- Email: [email protected]
Development of an in vitro test to evaluate cerumen dissolving properties of several veterinary ear cleansing solutions
J. DRUG DEL. SCI. TECH., 14 (3) 235-238 2004
Development of an in vitro test to evaluate cerumen dissolving properties of several veterinary ear cleansing solutions F. Nielloud1*, C.A. Rème2, R. Fortuné1, J.P. Laget1, G. Mestres1, H. Gatto2 Laboratoire de Technique pharmaceutique industrielle, Faculté de Pharmacie, Université Montpellier-I , Avenue Charles-Flahault, BP 14492, 34093 Montpellier Cedex, France 2 Laboratoires Virbac, 13e rue, LID, BP 27, 06511 Carros Cedex, France *Correspondence: [email protected]
1
The aim of the present study was to evaluate objectively the cleansing properties of four commercially available veterinary ear cleansing solutions. An in vitro test was developed, based on dissolution of artificial cerumen. In addition, the wetting properties of the solutions were evaluated using a standard normalized reference method, of common use in the cosmetic field. Formulations investigated (Otolane, Cerulane, Otoclean and Virbac Physiologic Ear Cleanser) are quite different in composition. In the cerumen dissolution test, artificial reconstituted dog cerumen (lanolin, fatty acids, hydrocarbons) was suspended in test products, under conditions approximating in vivo administration and undissolved cerumen was weighed after 10 min contact time. Two of the test products (Otolane and Cerulane) did not demonstrate any dissolving or wetting properties. By contrast, the two other formulations (Otoclean and Virbac Physiologic ear cleanser) demonstrated interesting cleansing properties, because of confirmed cerumen dissolving and wetting activity. Virbac Physiologic Ear Cleanser was the only test solution that demonstrated statistical greater ability for cerumen dissolution, as compared to distilled water, suggesting improved cleansing efficiency. Key words: Canine cerumen – Dissolution – Wetting properties – Cleansing properties – Veterinary commercial formulations – In vitro test.
Cerumen presents as a yellow wax, secreted by the glands of the ear canal to coat the aural integument, and protect the ear from injury and invasion by micro-organisms. Cerumen is composed of desquamated epidermal cells and ceruminous glands secretions. Canine cerumen was reported to enhance the growth of the yeast Malassezia pachydermatis, a frequent resident organism in the ear canal of dogs. Auricular gland hyperplasia, humidity and abnormal cell-mediated immunity are favourable factors for yeast proliferation [1, 2]. Accumulation of cerumen in the external ear canal may predispose to otitis externa in dogs. Successful treatment of otitis externa requires careful cleansing of the external auditory canal to remove lipid secretions and exudate, before allowing therapeutic topical preparations to contact the diseased epithelium and target micro-organisms. Several authors have studied ear wax composition in order to investigate the relationships between normal ear physiology and the pathogenesis of otitis externa [3-5]. They showed that cerumen viscosity and hydrophobic properties result from a complex combination of different lipid classes, suggesting the use of ear cleansers of appropriate composition for the prophylaxis of otitis externa. Numerous ear cleansers commercially available to veterinarians contain a variety of ingredients, such as alcohols, organic acids, propylene glycol, various peroxides, and detergents, while few published reports document the efficacy of such ingredients [6]. Several studies have investigated ceruminolytic agents tolerance in vivo, but no published data demonstrate ear cleansers efficacy for earwax removal in dogs. The present study aimed to assess and compare objectively the cleansing properties of different solutions. A satisfactory ear cleanser should demonstrate (i) good wetting characteristics, in order to come into close contact with the surface to be cleaned, and (ii) ability to dissolve or disperse dirt and prevent redeposit.
An in vitro test was developed to evaluate the ability of four commercially available veterinary auricular solutions to dissolve cerumen, as no reference method was available to date. The wetting characteristics of the solutions were analysed using the reference ISO 8022 (NF T 73-420) method. Joint study of the dissolution and wetting properties of the solutions aimed at providing a simple and reliable predicting method to test the efficacy of the ear cleanser products.
I. MATERIALS AND METHODS 1. Artificial cerumen composition
Several authors have elucidated the lipid composition of canine cerumen. Different lipid classes include: a complex mixture of diterpenoids and steroids (in particular cholesterol and cholesterol esters), saturated and unsaturated C16-C18 free fatty acids and C5-C25 straight-chain hydrocarbons. In accordance with this composition, an artificial cerumen was elaborated based on various lipids, with a melting point close to 37°C and a consistency very close to canine cerumen. Composition of the artificial reconstituted cerumen is detailed in Table I. Lanolin and the different fatty acids were provided by Merck Eurolab (Fontenay-sous-Bois, France) and the mineral oil was obtained from Shell Direct (Rognac, France). Waxes were melted and then homogenized to obtain artificial cerumen.
