belides - drskinspa derma/belides-daisy-flower.pdf · epidermis gets thicker, and production of...

1for fine cosmetics · since 1926

Belides™

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Contents

Dosage: 2.0–5.0%

Recommended pH: 4.5–6.5%INCI Name: Bellis Perennis (Daisy) Flower Extract

Skin-lightening agent derived from daisy flowers (Bellis perennis). Provides even pigmentation against age spots, pigmentation disorders and freckles, as well as general skin-lightening activity. Belides™ influences biochemical pathways involved in melanin synthesis by: inhibition of tyrosinase, transcriptional control of tyrosinase expression, reduction of the pro-melano-genic mediators endothelin, and α-MSH (melanin-stim-ulating hormone) as well as reduction of melanosome transfer to keratinocytes. Patent pending. Belides™ is stabilized by a natural preservation system and does not contain any preservatives in the sense of EU Cosmetic Directive 76/768 EEC, Annex VI, Part 1.

Belides™ NP

Introduction 3

Melanogenesis 4 – 5

Activity 6

Efficacy Studies 7 – 11

Application 12 – 13

Bibliography 14

Company Profile 15

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Sun-bathing, tanning booths, and self-tanning lotions – tanned skin stands for beauty in Europe and North America. Exactly the opposite applies to Asia. Asian people love aristocratic paleness. They prefer pale skin over their own yellow-brownish complexion and detest freckles. In former ages, bright skin often was regarded as beautiful and associated with gentility; this was also due to the fact that only someone who did not have to do outside work could maintain fair skin. In order to maintain fair skin, often powder and creams with bright pigments were applied. To this day, white skin signifies noblesse, prosperity, and feminity.Therefore there is great demand for skin-lightening cosmetic products with significant, immediate, and long-lasting effects. In former times, also skin lighteners were applied, that were harmful to health, such as mercury. In the meantime, however, health consciousness has grown considerably, and demand for natural, non-dama-ging skin lightening is expanding. In other parts of the world more and more skin-lightening actives are used, e.g., they are incorporated into anti-aging products in order to prevent age spots or to lighten up existing ones. In our modern competitve world, an even and youthful skin is becoming more and more important, therefore nowadays most creams for everyday skin care include lightening ingredients.

Introduction

CLR Product Groups

Anti-Aging

Moisturizer

Preventive Care

Sensitive & Problem Skin

Hair Care

Botanical Oils

Belides™

CLR‘s active Belides™ represents a unique principle of action which enables significant lightening of the skin in a gentle and efficient way; Belides™ counteracts age spots and balances hyperpigmentation. This product is produced from blossoms of wild-collected daisy flowers, which are submitted to careful extraction and fraction-ation. Belides™ is stabilized by a natural preservation system and does not contain any preservatives in the sense of EU Cosmetic Directive 76/768 EEC, Annex VI, Part 1.

The daisy derived its English name from the Anglo-Saxon term daes eage or day‘s eye, referring to the way this flower opens and closes with the sun. The Latin name bellis perennis is derived from bellus = “beautiful” and perennis = “lasting”; today the daisy flower is one of the most popular domestic plants.

According to Roman mythological legend, the nymph Belides, as she danced with the other nymphs at the edge of the forest, caught the eye of Vertumnus, the god of the orchards. To escape his unwanted attention, she transformed herself into the flower bellis, which is the daisy‘s botanical name. Generally speaking, daisies indicate innocence, purity, and gentleness.

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The Melanogenesis

When skin is exposed to UV rays for a longer period of time, it reacts with increased cell proliferation; the epidermis gets thicker, and production of melanin, the metabolic product of melanocytes, is increased. Apart from keratinocytes, the basal layer also contains a small number of melanocytes. The proportion of keratinocytes to melanocytes is approx. 10:1; this ratio, however, may vary considerably with respect to skin area and one`s ethnic skin type.

