Azeco Cosmeceuticals

Technical Information File Azeco Cosmeceuticals

When quality and reliability c ome first

In this document we collected the frequently asked questions from our customers.

Version 4.00

September 2023

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Index of Change

Version number Description

Date

1.00

Original Ingredient Information File

02-2020

2.00

Ingredient Information File update

04-2022

3.00

Revised text and additional information

06-2023

4.00

Change in ordering and formatting

09-2023

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Sommario Solubility 1. Question: How to improve the solubility of Azelaic acid?

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2. Question: How to dissolve Azelaic acid?

3. Question: Should we consider 10-15 % Azelaic acid in Propanediol?

4. Question: Is the solubilty of Azelaic acid in propanediol 1,3 similar to that of propylene glycol?

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5. Question: We have tried another source of Azelaic acid (not Azepur99®) and experienced some problems with crystallization for an skin prone to acne application.

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6. Question: Is Azepur99® is soluble in glycerin, 1.3-Butylene Glycol or Propanediol?

7. Question: When Azepur99® is dissolved in glycols or glycerin, what rheology modifier should be used to make a fully transparent gel of this solution? 8. Question: Except Hydroxypropylcellulose (HPC), and Sodium Polyacryloyldimethyl Taurate, can other rheology modifier be used?

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9. Question: What can be done to make the Azelaic accid penetrate even deeper into the skin?

Formulations 10. Question: We have developed an emulsion with 10% Azelaic acid, to increase the solubility of Azelaic acid, we need to increase pH of the aqueous phase before emulsification and before adding the Azelaic acid. Should the pH raised to 7 or more? To have good stability and to avoid the crystallization of Azelaic acid, what should be the optimum pH of the emulsion?

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11. Question: How to create a base formulation and add per skin type a different active

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ingredient?

12. Question: In the formulation we experienced some colouring due to oxidation. Could you recommend addition of an anti-oxidant in the formulation? 13. Question: Which combination can you recommend for Azelaic acid together with other ingredients to reinforce the whitening action: niacinamide? 14. Question: Question on the activity in the free acid or neutralized state: I’ve been pretty careful not to neutralize by post adding it to a PH of 5.5. Are there any differences in the free or salt state? 15. Question: How to formulate a peeling with 10-15 % Azelaic acid? The final product should be a liquid or a gel. 12 18. Question: We are developing a new formulation. We encountered an issue of solubility in water. Did an suggestion to use ethoxydiglycol. The condition improve a lot after the application of ethoxydiglycol. 13 19. Question: In a formulation we add Azelaic acid directly in the oil phase. The pH is between 4 and 5, is this the correct pH or should it be higher? 13 20. Question: Can a natural hydrocolloid (INCI: Carrageenan and Algin, pH 8-11), carbomer be used? 13 mulation? 10 10 11 12 16. Question: Can you recommend some formulation concepts?

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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21. Question: I premix my Azelaic acid (7%) in propanediol (15%) I get a white mixture, do I need to neutralize the acid with sodium hydroxide at pH 6-7? 13 22. Question: According to formula SC003, Is it possible to have a oil free serum at pH 4.5-5 with high bioavailability? 13 23. Question: Does Azelaic acid have a higher bioavailability at pH 6-7 or 5 in the finished 13 product? 13 24. Question: Why in your formulation example (SC003) of the oil free serum for skin with disorders you do not neutralize the Azelaic acid with sodium hydroxide at pH 6-7? 14 25. Question: For an O/W emulsion, can Azelaic acid be added in the aqueous phase (water + propanediol)? Or should it be neutralized with sodium hydroxide at pH 6-7? 14 26. Question: In your formulation example (SC001) anti acne - asian formula is there no recrystallization problem since your pH is at 4.9-5.6? 14 27. Question: Do you advise including Azelaic acid in haircolouring products in view of pH 14 grade and possible scalp irritation? 14 28. Question: For developing a 15% peel, but don’t use too much ethanol. How to get a good solution? 14 29. Question: Is the bioavailability of Azepur99® better in emulsion? 14 30. Question: What is the most effective level of use for treating androgenic alopecia? We have so far put the Azelaic acid into propylene glycol and glycerine with some ethanol, but this can be too heavy. What is the best mixture to use as a carrier? 15 31. Question: Which pH is applicable for peelings? 15 32. Question: At what stage Azelaic acid should be added in a formulation to obtain the texture of a product in cream or lotion? 15 33. Question: What would be the effect of a lower dosage (3%-5%) of Azelaic acid combined with other ingredients 16 in a skincare formulation? 34. Question: Are there any more detailed solubility data in various polar oils to share? 16 35. Question: Question regarding an emulsion with 15% Azelaic acid: after several tests we still have problems. 16 36. Question: What is the maximum pH in order to guarantee the effectiveness of Azelaic acid? 37. Question: Can Azelaic acid be solubilised using only Ethanol? What would be the recommended proportion Azelaic acid and Ethanol in case of adding another active ingredient? 16 38. Question: What is a good subsitute for Polysorbate-80 for COSMOS/ECOCERT products? 16 Dosage of finished forms 39. Question: What is the method of Azelaic acid assay in cream as finished product? 17 40. Question: Is the action of Azelaic acid dosage dependent? And is transdermal transport also a factor? 19 41. Question: What is the best recommended dosage of Azelaic acid for an anti acne 19 formulation. And what would be the best formulating pH range? 19 42. Question: What is the best recommended dosage of Azelaic acid for skin lightening and anti-dark spots formulation. What would be the best formulating pH range? 19 43. Question: Is the pH for a cream lower than for a serum? 19

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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44. Question: What percentage of Azelaic is recommended for professional use versus home care use?