2. Commercial formulations
Physiologic Ear Cleanser was provided by Virbac Laboratory (Carros, France). Otolane and Cerulane were purchased from TVM Laboratory (Lempdes, France), while Otoclean was purchased from Janssen-Cilag Santé Animale (Issy-les-Moulineaux, France). Distilled water was used as a control solution. Compositions of test products are specified in Table II, according to manufacturers labelling. Marked differences are easily noticeable in the composition of the four commercial 235
Development of an in vitro test to evaluate cerumen dissolving properties of several veterinary ear cleansing solutions F. Nielloud, C.A. Rème, R. Fortuné, J.P. Laget, G. Mestres, H. Gatto
J. DRUG DEL. SCI. TECH., 14 (3) 235-238 2004
the disc to drown was below 300 s, three subsequent dilutions were performed in order to plot a line and find the concentration necessary to obtain disc drowning in 100 s. If the time for the disc to drown was above 300 s, the product was considered as failing to demonstrate any wetting ability.
Table I - Composition of artificial cerumen. Lipids
Weight (%) 30 30 10 10 10 10
Lanolin Palmitic acid Myristic acid Oleic acid Linoleic acid Paraffin oil
II. RESULTS AND DISCUSSION 1. Dissolution assays: macroscopic observations
Table II - Composition of tested commercial ear cleansing formulations. Virbac Physiologic Ear Cleanser
Otolane (TVM)
Cerulane (TVM)
Otoclean (Janssen)
Sorbitan ester NaCl Sodium EDTA PEG glyceride Citrus extract
Chlorhexidine digluconate Acetic acid Neutral cleansing base Soothing agents
Purified sea water
Salicylic acid Propylene glycol Polyglycol Glycerol Ethoxydiglycol Lactic acid Cucumis sativus Cetraria islandica Mimosa tenuiflora Oleic acid
Macroscopical appearance of in vitro dissolution assays before and after vortex agitation is displayed in Figure 1. Before agitation, all five solutions remained clear while cerumen adhered to the tube walls. After 10 s of agitation, no detectable changes were recorded for control distilled water and Cerulane: cerumen still adhered to the tube and solutions remained clear. By contrast, substantial modifications were recorded in the test tubes containing the three other cleansing solutions. The Otoclean solution became turbid with homogeneous dispersion of cerumen. The Otolane solution became partially opaque with cerumen clearly detached from the tube walls and floating at the surface. The Virbac Physiologic Ear Cleanser solution became completely white and opaque, reflecting the constitution of an emulsion. The latter finding strongly argues for a greater quantity of cerumen dissolved by the corresponding solution. The above mentioned macroscopic observations were of valuable interest to visualize cerumen dissolution ability of tested solutions during the experiments.
products. One basic solution contains purified sea water. The other formulations are more complex and include organic acids, polyols, vegetal extracts or surfactants.
3. In vitro cerumen dissolution assay
Cerumen dissolution tests were performed at 37°C. One millilitre of each test solution was poured into a hæmolysis tube containing 100 mg of artificial cerumen. Contact time between the solution and artificial cerumen was 10 min. Moderate agitation was then applied (Vortex, 10 s) to mimic in vivo application that shears off some of the dirt and favours colloidal dispersion. The solution was then vacuum-filtered on a filter paper (Whatman, porosity 10 µm). Undissolved cerumen remaining in the tube or on the paper, was weighted after drying. Quantity of dissolved cerumen was calculated by difference from 100 mg. The experiment was repeated five times at least for each formulation. One-way analysis of variance was performed to detect differences in mean quantity of cerumen dissolved between formulations [7].
(a)
(b)
(c)
(d)
(e)
4. Wetting power assay
Normalized in vitro method ISO 8022 (NF T 73-420) was implemented [8]. According to this procedure, the wetting power of a product is given by the concentration needed to impregnate a cotton disc, so that the disk immersed in a bulk solution drown down to the bottom of a tube in approximately 100 s. Standardized cotton disc were immersed in surfactant solutions, maintained by mean of a special grip. Complete penetration of the solutions into the fabric was evidenced by drowning of the disk. The time to wetting for three concentrations of test products were determined. Standardized cotton discs were obtained from Tissus Filtrants (Provins, France). Experimental tube content was 100 ml. Three to five measurements were performed for each concentration of test solutions. The products were first tested pure, undiluted. If the time for
Figure 1 - In vitro cerumen dissolution test. Up: cerumen suspended in test solutions for 10 min at 37°C. Down: after 10 s vortex agitation. (a) Distilled water. (b) Cerulane. (c) Otoclean. (d) Otolane. (e) Virbac Physiologic Ear Cleanser.
