Performance and Tolerance of a Protocol for Idiopathic Chronic Greasy Seborrhea in 18 Dogs Using a Shampoo and Mousse Containing Plant Extracts

Simple Summary Greasy skin and excessive skin scaling accompany numerous dermatoses. Rapid and sustainable improvement of skin appearance and application practicability are important features to enhance the dog’s appearance and encourage owner compliance. The study aimed to evaluate the tolerance and performance of one shampoo and subsequent mousses applications containing plant extracts in dogs with greasy skin. Six dogs were washed with plain water on day (D)0 and served as negative controls. Twelve dogs were shampooed on D0 and received eight mousse applications at 48–72 h intervals from D2 to D18. Skin was evaluated on D0, D0 + 4 h, D7, D14 and D24. At baseline, there were no significant differences observed between groups. In the control group, skin appearance and lipid contents remained stable throughout the study. Skin lesions and malodour were significantly reduced in the test group from D7 with no side effects. Hydration and hair lipids levels decreased in the test group at D0 + 4 h and returned to baseline from D14 and D7 on, respectively. In conclusion, one shampoo and subsequent mousse applications rapidly and safely improved coat and skin appearance in dogs with greasy skin and dandruff without affecting water and hair lipid contents. Abstract The study aimed to evaluate the tolerance, performance and effect on hair lipids and skin hydration of a protocol combining applications of one shampoo and subsequent mousses containing plant extracts (Ophytrium and Seboliance) in dogs with an undiagnosed chronic greasy keratinisation disorder. Six dogs were washed with plain water on day (D)0. Twelve dogs were shampooed on D0 and received eight mousse applications at 48–72 h intervals from D2 to D18. Clinical score (CS), Natural Moisturizing Factors (NMF) and hair lipids (HL) were evaluated on D0, D0 + 4 h, D7, D14 and D24. At baseline, no significant differences were observed in CS, NMF and HL between groups. In the control group, CS and HL remained stable throughout the study while a slight decrease in NMF was observed at D0 + 4 h. CS was significantly reduced in the test group between D0 and D7 (−53%) which reached 91% at D24 (p < 0.05), with no side effects. NMF levels decreased in the test group at D0 + 4 h (−73%, p < 0.0001) and returned to baseline from D14. In conclusion, one shampoo and subsequent mousse applications rapidly and safely improved coat quality in dogs with an undiagnosed keratinisation disorder without affecting NMF and HL contents over the study period.


Design of the Study
This was a prospective, randomized, controlled 24-day study. Each dog was allocated a number from 1 to 18 for the duration of the study. Before beginning the study, the 18 dogs were randomly (simple randomization, Research Randomizer (Version 4.0) [24]) assigned to group A (negative control group; six dogs) or group B (DOUXO ® S3 SEB Shampoo and Mousse, Ceva Santé Animale, Libourne, France group; 12 dogs). The two groups were housed in separate kennels and never came into direct or indirect contact with dogs in the other group during the course of the study.
The six dogs in group A were hand washed with plain water (temperature of approx. 15 • C) on day (D) 0 for 10 min and received no further intervention. In group B, the 12 dogs were shampooed on D0 with the test shampoo (1 pump dose/2 kg for short-haired dogs and 1 pump dose/kg in dense and/or long-haired dogs). The shampoo was applied on a pre-wet haircoat, massaged and left on for ten minutes before rinsing. All dogs were left to air dry. Dogs subsequently received an application of DOUXO ® S3 SEB Mousse, Ceva Santé Animale, Libourne, France on D2, 4, 7, 9, 11, 14, 16 and 18 (1 pump dose/2 kg for short-Vet. Sci. 2023, 10, 95 4 of 12 haired dogs and 1 pump dose /kg in dense and/or long-haired dogs). All applications were carried out by trained staff who were not involved in the clinical evaluation. The owner was instructed to monitor any adverse event and to report to the investigator in due course. The study was conducted during the closed hunting season.