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45. Question: What is the highest % of Azelaic acid that can be used in cosmetics in EU and the USA.

Regulatory 46. Question: Is Azelaic acid standardized in the US Pharmacopeia or India Pharmacopeia?

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47. Question: The main CAS of the product is “123-99-9”, however, in section 11 there are 4 more CAS present (112-85-6, 57-11-4, 124-07-2, 68937-75-7). Kindly confirm are these 4 components are part of the product?

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48. Question: Is Azepur99® tested on animals?

49. Question: Is Azepur99® cosmetic grade TSCA (USA) and ENCS (Japan) registered?

Functionality 50. Question: If a consumer stops using the product with Azelaic acid for skin prone to acne, how long will the effects last after stopping? 51. Question: Can you share the MIC-value of Azelaic acid and what dosage is recommended? T his to indicate the antimicrobial effect.

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52. Question: Could you advise on the packaging material for formulations with Azelaic acid?

We have to check the stability in several materials sucha s PP or PE.

Applications 53. Question: Can Azelaic acid be used as a preservative or co-preservative in cosmetic products?

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54. Question: How long is it recommended to leave an exfoliant on the skin for it to penetrate? 23 55. Question: Azelaic acid can be used during pregnancy. Can it also be used during breast- feeding/lactancy period? 23 56. Question: If Azelaic acid can be used for hair (re)growth products, what consequence does this have for growing 23 facial hair (women) when used in skin products for the face? Azelaic acid and other ingredients 57. Question: Are there skincare ingredients that should not be combined with Azelaic acid? For example Retinol? 24 58. Question: We currently using Vitamin A and Vitamin C in a skin whitening application. Both can cause skin irritation. Is there a comparative table available where the different adverse effects are showed for Vitamin A and Vitamin C compared to Azelaic acid. 24

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Solubility

1. Question: How to improve the solubility of Azelaic acid?

Answer: Azelaic acid is poorly soluble in water, as described earlier by Brand-Garnys & Brand. The monosodium & disodium slats are slightly better soluble but as a result the pH of these solutions is alkaline, frequently a disadvantage. Mather & Stahl (WO 1996.039119) des cribed several combinations of solvent, frequently glycols that enable to properly dissolve Azelaic acid.

Example 1. 40 parts ethoxydiglycol

3 parts diisopropyl adipate 1-10% Azelaic acid Mix & stir until a clear solution has been obtained.

Example 2. 40 parts dipropylene glycol 1-10% Azelaic acid

Mix & stir while heating to 60°C until a clear solution has been obtained. Dipropylene glycol and ethoxydiglycol are excellent solvent systems for Azelaic acid. It is recommended to further dilute the solutions of example 1 and example 2 with other glycols, more par ticularly propanediol (1,3-dihydroxypropane), pentylene glycol (1,2- dihydroxypentane), glycerol, ethoxylated glycerol (glycereth-12, 18,20 & 26) and similar polyols. The solutions enable to dissolve up to 25% Azelaic acid, and may function as the basis of serum preparations for use in personal care & cosmetic preparations as well as medical device, as well as a starting product for e.g. emulsion & gel preparations.

For more information see the Azelaic acid essential guide, Chapter 6, page 28: Solubility of Azelaic acid.

2. Question: How to dissolve Azelaic acid?

Answer: The limited solubility of Azelaic acid in aqueous conditions requires some creativity. There are several methods to incorporate Azelaic acid in a final formulation using a premix. The most straight forward approach is to dissolve Azelaic acid in ethanol or isopropanol. For many preparations these alcohols are not the most preferred substances. In that case a suitable diol will do the job. We have tested propanediol (Zemea®; Dupont), as well as 1,3-butylene glycol and pentylene glycol. Pentylene offers the advantage that it also gives a massive microbiological protection. Pentylene glycol is available from petrochemical origin (Symrise, Minasolve), but also from vegetable origin, obtained by hydroge nation of furfuraldehyde (from wood processing; Minacare Pentiol Green/Minasolve). Also glycerin and glycerin/water mixtures are suitable solvent systems; the maximum concentration water in this solvent mixture are not properly defined and not reported in the literature. A second, but far more expensive option is to dissolve Azelaic acid is a mixture of phosphatidylcholine & isopropyl palmitate. In this case a premix is made of Azelaic acid in an aqueous glycerin solution. This procedure is only recommended when dissolution in glycols of glycerin/water mixtures fails.

