store in a refrigerator and protect from light.
20 mM imidazole
Dissolve 13.6 mg of imidazole in 10 mL of 20 mM NaPB.
Dilute the 2×10-2 M imidazole solution 100 times with 20 mM NaPB.
Store in a refrigerator and protect from light.
0.4 mM nitroblue tetrazolium chloride (NBT)
Dissolve 32.7 mg of NBT in 100 mL of 20 mM NaPB.
Store in a refrigerator and protect from light.

Test chemicals
Test chemicals are to be stored appropriately until termination of the study and their stability during
the test period is to be confirmed.
used.

One concentration level, 200 µM (final concentration), is to be

A 20-µM concentration can be used if precipitation before light exposure, coloration, orother interference is observed in the reaction mixture at 200 µM.

Preparation of test chemical solutions
The test chemical solutions are to be prepared using a solvent just before use.
to be weighed in a tube, and solvent added to a concentration 10 mM.
4Each test chemical is

The tube is to be mixed with

ROS Assay Protocol
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28November 2014a vortex mixer and sonicated for 5 to 10 minutes under UV-cut illumination or shade.
preparations are to be protected from light.
200 µM.

All

The final concentration in a reaction mixture is to be

When precipitation before light exposure, coloration, or other interference is observed in

the reaction mixture at 200 µM, a 1-mM solution (20 µM as the final concentration) is to beprepared using the solvent.

For chemicals that are not soluble in DMSO, 20 µL of DMSO is to becontained in the reaction mixture.

Positive and negative control chemicals
Quinine hydrochloride (CAS No. 6119-47-7) is to be used at 200 µM (final concentration) as apositive control.

Sulisobenzone (CAS No. 4065-45-6) is to be used at 200 µM (final concentration)

as a negative control.

Preparation of positive and negative control chemical solutions
Stock solutions of quinine and sulisobenzone are to be prepared at 10 mM each in DMSO (final
concentration of 200 µM) according to the above procedure, divided into tubes, and stored in afreezer (generally below -20˚C) for up to 1 month.

The stock solution is to be thawed just before

the experiment and used within the day.

Solvent
Use analytical grade DMSO at first.

For chemicals that are not soluble in DMSO, 20 mM of NaPB

is to be used. When a test chemical is insoluble in either DMSO or 20 mM NaPB, try bovine serum
albumin (BSA) or other solvent [7-8]. Prior to use of BSA or other solvent, however, perform afeasibility study (see Section 6) using the reference chemicals to determine appropriate test
conditions. The results of ROS assays using BSA or other solvents, however, are not suitable forregulatory purposes until these solvents have been properly evaluated.

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Test procedure
A tube (e.g. 1.5 mL micro tube) and a plastic clear flat bottomed 96-well microplate are to be used.
The reaction mixture is to be prepared by vortex mixing and/or sonication under UV-cut illuminationor shade.

The same volume of DMSO, 20 μL, is to be added in a blank instead of test chemicalsolution.
SO

SA

20 mM NaPB

480 μL

20 mM NaPB

855 μL

Imidazole

250 μLNBT

125 μLRNO

250 μL

Chemical

20 μL

Chemical

20 μL

Mix (Vortex and sonication for 5 to 10 min)Add 200 μL of mixture to each well (n=3)1
Check solubility using a microscope at 100×2 and coloration3
Pre-read Abs at 440 and 560 nm after shaking for 5 sec
Light exposure for 1 hr4
Read A440 and A560 after shaking for 1 min and check coloration3
Notes
1) Avoid using peripheral wells. More than one test chemical can be tested on a plate. A typical
96-well plate configuration is as follows:

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2) Some chemicals might precipitate in the reaction mixture.
solubility prior to irradiation.

It is therefore important to check

Solubility of each reaction mixture in its well is to be observedwith a microscope prior to irradiation. Test chemical concentrations are to be selected so as
to avoid precipitation or cloudy solutions.
3) The reaction mixture is to be checked for coloration with the naked eye.
4) The 96-well plate is to be placed in the quartz reaction container. A quartz cover is to be seton the plate and fastened with bolts.

Ensure that temperature and other ambient conditions

are stable when using the solar simulator.

Measure UVA intensity and temperature at theplate position using a UVA detector and thermometer both before and after irradiation.

5.

DATA AND REPORTING

Data analysis
Data from three wells for each chemical concentration is used to calculate mean and standard
deviation.
SO
Decrease of A440 ×1000 = [A440 (–) – A440 (+) – (a – b)] ×1000
A440 (–): Absorbance before light exposure at 440 nm
A440 (+): Absorbance after light exposure at 440 nm
a: Blank before light exposure (mean)b: Blank after exposure (mean)
SA
Increase of A560 ×1000 = [A560 (+) – A560 (–) – (b – a)] ×1000
A560 (–): Absorbance before light exposure at 560 nm
A560 (+): Absorbance after light exposure at 560 nm
a: Blank before light exposure (mean)b: Blank after exposure (mean)

Criteria for data acceptance
The following criteria are to be satisfied in each experiment.
No precipitation of test chemical in the reaction mixture before light exposure.
No coloration of test chemical in the reaction mixture before or after light exposure.
No technical problems, including prescribed temperature range, when collecting data set.
Raw OD value: 0.02 to 1.5
Historical positive and negative control values are to be developed by each laboratory based on a
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mean +/-2 SD.

