ROS Assay Protocol
Version 1.0
17 May 2013

REACTIVE OXYGEN SPECIES (ROS) ASSAY TO EXAMINEPHOTOREACTIVITY OF CHEMICALS

Issued by: ROS assay Validation Management Team
Date: 17 May 2013.

1.

INTRODUCTION

The purpose of this document is to recommended protocol for reactive oxygen species (ROS) assay
to examine photoreactivity of chemicals. Photoreactivity is defined as the property of chemicals
that react with another molecule in consequence of photon absorption. Excitation of molecules bylight can lead to generation of ROS including superoxide anion (SA) and singlet oxygen (SO)through energy transfer mechanisms.

ROS assay is not a phototoxicity test but a physicochemical

test similar to measurement of UV absorbance.

2.

INITIAL CONSIDERATION

Validation studies conducted by JaCVAM showed that ROS assay has 100% sensitivity forpredicting phototoxicants but a low specificity [1-3]. Based on the results of the validation studies,
conducting this assay would classify a test chemical into one of 3 criterion; “Non-photoreactive”,“Weakly photoreactive” or “Photoreactive”.

A “Non-photoreactive” result in this assay could

predict as a non-phototoxic chemical, however, further examination on phototoxicity is necessary for“Weakly photoreactive” or “Photoreactive” result.

When precipitation is found microscopically at

20 µM (final concentration), it is inadvisable to evaluate the chemical.

It is inadvisable to test

chemicals exhibiting peak absorbance at 440 or 560 nm, which might interfere with the ROS assay.

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PRINCIPLE OF THE TEST METHOD

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

Several classes of drugs including

antibacterials, thiazide diuretics, non-steroidal anti-inflammatory drugs, quinolones, and tricyclic
antidepressants, even though nontoxic by themselves, may become reactive under exposure toenvironmental light, leading to undesired side effects. The primary event in any photosensitization
process is the absorption of photons of the appropriate wavelength, which allows chromophore toreach an excited state. The excitation energy is often transferred to oxygen molecules, followed bygeneration of ROS: SA through type I reaction and SO through type II reaction by photo-excited
drug molecules. These appear to be the principal intermediate species in the phototoxic response.
From the standpoint of risk assessment, previous study demonstrated that determination of ROS
from pharmaceutical substances irradiated with UVA/B and visible light would be of help in
recognizing their phototoxic potential.
In the ROS assay, the 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, resulting in the formation of a trans-annular peroxide intermediate
capable of inducing the bleaching of RNO as follows;
SO+ Imidazole → [Peroxide intermediate] → Oxidized imidazole[Peroxide intermediate] + RNO → RNO + Products
The SA generation could be determined by the reduction of nitroblue tetrazolium (NBT) as indicatedbelow; NBT can be reduced by SA via a one-electron transfer reaction, yielding partially reduced (2e-) monoformazan (NBT+) as a stable intermediate [5]. Thus, SA can reduce NBT to NBT+, whose
formation can be monitored spectrophotometrically at 560 nm.
SA + NBT → O 2 + NBT+4.

DESCRIPTION OF THE TEST METHODTechnical equipment
・ Solar simulator: e.g. Suntest CP series (Atlas Material Technology, Chicago, IL, USA) or
SXL-2500V2 (Seric, Tokyo, Japan) with a fan and UVC cut filter (spectrum are shown inAppendix 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 its 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-treat flat-bottom)
・ Plastic- and glassware

Solar simulator
Appropriate solar simulator irradiating UV and visible light should be used. The irradiation power
distribution should be close to that of outdoor daylight by using an appropriate UVC cut filter.
Recommended solar simulators and UVA intensity on the plate position measured by UVA detector#0037 (Dr. Hönle) are shown as follows;・ Suntest CPS+ or CPS (Atlas) with UV cut filter (<290 nm): 1.8 to 2.2 mW/cm2 (e.g. theindicator 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 should be equipped with appropriate temperature control unit or fan to stabilize
the temperature during irradiation because ROS production is influenced by temperature.

When thesolar simulator has a temperature control unit, the temperature will be adjusted at 25°C.
Acceptable temperature range is 20 to 29˚C during irradiation. When a different solar simulator isused, the reference chemical set listed in section 6 should be tested prior to the test. Values ofsinglet oxygen (SO) and superoxide anion (SA) should be close to the values mentioned in section 6.

Quarts reaction container
A quarts reaction container is used to avoid the loss of UV by passing through a plastic lid andvaporizing of reaction mixture [6].
recommended.

The made-to-order container (Appendix 2), or its equivalent is

If a different container is used, a lid or seal with high UV transmittance must be

used. In this case, the feasibility study should be conducted using the reference chemicals todetermine appropriate condition of UV/visible light exposure.

Reagents
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The following reagents will be used and stored according to the instructions of manufacturers.
・ NaH 2 PO 4 ・2H 2 O (CAS No. 13472-35-0)
・ Na 2 HPO 4 ・12H 2 O (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 should 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 NaH 2 PO 4 ・2H 2 O and 5.8 g of Na 2 HPO 4 ・12H 2 O, add 900 mL ofpurified water, adjust with HCl to a pH of 7.4, dilute with purified water up to 1 L, andmix.
Stored 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.
Stored 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.
Stored 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.
Stored in a refrigerator and protect from light.

