EN ISO 24443:2012

Determination of sunscreen UVA photoprotection in vitro (ISO 24443:2012)

ISO 24443:2012 specifies an in vitro procedure to characterize the UVA protection of sunscreen products. Specifications are given to enable determination of the spectral absorbance characteristics of UVA protection in a reproducible manner.
In order to determine relevant UVA protection parameters, the method has been created to provide a UV spectral absorbance curve from which a number of calculations and evaluations can be undertaken. This method relies on the use of in vivo SPF results for scaling the UV absorbance curve.

In vitro Bestimmung des UVA-Schutzes von Sonnenschutzmitteln (ISO 24443:2012)

Diese Internationale Norm stellt ein in vitro Verfahren zur Charakterisierung des UVA-Schutzes von Sonnenschutzmitteln dar. Es werden Spezifikationen angegeben, für eine reproduzierbare Bestimmung der spektralen Extinktion als Charakterisierung des UVA-Schutzes.
Zur Bestimmung der relevanten Kenngrößen für den UVA-Schutz wird die Kurve der spektralen Extinktion im UV ermittelt, auf deren Grundlage eine Reihe von Berechnungen und Bewertungen vorgenommen werden können. Ergebnisse dieses Messverfahrens können für weitere Berechnungen, wie zum Beispiel für örtliche Aufsichtsbehörden, verwendet werden. Dies beinhaltet die Berechnung des UVA-Schutzfaktors (UVAPF) [korreliert mit dem in vivo UVA-Schutzfaktor aus dem Persistent Pigment Darkening-Prüfverfahren (PPD)], der kritischen Wellenlänge und des UVA-/UVB-Extinktionsverhältnisses. Diese Berechnungen sind optional und beziehen sich auf die örtlichen Anforderungen zur Kennzeichnung von Sonnenschutzmitteln. Diese Methode basiert auf der Verwendung der in vivo SPF-Ergebnisse für die Skalierung der UV-Extinktionskurve.
Diese Internationale Norm gilt nicht für pulverförmige Produkte, die zum Beispiel gepresst oder lose vorliegen.

Détermination in vitro de la photoprotection UVA (ISO 24443:2012)

L'ISO 24443:2012 d'essai spécifie un mode opératoire in vitro pour caractériser la protection UVA apportée par des produits de protection solaire. Des spécifications sont données pour permettre de déterminer les caractéristiques d'absorbance spectrale de la protection UVA de façon reproductible.
Afin de déterminer les paramètres de protection UVA pertinents, la méthode a été mise au point pour fournir une courbe d'absorbance spectrale UV à partir de laquelle des calculs et des évaluations peuvent être effectués. Cette méthode repose sur les résultats du FPS in vivo pour élaborer la courbe d'absorbance des UV.

Kozmetika - Določevanje zaščitnega faktorja UVA in vitro (ISO 24443:2012)

Ta preizkusna metoda zagotavlja postopek in vitro za zaščito pred žarki UVA varovalnih sredstev za zaščito kože pred soncem. Podane specifikacije omogočajo določitev lastnosti spektralne absorpcije zaščite pred žarki UVA na ponovljiv način. Za določitev ustreznih parametrov zaščite pred žarki UVA je bila ustvarjena metoda za zagotovitev krivulje spektralne absorpcije UV, iz katere je mogoče izpeljati številne izračune in ocene. Rezultati tega postopka merjenja se lahko po potrebi uporabijo za druge izračune za lokalne regulativne organe. Ti vključujejo izračun ultravijoličnega A zaščitnega faktorja (UVA-PF) (ki se navezuje na UVA-PF in vivo iz preizkusnega postopka obstojne pigmentacije), kritične valovne dolžine in razmerja absorpcije žarkov UVA/UVB. Ti izračuni so izbirni in se nanašajo na zahteve glede označevanja izdelkov za zaščito kože pred soncem.

General Information

• Status: Withdrawn
• Publication Date: 31-May-2012
• Withdrawal Date: 13-Apr-2025
• ICS: 11.100.10 - In vitro diagnostic test systems
• ICS: 71.100.70 - Cosmetics. Toiletries
• Technical Committee: CEN/TC 392 - Cosmetics
• Drafting Committee: CEN/TC 392 - Cosmetics
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Start Date: 15-Dec-2021
• Completion Date: 14-Apr-2025
• Mandate: M/389 - Standardisation mandate assigned to CEN concerning methods for testing efficacy of suncreen products
• Ref Project: SIST EN ISO 24443:2012 - Determination of sunscreen UVA photoprotection in vitro (ISO 24443:2012)