Tyrosinase is an enzyme that is activated by UV irradia-tion. It is responsible for the oxidative transformation of the amino acid tyrosine first into DOPA (dihydroxy-phenylalanine) and subsequently into melanin. Melanocytes are dendritic cells which transfer melanin via their dendrites to the above-located keratinocytes, where it is aggregated and deposited. The formation of new pigments is predominantly initiated by tyrosine and

UVB irradiation. This process takes several days, and the tanning effect produced lasts several weeks.Direct pigmentation is another possibility of melanogen-esis. During this process, which is initiated by UV light, the colorless melanin precursors are oxidated into dark-colored melanin by UVA irradiation. This process is reversible and therefore results only in a tan of short duration, vanishing quickly if exposure to UV light is reduced.Discrepancies among differently pigmented skin types can be explained by variable production, number, size, and composition, as well as distribution and decomposi-tion of the melanin-containing melanosomes.There are two melanin variations in humans. One, named eumelanin, is darkish brown. The second melanin variation, pheomelanin, is brighter and of yellow-reddish color. When skin is exposed to sunlight, the sulfur-containing pheomelanin is more likely to sensitize than

5

protect the skin. Irrespective of one`s ethnic skin type, the concentration of melanocytes per surface unit is the same (13% of the cell population in the epidermis). Without being stimulated by UV light, melanocytes in light-skinned humans show only slow action and produce small melanosomes (400 nm), which are arranged in small groups within the keratinocytes. These melanosomes filled with pheomelanin, which is the dominant type of melanin, are decomposed before they reach the stratum corneum.In contrast, melanocytes of dark-skinned humans con-stantly produce large melanosomes (800 nm) irrespec-tive of the influence of the sun. These melanosomes are distributed singularly within the cytoplasm and filled with eumelanin. These melanosomes virtually never decompose and occur in the complete epidermis and stratum corneum, which they reach in an intact state.Yellow-toned skin contains a mixture of small and large melanosomes.Hyperpigmentation, such as age spots or freckles, results from surplus production of melanin. UV light even intensifies this process. Furthermore, decomposi-tion of melanosomes in hyperpigmented areas is dis-turbed or completely interrupted. This explains why age spots do not disappear of their own accord.

A common way of down-regulating melanogenesis during synthesis consists in reducing the enzyme activity of tyrosinase. Tyrosinase catalyzes two oxidative steps in melanin synthesis. Depigmentation can be induced after melanin synthesis is finished by inhibiting the transfer of mature melanosomes to keratinocytes. Several studies have focused on the interaction between melanocytes and keratinocytes during the transfer process. The most common way to achieve lightening of naturally pigmented skin is to reduce efficiency of melanin synthesis, e.g., by using tyrosinase inhibitors and antioxidants. These different target fields of application illustrate the advantage of a whitening active which produces a cumulative effect by acting on different pigmentation pathways.We report on in vitro and in vivo test results obtained with our skin-lightening product Belides™, demon-strating the efficacy of skin-lightening activity and the mechanisms of action.

6

Activity

Belides™ interferes with different stages of melanogen-esis, i.e., prior, during, and after melanin formation. When the skin is exposed to UV light, the expression of various mediators is stimulated. Among these mediators are endothelin (ET-1) and the melanin-stimulating hormone and neuropeptide α-MSH, which are released, for example, by keratinocytes. The mediators bind to special receptors of the melanocytes and induce tyrosi-nase synthesis in the cells. Tyrosinase oxidizes the elemental amino acid tyrosine into melanin (Fig.1). The biochemical melanin synthesis takes place in the mela-nosomes and starts from tyrosine, which is transported by the blood flow. A sequence of oxidation processes involving tyrosine and controlled by tyrosinase results in the formation of DOPA, subsequently into an interme-diate substance (dopaquinone) and finally to melanin. Then, the melanosomes filled with melanin are trans-ferred by the melanocyte dendrites to the keratinocytes, which absorb the melanosomes and obtain their color.

Before melanogenesis starts, Belides™ inhibits ET-1 expression in the keratinocytes and blocks the receptor MC-1 (melanocortin receptor-1) at the melanocytes, the docking place for α-MSH. Furthermore, Belides™ decreases tyrosinase synthesis and reduces tyrosinase activity during melanin formation, thus reducing mel-anin production in the melanosomes. After mature melanosomes have been transferred to the

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keratinocytes via dendrites and after melanin synthesis is finished, Belides™ reduces the uptake of melano-somes by keratinocytes that have been stimulated by UV light. This unique principle of action that Belides™ offers could be demonstrated in vitro, and the significant fast skin-lightening effect could also be observed in vivo.