2. Quantification of cerumen dissolution
Several authors suggest that cerumen dissolution is of primary importance in the cleansing action of ear solutions [2, 4]. Mean quantities of cerumen dissolved by the different formulations are represented in Figure 2, together with standard deviations. A minimum of five measurements were performed for each solution. Homogeneity of variances was checked between the groups by use of the Cochranʼs C test (p = 0.502), validating 236
Development of an in vitro test to evaluate cerumen dissolving properties of several veterinary ear cleansing solutions F. Nielloud, C.A. Rème, R. Fortuné, J.P. Laget, G. Mestres, H. Gatto
40
450
quantity of dissolved cerumen wetting power index
35
log (time to cotton disk drowning)
g/l (wetting power)
mg/ml (dissolved cerumen)
400 350 300
30 25
250
20
200
15 10
150 100
5
50
0
0
Cerulane
Otolane
Distilled water
Otoclean
J. DRUG DEL. SCI. TECH., 14 (3) 235-238 2004
2,5 2 1,5 1 0,5 0 2,5
Virbac Physiologic Ear Cleanser
2,6
2,7
2,8
2,9
3
log (Concentration of test solution) Virbac Physiologic Ear Cleanser
Figure 2 - Mean quantities (±SD) of cerumen dissolved by the four solutions and control distilled water. Associated wetting power index.
Otoclean
Figure 3 - Wetting power of Otoclean and Virbac Physiologic Ear Cleanser as determined by ISO 8022 reference procedure.
the performance of analysis of variance to compare cerumen dissolution ability between test solutions. One-way ANOVA detected a significant difference for the quantity of cerumen dissolved between the ear cleansers (p = 0.033, 95% confidence level). A multiple comparison test was then performed to determine which means were significantly different. The method used to discriminate among the means was Fisherʼs least significant difference (LSD) procedure; results are recorded in Table III. Two homogeneous groups were identified using columns of Xs. Within each column, test solutions ticked with Xs formed a group with statistically homogeneous ceruminolytic activity. Mean quantities of cerumen dissolved by Cerulane, Otolane and Otoclean were no statistically different from that of control distilled water. As a matter of fact, Cerulane and Otolane dissolved nearly the same quantity of cerumen than distilled water. Otoclean dissolved a slightly greater quantity of cerumen (mean 14.72 mg/ml), although the difference from the control group was not statistically significant. Virbac Physiologic Ear Cleanser was the only test solution that demonstrated a greater statistical ability for cerumen dissolution (mean 23.24 mg/ml), as compared to distilled water, suggesting improved cleansing efficiency.
a function of the logarithm of the solution concentration. Linear plotting yielded correlation coefficients r always higher than 0.98. The linearity of the plots allowed using the equations to calculate the concentration (g/l) of each formulation necessary to obtain disk drowning in 100 s. The latter value represented the wetting power of the solution (Table IV). This parameter could not be calculated for Otolane and Cerulane, because the cotton disc did not drown before 300 s when immersed in undiluted solutions. Otolane and Cerulane therefore failed to demonstrate significant wetting ability. By contrast, Otoclean and Virbac Physiologic Ear Cleanser presented quite similar wetting power, around 420 g/l. Results of the wetting assays suggest that the latter solutions have singular ability to spread and being adsorbed on surfaces, which is of particular value to initiate the cleaning of dirty interfaces, like water-earwax in the ear canal. Calculated wetting power values for Otoclean and Virbac Physiologic Ear Cleanser were rather elevated and corresponded to values recorded for particular non-ionic agents used in the human cosmetic industry, particularly those with mild cleansing properties used for baby toiletry, with active surface action and very good tolerance profile on skin and mucosae.
Table III - Multiple range test to compare the quantity of cerumen dissolved by test products.
Table IV - Evaluation of the wetting properties of test solutions using ISO 8022-NF T 73-420 procedure.
Product
Cerulane Otolane Distilled water Otoclean Virbac Physiologic Ear Cleanser
Method: 95% LSD Count
Mean
Homogeneous groups
6 5 6 5 6
4.70 6.45 7.71 14.72 23.24
X X X X
X X
Product
Linear plot equation of log (time to cotton disk drowning) vs log (product conc.)
Correlation coefficient
Wetting power (g/l)
Otoclean Virbac Physiologic Ear Cleanser
Y = -2.954x + 9.768 Y = -1.015x + 4.662
0.9954 0.9998
426 419
4. Correlation between cerumen dissolution and wetting properties
3. Wetting power assay
Quantities of cerumen dissolved by the solutions and respective wetting power values are represented on the same graph (Figure 2). Products like Cerulane and Otoclean, which presented water-like cerumen dissolving properties, did not demonstrate either any significant wetting power (value set to 0, because both products failed to impregnate cotton disks). Yet, it is obvious that greasy dirt is not easily removed by water if not properly wetted, because not made readily soluble by surfactants.