Relations

• Replaced By: EN ISO 24443:2021 - Cosmetics - Determination of sunscreen UVA photoprotection in vitro (ISO 24443:2021, Corrected version 2022-02)
• Effective Date: 22-Dec-2021

Standards Content (Sample)

SLOVENSKI STANDARD
01-december-2012
.R]PHWLND'RORþHYDQMH]DãþLWQHJDIDNWRUMD89$LQYLWUR,62
Determination of sunscreen UVA photoprotection in vitro (ISO 24443:2012)
In vitro Bestimmung des UVA-Schutzes von Sonnenschutzmitteln (ISO 24443:2012)
Détermination in vitro de la photoprotection UVA (ISO 24443:2012)
Ta slovenski standard je istoveten z: EN ISO 24443:2012
ICS:
71.100.70 .R]PHWLND7RDOHWQL Cosmetics. Toiletries
SULSRPRþNL
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 24443
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2012
ICS 71.100.70
English Version
Determination of sunscreen UVA photoprotection in vitro (ISO
24443:2012)
Détermination in vitro de la photoprotection UVA (ISO In vitro Bestimmung des UVA-Schutzes von
24443:2012) Sonnenschutzmitteln (ISO 24443:2012)
This European Standard was approved by CEN on 24 May 2012.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 24443:2012: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
This document (EN ISO 24443:2012) has been prepared by Technical Committee ISO/TC 217 "Cosmetics" in
collaboration with 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 December 2012, and conflicting national standards shall be withdrawn
at the latest by December 2012.
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.
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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 24443:2012 has been approved by CEN as a EN ISO 24443:2012 without any modification.

INTERNATIONAL ISO
STANDARD 24443
First edition
2012-06-01
Determination of sunscreen UVA
photoprotection in vitro
Détermination in vitro de la photoprotection UVA
Reference number
ISO 24443:2012(E)
©
ISO 2012
ISO 24443:2012(E)
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved

ISO 24443:2012(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Terms and definitions . 1
3 Principle . 2
4 Apparatus . 2
4.1 UV spectrophotometer specifications . 2
4.2 Calibration of the UV spectrophotometer . 3
4.3 Calibration of the UV exposure source . 3
4.4 Monitoring of the UV exposure source . 3
4.5 Calibration of the UVA radiometer used to monitor the test sample irradiation . 4
4.6 Substrate/plate . 4
5 Test method . 4
5.1 Outline of the test procedure . 4
5.2 Equipment calibration and validation of test plates . 4
5.3 Absorption measurements through the plate . 5
5.4 Sample application . 5
5.5 Absorbance measurements of the product-treated plate . 5
5.6 Number of determinations . 5
5.7 Determination of initial calculated SPF (SPF ), “C” value, initial UVAPF(UVAPF ), and UV
in vitro 0
exposure dose . 5
5.8 UV exposure . 7
5.9 Measurement of final adjusted absorbance spectrum . 7
5.10 Calculation of UVAPF of plates after UV exposure of the sample . 8
6 Procedure using the spreadsheet in this International Standard . 8
7 Reference sunscreen formula S2 . 9
8 Test report . 9
Annex A (normative) Calibration of UV spectrophotometer and plate transmission test .10
Annex B (normative) Radiometer calibration to spectroradiometric irradiance procedure .14
Annex C (normative) Computation values: PPD and erythema action spectra and UVA and UV-SSR
spectral irradiances .16
Annex D (normative) PMMA test plate surface specifications .19
Annex E (normative) UVA reference sunscreen S2 .21
Annex F (informative) Statistical calculations .25
Bibliography .27
ISO 24443:2012(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International
Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
iv © ISO 2012 – All rights reserved

