SUN SET 50 SPF test

RESEARCH BASIS 

Product data 

The responsible person is responsible for conformity with declared qualitative and quantitative composition and microbiological purity of the delivered research samples. 

Methodology 

• All research of the preparation Zonnemist SPF 50 48-000-0994.08 was conducted under  supervision of doctors-specialists of dermatology. 
• The research was done in accordance with EN_ISO_24444:2022A. 
• The apparatus tests were performed using the Solar Light – UV Solar Simulator Multiport V601  S/N: 24303. 
• Characteristic of the UV source. 

 

All measurements were carried out in air-conditioned room of 22°C and relative air  humidity of 40 – 60%, in a group of subjects – volunteers participating in the tests. The UV protective factor study was performed in accordance with the Recommendation  of the Commission of the European Communities of 22 September 2006 on the efficacy of  sunscreen products and the claims made relating thereto (document number C(2006) 4089,  2006/647/EC) and ISO24444:2022A1. 

Sunscreen products should be sufficiently effective against UVB and UVA radiation to  provide a high level of health protection. For this purpose, a sunscreen product should provide  the minimum UVB and UVA protection. Protection against UVA and UVB radiation should be  interconnected. Scientific findings show that some of the biological damage to the skin can be  prevented or reduced if the level of SPF measured in the persistent pigment-darkening test.  Additionally, in order to provide comprehensive protection, dermatologists recommend a critical  wavelength of at least 370 nm. For the determination of MED and SPF the UV Solar simulator was  used containing UV spectrum of 290 - 400 nm. MED indicates lowest erythemal effective radiant  exposure (Her) that produces the first perceptible unambiguous erythema with defined borders  appearing over more than 50 % of UV exposure subsite, 16 h to 24 h after UV exposure. 

For a multiple output device, the intensity uniformity of each output beam shall be  determined by measuring at least 4 circles of equal area of each output beam as calculated Uniformity%=(1-(max-min) / (average))% 

Uniformity% =

1 − (max − min)
(average)%

The average uniformity of all beams for the multiple output device shall be ≥90% 

Reference standard to be used: 

• SPF Claim ≤24: P2, P3, P5, P6, or P8r eference standard (all subjects); 
• SPF ≥25 but less than SPF 50: P5 or P6 reference standard (on at least 5 subjects) and P2 or P3  on remaining subjects; 
• SPF ≥50: P8 reference standard (on at least 5 subjects) and P2 or P3 on the remaining subjects. If P5, P6, or P8 reference standard is used on a subject, there is no necessity to include a lower  SPF reference standard on that subject even though there may be lower SPF test products  included in the same test. 

Table 1. Mean SPF and acceptance limits for reference sunscreen formulations 

 

Before UV exposure of each test site, the UV irradiance shall be measured and recorded with an  erythema weighted radiometer cross-calibrated against a spectroradiometric measurement of the solar simulator output (There shall be a spectroradiometric check of the spectrum of each  solar simulator output port (UVA and UVB) and intensity made by the laboratory at least once  every 12 months or after 2 500 h of lamp running time and after changing any significant physical (optical) component (including the bulb) of the solar simulator. The simple use of specific filters  is not in itself adequate assurance that the UV output is of the correct quality. This periodical  inspection should be conducted by a trained, competent, and suitably qualified person (internal  or external) using a spectroradiometer that has been calibrated against a standard lamp that is  traceable to a national or an international calibration standard, with a band width of 2 nm or  smaller and having a dynamic range of at least 5 decades which is usually met by spectroradiometers equipped with double monochromator. Measurements shall be recorded at  1 nm increments). Optical alignment shall be configured to ensure accurate radiometer  alignment and reproduction of the simulator output at the same optical reference plane  measured with the spectroradiometer. A calibration factor Y for each radiometer shall be  determined by below formula:

Procedure:
1. Acclimatization period for the skin – test conditions should be stable with room temperature maintained between (23±3) oC
2. ITAo determination on the back of subject;
3. Delineation of test sites on the back of subject
- the test sites intended for UV exposure shall be free from blemishes and hair, and have an even colour tone with no variation in ITA° greater than 5° from each other or the MEDu test area. When necessary, hair shall be shaved more than three days prior to the test, but not thereafter. If necessary, hair may be clipped or cut with scissors on the test day,
- the minimum total area for a test site for product application shall be 30 cm2 and the
maximum shall be 60 cm2
- the positions of the test products and reference sunscreen test sites shall be distributed randomly on the backs of subjects over the whole test group in order to reduce error arising from anatomical differences in skin. The unprotected test site used to determine MEDu shall be randomized as one of the test sites across the test area and across subjects,
- there shall be a minimum distance of 1 cm between the borders of adjacent test sites,
- before product application, the test area may be cleaned by using a dry cotton pad or
equivalent,
- the test sites shall be delineated by a method which does not interfere with the test or harm the subject such as skin marker and/or a template made from non-absorbent material. The skin marker shall be indelible so as to be discernible at the time of MED evaluations 16 h to 24 h post UV-exposure,
4. Weighing of the product to the test site
- the amount of test product and reference sunscreen formulation applied to the skin after spreading shall be (2,00 ± 0,05) mg/cm2,