The wetting process is a preliminary stage in many interfacial phenomena, including detergency, and is thus of particular interest to evaluate cleansing properties of solutions [9]. Results of the determination of test solutions wetting power, in compliance with the reference ISO method ISO 8022 (NF T 73-420), are illustrated in Figure 3, in which the logarithm of time to cotton disc impregnation and drowning is represented as 237
J. DRUG DEL. SCI. TECH., 14 (3) 235-238 2004
Development of an in vitro test to evaluate cerumen dissolving properties of several veterinary ear cleansing solutions F. Nielloud, C.A. Rème, R. Fortuné, J.P. Laget, G. Mestres, H. Gatto
These findings correlated with the macroscopic appearance of test solutions after vortex agitation in the cerumen dissolution assay, notably for Cerulane, a product entirely based on purified sea water. Otolane( contains a neutral cleansing base that could explain the observed dirt detachment from tube walls. Virbac Physiologic Ear Cleanser and Otoclean demonstrated both cerumen dissolution properties and significant wetting power. These findings also correlated with macroscopic observations of cerumen dissolution assays. Formulation issues may account for differences in product activity. Main components in Otoclean are polyols, among which ethoxydiglycol, well known for its cerumen dissolution properties. However, this component has low documented wetting power which could explain overall intermediate level of cerumen dissolution observed macroscopically in tube assays. Virbac Physiologic Ear Cleanser composition includes two non-ionic surfactants. Non-ionic surfactants are good dissolving and wetting agents that lower liquid superficial surface tension and therefore favour liquid distribution on surfaces [10, 11]. An essential component of detergency is dirt detachment from supporting surfaces thanks to surfactant adhesive properties and associated phenomena, such as flocculation, emulsification and induced solubility. Indeed Virbac Physiologic Ear Cleanser produced emulsification of the earwax, as evidenced macroscopically by the dissolution tube assays. Thus, this formulation could be of special interest, because added surfactants minimize the oil-water and solid-water interfacial tensions, inducing spontaneous cerumen removal. This ear cleansing product could act by mesophase formation, especially if dirt contains polar material, as it is the case with cerumen, which is a mixture of fatty acids, waxes and sterols esters. Non-ionic detergents also are effective against dirt redeposit, as a result of strong hydration of polyethylene oxide chains [12]. These combined characteristics may explain why Virbac Physiologic Ear Cleanser was the single product for which statistically significant difference in global cleansing power was detected in test tubes, as compared to the control group (distilled water). Development of an in vitro cerumen dissolution test in the present study, associated with application of the standard ISO normalized method for wetting power determination, proved a practical and valuable model to assess and compare objectively the cleansing power of veterinary ear cleansers. The above mentioned methods suggested that two of the four formulations tested could be more effective on earwax removal. Virbac Physiologic Ear Cleanser was one solution that presented
synergistic characteristics of wetting activity and cerumen dissolving and emulsifying ability, resulting in overall good cleansing properties. Since non-ionic surfactants used in the latter formulation are well known for being particularly well tolerated, this physiologic ear cleanser may combine a marked cleansing activity with respect of skin and mucosal epithelium in vivo. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12.
HUANG H.P., FIXTER L.M., LITTLE C.J.L. - Lipid content of cerumen from normal dogs and otitic canine ears. - Veterinary Record, 134, 380-381, 1994. MELMAN S.A. - Principles of ear disease and therapy. II. - Veterinary Forum, l (8), 62-63,78, 1993. HUANG H.P., FIXTER L.M., LITTLE C.J.L. - Canine cerumen fatty acid composition. - Guoil Taiwan Daxue Nongxueyuan Yanjiu Baogao, 35 (3), 375-392, 1995. BURKHART C.N., BURKHART C.G., WILLIAMS S., ANDREWS P.C., ADAPPA V. - In pursuit of ceruminolytic agents: a study of earwax composition. - Am. J. Otology, 21 (2), 157-160, 2000. BURKHART C.N., KRUGE M.A., BURKHART C.G., BLCK C. - Cerumen composition by flash pyrolysis-gas chromatography/ mass spectrometry. - Otol. Neurotol., 22 (6), 715-722, 2001. GOTTHELF L., YOUNG S.E. - New treatment of Malassezia otitis externa in dogs. - Veterinary Forum, August 25, 1997. Statgraphics Plus 5. - Manugistics, Maryland, US, 2000. French Standard NF T 73-420, ISO 8022. - Agents de surface, Détermination du pouvoir mouillant par immersion - méthode par étalonnage. - Afnor, 1985. BUCKTON G. - Surfactants. - In : Interfacial Phenomena in Drug Targeting, Serie Drug Targeting and Delivery, A.T Florence and G. Gregoriadis Eds., Harwood Academic Publishers, Chur,1995, pp. 135-161. ADAMSON A.W., GAST A.P. - Wetting, flotation and detergency. - In: Physical Chemistry of Surfaces, John Wiley & Sons, Inc., New York, 1997, pp. 484-500. HUNTER R.J. - Characterisation of colloidal dispersions. - In : Introduction to Modern Colloid Science, Oxford University Press, New York, 1994, pp 1-31. SHAW D.J. - The solid-liquid interface. - In: Colloid & Surface Chemistry, Butterworth-Heinemann Lyd, Oxford, 1994, pp 151173.
ACKNOWLEDGEMENTS We are grateful to P. Peralta for his technical assistance.