INTERNATIONAL STANDARD ISO 24443:2012(E)
Determination of sunscreen UVA photoprotection in vitro
1 Scope
This International Standard specifies an in vitro procedure to characterize the UVA protection of sunscreen
products. Specifications are given to enable determination of the spectral absorbance characteristics of UVA
protection in a reproducible manner.
In order to determine relevant UVA protection parameters, the method has been created to provide a UV spectral
absorbance curve from which a number of calculations and evaluations can be undertaken. Results from
this measurement procedure can be used for other computations, as required by local regulatory authorities.
These include calculation of the Ultraviolet-A protection factor (UVAPF) [correlating with in vivo UVAPF
from the persistent pigment darkening (PPD) testing procedure], critical wavelength and UVA absorbance
proportionality. These computations are optional and relate to local sunscreen product labelling requirements.
This method relies on the use of in vivo SPF results for scaling the UV absorbance curve.
This International Standard is not applicable to powder products such as pressed powder and loose powder products.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
in vitro UVA protection factor
UVAPF
in vitro UVA protection factor of a sun protection product against UVA radiation, which can be derived
mathematically with in vitro spectral modelling
2.2
in vitro calculation of SPF
SPF
in vitro
protection factor of a sun protection product against erythema-inducing radiation calculated with spectral modelling
2.3
action spectrum for erythema
E(l)
relative effects of individual spectral bands of an exposure source for an erythema response
NOTE See References [1] and [2].
2.4
action spectrum for PPD
P(l)
relative effects of individual spectral bands of an exposure source for a persistent pigment response
NOTE See References [3] and [4].
2.5
monochromatic absorbance
A
l
sunscreen absorbance at wavelength, l, related to the sunscreen transmittance, T , by
l
A = - log (T )
l l
where transmittance, T , is the fraction of incident irradiance transmitted by the sunscreen film
l
ISO 24443:2012(E)
2.6
irradiance
I
fluence rate per unit area, expressed in W/m , for a defined range of wavelengths
EXAMPLE From 290 nm to 400 nm for UVA + UVB irradiance; from 320 nm to 400 nm for UVA irradiance.
2.7
spectral irradiance for SPF testing or PPD testing
I(l)
irradiance per unit wavelength, I(l) , expressed in W/m /nm
2.8
spectrophotometer
instrument that measures absorbance (or transmission) properties of a test medium as a function of wavelength
2.9
spectroradiometer
instrument that measures spectral irradiance (intensity in watts per unit area per nanometre) of
electromagnetic sources
NOTE Limited to ultraviolet, visible and short infrared ranges in this International Standard.
2.10
radiometer
instrument that measures broad band irradiance (intensity in watts per unit area) of electromagnetic sources
NOTE Limited to ultraviolet, visible and short infrared ranges in this International Standard.
3 Principle
The test is based on the assessment of UV-transmittance through a thin film of sunscreen sample spread on a
roughened substrate, before and after exposure to a controlled dose of radiation from a defined UV exposure
source. Because of the several variables that cannot be controlled with typical thin film spectroscopic techniques,
each set of sunscreen transmission data is mathematically adjusted so that the in vitro SPF data yield the same
measured in vivo SPF value that was determined by in vivo testing. Samples are then exposed to a specific
measured dose of UV radiation to account for the photostability characteristics of the test product. The resulting
spectral absorbance data have been shown to be a useful representation of both the width and height of the UVA
protection characteristics of the sunscreen product being tested. The mathematical modelling procedure has
been empirically derived to correlate with human in vivo (persistent pigment darkening) test results.
4 Apparatus
4.1 UV spectrophotometer specifications
The UV spectrophotometer wavelength range shall span the primary waveband of 290 nm to 400 nm. The
wavelength increment step shall be 1 nm.
A UV spectrophotometer that does not have a monochromator after the test sample should employ a
fluorescence rejection filter.
The UV spectrophotometer input optics should be designed for diffuse illumination and/or diffuse collection
of the transmitted irradiance through the roughened polymethylmethacrylate (PMMA) substrate, with and
without the sunscreen layer spread on its surface. The size of the diameter of the entrance port of the UV
spectrophotometer probe shall be smaller than the size of the light spot to be measured at the sample level (in
order to account for stray light). The area of each reading site should be at least 0,5 cm in order to reduce the
variability between readings and to compensate for the lack of uniformity in the product layer. The wavelength
should be accurate to within 1 nm, as checked using a holmium-doped filter (see Annex A). The ability of an
instrument to accurately measure absorbance is limited by the sensitivity of the instrument. The minimum
2 © ISO 2012 – All rights reserved