The following range was defined based on the 95% confidence interval (mean+/- 1.96SD) obtained from the validation data.

When a solar simulator other than arecommended model is used, establish modified criteria based on 95% confidence interval.
Positive control value at 200 μM (mean of 3 wells)
SO: 319 to 583
SA: 193 to 385
Negative control value at 200 μM (mean of 3 wells)
SO: -9 to 11
SA: -20 to 2

Criteria for judgment
Each test chemical is to be judged as follows:
Judgment1, 2Concentration3

SO (mean of 3 wells)

SA (mean of 3 wells)

Photoreactive

200 μM

≥25

and

≥70

<25 and/or I4

and

≥70

≥25

and

<70 and/or I4

Weakly photoreactive

200 μM<25

and≥20, <70

Non-photoreactive

200 μM

<25

and

<20

Inconclusive

The results do not meet the above-mentioned criterion.

Notes
1. A single experiment is sufficient for judging
Photoreactive

results, because the ROS assay shows good intraandinter-laboratory

reproducibility

in

70

the

validation studies.

SA

2. If precipitation, coloration, or other interference isobserved at both 20 and 200 μM, the chemical is

Weaklyphotoreactive
20
Non‐photoreactive
25

considered incompatible with the ROS assay andSOjudged as inconclusive.

3. Twenty μM can be used for judgment when precipitation or coloration is observed at 200 μM.
For regulatory purposes, the stability of the test chemical in the reaction mixture both before andafter light exposure is to be confirmed when results at 20 μM are used for judgment as a
non-photoreactive chemical for which no further phototoxicity testing is necessary.
4. Interference such as precipitation or coloration.

Data quality
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Studies for regulatory purposes are to be conducted to the highest of quality standards, with datacollection records readily available, in compliance with GLP/GMP regulations whenever possible,
and all documents checked by the Quality Assurance Unit of the laboratory.

Test report
The test report must include the following information:
Test chemical
Name and lot No.
Physical nature and purity
Storage condition
Stability during the test period
UV/vis absorption spectrum, maximum molar extinction coefficient at 290 to 700 nm, and/or
photostability, if known
Preparation of test chemical solution
Final concentrations tested
Control chemicals
Name, manufacturer, and lot No.
Physical nature and purity
Storage condition
Preparation of control chemical solutions
Final concentrations tested
Solvent
Name, manufacturer, and lot No.
Justification for choice of solvent
Irradiation condition
Manufacturer and type of the solar simulator used
Rationale for selection of the solar simulator used
UVA detector used
UVA irradiance, expressed in mW/cm2
UVA dose, expressed in J/cm2
Temperature before and after irradiation
ROS assay procedure
Acceptance and decision criteria
Results
Discussion
Conclusions
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Archives
The study report and all raw data is to be retained according to the SOP in the testing facility.

6.

REFERENCE CHEMICALS FOR THE FEASIBILITY STUDY

To perform a ROS assay, it is necessary first to ensure irradiation conditions that satisfy therecommended criteria using the positive and negative controls, after which reference chemicals areto be tested in a feasibility study.

The reference chemical set, as selected from the validation

studies, and acceptable value ranges are shown in Table 1 and Table 2.

It is important that thevalues for SO and SA obtained in the feasibility study be similar to the following values.
Table 1

Standard chemical set for laboratories to demonstrate proficiency using solar simulators ofSuntest CPS/CPS+ (Atlas) or SXL-2500V2 (Seric) and the acceptable range

No.

Chemical

CAS No.

SO

SA

Solvent

Concentration

11

Doxycyclinehydrochloride

10592-13-9

115 to 429

230 to 468

DMSO

200 µM

12

Norfloxacin70458-96-7

131 to 271

57 to 161

DMSO

200 µM

13

8-Methoxy
psoralen298-81-7

31 to 137

0 to 126

DMSO

200 µM

14

Fenofibrate

49562-28-9

77 to 203

-31 to 11

DMSO

20 µM

15

p-Aminobenzoicacid150-13-0-8 to 12

-11 to 7

DMSO

200 µM

16

Benzocaine

94-09-7-7 to 9-7 to 17DMSO

200 µM

17

Erythromycin

114-07-8-15 to 11-9 to 21DMSO

200 µM

18

Octyl salicylate

118-60-5-5 to 11-8 to 20

DMSO

20 µM

19

L-Histidine

71-00-1-8 to 128 to 120

NaPB

200 µM

The values were calculated as mean +/- 1.96 SD from the validation data.
Note: Underline parts were corrected because of errors in writing.

