Cosmetics - Analytical methods - HPLC/UV method for the identification and assay of hydroquinone, ethers of hydroquinone and corticosteroids in skin whitening cosmetic products

This European Standard specifies a HPLC/UV method for the assay of hydroquinone, 3 ethers of hydroquinone and 4 corticosteroids most frequently found in illegally sold skin whitening cosmetic products: clobetasol propionate, betamethasone dipropionate, fluocinonide and fluocinolone acetonide.
This standard also proposes HPLC/UV methods for the identification of 38 corticosteroids may be found in skin whitening products. Indeed, as corticosteroids could be deliberately introduced in skin whitening cosmetics, despite the fact that they are forbidden to use, an identification of the presence of one of this illicit compounds could be enough for a market survey control.
This standard is not dedicated to artificial nail products or soaps.

Kosmetische Mittel - Untersuchungsverfahren - HPLC/UV Verfahren für die Identifizierung und Bestimmung von Hydrochinon, Hydrochinonethern und Kortikosteroiden in hautaufhellenden kosmetischen Mitteln

Diese Europäische Norm legt ein HPLC/UV Verfahren zur Identifizierung und Quantifizierung von Hydrochinon, drei Hydrochinon¬ethern und vier Kortikosteroiden fest, die am häufigsten in entgegen den Vorschriften verkauften kosmetischen Mitteln zur Hautaufhellung vorgefunden werden: Clobetasolpro-pionat, Betamethasondipro¬pionat, Fluocinonid und Fluocinolonacetonid.
Diese Norm enthält außerdem HPLC/UV Verfahren zur Identifizierung von 38 Kortikosteroiden, die in Hautaufhellungsprodukten vorkommen können (siehe Anhang D).
Diese Norm ist nicht für künstliche Nagelpflegeprodukte oder Seifen vorgesehen.

Cosmétiques - Méthodes analytiques - Méthode de CLHP couplée à la détection UV pour l'identification et l'analyse de l'hydroquinone, de ses éthers et des corticostéroïdes dans les produits cosmétiques éclaircissants de la peau

La présente Norme européenne spécifie une méthode CLHP couplée à la détection UV pour l’identification et la quantification de l’hydroquinone, de trois de ses éthers et de quatre corticostéroïdes principalement présents dans les produits cosmétiques éclaircissants de la peau commercialisés illégalement : le propionate de clobétasol, le dipropionate de bétaméthasone, le fluocinonide et le fluocinolone acétonide.
La présente norme décrit également des méthodes CLHP couplées à la détection UV pour l’identification de trente-huit corticostéroïdes susceptibles d’être présents dans les produits cosmétiques éclaircissants de la peau (voir l’Annexe D).
La présente norme ne concerne pas les produits pour ongles artificiels ou les savons.

Kozmetika - Analizne metode - Metoda HPLC/UV za identifikacijo in določevanje hidrokinona, etrov hidrokinona in kortikosteroidov v kozmetičnih izdelkih za beljenje kože

ICS:   71.100.70
Ta evropski standard določa metodo HPLC/UV za določevanje hidrokinona, 3 etrov hidrokinona in 4 kortikosteroide, ki so najpogosteje prisotni v nezakonito prodajanih kozmetičnih izdelkih za beljenje kože: klobetazol propionat, betametazon dipropionat, fluocinonid in fluocinolon acetonid.
Ta standard podaja tudi metode HPLC/UV za identifikacijo 38 kortikosteroidov, ki jih je mogoče najti v izdelkih za beljenje kože. Ker je kortikosteroide mogoče namerno vnesti v kozmetične izdelke za beljenje kože kljub temu, da je njihova uporaba prepovedana, se lahko na podlagi ugotovljene prisotnosti ene od teh nezakonitih spojin uvede nadzor, ki temelji na raziskavi trga.
Uporaba tega standarda ni predvidena za umetne izdelke za nego nohtov ali mila.