Test chemicals
The test chemical should be stored appropriately until termination of the study and its stability
during the test period should be confirmed. Two concentration levels, 20 and 200 µM (final
concentration), should be used.

Preparation of test chemicals
The test chemicals will be prepared using DMSO just before use. Each test chemical will beweighed in a tube, and added DMSO at the concentration 10 mM at first. The tube will be mixed
with vortex mixer and sonicated for 5 to 10 min under UV-cut illumination or shade.
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preparations should be kept to protect from light.
be set at 200 µM.

The final concentration in a reaction mixture will

When precipitation is observed at 20 µM in the reaction mixture under amicroscope, 1 mM of the test chemical solution should be prepared using DMSO. In the case of
DMSO-insoluble chemical, the final concentration in the reaction mixture including 20 µL of DMSO
will be used at the maximum concentration without precipitation (20 or 200 µM).

Positive and negative control chemicals
Qunine HCl (CAS No. 6119-47-7) should be used at 200 µM (final concentration) as a positive
control.

Sulisobenzone (CAS No. 4065-45-6) should be used at 200 µM (final concentration) as anegative control.

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

The stock solution will be thawed just before

the experiment and used within the day.

Solvent
DMSO (analytical grade) should be used at first. In the case of DMSO-insoluble chemical, 20 mM
NaPB should be used. When a test chemical is insoluble in either DMSO or 20 mM NaPB, bovine
serum albumin (BSA) or other solvent might be helpful [7-8].

Prior to use of BSA or other solvent,a feasibility study (see section 6) should be conducted using the reference chemicals to determine
appropriate test conditions. However, classification for regulatory purposes should not be based on
a ROS assay using BSA or other solvent until these solvents have been properly evaluated.

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Test procedure
A tube (e.g. 1.5 mL of micro tube) and a plastic clear flat bottomed 96-well microplate will be used.
The reaction mixture should be prepared by vortex mixing and/or sonication under UV-cut
illumination or shade. The same volume of DMSO, 20μL, will be added in a blank instead of test
chemical solution.
【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 – 10 min)Add 200 μL of mixture to each well (n=3) 1)
Check solubility using a microscope (×100) 2)

Pre-read Abs at 440 nm after shaking for 5 sec
Light exposure for 1 hr 3)

Read A 440 and A 560 after shaking for 1 min
Notes
1) It should be avoided using peripheral wells. More than one test chemical could be tested on aplate. An example of 96-well plate configuration is shown as follows;

B: Blank (DMSO)
P: Quinine, 200 μM
N: Sulisobenzone，200 μM
T1-T3: Test chemical No. 1-3
L: 20 μM
H: 200 μM

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2) Some chemicals may precipitate in the reaction mixture. Therefore it is important to checkthe solubility before the irradiation. The solubility of each reaction mixture in a well should
be observed by using a microscope before irradiation. Test chemicals concentrations should
be selected so as to avoid precipitation or cloudy solutions.

3) The 96-well plate will be placed in to the Quartz reaction container. A quarts cover will be seton the plate and fasten with bolts.

The solar simulator and the temperature control unit (or its

equivalent) will be used under a stable condition. Before and after irradiation, UVA intensity
and temperature at the plate position will be measured using a UVA detector and thermometer.

5.

DATA AND REPORTING

Data analysis
Data from three wells for each chemical concentration will be used to calculate mean and standard
deviation.
・ SO
Decrease of A 440 ×1000 = [A 440 (–) – A 440 (+) – (a – b)] ×1000
A 440 (–): Absorbance before light exposure at 440 nm
A 440 (+): Absorbance after light exposure at 440 nm
a: Blank before light exposure (mean)b: Blank after exposure (mean)
・ SA
Increase of A 440 ×1000 = [A 560 (+) – A 560 (–) – (b – a)] ×1000
A 560 (–): Absorbance before light exposure at 560 nm
A 560 (+): 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 should be satisfied in each experiment.
・ No precipitation of test chemical in the reaction mixture before light exposure.
・ No technical problems when collecting data set (including temperature within prescribedrange).
・ Raw OD value: 0.02 to 1.5
・ Historical positive and negative control values should be developed by each laboratory based
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on a mean +/-2 SD. The following range was defined based on the 95% confidence interval(mean +/- 1.96SD) obtained from the validation data. When other solar simulators are 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

Judgment criteria
Each test chemical will be judged as follows;
Judgment1)

Concentration
judgedSO(mean of 3 wells)

Photoreactive

20 and/or 200
μM2)

≥25

and

≥70

<25 and/or P3)

and

≥70

≥25

and

<70 and/or P

SA(mean of 3 wells)

Weakly photoreactive

20 and 200 μM2)

<25

and

≥20, <70

Non-photoreactive

20 and 200 μM2)

<25

and

<70

Inconclusive

The results do not meet the above-mentioned criterion.4)

Notes1) It can be judged based on results of one experiment because the ROS assay shows good
reproducibility in the validation studies.
2) It would be judged at 20 μM only when precipitation is observed at 200 μM.
3) Precipitation before irradiation.
4) When precipitation is observed at 20 and 200 μM before irradiation, the compound isregarded incompatible with the ROS assay.

