Introduction

In recent years, establishment of regulations for animal testing have been encouraged from the viewpoints of 3Rs principles (replacement, reduction, and refinement) for animal testing and welfare. Especially, in EU, animal testing for cosmetics has been prohibited stepwise since 1993, and the EU directive¹ intended to enforce the total ban on marketing of cosmetics tested on animals after 2013 was issued in 2003. The relevant regulations were established² in 2009.

In such situations, development of alternatives to animal experiments has been required worldwide. In Japan, the Japanese Center for the Validation of Alternative Methods, National Institute of Health Science (JaCVAM) was founded in 2005. Since then, many alternative testing methods developed in Japan have been listed in the test guidelines issued by the Organisation for Economic Co-operation and Development (OECD) (OECD Test Guidelines).³

For details of ADRA and differences from the existing technique of Direct Peptide Reactivity Assay (DPRA), see the references.^5-9

Testing method

In this document, the testing was performed with the ADRA kit (code No. 296-80901) in accordance with the attached protocol. In this testing, however, polypropylene microtubes were used for the reaction instead of a 96-well plate indicated in the original ADRA. Table 1 shows liquid chromatography (LC) conditions. It should be noted that the equilibration time after the retention time of 13.5 minutes in each run may differ because it depends on configuration of instruments and mixer volume.

Table 1. LC conditions

Instrument	Agilent UHPLC 1290 infinity Ⅱ (Agilent Corporation）
Column	Wakopak® Core C18 ADRA 3.0 x 150 mm (D)
Mobile phase A	0.1% Trifluoroacetic acid in water
Mobile phase B	0.1% Trifluoroacetic acid in acetonitrile
Gradient condition for NAC	0 min. (A：B= 70: 30)→ 9.5 min. (45: 55)→ 10.0 min (0: 100)→ 13.0 min. (0: 100)→ 13.5 min. (70: 30)→ 25.0 min. (70: 30)
Gradient condition for NAL	0 min. (A：B= 80: 20)→ 9.5 min. (55: 45)→ 10.0 min (0: 100)→ 13.0 min. (0: 100)→ 13.5 min. (80: 20)→ 25.0 min. (80: 20)
Flow rate	0.3 mL/min.
Mixer volume	35 µL
Column temperature	40 ℃
Wavelength	UV 281 nm
Injection volume	10 µL

Results

Table 2 shows evaluation results on proficiency substances, and Figure 2 shows representative chromatograms. The decreased rates for the proficiency substances obtained with the Wakopak^® Core C18 ADRA fell within the ranges presented in the guideline for ADRA.

FUJIFILM Wako's website offers videos for testing operations with the kit and the protocols. We are always pleased to help your ADRA operations with our products and information.

　

Table 2. List of proficiency substances and evaluation results

No.	Test chemical	CAS RN®	Description	Molecular weight	Solvent	Prediction by ADRA	Known decreased rate (%)		Measured decreased rate (%)
							NAC	NAL	NAC	NAL
1	p-Benzoquinone	106-51-4	Solid	108.09	Water	Positive	90-100	40-70	100	61
2	Sodium p-toluenesulfonchloramide trihydrate (Chloramine T)	7080-50-4	Solid	281.69	Water	Positive	90-100	90-100	100	97
3	Cinnamaldehyde	14371-10-9	Liquid	132.16	Acetonitrile	Positive	40-100	≦ 20	43	1
4	Palmitoyl chloride	112-67-4	Liquid	274.87	Acetonitrile	Positive	≦ 10	50-100	2	93
5	Imidazolidinyl urea	39236-46-9	Solid	388.29	Water	Positive	10-45	≦ 10	35	1
6	Farnesal	19317-11-4	Liquid	220.35	Acetonitrile	Positive	10-40*	≦ 15	15	2
7	Glycerin	56-81-5	Liquid	92.09	Water	Negative	≦ 7	≦ 7	0	0
8	Benzyl alcohol	100-51-6	Liquid	108.14	Acetonitrile	Negative	≦ 7	≦ 7	0	0
9	Dimethyl isophthalate	1459-93-4	Solid	194.19	Acetonitrile	Negative	≦ 7	≦ 7	0	0
10	Propyl p-Hydroxybenzoate	94-13-3	Solid	180.20	Acetonitrile	Negative	≦ 7	≦ 7	0	0

* The range will be revised, although it is different from the guideline.

References

1. European Commission : "DIRECTIVE 2003/15/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 27 February 2003, amending Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products, Official J. European Union, L66/26(2003).
2. European Commission : "REGULATION(EC) No 1223/2009 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 30 November 2009 oncosmetic products", Official J. European Union, L 342/59(2009).
3. Miyazaki, H. and Yoshiyama, Y.: Folia Pharmacologica Japonica, 151,48(2018).
4. OECD："In Chemico Skin Sensitisation : Amino acid Derivative Reactivity Assay(ADRA)", OECD Guideline for the Testing of Chemicals 442C(2019).
5. Yamamoto, Y. et al.: Wako Junyaku Jiho 87(1), 12(2019).
6. Fujita, M. et al. : J. Pharmacol. Toxicol. Methods, 70, 94(2014).
7. Yamamoto, Y. et al. : J. Appl. Toxicol., 35, 1348(2015).
8. Fujita, M. et al. : J. Appl. Toxicol., 39, 191(2019).
9. Fujita, M. et al. : Toxicol. in Vitro, 59, 161(2019).