General Information

Status
Published
Public Enquiry End Date
02-Mar-2016
Publication Date
08-Oct-2017
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
06-Sep-2017
Due Date
11-Nov-2017
Completion Date
09-Oct-2017

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Kosmetische Mittel - Untersuchungsverfahren - HPLC/UV Verfahren für die Identifizierung und Bestimmung von Hydrochinon, Hydrochinonethern und Kortikosteroiden in hautaufhellenden kosmetischen MittelnCosmétiques - Méthodes analytiques - Méthode de CLHP couplée à la détection UV pour l'identification et l'analyse de l'hydroquinone, de ses éthers et des corticostéroïdes dans les produits cosmétiques éclaircissants de la peauCosmetics - Analytical methods - HPLC/UV method for the identification and assay of hydroquinone, ethers of hydroquinone and corticosteroids in skin whitening cosmetic products71.100.70SULSRPRþNLCosmetics. ToiletriesICS:Ta slovenski standard je istoveten z:EN 16956:2017SIST EN 16956:2017en,fr,de01-november-2017SIST EN 16956:2017SLOVENSKI

STANDARD
SIST EN 16956:2017
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16956
September
t r s y ICS
y sä s r rä y r English Version

Cosmetics æ Analytical methoidentification and assay of hydroquinoneá ethers of hydroquinone and corticosteroids in skin whitening cosmetic products Cosmétiques æ Méthodes analytiques æ Méthode de CLHP couplée à la détection UV pour l 5identification et l 5analyse de l 5hydroquinoneá de ses éthers et des corticostéroïdes dans les produits cosmétiques éclaircissants de la peau

Kosmetische Mittel æ Untersuchungsverfahren æ Bestimmung von Hydrochinoná Hydrochinonethern und Kortikosteroiden in hautaufhellenden kosmetischen Mitteln This European Standard was approved by CEN on

s { June
t r s yä

egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä

translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä

CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä

EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels

t r s y CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN

s x { w xã t r s y ESIST EN 16956:2017
EN 16956:2017 (E) 2

Contents Page European foreword ....................................................................................................................................................... 3 Introduction .................................................................................................................................................................... 4 1 Scope .................................................................................................................................................................... 5 2 Principle ............................................................................................................................................................. 5 3 Reagents ............................................................................................................................................................. 5 4 Apparatus and equipment ........................................................................................................................... 8 5 Procedure........................................................................................................................................................... 9 5.1 Sample preparation ........................................................................................................................................ 9 5.2 Liquid chromatography measurement conditions ............................................................................. 9 5.3 Detection ......................................................................................................................................................... 10 6 Evaluation ....................................................................................................................................................... 10 6.1 Identification ................................................................................................................................................. 10 6.2 Quantitative determination ..................................................................................................................... 10 6.3 Result expression ......................................................................................................................................... 11 7 Test report ...................................................................................................................................................... 11 Annex A (informative)

Example of chromatograms obtained .................................................................. 12 Annex B (informative)

Validation Data for the quantitative method hydroquinone and its three ethers .................................................................................................................................................... 13 Annex C (informative)

Validation data for the quantitative method for the 4 most frequently found corticosteroids ........................................................................................................... 18 Annex D (normative)

Screening methods for the identification of hydroquinone, 3 ethers of hydroquinone and 38 corticosteroids .................................................................................................. 24 Bibliography ................................................................................................................................................................. 37

SIST EN 16956:2017
EN 16956:2017 (E) 3

European foreword This document (EN 16956:2017) has been prepared by Technical Committee CEN/TC 392 “Cosmetics”, the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by March 2018, and conflicting national standards shall be withdrawn at the latest by March 2018. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. The existing peer review validation data for hydroquinone are preliminary and will be supplemented by inter-laboratory test data if available. According to the CEN-CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 16956:2017

EN 16956:2017 (E) 4

Introduction Hydroquinone is not allowed for use in cosmetic products for skin whitening and depigmentation of dermal spots or imperfections. Due to its cytotoxic effects its use has been regulated. Hydroquinone and 3 of its ethers (hydroquinone monomethylether (MME), hydroquinone monoethylether (MEE) and hydroquinone monobenzylether (MBE)) are regulated by the cosmetic regulation 1223/2009. Nowadays the use of these substances is prohibited in skin whitening cosmetic products. Depigmentation is a side effect of topical steroids, in this way corticosteroids might be used as compounds in products illegally sold as cosmetics. Corticosteroids most commonly found in these products are clobetasol propionate, fluocinonide, betamethasone dipropionate, and fluocinolone acetonide (see Figure 1). Corticosteroids are listed in Regulation 1223/2009 Annex II “List of substances prohibited in cosmetic products” (reference number 300), and their use is also prohibited in cosmetic products.