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Solubility

3. Question: Should we consider 10-15 % Azelaic acid in Propanediol?

Answer: A base formulation can be generated by dissolution of Azelaic acid in propanediol (Zemea, ex.Dupont), chemically identified as 1,3-propanediol. The solution of Azelaic acid in propanediol does not require preservation and may be thickened using 3-4% hydroxy propylcellulose (HPC; Ashland). To the obtained gel other active ingredients can be added, provided the additive is miscible with the Azelaic acid/propanediol gel. That is the case for hydrophilic extracts, but also oil-in-water emulsions. The solubility of Azelaic acid in propanediol is relatively high. Solutions of 10-15% are frequently used. The maximum solubility of Azelaic acid in propanediol is not determined and would need to be researched. Note that gelation of propanediol with HPC is a tedious process. 4. Question: Is the solubilty of Azelaic acid in propanediol 1,3 similar to that of propylene gly col? We are planning to experiment with solubilizing in propanediol then suspending in sclero tium gum adjusted to 3.8 pH (as part of a combination AHA formulation). Would this adversely affect bio-availability and/or risk crystallization? Answer: 1. The solubility of Azelaic acid in 1,2-propylene glycol (INCI: Propylene Glycol) is comparable with 1,3-propylene glycol (INCI: Propa nediol). I understand where your client s coming from as 1,2- propylene glycol is from petrochemical origin and 1,3-propylene glycol is obtained by fermentation of corn sugar and has an EcoCert/Cosmos approval. Having said that, the solubility of Azelaic acid in both solvents is rather limited indeed. The solubility of Azelaic acid in both solvents can be greatly increased as follows: a. Add ethoxydiglycol or ethoxytriglycol to the glycols to further increase the solubility. Ethoxydiglycol is limited in concentration for personal care & cosmetics by the EU Legislator; ethoxy triglycol can be used freely without concentration limitations. For the USA the FDA has not put any constraints to both solvents. 2. Azelaic acid is well tolerated by sclerotium gum (Alban Muller) and vice versa. While working in an anhydrous environment there is no concern for the pH of the system. Having said that, quantitative data on the acid/base behavior in non-aqueous solvents have not been published. When following the instructions as laid down in §1 the risk of crystallization will be minimized (but not zero!), to be confirmed by a shelf life study. Usually crystallization phenomena pop up already after a few days. 5. Question: We have tried another source of Azelaic acid (not Azepur99®) and experienced some problems with crystallization for an skin prone to acne application. Now we are going to try the Azepur99® sample. Solvent used is PEG-400, which other solvents do you recom mend? Answer: It is better to add also propylen glycol, butylen glycol or other low molecular weight glycols. For more information can be found on the Essential Guide chapter 6 page 28. 6. Question: Is Azepur99® is soluble in glycerin, 1.3-Butylene Glycol or Propanediol? b. Azelaic acid can properly be dissolved in these glycols using chitosan in a (semi)-stoichiometric quantity. Reference is made to your booklet and the publication in SPC.

Answer: Yes in a mixture 50/50 of 1,3 butylene glycol/propanediol it is possible to solubilise up to 10% of Azelaic acid.

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Solubility

7. Question: When Azepur99® is dissolved in glycols or glycerin, what rheology modifier should be used to make a fully transparent gel of this solution? Answer: Glycerin is very viscous so, even if the product is soluble in a good quantity, it is very difficult to achieve an homogeneous product. It is always recommended to mix glycerin with different glycols such as propylen glycol or 1,3-Butylen glycol. 8. Question: Except Hydroxypropylcellulose (HPC), and Sodium Polyacryloyldimethyl Taurate, can other rheology modifier be used? Answer: The rheology modifier to be used must be soluble and/or dispersible in the chosen solvent. Both of them you have indicated work in water phases. In these media also xanthan gum works well. 9. Question: What can be done to make the Azelaic accid penetrate even deeper into the skin? Answer: The penetrating action is important for the main action of Azelaic acid (antiacne, whitening, etc.). In any case to better penetrate into the skin it must be well solubilized, for example in glycols that can act as a vehicle. 10. Question: We have developed an emulsion with 10% Azelaic acid, to increase the solubility of Azelaic acid, we need to increase pH of the aqueous phase before emulsification and before adding the Azelaic acid. Should the pH raised to 7 or more? To have good stability and to avoid the crystallization of Azelaic acid, what should be the optimum pH of the emulsion? Answer: The increase of the water phase pH can help only in o/w emulsions. It is not required for w/o emulsions. This information is also reported in the Essential Guide. In case of a o/w emulsion, as the final pH of a skincare emulsion it is better to stay below 6, the solu bility of Azelaic acid up to 10% must been increased by using the right ingredients glycols, polyglycols, glycerin, glyceryl derivatives or ethoxylated sorbitane derivatives.

For more information, please download the Cosmos Listed INCI names that improve the solibity of Azelaic acid via the website: azeco-cosmeceuticals.com/downloads/cosmos-listed-inci-names-improve-solubility-Azelaicacid/.

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Formulations

11. Question: How to create a base formulation and add per skin type a different active ingredient? Answer Such a base formulation can be generated by dissolution of Azelaic acid in propanediol (Zemea, ex.Dupont), chemically identified as 1,3-propanediol. The solution of Azelaic acid in propanediol does not require preservation and may be thickened using 3-4% hydroxy propylcellulose (HPC; Ashland). To the obtained gel other active ingredients can be added, provided the additive is miscible with the Azelaic acid/propanediol gel. That is the case for hydrophilic extracts, but also oil-in-water emulsions. The solubility of Azelaic acid in propanediol is relatively high. We frequently use solutions of 10-15%. We do not know what the maximum solubility of Azelaic acid in propanediol would be. That would need to be researched. Note that gelation of propanediol with HPC is a tedious process. 12. Question: In the formulation we experienced some colouring due to oxidation. Could you recommend addition of an anti-oxidant in the formulation? Answer: It is difficult to suggest an antioxidant without having information on the product composition and some tests done to understand wich is the cause of the discoloration. 13. Question: Which combination can you recommend for Azelaic acid together with other ingredients to reinforce the whitening action: niacinamide? Answer: As a whitening booster they can use for example glycolic acid (about 3%), as a low price ingredient, or niacinamide at 3-4%. Also a low amount of allantoin (0,5 %) can help.