MANUSCRIPT Received 29 July 2003, accepted for publication 23 February 2004.
238
Development of an in vitro test to evaluate cerumen dissolving properties of several veterinary ear cleansing solutions F. Nielloud1*, C.A. Rème2, R. Fortuné1, J.P. Laget1, G. Mestres1, H. Gatto2 Laboratoire de Technique pharmaceutique industrielle, Faculté de Pharmacie, Université Montpellier-I , Avenue Charles-Flahault, BP 14492, 34093 Montpellier Cedex, France 2 Laboratoires Virbac, 13e rue, LID, BP 27, 06511 Carros Cedex, France *Correspondence: [email protected]
1
The aim of the present study was to evaluate objectively the cleansing properties of four commercially available veterinary ear cleansing solutions. An in vitro test was developed, based on dissolution of artificial cerumen. In addition, the wetting properties of the solutions were evaluated using a standard normalized reference method, of common use in the cosmetic field. Formulations investigated (Otolane, Cerulane, Otoclean and Virbac Physiologic Ear Cleanser) are quite different in composition. In the cerumen dissolution test, artificial reconstituted dog cerumen (lanolin, fatty acids, hydrocarbons) was suspended in test products, under conditions approximating in vivo administration and undissolved cerumen was weighed after 10 min contact time. Two of the test products (Otolane and Cerulane) did not demonstrate any dissolving or wetting properties. By contrast, the two other formulations (Otoclean and Virbac Physiologic ear cleanser) demonstrated interesting cleansing properties, because of confirmed cerumen dissolving and wetting activity. Virbac Physiologic Ear Cleanser was the only test solution that demonstrated statistical greater ability for cerumen dissolution, as compared to distilled water, suggesting improved cleansing efficiency. Key words: Canine cerumen – Dissolution – Wetting properties – Cleansing properties – Veterinary commercial formulations – In vitro test.
Cerumen presents as a yellow wax, secreted by the glands of the ear canal to coat the aural integument, and protect the ear from injury and invasion by micro-organisms. Cerumen is composed of desquamated epidermal cells and ceruminous glands secretions. Canine cerumen was reported to enhance the growth of the yeast Malassezia pachydermatis, a frequent resident organism in the ear canal of dogs. Auricular gland hyperplasia, humidity and abnormal cell-mediated immunity are favourable factors for yeast proliferation [1, 2]. Accumulation of cerumen in the external ear canal may predispose to otitis externa in dogs. Successful treatment of otitis externa requires careful cleansing of the external auditory canal to remove lipid secretions and exudate, before allowing therapeutic topical preparations to contact the diseased epithelium and target micro-organisms. Several authors have studied ear wax composition in order to investigate the relationships between normal ear physiology and the pathogenesis of otitis externa [3-5]. They showed that cerumen viscosity and hydrophobic properties result from a complex combination of different lipid classes, suggesting the use of ear cleansers of appropriate composition for the prophylaxis of otitis externa. Numerous ear cleansers commercially available to veterinarians contain a variety of ingredients, such as alcohols, organic acids, propylene glycol, various peroxides, and detergents, while few published reports document the efficacy of such ingredients [6]. Several studies have investigated ceruminolytic agents tolerance in vivo, but no published data demonstrate ear cleansers efficacy for earwax removal in dogs. The present study aimed to assess and compare objectively the cleansing properties of different solutions. A satisfactory ear cleanser should demonstrate (i) good wetting characteristics, in order to come into close contact with the surface to be cleaned, and (ii) ability to dissolve or disperse dirt and prevent redeposit.
An in vitro test was developed to evaluate the ability of four commercially available veterinary auricular solutions to dissolve cerumen, as no reference method was available to date. The wetting characteristics of the solutions were analysed using the reference ISO 8022 (NF T 73-420) method. Joint study of the dissolution and wetting properties of the solutions aimed at providing a simple and reliable predicting method to test the efficacy of the ear cleanser products.
I. MATERIALS AND METHODS 1. Artificial cerumen composition
Several authors have elucidated the lipid composition of canine cerumen. Different lipid classes include: a complex mixture of diterpenoids and steroids (in particular cholesterol and cholesterol esters), saturated and unsaturated C16-C18 free fatty acids and C5-C25 straight-chain hydrocarbons. In accordance with this composition, an artificial cerumen was elaborated based on various lipids, with a melting point close to 37°C and a consistency very close to canine cerumen. Composition of the artificial reconstituted cerumen is detailed in Table I. Lanolin and the different fatty acids were provided by Merck Eurolab (Fontenay-sous-Bois, France) and the mineral oil was obtained from Shell Direct (Rognac, France). Waxes were melted and then homogenized to obtain artificial cerumen.
