1. Efficacy Methodology and Assessment of Cosmetics
R. Randall Wickett
University of Cincinnati
College of Pharmacy

2. Outline of Topics Non-invasive measurements of skin function
Transepidermal Water Loss
Electrical Measurements
Mechanical Measurements
Skin Color
Surface Analysis
Protocols for Efficacy Assessment
Moisturizer Efficacy
Cleanser Mildness
Anti-aging products

3. Utility of Non-invasive Bioengineering Measurements
Why not just rely on visual grading?
Grading is not “objective”
Grading is not “reproducible” at an exact level. Agreement within ± 0.5 grades units is considered very good.
Bioengineering Tools can give additional information. For instance visual grades will not necessarily tell you if skin is more or less elastic or if the skin barrier is perturbed.
Bioengineering measurements can be used to support claims and for guiding the product development process
Training and experience are required to obtain valid and reproducible bioengineering measurements

4. Ronald Marks, M.D.
One of the “Godfathers” of bioengineering measurement on skin
Organized the first symposium on noninvasive methods and the skin in 1976 and founded ISBS

5. Albert M. Kligman, M.D. A fool with a tool is still a fool
Another Godfather of Skin Bioengineering – first talked about the possibility of admitting a “blind person” as a dermatology resident.
A fool with a tool is still a fool

6. General Considerations for non-invasive measurements on skin
There are no published standard protocols similar to ASTM (American Society for Testing Materials)
“Guidances” are published by EEMCO (European group on Efficacy Measurements of COsmetics and other topical products) for various categories of methods

7. General Recommendations for instrumental measurements on Skin
Some control of testing environment is necessary to prevent sweating
Normally 20 ± 1 C, 30-45% RH
Subjects should equilibrate 30 minutes if possible
Subjects should avoid drinking hot coffee or tea or eating for at least 1 hour prior to measurement
Subjects should avoid uncontrolled use of cosmetic products on the measurement sites for at least 1 week prior to measurement
Subjects with any obvious skin disease, tattoos or injury in the treatment area will be excluded.

8. Transepidermal Water Loss
Water transport through the skin by passive diffusion
Rate is 5-10 g/m2/hr in healthy skin
Increases with surfactant damage
Used to measure occlusion by products
Used extensively in barrier disruption and repair studies
Panelists must not be sweating. Equilibrate 30 min. at ~21C RH<50%

9. The Dermalab Evaporimeter

10. The C+K devices Corneometer probe Cutometer probe TEWA Meter probe
Sebumeter

11. The operating principle of open chamber TEWL devices

12. Forearm Controlled Application Test (FCAT)
Lather wetted bar for 6 seconds with Masselin towel
Transfer lather to site and wash for 10 seconds
Leave lather in place 90 seconds
Rinse for 20 seconds
Repeat 4X daily for one week

13. Forearm washing

14. Washing and rinsing

15. Forearm wash test bars Bar 1 mild syndet + oatmeal
Bar 4 Soap pure soap
Bar 5 Soap + humectant

16. Measuring TEWL after a forearm wash test

17. TEWL: Forearm wash test
RR Wickett, Forearm wash testing of mild soap bars containing colloidal oatmeal, Canadian Chemical News, 49(1), (1997).

18. Redness grade versus TEWL: Forearm Wash Test

19. Electrical Measurements of Skin
Skin lipids have a low dielectric constant, water has a high dielectric constant.
When skin is hydrated conductance and capacitance (reactance) increase and impedance decreases.
Used to estimate water content of stratum corneum.

20. The NOVA DPM

21. The Skicon

22. Correlation with dry skin grades
Dry skin is lacking in moisture.
Several studies have shown that electrical measurements correlate with dry skin grades as expected
Morrison and Scala correlated Correlated Corneometer and Skicon readings to flaking grades on dry hands scores that developed as subjects washed their hands for 1 minute five times a day for 4 days.
We also have data on correlation of readings of all three instruments to dry skin grades on the legs.