- the balance used to weigh the products should be capable of weighing to the nearest
0,1 mg,
- all products shall be homogeneous and shall be shaken before weighing, to ensure uniform dispersion,
- when handling the product during weighing or before application to the skin, take
appropriate measures to prevent evaporative loss of the volatile components. It is important that the total quantity of weighed product is transferred to the product application site. The amount of product to be applied shall be weighed in a syringe or in another device such as a watch glass. A method of weighing by loss is required,

5. Application of the product to the test site
- the use of a finger cot (i.e. latex, nitrile, etc.) shall be required except in cases when use of a finger cot interferes with even application of the product. A new finger cot shall be used for each new application of product and shall not be pre-saturated with the test product. When a naked finger, is used a maximum of 2,1 mg/cm2 (additional 5 %) shall be applied to the test area to account for the additional area of the application finger, and the finger shall be cleaned between product applications with an alcohol wipe,
- there are 3 application techniques dependent on the product type:

• Method A: Fluid products (lotion, cream, oil, liquid, gel, pump spray, roll on, aerosol
  spray/degassed first/.
  To aid uniform coverage, droplets (at least 15 per 30 cm2 , 30 per 60 cm2 of the product should be deposited within the test site using a syringe/pipette at one time, then spread over the whole test site, first with circular movements to gather the droplets and secondin horizontal and vertical directions using light pressure. It is recommended that during the whole process, the application finger stays in contact with the skin. Spray products provided in a pressurized container should first be degassed by puncturing a very small pinhole in the container to relieve all of the pressure. Degassing shall be done with appropriate safety precautions by securing the can within a ventilated safety hood with appropriate personnel safety equipment. The degassed can shall be allowed to rest for 24 h at room temperature when the product shall be decanted into a separate closed container with minimal headspace to minimize evaporation.
• Method B: Non-flowing viscous liquids and semi-solids. (stick, balm, roll on)
  Test product should be measured into a weigh boat and applied by finger in multiple areas of the test site, first with circular movements to gather the material and second in horizontal and vertical directions using light pressure. It is recommended that during the whole process, the application finger stays in contact with the skin.
  
• Method C: Powders. Aliquots of powder should be transferred to the skin in a grid-like manner, using a spatula, sponge, or finger. The accumulated powder shall be tapped and then spread over the whole test site using a finger with or without a finger cot. Alternatively, the tip of a pre-loaded cosmetic applicator puff may be used instead of a finger. In this case, it is important to verify that (2,00 ± 0,05) mg/cm2 of test powder product remains on the skin after spreading, by weighing the powder remaining on the tip of the applicator puff.
  Purified water or another suitable solvent that has no UV protection properties may be applied on the skin before the powder application to help the sample adhere to the application site. Water or solvent should not transform the powder into a paste and thus influence its SPF value.
• Method D: Foaming formulations. For samples which are presented as foams and where the contents cannot be extracted or dispensed other than as a foam, the test product should be measured into a weighing boat and then the sample allowed to degas or deaerate until they can be easily applied to the skin. Application will be subsequently accomplished following Method B.
• Spreading time should be in the range of (35 ± 15) s depending on the surface and ease of spreading of the product. Volatile liquids should be spread without delay
• After application is completed, and before commencement of the UV exposure doses, the application shall be checked with an ultraviolet-A “Woods” lamp with at least 6W of power, that is capable to visualize the uniformity of the application. If noticeable non-uniformity or streaking of the product is noted, the test site shall be rejected and may not be used for the test. If another test site is available, a new application may be attempted.