Clinical Assessment
After a general clinical examination, the dogs were dermatologically evaluated on D0, D7, D14 and D24. A scoring index, adapted from Viaud et al. [25] was used which allowed attribution of a clinical score (CS). Briefly, the following parameters were assessed on a 0-3 scale: malodour, scaling, greasiness, haircoat quality and extent of the affected area ( Table 1). The maximum possible CS was 15. The intensity of pruritus was evaluated by the owner using a visual analogue scale [26] on the same days.

Skin Surface Cytology
To evaluate an eventual proliferation of bacteria and/or yeasts on the skin surface, surface cytologies were performed. Acetate tape preparations were performed on D0, 7, 14 and 24 by repeatedly pressing a 1 × 1 cm area of a strip of clear acetate tape (Scotch ® Crystal; 3 M; Cergy-Pontoise, France) against the most affected site of the skin for five seconds before removing it. Collected samples were analysed microscopically after staining (RAL555 ® ; RAL Diagnostics; Site Montesquieu-Martillac, France). Ten high power fields (HPF) were examined at ×1000 magnification and cytological findings were scored semi-quantitatively using a 0-4 scale as described by Bouassiba et al. [27].

Natural Moisturising Factor (NMF) Content
In order to evaluate a possible variation in skin hydration during the study, the content of NMFs was measured, as NMFs are hydrophilic markers of stratum corneum [28]. Skin surface samples were taken for NMFs analysis on D0, D0 + 4 h, D7, D14 and D24 by rubbing a 5-cm line on the skin with two swabs (Dryswab ® MWE, Medical Wire & Equipment, Wiltshire, UK) previously soaked in an aqueous non-ionic surfactant solution (QIMA, Labège, France-proprietary method), using a standardised, previously validated, sampling technique (unpublished data). The swab heads were then removed, placed in dry Eppendorf tubes (Safe-Lock Tubes 1.5 mL, Eppendorf AG, Hamburg, Germany) and frozen at −20 • C until analysis.
NMFs of interest (urocanic acid (cis/trans-UCA), pyrrolidone carboxylic acid (PCA) and serine were extracted using an aqueous solution, then analysed by a LC/MS system (UltiMate 3000 liquid chromatography system (ThermoScientific, Sunnyvale, CA, USA) coupled with a MSQ Plus Mass detector (ThermoScientific, Sunnyvale, CA, USA). Results are expressed in µg per sample.

Hair Surface Lipids
To assess the impact of products applications on the amounts of lipids in the coat, hair surface lipids were quantified. Hair samples were collected from the dorsum of each dog by shaving hair shafts with a clipper (Aesculap Isis clipper GT421, B Braun Medical; Saint Vet. Sci. 2023, 10, 95 5 of 12 Cloud, France) on D0, D0 + 4 h, D7, D14 and D24. An area of approximately 480 mm 2 was clipped on the dorsum of each dog on each sampling occasion. Samples were conserved in plastic vials (Greiner Bio-one CELLSTAR ® , Greiner Bio-One, Kremsmünster, Austria) at −20 • C until analysis.
Before analysis, 100 mg of hair fibres were cut and washed in a mixture of water, sodium dodecylsulphate and hexane. Total lipids were extracted using the method of Bligh and Dyer [29]. Mass fraction results are expressed in µg of total lipids per mg of hair fibres.

Tolerance
After the shampoo was applied, tolerance was evaluated on the following in-house scale: very bad (withdrawal movement, defence movement, or pruritic behaviours with vocalization), bad (withdrawal movement, defence or pruritic behaviours), medium (pruritic behaviours after application), good (rare pruritic behaviours after application) or excellent (no pruritic behaviours after application). Adverse events occurring in between visits were recorded.

Investigator and Owner Satisfaction
At the end of the study, the following items were evaluated on a 0-4 scale (0-very bad, 1-bad, 2-average, 3-good, 4-excellent): overall assessment of tolerance by the investigator; overall assessment of tolerance by the owner; overall assessment of performance by the investigator and overall assessment of performance by the owner.

Statistical Methods
The Shapiro-Wilk normality test was used to test data for normality distribution. When the distribution of the data was normal, Bartlett's test was used to verify homogeneity of variance and data collected on the different sampling days were compared using the parametric ANOVA 1 test. For data that showed statistically significant differences over time, Bonferroni's multiple comparison test was computed to compare individual time points within the group. In the absence of a normal distribution at all-time points, nonparametric Mann-Whitney tests were used. Statistical analyses were performed using XLSTAT software (Microsoft ® , version Base 2019.4.1.63305) and a two-sided p-value < 0.05 was considered statistically significant.

