1. Prepared by : Dr. Mariani Abdul Hamid

2. STABILITY TEST WHAT, WHY WHEN AND HOW 3. INTRODUCTION There is such a big difference between what you experienced in your college organic chemistry lab versus a cosmetic formulation lab. In an organic lab, you mix chemicals together and hope something happens. Ideally, you get a chemical reaction you expect. As a formulating chemist, you mix chemicals together and hope nothing happens. Cosmetics are mixtures of chemicals that mostly arent supposed to react with each other. 4. Unfortunately, they often do react (or otherwise change) so you need to test your formulas to see how long they will last. This is called Stability Testing and is something a cosmetic scientist spend much of her time doing. In this lecture, Ill give a brief description of the test and suggest when, why and how it should be done. 5. What is stability testing? Stability testing is simply an experiment in which you create a batch of your formula and put samples of it at different environmental conditions for a set period of time. These conditions vary in temperature and light levels and are meant to simulate what will happen to the product during its life cycle. 6. At select intervals you evaluate your samples for various physical, chemical and performance characteristics to see how they have changed. If the changes are minimal according to your company standards, then your formula is said to have passed stability testing. This means you can have confident that when the formula is shipped to stores and ultimately customers, it will still be as good as when it was first manufactured. What is stability testing? 7. The underlying assumption in stability testing is that increasing storage temperature speeds up any aging reactions that will occur. A handy rule of thumb is that a sample stored at 45C for 8 weeks is equivalent to one that is stored at room temperature for one year. This isnt an exact predictor, but is good enough for the purposes of cosmetic products. What is stability testing? 8. When do you perform stability testing? Since youll be making hundreds or thousands of prototypes during your career, it wont be practical to run a stability test on all of them. Youll also find that changes happen so rapidly at your company, you wont have time to properly test many of your formulas. But there are times when you need to do stability testing. Here is a short list of some of the most important times to conduct a stability test. 9. 1. New prototypes Whenever you make a new formula and are satisfied with the way it performs, youll want to do a stability test to ensure that it will stay together. Dont bother testing all your prototypes, just the ones that work the way you want. 2. New raw materials Whenever you have to change the fragrance, color, or other raw material in a formula, youll have to do a stability test to make sure there arent unacceptable changes. Also, when you have a new raw material source (or supplier) youll want to run a test. 10. 3. New manufacturing procedure Manufacturing is always trying to find faster ways to make formulas. This often means they change some order of addition or shorten mixing time. Whenever changes like these happen, it could affect your formula. Run a stability test to see if the change is acceptable. 4. New packaging Cosmetic products change their look almost yearly so packaging is constantly being modified. Whenever you get a new package, youll have to determine if the formula continues to be compatible. Stability testing helps ensure that it is. 11. Stability Testing of Cosmetics The purpose of stability testing cosmetic products is to ensure that a new or modified product meets the intended physical, chemical and microbiological quality standards as well as functionality and aesthetics when stored under appropriate conditions. Because the development cycle of cosmetic products is relatively short each manufacturer should design their own stability testing program such that it is economically reasonable and efficiently addresses the testing required. 12. Manufacturers require the flexibility to modify testing protocols and to build a sound scientific basis for assessing stability of their own products. Thus, specific tests may be developed in order to address new or unusual technologies, or to be adapted to products having extended shelf lives. In general, stability tests can be conducted in real time or under accelerated conditions and should address the stability of a product under appropriate conditions of storage, transport and use. Stability Testing of Cosmetics 13. Stability Tests Physical and chemical integrity tests: evaluate color, odor / fragrance, pH value, viscosity, texture, flow, and emulsion stability (signs of separation) Microbiological stability tests: evaluate the degree of contamination with bacteria, mold, and yeast. Packaging stability tests: evaluate the impact of packaging on the contained product. 14. Physical / Chemical Stability Tests These describes approaches to predicting how well cosmetics will resist common stresses such as temperature extremes and light. Typically, manufacturers determine whether to perform such specialized testing based on the vulnerabilities of the particular cosmetic product and its anticipated shipping, storage display and use conditions. 15. Common Test Procedures Temperature variations: High temperature testing is now commonly used as a predictor of long-term stability. Most companies conduct their high temperature testing at 37oC (98F) and 45oC (113F). If a product is stored at 45oC for three months (and exhibits acceptable stability) then it should be stable at room temperature for two years. Of course, the product must be stored at 25oC (77F) for a period of one year. A good control temperature is 4oC (39F) where most products will exhibit excellent stability. The product should also be subjected to -10oC (14F) for three months. 