6. Waiting period (15 to 30 minutes) before UV Exposure of the test site; Any extraneous
exposure of the test sites to UV light (artificial or natural) shall be avoided during this period and for a period of 24 h after exposure

7. UV exposure:
- the minimum area of each exposure sub-site shall be 0,5 cm2,
- the minimum distance between borders of each exposure sub-site (spots) shall be at least 0,8 cm,
- the distance between any exposure sub-site and any edge of the test site shall be at least 1 cm,
- the minimum number of exposure sub-sites used shall be five for unprotected MED
(MEDu) and five for protected MED (MEDp)

8. MED assessment
- Provisional MEDiu - before starting the main test, it may be necessary to determine
a provisional MEDiu in order to centre the UV dose ranges for the exposures of MEDiu and MEDip. A provisional MEDiu is a pre-test in which the MEDiu of a subject should be
determined prior to establishing the test MEDiu. This is performed by applying
a preliminary series of UV exposures up to one week before the test. The provisional MEDiu shall be determined by the colorimetric ITA° technique using the UV dose range in
Table 2.
- Estimated MEDiu - if the provisional MEDiu has not been determined before the product
test day, the MEDiu can be estimated by colourimetric technique (ITA°) without UV
exposure on the same day as the test. The estimated MEDiu exposure dose shall be
determined using the colometric ITA° technique and the UV dose range from Table 2.
Otherwise, use the provisional MEDiu previously determined above.
- For each subject, the unprotected MEDiu shall be determined on the same day as the
test product protected MEDip

For the unprotected site, the range of UV doses applied shall be established using the
subject's provisional MEDiu, or the estimated MEDiu based on the ITA°. A minimum of five
sub-sites centred on or close to the provisional/estimated MEDiu shall be exposed with
incremental UV doses using a recommended geometric progression of 1,25×. Other
geometric progressions of less than 1,25× may be used (such as 1,2; 1,15; 1,12) but shall be consistent throughout the test (same progression used for unprotected and protected
sites). Exposure times may be rounded to the closest integer seconds,

- For the product protected sites, the UV doses delivered are defined by the expected
MEDip, which is the multiple of the expected SPF of the test product (as determined by
the test sponsor or previous data) and the provisional or estimated MEDiu for the subject. A minimum of five sub-sites centred on or close to the expected MEDip shall be exposed with incremental UV doses using a recommended geometric progression of 1,25×. Other geometric progressions may be used (such as 1,2, 1,15 or 1,12). A maximum geometric progress of 1,15 shall be used for expected SPF ≥25. Smaller geometric progressions (such as 1,12) may be used but shall also be consistent throughout the exposure procedure (same progression used for unprotected and protected sites).
- The expected value of the SPF may be changed during the testing of the product
between test subjects as requested by the test sponsor or the laboratory management
to prevent test failures or overexposure of subjects.
- The minimal erythemal dose for individual unprotected skin (MEDiu), for the test product individual protected skin (MEDip), and the MEDip for the reference sunscreen formulation, shall all be determined on the same day.
- The MED(s) shall be assessed 20 h ± 4 h after UV exposure (between 16 h and 24 h) as
measured from the end of the last exposure period. During the time interval between UV
exposure and MED assessment, the subject shall avoid any extra UV exposure (artificial
UV light or sunlight) to the exposed area. Any additional UV exposure (natural or artificial) to the test area of an individual will invalidate the data from that individual and that data shall be rejected from the test results and not count against the total allowable rejected subjects.
- The responses observed at the exposure sites may be pink/red (erythema), grey/brown
(pigmentation), or a mixture of both. If the exposure site has grey/brown colouration, the
surface of the skin shall be lightly pressed with a glass slide to determine if there is also
erythema present. If erythema is present, there will be a slight blanching of the any
redness of the skin with the pressure and colour will return after removal of the pressure.
The site shall be scored as having unambiguous erythema provided that the returning
redness in the exposed sub-site is more than that of the unexposed surrounding skin, and the erythema occurs over more than 50% of the exposure sub-site. Pigmentation
responses (with no erythema) shall not be considered as a qualifying response for MEDu
or MEDp
- The grading scale for UV exposed test subsites shall be:
0: no erythema present
0.5: ambiguous erythema, and/or no clear border, and/or not filling more than 50 % of the
exposure subsite
1: Perceptible unambiguous erythema with defined borders filling more than 50 % of the
exposure subsite (MED if it is the lowest exposure dose with grade 1)
2: Moderate to intense erythema

9. Calculations
- The SPFi of both the reference sunscreen and the product under test for each subject
shall be calculated as shown blow and expressed to one decimal place by truncation:

where MEDip is the MED of sunscreen protected skin for an individual
MEDiu is the MED of unprotected skin for an individual
- The SPF result for the test product and for the reference sunscreen formulation shall be
calculated as the arithmetic mean of all valid individual SPFi values,
- The minimum number of valid SPFi values shall be ten and the maximum number of
valid SPFi values twenty. A maximum of five results may be excluded from the calculation
of the mean SPF, but each exclusion shall be justified according to rejection criteria
explained below or if protocol non-compliance has occurred. A sixth invalid result
automatically invalidates the whole test for that test product and no SPF can be
calculated for it.
- The mean SPF of the reference sunscreen formulation used in the test shall fall within the acceptance limits shown in table 1.