ISO 24443:2012(E)
required dynamic range for this methodology is 2,2 absorbance units as determined according to Annex A.
The maximum measured absorbance should be within the dynamic range of the device used. If the test
measurements yield absorbance curves that exceed the determined upper limit of the UV spectrophotometer,
the product should be re-tested using an instrument with increased sensitivity and dynamic range.
The lamp in the UV spectrophotometer that is used to measure the transmittance shall emit continuous
radiation over the range of 290 nm to 400 nm, and the level of irradiance should be sufficiently low, so that the
photostability of the product is not unduly challenged (a xenon flash lamp is a convenient solution). Therefore
the UV dose during one measurement cycle should not exceed 0,2 J/cm .
NOTE A UV spectrophotometer is used to measure the absorbance properties of the sunscreen on the test plates. A
spectroradiometer is used to measure the spectral energy distribution and intensity of the UV exposure source or the UV
spectrophotometer during the absorbance measurement of the sunscreen on the test plate.
4.2 Calibration of the UV spectrophotometer
The UV spectrophotometer shall be validated at regular intervals (recommended at least every month) by
measurements of reference materials.
A three-fold test is required, as described in Annex A:
— dynamic range of the UV spectrophotometer;
— linearity test of the UV spectrophotometer;
— wavelength accuracy test.
4.3 Calibration of the UV exposure source
The spectral irradiance at the exposure plane of the UV exposure source that is used for irradiation (to take into
account any photoinstability) shall be as similar as possible to the irradiance at ground level under a standard
[5] [6] [7]
zenith sun as defined by COLIPA or in DIN 67501 . The UV irradiance shall be within the following
acceptance limits (measured at sample distance).
Table 1 — UV exposure source specifications
UV exposure source specifications as measured with a spectroradiometer
2 2
Total UV irradiance (290 nm to 400 nm) 40 W/m to 200 W/m
a b
Irradiance ratio of UVA to UVB 8:22
a
320 nm to 400 nm.
b
290 nm to 320 nm.
The UV exposure source device should have the ability to maintain samples within the range of 25 °C to
35 °C. It is important that the temperature of the sample itself be measured and not just the surrounding air
temperature. To maintain samples at a temperature less than or equal to 35 °C, a filter system that particularly
reduces IR radiation should be used to achieve the specified temperature range. Cooling trays for the sample
plates or ventilators should be used to maintain a temperature below 35 °C and warming devices to maintain
samples at or above 25 °C.
4.4 Monitoring of the UV exposure source
The emission of the UV exposure source used for exposure shall be checked for compliance with the given
acceptance limits by a suitably qualified expert (at least) every 18 months, or after 3 000 hours of lamp running
time. The inspection should be conducted with a spectroradiometer that has been calibrated against a standard
lamp that is traceable to a national or an international calibration standard. In addition to the spectroradiometric
inspection, the intensity of the UV exposure source used for exposure shall be checked prior to each use. This
can be done using either a spectroradiometer or a radiometer with sensitivity in the UVA, calibrated for the
ISO 24443:2012(E)
same UV exposure source spectrum used for the exposure step of the procedure, applying the coefficient of
calibration to adjust for variance between the UVA radiometer and the reference spectroradiometer.
4.5 Calibration of the UVA radiometer used to monitor the test sample irradiation
If a UVA radiometer is used, this device shall have been suitably calibrated. This requires that it be calibrated
to the spectroradiometer used to measure the exposure source (as during annual solar simulator calibration).
Calibration shall be conducted in terms of UVA irradiance (320 nm to 400 nm) and shall be at the same level
at which the test plates are exposed. Once calibrated with the spectroradiometer, the UVA radiometer may be
used to determine the UV doses to be used during the exposure procedure on a day-to-day basis. Annex B
provides the step-by-step calibration procedure.
4.6 Substrate/plate
The substrate/plate is the material to which the test product is to be applied. For this method, PMMA plates
with one rough side of the substrate are to be used and are commercially available. One specific plate has
[9]
been validated for this test method; the specifications and preparation of this type of plate are described in
Annex D. The size of the substrate should be chosen such that the application area is not less than 16 cm .
5 Test method
5.1 Outline of the test procedure
5.1.1 Conduct the calibration and validation of the test equipment, including the UV spectrophotometer
used for transmission/absorbance measurements and the UVA radiometer (or spectroradiometer)
used to measure the UV exposure source, and verify the transmission properties of the test plates
as described in Annex D.
5.1.2 Conduct blank measurements of a glycerin-treated plate for the reference “blank”, which will be
used in the subsequent absorbance measurements.
5.1.3 Conduct in vitro absorbance measurements of the sunscreen product spread on a PMMA plate,
prior to any UV irradiation. Acquire the initial UV absorbance spectrum with A (l) data.
5.1.4 Conduct the mathematical adjustment of the initial UV absorbance spectrum using coefficient “C”
(see the calculation in 5.7.2) to achieve an in vitro SPF (no UV dose) equal to the in vivo SPF. Initial
UVAPF is calculated using A (l) and C.
0 0
5.1.5 A single UV exposure dose, D, is calculated, equal to 1,2 × UVAPF in J/cm .
5.1.6 Conduct UV exposure of the same sample as in 5.1.3, according to the calculated UV exposure
dose D.
5.1.7 Measure the in vitro absorbance of the sunscreen product after UV exposure. Acquire the second
UV spectrum with A(l) data.
5.1.8 Conduct the mathematical adjustment of the second absorbance spectrum (following UV
exposure) by multiplying with the same “C” coefficient, previously determined in 5.1.4. The resulting
absorbance curve is the final adjusted absorbance values.
NOTE For calculations, UV absorbance values shall be used.
5.2 Equipment calibration and validation of test plates
Test procedures as described in Annex A are to be completed to validate the wavelength accuracy, linearity and
absorbance limits of the UV spectrophotometer/spectroradiometer to be used for the test procedure. Validation
of the UV properties of the test PMMA plates shall also be conducted as described in Annex D.
4 © ISO 2012 – All rights reserved