2. Commercial formulations
Physiologic Ear Cleanser was provided by Virbac Laboratory (Carros, France). Otolane and Cerulane were purchased from TVM Laboratory (Lempdes, France), while Otoclean was purchased from Janssen-Cilag Santé Animale (Issy-les-Moulineaux, France). Distilled water was used as a control solution. Compositions of test products are specified in Table II, according to manufacturers labelling. Marked differences are easily noticeable in the composition of the four commercial 235
Development of an in vitro test to evaluate cerumen dissolving properties of several veterinary ear cleansing solutions F. Nielloud, C.A. Rème, R. Fortuné, J.P. Laget, G. Mestres, H. Gatto
J. DRUG DEL. SCI. TECH., 14 (3) 235-238 2004
the disc to drown was below 300 s, three subsequent dilutions were performed in order to plot a line and find the concentration necessary to obtain disc drowning in 100 s. If the time for the disc to drown was above 300 s, the product was considered as failing to demonstrate any wetting ability.
Table I - Composition of artificial cerumen. Lipids
Weight (%) 30 30 10 10 10 10
Lanolin Palmitic acid Myristic acid Oleic acid Linoleic acid Paraffin oil
II. RESULTS AND DISCUSSION 1. Dissolution assays: macroscopic observations
Table II - Composition of tested commercial ear cleansing formulations. Virbac Physiologic Ear Cleanser
Otolane (TVM)
Cerulane (TVM)
Otoclean (Janssen)
Sorbitan ester NaCl Sodium EDTA PEG glyceride Citrus extract
Chlorhexidine digluconate Acetic acid Neutral cleansing base Soothing agents
Purified sea water
Salicylic acid Propylene glycol Polyglycol Glycerol Ethoxydiglycol Lactic acid Cucumis sativus Cetraria islandica Mimosa tenuiflora Oleic acid
Macroscopical appearance of in vitro dissolution assays before and after vortex agitation is displayed in Figure 1. Before agitation, all five solutions remained clear while cerumen adhered to the tube walls. After 10 s of agitation, no detectable changes were recorded for control distilled water and Cerulane: cerumen still adhered to the tube and solutions remained clear. By contrast, substantial modifications were recorded in the test tubes containing the three other cleansing solutions. The Otoclean solution became turbid with homogeneous dispersion of cerumen. The Otolane solution became partially opaque with cerumen clearly detached from the tube walls and floating at the surface. The Virbac Physiologic Ear Cleanser solution became completely white and opaque, reflecting the constitution of an emulsion. The latter finding strongly argues for a greater quantity of cerumen dissolved by the corresponding solution. The above mentioned macroscopic observations were of valuable interest to visualize cerumen dissolution ability of tested solutions during the experiments.
products. One basic solution contains purified sea water. The other formulations are more complex and include organic acids, polyols, vegetal extracts or surfactants.
3. In vitro cerumen dissolution assay
Cerumen dissolution tests were performed at 37°C. One millilitre of each test solution was poured into a hæmolysis tube containing 100 mg of artificial cerumen. Contact time between the solution and artificial cerumen was 10 min. Moderate agitation was then applied (Vortex, 10 s) to mimic in vivo application that shears off some of the dirt and favours colloidal dispersion. The solution was then vacuum-filtered on a filter paper (Whatman, porosity 10 µm). Undissolved cerumen remaining in the tube or on the paper, was weighted after drying. Quantity of dissolved cerumen was calculated by difference from 100 mg. The experiment was repeated five times at least for each formulation. One-way analysis of variance was performed to detect differences in mean quantity of cerumen dissolved between formulations [7].
(a)
(b)
(c)
(d)
(e)
4. Wetting power assay
Normalized in vitro method ISO 8022 (NF T 73-420) was implemented [8]. According to this procedure, the wetting power of a product is given by the concentration needed to impregnate a cotton disc, so that the disk immersed in a bulk solution drown down to the bottom of a tube in approximately 100 s. Standardized cotton disc were immersed in surfactant solutions, maintained by mean of a special grip. Complete penetration of the solutions into the fabric was evidenced by drowning of the disk. The time to wetting for three concentrations of test products were determined. Standardized cotton discs were obtained from Tissus Filtrants (Provins, France). Experimental tube content was 100 ml. Three to five measurements were performed for each concentration of test solutions. The products were first tested pure, undiluted. If the time for
Figure 1 - In vitro cerumen dissolution test. Up: cerumen suspended in test solutions for 10 min at 37°C. Down: after 10 s vortex agitation. (a) Distilled water. (b) Cerulane. (c) Otoclean. (d) Otolane. (e) Virbac Physiologic Ear Cleanser.