16. Cycle testing: The product should pass three cycles of temperature testing from -10oC (14F) to 25oC (77F). Place the product at -10oC for 24 hours and place it at room temperature (25oC) for 24 hours. This completes one cycle. If the product passes three cycles then you can have a good degree of confidence in the stability of the product. An even more rigorous test is a -10oC to 45oC five-cycle test. This puts emulsions under a tremendous stress and, if it passes the test, indicates that you have a really stable product. 17. Centrifuge testing: The dispersed phase (of an oil-in-water emulsion) has a tendency to separate and rise to the top of the emulsion forming a layer of oil droplets. This phenomenon is called creaming. Creaming is one of the first signs of impending emulsion instability and should be taken seriously. A good test method to predict creaming is centrifugation. Heat the emulsion to 50oC (122F) and centrifuge it for thirty minutes at 3000 rpm. Then inspect the resultant product for signs of creaming. This test is an absolute necessity for those products that contain powders of any kind such as liquid/cream make- up. 18. Light testing: Both formulas and packaging can be sensitive to the UV radiation. All products should be placed, in glass and the actual package, in the window and if its available a light box that has a broad-spectrum output. Place another glass jar completely covered with aluminum foil in the window to serve as a control. All too often we will see significant discoloration of the product and sometimes of the package also. This discoloration may be due to the fragrance or some other sensitive ingredient. Usually all that is needed is the addition of a UV absorber (e.g. 0.1% of benzophenone). 19. Mechanical shock testing: In order to determine whether or not shipping movements may damage the cosmetic and its packaging mechanical shock testing is often conducted. Vibration testing (e.g. on a pallet shaker) can help to determine whether de-mixing (separation) of powders or granular products is likely to occur. 20. Parameters to monitor: For all the above mentioned tests you should monitor the color, odor / fragrance, viscosity, pH value, and, if available, particle size uniformity and/or particle agglomeration under the microscope. 21. Microbiological Stability Tests Microbial contaminants usually come from two different origins: during production and filling, and during the use of the cosmetic by the consumer. From the moment the cosmetic unit is opened by the consumer, a permanent microbial contamination of the cosmetic is introduced caused by contact with the consumers hands and body. 22. Microbial preservation of cosmetics is important to ensure the microbial safety of cosmetics for the consumer, maintain the quality of the product, and confirm hygenic and high-quality handling. Although only a small number of cases of microbial infections of the consumer has been reported, microbial contamination of cosmetic products may spoil them or seriously reduce the intended quality. 23. Therefore, it is necessary to carry out routine microbiological analysis of each batch of the finished product coming on the market. Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans are considered the main potential pathogens in cosmetic products. These specific potential pathogens must not be detectable in 0.1 g or 0.1 ml of a cosmetic product. The parameters examined, the criteria and methods used, and the results obtained per batch should be documented. 24. Common Test Procedures Screening tests: There are various easy testing kits available on the market (e.g. dip-slides or plate counts) which provide quick and semi-quantitative results whether a cosmetic product is significantly contaminated or not. Sampling and evaluation of the results is simple and can be performed also by personnel without any microbiological training. 25. Quantitative tests: Quantitative tests determine the actual count level of bacteria, mold and yeast in a cosmetic product. These tests are very sophisticated and laborious and can be performed only by professional microbiological testing laboratories. Typically, methods for isolation of microorganisms from cosmetic products include direct colony counts and enrichment culturing. 26. Packaging Stability Tests Packaging can directly affect finished product stability because of interactions which can occur between the product, the package, and the external environment. For example, product constituents may be absorbed into the container or may chemically react with the container. In addition, the container may not fully protect the product from the adverse effects of atmospheric oxygen and/or water vapor, or volatile product constituents (e.g. fragrances) may evaporate through the container. 27. Glass tests: Glass is the most inert material and does not react with a cosmetic product in any way. For this reason all testing should be done in glass and the actual packaging. In this way you can determine if the cause of product failure is the formula or the package. Leaking tests: It may be advisable to test the packaged product in various orientations (upright, inverted, on its side, etc.) to determine whether the packaging may leak (especially during transport). Common Test Procedures 28. Weight loss tests: To determine evaporation (water loss through the container wall or closure gaps) weight loss evaluation is one of the most important tests that must be conducted. This testing (performed in the actual package with the cap torqued to 100% of target torque) is done at room temperature and at 45oC (113F) for a period of three months. The weight loss should not exceed 1% per month for the package to be considered acceptable. 29. Formulating for Efficacy Active ingredients have been popular for more than a decade, and new actives are continuously being identified, studied and promoted. Many of these are supported by good in vitro efficacy data, and there is an increasing number of ingredients for which also good in vivo efficacy evidence is available. Based on this, one would expect to find many active cosmetic products in the market place, but unfortunately this is not the case. 30. Assuming that the efficacy data provided is robust (i.e., the active ingredient has indeed its claimed cosmetic activity), questions arise about the formulation development process that should assure that the efficacy of an active ingredient is transformed to an efficacious cosmetic product. Cosmetic formulators should therefore select their ingredients and manufacturing procedures in such a way that cosmetic efficacy is obtained. In other words, they should formulate for efficacy. In many cases, however, this does not happen. 