Fig. 1: Biosynthesis of melanin

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Fig. 2: Reduction of melanin biosynthesis

In vitro assays

Influence on melanin biosynthesis

Depending on skin type, melanocytes produce a certain amount of melanin; melanin synthesis is stimulated and clearly intensified by UV light.The influence of Belides™ on melanin formation of melanocytes was determined. Belides™ significantly reduced the biosynthesis of melanin in non-stimulated human melanocytes. Values were related to cell number and to their appropriate controls (Fig. 2).

Influence on ET-1 expression

The influence on inhibition of ET-1 expression on keratinocytes with and without stimulation was deter-mined. Human keratinocytes were stimulated with 40 ng/ml Interleukin-1.Sunlight triggers a cascade in the human skin which also induces increased release of promelanogenetic mediators (ET-1, α-MSH). Interleukin-1 (IL-1) is involved in this cascade. Therefore, IL-1 was used to stimulate the natural reaction of the skin after exposure to UV light. Belides™ significantly reduced the expression of ET-1 in human keratinocytes, irrespective of whether or not they were stimulated with IL-1 (Fig. 3). Values were related to their appropriate controls.

The binding capacity of α-MSH on the MC-1 receptor of melanocytes was determined. Belides™ itself bound to MC-1R, therefore the Ligand–Receptor Interaction with α-MSH was decreased. Values were related to their appropriate controls.

Fig. 4: Reduction of α-MSH binding capacity

Efficacy Studies

Fig. 3: Inhibition of ET-1 expression

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Influence on the binding capacity of α-MSH on MC-1R

8

Influence on tyrosinase expression

Tyrosinase is an enzyme that is essential for the bio-chemical synthesis of melanin. The process of tyrosinase synthesis includes ribosomes, the endoplasmatic reticulum and the Golgi complex, from which they are released as microvesicles.

After cultivation of melanocytes in the presence of the active, the supernatant including Belides™ was removed before cells were lysed in order to release the produced tyrosinase. Quantification of tyrosinase was done by measuring its activity (Fig. 5).Belides™ clearly decreased tyrosinase synthesis.

Influence on tyrosinase enzymatic activity

L-Tyrosine was incubated with tyrosinase and the L-DOPA chrome formation was measured. Belides™ significantly reduced enzymatic activity (Fig. 6).

Endocytosis

After being stimulated by UV light, a keratinocyte is able to take up melanosomes. The melanocyte dendrites dock to the keratinocytes. The mature melanosomes migrate through the dendrites to the skin cells; this process is called endocytosis. After the keratinocyte has absorbed the melanosomes, the tanning of the skin becomes visible (Fig. 7). Reduction of endocytosis leads to lightening of skin tone.

Fig. 7: UV-stimulated uptake of melanosome by keratinocytes

Fig. 5: Influence on tyrosinase expression

Fig. 6: Reduction of tyrosinase activity

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9

Influence on melanosome transfer

The melanosomes produced from melanocytes are transferred to the surrounding keratinocytes via the melanocyte dendrites by a process called endocytosis. One melanocyte is able to supply 36 keratinocytes in this way.To study the influence of Belides™ on melanosome transfer, a FluoSphere® (Molecular Probes) beads model was used. For simulation of melanosome uptake, cells were stimulated with UV light to induce endocytosis. Belides™ significantly reduced the FluoSphere® uptake, compared to control cultures (100% uptake) (Fig. 8).

Furthermore, microscopic images taken from control cultures and cultures preincubated with Belides™ showed that fewer FluoSphere® beads were accumu-lated around the cell nuclei when the keratinocytes were treated with Belides™ (Fig. 9a/b).