Animal Population
Group A (negative control group) included five male dogs and one female (five Griffons and one Bruno du Jura). The median age was 4 years (range 2-5) and median weight 27.3 kg (range [24][25][26][27][28][29][30][31][32][33]. Group B (DOUXO ® S3 SEB group) comprised 10 male dogs and 2 females (six Griffons, three Bruno du Jura and three Bleu de Gascogne), median age 5 years (range 1-8), median weight 27.0 kg (range 20-29). The differences in breed, sex, age or weight between the two groups were not statistically significant. Details are given in Table 2. Table 2. Details of the distribution of the animals between the two groups of dogs with keratinization disorders that were washed in plain water (group A) or received DOUXO ® S3 SEB shampoo and mousse (Ceva Santé Animale, France) (group B).

Clinical Score
The baseline clinical scores were similar in the two groups (median 8, min 4, max 10 and 13, group A and B, respectively) and in group A, the clinical scores remained steady throughout the study (median between 7 and 8.5, min 4 or 5 and max 9 or 10, p > 0.05). Conversely, a statistically significant reduction in CS was observed in group B on D7 compared to on D0 (median 3.5, min 1, max 8) (p = 0.002), reaching the lowest score on D24 with 91% of reduction (median 1, min 0, max 2) (p < 0.001)- Table 3 and Figure 1. Table 3. Results of the clinical and biochemical parameters at different time-points of the two groups of dogs with keratinization disorders that were washed in plain water (group A) or received DOUXO ® S3 SEB shampoo and mousse (Ceva Santé Animale, France) (group B).

Clinical Score
The baseline clinical scores were similar in the two groups (median 8, min 4, max 10 and 13, group A and B, respectively) and in group A, the clinical scores remained steady throughout the study (median between 7 and 8.5, min 4 or 5 and max 9 or 10, p > 0.05). Conversely, a statistically significant reduction in CS was observed in group B on D7 compared to on D0 (median 3.5, min 1, max 8) (p = 0.002), reaching the lowest score on D24 with 91% of reduction (median 1, min 0, max 2) (p < 0.001)- Table 3 and Figure 1.    Changes were observed in all five parameters of the CS (Table 4), the most dramatic changes were in the malodour score (−93% on D7 and −100% on D14).

Pruritus
On D0, pruritus was very mild and similar in the two groups (median 0.5/10, min 0.4 and 0.2, max 1.1 and 1.0, group A and B, respectively). Its severity remained very low (<0.4) throughout the study in the two groups (Table 3).

Skin Surface Cytology
No evidence of microbial elements was found on D0 or at any time point throughout the study.

NMF Content
At baseline, NMF contents did not significantly differ between the two groups. In the control group, they decreased slightly at D0 + 4 h, then returned to normal at D14, then decreased again between D14 and D24. NMF levels decreased in the test group at D0 + 4 h (−73%, p < 0.0001), returned to baseline from D14 on and were then slightly higher than in the control group at D24. Details are given in Table 3 and Figure 2.
Changes were observed in all five parameters of the CS (Table 4), the most dramatic changes were in the malodour score (−93% on D7 and −100% on D14).

Pruritus
On D0, pruritus was very mild and similar in the two groups (median 0.5/10, min 0.4 and 0.2, max 1.1 and 1.0, group A and B, respectively). Its severity remained very low (<0.4) throughout the study in the two groups (Table 3).

Skin Surface Cytology
No evidence of microbial elements was found on D0 or at any time point throughout the study.

NMF Content
At baseline, NMF contents did not significantly differ between the two groups. In the control group, they decreased slightly at D0 + 4 h, then returned to normal at D14, then decreased again between D14 and D24. NMF levels decreased in the test group at D0 + 4 h (−73%, p < 0.0001), returned to baseline from D14 on and were then slightly higher than in the control group at D24. Details are given in Table 3 and Figure 2.