2. Quantification of cerumen dissolution
Several authors suggest that cerumen dissolution is of primary importance in the cleansing action of ear solutions [2, 4]. Mean quantities of cerumen dissolved by the different formulations are represented in Figure 2, together with standard deviations. A minimum of five measurements were performed for each solution. Homogeneity of variances was checked between the groups by use of the Cochranʼs C test (p = 0.502), validating 236
Development of an in vitro test to evaluate cerumen dissolving properties of several veterinary ear cleansing solutions F. Nielloud, C.A. Rème, R. Fortuné, J.P. Laget, G. Mestres, H. Gatto
40
450
quantity of dissolved cerumen wetting power index
35
log (time to cotton disk drowning)
g/l (wetting power)
mg/ml (dissolved cerumen)
400 350 300
30 25
250
20
200
15 10
150 100
5
50
0
0
Cerulane
Otolane
Distilled water
Otoclean
J. DRUG DEL. SCI. TECH., 14 (3) 235-238 2004
2,5 2 1,5 1 0,5 0 2,5
Virbac Physiologic Ear Cleanser
2,6
2,7
2,8
2,9
3
log (Concentration of test solution) Virbac Physiologic Ear Cleanser
Figure 2 - Mean quantities (±SD) of cerumen dissolved by the four solutions and control distilled water. Associated wetting power index.
Otoclean
Figure 3 - Wetting power of Otoclean and Virbac Physiologic Ear Cleanser as determined by ISO 8022 reference procedure.
the performance of analysis of variance to compare cerumen dissolution ability between test solutions. One-way ANOVA detected a significant difference for the quantity of cerumen dissolved between the ear cleansers (p = 0.033, 95% confidence level). A multiple comparison test was then performed to determine which means were significantly different. The method used to discriminate among the means was Fisherʼs least significant difference (LSD) procedure; results are recorded in Table III. Two homogeneous groups were identified using columns of Xs. Within each column, test solutions ticked with Xs formed a group with statistically homogeneous ceruminolytic activity. Mean quantities of cerumen dissolved by Cerulane, Otolane and Otoclean were no statistically different from that of control distilled water. As a matter of fact, Cerulane and Otolane dissolved nearly the same quantity of cerumen than distilled water. Otoclean dissolved a slightly greater quantity of cerumen (mean 14.72 mg/ml), although the difference from the control group was not statistically significant. Virbac Physiologic Ear Cleanser was the only test solution that demonstrated a greater statistical ability for cerumen dissolution (mean 23.24 mg/ml), as compared to distilled water, suggesting improved cleansing efficiency.
a function of the logarithm of the solution concentration. Linear plotting yielded correlation coefficients r always higher than 0.98. The linearity of the plots allowed using the equations to calculate the concentration (g/l) of each formulation necessary to obtain disk drowning in 100 s. The latter value represented the wetting power of the solution (Table IV). This parameter could not be calculated for Otolane and Cerulane, because the cotton disc did not drown before 300 s when immersed in undiluted solutions. Otolane and Cerulane therefore failed to demonstrate significant wetting ability. By contrast, Otoclean and Virbac Physiologic Ear Cleanser presented quite similar wetting power, around 420 g/l. Results of the wetting assays suggest that the latter solutions have singular ability to spread and being adsorbed on surfaces, which is of particular value to initiate the cleaning of dirty interfaces, like water-earwax in the ear canal. Calculated wetting power values for Otoclean and Virbac Physiologic Ear Cleanser were rather elevated and corresponded to values recorded for particular non-ionic agents used in the human cosmetic industry, particularly those with mild cleansing properties used for baby toiletry, with active surface action and very good tolerance profile on skin and mucosae.
Table III - Multiple range test to compare the quantity of cerumen dissolved by test products.
Table IV - Evaluation of the wetting properties of test solutions using ISO 8022-NF T 73-420 procedure.
Product
Cerulane Otolane Distilled water Otoclean Virbac Physiologic Ear Cleanser
Method: 95% LSD Count
Mean
Homogeneous groups
6 5 6 5 6
4.70 6.45 7.71 14.72 23.24
X X X X
X X
Product
Linear plot equation of log (time to cotton disk drowning) vs log (product conc.)
Correlation coefficient
Wetting power (g/l)
Otoclean Virbac Physiologic Ear Cleanser
Y = -2.954x + 9.768 Y = -1.015x + 4.662
0.9954 0.9998
426 419
4. Correlation between cerumen dissolution and wetting properties
3. Wetting power assay
Quantities of cerumen dissolved by the solutions and respective wetting power values are represented on the same graph (Figure 2). Products like Cerulane and Otoclean, which presented water-like cerumen dissolving properties, did not demonstrate either any significant wetting power (value set to 0, because both products failed to impregnate cotton disks). Yet, it is obvious that greasy dirt is not easily removed by water if not properly wetted, because not made readily soluble by surfactants.