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Table 2

Recommended chemical set for the other solar simulators and the acceptable range

No.

Chemical

CAS No.

SO

SA

Solvent

Concentration

21

Acridine

260-94-6

182 to 328

121 to 243

DMSO

200 µM

22

Chlorpromazinehydrochloride

69-09-0-56 to 70

66 to 106DMSO

200 µM

23

Diclofenac

15307-79-6

34 to 416

47 to 437

DMSO

200 µM

24

Doxycyclinehydrochloride

10592-13-9

115 to 429

230 to 468

DMSO

200 µM

25

Furosemide

54-31-931 to 225

-7 to 109

DMSO

200 µM

26

Ketoprofen

22071-15-4

120 to 346

77 to 151

DMSO

200 µM27

8-Methoxy
psoralen298-81-7

31 to 137

0 to 126

DMSO

200 µM

28

Nalidixic acid389-08-2

54 to 246

88 to 470

DMSO

200 µM

29

Norfloxacin70458-96-7

131 to 271

57 to 161

DMSO

200 µM

30

Omeprazole

73590-58-6-221 to 103

30 to 216

DMSO

200 µM

31

Promethazinehydrochloride

58-33-3

20 to 168

-3 to 77

DMSO

200 µM

32

Fenofibrate

49562-28-9

77 to 203

-31 to 11

DMSO

20 µM

33

p-Aminobenzoicacid150-13-0-8 to 12

-11 to 7

DMSO

200 µM

34

Benzocaine

94-09-7-7 to 9-7 to 17DMSO

200 µM

35

Erythromycin

114-07-8-15 to 11-9 to 21DMSO

200 µM

36

Octyl salicylate

118-60-5-5 to 11-8 to 20

DMSO

20 µM

37

L-Histidine

71-00-1-8 to 128 to 120

NaPB

200 µM

The values were calculated as mean +/- 1.96 SD from the validation data.

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7.

GLOSSARY

ROS: Reactive Oxygen Species, including superoxide anion (SA) and singlet oxygen (SO).
3T3 NRU-PT: In vitro 3T3 neutral red uptake phototoxicity test.
Irradiance: The intensity of UV or visible light incident on a surface, measured in W/m2 or
mW/cm2.
Dose of light: The quantity [= intensity × time (seconds)] of UV or visible light incident on asurface, expressed in J/m2 or J/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) and reflects
the efficiency with which a molecule can absorb a photon (typically expressed as L mol-1 cm-1).
Photoreactivity: The property of chemicals that react with another molecule as a consequence ofabsorption of photons.
Phototoxicity: An acute light-induced tissue response to a photoreactive chemical.
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).

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REFFERENCES[1] 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), 2013.
[2] 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), 2013.
[3] Onoue S, Hosoi K, Wakuri S, Iwase Y, Yamamoto T, Matsuoka N, Nakamura K, Toda T,
Takagi H, Osaki N, Matsumoto Y, Kawakami S, Seto Y, Kato M, Yamada S, Ohno Y, Kojima
H. Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen
species assay for chemical photosafety evaluation. J Appl Toxicol, Article first published online,
13 June, 2012 (DOI: 10.1002/jat.2776).
[4] I. Kraljić, S. El Mohsni. A new method for the detection of singlet oxygen in aqueous solutions.
Photochem Photobiol, 28, 577-81, 1978.
[5] Pathak MA, Joshi PC. Production of active oxygen species (1O2 and O2-.) by psoralens andultraviolet radiation (320-400 nm). Biochim Biophys Acta, 798, 115-26, 1984.
[6] Onoue S, Igarashi N, Yamada S, Tsuda Y. High-throughput reactive oxygen species (ROS)
assay: an enabling technology for screening the phototoxic potential of pharmaceuticalsubstances. J Pharm Biomed Anal, 46, 187-93, 2008.
[7] Onoue S, Yamauchi Y, Kojima T, Igarashi N, Tsuda Y. Analytical studies on photochemical
behavior of phototoxic substances; effect of detergent additives on singlet oxygen generation.
Pharm Res, 25, 861-868, 2008.
[8] Onoue S, Kato M, Yamada S. Development of an albuminous reactive oxygen species assay forphotosafety evaluation under experimental biomimetic conditions. J Appl Toxicol, Article firstpublished online, 28 Jan, 2013 (DOI: 10.1002/jat.2846).

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Appendix 1

Spectrum of solar simulators used in the validation studies

Suntest CPS/CPS+(Atlas)SXL-2500V2(Seric)Sun light

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Appendix 2

Quartz reaction container used in the validation studies
166 mm

86 mm

24 mm

14.5 mm

127 mm

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Appendix 3

Amendment

Version 3.2
Page 10: The SA values of benzocaine and erythromycin were corrected in Table 1 because of errors
in writing.
Incorrect

Correct

Benzocaine

7 to 17-7 to 17

Erythromycin

9 to 21-9 to 2116