Hair Lipid Analysis
At baseline, lipid contents did not differ significantly between the two groups. In the control group, lipid contents remained stable throughout the study. Lipid levels decreased in the test group at D0 + 4 h (−50%, p = 0.14) and remained lower than those of the hair of control dogs at D7 and D14, but the difference was not significant (p = 0.08 and p = 0.494). At D14, the levels were back to baseline. Details are given in Table 3 and Figure 3. during the study. Interpretation of the box and whisker plots: lower and upper box boundaries 25th and 75th percentiles, respectively, line inside box median, lower and upper whiskers minimum and maximum scores, respectively. X represents the mean. * represents significant differences at p = 0.05, compared to baseline values.

Hair Lipid Analysis
At baseline, lipid contents did not differ significantly between the two groups. In the control group, lipid contents remained stable throughout the study. Lipid levels decreased in the test group at D0 + 4 h (−50%, p = 0.14) and remained lower than those of the hair of control dogs at D7 and D14, but the difference was not significant (p = 0.08 and p = 0.494). At D14, the levels were back to baseline. Details are given in Table 3 and Figure 3.

Tolerance and Feedback from the Users
The shampoo was well tolerated. No adverse reaction was reported after shampoo and mousse applications. Tolerance and the cosmetic effect were scored 4/4 by both the owner and the investigator. Efficacy and practicability were scored 3/4 by both the owner and the investigator.

Discussion
In this study, the combination of one shampoo and subsequent mousse applications at 48-72 h intervals was a convenient protocol to quickly reduce greasiness, scaling, malodour and improve subjectively hair coat quality in dogs with an undiagnosed keratinisation disorder. Canine keratinization disorders may improve with regular appropriate shampoos [6], but today simpler and/or easier to implement protocols are desired, especially when long-term management is required. The application of a mousse is simple through massage, it does not require prior wetting of the skin, nor rinsing or drying afterwards, thus allowing longer direct skin contact and prolonged action [30]. In recent years, mousse formulations, containing either phytosphingosine and pseudofilaggrin [31], or plant extracts [32] have been used in the management of canine atopic dermatitis, in spontaneous cases, improving skin lesions and pruritus. In another study, the authors showed that a mousse containing phytosphingosine and pseudofilaggrin could decrease the skin pH and inflammation in an experimental model of impaired skin barrier [33]. Other

Tolerance and Feedback from the Users
The shampoo was well tolerated. No adverse reaction was reported after shampoo and mousse applications. Tolerance and the cosmetic effect were scored 4/4 by both the owner and the investigator. Efficacy and practicability were scored 3/4 by both the owner and the investigator.

