Sederma TB Mediatone

MEDIATONE™
ENGLISH
03/18 PCS TB 014 V2 EN
MEDIATONE™
Patent and publications: FR 3 024 037 ; WO 2016/012973 ; US 9,918,916 ; CN 06794124 ; HK 17106904.30
HIGHLY PURIFIED
SKIN TONE MEDIATOR
Any use of this file for commercial or advertisement purposes is subject to the prior
written consent of SEDERMA.
Non-Warranty: The information in this publication is believed to be accurate and is given in good faith, but no representation or
warranty as to its completeness or accuracy is made. Suggestions for uses or applications are only opinions. Users are
responsible for determining the suitability of these products for their own particular purpose. No representation or warranty,
expressed or implied, is made with respect to information or products including, without limitation, warranties of merchantability,
fitness for a particular purpose, non-infringement of any third party patent or other intellectual property rights including, without
limit, copyright, trademark and designs. Any trademarks identified herein, unless otherwise noted, are trademarks of the Croda
group of companies.
Part of Croda International Plc, © 2018 Sederma, All rights reserved.

Member of Croda International Plc, © 2016 Sederma, All rights reserved.
MEDIATONE™
SYNOPSIS
Description 9-octadecenedioic acid obtained by metathesis, skin tone mediator, acting at all
levels of the skin pigmentation development.
INCI Name Octadecenedioic Acid.
Proven cosmetic activity
In vitro
All tests were carried out on pure octadecenedioic acid.
Melanin production
• Melanin synthesis (NHM*) ................................................................................. -51% (p<0.01)

• General tyrosinase activity (NHM*) ................................................................... -27% (p<0.01)
• Tyrosinase: mRNA, Protein (B16F1/Inserm-CNRS) ......................................... -54% / -52%
• PPARs binding, «gene reporter» technology (HeLa/Inserm-CNRS) ................. Yes
*: NHM: normal human melanocytes.
Melanin diffusion: melanosome phagocytosis
• ± UVB induction (NHK*) ..................................................................... -53 / -59% (both: p<0.01)

• ± KGF induction (NHK*) ..................................................................... -78 / -85% (both: p<0.01)
• KGF receptor (explants) .................................................................... -40% (p<0.01 vs placebo)
*: NHK: normal human keratinocytes.
Pro-pigmenting agents
• PGE2, pro-pigmenting lipid, UVB induction (NHK) ..............................................-69% (p<0.01)

• Endothelin-1, pro-pigmenting peptide, UVB induction (NHK) ..............................-57% (p<0.01)
• Reactive oxygen species (ROS) (NHM/NHF) ................................. -38% / -54% (both: p<0.01)
Strengthening of the skin barrier
• Keratinocyte differentiation (visual effect) ............................................................. Improvement

• Involucrin, loricrin, ceramides (NHK) ............................. +841%, +385%, +778% (all 3: p<0.01)
Hyaluronic acid synthesis
• Hyaluronic acid (HK) ........................................................................................... +72% (p<0.01)
In vivo
Two independent clinical studies conducted to evaluate the action of MEDIATONE™. The first on Asian
skin types, 2.5% cream vs placebo; the second on sub-Saharan skin types, 4% cream vs placebo.
Evaluation on Asian skin (Spincontrol)
• ITA° after 3 weeks ................................................................ +16.5% vs T0 (p<0.05 vs placebo)

• Clarity L* after 3 weeks ......................................................... +1.9% vs T0 (p<0.05 vs placebo)
Evaluation on sub-Saharan African skin (Spincontrol)
• ITA° after 1 and 2 months .............. +26.2% and 30.3% vs T0 (p<0.03 and p<0.01 vs placebo)
• L* clarity after 1 and 2 months ............... +4.0 and +4.6% vs T0 (p<0.03 et p<0.01 vs placebo)
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MEDIATONE™
Recommended use:
General information:
• Recommended pH: 3.50 - 8.00

• Melt MEDIATONE™ at 85°C in the oily phase or humectant phase before formation of the
emulsion, depending on the type of formulation.
• Solubility: good in highly polar emollients and humectants such as Arlasolve DMI™ (Dimethyl
Isosorbide) and Pentylene Glycol.
• Compatible with carbomers
Note: MEDIATONE™ is not soluble in water or glycerin, or in apolar emollients such as silicone oils, mineral oil or
squalane.
Toxicology: Patch test

In vitro ocular tolerance (BCOP)*
In vitro ocular irritation SkinEthic model (rHCE)
In vitro cutaneous tolerance SkinEthic model
HRIPT (100 volunteers)
Direct peptide reactivity assay (DPRA)
Ames test
In vitro micronucleus test on human lymphocytes
Phototoxicity
Expert Toxicologist Certificate
*: performed instead of HetCam and Red Neutral Release Method for reasons relating to the solubility of
the product in an aqueous medium.
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SUMMARY OF THE TRADEMARK AND PATENT SITUATION OF MEDIATONE™

MEDIATONE™ is a Sederma trademark designating the product Octadecenedioic Acid (C18:1
dicarboxylic acid), sold as a raw material for cosmetic and dermatological applications worldwide.
MEDIATONE™, its uses and preparation method are currently the subject matters of pending patent
applications filed by Croda and Sederma.
There is a number of pending patent applications or patents of various companies on specific
formulations containing Octadecenedioic Acid. Many of these have very broad claims which clearly lack
novelty and/or inventive step with respect to prior art, as clearly reflected by their published search reports.
The following documents constitute a solid body of "Prior Art" which invalidates most of the
claims mentioned in these pending applications:
• Uniqema patent family comprising European EP 0 662 946, US 5,753,704 and WO94/07837;
• Research Disclosure of April 2001, Vol. 444, No. 444077;
• Research Disclosure of November 2003, Vol. 475, No. 475117;
• Research Disclosure of January 2004, Vol. 477, No. 477003;
• As well as numerous scientific articles published in journals of the cosmetic domain (C&T, SOFW,
IFSCC).
The currently relevant patents and patent applications are listed below:
THOREL: granted FR 2 857 266 and Composition against acne and for the treatment of skin
KR 101161682 disorders related to the formation of blackheads comprising
Octadecenedioic Acid and a hydrophilic antioxidant chosen
from mannitol, vitamin C, lysine azelate, rutin and quercetin.
UNILEVER: granted US 6,171,582 and Cosmetic method for reducing or preventing body malodor by
US 6,183,731 topically applying Octadecenedioic Acid to skin.
UNILEVER: Granted: EP 2 285 342, Cosmetic composition comprising a skin lightening additive
CN 102065829, JP 5620908 and which is 12-hydroxystearic acid and a dioic acid (US, AU et
AU 2009259483 ; CN).
US 9227090 and KR 101617478 In EP, JP and KR: composition further comprising niacinamide.
Although patent offices may grant a few very specific combinations or uses of Octadecenedioic
Acid, these patents or applications do not constitute an obstacle to formulating skin whitening
products with MEDIATONE™ on a broad base of classical or novel ingredients!
Sederma is closely monitoring the status of these granted patents and applications.
An advice can be obtained from Sederma for any particular case.
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CONTENT
1. INTRODUCTION ................................................................................................................... 9
2. EFFICACY STUDIES .......................................................................................................... 17

2.1. In vitro studies ............................................................................................................................... 17
2.1.1. Pigmentation control .............................................................................................................. 17
2.1.2. Indirect control of pigmentation inducers ............................................................................... 22
2.1.3. Strengthening of the skin barrier ............................................................................................ 25
2.1.4. Conclusion of the in vitro studies ........................................................................................... 28
2.2. In vivo studies ............................................................................................................................... 31
2.2.1. Evaluation by spectrophotometry/Asian skin ......................................................................... 32
2.2.2. Evaluation by colorimetric analysis on photographs/sub-Saharan-type skin ......................... 35
3. CONCLUSION ..................................................................................................................... 39
4. REFERENCES .................................................................................................................... 41
5. APPENDICES ..................................................................................................................... 45
5.1. Formula of products used for in vivo evaluation “Asian skin” ........................................................ 45
5.2. Formula of products used for in vivo evaluation “Sub-saharan skin” ............................................ 46
5.3. Formulation advices with MEDIATONE™..................................................................................... 47
5.3.1. Solubilisation of active ingredient........................................................................................... 47
5.3.2. Choice of surfactants ............................................................................................................. 47
5.3.3. Other raw materials................................................................................................................ 48
5.3.4. Conclusion ............................................................................................................................. 48
5.3.5. Appendix 1: Examples of MEDIATONE™ solubilisers .......................................................... 49
5.3.6. Appendix 2: Examples of surfactants compatible with MEDIATONE™ ................................. 50
5.3.7. Appendix 3: Examples of finished products’ formula ............................................................. 51
04/2017/V2
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1. INTRODUCTION
With the exception of European-type populations who have favoured tanned skin for several decades,
Indian, Asian, African and South-American populations endeavour to lighten their complexions and reduce
the unsightly appearance of pigment disorders such age spots or melasma. A light complexion and
blemish-free skin are still a sign of youth, purity, nobility and high social status in many countries.
For instance, in India, social customs have been built around skin
colour for more than 2000 years. More light-skinned people are in
higher castes compared to others. Other Asian populations,
particularly women, also take great care of their complexion to
make it as light as possible, with a view to enhancing their marriage
or career prospects. Pigmentation may be limited by avoiding
exposure to sun, or by using brightening treatments, which are in
Figure 1: Face-kini on a
increasing demand. Exposure to sun can be avoided by extreme
Chinese beach
behaviour, as seen on certain beaches in China where face-kinis
(Figure 1) are now widely observed, or, more reasonably, by using sunscreens and filters. Skin can be
brightened using laser treatments or creams containing toxic products: mercury salts and hydroquinone.
There are also approaches which are more gentle to the skin, brightening cosmetic products containing
agents with proven, tried and tested safety.
The global skin lightening market predicted to reach up to US $10 billion by 2015 and it is projected to
reach US $23 billion by 2020, driven by the desire among both women and men to have even-tone, light-
coloured skin. In Asia, about one fourth of cosmetic products contain sunscreens and/or brightening
agents.
To curb skin pigmentation, the preferred target action is the reduction of melanin pigment production and
distribution. This can be achieved in several ways: less melanin production in the melanocyte, lesser
transfer of pigment from the melanocyte to neighbouring keratinocytes, reduction of radical, inflammatory
and peptide production promoting pigmentation, acceleration of epidermis renewal, and improvement in
barrier function which is sometimes impaired in the event of hyperpigmentation due to melasma and age
spots.
Pigmentation, general information
The first Hominids had white skin, unpigmented due to being protected by fur. As the human species
moved through the hot, dry savannah, they developed into a "naked ape" (MORRIS, 1967) to promote the
dissipation of body heat. Melanocytes gradually populated the epidermis, undoubtedly from the
infundibulum of hair, to protect the skin barrier from the dry and aggressive savannah environment, and
also from the sun's ultraviolet rays (ELIAS et al., 2009).
Then, as humankind migrated across the planet, and their pigmentation adapted to the environment in
which they evolved. A graduated scale from the darkest pigmentation in hot regions (sub-Saharan Africa,
Australia and Deccan Plateau) to the lightest pigmentation in regions with little sun (Scotland, Ireland and
Finland) is thus observed.
However, a social dimension is associated with skin colour in countries where light complexions are highly
prized. With the recent exception of Caucasian-type populations, a porcelain complexion has always been
sought after by most of the world's populations, as a symbol of youth, purity and high social standing.
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MEDIATONE™
In addition to brightening the complexion, consumers attempt to reduce the unsightly effects of skin
hyperpigmentation. This includes age spots and melasma, marks and larger areas respectively, which
increase with age among men and women, and which are caused by or exacerbated by sunlight.
Pigmentation biology
MELANOGENESIS
Skin colour is due to several factors, including haemoglobin and carotenoids, but is mainly related to the
production of melanin by melanocytes and its distribution into the surrounding keratinocytes. Melanocytes
are established in the epidermal floor, known as the basal layer, each one being surrounded by
approximately 36 keratinocytes thereby constituting a melanocyte unit.
Melanocytes are cells with a long life span which produce small
extensions, known as dendrites, which wind between the
keratinocytes to supply them with melanin. These dendrites may
extend or retract based on the stresses and stimuli received,
thereby modulating the number of contacts with keratinocytes and
the quantity of melanin supplied.
Melanin is a natural polymer produced in the melanocyte at the end

of a complex process. It is produced by the activity of different
enzymes including tyrosinase and TRP (tyrosinase-related proteins)
within a small carrier unit known as a melanosome. Figure 2: Tyrosinase
Tyrosinase is the enzyme which triggers melanin formation, hence, a reduction in its production or its
activity is thus a limiting factor for melanogenesis. An approximately 60-70 kDa glycoprotein contributes to
the formation of two major types of melanin: brown eumelanin and red pheomelanin. Tyrosinase catalyses
the hydroxylation of tyrosine into o-diphenol3,4-
dihydroxyphenylalanine (or DOPA) then the oxidation
of DOPA into DOPA-quinone, a precursor of melanin.
Modulation of tyrosinase activity involves a reduction
in its mRNA production (see next section on PPAR),
interference at its active sites, defective maturation
(glycosylation) or an increase in its destruction.
In general, the skin contains a mixture of pheomelanin
and eumelanin which determines the different colours
that may be observed in an individual. Furthermore,
melanosomes are free to varying degrees in
keratinocytes according to ethnic type.
Once formed in the melanosome, melanin is

transferred to keratinocytes via dendrites by means of
a small biological mechanism guided by a tubular Figure 3: Melanocytes, melanosomes and
transport structure. Melanosomes reaching the tip of keratinocytes
the dendrite are released from the melanocytes and are then subjected to phagocytosis by neighbouring
keratinocytes (Figure 3). This transfer is influenced by a large number of factors (see below), and the
reduction thereof represents another interesting approach to control of hyperpigmentation.
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FACTORS AMPLIFYING PIGMENTATION