23. Correlation between Flake Grade and Corneometer CM 820 values
Morrison and Scala(1996) J. Toxicol-Cut.& Ocular Toxicol, 15:

24. Skicon versus mean flaking grade
Morrison and Scala(1996) J. Toxicol-Cut.& Ocular Toxicol, 15:

25. Grading scale for dry skin, half grades are allowed
Description
No scaling
1.0
Slight powdery appearance and occasional patches of small scales widely distributed
2.0
Small scales in combination with a few larger scales, slight roughness, whitish appearance
3.0
Small and large scales uniformly distributed, definite roughness and possible a few superficial cracks
4.0
Dominated by large scale, rough, redness present and eczematous changes
F Li, MO Visscher, E Conroy and RR Wickett, J. Cosmet. Sci. 52, (2001).

26. Scatter plot of Skicon reading versus Dry Skin Grade
R. R. Wickett and F. Li Unpublished Data

27. Average values versus dry skin grades Skicon and Corneometer
R. R. Wickett and F. Li Unpublished Data

28. Average values versus dry skin grades NOVA meter
R. R. Wickett and F. Li Unpublished Data

29. Brancaleon et al. correlated Skicon readings with ATR-FTIR measurements of water in the SC. “Our attempt to link the hydration measured with ATR-FTIR to the hydration measured with the Skicon hygrometer shows a high degree of correlation. This is not unexpected as both instruments record superficial hydration.”

30. Correlations among instrumental measurements
F. Li, et. Al J. Cosmet. Sci. 52, 13-22(2001).

31. Correlations between instruments
Clarys et. al. Fluhr et. al. Our work*
NOVA vs Skicon   NOVA vs. CM   Skicon vs. CM  
Clarys, P., et al Skin Res Technol, 5, 14(1999).
Fluhr, J. W., et al Skin Res. and Technol., 5, 171 (1999).
Li, F. et al J. Cosmet Sci 52, (2001)

32. Electrical measurements and moisturizer claims
For more than 20 years electrical measurements made within 30 minutes to a few hours of treatment have been used to substantiate claims of moisturizing efficacy.
We have reported that measurements made at one hour predicted moisturizing in a two week test for formulations containing glycerin from 0 to 15% concentration. (Li et al J Cosmetic Science, January 2001)

33. Effect of moisturizers on DPM (Formulations from ~ 1990)

34. Correlation Test One hour Test: 9 subjects Two week test: 20 subjects
Goal determine the correlation between one hour device readings and effects on skin grades in a dry skin test:
One hour Test: 9 subjects
Two week test: 20 subjects
subjects were female, between the ages of 18 and 57 and in good general health with no known skin diseases.
Products contained 0, 5, 10 and 15% Glycerin in identical base
Each subject used each of the 4 products on their legs

35. DEVICE PREDICTION CAPABILITY: SKICON

36. Regression equations of one week skin grade versus one hour electrical measurements
 Device
Regression equation
R-square p
NOVA
G = x
0.92
0.04
SKICON
G = x
0.97
0.02
CM 825
G = x+ 0.76
0.84
0.08
We do not consider the difference between instruments to be meaningful.

37. Conclusion
Single point or short term electrical measurements have been shown to be predictive of long term moisturization for simple formulations using on Glycerin as a moisturizer.
F Li, MO Visscher, E Conroy and RR Wickett, J. Cosmet. Sci. 52, (2001).

38. Mechanical Properties of Skin
There are two fundamental ways to measure the mechanical properties of an object
Apply a known strain and measure the stress
Apply a known stress and measure the strain
Most skin instruments use the second approach

39. The Cutometer 575

40. The Cutometer probe
An optical system is used to measure the height that the skin rises into the probe

41. The optical system of the Cutometer
Picture courtesy of Courage + Khazaka GmbH

42. The Cutometer 575 probe

43. Cutometer curves

44. Cutometer Parameters
UE is the elastic deformation of the skin due to the application of stress (vacuum or torque) by the instrument; 
UV is the viscoelastic creep occurring after the elastic deformation; 
UF is the total extensibility of the skin;
UR is the elastic deformation recovery due to stress removal; 
UA is the total deformation recovery at the end of the stress-off period;
R is the amount of deformation not recovered by the end of the stress-off period

45. Cutometer curves

46. An illustration of the Cutometer curve and analysis (from C+K)
See RR Wickett, Stretching the Skin Surface Cosmetics and Toiletries 116(3), 47-54(2001) for details of Cutometer and DTM analysis

47. Ratio Parameters
UA/UF is the overall elasticity of the skin including creep and creep recovery;
UR/UE is the pure elasticity ignoring viscoelastic creep; 
UV/UE is the ratio of viscoelastic to elastic extension called the viscoelastic ratio;
UR/UF is the ratio of elastic recovery to total deformation.