a) Clobetasol propionate b) Fluocinonide c) Betamethasone dipropionate d) Fluocinolone Acetonide Figure 1 — Corticosteroids most commonly found in illegal cosmetics All these substances work on the same principle as hydroquinone which mainly consists of inhibition of melanin synthesis. The cosmetic directive 95/32/EC [2] gives an analytical method for the assay of hydroquinone and 3 of its ethers (hydroquinone monomethylether (MME), hydroquinone monoethylether (MEE) and hydroquinone monobenzylether (MBE)) in cosmetic products for lightening the skin. In order to update and extend this official method to the identification and assay of corticosteroids in cosmetic products, this standard describes an HPLC/UV method for the identification and assay of hydroquinone, ethers of hydroquinone and corticosteroids in cosmetic products. SIST EN 16956:2017

EN 16956:2017 (E) 5

1 Scope This European Standard specifies a HPLC/UV method for the identification and quantification of hydroquinone, 3 ethers of hydroquinone and 4 corticosteroids most frequently found in illegally sold skin whitening cosmetic products: clobetasol propionate, betamethasone dipropionate, fluocinonide and fluocinolone acetonide. This standard also gives HPLC/UV methods for the identification of 38 corticosteroids that may be found in skin whitening cosmetic products (see Annex D). This standard is not dedicated to artificial nail products or soaps. 2 Principle The sample is extracted by a mixture of water/methanol and gently warmed in order to extract compounds present in the product. The obtained mixture is filtered. The quantitation of present compounds in solution is made by reversed phase HPLC with DAD (Diode Array Detector) detection. 3 Reagents If not otherwise specified, analytical-grade chemicals shall be used; the water shall be distilled or of a corresponding purity. “Solution” shall be understood as an aqueous solution unless otherwise specified. 3.1 Methanol, HPLC grade. 3.2 Water, HPLC grade. 3.3 Extraction solution, methanol/water (1/1). Mix 500 ml of methanol (3.1) and 500 ml of water (3.2) in a 1 000 ml conical flask. 3.4 Compounds considered, see Table 1. Table 1 — Compounds considered Compound

CAS
Manufacturera

Purity % Used method – clause Alclometasone dipropionate (ACD) 66734–13–2 USP 99,2 Annex D Amcinonide (AMC) 51022–69–6 Sigma 97,9 Annex D Beclomethasone dipropionate (BCD) 5534-09-8 Sigma 99,0 Annex D Betamethasone acetate (BMA) 987–24–6 Sigma 98,6 Annex D Betamethasone (BM) 378–44–9 Sigma 98,4 Annex D Betamethasone dipropionate (BMD) 5593–20–4 Sigma 98,6 2/ Annex D Betamethasone valerate (BMV) 2152–44–5 Sigma 98,1 Annex D Budesonide (BUD) 51333–22–3 Ph. Eur. 99,7 Annex D Clobetasol propionate (CP) 25122–46–7 Sigma 98,8 2/ Annex D Clocortolone pivalate (CLP) 34097–16–0 USP 98,9 Annex D Cortisone (CS) 53–06–5 Sigma 98,3 Annex D Desonide (DSN) 638–94–8 Cil 98,0 Annex D SIST EN 16956:2017