Melanin production is controlled by exogenous or endogenous factors. Ultraviolet exposure (UV)
undoubtedly represents the most common exogenous factor for hyperpigmentation although hormonal
factors are also brought into play. UV radiation acts on melanocytes, keratinocytes and fibroblasts. All of
these cells produce and exchange numerous messengers, for example, such as endothelin-1, PGE2
(prostaglandin E2) and KGF (or Keratinocyte Growth Factor). These messengers modulate pigmentation
by acting on melanocyte multiplication, melanin production, dendricity and phagocytosis.
Endothelin-1 (Figure 4) is an oligopeptide, produced by keratinocytes, which

activates melanocyte receptor EDNRB. Via the PKC/ERK1,2 pathway, it
induces an increase in tyrosinase enzyme production, hence its overall
activity, and, ultimately, melanogenesis. At the same time, endothelin
promotes melanin distribution through elongation of melanocyte dendrites
(IMOKAWA et al., 1995; HYTER et al., 2013). Endothelin, the production of
which increases with UV radiation, is therefore one of the molecules which
liaises between keratinocytes and melanocytes and promotes pigmentation.
Figure 4: Endotheline-1
PGE2 (Figure 5) are complex lipids resulting from the

transformation of precursors originating from keratinocyte,
melanocyte and fibroblast membrane phospholipids. UV
exposure, micro-inflammatory or radical phenomena
stimulate their production. The binding of PGE2 with EP1
and EP3 receptor strongly stimulates melanogenesis, the Figure 5: Prostanglandine E2
development of dendrites and leads to the installation of
hyperpigmented areas (STARNER et al., 2010, SCOTT et al., 2004).
KGF is a protein for which fibroblast production increases further to UV exposure; it then stimulates the
keratinocyte receptor (KGF-R) and melanosome phagocytosis (LIN et al., 2010). Furthermore, the authors
showed that the KGF receptor was expressed to a greater extent in hyperpigmented lesions corresponding
to age spots in the early stages, which promotes melanin deposits.
Moreover, UV radiation and certain micro-inflammatory phenomena induce the formation, within each cell,
of a large quantity of reactive oxygen species (or ROS) including H2O2 (CHENG et al., 2005; VALENCIA
and KOCHEVAR, 2008). This leads to overproduction of pro-pigmenting signals such as PGE2 via chain
reactions in the cell.
ACQUIRED HYPER-PIGMENTED LESIONS

Age spots, also known as liver spots, mainly appear on the hands, face, chest and forearms from the age
of 50. They form more or less even brown spots which have a tendency to grow in size. Repeated
exposure to sun is clearly implicated in the emergence and progression of these lesions; boosted
tyrosinase activity is also observed.
A number of authors also highlight the existence of a micro-inflammatory environment, overproduction of

endothelin and overexpression of KGF-R in these disorders (AOKI et al., 2007; LIN et al., 2010). AOKI et
al., report the overexpression of genes involved in the metabolism of arachidonic acid, a precursor of
PGE2 and other pro-pigmenting lipids, together with the reduction in the expression of the gene for
involucrin, involved in barrier function.
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Melasma is a fairly common hyperpigmentation disorder in women,

which has a major psychological impact on those affected. Melasma
presents as wide, symmetrical zones, on areas of the face exposed to
sunlight (Figure 6). In addition, these zones present lesions
characteristic of actinic elastosis, a weak stratum corneum and
impaired barrier function (LEE et al., 2012).
Conventional treatments involve hydroquinone, tretinoin,

corticosteroids and resurfacing agents (SCHERDIN et al., 2008; Figure 6: Melasma
TIRADO-SANCHEZ et al., 2009). These treatments aim to limit melanin production and diffusion, and
exert a "peeling" effect. They are often aggressive and cause irritation; tretinoin thus induces dermatosis in
2/3 of users, and this proportion increases when used in combination with hydroquinone, banned in
Europe.
Less harsh products able to curb pigmentation in these zones and help reconstruct an effective barrier are
thus necessary. The latter activity prevents the penetration of irritants and allergens which can produce
endogenous pro-pigmenting compounds (MAN et al., 2014).
Pigmentation and barrier function, role of PPARs
PPARs (Peroxisome Proliferator-Activated Receptors) are a group of proteins which naturally bind to
certain fatty acids, their metabolites and eicosanoids (BERGER and MOLLER, 2002). Binding modifies the
conformation of PPARs, allows them to fix onto RXR (another receptor) and triggers, or prevents, the
production of target proteins via binding to DNA.
The three types of PPARs (identified as

α, δ and γ) are found in different skin
cell types (Figure 7) and are described
in numerous publications in their effect
on skin homoeostasis and on the
regulation of skin disorders (DI-POI et
al., 2004; MICHALIK and WAHLI,
2007).
These encourage keratinocyte

differentiation, improve barrier function,
and may promote loricrin and
involucrin production. Lipids bind to
them in some cases, but have no
effect on a given target highlighting the
complex nature of the phenomenon
(YAN et al., 2015). PPARs are Figure 7: Distribution and roles of the different PPARs in skin.
involved in skin lipid metabolism which
is again a favourable element for barrier function (MAN et al., 2006; MAN et al., 2008).
They also have an anti-inflammatory action by curbing the production of IL-1α and PGE2
(SUBBARAMAIAH et al., 2001). In addition to their positive action on melanoma control (MÖSSNER et al.,
2002; PLACHA et al., 2003), depending on the ligands used, they may stimulate or, on the contrary,
reduce the production and stimulate the degradation of tyrosinase (WIECHERS et al., 2005). In this
respect, they may play a part in strategies aiming to brighten the complexion and curb hyperpigmentation.
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Barrier function renewal
The skin protects the body from the dry environment in order to prevent fatal dehydration. An external
structure forming a very thin barrier, known as the stratum corneum, is thus constantly produced by the
epidermis (FEINGOLD et al., 2007). This is made up of cell bodies known as tightly bound corneocytes,
highly resistant due to the formation of a protein shell (loricrin, involucrin, etc.; Figure 8), and lipid cement
mainly consisting of cholesterol and ceramides (ELIAS and MENON; 1991). When this structure is
defective, it leads to numerous skin disorders as it also protects the body from invasion by allergens and
pro-inflammatory irritants, and acts as a UV filter.
Barrier defects have been observed to exist in certain hyperpigmentation disorders although it is not
known whether these are the cause or consequence of hyperpigmentation. In addition to data on age
spots or melasma, a number of authors have also observed defective barrier renewal after lesions in
depigmented zones (vitiligo) (LIU et al., 2010).
Figure 8: Structure of the stratum corneum.
Despite the few obvious links between pigmentation and barrier function, it is clear that numerous skin
pigmentation disorders are also associated with a barrier function disorder. Stimulating good barrier
formation should therefore be considerable interest. Further, promoting epidermal renewal by stimulating
keratinocyte differentiation serves to eliminate melanosomes stored in keratinocytes more rapidly.
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MEDIATONE™
THE SEDERMA CONCEPT

SEDERMA offers a safe, experienced solution to control pigmentation and clarity of the complexion,
thanks to a patented active ingredient derived from sunflower oil.
MEDIATONE™
is a modified technical cosmetic active ingredient, based on natural

ingredients derived from sunflower seeds (or Helianthus annuus). It
is obtained using original patented technology known as
metathesis, and fulfils current regulatory requirements. It is supplied
as a white pellet containing 90 to 100% of C18:1α,ω dicarboxylic
acid (Figure 9). Figure 9: 9-octadecenedioic acid
(9OA) derived from Helianthus
annuus
MEDIATONE™
• Curbs melanin production by melanocytes.
• Limits the production of tyrosinase.
• Acts on PPARs
• Reduces production of endothelin-1, pro-pigmenting peptide
• Reduces melanosome phagocytosis stimulated by UV or KGF
• Curbs production of KGF receptors
• Curbs the intracellular production of ROS and PGE2, pro-pigmenting lipids
• Strengthens the skin barrier, regulates the maturation of keratinocytes.
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PRESENTATION OF MEDIATONE™
MEDIATONE™ (Figure 10) is derived from purified, natural oleic acid, from sunflower seed oil guaranteed
GMO-free. This substrate is subjected to an olefin metathesis reaction to yield 9-octadecenedioic acid (or
dioic acid; Figure 12).
Figure 10: Helianthus annuus, seeds and flowers.
This complex lipid displays a beneficial cosmetic action. It is notably known to have a positive regulating
effect on skin pigmentation conditions (WIECHERS et al., 2002; THIRION et al., 2006; SCHERDIN et al.,
2008; TIRADO-SANCHEZ et al., 2009; MERINVILLE et al., 2012).
These different authors were able to demonstrate, in clinical tests, the brightening effects of creams
containing dioic acid on the skin of 143 European or Mexican volunteers with melasma (THIRION et al.,
2006; SCHERDIN et al., 2008; TIRADO-SANCHEZ et al., 2009), 71 Indian volunteers (MERINVILLE et al.,
2012) and also Indian-Pakistani volunteers (WIECHERS et al., 2002). Furthermore, no irritant effects were
observed by these authors.
It promotes the modulation of skin pigmentation by limiting the production of tyrosinase protein messenger
RNA via binding to PPARs (Figure 11, WIECHERS et al., 2005, also see in vitro results §2.1.1.a); but also
plays a role at different levels in melanin pigment production and distribution in skin. Furthermore, it
positively stimulates the production of skin barrier and moisturising constituents.
Figure 11: Effect of MEDIATONE™ on the formation of tyrosinase mRNA.
METATHESIS
Metathesis is a controlled low-energy-consuming catalytic reaction, which rearranges unsaturated
molecules at their double binding site. It yields highly pure products. Chauvin, Grubbs and Schrock were
awarded the Nobel Prize in Chemistry in 2005 for their research into metathesis.
No solvents are used throughout the MEDIATONE™ production process, and all compounds obtained are
either recycled or ultimately developed. To produce MEDIATONE™, two oleic acid molecules are used,
after being transiently modified in methyl oleate, subjected to a metathesis reaction which yields dimethyl
octadecenedioate and 9-octadecene (Figure 12). These two forms are separated and dimethyl is hot
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MEDIATONE™
saponified to yield 9-octadecenedioic acid (or MEDIATONE™). Simple distillation then gives rise to a high
yield of the desired molecule.
O
O
O
O
Metathesis reaction
+
+
O
O
O
O
Figure 12: Metathesis reaction yielding the intermediate product dimethyl octadecenedioate and
9-octadecene.
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MEDIATONE™
2. EFFICACY STUDIES
2.1. In vitro studies
MEDIATONE™ can be tested on cells in culture after dissolution in ethanol or DMSO, and sometimes
using 0.5 to 2% bovine serum albumin (BSA) (HANLEY et al., 1998; MERINVILLE et al., 2012). It can thus
be tested in contact with cells from 10 to 150 ppm. It can only be tested at 2.5 to 4% when formulated as a
cream.
2.1.1. Pigmentation control
a. Decrease of melanin production and tyrosinase activity
PROTOCOL
(1)
Strongly pigmented , confluent normal human melanocytes (NHM) were placed in contact with
(2)
MEDIATONE™ for 10 days. At the end of the contact period, melanins are extracted from the cells after
milling, and assayed by spectrophotometry. Cell viability is also estimated at the end of culture by protein
(3)
assay, as per the BCA method. Likewise, moderately pigmented NHM were milled at the end of an
equivalent contact period and tyrosinase activity was determined as per the method described by WINDER
and HARRIS (1991), the results being weighted in terms of the quantity of proteins.
(1) ®
: Strongly pigmented NHM (male donor phototype estimated as V-VI; Cascade™ Biologics ).
(2)
: % BSA was added to the culture medium already containing 5% calf serum to promote the bioavailability of
MEDIATONE™. MEDIATONE™ was then incorporated in this medium.
(3) ®
: Moderately pigmented NHM (male donor phototype estimated as III-IV; Cascade™ Biologics ).
RESULTS
Control MEDIATONE™ 150 ppm
Figure 13: Modulation of melanin production in NHM.
Table 1: Melanin production by NHM, effect of MEDIATONE™ (n=4).
Melanin
6 Variation (%)
(ng/10 cell.)
Control 180.5 ± 8.4 Reference
MEDIATONE™ 30 ppm 152.7 ± 1.6 -15%; p<0.01
MEDIATONE™ 100 ppm 118.5 ± 3.5 -34%; p<0.01
MEDIATONE™ 150 ppm 88.2 ± 2.6 -51%; p<0.01
No toxicity was observed.
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Table 2: Tyrosinase activity of NHM, effect of MEDIATONE™ (n=4).
Tyrosinase
Variation (%)
(U/100µg prot.)
MEDIATONE™ 30 ppm 48.24 ± 4.45 -18%; p<0.05
MEDIATONE™ 100 ppm 42.87 ± 1.96 -27%; p<0.01