ISO 24443:2012(E)
5.3 Absorption measurements through the plate
It is necessary to first determine the absorbance of UV radiation through a “blank” PMMA plate. Prepare a
“blank” plate by spreading a few microlitres of glycerin on the roughened side of the plate. Choose the amount
of glycerin such that the entire surface is just completely covered (approximately 15 µl for a 50 × 50 mm plate).
Any excess of glycerin should be avoided. Measure the absorbance through this “blank” plate and use this as
the baseline measurement for subsequent absorbance measurements.
NOTE Many spectrophotometers have “baseline” functions to automatically incorporate this baseline measurement
into the calculations of subsequent absorbance measurements.
5.4 Sample application
The sunscreen product is applied to a new untreated roughened PMMA plate (with the roughened side
uppermost) by mass, at an application rate of 1,3 mg/cm . To ensure dose accuracy and repeatability, the
application area should be not less than 16 cm . The application dose may be determined by measuring the
mass loss of the pipette before and after application of the product; alternatively, it may be applied based
on volumetric measurements with consideration of the specific gravity of the test sample. Where possible, a
positive-displacement automatic pipette should be used for this purpose.
The sunscreen is applied as a large number of small droplets of approximate equal volume, distributed evenly
over the whole surface of the plate. Finger cots should not be used to spread the product on the plate. The
fingertip used for spreading should be dipped into the test product and then wiped to remove excess product
before spreading the test product applied to the plate. The fingertip used to spread the product shall be cleaned
between applications of different test products.
After the sunscreen product is deposited on the surface of the plate, it shall be spread immediately over the
whole surface using light strokes with a fingertip (without finger cot). Spreading should be completed in a
two-phase process. First, the product should be distributed over the whole area as quickly as possible (less
than 30 seconds) using small circular motions with minimal pressure. Then the sample should be rubbed on the
plate surface using alternating horizontal and vertical strokes with increased moderate pressure. The second
phase should take 20 to 30 s.
This treated sample shall be allowed to dry for at least 30 min in the dark at the same temperature that will be
experienced under the UV exposure conditions (i.e. if UV source exposure conditions will be 35 °C, then the
drying conditions should also be at 35 °C; or if the UV source exposure conditions will be 25 °C, then the drying
conditions should also be 25 °C).
5.5 Absorbance measurements of the product-treated plate
The product-treated plate is placed in the light-path of the UV spectrophotometer and the absorbance of UV
radiation through the sample is determined for each wavelength, from 290 nm to 400 nm, in 1 nm steps. One or
more observations of absorbance may be made per plate and the mean value shall be determined for each plate.
5.6 Number of determinations
At least four plates prepared with the test sunscreen shall be used to establish the protection aspects of the
test sample. Additional plates shall be added to the sampling if the 95 % confidence interval (CI) is greater than
17 % of the mean value of the UVAPF value, until the 95 % CI is less than 17 % of the mean UVAPF value.
Calculation procedures for this are described in Annex F.
5.7 Determination of initial calculated SPF (SPF ), “C” value, initial UVAPF(UVAPF ),
in vitro 0
and UV exposure dose
5.7.1 Determination of SPF
in vitro
The UV solar simulator radiation (UV-SSR) source spectrum, I(l), (see Annex C) is multiplied with the corresponding
erythema action spectrum sensitivity value, E(l), (see Annex C) at that wavelength to yield the sunburning effective
energy at that wavelength. The resulting sunburning effective irradiance is integrated over the 290 nm to 400 nm
ISO 24443:2012(E)
range. The sunscreen transmission values at each wavelength are multiplied with the erythemal effective energy
at that wavelength and integrated over the same interval to yield the effective sunburning energy transmitted
through the test product. The ratio of these two integrals is the in vitro calculated SPF value.
Calculation of SPF is shown in Equation (1):
in vitro
λ = 400
EI()λλ××() dλ
∫
λ =290
SPF = (1)
in vitro
λ = 400
−A ()λ
EI()λλ××() 10 ×dλ
∫
λ =2990
where
[1]
E(l) is the erythema action spectrum (see Annex C);
I(l) is the spectral irradiance received from the UV source (SSR for SPF testing) (see Annex C);
A (l) is the mean monochromatic absorbance of the test product layer before UV exposure;
dl is the wavelength step (1 nm).
NOTE This calculated SPF value cannot be used as an SPF result.
in vitro
5.7.2 Determination of “C” value
The initial absorbance curve values are multiplied by a scalar value “C” until the in vitro calculated SPF values
are equal to the in vivo measured SPF. This is accomplished in an iterative calculation process. The initial
absorbance values multiplied by this “C” value become the adjusted sunscreen absorbance curve that is used
for determination of the initial UVAPF value, and the exposure dose. Equation (2) shows the calculation of the
adjusted in vitro SPF (SPF ) and determination of the coefficient of adjustment “C”:
in vitro,adj
λ = 400
EI()λλ××() dλ
∫
λ = 290
SPF ==SPF (2)
in vitroi,adj nvitro
λ = 400
−AC()λ
EI()λλ× ()×××10 dλ
∫
λ = 290
where
E(l), I(l), A (l) and dl are as defined in Equation (1).
– cd
This calculation is based on Lambert-Beer’s law E = E e which is related to ideal solutions. While
sunscreens in thin film do not behave as ideal solutions, this calculation has been proven satisfactory for this
[10][11]
specific application .
The “C” value typically lies between 0,8 and 1,6 for valid interpretation. If it is outside this range, new samples
should be prepared to validate the original observations. The “C” value for the reference S2 shall lie in this
range 0,8 to 1,6 or the application procedure should be modified to achieve it.
5.7.3 Determination of initial UVA protection factor before UV exposure (UVAPF )
The initial UVAPF value is calculated for the purpose of determining the UV exposure dose. It is calculated in
a manner similar to the calculation of the initial SPF . The intensity spectrum for a UVA radiation source,
in vitro
I(l), (as described in Annex C) is multiplied at each wavelength with the persistent pigment darkening action
spectrum sensitivity values, P(l), to yield the pigment darkening energy at that wavelength. The resulting
pigment darkening effective irradiance is integrated over the 320 nm to 400 nm range. The initial absorbance
values from the test product at each wavelength are used to calculate the effective intensity at each wavelength
6 © ISO 2012 – All rights reserved