EN 16956:2017 (E) 6
Compound
CAS
Manufacturera

Purity % Used method – clause Desoximetasone (DXM) 382–67–2 Sigma 99,0 Annex D Dexamethasone phosphate (DMPS) 2392–39–4 Sigma 100,2 Annex D Dexamethasone acetate (DMA) 1177–87–3 Sigma 99,0 Annex D Diflorasone diacetate (DFD) 33564–31–7 USP 99,8 Annex D Diflucortolone valerate (DFCV) 59198–70–8 Schering / Annex D Difluprednate (DFP) 23674–86–4 Sigma 99,5 Annex D Flumethasone pivalate (FMP) 2002–29–1 Farmabios 100,4 Annex D Fluocinolone acetonide (FCA) 67–73–2 Sigma 99,6 2/ Annex D Fluocinonide (FCAA) 356–12–7 Sigma 99,0 2/ Annex D Fluocortolone –hexanoate (FCH) 303–40–2 Schering / Annex D Fluocortolone pivalate (FCP) 29205–06–9 Ph. Eur. / Annex D Flurandrenolide (FDL) 1524–88–5 USP / Annex D Halcinonide (HAL) 3093–35–4 Sigma 99,0 Annex D Hydrocortisone (HC) 3093–25–4 Sigma 98,0 Annex D Hydrocortisone aceponate (HCAP) 74050–20–7 Toronto Research Chemicals 98,0 Annex D Hydrocortisone butyrate (HCB) 13609–67–1 Sigma 99,1 Annex D Hydrocortisone valerate (HCV) 57524–89–7 Sigma 99,0 Annex D Methylprednisolone acetate (MPLA) 53–36–1 USP 99,8 Annex D Mometasone furoate (MMF) 83919–23–7 USP 99,8 Annex D Prednicarbate (PCN) 73771–04–7 Ph. Eur. / Annex D Prednisolone (PL) 50–24–8 Aventis Pharma 98,0 Annex D Prednisolone acetate (PLA) 52–21–1 Dr Ehrenstrofer 99,4 Annex D Prednisolone hexanoate (PLH) 69164–69–8 Schering / Annex D Prednisolone sulfobenzoate Na (PLSB) 630–67–1 Pharmaceutical drug / Annex D Triamcinolone (TRI) 124–94–7 Sigma 98,2 Annex D Triamcinolone acetonide (TRA) 76–25–5 Sigma 99,6 Annex D Hydroquinone (HQ) 123–31–9 Sigma > 99,9 2/ Annex D Hydroquinone monomethylether (MME) 150–76–5 Sigma 99,7 2/ Annex D Hydroquinone monoethylether (MEE) 622–62–8 Sigma 99,9 2/ Annex D Hydroquinone monobenzylether (MBE) 103–16–2 Sigma 99,5 2/ Annex D a Examples of manufacturer. SIST EN 16956:2017

EN 16956:2017 (E) 7

3.5 Mobile phase for HPLC, mobile phase A: methanol (3.1); mobile phase B: water (3.2). 3.6 Reference solutions. Methanol (3.1) is used as a solvent for the preparation of stock solutions. Standard solutions are prepared by dilution in a methanol / water solution (3.3). 3.6.1 Standard Stock Solution of hydroquinone and its 3 ethers (between 1,2 mg/ml and 2,4 mg/ml). Weigh approximately into a 25 ml volumetric flask: — 0,03 g of hydroquinone; — 0,04 g of monomethylether of hydroquinone; — 0,05 g of monoethylether of hydroquinone; — 0,06 g of monobenzylether of hydroquinone. Firstly, dissolve in 15 ml of methanol (3.1), if necessary use a shaker and then fill up to the calibration mark with methanol. This solution shall be prepared daily. 3.6.2 Standard Stock Solutions (1 mg/ml) and Standard Working Solution of 4 most frequently found corticosteroids (10 µg/ml). Weigh separately and approximately into a 10 ml volumetric flask: — 10 mg of betamethasone dipropionate; — 10 mg of clobetasol propionate; — 10 mg of fluocinonide; — 10 mg of fluocinolone acetonide. Firstly, dissolve in 5 ml of methanol (3.1), if necessary use a shaker and then fill up to the calibration mark with methanol. Theses 4 solutions can be stored during 6 months at 4 °C. Then prepare a daughter solution of 4 corticosteroids by introducing 1 ml of each standard stock solution with a glass pipette in a 100 ml volumetric flask. Fill up to the mark with mixture methanol/water (3.3). These solution can be stored during 6 weeks at 4 °C. 3.6.3 Calibration Solutions of hydroquinone and its 3 ethers The calibration solutions shall be freshly prepared (see Table 2). With a glass pipette, introduce the volume of standard stock solution (3.6.1.) in a volumetric flask then fill up to the mark with the mixture methanol/water (3.3). SIST EN 16956:2017