CONCLUSION
The results (Table 1; Figure 13) show that MEDIATONE™ gives rise to a dose-dependent and significant
reduction in melanin production (-15% to -51%), without any cytotoxic effects. The reduction in melanin
production observed in this case corroborates the results obtained by WIECHERS et al., (2005) on B16F1
and MERINVILLE et al., (2012) on NHM. Furthermore, the tyrosinase activity of NHM after contact with
MEDIATONE™ is also reduced in a dose-dependent manner (-18 to -27%, Table 2).
b. Variation of tyrosinase mRNA expression
PRINCIPLE /PROTOCOL (INSERM U540; CNRS UMR 6061, PROCLAIM STUDY)

B16F1 pigment cells in culture were placed in contact with MEDIATONE™ then total RNA was extracted
using appropriate methods. Tyrosinase mRNA expression was evaluated by qRT-PCR following the Ct*
(WIECHERS et al., 2005).
* The up and down primers were as follows:
for tyrosinase: 5'-TCCTTCTGTCCAGTGCACCAT-3' and 5'-CACAGAGGGCCAGGACTCA-3';
for the in-house reference (or housekeeping gene): 5'-AGGTTCTGGCCAACGGTCTAG-3' and
5'-CCCTCTATGGGCTCGAATTTT-3'.
RESULTS / CONCLUSION
Figure 14 shows the variation of the quantity of tyrosinase mRNA in the cells in the presence of
MEDIATONE™ or the control. This production is approximately 50% lower than the control values. This
indicates that less coding material is present to produce the new tyrosinase enzyme, a limiting factor in
melanin formation.
100
Quantity of tyrosinase mRNA
80 -54%
(% vs control)
60
40
20
0
Negative control MEDIATONE™ 62.5 ppm 4 days later
Figure 14: Tyrosinase mRNA production in B16F1.

Effect of 62.5 ppm MEDIATONE™ vs control.
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MEDIATONE™
c. Variation of tyrosinase protein production (Western Blot)
PRINCIPLE / PROTOCOL (INSERM U540; CNRS UMR 6061, PROCLAIM STUDY)

B16F1 pigment cells in culture were placed in contact for 24 hours with MEDIATONE™ then washed twice
with PBS before being milled and centrifuged in Laemmli buffer*. Total proteins were separated by
electrophoresis then transferred to a membrane. The tyrosinase proteins or tubulin (stable reference) were
then labelled on the membrane using appropriate antibodies (WIECHERS et al., 2005).
* 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.125M Tris HCl, pH 6.8.
Figure 15 below shows the variation in the quantity of tyrosinase observed in the cells in the presence of
MEDIATONE™ or the control (DMSO only). This production is approximately 50% lower than the control
values. This reduction is consistent with that observed for mRNA production and is correlated with the
experimental results presented in section 2.1.1.a.
100
Negative control
Quantity of tyrosinase mRNA

MEDIATONE™
80
-52%
(% vs control)
60
40
20
0
Negative control MEDIATONE™ 9.4 ppm
Figure 15: Tyrosinase protein production in B16F1 cells.

Effect of 9.4ppm MEDIATONE™ vs control. (Left: Western Blot).
d. PPAR binding, reporter gene technology
PRINCIPLE
In order to demonstrate the affinity of MEDIATONE™ for PPAR, three HeLa cell lines were established,
one per type of PPAR. For this purpose, the receptor parts of PPARα, PPARδ, or PPARγ were associated
with a GAL4 structure having a strong affinity for promoter gene GAL4. Five copies of the latter were
inserted into the DNA of cells with a copy of the luciferase gene serving as a signal; this corresponds to
reporter gene technology (JOYEUX et al., 1997). Hence, a molecule having an affinity for at least one of
the PPAR will trigger cascade binding of GAL4 to DNA and immediate formation of luciferase. The latter, in
the presence of luciferin, will then produce oxyluciferin and a photon which is quantified.
- 19 -

MEDIATONE™
PROTOCOL (INSERM U540; CNRS UMR 6061, PROCLAIM STUDY)

The three cell types were incubated for 16 hours in the presence of MEDIATONE™ in a DMEM medium
containing 6% foetal calf serum (WIECHERS et al., 2005). At the end of incubation, the medium was
removed and replaced by a culture medium containing luciferin. The luminescent signal was measured
after 5 minutes using a Microbeta luminometer (Wallac, Turku, Finland). All tests were conducted four
times.
The specificity of the three cell lines was first validated using pharmacological ligands for the 3 PPAR iso-
types. As expected, these compounds activated the chimeric receptors and produced luminescence for
-9 -10
very low concentrations close to 10 to 10 M (WIECHERS et al., 2005).
The affinity of MEDIATONE™ for the receptors was evaluated on the three cell lines. The results show
that MEDIATONE™ has an affinity for the three receptors; this was obtained for PPARγ at a concentration
10 times lower than for PPARα and δ (Figure 16).
The effect of MEDIATONE™ is not, however, totally comparable to that of the pharmacological ligands,
and not only in terms of sensitivity. While certain ligands can stimulate melanin synthesis (LEE et al.,
2007), the INSERM/CNRS/Proclaim study (WIECHERS et al., 2005) showed, on B16, that this affinity, on
the contrary, gave rise to a reduction in tyrosinase production (mRNA and protein).
120
100
Luciferase expression
80
PPARα
60
PPARδ
40 PPARγ
20
0
-7 -6,5 -6 -5,5 -5
Log [9-octadecenedioic acid] (M)
Figure 16: Affinity of MEDIATONE™ for PPAR receptors.
The well-known pharmacological agonists of PPAR-γ are recognised to have very different effects on a
given target. For example, they may or may not activate epidermal differentiation (filaggrin and loricrin
production; YAN et al., 2015), which is explained by the complex nature of the PPAR/RXR system. It is
therefore highly likely that MEDIATONE™, despite being a PPAR agonist, will have an effect different to
other activators on the tyrosinase target. Furthermore, activation of proteasome by fatty acids could also
explain the reduction in general tyrosinase activity (ANDO et al., 2006; MERINVILLE et al., 2012).
We will see below that MEDIATONE™ has a marked effect on epithelial barrier formation, a characteristic
also observed in certain stimulators of PPAR-α, β/δ and γ (MAN et al.; 2006).
- 20 -

MEDIATONE™
e. Reduction of melanosome phagocytosis
PRINCIPLE
Type B ultraviolet radiation accelerates skin pigmentation and melanosome transfer from melanocytes to
neighbouring keratinocytes by means of a phagocytotic phenomenon, promoting skin pigmentation.
Likewise, keratinocyte growth factor (KGF) accelerates phagocytosis (CARDINALI et al., 2005). UVB and
KGF are also said to play a key role in the first stages of age spots both by stimulating phagocytosis, but
also by directly activating tyrosinase synthesis in the melanocyte (CHEN et al., 2010). Curbing this
phagocytosis is known to promote brightening of the skin (SEIBERG et al., 2000).
PROTOCOL
Confluent normal human keratinocytes (NHK) were rinsed with PBS containing 0.5% BSA, then placed in
K-SFM (Gibco) medium for 24 hours. The cells were exposed to UVB radiation in an HBSS buffer then
placed in culture for 48 hours with MEDIATONE™ in the K-SFM culture medium. Phagocytotic capacity
was tested by adding fluorescent microbeads (as per CARDINALI et al., 205). After rinsing to eliminate the
beads not having undergone phagocytosis, photographs were taken by fluorescence microscopy and
examined by image analysis. The cell count was estimated as per the Hoechst 33258 method (LABARCA
et al., 1980).
To stimulate phagocytosis by KGF, a protocol identical to that described above was used, with the cells in
contact with KGF instead of being subjected to irradiation (Figure 17).
RESULTS
Control MEDIATONE™
Figure 17: Microbeads phagocytosis (in green) by NHK induced by KGF.
- 21 -

MEDIATONE™
Table 3: Phagocytosis by NHK under UVB or KGF induction;

Effect of MEDIATONE™ (n=4).
[microbeads]
3 Variation (%) Variation (%)
(AFU*/10 cells)
Control 83834 ± 14239 Reference (-) UVB
(-) UVB
MEDIATONE™ 20 ppm 39572 ± 19398 -53%; p<0.01
Control 144380 ± 13506 +72%; p<0.01 Reference UVB
UVB
MEDIATONE™ 20 ppm 59107 ± 19251 -59%; p<0.01
Control 123188 ± 21964 Reference (-) KGF
(-) KGF
MEDIATONE™ 20 ppm 26981 ± 6893 -78%; p<0.01
Control 184175 ± 16618 +50%; p<0.01 Reference KGF
KGF
MEDIATONE™ 20 ppm 27864 ± 4831 -85%; p<0.01
* AFU: Arbitrary Fluorescence Unit; no toxicity was observed, 36 photographs/case.
CONCLUSION
As expected, the results (Table 3) (CARDINALLI et al., 2005) show that UVB and KGF significantly
increase microbeads phagocytosis (equivalent to melanosomes) by +72% and +50% respectively.
MEDIATONE™ reduces phagocytosis both at basal level and that induced in NHK. For UVB, the reduction
respectively corresponds to -53% and -59% (p<0.01); for KGF, the reduction respectively corresponds to
-78% and -85% (p<0.01).
We used the skin explant model (abdomen, female, 70 years, Caucasian type) in order to obtain broader
results. The cream containing 4% MEDIATONE™ (or placebo; see appendix) was applied to the skin
surface for 7 days. Immuno-histochemical labelling was then performed on thin sections (8 µm) in order to
evaluate the presence of the KGF receptor (KGF-R) in the epidermis. 15 photographs/explant were taken
then KGF-R labelling of the epidermis was quantified by image analysis. The results show that the cream
containing MEDIATONE™ reduces KGF-R expression by -40% (p<0.01) vs the placebo cream. These
results therefore indicate that one of the constituents involved in melanosome phagocytosis is reduced
thanks to MEDIATONE™.
Hence, these results on NHM, NHK or explants demonstrate reduction of melanin production, together
with a reduction in residual tyrosinase activity and phagocytosis, three clear indicators that MEDIATONE™
should promote brightening of the skin (SEIBERG et al., 2000).
2.1.2. Indirect control of pigmentation inducers
a. Reduction of pro-inflammatory and pro-pigmenting lipids
PROTOCOL
Sub-confluent NHK were placed in contact with MEDIATONE™ in the cell test medium for 24 hours. After
being rinsed with a buffer, the cells are then irradiated with UVB in the same buffer. At the end of this
phase, the cells once again received MEDIATONE™ for 24 hours. Lastly, the media were recovered and
the quantities of PGE2 synthesised were measured using an ELISA method. The cell layers were used to
estimate viability by the MTT method (DENIZOT and LANG, 1986).
- 22 -