Fig. 8: Uptake of FluoSpheres® by keratinocytes

Fig. 9: Microscopic images showing (a) the FluoSphere ® uptake (red FluoSphere ® beads, 1.0 µm size) in control and (b) in Belides™-treated keratinocytes. For visualization of the endocytosis, cells were counter-stained with DAPI (blue nuclei).

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In vivo assays

Our in vitro results demonstrate that Belides™ acts as a functional ingredient at different stages of melanogen-esis. By influencing pathways of action before, during, and after melanin synthesis, Belides™ effectively reduces skin pigmentation.For demonstration that Belides™ produces a noticeable lightening effect on human skin, two in vivo studies were carried out.

Pilot study

Initially a pilot study on 5 volunteers from the Philippines (age: 19–39; 4 females, 1 male) was conducted over a period of 4 weeks. Twice daily two formulations con-taining 2% Belides™ and 2% α-Arbutin were applied on the inner forearm.

The first study was made with the same people but in the winter without any UV stimulus. An amount of 2% α-Arbutin was used as a positive control. The skin color was determined with a chromameter after 14 days and 28 days. The application was stopped after four weeks, then the areas remained untreated for one week. After this one-week break the skin color was re-recorded with a chromameter, to determine how long the effect lasted.The effect of α-Arbutin was comparable, but the result with Belides™ lasted longer.

The study was repeated with the same people but in the summer, with a natural UV stimulus. Again twice daily two formulations containing 2 and 5% Belides™ and a placebo were applied. The lightening effect was measured with a chromameter and compared to an untreated area (Fig. 10).

After only 14 days of application, a tremendous effect could be observed. Skin lightening was significantly increased. These results show that the lightening process is complete after only 14 days and that continued appli-cation of the active does not further lighten the skin.

It could be observed that the lightening activity of Belides™ on brighter skin in the wintertime was weaker. This shows that Belides™ is the more active the darker the skin and the higher the stimulation is. This led to the conclusion that after application of a cosmetic product containing Belides™, no lighter circles will result around darker areas (e.g., age spots).

Fig. 11: Skin-lightening effect of Belides™ with natural UV stimulus

Efficacy Studies

Fig. 10: Skin-lightening effect of Belides™ without natural UV stimulus

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11

Summary

Fig. 12: Reduction of intensity of age spots after 28 and 50 days

Belides™ is obtained from daisy flower blossoms (Bellis perennis) and contains, among other ingredients, saponins, polyphenols, and polysaccharides. Belides™ interferes with different mechanisms involved in melanogenesis (patent pending).An effective way of inhibiting melanogenesis consists in influencing the mediators ET-1 and α-MSH. After UV stimulation these mediators are released by keratinocytes. It could be shown that Belides™ signifi-cantly inhibits ET-1 expression and reduces the α-MSH receptor MC-1. Thus, induction of melanin synthesis is blocked even before it starts. In this context it could also be demonstrated that transcription of tyrosinase, i.e., tyrosinase synthesis, is reduced. When melanin synthesis is finished, mature melanosomes are trans-ferred from melanocytes to keratinocytes. This transfer is clearly reduced by Belides™. The remarkable light-ening effect of Belides™ was also demonstrated in vivo on human volunteers.

Influence on age spots Age spots mostly occur in light-skinned humans aged over 40. They are caused by a surplus of melanin pro-duction and an additional decrease in decomposition of melanosomes. Furthermore, they never disappear.Formation and darkening of age spots is promoted by UV irradiation. In a study with 20 volunteers (aged 50–60) 5% Belides™ in an O/W cream was applied twice daily on the back of the hand. The initial color of the existent age spots was determined with a chromameter. After 28 and 50 days the skin color was recorded again. Values for 28 and 50 days shown in the graph are related to initial values. It is obvious that Belides™ has a significant effect in the reduction of brown color of age spots. It has to be noted, however, that the impact of Belides™ varies considerably, which is due to the fact that during the application period the volunteeres deliberately did not protect their hands against UV rays. Everyone who wants to fight formation and darkening of age spots, however, should make sure not to expose these skin sections to sunlight. Each age spots reacts to lightening treatment in a different way. Thus, it has to be taken into consideration that efficacy differs individually.