EN 16956:2017 (E) 8

Table 2 — Calibration solutions Number of calibration solution Volume of Standard Stock Solution ml Volume of flask ml Concentration of calibration solutions µg/ml 1 0,5 100 6 – 8 – 10 – 12 2 1,0 50 24 – 32 – 40 – 48 3 5,0 120 – 160 – 200 – 240 4 10,0 240 – 320 – 400 – 480 5 20,0 480 – 640 – 800 - 960 3.6.4 Calibration Solutions of 4 most frequently found corticosteroids The calibration solutions shall be freshly prepared (see Table 3). With a glass pipette, introduce the volume of standard daughter solution (3.6.2) in a volumetric flask then fill up to the mark with the mixture methanol/water (3.3). Table 3 — Calibration solution Number of calibration solution Volume of Standard Stock Solution ml Volume of flask ml Concentration of calibration solutions µg/ml 6 5 100 0,5 7 5 50 1,0 8 10 2,0 9 20 4,0 10 25 5,0 4 Apparatus and equipment In addition to the usual laboratory equipment, the following is required. 4.1 Analytical balance, with a precision of 0,1 mg. 4.2 Laboratory shaker. 4.3 Water-bath, allowing to heat at 60 °C. 4.4 High Performance Liquid Chromatography, consisting of: — sampling device; — pump system with gradient function; — degasser; SIST EN 16956:2017

EN 16956:2017 (E) 9

— column oven; — photodiode array detector; — evaluation system. 4.5 Membrane filter, for sample filtration e.g. polypropylene, 0,45

4.6. Analytical separation column, e.g. Thermo Fisher Scientific Hypurity Aquastar C181) length = 0,25 m, internal diameter = 4,6 mm, diameter particle size = 5

4.7 Paper filter, for sample filtration, e.g. filter quality medium folded 210 mm diameter. 5 Procedure 5.1 Sample preparation Weigh accurately 0,5 g of sample in a volumetric flask of 100 ml. Add 50 ml of extraction solution (3.3) and shake it with a laboratory shaker until a homogeneous suspension is formed. Place the mixture into a water-bath at 60 °C for 5 min to improve solution. If necessary repeat shaking with a laboratory shaker. Cool the flask to room temperature and adjust to 100 ml with the extraction mixture (3.3). Filter the extract through a paper filter and then through a membrane filter (0,45 µm). Inject the filtrate within the following 24 h into a HPLC system according to 5.2. 5.2 Liquid chromatography measurement conditions When using the apparatus (4.4) and column (4.6), the following conditions have shown to be useful: — Flow rate: 1,5 ml/min — Acquisition time: 25 min — Injection volume: 10

u Oven temperature: 36 °C

± 2 °C u Detection: Diode Array Detection with wavelength: 240 nm (corticosteroids) and 295 nm (hydroquinone and its ethers) u Mobile phases: Mobile Phase A: Methanol Mobile Phase B: Water See Table 4 for gradient separation.

1) This is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16956:2017

EN 16956:2017 (E) 10

Table 4 — Gradient separation Time min Volume fraction Mobile Phase A % Volume fraction Mobile Phase B % 0 5 95 20 80 20 21 5 95 25 5 95 5.3 Detection The detection and quantitative determination can be performed by DAD

= 240

= 295 nm). 6 Evaluation 6.1 Identification Hydroquinone, ethers of hydroquinone and corticosteroids are identified by comparing the retention times and the UV spectra of the sample with those of standard solution substances. Table 5 gives chromatographic parameters for the identification of compounds using the analytical method proposed in 5.2. Annex A gives chromatograms of standard solutions at the two studied wavelengths. Table 5 — Chromatographic parameters for the identification of compounds Compound Rt min

nm Hydroquinone 4,24 295 Hydroquinone monomethylether (MME) 9,34 295 Hydroquinone monoethylether (MEE) 12,08 295 Hydroquinone monobenzylether (MBE) 17,54 295 Fluocinolone acetonide (FCA) 17,73 240 Fluocinonide (FCAA) 19,60 240 Clobetasol propionate (CP) 20,55 240 Betamethasone dipropionate (BMD) 21,44 240 6.2 Quantitative determination The quantitative determination is done by linear regression based on peak areas of the external standard solutions. The calibration curve shall be linear and the correlation coefficient shall be upper or equal to 0,995. SIST EN 16956:2017

EN 16956:2017 (E) 11

The mass fraction of the compound in g /100 g in the sample is calculated with the following formula: ⋅⋅⋅=⋅1001000CVFRSW where R is the result of concentration in percent % (g/100 g); C is the concentration in g/l or in mg/ml of compounds detected in the sample solution given by the HPLC evaluation; V is the volume of dilution for the sample in ml; SW is the weight of sample in g; F is the possible dilution factor. 6.3 Result expression The amount is expressed in % or in g / 100 g and is rounded up to the nearest tenth of a percent. Annexes B and C give validation data. 7 Test report The test report should contain the data according to EN ISO/IEC 17025 [1] and at least the following information: a) all information necessary for the identification of the sample (kind of sample, origin of sample, designation); b) a reference to this European Standard; c) the date and type of sampling procedure (if known); d) the date of receipt; e) the date of test; f) the test results and the units in which they have been expressed; g) any particular points observed in the course of the test; h) any operations not specified in the method or regarded as optional, which might have affected the results. SIST EN 16956:2017