MEDIATONE™
Table 4: Production of PGE2 by NHK under UVB induction;

PGE2
(pg/10 cells)
(-) UVB Control (-) UVB 415 ± 83 Reference (-) UVB
Control UVB 1978 ± 244 +377%; p<0.01 Reference UVB
UVB MEDIATONE™ 10 ppm 1043 ± 117 -47%; p<0.01
MEDIATONE™ 30 ppm 616 ± 129 -69%; p<0.01
As expected, the results (Table 4) show that UVB stress significantly increases PGE2 production by
+377% (p<0.01).
MEDIATONE™ reduces this production in NHK by -47% and -69% for 10 and 30 ppm, respectively
(p<0.01). This serves to control a major although indirect contributor to skin pigmentation.
b. Endothelin-1 production
PRINCIPLE / PROTOCOL
A protocol similar to that described above was used. Assay of endothelin-1 was performed by means of an
ELISA test.
Table 5: Production of endothelin-1 by NHK under UVB induction;

Endotheline-1
(pg/10 cells)
Control (-) UVB 1638 ± 32 Reference (-) UVB
(-) UVB
MEDIATONE™ 30 ppm 581 ± 25 -65%; p<0.01
Control UVB 2003 ± 257 +22%; p=0.07 Reference UVB
UVB
MEDIATONE™ 30 ppm 855 ± 71 -57%; p<0.01
As expected, the results (Table 5) show that UVB stress significantly increases endothelin-1 production by
+22% (p=0.07).
MEDIATONE™ reduces this production in NHK, both at basal level or further to induction, by -65% and
-57% (p<0.01 in both cases). As observed for PGE2 lipids, MEDIATONE™ serves to control another
major contributor to skin pigmentation.
c. Production of free radicals
PRINCIPLE / PROTOCOL
UV radiation and inflammatory aggression on the skin induce reactive oxygen species (ROS) which, in
return, promote hyperpigmentation of the zones concerned, among other effects (CHENG et al., 2005;
VALENCIA and KOCHEVAR, 2008).
- 23 -

MEDIATONE™
Tests were initially conducted on normal human fibroblasts (NHF). Once attached to their support, the
NHF were placed in contact with MEDIATONE™ then the DCFH-DA sensor for 30 min. (ROSENKRANZ
et al., 1992). After rinsing, the cells were again placed in contact with MEDIATONE™ for 24 hours,
together with the stress agent (H2O2). The ROS thus produced turned the sensor fluorescent; the signal is
recorded and compared to the control free from MEDIATONE™.
Table 6: ROS production in NHF, effect of MEDIATONE™

in the presence or absence of oxidative stress (n=3).
ROS / DCFH
(AFU*/10 cells)
Control (-) H2O2 2422 ± 102 Reference (-) H2O2
MEDIATONE™ 10 ppm 2179 ± 70 -10%; p<0.01
(-) H2O2
MEDIATONE™ 20 ppm 2053 ± 46 -15%; p<0.01
MEDIATONE™ 30 ppm 1677 ± 38 -31%; p<0.01

Control H2O2 5559 ± 159 +130%; p<0.01 Reference H2O2
MEDIATONE™ 10 ppm 3913 ± 91 -30%; p<0.01
H 2O 2
MEDIATONE™ 20 ppm 3282 ± 182 -41%; p<0.01
MEDIATONE™ 30 ppm 2534 ± 160 -54%; p<0.01
* AFU: Arbitrary Fluorescence Unit; no toxicity was observed.
As expected, the results (Table 6) show that oxidative stress significantly increases ROS production
+130% (p<0.01).
MEDIATONE™ reduces this production both at basal level and induced in NHF, by -31% and -54%
respectively (p<0.01) at 30 ppm.
These tests were supplemented by tests on NHM.
Table 7: ROS production in NHM, effect of MEDIATONE™

in the presence or absence of oxidative stress (n=4).
ROS / DCFH
(AFU*/10 cells)
Control (-) H2O2 1455 ± 154 Reference (-) H2O2
Control H2O2 35357 ± 2808 x 24; p<0.01 Reference H2O2

H2O2 + MEDIATONE™ 30 ppm 21936 ± 1712 -38%; p<0.01
* AFU: Arbitrary Fluorescence Unit; no toxicity was observed.
Table 7 shows that oxidative stress model (H2O2) generates free radicals in melanocytes (x24, p<0.01);
the reduction in formation is highly significant thanks to the use of MEDIATONE™ (-57%, p<0.01) which is
capable of curbing ROS production even in NHM.
- 24 -

MEDIATONE™
2.1.3. Strengthening of the skin barrier
Certain pigmentation disorders (age spots, melasma, vitiligo) have an impact on barrier function (see
introduction). Strengthening this barrier is therefore crucial. Keratinocytes are known to play a vital role in
controlling skin homoeostasis in addition to its important, direct role on melanocytes (phagocytosis,
transfer of cytokines, peptides, etc.). Furthermore, accelerated epidermal differentiation activates the
transfer of melanin to the stratum disjunctum and its elimination.
a. Accelerated keratinocyte differentiation
PROTOCOL
Sub-confluent NHK were placed in contact with MEDIATONE™ in the cell test medium for 7 days during
which their differentiation rate was scored using a microscope (Figure 18, n=3).
Figure 18: NHK differentiation after 7 days, effect of MEDIATONE™ vs control.
These photographs show that MEDIATONE™ activates keratinocyte maturation. The cell layer shows
several signs of more differentiated cells.
In order to estimate this differentiation, a score corresponding to the state of cell differentiation
(0 undifferentiated cells/5 highly differentiated cells was allocated by a panel of 10 experts after viewing
the photographs.
NHK differentiation in the presence of MEDIATONE™
3,00
2,50
differentiation score
2,00
1,50 Control
Contrôle
MEDIATONE™
MEDIATONE™
1,00
0,50
0,00
D2 D5 D7
-0,50
Time (day)
- 25 -

MEDIATONE™
b. Markers of epidermal differentiation: involucrin, loricrin, ceramides
The same cells as those described previously were set and immunolabelled in order to observe synthesis
of the various epidermal differentiation markers: involucrin, loricrin and ceramides (see introduction). Five
photographs were taken for each labelling operation, using a fluorescence microscope on each of the
three replicates. Labelling was quantified by image analysis on each series of 15 photographs. Finally,
counter-staining of the nuclei with Hoechst stain enabled homogeneous data to be obtained.
RESULTS
INVOLUCRIN
Figure 19: NHK differentiation after 7 days, effect of MEDIATONE™

vs control on involucrin synthesis (green; n=3; x 200).
Table 8: Modulation of involucrin expression by keratinocytes

in contact with MEDIATONE™ (15 photographs/case).
Involucrin
Variation (%)
(AFU*/ cells)
MEDIATONE™ 15 ppm 452.2 ± 321.0 +842%; p<0.01
MEDIATONE™ 20 ppm 451.8 ± 341.7 +841%; p<0.01

* AFU: Arbitrary Fluorescence Unit; no toxic effect was observed.
- 26 -

MEDIATONE™
LORICRIN
Figure 20: NHK differentiation after 7 days, effect of MEDIATONE™

vs control on loricrin synthesis (green; n=3; x 200).
Table 9: Modulation of loricrin expression by keratinocytes

Loricrin
Variation (%)
(AFU*/ cells)
MEDIATONE™ 15 ppm 231.1 ± 93.0 +159%; p<0.01
MEDIATONE™ 20 ppm 433.8 ± 272.8 +385%; p<0.01

* AFU: Arbitrary fluorescence unit; no toxic effect was observed.
CERAMIDES
Figure 21: NHK differentiation after 7 days,

effect of MEDIATONE™ vs control on ceramide synthesis (in green; n=3; x 200).
- 27 -

MEDIATONE™
Table 10: Modulation of ceramides expression by keratinocytes

Ceramides
Variation (%)
(AFU*/ cells)
MEDIATONE™ 15 ppm 122.2 ± 208.1 +623%; p<0.05
MEDIATONE™ 20 ppm 148.3 ± 167.3 +778%; p<0.01

* AFU: Arbitrary Fluorescence Unit; no toxic effect was observed.
These results show that MEDIATONE™ actively promotes the production of epidermal differentiation and
barrier function markers in a dose-dependent and significant manner: involucrin, loricrin and ceramides.
These increases reach +841%; +385% and +778% respectively (all p<0.01).
c. Hyaluronic acid synthesis
Sub-confluent human keratinocytes were cultured then placed in contact with MEDIATONE™. At the end
of the contact period, the culture supernatants were assayed to determine their hyaluronic acid content
using an ELISA-type kit. Estimation of the quantity of cells using the Hoechst method gave rise to
homogeneous results.
Table 11: Modulation of hyaluronic acid production by keratinocytes

in contact with MEDIATONE™ (n=4 or 5).
Hyaluronic acid
6 Variation (%)
(ng/10 cells)
MEDIATONE™ 10 ppm 2585.8 ± 599.6 +66%; p<0.01
MEDIATONE™ 30 ppm 2681.8 ± 608.8 +72%; p<0.01

Positive control: 1 µM retinoic acid: +59% (p<0.01). No toxic effect was observed.
These results show that MEDIATONE™ strongly stimulates hyaluronic acid production in keratinocytes.
This increase reaches +72% (p<0.01).
2.1.4. Conclusion of the in vitro studies
It is only recently that the Peroxisome Proliferator-Activated Receptors factors (PPAR) have been
identified as influencing a multitude of biochemically regulated processes in human epidermis.
Using the reporter gene technology, we are able to show that the 9-octadecenedioic acid binds to PPAR
especially PPARγ.
Since the expression of PPARγ has been described in the literature, we investigated whether the
stimulation of PPARγ by 9-octadecenedioic acid also resulted in a reduction of mRNA of tyrosinase
(transrepression mechanism) which in turn leads to reduce the tyrosinase protein levels and thereby
reduces the production of melanin in melanocytes B16F1 cells.
- 28 -

MEDIATONE™
To confirm that it is possible to reduce melanogenesis via stimulation of PPARγ, we have also
incorporated pioglitazone, well known PPARγ agonist. Melanin production is reduced by 40 and 80%
respectively for 9-octadecenedioic acid and pioglitazone (with 20 µM each).
We therefore believe that the mechanism of action by which the 9-octadecenedioic acid lightens the skin is
directly or indirectly done through the PPAR.
- 29 -

MEDIATONE™
- 30 -

MEDIATONE™
2.2. In vivo studies
We observed in the introduction that this lipid is known to have a positive regulating effect on skin
pigmentation conditions (WIECHERS et al., 2002; THIRION et al., 2006; SCHERDIN et al., 2008;
TIRADO-SANCHEZ et al., 2009; MERINVILLE et al., 2012). These different authors were able to
demonstrate, in clinical tests, the brightening effects of creams containing 9-octadecenedioic acid on the
skin of 143 European or Mexican volunteers with melasma (THIRION et al., 2006; SCHERDIN et al., 2008;
TIRADO-SANCHEZ et al., 2009) 71 Indian volunteers (MERINVILLE et al., 2012) and also Indian-
Pakistani volunteers (WIECHERS et al., 2002). We carried out two tests on populations with pigmented
skin to validate these effects.
PRINCIPLE
Two independent tests were carried out. The first, carried out on volunteers with Asian-type skin, with a
cream containing 2.5% MEDIATONE™ or its placebo. The second carried out on volunteers with sub-
Saharan African-type skin, with a cream containing 4% MEDIATONE™ or its placebo.
PROTOCOL
STUDY TYPES AND DURATION
Both studies were conducted on a total of 48 volunteers:
• The first evaluation was carried out on Asian-type volunteers. 25 women, mean age 47 [21-60 years],
completed the study. This study was conducted versus placebo. During this study, face colour was
measured using a spectrophotometry technique (SPINCONTROL - Thailand; January 2015/February
2015).
• The second study was conducted on volunteers with sub-Saharan African-type skin. 23 volunteers,
mean age 32 [22-43 years], completed the study. This study was conducted versus placebo. During
this study, face colour was measured by means of a colorimetric analysis on photographs
(SPINCONTROL - Canada; December 2013/February 2014).
These studies were conducted under single-blind conditions on the face. Treatment involved using a
cream containing 2.5% MEDIATONE™ at least twice daily for 3 weeks (Asian skin study), or a cream
containing 4% MEDIATONE™ for 2 months (study on black skin), or their respective placebos under
contralateral conditions (see formulations in appendices).
The synopsis of the study on Asian skin can be summarised as follows:
T0 T3 weeks
• Spectrophotometry • Spectrophotometry
The synopsis of the study on "sub-Saharan skin" can be summarised as follows:
T0 T1 month T2 months
• Facial photographs by • Facial photographs • Facial photographs by