48. Issues with mechanical measurements
What part of the skin are we measuring?
General principle: to measure the stratum corneum use the smallest feasible deformation
To measure the dermis use a larger deformation
Interpretation of data
For moisturization we can look increased Ue and Ur reflecting increased elasticity.
Many anti-aging treatments claim “firmness” but there is no engineering definition of firmness

49. Extensibility of shoulder skin v. age Cutometer Uf 6 mm diameter probe
Smalls LK, Wickett RR, Visscher M, Effect of Dermal Thickness, Tissue Composition, and Body Site on Skin Biomechanical Properties, Skin Research and Technology, (2006)
Lola Kelly Smalls Ph.D. dissertation University of Cincinnati

50. Effect of vacuum level on Cutometer sensitivity to 1 week of treatment with a moisturizer with limited efficacy
Parameter
Pre-treat
Post-Treat
P-value
NOVA DPM
98.8
116.2
0.001
Ue (200)
0.036
0.048
0.02
Ue (500)
0.068
0.079
0.46
Uv (200)
0.015
0.024
0.07
Uv (500)
0.025
0.029
0.60
Ur (200)
0.026
0.039
0.03
Ur (500)
0.050
0.061
0.26
Wickett, RR (2001) Cosmetics and Toiletries 116, 47-54

51. D’Squames® are an easy way in investigate skin scaling and flaking

52. They can be graded visually or analyzed by computer

53. The computer can calculate the Desquamation index (D.I.)
A = percent area covered by scales
Tn = percentage of scale at each thickness level
n = thickness level 1 to 5
Schatz, et al. J. Soc Cosmet. Chem. 44, 53-63, 1993

54. Warrier(1996) et al J. Soc. Cosmet. Chem. 47, 229-240
We found blacks to have higher visual dryness scores than whites, especially on legs
Warrier(1996) et al J. Soc. Cosmet. Chem. 47,

55. Warrier(1996) et al J. Soc. Cosmet. Chem. 47, 229-240
D.I. showed the opposite
Warrier(1996) et al J. Soc. Cosmet. Chem. 47,

56. The Minolta Chromameter
Color is measured on the L* a* b* scale.
L* = white 0 to 100
a* = red – green 60 to –60
b* = blue – yellow –60 to 60

57. Using the Chromameter

58. The color wheel

59. The color solid

60. Minolta a* in forearm wash test
RR Wickett, Canadian Chemical News, 49(1), (1997).

61. Correlation between a* and erythema grade in the FCAT

62. Chromameter L values for various ethnic groups
Alaluf et al (2002) Pigment Cell Res.

63. Analysis of skin surface texture Silicone replicas are most commonly used

64. Obtaining a replica from the peri-orbital (crows foot) area
Photo courtesy of Ken Marenus

65. Image analysis can quantify wrinkle depth by the shadow principle
Corcuff et. Al. JSCC (1983)

66. Replicas of young and old forearm skin montage

67. Replicas from the “crows foot” area of the eye

68. Crow’s foot wrinkle depth versus age
Corcuff et. Al. JSCC (1983)

69. Visiometer Replicas are died blue
Light transmission is measured to determine the depth of the lines

70. Visiometer

72. Microrelief of the skin determined by the visiometer

73. Fringe Projection (PRIMOS)
Phase Shifting Rapid In vivo Measurement of Skin
Digital micromirror device used to project fringe pattern on the skin surface
CCD camera records image
Shifting of the fringe patterns by surface contour allows 3D imaging

75. Primos measurement on the face

76. Distortion of fringes by wrinkles

77. Grey scale coded 3D image

78. Line profile from the Primos

79. The VISIA imaging system by Canfield allows for capture of high quality images of the face using different types of light Image analysis can be performed to pick out hyperpigmented spots and other features of photoaging