EN 16956:2017 (E) 12
Annex A (informative)
Example of chromatograms obtained

Key X Rentention time [min] 2 Methyl hydroquinone (9,337 min) Y Absorbance [mAU] 3 Ethyl hydroquinone (12,081 min) 1 Hydroquinone (4,235 min) 4 Benzyl hydroquinone (17,544 min) Figure A.1 — Example chromatogram obtained for between 0,5 and 1 mg/ml standard solutions of hydroquinone and its ethers

Key X Rentention time [min] 3 Fluocinolone acétonide (17,726 min) Y Absorbance [mAU] 4 Fluocinonide (19,597 min) 1 Arbutine (3,765 min) 5 Clobetasol propionate (20,551 min) 2 Acide kojic (8,898 min) 6 Betamethasone dipropionate (21,438 min) Figure A.2 — Example chromatogram obtained for 5

SIST EN 16956:2017
EN 16956:2017 (E) 13
Annex B (informative)

Validation Data for the quantitative method hydroquinone and its three ethers These validation data have been obtained by Service Commun des Laboratoires (SCL) du Ministère de l'Economie et des Finances – Laboratoire de Lyon-Oullins – Unité Produits – Section Cosmétiques. Limits of detection: 0,01 % (w/w) validated from standard solutions on three HPLC.

Key X Rentention time [min] 2 Methyl hydroquinone (9,458 min) Y Absorbance [mAU] 3 Ethyl hydroquinone (12,214 min) 1 Hydroquinone (4,299 min) 4 Benzyl hydroquinone (17,665 min) Figure B.1 — Chromatogram of hydroquinone and its three ethers standard solution at 0,01 % (w/w) Table B.1 — Limits of quantification (LOQ) validated from spiked matrixes analysed by 4 analysts on three HPLC during five days Compound Matrix: Whitening cream Matrix: Whitening oil LOQ % (w/w) RSD of repeatability % RSD of internal Reproducibility % LOQ % (w/w) RSD of repeatability % RSD of internal Reproducibility % Hydroquinone 0,1 2,3 12,6 0,1 2,8 25,3 Methyl ether 0,2 0,0 9,2 0,2 3,0 27,1 Ethyl ether 0,2 2,2 10,9 0,2 2,6 21,0 Benzyl ether 0,3 1,3 7,0 0,2 3,5 15,6 SIST EN 16956:2017

EN 16956:2017 (E) 14

Table B.2 — Linearity study validated from 8 calibration levels (standard solutions) analysed by 4 analysts on two different HPLC (n° 2 and n° 3) during five days Compound Maximal Relative Bias Obtained % Level 1 (LOQ) Level 2 Level 3 Level 4 Level 5 Level 6 Level 7 Level 8 Hydroquinone 6,3 ml 12,5 ml 25,0 ml 49,9 ml 124,9 /ml 249,7 /ml 374,6 /ml 499,4 /ml 27,7 10,7 3,7

«2,9
«1,6 1,7 1,5

«0,6 Methyl ether 8,1 ml 16,1 ml 32,2 ml 64,4 ml 161,1 /ml 322,1 /ml 483,2 /ml 644,2 /ml 11,1

«7,5
«2,5
«1,7 1,8 1,8 1,5

«0,8 Ethyl ether 10,1 ml 20,2 ml 40,5 ml 81 l 202,5 /ml 405,1 /ml 607,6 /ml 810,2 /ml

«11,0
«7,0 3,0
«1,8 1,8 1,7 1,5

«0,8 Benzyl ether 12,2 ml 24,5 ml 48,9 ml 97,9 ml 244,8 /ml 489,6 /ml 734,3 /ml 979,1 /ml