The wetting process is a preliminary stage in many interfacial phenomena, including detergency, and is thus of particular interest to evaluate cleansing properties of solutions [9]. Results of the determination of test solutions wetting power, in compliance with the reference ISO method ISO 8022 (NF T 73-420), are illustrated in Figure 3, in which the logarithm of time to cotton disc impregnation and drowning is represented as 237
J. DRUG DEL. SCI. TECH., 14 (3) 235-238 2004
Development of an in vitro test to evaluate cerumen dissolving properties of several veterinary ear cleansing solutions F. Nielloud, C.A. Rème, R. Fortuné, J.P. Laget, G. Mestres, H. Gatto
These findings correlated with the macroscopic appearance of test solutions after vortex agitation in the cerumen dissolution assay, notably for Cerulane, a product entirely based on purified sea water. Otolane( contains a neutral cleansing base that could explain the observed dirt detachment from tube walls. Virbac Physiologic Ear Cleanser and Otoclean demonstrated both cerumen dissolution properties and significant wetting power. These findings also correlated with macroscopic observations of cerumen dissolution assays. Formulation issues may account for differences in product activity. Main components in Otoclean are polyols, among which ethoxydiglycol, well known for its cerumen dissolution properties. However, this component has low documented wetting power which could explain overall intermediate level of cerumen dissolution observed macroscopically in tube assays. Virbac Physiologic Ear Cleanser composition includes two non-ionic surfactants. Non-ionic surfactants are good dissolving and wetting agents that lower liquid superficial surface tension and therefore favour liquid distribution on surfaces [10, 11]. An essential component of detergency is dirt detachment from supporting surfaces thanks to surfactant adhesive properties and associated phenomena, such as flocculation, emulsification and induced solubility. Indeed Virbac Physiologic Ear Cleanser produced emulsification of the earwax, as evidenced macroscopically by the dissolution tube assays. Thus, this formulation could be of special interest, because added surfactants minimize the oil-water and solid-water interfacial tensions, inducing spontaneous cerumen removal. This ear cleansing product could act by mesophase formation, especially if dirt contains polar material, as it is the case with cerumen, which is a mixture of fatty acids, waxes and sterols esters. Non-ionic detergents also are effective against dirt redeposit, as a result of strong hydration of polyethylene oxide chains [12]. These combined characteristics may explain why Virbac Physiologic Ear Cleanser was the single product for which statistically significant difference in global cleansing power was detected in test tubes, as compared to the control group (distilled water). Development of an in vitro cerumen dissolution test in the present study, associated with application of the standard ISO normalized method for wetting power determination, proved a practical and valuable model to assess and compare objectively the cleansing power of veterinary ear cleansers. The above mentioned methods suggested that two of the four formulations tested could be more effective on earwax removal. Virbac Physiologic Ear Cleanser was one solution that presented
synergistic characteristics of wetting activity and cerumen dissolving and emulsifying ability, resulting in overall good cleansing properties. Since non-ionic surfactants used in the latter formulation are well known for being particularly well tolerated, this physiologic ear cleanser may combine a marked cleansing activity with respect of skin and mucosal epithelium in vivo. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12.
HUANG H.P., FIXTER L.M., LITTLE C.J.L. - Lipid content of cerumen from normal dogs and otitic canine ears. - Veterinary Record, 134, 380-381, 1994. MELMAN S.A. - Principles of ear disease and therapy. II. - Veterinary Forum, l (8), 62-63,78, 1993. HUANG H.P., FIXTER L.M., LITTLE C.J.L. - Canine cerumen fatty acid composition. - Guoil Taiwan Daxue Nongxueyuan Yanjiu Baogao, 35 (3), 375-392, 1995. BURKHART C.N., BURKHART C.G., WILLIAMS S., ANDREWS P.C., ADAPPA V. - In pursuit of ceruminolytic agents: a study of earwax composition. - Am. J. Otology, 21 (2), 157-160, 2000. BURKHART C.N., KRUGE M.A., BURKHART C.G., BLCK C. - Cerumen composition by flash pyrolysis-gas chromatography/ mass spectrometry. - Otol. Neurotol., 22 (6), 715-722, 2001. GOTTHELF L., YOUNG S.E. - New treatment of Malassezia otitis externa in dogs. - Veterinary Forum, August 25, 1997. Statgraphics Plus 5. - Manugistics, Maryland, US, 2000. French Standard NF T 73-420, ISO 8022. - Agents de surface, Détermination du pouvoir mouillant par immersion - méthode par étalonnage. - Afnor, 1985. BUCKTON G. - Surfactants. - In : Interfacial Phenomena in Drug Targeting, Serie Drug Targeting and Delivery, A.T Florence and G. Gregoriadis Eds., Harwood Academic Publishers, Chur,1995, pp. 135-161. ADAMSON A.W., GAST A.P. - Wetting, flotation and detergency. - In: Physical Chemistry of Surfaces, John Wiley & Sons, Inc., New York, 1997, pp. 484-500. HUNTER R.J. - Characterisation of colloidal dispersions. - In : Introduction to Modern Colloid Science, Oxford University Press, New York, 1994, pp 1-31. SHAW D.J. - The solid-liquid interface. - In: Colloid & Surface Chemistry, Butterworth-Heinemann Lyd, Oxford, 1994, pp 151173.
ACKNOWLEDGEMENTS We are grateful to P. Peralta for his technical assistance.
MANUSCRIPT Received 29 July 2003, accepted for publication 23 February 2004.
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