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12

*) This formula has been manufactured and stability-tested using a special preservative, but has not been subjected to microbiological challenge tests.

Application

Manufacture: Heat A and B separately to 70°C. Add A to B, then homogenize for 2 minutes. Cool down to 30°C under stirring. Add C to AB and stir to obtain a homogeneous emulsion. Adjust pH to approx. 4.0–5.0 with D. Add E to the emulsion.

Belides™ is a translucent, cognac-colored liquid with a characteristic odor. The product may form a slight deposit over time, which does not affect its bioactivity.Belides™ is clearly soluble in water at any proportion, but incompatible with alcohol. It is possible to formulate clear gels with the product.

Belides™ should be added to the formulation during the cooling process, because only short heating up to 40°C does not affect activity. The pH-value of formulations containing Belides™ should be adjusted to the range between 4.5 and 6.5.If kept at a temperature between 10°C and 20°C in well- closed containers, Belides™ is stable for at least 1 year. The product packs should be used without leftovers.

Belides™ in Cosmetic Formulations

Lightening Day Cream, Type O/W

Phase Trade Name INCI Name w/w % Supplier

A Tego Care PS Cetearyl Glucoside 4.0 Goldschmidt

Arlamol HD Isohexadecane 6.0 Uniqema

Tegosoft MM Myristyl Myristate 1.0 Goldschmidt

Tegosoft OS Octyl Stearate 6.0 Goldschmidt

Lanette O Cetearyl Alcohol 2.0 Cognis

Abil 350 Dimethicone 3.0 Goldschmidt

B Water Aqua ad 100

Glycerin Glycerin 2.0

C Phenonip* Phenoxyethanol, Methylparaben, Ethylparaben, Butylparaben, Propylparaben, Isobutylparaben

0.6 Clariant

Aerosil 200 Silica 1.0 Degussa

D Citric Acid (10%) Citric Acid q.s.

E Belides™ Bellis Perennis (Daisy) Flower Extract 5.0 CLR

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Manufacture: Heat up A and B to 85°C. Add A to B and homogenize for 3 minutes. Cool down under stirring, add C and D at room temperature.

Manufacture: Disperse the hydroxyethylcellulose in water, and add Abil B 88183. Mix B, and add it to A. Adjust the pH to approx. 8.5 with NaOH (10%) to obtain a clear gel. Then adjust the pH to approx. 4.0–5.0 with citric acid. Add D under stirring. Add E and mix to obtain a homogeneous gel.

Silky Face Cream, Type O/W


A Water Aqua ad 100


Cosmedia SP Sodium Polyacrylate 1.0 Cognis

B Emulgade PL 68/50 Cetearyl Glucoside, Cetearyl Alcohol 1.0 Cognis

Cetiol CC Dicaprylyl Carbonate 5.0 Cognis

Cetiol 868 Ethylhexyl Stearate, Octylstearate 5.0 Cognis

Myritol 312 Caprylic/Capric Triglyceride 3.0 Cognis

Copherol 1300 D-alpha Tocopherol 0.5 Cognis

Abil 350 Dimethicone 0.5 Goldschmidt

C Belides™ Bellis Perennis (Daisy) Flower Extract 5.0 CLR

D Euxyl K702* Benzoic Acid, Dehydroacetic Acid, Phenoxyethanol

0.8 Schuelke&Mayr

Skin Lightening Gel


A Water Aqua ad 100

Elfacos CD 481 Hydroxyethylcellulose 1.5 Akzo Nobel

Abil B 88183 PEG/PPG-20/6 Dimethicone 2.0 Goldschmidt

B Glycerin Glycerin 3.0

Phenonip* Phenoxyethanol, Methylparaben, Ethylparaben, Butylparaben, Propylparaben, Isobutylparaben

0.6 Clariant

C NaOH (10%) Sodium Hydroxide q.s.

Citric Acid (10%) Citric Acid q.s.