«10,9
«9,0
«5,6
«1,8 2,5 1,6 1,5

«0,8 Tfor all calibration analytes. Table B.3 — Linearity study validated from 5 calibration levels (standard solutions) analysed by 4 analysts on two different HPLC (n° 1 and n° 2) during five days Compound Maximal Relative Bias Obtained % Level 1 (LOQ) Level 2 Level 3 Level 4 Level 5 Hydroquinone 6,4

25,5
127,7
251,7
510,6
«40,6
«13,2
«3,1 6,9 - 1,3 Methyl ether 8,1
16,1
32,2
64,4
161,1
«35,3 - 6,1 2,7 - 1,7 0,4 Ethyl ether 10,1
20,2
40,5
202,5
- 31,7 - 3,9 2,6 - 1,7 0,4 Benzyl ether 12,2
24,5
48,9
97,9
244,8
26,7 - 5,0 1,9 - 1,8 0,4 Tfor all calibration analytes. SIST EN 16956:2017
EN 16956:2017 (E) 15

Table B.4 — Efficiency study validated from spiked matrix whitening cream at two levels analysed by 4 analysts on three different HPLC during five days Compound Matrix: Whitening cream Level (LOQ):

0,1 % for hydroquinone 0,2 % for methyl and ethyl 0,3 % for benzyl ether Level: 1 % (w/w) for ethers 2 % (w/w) for hydroquinone Efficiency RSD of repeatability RSD of internal reproducibility Efficiency RSD of repeatability RSD of internal reproducibility % % % % % % Hydroquinone 112 2,1 12,2 99 1,3 1,6 Methyl ether 97 1,6 9,1 100 1,4 2,7 Ethyl ether 98 1,9 10,8 101 1,5 3,4 Benzyl ether 96 2,1 7,9 100 1,7 5,5 Table B.5 — Efficiency study validated from spiked matrix whitening oil at two levels analysed by 4 analysts on three different HPLC during five days Compound Matrix: Whitening oil Level (LOQ):

0,1 % for hydroquinone 0,2 % for methyl and ethyl and benzyl ether Level: 1 % (w/w) for ethers 2 % (w/w) for hydroquinone Efficiency

RSD of repeatability
RSD of internal reproducibility Efficiency
RSD of repeatability

RSD of internal reproducibility % % % % % % Hydroquinone 95 2,8 25,3 100 1,5 3,5 Methyl ether 89 3,0 27,1 98 2,2 6,9 Ethyl ether 94 2,6 21,0 97 2,0 10,2 Benzyl ether 118 3,5 15,6 100 2,7 17,6 SIST EN 16956:2017

EN 16956:2017 (E) 16

Table B.6 — Maximal repeatability differences (MRD) in absolute value between two results from 30 obtained differences Compound Matrix: Cream Matrix: Oil MRD obtained

Directive CE n°95–32 criteria MRD obtained

Directive CE n°95–32 criteria % (w/w) % (w/w) % (w/w) % (w/w) Hydroquinone at 2 % (w/w) 0,08 0,13 0,12 0,13 Methyl ether at 1 % (w/w) 0,04 0,10 0,08 0,10 Ethyl ether at 1 % (w/w) 0,05 0,11 0,09 0,11 Benzyl ether at 1 % (w/w) 0,07 0,11 0,14a 0,11 a 29 on 30 differences are under or equal to 0,08, only one is equal to 0,14. Table B.7 — Maximal internal reproducibility differences (MIRD) in absolute value between two results from 40 obtained differences Compound Matrix: Cream Matrix: Oil MIRD obtained

Directive CE n°95–32 criteria MIRD obtained

Directive CE n°95–32 criteria % (w/w) % (w/w) % (w/w) % (w/w) Hydroquinone at 2 % (w/w) 0,12 0,37 0,22 0,37 Methyl ether at 1 % (w/w) 0,09 0,21 0,09 0,21 Ethyl ether at 1 % (w/w) 0,10 0,19 0,24 0,19 Benzyl ether at 1 % (w/w) 0,17 0,11a 0,42 0,11a a This criteria is strange by comparison with the others criteria. SIST EN 16956:2017

