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Gold standard “in vitro” procedure to evaluate safety and efficacy of nail care products ¹Marcello Bracchi, ¹Giorgio Musitelli, ¹Priscilla Capra, 2Mariella Bleve, ¹,2Paola Perugini, 1Department of Drug Sciences, 2EticHub s.r.l., Academic Spin-off, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy, e-mail: [email protected]
Sample Theoretical A B C
100
Actual (S.D.) 128.25 (4.87)
200
230.17 (39.37)
400
426.65 (28.75)
Production of bovine hoof membranes: this method allows to
Conclusions
choose thickness and diameter of the membranes.
Freshly slaughtered bovine hooves are dipped in liquid nitrogen, cored according to the chosen diameter and cutted in slices. The operator set the thickness of the slices. Samples are stored in ethanol 70% v/v. More than 263 membranes of 100 µm, 134 membranes of 200 µm and 66 membranes of 400 µm have been produced.
1
Preparation before the analysis: hoof membranes are dried and fixed on a PTFE support to reproduce the human nail shape. Different supports with different diameters (from 6 to 15 mm) are used in order to represent the bend of the human nails.
The access to intact human nails is often limited, so animal material has been used as substitute to study nail permeability and the effect of substances in contact with nails. Furthermore, using bovine hooves, the evaluation of safety and efficacy of ingredients and cosmetic products can be achieved. However, differences between animal hooves and human nails are reported in the literature and so caution is required in extrapolating such data to predict ungual effect on humans.
6
• • • • •
Evaluation of bovine hoof membrane density Density is calculated dividing the membrane weight for its volume. Density is a property that is similar between membranes that comes from the same part of the bovine hoof core. This parameter is very important cause similar density means similar tissue structure. Every membrane is being classified according to its density and structural firmness Membrane classification on the base of FI and density values
Aim of this work is to define a
Samples are stored in a climatic chamber (30°C and 50% R.H .)
2
Set up of a new reliable and reproducible method for bovine hoof membranes production. Morphological and mechanical analysis of the substrates. Desquamation and structural firmness are inversely proportional. Firmness and density are related. Membranes with 100 µm of thickness seemed to be very useful for permeability studies instead of 200-400 µm are the best thickness range to simulate in vivo response in studies concerning the efficacy and the safety of ingredients and final products.
methodological approach to standardize samples obtained from animals to improve reliability and reproducibility of the data in order to predict human nail responses.
5
Group
Firmness Index (D.S.)
A1
3.74 (0.57)
0.16
A2
7.45 (1.13)
0.19
A3
18.24 (1.72)
0.21
Density (mg/mm³)
THICKNESS (µm)
Density (mg/mm³)
y = 0.0317ln(x) + 0.124 R² = 0.9178
0
Mechanical Characterization: Nail StrainStress Meter NM 100 provides data for the classification of membranes. Two different tests are performed on every membrane, each test is repeated 5 times.
Firmness Index (FI) The sample is compressed punctually. The slope of the linear regression curve indicates the structural firmness of the membrane. The increase of this parameter means that the sample is more resistant to deformation. Thickness: The thickness of the membrane is directly determined by the compression analysis .
3
4
Membranes with the same rate of desquamation show similar Firmness Index.
ROUGHNESS ++++ (A) +++(B)
99.49 (28.31)
++(C)
113.64 (32.98)
+(D)
140.75 48.16)
30
B
Firmness Index (S.D.) 45.72 (13.38)
20 Firmness Index
A
ROUGHNESS and FIRMNESS INDEX correlation Membranes are divided according to their roughness, evaluated by morphological analysis.
10
C
D
References
Musitelli G. et al., TO2013A001064 , PCT/IB2014/067260 D. Monti et al, Br. J. Dermatol., vol. 165, no. 1, pp. 99–105, 2011. D. Miron et al., Eur. J. Pharm. Sci., vol. 51, pp. 20–25, 2014.
Sample Theoretical A B C
100
Actual (S.D.) 128.25 (4.87)
200
230.17 (39.37)
400
426.65 (28.75)
Production of bovine hoof membranes: this method allows to
Conclusions
choose thickness and diameter of the membranes.
Freshly slaughtered bovine hooves are dipped in liquid nitrogen, cored according to the chosen diameter and cutted in slices. The operator set the thickness of the slices. Samples are stored in ethanol 70% v/v. More than 263 membranes of 100 µm, 134 membranes of 200 µm and 66 membranes of 400 µm have been produced.
1
Preparation before the analysis: hoof membranes are dried and fixed on a PTFE support to reproduce the human nail shape. Different supports with different diameters (from 6 to 15 mm) are used in order to represent the bend of the human nails.
The access to intact human nails is often limited, so animal material has been used as substitute to study nail permeability and the effect of substances in contact with nails. Furthermore, using bovine hooves, the evaluation of safety and efficacy of ingredients and cosmetic products can be achieved. However, differences between animal hooves and human nails are reported in the literature and so caution is required in extrapolating such data to predict ungual effect on humans.
6
• • • • •
Evaluation of bovine hoof membrane density Density is calculated dividing the membrane weight for its volume. Density is a property that is similar between membranes that comes from the same part of the bovine hoof core. This parameter is very important cause similar density means similar tissue structure. Every membrane is being classified according to its density and structural firmness Membrane classification on the base of FI and density values
Aim of this work is to define a
Samples are stored in a climatic chamber (30°C and 50% R.H .)
2
Set up of a new reliable and reproducible method for bovine hoof membranes production. Morphological and mechanical analysis of the substrates. Desquamation and structural firmness are inversely proportional. Firmness and density are related. Membranes with 100 µm of thickness seemed to be very useful for permeability studies instead of 200-400 µm are the best thickness range to simulate in vivo response in studies concerning the efficacy and the safety of ingredients and final products.
methodological approach to standardize samples obtained from animals to improve reliability and reproducibility of the data in order to predict human nail responses.
5
Group
Firmness Index (D.S.)
A1
3.74 (0.57)
0.16
A2
7.45 (1.13)
0.19
A3
18.24 (1.72)
0.21
Density (mg/mm³)
THICKNESS (µm)
Density (mg/mm³)
y = 0.0317ln(x) + 0.124 R² = 0.9178
0
Mechanical Characterization: Nail StrainStress Meter NM 100 provides data for the classification of membranes. Two different tests are performed on every membrane, each test is repeated 5 times.
Firmness Index (FI) The sample is compressed punctually. The slope of the linear regression curve indicates the structural firmness of the membrane. The increase of this parameter means that the sample is more resistant to deformation. Thickness: The thickness of the membrane is directly determined by the compression analysis .
3
4
Membranes with the same rate of desquamation show similar Firmness Index.
ROUGHNESS ++++ (A) +++(B)
99.49 (28.31)
++(C)
113.64 (32.98)
+(D)
140.75 48.16)
30
B
Firmness Index (S.D.) 45.72 (13.38)
20 Firmness Index
A
ROUGHNESS and FIRMNESS INDEX correlation Membranes are divided according to their roughness, evaluated by morphological analysis.
10
C
D
References
Musitelli G. et al., TO2013A001064 , PCT/IB2014/067260 D. Monti et al, Br. J. Dermatol., vol. 165, no. 1, pp. 99–105, 2011. D. Miron et al., Eur. J. Pharm. Sci., vol. 51, pp. 20–25, 2014.