31. Many companies have a number of standard formulations to which the latest new active ingredient is simply added. Following stability testing and elimination of those failing the stability tests, small clinical trials are performed with the remaining formulations to assess whether the claimed efficacy of the active ingredient is maintained in the standard formulation. In most cases, no efficacy is seen and after some additional work, the active ingredient is discarded. Whereas the reasons for using standard formulations are very understandable, this strategy does not lead to the best possible product because it completely ignores the principles that underpin the skin delivery of the active ingredient. 32. This chapter describes the selection criteria for ingredients in cosmetic formulations that help to optimize the delivery of the active ingredient into the skin. As formulations can be very complicated, many factors need to be taken into account. To date only a few have been systematically studied. The guidelines described in this chapter are, therefore, only guidelines but the guideline recipe will be a lot closer to an efficacious cosmetic formulation than a random choice from a selection of standard formulations. As further results from new work become available, the system will be further refined. 33. Theoretical considerations for the skin delivery of cosmetics As illustrated in Figure 1, Barry described the skin penetration process as a series of consecutive steps, each of which can potentially be rate limiting. First, the chemical needs to diffuse within the formulation to the skin surface. There is partitions into the skin, diffuses through the stratum corneum, partitions into the viable epidermis and diffuses through the viable epidermis. It then partitions into and diffuses through the dermis before partitioning into the fat deposits or it partitions into the blood capillaries just beneath the viable epidermis/dermis interface. 34. http://en.wikipedia.org/wiki/Skin Structure of Human Skin 35. Skin layer Description Epidermis The external layer mainly composed of layers of keratinocytes but also containing melanocytes, Langerhans cells and Merkel cells. Basement membrane The multilayered structure forming the dermoepidermal junction. Dermis The area of supportive connective tissue between the epidermis and the underlying subcutis: contains sweat glands, hair roots, nervous cells and fibres, blood and lymph vessels. Subcutis The layer of loose connective tissue and fat beneath the dermis. Layers of the skin 36. Layers of Epidermis 37. Figure 1. The various subsequent steps that an active ingredient will have to undertake during its journey from the formulation in which it is incorporated to its site of action. reproduced with the permission of the author and Marcel Dekker Inc., New York, NY, USa. 38. From this, it can be concluded that both partition and diffusion are very important in determining skin penetration. They are normally combined in the permeability coefficient according to the formula : in which kp is the permeability coefficient, Koct/water the octanol/water partition coefficient, D the diffusion coefficient and L the length of the pathway of diffusion of the penetrating molecule 39. The unit of the permeability coefficient, cm/s, indicates that this parameter basically reflects the speed with which a chemical diffuses through the stratum corneum. However, in order to obtain efficacy a sufficiently high concentration of the active ingredient needs to be reached at the site of action and maintained for a sufficiently long period of time. Absolute amounts are therefore also important but here some conflicting evidence is obtained from skin penetration theory. 40. The most logical way to increase the degree of skin penetration is to increase the concentration of the active ingredient in the formulation, according to the well-known formula (eq 1): in which is the concentration difference of the penetrating molecule over the stratum corneum, i.e., the difference in concentrations between the formulation and the deepest layers of the stratum corneum. 41. The larger this concentration difference, the greater the flux through the stratum corneum. At the same time, the more soluble an active ingredient is in the formulation, the more active ingredient can be contained in the formulation and the more can therefore penetrate into the stratum corneum. But difficulties arise when increasing the solubility of the active ingredient in the formulation. 42. According to the definition of the partition coefficient, the Ksc/form of the penetrating molecule, the solubility of the active ingredient in the stratum corneum is related to its solubility in the formulation as expressed in Equation 3: in which C penetrant represents the solubility of the penetrat-ing molecule in either the stratum corneum or the formulation 43. Because this K is the same as those in Equations 1 and 2, the quantity of penetrating molecules into the stratum corneum can be increased by increasing the solubility of the penetrating molecule in the stratum corneum or by reducing its solubility in the formulation. One therefore needs to increase the solubility of the active ingredient in the formulation in Equation 2 to achieve sufficiently high quantities to obtain efficacy in the skin, but one needs to reduce the same solubility in order to force the material to leave the formulation and partition into the stratum corneum. The remainder of this chapter will describe how can one increase and reduce the solubility of the active ingredient in the formulation.