ISO 24443:2012(E)
to yield the effective pigment darkening energy transmitted through the test product as shown in Equation (3)
below. The ratio of these two integrals is the initial in vitro UVAPF value:
λ =400
PI()λλ××() dλ
∫
λ =320
UVAPF = (3)
λ ==400
−AC()λ
PI()λλ××() 10 ×dλ
∫
λ =320
where
P(l) is the PPD action spectrum (see Annex C);
I(l) is the spectral irradiance received from the UVA source (UVA 320 nm to 400 nm for PPD testing)
(see Annex C);
A (l) is the mean monochromatic absorbance of the test product layer before UV exposure;
C is the coefficient of adjustment, previously determined in Equation (2);
dl is the wavelength step (1 nm).
5.7.4 Determination of the UV exposure dose
The UV exposure dose, D, is the UVAPF value multiplied by a factor of 1,2, in Joules/cm :
D = UVAPF × 1,2 (4)
The sample is exposed to full spectrum UV radiation but the dose is being defined by the UVA content.
2 [8]
NOTE The 1,2 J/cm factor is based on ISO ring test validation study results .
5.8 UV exposure
WARNING — Personnel working with this irradiator system should be protected adequately against
UV rays (glasses, gloves, etc.).
Expose the sample plates to the radiation from the UV exposure source. During the exposure the samples
should be maintained at a temperature between 25 °C and 35 °C, and at the same temperature used for the
drying period. The PMMA plates should be fixed above a non-reflective UV background behind each plate to
reduce back exposure. Ensure that the UV exposure source does not switch off while placing samples under the
lamp (in this case, ensure the output irradiance is the same on restart as it was before the lamp was turned off).
5.9 Measurement of final adjusted absorbance spectrum
After the UV exposure, re-measure the absorbance of the test samples on the same spots as measured before
the UV exposure, as in 5.5. The final absorbance values are equal to the observed absorbance values after the
UV exposure, multiplied by the “C” value determined in 5.7.2.
A (l) = A (l)C
f e
where
A is the mean monochromatic absorbance of the test product layer after UV exposure;
e
A is the mean final monochromatic absorbance of the test product.
f
ISO 24443:2012(E)
5.10 Calculation of UVAPF of plates after UV exposure of the sample
The UVAPF shall be calculated according to Equation (5) for each individual plate, using the single observation
value or the mean of multiple observations on that plate.
λ =400
PI()λλ××() dλ
∫
λ =320
UVAPF = (5)
λ =4400
−AC()λ
e
PI()λλ××() 10 ×dλ
∫
λ =320
where
P(l) is the PPD action spectrum (see Annex C);
I(l) is the spectral irradiance received from the UVA source (UVA 320 nm to 400 nm for PPD testing)
(see Annex C);
A (l) is the mean monochromatic absorbance of the test product layer after UV exposure;
e
C is the coefficient of adjustment, previously determined in Equation (2);
dl is the wavelength step (1 nm).
Other protection parameters may be calculated from the final absorbance curve in 5.9 as desired.
6 Procedure using the spreadsheet in this International Standard
6.1 The calculations given in 5.1.4 to 5.1.8 can be performed automatically using the calculation spreadsheet
in this International Standard and the following steps.
6.2 Enter the name, date, operator identification and in vivo SPF of the test product, the spectroanalyser
type, UV exposure device type, the raw UVA exposure irradiance of the UV exposure source, and the irradiance
correction value “Y” (from Annex B) into the test spreadsheet on the “Start here” tab.
6.3 Measure and input the absorbance data for the first four unirradiated sample plates into the spreadsheet
on the tabs named “Plate #0”. Click on the “OK, data entered, let’s proceed” button after each entry.
6.4 The UV exposure irradiance and exposure time are reported on the “Results #” tab for each individual plate.
6.5 Expose the sunscreen treated plate for the prescribed time to achieve the UV exposure dose for each plate.
6.6 Measure the absorbance of each of the individual UV exposed plates. The measurements conducted
after UV exposure should be on the same spot or spots as measured before the UV exposure.
6.7 Input the post-UV exposure absorbance measurements for each plate into their respective spreadsheet
tabs “Plate #UV”. Click on the “OK, data entered, let’s proceed” button.
6.8 The “RESULTS (Plate #)” spreadsheet tab will show the results data for each individual plate.
6.9 When the full data input for the first four plates is complete, the “Report” tab spreadsheet will appear
giving the summary results for the test sample. If the 95 % CI of the UVAPF values is less than 17 % of the mean
UVAPF, no further plates are required and the final results are displayed in graphic and tabular form. Otherwise,
additional samples will need to be added sequentially as above. Additional data sheets for additional plates will
appear and be completed as above until the test criterion is satisfied.
8 © ISO 2012 – All rights reserved