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Formulations

14. Question: Question on the activity in the free acid or neutralized state: I’ve been pretty careful not to neutralize by post adding it to a PH of 5.5. Are there any differences in the free or salt state? Answer: Azelaic acid is a dicarboxylic acid and has therefore two dissociation constants. pKA1 = 4,55 & pKA2 = 5,50. As a consequence of being a weak dicarboxylic acid the ratio Azelaic acid/monosodium azelate/disodium azelate is pH dependant. To give an example of the calculation for pH=5,0 ([H3O+]= 1x10-5).

Two equilibrium equations: H2A + H2O ç è HA- + H3O+ HA- + H2O çè A2- + H3O+

With the two dissociation constants. KA1=[H3O+]x[HA-]/[H2A] = 2,82x10-5 KA2=[H3O+]x[A2-]/[HA-] = 3,16x10-6

Furthermore the mass balance reads: [H2A] + [HA-] + [A2-] = C After substitution of [H3O+]= 1x10-5): we are left with three equations with three unknowns, and that gives only one solution. Calcu lus gives: [H2A] = 0,213xC

[HA-] = 0,598xC [A2-] = 0,189xC

In other words: at pH=5,00 21,3% of the amount of Azelaic acid present as the free dicarboxylic acid, 59,8% is present as the mono sodium salt and 18,9% is present as the disodium salt. When another pH value would have been chosen another concentration profile is applicable. There is no picture for Azelaic acid, but below is q picture for another dicarboxylic acid: fumaric acid.

The picture shows that the concentration of the three species is pH dependant. At very low pH (e.g. pH=1,5 most fumaric acid is pre sent as the free acid. At very high pH the disodium salt is dominant. At pH=3 we have almost equal quantities of the free acid and the monosodium salt, and at pH=5 we have almost equal amounts of the monosodium salt and disodium salt, and only a small quantity of the free acid. So, the pH determines how much of each species is present. That is valid for fumaric acid, but also for Azelaic acid.. Does this affect the functionality of Azelaic acid ? No ! In the tallow gland where Azelaic acid must do the job all species act equally. However, at elevated pH the solubility of Azelaic acid increase.

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Formulations

15. Question: How to formulate a peeling with 10-15 % Azelaic acid? The final product should be a liquid or a gel. Answer: Azelaic acid is poorly soluble in water (2.1 g/l [20ºC]). The solubility increases with (partial neutralisation). Azelaic acid is a diprotic acid, with pKA1= 4,55 and pKA2=5,50. The solubility of disodium azelate is quite good: dissolved in water a translucent solution will be obtained. However, the pH of disodium azelate solutions is rather high (>7,0).

To dissolve a relatively high concentration Azelaic acid requires a thorough choice of the solvent(s)/emollient(s). There are several possibilities to achieve proper dissolution.

1. Probably the best solvent for Azelaic acid is ethoxydiglycol (diethylene glycol monoethyl ether). The solubility of Azelaic acid in ethoxydiglycol is better than 250 g/l. However, according to EU Regulation 1223/2009 this solvent is subject to concentration limitations (page 291, item #297). In cosmetic & personal care stay-on products only 2,6%. For medical devices and/or pharmaceutical products a higher concentration ethoxydiglycol is not a constraint. Ethoxydiglycol is commercially available from Gattefossé as Trans cutol® P. 2. The solubility of disodium azelate is relatively high (~180 g/l), but this results in a relatively high pH. For personal care pre parations this is considered as less appropriate. Neutralisation with a weak organic base leads to a lower pH value that simultaneously acts as a pH buffer. To be mentioned are TRIS (2-amino-2- (hydroxymethyl)propaan-1,3-diol) or AMP (aminomethyl propanol), both are available as a pharmaceutical grade. An even more elegant method is by using amino acids, more particularly arginine (2-amino-5- guanidinopentanoic acid) that also aids in wound healing. 3. The use of aminosugars, glucosamine being an exponent thereof, is a very elegant method as well, using a natural product that also enables irritation quenching. A cheap and effective method. In emulsion systems particular emollients may beneficially be used as well, more particularly the used of dimethyl or diisopropyl esters of adipic or sebacic acid. These are also powerful solvents that enhance the solubility of Azelaic acid. Particular attention shall also be given to triglycol ethyl ether, PPG-2 methyl ether acetate. propylene glycol butyl ether and propylene glycol propyl ether. These products are known with their tradename Dowanol®, commercially made available from DOW Chemical. Finally, the use of micro-emulsions base on polyglyceryl ethers is recommended, such as {polyglyceryl-4 laurate + isopropyl palmitate + demineralised water + pentylene glycol}, whereby polyglyceryl-4 oleate is a good alternative for polyglyce ryl-4 laurate. This system enables even to dissolve hard-to-dissolve ceramides. 16. Question: Can you recommend some formulation concepts? Answer: Formulation for skincare, (serum, geld, cream, lotion) shampoo & lotion and high dosage. We would like to refer to the Azepur99® skincare and haircare brochure. Here, all ingredients are presented and can be requested. 17. Question: We have taken trials with Azepur99® and we have observed crystallization in the formula. At the time of addition we are following the process as per the guidelines procedure given in the formulations from the skincare information file. Answer: Try to decrease the water content, substituting part of it with propylen/butylen glycol and a more polar emollient (synthetic esters with a better affinity with azelaic acid).