Visia®-CR by Visia®-CR Visia®-CR
• Study by colorimetric • Study by colorimetric • Study by colorimetric
analysis analysis analysis
- 31 -

MEDIATONE™
Statistical testing was performed using the Student's t test or, if needed, a Wilcoxon signed-rank test. Two-
sided tests were carried out on paired series.
SPECIFIC INCLUSION CRITERIA

For the evaluation on Asian skin, the volunteers were required to present spots on their cheeks, with at
least one 3 mm in diameter minimum on each side. All skin types were permitted (dry, oily or normal).
During the test, strict sun protection was required (hat, umbrella) and the use of a sun protection product
or a change in sun protection factor for regular sun protection users was prohibited.
For the second test, the volunteers were required to have sub-Saharan African origins (even distant) and
correspond to phototype V or VI according to the Fitzpatrick scale. Phototype V was to represent not more
than 30% of the panel.
2.2.1. Evaluation by spectrophotometry/Asian skin
A CM700d spectrophotometer (Konica Minolta, Japan, Figure 23) was used for the study on Asian skin.
This device, widely used in cosmetology, measures light reflection between 400 and 700 nm (visible light).
It makes it possible to operate within the reference CIE L*a*b* colorimetric range, as defined by the
International Commission on Illumination (1976) which defines colour by 3 coordinates:
• L*: 0 (black) to 100 (white)
• a*: 100 (red) to -100 (green)
• b*: 100 (yellow) to -100 (blue).
For brightening effects, L* and b* are combined so as to calculate the parameter ITA° (Individual Typology
Angle; CHARDON et al., 1991) as per the following formula:
ITA° = Arctg [(L*-50)/b*]x(180/π)
Hence, an increase in L* and ITA° are expected within the context of a study on a brightening product.
Figure 22: CM700d spectrophotometer, colorimetric range L*a*b*, parameter ITA°.
During this study, 5 acquisitions were performed for each site with a 3-mm diameter tip. Table 12 below
presents the mean ± standard deviation for these values.
- 32 -

MEDIATONE™
Table 12: Variation of ITA° parameter after 3 weeks of applying 2.5% MEDIATONE™,
or its placebo, to a hyperpigmented zone on the face
(n= 25 volunteers, n= 5 measurements /site).
2.5% MEDIATONE™ Placebo Non-treated
T0 T21 days T0 T21 days T0 T21 days
Mean 17.48 20.61 18.76 20.04 18.04 18.30

± SD ± 7.71 ± 8.09 ± 7.33 ± 6.13 ± 9.57 ± 9.60
Variation vs T0 (%) 17.9% 6.8% 1.5%
Variation vs T0
corrected, control zone 16.5% 5.4%
(%)
Significance vs T0 p<0.01 nsd
Responders 88%
Significance vs placebo p<0.05
nsd: non-significant difference.
18 16.5
16
**$
14
Variation (%)
12
10 Placebo
8 MEDIATONE™
5.4
6
0
Placebo MEDIATONE™
**: Significant variation relative to T0 with p<0.01.
$: Significant variation relative to placebo with p<0.05.
Analysis of the results for parameter ITA° reveals brightening in the hyperpigmented zone (spot) after only
3 weeks of applying the cream containing 2.5% MEDIATONE™ compared to the zone receiving the
placebo cream.
An increase in parameter ITA° is observed corresponding to +16.5% (p<0.01 vs T0) whereas, during the
same period, application of the placebo cream gives rise to a small non-significant change corresponding
to +5.4%.
The difference between the two treatments is significant (p<0.05) and in favour of the cream containing
2.5% MEDIATONE™.
- 33 -

MEDIATONE™
Table 13: Variation of L* parameter after 3 weeks of applying 2.5% MEDIATONE™,

or its placebo, to a hyperpigmented zone of the face
(n= 25 volunteers, n= 5 measurements/site).
2.5% MEDIATONE™ Placebo Non-treated
T0 T21 days T0 T21 days T0 T21 days
Mean 56.15 ± 57.41 ± 56.47 ± 57.23 ± 55.91 ± 56.13 ±

± SD 2.67 2.80 2.56 2.38 3.05 3.06
Variation vs T0 (%) 2.2% 1.4% 0.4%
Variation vs T0 corrected,
1.9% 1%
control zone (%)
Significance vs T0 p<0.01 p<0.05
Responders 96%
Significance vs placebo p<0.05
2 1.9
1,8
1,6 **$
1,4
Variation (%)
1,2
1 Placebo
1
* MEDIATONE™
0,8
0,6
0,4
0,2
0
Placebo MEDIATONE™
*(*): Significant variation relative to T0 with p<0.05 (p<0.01).
$: Significant variation relative to placebo with p<0.05.
As for parameter ITA°, clarity parameter L* increases significantly after 3 weeks of applying the cream
containing 2.5% MEDIATONE™ compared to the zone receiving the placebo cream.
Brightening of the hyperpigmented zone is observed, corresponding to +1.9% (p<0.01 vs T0) whereas,
during the same period, application of the placebo cream gives rise to a smaller change corresponding to
1% (p<0.05 vs T0).
The difference between the two treatments is significant where p<0.05 and in favour of the cream
containing 2.5% MEDIATONE™.
In order to provide a visual representation of the spectrophotometry results obtained, the mean colour
obtained for the pigmented zone before and after treatment is shown below (Figure 23). An example
showing the mean observed for the whole panel, together with an individual example are provided.
- 34 -

MEDIATONE™
Volunteer 24 Mean
Figure 23: Representation of the mean colour in hyperpigmented zones.
2.2.2. Evaluation by colorimetric analysis on photographs/sub-Saharan-type skin

®
For the study on sub-Saharan-type skin, standardised photographs were taken using a Visia -CR (Figure
24), in crossed polarised light at a 45° angle. Each photograph was then analysed using a specific
software. As described above, parameters L* and ITA° were analysed at T1 month and T2 months.
®
Figure 24: Visia -CR
- 35 -

MEDIATONE™
Table 14: Variation of ITA° parameter after 1 and 2 months of applying

4% MEDIATONE™, or its placebo, to the face;
(n= 23 volunteers).
4% MEDIATONE™ Placebo
T1 T2
T0 T1 month T2 months T0
month months
Mean -23.35 ± -17.24 ± -16.28 ± -21.34 ± -19.06 ± -20.60 ±
± SD 19.74 21.47 19.77 20.30 20.82 20.68
Variation vs T0 (%) 26.2% 30.3% 10.7% 3.5%
Significance vs T0 p<0.01 p<0.01 p<0.05 nsd

Responders 70% 74%
Significance (Tx-T0) vs
p<0.03 p<0.01
placebo
35
30.3
30
26.2 **$$
25
**$
Variation (%)
20
Placebo
15 MEDIATONE™
10.7
10
*
5 3.5
0
T1 month T2 months
$($): Significant variation relative to placebo with p<0.05 (p<0.01).
Analysis of the results for parameter ITA° reveals brightening in the facial zone of sub-Saharan African-
type volunteers after only 1 month of applying the cream containing 4% MEDIATONE™ compared to the
zone receiving the placebo cream.
At 1 month, an increase in parameter ITA° is observed corresponding to +26.2% (p<0.01 vs T0) whereas,
during the same period, application of the placebo cream gives rise to a smaller change corresponding to
+10.7% (p<0.05 vs T0).
At 2 months, a greater increase in parameter ITA° is observed, corresponding to +30.3% (p<0.01 vs T0)
whereas no change is observed for placebo (+3.5%, nsd vs T0).
The difference between the two treatments is significant at 1 month (p<0.03) and 2 months (p<0.01) in
favour of the cream containing 4% MEDIATONE™.
- 36 -

MEDIATONE™
Table 15: Variation of L* parameter after 1 and 2 months of applying 4% MEDIATONE™

or its placebo, to the face;
(n= 23 volunteers).
4% MEDIATONE™ Placebo
T1 T2
T0 T1 month T2 months T0
month months
Mean 43.48 ± 45.21 ± 45.48 ± 44.13 ± 44.74 ± 44.26 ±
± SD 5.68 6.12 5.65 5.82 5.98 5.91
Variation vs T0 (%) 4.0% 4.6% 1.4% 0.3%
Significance vs T0 p<0.01 p<0.01 p<0.05 nsd

Responders 70% 74%
Significance (Tx-T0) vs
p<0.03 p<0.01
placebo
5 4.6
4,5
4.0
4 **$$
3,5 **$
Variation (%)
3
Placebo
2,5
MEDIATONE™
2
1.4
1,5
1
*
0,5 0.3
0
T1 month T2 months
$($): Significant variation relative to placebo with p<0.05 (p<0.01).
As for parameter ITA°, clarity parameter L* increases significantly after 1 and 2 months of applying the
cream containing 4% MEDIATONE™ compared to the zone receiving the placebo cream
Brightening in the facial zone of sub-Saharan African-type volunteers is observed corresponding to +4.0%
(p<0.01 vs T0) whereas, during the same period, application of the placebo cream gives rise to a smaller
change corresponding to +1.4% (p<0.05 vs T0).
At 2 months, a greater increase in parameter L* is observed, corresponding to +4.6% (p<0.01 vs T0)

whereas no change is observed for placebo (+0.3%, nsd vs T0).
The difference between the 2 treatments is significant at 1 month (p<0.03) and 2 months (p<0.01) in favour
of the cream containing 4% MEDIATONE™.
- 37 -

MEDIATONE™
An example of the change obtained for a volunteer following application of a cream containing 4%
MEDIATONE™ is shown below.
T0 Volunteer 22 T1 month
- 38 -

MEDIATONE™
3. CONCLUSION
A light complexion blemish-free skin is for many consumers a hint of youth and highly sought beauty. If in
Asia, this research is done through highly sophisticated products, in Africa, the use of aggressive
cosmetics can affect the health of the skin and even the consumer.
MEDIATONE™ is a product of natural origin, perfectly harmless, obtained by the metathesis reaction. This
eco-designed method, which was awarded the Nobel Prize for Chemistry in 2005, makes it possible to
manufacture an ultra-purified 9-octadecenedioic acid.
MEDIATONE™ provides a customised approach allowing ethnic skin to have a clearer and more uniform
tone. By regulating the genetic pigmentation and controlling the course of melanin in the skin,
MEDIATONE™ acts as a skin tone mediator.
In vitro studies have shown MEDIATONE™’s action at three levels:
REGULATION OF THE GENETIC PIGMENTATION

• Regulation of tyrosinase gene transcription:
Inhibitory effect of PPAR binding on tyrosinase mRNA expression: -54%.
• Decrease of tyrosinase and melanin production:
Tyrosinase: -27%, p<0.01; Melanin: -51%, p<0.01.
CONTROL OF THE MELANOSOME TRANSFER

• Limitation of melanocyte dendrite expansion and reduction of melanosome transfer:
Mediator of melanocyte dendricity- Endothelin-1: -57%, p<0.01 vs UVB-stressed control.
Phagocytic receptor - Keratinocyte Growth Factor Receptor (KGF-R): -40%, p<0.01 vs placebo.
Phagocytic uptake into keratinocytes: -59%, p<0.01 vs UVB-stressed control.
REGULATION OF THE STRESS-INDUCED PIGMENTATION