ISO 24443:2012(E)
7 Reference sunscreen formula S2
The method is controlled by the use of a reference sunscreen formulation to verify the test procedure. Reference
S2 sunscreen formula as described in Annex E shall be used. The UVAPF test results of the reference S2 shall
lie between the upper and lower limits, as determined from in vivo testing results listed below, or else the test is
invalid and shall be repeated. SPF 16 is to be used as the in vivo SPF value for S2 for computation purposes.
Table 2 — Limits of UVAPF testing results
Parameter Lower limit Upper limit
UVAPF 10,7 14,7
The frequency of testing of the S2 standard shall be in accordance with the user’s internal procedures and/or
ISO/IEC 17025.
8 Test report
The test report on the determination of the absorbance spectrum of a sun protection product should contain
at least the following information:
a) description of the instruments used, manufacturer and instrument model with the system calibration
summary in this International Standard as per the format in A.6;
b) the calibration factor “Y” used to adjust the UVA radiometer measurement with the reference
spectroradiometer measurement of the UV exposure source (4.5 and B.3.10);
c) plate manufacturer and batch code;
d) mean UV absorbance values at each 1 nm wavelength increment for the test sample (a graph of absorbance
values, pre-exposure and post-exposure may be provided);
e) statement of the measured in vivo sun protection factor (SPF) used for calculations;
f) constant “C”;
g) UVA irradiance (W/m ) and mean UVA exposure dose used to irradiate the test sample;
h) reference data for Reference S2 material with date of testing;
i) sample identification;
j) identification of the individual conducting the test;
k) other informative calculations derived from the absorbance values [see 8 d)] may be reported.
ISO 24443:2012(E)
Annex A
(normative)
Calibration of UV spectrophotometer and plate transmission test
A.1 General
This procedure describes the requirements for wavelength accuracy, linearity and dynamic range of the UV
spectrophotometer. For clarity, and in order to standardize the report format, a spreadsheet is provided as part
of this International Standard.
A.2 Wavelength accuracy
A.2.1 Holmium oxide filter
The filter should be no more than 3 mm in thickness and dosed with holmium oxide in order to provide absolute
wavelength calibration using an absorbance peak of 361 nm.
A.2.2 Method
A.2.2.1 Place the holmium oxide filter in the sample path and scan the absorbance in the range between
290 nm and 430 nm. Measure against air in the blank light path. Repeat the scan for 3 replicates. Accumulate
the data and transfer absorbance values to the “Holmium Wavelength Accuracy” tab in the attached spreadsheet
“ISO 24443 UVSpectcalib.xls” in Columns B to D. Click on the macro button at cell “P28” to activate the peak
check function. The results will automatically appear in the system calibration summary sheet in this International
Standard (see Figure A.2) and be similar to Figure A.1 below.
A.2.2.2 The deviations of the measured band from the reference value in the UV range of the instrument
should not exceed 1 nm. An example of a measured calibration spectrum is shown in the graph below. The
reported peak wavelength shall be either 360, 361 or 362, or else the instrument shall be recalibrated to achieve
one of these wavelength values.
10 © ISO 2012 – All rights reserved