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Formulations

18. Question: We are developing a new formulation. We encountered an issue of solubility in water. Did an suggestion to use ethoxydiglycol. The condition improve a lot after the applicati on of ethoxydiglycol. However, there is still some crystal precipitation after 2 days. We wonder if there is anything we could do to improve the stability. Do you have any suggestions? Answer: We recommended to substitute at least part of the water with an organic solvent. You can use ethanol, propylen glycol, butylen gly col, starting from the substitution of a smal part (for example 5%), try until you will obtain a stable product. 19. Question: In a formulation we add Azelaic acid directly in the oil phase. The pH is between 4 and 5, is this the correct pH or should it be higher? Answer: The higher the pH, the higher the stability is. But considering the skin products cannot be alkaline, a pH between 5 and 6 will be better. 20. Question: Can a natural hydrocolloid (INCI: Carrageenan and Algin, pH 8-11), carbomer be used? Answer: The ones you have indicated have not resistance to electrolytes. So they are low performant in products very rich in Azelaic acid. Probably better carrageenan and Algin than carbomer. 21. Question: I premix my Azelaic acid (7%) in propanediol (15%) I get a white mixture, do I need to neutralize the acid with sodium hydroxide at pH 6-7? Answer: The addition of sodium hydroxide can only cause more precipitation in this solvent. Sodium azelate is more soluble in water but for sure less soluble than Azelaic acid in organic solvents. 22. Question: According to formula SC003, Is it possible to have a oil free serum at pH 4.5-5 with high bioavailability? Answer: Until the pH is neutral (up to 7) the bioavailability of Azelaic acid is good enough. The best choice is to stay at the lowest pH able to give the finished product enough stability. 23. Question: Does Azelaic acid have a higher bioavailability at pH 6-7 or 5 in the finished product? Answer: The bioavailability is higher for the acid form than for the sodium salt. So lower pH means higher availability, but for sure in aqueous media we need to maintain a compromise, using a pH not so low to improve the solubility in water of this active.

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Formulations

24. Question: Why in your formulation example (SC003) of the oil free serum for skin with di sorders you do not neutralize the Azelaic acid with sodium hydroxide at pH 6-7? In this formula does the Azelaic acid have a high bioavailability? Why your pH of the finished product is bet ween 3.4 - 3.6 is there no recrystallization problem? Answer: In this case the dispersion of Azelaic acid is strongly improved by the addition of a high amount of emulsifier (microemulsion) Sepiclear G7. The bioavalaibility is high because Azelaic acid is present in the neutral form (acidic). 25. Question: For an O/W emulsion, can Azelaic acid be added in the aqueous phase (water + propanediol)? Or should it be neutralized with sodium hydroxide at pH 6-7? Answer: If water is present, a small amount of sodium hidroxide can increase the solubility. For more information see our Skincare brochure. https://azelaicacid.cld.bz/Azeco-Cosmeceuticals-Library-download/Azeco-Cosmeceuticals11 26. Question: In your formulation example (SC001) anti acne - asian formula is there no recrys tallization problem since your pH is at 4.9-5.6? Answer: The product was stable for all the storage stability test (6 months at 40°C). 27. Question: Do you advise including Azelaic acid in haircolouring products in view of pH grade and possible scalp irritation? Answer: The addition of Azelaic acid in hair color cosmetics is not recommendable because their pH is very high, and this can eliminate any positive action of Azelaic acid. At that pH, all Azelaic acid is transformed in sodium and or MEA azelate, that is not absorbed by the skin). 28. Question: For developing a 15% peel, but don’t use too much ethanol. How to get a good solution? Answer: Glycols are better solubilizers than ethanol. You can use for example propylen glycol or butylen glycol in substitution of part of ethanol. 29. Question: Is the bioavailability of Azepur99® better in emulsion?

Answer: No it does not relate to this, in general it is the presence of glycols that is able to improve bioavailability.

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Formulations

30. Question: What is the most effective level of use for treating androgenic alopecia? We have so far put the Azelaic acid into propylene glycol and glycerine with some ethanol, but this can be too heavy. What is the best mixture to use as a carrier? Do you have a sample formulation available as a guide? Answer: This answer relates to Azelaic acid for the treatment of androgenic alopecia. To give a certified answer to your question is impossi ble: by all means, it all depends on the bio-availability of Azelaic acid how effective the product will be. If you would be able to make 100% of the amount of Azelaic acid bio-available you would need a concentration of 1,0-1,5% calculated on the basis of a gel-like preparation. 1. Add 2% ethoxydiglycol. This is a superb solvent for Azelaic acid and improves the dryness of the product when applied to the scalp. Ethoxydiglycol enables transdermal transport in the follicle, with a high degree of efficacy. For personal care & cosmetic products a maximum of ethoxydiglycol of 2,6% applies. This is not applicable for medical devices, given the “mode of action” of the medical device has been properly defined and that the consumer product is not subject to cytotoxicity. Ethoxydiglycol is supplied by Gattefosse as Transcutol CG. 2. Cytotoxicity can be suppressed by using 5% betaine (trimethylglycine), supplied by Dupont under the tradename Genencare OSMS BA. 3. It would properly better to use Zemea (INCI: propanediol; chemical name 1,3-propylene glycol). This product reduces the skin irritation induced by 1,2-propylkene glycol. Zemea is commercially made available by Dupont. 4. A system as applicable to your wishes is best thickened by 2-3% hydroxypropylcellulose (HPC). This polymer does not give a resident feel on the scalp. To prepare a gel the polymer shall first be dissolved in the final HPC before adding other products to be dissolved to avoid dissolution competition. HPC is commercially made available by Ashland as Klucel. 31. Question: Which pH is applicable for peelings? Answer: The pH of peelings is regulated by the SCCP opinion according to the used active ingredient. For example: - glycolic acid may be used safely at a level of up to 4% and a pH ≥ 3.8 - lactic acid up to a maximum level of 2.5% and a pH ≥ 5.0. But for Azelaic acid, considering is not a peeling active ingredient there are not indications by SCCP. In any case to have a stable product, the pH cannot be too low (>5). 32. Question: At what stage Azelaic acid should be added in a formulation to obtain the textu re of a product in cream or lotion? Answer: If predissolved in glycols or glycol based mixture, it can be added after the emulsioning step. In case of a w/o emulsion, Azelaic acid can be also added after the emulsioning step at a temperature of about 60°C (see SC005). In case of o/w emulsions it can be added in the oil phase once it is warmed (see SC001 and SC002 for more indications). The current system based on propylene glycol, glycerin & ethanol is rather resident on the scalp indeed, but it is a well-chosen sol vent for Azelaic acid. The system can be greatly improved using the following system:

Mussenberg 1 • 6049 GZ Herten • The Netherlands

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Formulations

33. Question: What would be the effect of a lower dosage (3%-5%) of Azelaic acid combined with other ingredients in a skincare formulation? Answer: It can in any case give a purifying effect on skin. Not enough for a whitening effect. 34. Question: Are there any more detailed solubility data in various polar oils to share? Answer: Azelaic acid is not soluble in any polar oil. So the recommendation is to prepare a premix of Azelaic acid with glycols, glycerin, glyce ryl derivatives etc., and after add it to the oil phase. For sure, the use of polar oils is preferable to the use of unpolar in the oil phase of the emulsion. 35. Question: Question regarding an emulsion with 15% Azelaic acid: after several tests we still have problems. Either the emulsion does not solidify, or the formula is not stable in a crach test at 50°C, or the Azelaic acid clumps together after 1 day. Have you conducted tests with natural ingredients? Indeed, I wonder if my choice of emulsifiers or gelling agents of natural origin is solid. Answer: Please download the Cosmos Listed INCI names that improve the solubility of Azelaic acid via the website: azeco-cosmeceuticals.com/downloads/cosmos-listed-inci-names-improve-solubility-Azelaicacid/ 36. Question: What is the maximum pH in order to guarantee the effectiveness of Azelaic acid? 37. Question: Can Azelaic acid be solubilised using only Ethanol? What would be the recom mended proportion Azelaic acid and Ethanol in case of adding another active ingredient? Answer: Azelaic acid is soluble in ethanol but not in high percentage at room temperature: about 1g of Azelaic acid can be solubilised in 100 ml of ethanol. 38. Question: What is a good subsitute for Polysorbate-80 for COSMOS/ECOCERT products? Answer: Between 5.5 and 6.5. pH is an applicable parameter only for water based products or for o/w emulsions.

Answer: A good substitute of Polysorbate-80 for COSMOS/ECOCERT products is Polyglyceryl-10 Laurate, that is COSMOS listed.

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Dosage of finished forms

39. Question: What is the method of Azelaic acid assay in cream as finished product?

Answer: There is no formal standardised method of analysis available for the determination of the assay of Azelaic acid in consumer products. However, to determine the assay of Azelaic acid is common practice for the skilled analytical chemist. The methods described here are not unique but are merely an interpretation of the current standards in analytical chemistry. It is important to distinguish between the vehicles used for Azelaic acid. This may be gels or emulsions (oil-in-water or water-in-oil); usually gels are more easy to analyse than emulsion systems. 1. Gels Gels may contain 1-25% Azelaic acid. The commercially available gels are either configured around acrylic acid- based polymers (carbomers, acrylic acid copolymers), exo-polysaccharides (xanthan gum, sclerotium gum, carrageenans, algins, karaya gum), and/or commercially available cellulose ethers. 1.1. Acrylic acid-based polymers Gels made of cross-linked acrylic acid-based homopolymers are usually identified as carbomers. Direct quantitative determination of Azelaic acid in these gels is not possible; the first step must be to break the gel structure. The matrix has to be opened up and this is best done using a saturated NaCl solution. Carbomers are highly sensitive for electrolytes and the gel particles will “implode”. The viscosity of the original gel is completely eliminated. As it is frequently seen that Azelaic acid is present in those gels as Azelaic acid crystals it is very well possible that the precipitate also will contain Azelaic acid crystals. To avoid that this quantity is not quantitatively determined ethoxydiglycol (Trancutol CG; Gattefossé) is added to the aliquot, double the weight of the sample, to dissolve crystalline Azelaic acid. Subsequently the sample is centrifuged to eliminate the precipitated carbomer, using a standard centrifuge @10,000 rpm during eight minutes. The sample is decanted and the aliquot is than subjected to further analysis. It shall be obvious that the aliquot may also contain other products than Azelaic acid only, and the composition may be rather complex indeed. Quantitative analysis is best done by HPLC, if possible combined with MS (although not strictly necessary). A C18 reversed phase column, 20 cm, is preferred; detection is best done using an UV detector @253,9 nm. To quantitatively determine the assay of Azelaic acid the technique of standard addition is used. For that reason a calibration is made using several, preferably five, concentrations of Azelaic acid dissolved in ethoxy diglycol to determine the response of the detection unit. Subsequently a well-known quantity Azelaic acid is added to the aliquot. Using the calibration curve the quantity Azelaic acid is than easily determined. The methodology is not only valid for carbomers, but is also applicable for all other acrylic & methacrylic acid polymers, usually copolymers with other monomers, provided that the polymers are electrolyte-sensitive. This methodology described for acrylic acid-based polymers is not applicable for exo-polysaccharides and cellulosics as these gels are only limited sensitive for electrolytes. 1.2. Exo-polysaccharides & cellulose ethers The most frequently used exo-polysaccharides (EPS’s) are xanthan gum, carrageenans and alginates. These biopolymers are usually insoluble in lower alcohols such methanol, ethanol, propanol and isopropanol. Gels based on exo-polysaccharides are broken with a lower alcohol of choice; the alcohol shall not be denatured as the denaturant would complicate the product mixture. The amount alco hol needed must be determined on a case-to-case basis. To the mixture of the precipitated EPS an amount ethoxydiglycol double the weight of the sample is added to the aliquot to dissolve crystalline Azelaic acid. From this point onwards the analytical procedure as described in § 1.1 is followed. This methodology is usually also applicable for cellulose ethers: methylcellulose (MC), hydroxyethylcel lulose (HEC), hydroxypropyl methylcellulose (HPMC), but is not applicable for hydroxypropylcellulose (HPC). HPC is a polymer mostly used to gel anhydrous hydrophilic solvents such as ethanol, low molecular weight polyethylene glycols (n=6,8) or diacetin/triacetin.