• Neutralisation of stress conditions promoting hyperpigmentation:
Oxidative stress: ROS: -57%, p<0.01 vs H2O2-stressed control
Inflammation: PGE2: -69%, p<0.01 vs UVB-stressed control
• Regulation of keratinocyte maturation:
Expression of maturation markers: involucrin: +841%, p<0.01; loricrin: +385%, p<0.01;
ceramides: +778%, p<0.01; hyaluronic acid: +72%, p<0.01.
MEDIATONE™ acts all along the path of melanin, from the melanocyte nucleus by regulating the
transcription of the tyrosinase gene, to the surface of the skin, by reducing the pro-pigmenting conditions
via the melanocyte environment, by controlling the transfer of melanosomes.
MEDIATONE™ shows in vivo effects on dark skin (black skin) and Asian skin in the short term and long
term.
The product was tested at 4% on black skin. The global brightening (ITA°) is +6.11°, or an increase of
26.2%, and the luminosity increased by 1.73 units L* after 4 weeks for 70% of the responders. In the long
term, after 8 weeks of use, the product increased skin brightness by +7.07° and luminosity by 2.00 units.
Three quarters of the panel achieved a reduction in skin pigmentation long term.
The product was tested at 2.5% for Asian skin with clearer skin type. The global brightening (ITA°) is
+3.13°, or an increase of 16.5%, and the luminosity increased by 1.26 L* units after only 3 weeks with
approximately 90% of responders.
For both panels, the study of images reveals a significant effect on the spots particularly due to an
inflammatory hyperpigmentation.
MEDIATONE™ is in compliance with Chinese regulation for cosmetic ingredients.
Recommended use level: 2.5% to 4%.
- 39 -

MEDIATONE™
- 40 -

MEDIATONE™
4. REFERENCES
ANDO H., WEN Z.M., KIM H.Y., VALENCIA J.C., COSTIN G.E., WATABE H., YASUMOTO K., NIKI Y.,
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AOKI H., MORO O., TAGAMI H., KISHIMOTO J., “Gene expression profiling analysis of solar lentigo in
relation to immunohistochemical characteristics”. Brit. J. Dermatol., 2007, 156, p. 1214-1223.
BERGER J., MOLLER D.E., “The mechanisms of action of PPARs”, Annu. Rev. Med., 2002, 53, p. 409-
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CARDINALI G., CECCARELLI S., KOVACS D., ASPITE N., LOTTI L.V., TORRISI M.R., PICARDO M.,
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CHARDON A., CRETOIS I, HOURSEAU C., “Skin colour typology and suntanning pathways”. Int. J.
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CHEN N., HU Y., LI W.H., EISINGER M., SEIBERG M., LIN C.B., “The role of keratinocyte growth factor
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CHENG T.H., SHIH N.L., CHEN C.H., LIN H., LIU J.C., CHAO H.H., LIOU J.Y., CHEN Y.L., TSAI H.W.,
CHEN Y.S., CHENG C.F., CHEN J.J., “Role of mitogen-activated protein kinase pathway in reactive
oxygen species-mediated endothelin-1-induced beta-myosin heavy chain gene expression and
cardiomyocyte hypertrophy”, J. Biomed. Sci., 2005, 12, p. 123-133.
DENIZOT F., LANG R., “Rapid colorimetric assay for cell growth and survival. Modifications to the
tetrazolium dye procedure giving improved sensitivity and reliability”. J. Immunol. Methods., 1986, 89,
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DI-POÏ N., MICHALIK L., DESVERGNE B., WAHLI W., “Functions of peroxisome proliferator-activated
receptors (PPAR) in skin homeostasis”, Lipids, 2004, 39, p. 1093-1099.
ELIAS P.M., MENON G.K., “Structural and lipid biochemical correlates of the epidermal permeability
barrier”, Adv. Lipid Res., 1991, 24, p. 1-26.
ELIAS P.M., MENON G., WETZEL B.K., WILLIAMS J.J., “Evidence that stress to the epidermal barrier
influenced the development of pigmentation in humans”, Pigment Cell. Melanoma Res., 2009, 22, p. 420-
434.
FEINGOLD K.R., SCHMUTH M., ELIAS P.M., “The regulation of permeability barrier homeostasis”, J.
Invest. Dermatol., 2007, 127, p. 1574-1576.
HANLEY K., JIANG Y., HE S.S., FRIEDMAN M., ELIAS P.M., BIKLE D.D., WILLIAMS M.L., FEINGOLD
K.R., “Keratinocyte differentiation is stimulated by activators of the nuclear hormone receptor PPARalpha”,
J.Invest. Dermatol., 1998, 110, p. 368-375.
HYTER S., COLEMAN D.J., GANGULI-INDRA G., MERRILL G.F., MA S., YANAGISAWA M., INDRA
A.K., “Endothelin-1 is a transcriptional target of p53 in epidermal keratinocytes and regulates ultraviolet-
induced melanocyte homeostasis”. Pigment Cell Melanoma Res., 2013, 26, p. 247-258.
IMOKAWA G., MIYAGISHI M., YADA Y., “Endothelin-1 as a new melanogen: coordinated expression of
its gene and the tyrosinase gene in UVB-exposed human epidermis”, J. Invest. Dermatol., 1995, 105,
p. 32-37.
JOYEUX A., BALAGUER P., GERMAIN P., BOUSSIOUX A.M., PONS M., NICOLAS J.C., “Engineered
cell lines as a tool for monitoring biological activity of hormone analogs”, Anal. Biochem., 1997, 249,
p. 119-130.
LABARCA C., PAIGEN K., “A simple, rapid, and sensitive DNA assay procedure”, Anal. Biochem., 1980,
102, p. 344-352.
LEE J.S., CHOI Y.M., KANG H.Y., “PPAR-gamma agonist, ciglitazone, increases pigmentation and
migration of human melanocytes”, Exp. Dermatol., 2007, 16, p. 118-123.
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MEDIATONE™
LEE D.J., LEE J., HA J., PARK K.C., ORTONNE J.P. KANG H.Y., “Deffective barrier function in melasma
skin”, J. Eur. Acad. Dermatol. Venerol., 2012, 26, p. 1533-1537.
LIN C.B., HU Y., ROSSETTI D., CHEN N., DAVID C., SLOMINSKI A., EIBERG M., “Immuno-
histochemical evaluation of solar lentigines: The association of KGF/KGFR and other factors with lesion
development“. J. Dermatol. Sci., 2010, 59, p. 91-97.
LIU J., MAN W.Y., LV C.Z., SONG S.P., SHI Y.J., ELIAS P.M., MAN M.Q. “Epidermal permeability barrier
recovery is delayed in vitiligo-involved sites”, Skin Pharmacol. Physiol., 2010, 23, p. 193-200.
MAN M.Q., CHOI E.H., SCHMUTH M., CRUMRINE D., UCHIDA Y., ELIAS P.M., HOLLERAN W.M.,
FEINGOLD K.R., “Basis for improved permeability barrier homeostasis induced by PPAR and LXR
activators: liposensors stimulate lipid synthesis, lamellar body secretion, and post-secretory lipid
processing”, J. Invest. Dermatol., 2006, 126, p. 386-392.
MAN M.Q., BARISH G.D., SCHMUTH M., CRUMRINE D., BARAK Y., CHANG S., JIANG Y., EVANS
R.M., ELIAS P.M., FEINGOLD K.R., “Deficiency of PPARβ/δ in the epidermis results in defective
cutaneous permeability barrier homeostasis and increased inflammation”, J. Invest. Dermatol., 2008, 128,
p. 370-377.
MAN M.Q., LIN T.K., SANTIAGO J.L., CELLI A., ZHONG L., HUANG Z.M., ROELANDT T., HUPE M.,
SUNDBERG J.P., SILVA K.A., CRUMRINE D., MARTIN-EZQUERRA G., TRULLAS C., SUN R.,
WAKEFIELD J.S., WEI M.L., FEINGOLD K.R., MAURO T.M., ELIAS P.M., “Basis for enhanced barrier
function of pigmented skin”, J. Invest. Dermatol., 2014, 134, p. 2399-2407.
MERINVILLE E., BYRNE A.J., VISDAL-JOHNSEN L., BOUVRY G., GILLBRO J.M., RAWLINGS A.V.,
LALOEUF A., “Clinical evaluation of a dioic acid based formulation on facial skin in an Indian population”,
Int. J. Cosmet. Sci., 2012, 34, p. 575-581.
MICHALIK L., WAHLI W., “Peroxisome proliferator-activated receptors (PPARs) in skin health, repair and
disease”, Biochem. Biophys. Acta, 2007, 1771, p. 991-998.
MORRIS D., (1967) “Le singe nu”.
MÖSSNER R., SCHULZ U., KRÜGER U., MIDDEL P., SCHINNER S., FÜZESI L., REICH K., “Agonists of
peroxisome proliferator-activated receptor gamma inhibit cell growth in malignant melanoma”, J .Invest.
Dermatol., 2002, 119, p. 576-582.
PLACHA W., GIL D., DEMBIŃSKA-KIEĆ A., LAIDLER P., “The effect of PPARgamma ligands on the
proliferation and apoptosis of human melanoma cells”, Melanoma Res., 2003, 13, p.447-456.
ROSENKRANZ A.R., SCHMALDIENST S., STUHLMEIER K.M., CHEN W., KNAPP W., ZLABINGER
G.J., “A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate”,
J. Immunol. Methods, 1992, 156, p. 39-45.
SCHERDIN U., BÜRGER A., BIELFELDT S., FILBRY A., WEBER T., SCHÖLERMANN A., WIGGER-
ALBERTI W., RIPPKE F., WILHELM K.P., “Skin-lightening effects of a new face care product in patients
with melasma”, J. Cosmet. Dermatol., 2008, 7, p. 68-75.
SCOTT G., LEOPARDI S., PRINTUP S., MALHI N., SEIBERG M., LAPOINT R., “Proteinase-activated
receptor-2 stimulates prostaglandin production in keratinocytes: analysis of prostaglandin receptors on
human melanocytes and effects of PGE2 and PGF2alpha on melanocyte dendricity”, J. Invest. Dermatol.,
2004, 122, p. 1214-1224.
SEIBERG M., PAINE C., SHARLOW E., ANDRADE-GORDON P., COSTANZO M., EISINGER M.,
SHAPIRO S.S., “Inhibition of melanosome transfer results in skin lightening”, J. Invest. Dermatol., 2000,
115, p. 162-167.
STARNER R.J., MCCLELLAND L., ABDEL-MALEK Z., FRICKE A., SCOTT G., “PGE(2) is a UVR-
inducible autocrine factor for human melanocytes that stimulates tyrosinase activation”, Exp. Dermatol.
2010, 19, p. 682-684.
SUBBARAMAIAH K., LIN D.T., HART J.C., DANNENBERG A.J., “Peroxisome proliferator-activated
receptor gamma ligands suppress the transcriptional activation of cyclooxygenase-2. Evidence for
involvement of activator protein-1 and CREB-binding protein/p300“, J. Biol. Chem., 2001, 276, p. 12440-
12448.
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MEDIATONE™
THIRION L., PIÉRARD-FRANCHIMONT C., PIÉRARD G.E., “Whitening effect of a dermocosmetic

formulation: a randomized double-blind controlled study on melasma”, Int. J. Cosmet. Sci., 2006, 28,
p. 263-267.
TIRADO-SANCHEZ A., SANTAMARIA-ROMAN A., PONCE-OLIVERA R.M., “Efficacy of dioic acid

compared with hydroquinone in the treatment of melisma”, Int. J. Dermatol., 2009, 48, p. 893-895.
VALENCIA A., KOCHEVAR I.E., “Nox1-based NADPH oxidase is the major source of UVA-induced
reactive oxygen species in human keratinocytes”, J. Invest. Dermatol., 2008, 128, p. 214-222.
WIECHERS J.W., GROENHOF F.J., WORTEL V.A.L., HINDLE N.A., MILLER R.M., “Efficacy studies
using octadecenedioic acid, a new nature-derived ingredient to even Asian skin tone”, SÖFW, 2002, 128,
p. 2-8.
WIECHERS J.W., RAWLINGS A.V., GARCIA C., CHESNE C., BALAGUER P., NICOLAS J.C., CORRE
S., GALIBERT M-D., “A new mechanism of action for skin whitening agents: binding to the peroxisome
proliferator-activated receptor”, Int. J. Cosmet. Sci., 2005, 27, p. 123-132.
YAN Y., FURUMURA M., NUMATA S., TEYE K., KARASHIMA T., OHYAMA B., TANIDA N.,
HASHIMOTO T., “Various peroxisome proliferator-activated receptor (PPAR)-γ agonists differently induce
differentiation of cultured human keratinocytes”, Exp. Dermatol., 2015, 24, p. 62-65.
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tyrosinase”, Eur. J. Biochem., 1991, 198, p. 317-326.
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MEDIATONE™
- 44 -