80. Image of face with spots highlighted
Photo courtesy of Greg Hillebrand, used with permission

81. Moisturizer Testing
Regression and “mini” regression tests

82. Regression testing of moisturizers
The ‘Gold Standard’ test of moisturizer efficacy
Three weeks of treatment followed by two weeks of regression
The goal is to separate out the visual effects of the product and show only the real effect on skin physiology
Better moisturizers will be better in the regression phase
Kligman, Cosmetics and Toiletries April
Rogers et al JAAD (1989)

83. Regression test with Petrolatum
Kligman, Cosmetics and Toiletries April

84. Regression test with “Cosmetic” and “Therapeutic” Moisturizers
From Rawlings et al Derm Ther (2004)

85. Pretreatment
The ‘mini’ regression test: One or two week treatment phase, one week to a few days regression. By comparing products on the same panelist and using instruments better sensitivity can be achieved with fewer subjects and a shorter treatment time.

86. End of treatment End of Regression

87. Grove has used 4 day “mini-regression” protocols with grading and conductance measurements on dry legs
Products applied to 2x2 cm area by trained staff
Conductance measured with Skicon
19 female subjects
Two week washout
Lotions separated in conductance readings but were not significantly different by skin grade
G. Grove et al poster presentation product names removed used with permission

88. Rawlings combined visual dryness with Corneometer readings in a 3 day mini regression test
Rawlings et al Derm Ther (2004)

89. Testing “Ant-Aging” products

90. Clinical Features of Photo-Aging
Lines and Wrinkles
Loss of Elasticity
Elasticity becomes more anisotropic
Appearance of “Age Spots”

91. Methods for evaluation of anti-aging products
Clinical grading directly on panelists
Grading of high quality clinical photographs
Analysis of replicas
Mechanical measurements
Primos system to analyze wrinkles
VISIA System
Biopsies to evaluate changes in dermal histology

92. Descriptive grading scale for photoaging
Griffiths et. Al. (1992) Arch Dermatol.;128:

93. Photographic Grading Scales
There are several different photographic grading scales
Griffiths et. Al. (1992) Arch Dermatol.;128:
Larnier et. Al. (1994) Brit. J. Dermatol
Chung (2003)Photodermatol Photoimmunol Photomed 19: 109–121

94. The grading scale by Griffiths et. Al
The grading scale by Griffiths et. Al. is probably the most commonly used.

95. The Larnier scale 4 -6

96. Chung’s scale with Korean subjects

97. Anti-Aging Studies: General Considerations
Anti-aging effects take time to demonstrate.
Studies usually run for about 24 weeks and 12 weeks seems to be a minimum.
Facial studies are usually monadic. That is each subject tests only one product.
Studies on forearms may be done as paired comparisons.
Studies must be done double blind.
Vehicle (placebo) Effects can be quite large. Moisturizing vehicles will produce noticeable effects on fine wrinkles and avoiding sun will improve grades.

98. A reason for not doing split-face studies on anti-aging products
I’ve been part of a 15 year study on sunscreens. Every day I put SPF 30 on one side of my body and nothing on the other side

99. Outline for anti-aging test on facial product
60 female subjects with moderate photodamage on the face.
Randomized or balanced into two groups of 30, active and vehicle or placebo.
Subjects and graders/measurement technicians are blind to the treatment
Treatment is to whole face
Forearms are also treated for biopsies
Measurements made at baseline and after 3 and 6 months of treatment.

100. Measurements anti-aging test
Grading.
Clinical photographs or VISIA system.
Cutometer readings on cheek.
Replicas taken from crows foot area of eye for image analysis or PRIMOS measurements made in eye area.
Biopsies taken from forearm at baseline and 6 months only for collagen and elastin measurement (this is definitely optional)

101. Minimum requirements
The test I have described could be called a Cadillac test as it has a lot of options
At the minimum grading and clinical photographs should be obtained.
Some studies run for only 3-4 months and occasionally they are done split face
Pilot studies may be done on photodamage forearms using both male and female subjects and in this case one arm will get vehicle and the other treatment.