ISO 24443:2012(E)
Key
X wavelength (nm)
Y absorbance
Figure A.1 — Holmium oxide actual
A.3 Linearity
A.3.1 Standard reference plates
The plates are cut from a large sheet of standard cast, UV-stabilized PMMA (helping ensure the same optical
properties for each plate). The plates are made in a way as to match the absorption spectra of a range of
common sunscreens. The casting process enables a very homogeneous distribution of UV absorbing material,
relative to a manually applied film of a test emulsion.
Because of their stable and standardized absorption and diffuse-scattering properties, they are very suitable
as “reference emulsions” to check and compare instruments used for in vitro determination of UV protection,
for intra- as well as inter-laboratory purposes.
A.3.2 Linearity assessment
Select two of the transparent UV-stabilized PMMA reference plates. The absorbance peak of these reference
plates at 340 nm shall be between 1,1 and 1,5 absorbance units (AU).
Designate the first plate as Slide A and place it in the light path of the UV spectrophotometer. Measure against air
in the blank light path. Run a duplicate (290 nm to 380 nm) and transfer data to the “Linearity Test” spreadsheet
tab, cells B8:C98.
Designate the second plate as Slide B and place it in the light path of the UV spectrophotometer. Measure
against air in the blank light path. Run in duplicate and transfer data to the “Linearity Test” spreadsheet tab,
cells D8:E98. Place both slides (A and B) on top of each other with their roughened sides towards one another
into the light path and measure the combined absorbance (290 nm to 380 nm). Measure against air in the blank
light path. Run 4 replicates and transfers data to the “Linearity Test” spreadsheet tab, cells F8:I98. The results
will automatically appear in the system calibration summary sheet in this International Standard (see Figure A.2).
ISO 24443:2012(E)
A.4 Dynamic absorbance range limit determination
The spreadsheet will also calculate the maximum absorbance range limit of the UV spectrophotometer,
based on deviation from additivity of the two plates. When the deviation exceeds 0,1 AU, the dynamic range
limit is determined and the results will automatically appear in the system calibration summary sheet in this
International Standard (see Figure A.2). The minimum range limit is 2,2 AU.
A.5 PMMA test plate qualification
A.5.1 General
The PMMA plates used as substrate for the sunscreen testing shall pass minimum transmission specifications.
A.5.2 Method
Set the baseline of the UV spectrophotometer with an air blank (no sample). Apply approximately 15 mg of
glycerin or modified glycerin solution to the rough surface of the PMMA plate to make the surface clear using
a finger tip. Remove any excess glycerin with the fingertip. Place the prepared plate in the measurement
position and measure the absorbance (or % transmission) of the plate. Record and transfer the data to the
spreadsheet tab marked “Plate Transmission” in the appropriate column B for absorbance values or column C
for % transmission values. The plate shall record > 60 % transmission at 290 nm, > 69 % at 300 nm, and > 81 %
at 320 nm. The results will automatically appear in the system calibration summary sheet in this International
Standard as shown in Figure A.2.
A.6 Reporting
The results of the calibration should be recorded in the format indicated on the spreadsheet “Summary”.
Instrument: Tested by:
0  0
Date: March 31, 2011  Sig: 0
Plate Transmission Test Plate Manuf: 0 Lot # : 0
nm Limits
290 >60% 65.5% Pass
300 >69% 70.9% Pass
320 >81% 82.1% Pass
Spectrophotometer Wavelength Accuracy
Peak 1
Reference Wavelength 361
Measured Wavelength 361
Peak Value
0.433
Limit ±1 TRUE
Figure A.2 — System calibration summary sheet (1 of 2)
12 © ISO 2012 – All rights reserved
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