MEDIATONE™
5. APPENDICES
5.1. Formula of products used for in vivo evaluation “Asian skin”
Product (%) Product (%)

Raw material INCI Name Suppliers
Placebo Active
Phase A
H2 O Water qs 100 qs 100
Carbopol Ultrez 10 Carbomer 0.30 0.30
Phase B
Brij S2-SS-(RB) Steareth-2 CRODA 0.40 0.40
Brij S10-SO-(RB) Steareth-10 CRODA 1.20 1.20
Crodafos CES-PA-(RB) Cetearyl Alcohol (and) CRODA 4.00 4.00
Dicetyl Phosphate
(and) Ceteth-10
Phosphate
Laurocapram Laurocapram 2.50 2.50
Cyclopentasiloxane (and) Cyclopentasiloxane 2.00 2.00
Cyclohexasiloxane (and)
Cyclohexasiloxane
Crodamol OSU-LQ-(JP) Diethylhexyl Succinate CRODA 4.00 4.00
Crodamol AB-LQ-(RB) C12-15 Alkyl CRODA 3.00 3.00
Benzoate
MEDIATONE™ - SEDERMA - 2.50
H2 O Water 2.50 -
Phase C
Glycerin Glycerin 4.00 4.00
Octanediol Caprylyl Glycol 0.50 0.50
Phase D
Phenoxyethanol Phenoxyethanol qs qs
Phase E
Potassium Sorbate Potassium Sorbate qs qs
Phase F
H2 O Water 4.00 4.00
NaOH 30% Sodium Hydroxide 0.40 0.40
Phase G
Perfume Fragrance Expressions Parfumées 0.10 0.10
Operating procedure: (Laboratory preparation)

Step 1. Weigh phase A and let it swell without agitation for 30 min.
Step 2. Heat phase A to 85°C in a water bath.
Step 3. Heat phase B to 85°C in a water bath. Mix well.
Step 4. Weigh phase C and melt it at a temperature of 45°C. Mix well.
Step 5. Put phase D into already-cooled phase C and mix.
Step 6. Transfer phase C+D to phase A under agitation in a rotor stator mixer at a speed of
500 rpm. Homogenise well.
Step 7. Add phase B into the previous phase under agitation in a rotor stator mixer at a speed of
1000 rpm.
Step 8. Add phase E extemporaneously in the previous phase under agitation in a rotor stator mixer
at a speed of 1000 rpm. Homogenise well.
Step 9. Add phase F. Homogenise well.
Step 10. Add phase G. Homogenise well.
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MEDIATONE™
5.2. Formula of products used for in vivo evaluation “Sub-saharan skin”
Product (%) Product (%)

Raw material INCI Name Suppliers
Placebo Active
Phase A
H2 O Water qs 100 qs 100
Potassium Sorbate Potassium Sorbate qs qs
Phase B
Butylene Glycol Butylene Glycol 3.00 3.00
Octanediol Caprylyl Glycol 0.50 0.50
Phase C
Phenoxyethanol Phenoxyethanol qs qs
Keltrol CG-SFT Xanthan Gum 0.60 0.60
Supercol GF Cyamopsis 0.20 0.20
Tetragonoloba (Guar)
Gum
Phase D
Brij S2-SS-(RB) Steareth-2 CRODA 1.00 1.00
Crodafos MCA-SO-(RB) Cetyl Phosphate CRODA 2.00 2.00
Stearic acid Stearic Acid 2.50 2.50
Laurocapram Laurocapram 2.00 2.00
Cyclopentasiloxane (and) Cyclopentasiloxane 3.00 3.00
Cyclohexasiloxane (and)
Cyclohexasiloxane
Arlacel 170-PA-(RB) Glyceryl Stearate (and) CRODA 3.00 3.00
PEG-100 Stearate
Crodamol GTCC-LQ-(MV) Caprylic/Capric CRODA 3.00 3.00
Triglyceride
MEDIATONE™ SEDERMA - 4.00
H2 O Water 4.00 -
Phase E
H2 O Water 4.50 4.50
NaOH 30% Sodium Hydroxide 0.90 0.90
Phase F
Perfume Fragrance Expressions Parfumées 0.10 0.10
Operating procedure: (Laboratory preparation)

Step 1. Weigh phase A.
Step 2. Weigh phase B and heat at 45°C. Homogenise well.
Step 3. Cool phase B to 25°C
Step 4. Add the ingredients of phase C one by one to phase B. Homogenise well.
Step 5. Transfer phase B+C to phase A under agitation in a rotor stator mixer at a speed of
500 rpm. Homogenise well.
Step 6. Heat phase A+B+C to 85°C in a water bath.
Step 7. Weigh phase D and heat at 85°C. Homogenise well.
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MEDIATONE™
5.3. Formulation advices with MEDIATONE™
5.3.1. Solubilisation of active ingredient
MEDIATONE™ should be melted at 85°C minimum, in a mixture of chosen excipients according to the
final product. To formulate emulsions, humectants and emollients capable of solubilising the
9-octadecenedioic acid were searched and determined.
A table of solubility of the active ingredient in emollients and humectants has been created (see Appendix 1).
The results of this study show that MEDIATONE™ is preferentially soluble in Pentylene Glycol and
Dipropylene Glycol, as well as in Arlasolve DMI™ [Dimethyl Isosorbide].
The active ingredient is soluble in polar emollients (Ester) at a lower concentration (+/- 2%).
MEDIATONE™ is not soluble in water or silicones oils or paraffin oil (Mineral Oil) or Glycerin.
5.3.2. Choice of surfactants
A series of surfactants, often associated by 2 or 3 were tested to determine the most appropriate
combinations to formulate MEDIATONE™ and propose different textures of finished products.
Nonionic and anionic surfactants were tested and the MEDIATONE™ formulates well with nonionic
surfactants.
a. Aqueous solution
MEDIATONE™ is compatible with Polysorbate 60 and Polysorbate 20 to form lotions or oil-free serums,
with a choice of compatible humectants.
Example of surfactants: 2.00% Polysorbate 20 - 2.00% Polysorbate 60.
Emollient concentration: 2.50% Dimethyl Isosorbide - 5.00% ethanol.
Humectant: 20.00%.
b. O/W emulsion
A minimum of 8-10% oily phase is needed to melt the active ingredient at 85°C with compatible emollients
to its dissolution. Steareth-based surfactants are good emulsifiers to stabilise the active in emulsion.
For example: 5.00% Steareth-21/1.00% Steareth-2.
From a placebo emulsion, by adding 2.50% of MEDIATONE™, it is recommended to increase the

concentration of surfactant already chosen or add a Steareth (eg. Steareth-20: 1.00 to 2.00%).
Nonionic surfactants derived from sugar are compatible to stabilise MEDIATONE™:

Example of surfactant: 4.00% [Sorbitan Cocoate and Sucrose Stearate].
It is possible to use anionic surfactants in combination with nonionic surfactants.

Example of surfactants:
• 2.00% Cetyl Phosphate/ 5.00% [Glyceryl Stearate and PEG-100 Stearate]/1.00%
Steareth-2/1.00% Steareth-10.
• 4.00% [Cetearyl Alcohol (and) Dicetyl Phosphate and Ceteth-10 Phosphate]/0.40%
Steareth-2/1.20% Steareth-10.
Stearic acid-based creams can be formulated by coupling it with an anionic surfactant.

Example: 2.00% PEG-40 Stearate/19.00% Stearic Acid.
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MEDIATONE™
c. W/O emulsion
The emulsions in a continuous oil phase are difficult to stabilise, because of the high polar characteristics
of MEDIATONE™. In order to stabilise W/O emulsions, non-polar emollients are favorable. However, to
solubilise the active ingredient, it is necessary to melt it into polar emollients.
The surfactant PEG-30 Dipolyhydroxystearate (Cithrol DPHS™) can be used to make emulsions with polar
oils. The Cithrol DPHS™ coupled with Polyglyceryl-3 Diisostearate and by adding the co-surfactant
Propylene Glycol Isostearate, allows the stability of these emulsions.
Examples of surfactants: 6.00% (PEG-30 Dipolyhydroxystearate)/ 2.50% Polyglyceryl-3 Diisostearate/
4.00% Propylene Glycol Isostearate.
MEDIATONE™ is not soluble in silicone oils because of its high polar characteristics, emulsions based on
silicone surfactants (very non-polar) are not recommended.
d. Anhydrous product
It is possible to formulate a anhydrous oily and clear product by choosing emollients that is compatible with
MEDIATONE™ such as Dimethyl Isosorbide and Isostearic Acid (see Appendix 1):
An example of oily stick formula is available in appendix 3.
5.3.3. Other raw materials
a. Jellifying agents
MEDIATONE™ is compatible with carbomers and polyacrylate polymers. However, in the presence of
Acrylates/C10-30 Alkyl Acrylate Crosspolymer, a pearlescent final appearance is obtained.
MEDIATONE™ is compatible with jellifying natural agents.
Examples: Xanthan Gum; Cyamopsis Tetragonoloba (Guar) Gum.
b. Suncare
MEDIATONE™ is compatible with organic and inorganic UV filters, associating them with the appropriate
emollients.
Example: 2.00% Ethylhexyl Methoxycinnamate; 2.00% Benzophenone-3;
3.00% [Titanium Dioxide and Caprylic/Capric Triglyceride and Polyhydroxystearic Acid and Stearic Acid
(and) Alumina].
5.3.4. Conclusion
MEDIATONE™ should be melted at 85°C minimum in a suitable excipient, in an oily phase or in a

humectant phase.
MEDIATONE™ is very soluble in Pentylene Glycol and Dipropylene Glycol, and also in Arlasolve DMI™
(Dimethyl Isosorbide). MEDIATONE™ is soluble in polar solvents.
MEDIATONE™ is not soluble in water, silicone oils, liquid paraffin or glycerin.
To dissolve MEDIATONE™ in the oily phase, the latter should represent about 10% of the total formula
and be composed of suitable emollients (see Appendix 1).
There must be a sufficient amount of surfactants to emulsify MEDIATONE™ properly (≈4.00%).
For aqueous solutions, the best solubilisers for MEDIATONE™ are Polysorbabe 60 and Polysorbate 20,
alone or in combination (about 3%).
The choice of jellifying agents or the preservative system does not interfere with the solubility of
MEDIATONE™.
Appendix 1: Examples of MEDIATONE™’s solubilisers.
Appendix 2: Examples of surfactants compatible with MEDIATONE™.
Appendix 3: Examples of finished products’ formula.
- 48 -

MEDIATONE™
5.3.5. Appendix 1: Examples of MEDIATONE™ solubilisers
Purpose: To define the most compatible excipients to formulate the active ingredient at the recommended
dose in cosmetic finished products.
Protocol
MEDIATONE™ is melted at 85°C in the excipient and then cooled at room temperature.
The maximum concentration of MEDIATONE™ that allows to maintain a clear appearance at t=1 day at
room temperature is indicated in the table below.
Table of solubility
Excipient INCI Name MEDIATONE™