D Polyglycol 400 PEG-400 5.0 Clariant

E Belides™ Bellis Perennis (Daisy) Flower Extract 5.0 CLR

Lightening Body Cream Gel, Type O/W


A Tegosoft EE Octyloctanoate, Ethylhexyl Ethylhexanoate 5.0 Goldschmidt

Arlamol HD Isohexadecane 2.0 Uniqema

Tegosoft DEC Diethylhexyl Carbonate 5.0 Goldschmidt

Dow Corning 200 fluid 350 cst

Trisiloxane 1.0 Dow Corning

B Aristoflex AVC Ammonium Acryloyldimethyltaurate / VP Copolymer

1.2 Clariant

C Water Aqua ad 100

Edeta B Tetrasodium EDTA 0.05 BASF


Euxyl PE 9010 Phenoxyethanol, Ethylhexylglycerin 0.6 Schuelke&Mayr

D Belides™ Bellis Perennis (Daisy) Flower Extract 5.0 CLR

Manufacture: Heat up A and B to 85°C. Add A to B and homogenize for 3 minutes. Cool down under stirring, add C and D at room temperature.

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Bibliography

ß Imokawa G, Miyagishi M, Yada Y: J. Invest. Dermatol. 105 (1995) 32-37. Endothelin-1 as new melanogen: coordinated expression of its gene and the tyrosinase gene in

UVB-exposed human epidermis.

ß Peyrefitte G: Verlag Simep, Masson, Paris 1997 Biologie de la peau.

ß Seiberg M, Paine C, Sharlow E, Costanzo M, Andrade-Gordon P: J. Invest. Dermatol. 115 (2000) 162-167. Inhibition of melanosome transfer results in skin lightening.

ß Umbach W: Georg Thieme Verlag, Stuttgart 1995 Kosmetik:Entwicklung, Herstellung und Anwendung kosmetischer Mittel.

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Company Profile

Efficient actives and concepts for cosmetics

CLR Chemisches Laboratorium Dr. Kurt Richter GmbH is an independent, private company based in Berlin, Germany. We have a long tradition of manufacturing and internationally marketing cosmetic actives. Founded in 1926, we have concentrated our efforts on the manufacture of bioactive cosmetic ingredients from the early 1950s onwards, and since then have marketed various innovative products.

Our strength lies in the research and development of new trends presented with an extensive proof of effectiveness and providing comprehensive assistance in formulating personal care products. Our products are subject to high standards of quality and safety throughout the entire development process, including research, the translation of data into innovative concepts, and production.

Following these standards we have developed many high-class biological actives obtained from natural raw materials of vegetable, animal, or biotechnological origin. CLR‘s safe and state-of-the-art cosmetic actives comply with the diverse requirements of skin and hair: protection, stimulation, regeneration, soothing, moisturization, and nutrition.

DisclaimerThis brochure, in whole and in part, is copyright-protected. All rights arising therefrom are reserved. CLR™ and our product names mentioned in this

brochure, namely Belides™, are national or international trademarks. They are CLR‘s property and may be used in connection with personal care

products containing our cosmetic actives only with our written consent based on mutual agreements to be concluded.

The use of trade names, trademarks, and product names of third parties in this brochure does not necessarily permit others to use them. These

names may be protected or registered trademarks, even if not necessarily labeled as such. Any information, suggestions, and guide formulations in

this brochure are given to the best of our knowledge at the time of printing. The information provided in this brochure on the uses of our cosmetic

actives does not constitute any liability on our part. CLR does not assume any responsibility or any other legal liability if the user of our products

adopts these data and results without independent reconfirmation of the findings by his or her own tests for the intended conditions of use.

It is the entire and sole responsibility of the users of our suggestions, information, and guide formulations, as well as of the users of our cosmetic

actives, to follow any existing legal or other regulations, including specific national laws or pertinent directives, and any possibly existing patent

rights and other proprietary rights of third parties.

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Chemisches Laboratorium Dr. Kurt Richter GmbH Bennigsenstraße 25 · 12159 Berlin · GermanyP.O.Box 4104 80 · 12114 Berlin · Germany Tel +49 (0)30 8510 26-0 · Fax +49 (0)30 8510 [email protected] · www.clr-berlin.com certified by DQS according to DIN EN ISO 9001

QUALITY MANAGEMENT SYSTEM

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