The aim of study

• The aim is to confirm expected sun protection.
  

Rejection criteria
Panelist's results are rejected and the panelist replaced if:

• The series of UV exposures on a subject fails to elicit an erythemal response on any sub-site,16 to 24 hours after exposure.
  
• The erythemal responses within an exposure series are randomly absent 16 to 24 hours after exposure, indicating uneven product spreading, non-constant light irradiance or unstable product.
• All sub-sites in the exposure series show an erythemal response 16 to 24 hours after exposure - thus prohibiting any MED calculation.
• When one of the above criteria applies to the exposure series on unprotected skin or to the reference sunscreen formulation exposure sites, then all data for all products on that subject are invalid and shall be rejected. The test is considered a technical failure even if the MED response is observed in the protected site.
• When one of the above criteria applies to a test product treated exposure series, then all data for that test product on that subject are invalid and shall be rejected.
• If invalid data (whether MEDu or MEDp) have to be rejected for any one product on more than five subjects, then the whole test for that product is invalid and shall be rejected.
• If invalid data have to be rejected for the reference sunscreen on more than five subjects, then the whole test is considered invalid and shall be rejected.

Subject – volunteers’ selection
· The selection of probands – volunteers was conducted by a dermatologist according to the Declaration of Helsinki of 1964 (with subsequent amendments), Polish laws, Cosmetics Europe directives with applying exclusion criteria as mentioned below:
a) children and persons below 18 years old or >70 years;
b) pregnant or lactating women;
c) subjects using medication with photo-sensitizing potential;
d) subjects using anti-inflammatory medication;
e) subjects with systemic dermatological conditions (including dysplastic nevi);
f) subjects with a history of abnormal response to the sun;
g) subjects who have used tanning beds in the previous eight weeks prior to SPF testing;
h) subjects having had sun exposure on the back area in the previous eight weeks prior to SPF testing;
i) subjects having marks, blemishes or nevi in the test area;
j) subjects presenting with existing sun damage in the test area;
k) subjects having excessive hair in the area on the test on the day of testing (may be
shaved up to 3 days prior to the test day);
l) subjects having skeletal protrusions and extreme areas of curvature in the test area.

Subjects shall be selected using the colourimetric ITA° value. The skin of subjects shall have a colourimetric ITA° value of subjects of ≥28°.
· Colourimetric ITA° values and skin colour categories are defined by the colourimetric
descriptors of Chardon et al. [9] using the CIE (1976) L*a*b* colour space.

• Subjects may participate in a test provided that at least 8 weeks have elapsed since they participate in a previous UV exposure study (i.e. SPF, UVA-PF, photoallergy, phototoxicity
  test), and all skin tanned marks from previous tests have cleared from the test sites on the back and are no longer visible.
• 10 people took part in the study who met the requirements for entering the study and agreed to informed consent to participate in the study. The skin at the selected area was normal, without any lesions. Subjects were informed not to use any kinds of antihistamines or pharmacological agents at the time of test, which may affect the tests’ results.

Table 4. Characteristic of subjects. 

 

Results
In 10 subjects, SPF of the product: Zonnemist SPF 50 48-000-0994.08 on average was 54,3. The protective factor in the Cosmetics Europe P3 control sample was 16,4 on average. The protective factor in the Cosmetics Europe P8 control sample was 60,3 on average. (see appendix 1)

CONCLUSION

• The study was conducted in accordance with recommendation of Cosmetics Europe and in accordance with International Standard ISO 24444:2019 the Sun Protection Factor (SPF).
• The tested material Zonnemist SPF 50 48-000-0994.08 when tested on ten subjects as described herein under static conditions yielded the mean SPF value 54,3.

Stamp and Signature of investigator 

1. The report may be reproduced only in its entirety. Another form of copying requires the written consent of the Contractor. 2. Report from research carried out in two identical copies (copy 1 – Customer, copy 2 –Diagnostic-Test). 3. The results refer only to the product of the composition given by the Principal.