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Dosage of finished forms

2. Emulsions Emulsion systems are less easily handled than gel systems. It is not always easy to break the emulsion: the emulsifiers used for making the emulsions determine what chemicals must be used to break the emulsion. 2.1. Emulsions based on non-ionic emulsifiers These emulsions are stabilised by the formation of a liquid crystalline system (Israelachvili et.al., 1975) and that is frequently translated in the HLB value of the emulsifier(s): the HLB value is usually 9-11 for stable emulsions. The HLB can be greatly increased to vales > 20 using an anionic emulsifier such as sodium lauryl sulfate or an aliphatic amines such as hexylamine. The addition of alcohol (30% w/w) is usually required to improve the kinetics of emulsion breaking (and to deactivate exo-polysaccharides and/or cellulosics), and a satu rated NaCl is added to deactivate acrylic acid-based polymers. The relative amount of the emulsion destabilisers must be determined on a case-to case basis. Phase separation may be tedious and time consuming, and therefore the test sample is centrifuged using a standard centrifuge @10,000 rpm during eight minutes. The oil/water interface must be sharp. The two phases are separated and the oil phase is dried with anhydrous sodium sulphate. Both the oil phase and the water phase may contain Azelaic acid and must be analysed according to the methodology as described in § 1.1. 2.2.1. Anionic emulsions are best broken by the addition of sodium chloride (salting out). The amount of sodium chloride may be substantial. Emulsion breaking can further be accelerated by the addition of ethanol. Phase separation may be tedious and time con suming, and therefore the test sample is centrifuged using a standard centrifuge @10,000 rpm during eight minutes. The oil/water interface must be sharp. The two phases are separated and the oil phase is dried with anhydrous sodium sulphate. Both the oil phase and the water phase may contain Azelaic acid and must be analysed according to the methodology as described in § 1.1. 2.2.2. Cationic emulsions usually have an acidic pH. These emulsions are best broken by adjustment of the pH to alkaline values (pH>10). The processing should be done quickly as other ingredients may be subjected to hydrolysis. Also here the oil/water interface must be sharp. The two phases are separated and the oil phase is dried with anhydrous sodium sulphate. Both the oil phase and the water phase may contain Azelaic acid and must be analysed according to the methodology as described in § 1.1. Polymeric emulsifiers behave in a similar fashion as acrylic acid-based polymers (see § 1.1) and these emulsions are easily broken using a saturated NaCl solution. The test sample is centrifuged using a standard centrifuge @10,000 rpm during eight minutes. The oil/water interface must be sharp. The two phases are separated and the oil phase is dried with anhydrous sodium sulphate. Both the oil phase and the water phase may contain Azelaic acid and must be analysed according to the methodology as described in § 1.1. There is no general procedure available for the quantitative determination of the assay of Azelaic acid in consumer products; a general procedure is also not possible.. Good laboratory practice and creativity of the analytical chemist(s) are prerequisites for the performance of the quantification of Azelaic acid in consumer products. 2.2. Emulsions based on anionic/cationic emulsifiers. 2.3. Emulsions based on polymeric emulsifiers.

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Dosage of finished forms

40. Question: Is the action of Azelaic acid dosage dependent? And is transdermal transport also a factor? Answer: Both the factors are important: dosage and transdermal absorption. The action relates to the dosages, to have a product with only claims of antimicrobial action, to maintain clean the skin, low amounts can be added (about 3-5%). For claims relative to anti acne, anti rosacea or whitening action, the range increases to 10-15%. The use of good solvents of Azelaic acids (glycols) increases the transdermal absorption, as the preparation on creams based on liposomes or similar structure (organogel). 41. Question: What is the best recommended dosage of Azelaic acid for an anti acne formulation. And what would be the best formulating pH range? Answer: It is better to stay around 10% if possible. The best pH for stability issues is 5.5-6.5. 42. Question: What is the best recommended dosage of Azelaic acid for skin lightening and anti-dark spots formulation. What would be the best formulating pH range?

Answer: For skin lightening 10% to 15% is recommended and pH between 5.5 and 6.5. 43. Question: Is the pH for a cream lower than for a serum?

Answer: In theory not, in general both of them must have a pH as much as possible (depending on product stability) similar to the skin pH: about 5.5. Lower pH are some times possible for peel to increase the action of the active. But please consider that if a serum is mainly based on organic phase (glycols, silicon based fluids, ethanol, etc.) the measurement of pH some times is not possible and if in any case measured, the registered values could be not reliable. 44. Question: What percentage of Azelaic is recommended for professional use versus home care use?