Data quality
For a regulatory purpose, the study should be conducted under high-quality standards with datacollection records readily available, preferably in compliance with GLP/GMP regulations and all
documents should be checked by the Quality Assurance Unit of the laboratory.

Test report
The test report must include the following information:
・ Test chemical
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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

Archives
The study report and all raw data will be retained according to the SOP in the testing facility.

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6.

REFERENCE CHEMICALS FOR THE FEASIBILITY STUDY

For establishing ROS assay, irradiation condition to satisfy the recommended criteria should bedetermined using the positive and negative controls at 200 µM. The reference chemicals should betested at 200 µM in a feasibility study prior to test.

The reference chemicals should be selectedfrom the following 21 chemicals judged at 200 µM in the validation studies. Recommendedreference chemical set and acceptable range are shown in Table 1 and Table 2. Values of SO andSA should be close to the values.
・

Photoreactive chemicals
Acridine, acridine hydrochloride, chlorpromazine hydrochloride, diclofenac, doxycyclinehydrochloride, furosemide, ketoprofen, 6-methylcoumarine, 8-methoxy psoralen (8-MOP),
nalidixic acid, nalidixic sodium salt, norfloxacin, ofloxacin, omeprazole, promethazinehydrochloride, and tetracycline.

・Non-photoreactive chemicals
Aspirin, benzocaine, camphor sulfonic acid, erythromycin, and p-aminobenzoic acid
(PABA).

Table 1 Recommended chemical set for solar simulators used in the validation studies and theacceptable range at 200 µM: 3 photoreactive chemicals showing strong (No. 11), moderate(No. 12), and weak (No. 13) responses and 3 non-photoreactive chemicals (No. 14-16)
No.

Chemical

CAS No.

SO

SA

Photoreactive chemicals
11

Doxycycline hydrochloride

10592-13-9≥115 (115 to 429)

≥230 (230 to 468)

12

Norfloxacin70458-96-7≥131 (131 to 271)

≥57 (57 to 161)

13

8-MOP

298-81-7≥31 (31 to 137)

≥20 (0 to 126)

Non-photoreactive chemicals
14

Benzocaine

94-09-7<25 (7 to 9)<20 (7 to 17)

15

Erythromycin

114-07-8<25 (-15 to 11)<20 (9 to 19)

16

PABA

150-13-0<25 (-8 to 12)<20 (-11 to 7)

DMSO should be used for preparation of the chemicals.
The values in parenthesis were calculated as mean +/- 1.96 SD from the validation data.

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Table 2 Recommended chemical set for the other solar simulators and the acceptable range at 200µM: 11 photoreactive chemicals (No. 21-31) and 3 non-photoreactive chemicals (No.
32-34)
No.

Chemical

CAS No.

SO

SA

Photoreactive chemicals
21

Acridine

260-94-6≥182 (182 to 328)

≥121 (121 to 243)

22

Chlorpromazine hydrochloride

69-09-0

Usually <25≥66 (66 to 106)23

Diclofenac

15307-79-6≥34 (34 to 416)

≥47 (47 to 437)

24

Doxycycline hydrochloride

10592-13-9≥115 (115 to 429)

≥230 (230 to 468)

25

Furosemide

54-31-9≥31 (31 to 225)

≥20 (-7 to 109)

26

Ketoprofen

22071-15-4≥120 (120 to 346)

≥77 (77 to 151)

27

8-MOP

298-81-7≥31 (31 to 137)

>20 (0 to 126)

28

Nalidixic acid389-08-2≥54 (54 to 246)

≥88 (88 to 470)29

Norfloxacin70458-96-7≥131 (131 to 271)

≥57 (57 to 161)

30

Omeprazole

73590-58-6

Usually <25≥30 (30 to 216)

31

Promethazine hydrochloride

58-33-3≥25 (20 to 168)

≥20 (-3 to 77)

Non-photoreactive chemicals
32

Benzocaine

94-09-7<25 (-7 to 9)<20 (-7 to 17)

33

Erythromycin

114-07-8<25 (-15 to 11)<20 (-9 to 19)

34

PABA

150-13-0<25 (-8 to 12)<20 (-11 to 7)

DMSO should be used for preparation of the chemicals.
The values in parenthesis 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 B (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 (1O 2 and O 2 -.) 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

Relative light intensity (%)

100
90
80
70
60
50

Suntest CPS/CPS+(Atlas)SXL-2500V2(Seric)40
30
Sun light

20
10
0
300

400

500

600

Wavelength (nm)

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Appendix 2

Quarts reaction container used in the validation studies
166 mm

86 mm

24 mm

14.5 mm

127 mm15