Discussion
In this study, the combination of one shampoo and subsequent mousse applications at 48-72 h intervals was a convenient protocol to quickly reduce greasiness, scaling, malodour and improve subjectively hair coat quality in dogs with an undiagnosed keratinisation disorder. Canine keratinization disorders may improve with regular appropriate shampoos [6], but today simpler and/or easier to implement protocols are desired, especially when long-term management is required. The application of a mousse is simple through massage, it does not require prior wetting of the skin, nor rinsing or drying afterwards, thus allowing longer direct skin contact and prolonged action [30]. In recent years, mousse formulations, containing either phytosphingosine and pseudofilaggrin [31], or plant extracts [32] have been used in the management of canine atopic dermatitis, in spontaneous cases, improving skin lesions and pruritus. In another study, the authors showed that a mousse containing phytosphingosine and pseudofilaggrin could decrease the skin pH and inflammation in an experimental model of impaired skin barrier [33]. Other mousse formulations have shown an in vitro residual effect against Staphylococcus pseudintermedius after application on the hair coat [34]. To the best of the authors' knowledge, this is the first controlled study of the use of a mousse combined with a shampoo in the management of a keratinisation disorder in dog and confirms the results obtained with the same combination in a non-controlled study [35]. The clinical score improved from the first recheck (day 7), and continued to improve until the end of the study (day 24). Among all the criteria evaluated, the biggest change was in the malodour score. This criterion is considered by the owners as one of the main complaints in keratinisation disorders and one of the major reasons for using a shampoo [36]. Clinical signs were completely resolved in all but one dog in the tested group, achieving a total reduction of 95% on D24. In the present study, all the dogs of the tested group initially presented with scaling; the amount of scales decreased over the course of the study and disappeared in most of the treated dogs. The remaining dogs had a maximum score of 1 out of 3.
Plant compounds are very popular in human cosmetics, and some are also incorporated in formulations for animals [37]. However, few scientific data are available concerning their effectiveness [22,[38][39][40][41][42]. In the present study, both the shampoo and the mousse formulations contained two ingredients of plant origin: Ophytrium is a specific extract from the tuberous roots of O. japonicus and Seboliance is a specific extract from the peel of P. granatum. The presence of scales in dermatological diseases is explained by abnormalities during the keratinisation and/or desquamation steps [5]. Shampooing probably helps as it mechanically removes some of the scales. However, in the present study, as only one shampooing was performed, it is hypothesized that anti-keratinocyte proliferation action of P. granatum extract played a role [21]. Nevertheless, to demonstrate this effect, a group of dogs receiving a shampoo similar to group B but lacking the Ophytrium and Seboliance would have been necessary.
Both formulations, shampoo and mousse, were well tolerated by all the treated dogsno side effects were observed by the investigators during the study. The negative impact of harsher surfactants on the skin barrier in some antiseborrhoeic shampoos is sometimes a concern for prescribers and users [43]. This action leads to a notable loss of lipids, most often related to the surfactants contained in certain products [44,45]. In the present study, shampooing (D0) resulted in a moderate reduction in the amount of lipids, as expected after use of an anti-seborrheic shampoo. The amount of lipids found on the hair of tested dogs remained lower than on the hair of control dogs at D7 and D14; no rebound effect was observed. NMFs are the degradation products of the skin protein filaggrin, and consist primarily of amino acids or their derivatives such as PCA and UCA, together with lactic acid, urea, citrate and sugars [46]. NMFs regulate stratum corneum hydration, its pH and activity of enzymes involved in key homeostatic processes in the stratum corneum including lipid synthesis, maturation of keratinocyte cornified envelope and keratinocyte desquamation. In humans, reduced NMFs are associated with increased transepidermal water loss secondary to skin barrier dysfunction [47]. In the present study, after shampooing (D + 4 h), NMFs content helped (i) monitor the progression of the integrity of the skin barrier, which can sometimes be impaired by excessive or repeated cleaning actions, and (ii) assess the hydration performance of the products. Whether due to the shampoo (tested group) or to simply rinsing with plain water (control group), the quantities of NMFs decreased at D0 + 4 h, logically more markedly in the tested group than in the control group because of the surfactant action of the shampoo. The amounts of NMFs then gradually increased again, more markedly in the tested group than in the control group, to reach baseline levels from D14 on. It could suggest the mousse may have contributed to the restoration of the skin barrier by supplying hydrating and protecting ingredients, although only a separate study looking at shampoo only vs shampoo with mousse would be required to support adequately this statement. The results of the lipid and NMF assays confirm-at the biochemical level-the immediate and lasting tolerance to shampooing combined with multiple applications of mousse.
The present study has several limitations. As previously stated, it would have been interesting to have, in addition to the actual control group, a control group on which a similar shampoo without Ophytrium and Seboliance would have been tested. Similarly, a control mousse without the active ingredients would have been interesting to test. The number of dogs included in the study is relatively low. The severity of the skin disease based on the clinical score is fairly low, which may have affected the final scores obtained. Further studies, conducted on more severe cases are advisable. Finally, the study was single blinded (investigators were blinded but not the owner).

Conclusions
The combination of one shampooing and subsequent mousse, containing Ophytrium and Seboliance, applications every 48-72 h, was found as to be a convenient, significantly effective and safe protocol to rapidly reduce malodour, greasiness, scaling and improve subjectively hair coat quality in dogs with an undiagnosed keratinisation disorder, without dehydrating their skin. Clinical observations were supported by biochemical assays showing that this protocol, in addition to being effective, did not damage the skin and is likely suitable for a long-term use.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available from the corresponding author upon request. The data are not publicly available due to the need to maintain patient confidentiality.