Answer: For home use treatments it starts at 8% and professional use is higher about 10% to 15%.

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Regulatory

45. Question: What is the highest % of Azelaic acid that can be used in cosmetics in EU and the USA. Answer: In Europe, Azelaic acid is not listed in the regulation. It can be freely used in cosmetic products without restriction. 1223/2009 EC. In the USA, Azelaic acid can be freely used in cosmetic products without restriction.

For information about the cosmetic regulatory status per country, is available upon request.

46. Question: Is Azelaic acid standardized in the US Pharmacopeia or India Pharmacopeia?

Answer: The Indian Pharmacopoeia (IP) is the only one having a monograph for Azelaic acid. In any case Azepur99® is in compliance in terms of characteristics (parameter ranges) with this monograph. 47. Question: The main CAS of the product is “123-99-9”, however, in section 11 there are 4 more CAS present (112-85-6, 57-11-4, 124-07-2, 68937-75-7). Kindly confirm are these 4 com ponents are part of the product? Answer: These four components are not present in the product. They are reported in the section 11 for the only reason that the toxicological data of those substance were used in the EU REACH registration dossier of the substance Azelaic acid to justify the covered end points.

The only classified ingredient present in the product are the ones reported in Section 3 of the SDS: then only Azelaic acid ≥ 99%.

48. Question: Is Azepur99® tested on animals?

Answer: Azepur99® was not tested on animals. A statement is available, which can be requested at any time. 49. Question: Is Azepur99® cosmetic grade TSCA (USA) and ENCS (Japan) registered?

Answer: Yes, a statement reporting the inventory status of Azelaic acid is available.

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Functionality

50. Question: If a consumer stops using the product with Azelaic acid for skin prone to acne, how long will the effects last after stopping? Answer: It will depend on factors regarding the health of the skin a general answer is not possible. 51. Question: Can you share the MIC-value of Azelaic acid and what dosage is recommended? This to indicate the antimicrobial effect. Answer: In the tables below some MIC values are reported. Other data are available in the attached articles. In any case the recommended dosage cannot be below 5-6 %. Better if 10%.

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Packaging of finished products

52. Question: Could you advise on the packaging material for formulations with Azelaic acid? We have to check the stability in several materials such as PP or PE.

Answer: No difference between PP and HDPE in the case of products containing Azelaic acid. Both the materials are compatible.

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Applications

Applications

53. Question: Can Azelaic acid be used as a preservative or co-preservative in cosmetic products? Answer: Azelaic acid, even if it has antimicrobial activity is not a preservative and cannot be used as a preservative. Preservatives must be soluble in the water phase and Azelaic acid is very low soluble in water. 54. Question: How long is it recommended to leave an exfoliant on the skin for it to penetrate? Answer: Exfoliant works on the surface of the skin, not high penetration is required. In general 15 minutes are sufficient, otherwise, for a concentration higher than 10% of Azelaic acid, slight skin irritation can occur. 55. Question: Azelaic acid can be used during pregnancy. Can it also be used during breast feeding/lactancy period? Answer: In general yes, because Azelaic acid is a fatty acid occuring in nature and already present in the diet of animals and humans. But there are not sufficient studies to give more details on this. 56. Question: If Azelaic acid can be used for hair (re)growth products, what consequence does this have for growing facial hair (women) when used in skin products for the face? Answer: In hair products to have enough results for the hair regrowth, a combination with Vitamin B6 and Zinc has to be used. This combination is not present in the product for skin care. In addition, the growth of facial hair is following a different mechanism than the growth of hair.

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Azelaic acid and other ingredients

57. Question: Are there skincare ingredients that should not be combined with Azelaic acid? For example Retinol? Answer: In consideration of the chemical nature of retinol, it is compatible with Azelaic acid. It is unknown if there is affectivily interaction. In any case, being two high performant ingredient it is always reccomended to use them separately. 58. Question: We currently using Vitamin A and Vitamin C in a skin whitening application. Both can cause skin irritation. Is there a comparative table available where the different adverse effects are showed for Vitamin A and Vitamin C compared to Azelaic acid. Is Azelaic acid less irritating than the named vitamins?

Answer: Comparative table among Azelaic acid and Vitamin C and Vitamin A.

Legend: (++ Very strong effects) (+ Noticable effects) (+/- Hardly noticable) (- Not noticable)

Vitamin C is a naturally occurring drug with multiple desirable effects. With an excellent safety profile, it finds increasing use in pho toageing, hyperpigmentation, tissue inflammation and promotion of tissue healing. It can also be used for the treatment of hyperpig mentation. Its main drowback is that it is unstable and it is also difficult to deliver it into the dermis in the optimum dosage. So it is more difficult to vehiculate Vitamin C than Azelaic acid. Vitamin C stability in finished products is also not so good. It is easily oxidated by the oxygen in the air. So Vitamin C is a good antioxidant. Vitamin C interacts with copper ions at the tyrosinase‑active site and inhibits action of the enzyme tyrosinase, thereby decreasing the melanin formation. Vitamin C also acts on the perifollicular pigment [5,7,8]. However, Vitamin C is an unstable compound. It is therefore often combined with other depigmenting agents such as soy and liquorice for better depigmenting effect. So probably to have a sufficient and stable depigmenting product, Vitamin C could be used in combination with Azelaic acid.

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