EN 16956:2017 (E) 17

Table B.8 — Accuracy study validated from two spiked matrixes at two levels analysed by 4 analysts on three different HPLC during five days Compound Matrix: Whitening cream Matrix: Whitening oil Level (LOQ): 0,1 % for hydroquinone 0,2 % for methyl and ethyl ethers 0,3 % for benzyl ether Level: 1 % (w/w) for ethers 2 % for hydroquinone Level (LOQ): 0,1 % for hydroquinone 0,2 % for methyl and ethyl and benzyl ether Level: 1 % (w/w) for ethers 2 % for hydroquinone FIDELITY Bias Error FIDELITY Bias Error FIDELITY Bias Error FIDELITY Bias Error RSDR RSDIR RSDR RSDIR RSDR RSDIR RSDR RSDIR % % % % % % % % % % % % Hydro-quinone 2,1 12,2 13,9 1,3 1,6

«1,5 28 25,3
«5,0 1,5 3,5 0,5 Methyl ether 1,6 9,1
«3,4 1,4 2,8 0,1 3,0 27,1
«10,9 2,2 6,9
«2,5 Ethyl ether 1,9 10,8
«1,7 1,5 3,4 0,6 2,6 21,0
«6,5 2,0 10,2
«3,3 Benzyl ether 2,1 7,9
«3,7 1,7 5,5 0,3 3,5 15,6 17,7 2,7 17,6

«0,3 NOTE RSDR: RSD of repeatability; RSDIR: RSD of internal reproducibility. Table B.9 — LGC Standard ring test: cream matrix with hydroquinone Round Number of laboratories Assigned value Result obtained Standard Deviation of reproducibility Z-score

% (m/m) % (m/m)
2012–03–05 10 2,00 1,90 0,156 - 0,64 2013–02–09 10 0,80 0,77 0,022

«1,36 2013–09–11 7 0,50 0,51 0,05 0,20 2014–03–13 3 1,25 1,26 0,125 0,08 2015–03–17 7 0,91 0,90 0,091

«0,11 2016–03–11 10 3,11 3,15 0,311 0,13 Table B.10 — NVWA Reference Material Matrix Compound Certified value Uncertainty Result obtained

% (w/w) % (w/w) % (w/w) CHEK RM 565 Body Milk Hydroquinone 2,57 0,06 2,63 CHEK RM 604 Body Lotion Hydroquinone 2,55 0,06 2,52 SIST EN 16956:2017

EN 16956:2017 (E) 18
Annex C (informative)

Validation data for the quantitative method for the 4 most frequently found corticosteroids These validation data were obtained by the Service Commun des Laboratoires (SCL) du Ministère de l'Economie et des Finances – Laboratoire de Lyon-Oullins – Unité Produits – Section Cosmétiques and by the Agence Nationale de Sécurité des Médicaments et des produits de santé (ANSM) du Ministère de la Santé – Laboratoire de Montpellier-Vendargues – Unité Contrôle Physico-chimie des médicaments chimiques et autres produits de santé. Limits of detection: 0,004 % (w/w) validated from standard solutions on three HPLC.

Key X Rentention time [min] 2 Fluocinonide (18,997 min) Y Absorbance [mAU] 3 Clobetasol propionate (19,925 min) 1 Fluocinolone acétonide (17,069 min) 4 Betamethasone dipropionate (20,885 min) Figure C.1 — Chromatogram of the 4 corticosteroids standard solution at 0,004 %(w/w) SIST EN 16956:2017

EN 16956:2017 (E) 19

Table C.1 — Limits of quantification (LOQ) validated from spiked matrix analysed by 4 analysts on three HPLC during five days in SCL and 1 analyst on two HPLC during five days in ANSM Compound Matrix: Whitening cream LOQ % (w/w) RSD of repeatability % RSD of internal Reproducibility % Fluocinolone acétonide 0,01 SCL: 9,1 SCL: 11,6 ANSM: 4,5 ANSM: 17,4 Fluocinonide 0,01 SCL: 5,1 SCL: 11,8 ANSM: 5,7 ANSM: 28,2 Clobetasol propionate 0,01 SCL: 8,4 SCL: 16,2 ANSM: 4,3 ANSM: 10,2 Betamethasone dipropionate 0,01 SCL: 8,2 SCL: 8,8 ANSM: 10,3 ANSM: 12,6 Table C.2 — Linearity study validated from 6 levels calibration (standard solutions) analysed by 4 analysts on three HPLC during five days in SCL and 1 analyst on two HPLC during five days in ANSM Compound Maximal Relative Bias Obtained % Level 1 (LOQ) Level 2 Level 3 Level 4 Level 5 Level 6

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