Max. concentration (%)
Pentylene Glycol Pentylene Glycol 10
Dipropylene Glycol Dipropylene Glycol 10
Arlasolve DMI-LQ-(MH) Dimethyl Isosorbide <10
Ethanol 96° Alcohol >4
Butylene Glycol Butylene Glycol <4
Prisorine 3505-LQ-(GD) Isostearic Acid <4
Zemea Propanediol <4
Eutanol G Octyldodecanol <4
Arlamol PS15E-LQ-(RB) PPG-15 Stearyl Ether 2
Prisorine 3515-LQ-(GD) Isostearyl Alcohol 2
Cithrol PGMIS-LQ-(GD) Propylene Glycol Isostearate 2
Cithrol GMIS40-LQ-(MV) Glyceryl Isostearate 2
Cithrol PG32IS-LQ-(MV) Polyglyceryl-3 Diisostearate 2
Crodamol PC-LQ-(MV) Propylene Glycol Dicaprylate/Dicaprate 2
Crodamol OC-LQ-(MV) Ethylhexylcocoate 2
Crodamol GTCC-LQ-(MV) Caprylic/Capric Triglyceride 2
Crodamol ISIS-LQ-(MV) Isostearyl Isostearate 2
Crodamol TN-[EU]-LQ-(JP) Isotridecyl Isononanoate 2
Crodamol AB-LQ-(RB) C12-15 Alkyl Benzoate 2
Crodamol OSU-LQ-(JP) Diethylhexyl Succinate 2
Crodamol CSO-LQ-(LK) Cetearyl Ethylhexanoate 2
Crodamol IPM-LQ-(MV) Isopropyl Myristate 2
Crodamol DA-LQ-(RB) Diisopropyl Adipate 2
Crodamol OP-LQ-(RB) Ethylhexyl Palmitate 2
Crodamol STS-LQ-(MH) PPG-3 Benzyl Ether Myristate 2
Crodamol OHS-LQ-(RB) Ethylhexyl Hydroxystearate 2
Procas H3-LQ-(RB) PPG-3 Hydrogenated Castor Oil 2
Crodamol SFX-LQ-(MH) PPG-3 Benzyl Ether Ethylhexanoate <2
Crodamol IPIS-LQ-(MV) Isopropyl Isostearate <2
Crodamol GTIS-LQ-(MV) Triisostearin <2
Arlamol HD-LQ-(RB) Isohexadecane <2
Crodamol GTEH-LQ-(MV) Triethylhexanoin <2
Cromollient DP3-LQ-(MH) Di-PPG-3 Myristyl Ether Adipate <2
BRB CM 56 Cyclopentasiloxane (and) Cyclohexasiloxane <1
Paraffin oil Mineral Oil <1

Pripure 3759-LG-(GD) Squalane <1
Glycerin Glycerin <1
- 49 -

MEDIATONE™
5.3.6. Appendix 2: Examples of surfactants compatible with MEDIATONE™
Product HLB INCI Name Function

Citrhol DPHS-SO-(MV) 1.50 PEG-30 Dipolyhydroxystearate Nonionic surfactant
Cithrol PGMIS-LQ-(GD) 1.50 Propylene Glycol Isostearate Nonionic co-surfactant
Cithrol PG32IS-LQ-(MV) 2.00 Polyglyceryl-3 Diisostearate Nonionic surfactant
Arlacel 1689-LQ-(MV) 3.50 Sorbitan Oleate (and) Polyglyceryl-3 Nonionic surfactant
Polyricinoleate
Arlacel 986-SO-(MV) 4.50 Sorbitan Isostearate (and) Hydrogenated Nonionic surfactant
Castor Oil (and) Cera Alba (and) Stearic
Acid
Crodafos CES-PA-(RB) NC Cetearyl Alcohol (and) Dicetyl Phosphate Anionic surfactant
(and) Ceteth-10 Phosphate
Crodafos MCA-SO-(RB) NC Cetyl Phosphate Anionic surfactant
Crodex A-PA-(RB) NC Cetearyl Alcohol (and) Sodium Lauryl Anionic surfactant
Sulfate
Stearic acid NC Stearic Acid Anionic surfactant
Arlacel 170-PA-(RB) 4.40 Glyceryl Stearate (and) PEG-100 Stearate Nonionic surfactant weakly
irritant
Brij S2-SS-(RB) 4.90 Steareth-2 Nonionic surfactant
Brij S10-SO-(RB) 12.40 Steareth-10 Nonionic surfactant
Brij S721-PA-(SG) 15.50 Steareth-21 Nonionic surfactant
Arlacel 2121-FL-(MV) 6.00 Sorbitan Stearate (and) Sucrose Cocoate Nonionic surfactant
Crodacol CS90-PA-(RB) NC Cetearyl Alcohol Nonionic co-surfactant
Polawax NF-PA-(RB) NC Cetearyl Alcohol (and) Polysorbate 60 Nonionic surfactant
Tween 60-LQ-(MV) 14.90 Polysorbate 60 Nonionic surfactant
Tween 20-LQ-(MV) 16.70 Polysorbate 20 Nonionic surfactant
Myrj S40-PA-(RB) 16.70 PEG-40 Stearate Nonionic surfactant
- 50 -

MEDIATONE™
5.3.7. Appendix 3: Examples of finished products’ formula
SKIN TONE MEDIATOR LOTION WITH MEDIATONE™

Product Suppliers % by wt
Phase A
Water deionised - qs 100
Volarest FL-LQ-(RB) [Acrylates/Beheneth-25 Methacrylate Croda 1.00
Copolymer]
Phase B
Pentylene Glycol - 5.00
Phenoxyethanol - qs
Xanthan Gum - 0.40
Phase C
Brij S721-PA-(SG) [Steareth-21] Croda 5.00
Brij S2-MBAL-SS-(RB) [Steareth-2] Croda 1.00
Crodamol W-SS-(LQ) [Stearyl Heptanoate (and) Stearyl Caprylate] Croda 1.00
Octyldodecanol - 1.50
Crodamol STS-LQ-(MH) [PPG-3 Benzyl Ether Myristate] Croda 1.00
Arlamol HD-LQ-(RB) [Isohexadecane] Croda 1.50
Crodamol AB-LQ-(RB) [C12-15 Alkyl Benzoate] Croda 5.00
Prisorine 3505-LQ-(GD) [Isostearic Acid] Croda 2.00
Phase D
Mediatone™ [Octadecenedioic Acid] Sederma 2.,50
Phase E
Potassium Sorbate - qs
Phase F
H2 O - 1.30
NaOH 30% - 0.13
Phase G
Fragrance (women anti-age) - 0.10
ANTI-DARK SPOTS OILY STICK WITH MEDIATONE™

Part A
Crodamol AB-LQ-(RB) [C12-15 Alkyl Benzoate] Croda qs 100
Oleocraft LP-20-PA (MV) [Polyamide-8] Croda 28.00
Tween 60-LQ-(MV) [Polysorbate 60] Croda 4.00
Arlasolve DMI-LQ-(MH) [Diimethyl Isosorbide] Croda 5.00
Mediatone™ [Octadecenedioic Acid] Sederma 2.50
Part B
Phenoxyethanol - qs
Fragrance (Lotus) Expressions Parfumées 0.20
- 51 -

MEDIATONE™
ECONOMIC BRIGHTENING DAY CREAM WITH MEDIATONE™

Part A
Water deionised - qs 100
Part B
Crodex A-PA-(RB) [Cetearyl Alcohol (and) Sodium Lauryl Sulfate] Croda 7.00
Crodacol CS90-PA-(RB) [Cetearyl Alcohol] Croda 2.00
Mineral Oil - 5.00
Part C
Glycerin - 8.00
Phenoxyethanol - qs
Part D
Fragrance (Volupté) Expressions Parfumées 0.20
BRIGHTENING AND ANTI-AGEING CREAM WITH MEDIATONE™ AND MEIRITAGE™

Part A
Water deionised
Carbomer - qs 100
Part B - 0.25
Arlacel 2121-FL-(MV) [Sorbitan Stearate (and) Sucrose Cocoate] Croda 3.50
Part C
Pentylene Glycol - 5.00
Phenoxyethanol - qs
Part D
Crodamol IPIS-LQ-(MV) [Isopropyl Isostearate] Croda 2.00
Crodamol ISIS-LQ-(MV) [Isostearyl Isostearate] Croda 4.00
Crodamol AB-LQ-(RB) [C12-15 Alkyl Benzoate] Croda 3.00
Part E
Part F
Water deionised - 2.00
NaOH 30% - 0.20
Part G
Meiritage™ [Glycerin (and) Astragalus Membranaceus Root Extract Sederma 3.00
(and) Atractyloides Macrocephalia Root Extract (and) Bupleurum
Falcatum Root Extract]
Part H
Fragrance (Lotus) Expressions Parfumées 0.10
- 52 -

SEDERMA SAS
29 rue du Chemin Vert
F-78612 Le Perray en Yvelines cedex
tel ++ 33 1 34 84 10 10 fax ++ 33 1 34 84 11 30
sederma@sederma.fr www.sederma.com
SEDERMA Inc
300 Columbus Circle
Edison NJ 08837 USA
tel ++ 1 (732) 692 1652 fax ++ 1 (732) 417 0804
sederma-usa@croda.com www.sederma.com
SEDERMA GmbH
Herrenpfad-Süd 33
41334 Nettetal Germany
tel ++ 49 21 57 817318 fax ++ 49 21 57 817361
sederma@sederma.de www.sederma.com
Non-warranty: The information in this publication is believed to be accurate and is given

in good faith, but no representation or warranty as to its completeness or accuracy is
made. Suggestions for uses or applications are only opinions. Users are responsible for
determining the suitability of these products for their own particular purpose. No
representation or warranty, expressed or implied, is made with respect to information or
products including, without limitation, warranties of merchantability, fitness for a particular
purpose, non-infringement of any third party patent or other intellectual property rights
including, without limit, copyright, trademark and designs. Any trademarks identified
herein, unless otherwise noted, are trademarks of the Croda group of companies.
©2018 Sederma
03/18 PCS TB 014 V2 EN

Sederma TB Mediatone

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MEDIATONE™

Part of Croda International Plc, © 2018 Sederma, All rights reserved.

INCI Name Octadecenedioic Acid.

Proven cosmetic activity

• Melanin synthesis (NHM*) ................................................................................. -51% (p<0.01)

Melanin diffusion: melanosome phagocytosis

• ± UVB induction (NHK*) ..................................................................... -53 / -59% (both: p<0.01)

• PGE2, pro-pigmenting lipid, UVB induction (NHK) ..............................................-69% (p<0.01)

Strengthening of the skin barrier

• Keratinocyte differentiation (visual effect) ............................................................. Improvement

Hyaluronic acid synthesis

• Hyaluronic acid (HK) ........................................................................................... +72% (p<0.01)

Evaluation on Asian skin (Spincontrol)

• ITA° after 3 weeks ................................................................ +16.5% vs T0 (p<0.05 vs placebo)

Evaluation on sub-Saharan African skin (Spincontrol)

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• Recommended pH: 3.50 - 8.00

Toxicology: Patch test

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SUMMARY OF THE TRADEMARK AND PATENT SITUATION OF MEDIATONE™

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2. EFFICACY STUDIES .......................................................................................................... 17

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Pigmentation, general information

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Melanin is a natural polymer produced in the melanocyte at the end

Once formed in the melanosome, melanin is

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FACTORS AMPLIFYING PIGMENTATION

Endothelin-1 (Figure 4) is an oligopeptide, produced by keratinocytes, which

PGE2 (Figure 5) are complex lipids resulting from the

ACQUIRED HYPER-PIGMENTED LESIONS

A number of authors also highlight the existence of a micro-inflammatory environment, overproduction of

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Melasma is a fairly common hyperpigmentation disorder in women,

Conventional treatments involve hydroquinone, tretinoin,

Pigmentation and barrier function, role of PPARs

The three types of PPARs (identified as

These encourage keratinocyte

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Barrier function renewal

Figure 8: Structure of the stratum corneum.

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THE SEDERMA CONCEPT

is a modified technical cosmetic active ingredient, based on natural

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Figure 10: Helianthus annuus, seeds and flowers.

Figure 11: Effect of MEDIATONE™ on the formation of tyrosinase mRNA.

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2.1. In vitro studies

2.1.1. Pigmentation control

a. Decrease of melanin production and tyrosinase activity

Control MEDIATONE™ 150 ppm

Figure 13: Modulation of melanin production in NHM.

Table 1: Melanin production by NHM, effect of MEDIATONE™ (n=4).

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Table 2: Tyrosinase activity of NHM, effect of MEDIATONE™ (n=4).

MEDIATONE™ 30 ppm 48.24 ± 4.45 -18%; p<0.05

MEDIATONE™ 100 ppm 42.87 ± 1.96 -27%; p<0.01

b. Variation of tyrosinase mRNA expression

• Facial photographs by • Facial photographs • Facial photographs by

ITA° = Arctg [(L-50)/b]x(180/π)

Figure 22: CM700d spectrophotometer, colorimetric range Lab*, parameter ITA°.