The Effects of Four Acidifying Sprays, Vinegar, and Water on Canine Cutaneous pH Levels

Introduction

The skin is relatively unique among organ systems, because it is accessible to both systemic and topical medications. Topical products are useful adjunctive therapies for many dermatological diseases, such as keratinization disorders, ectoparasite infestations, and bacterial or fungal infections. Although topical medications alone rarely cure dermatological disease, they may speed resolution of a disorder, help maintain improvements gained with systemic therapy, increase patient comfort, or decrease the amount of systemic medication required.

Many veterinary shampoos, conditioners, and ear drops contain medications that target microorganisms. Additionally, some products contain acidifying agents, such as lactic acid or acetic acid. Although these agents do not have well-defined antimicrobial activities, acidification of human skin or canine ear canals theoretically creates a hostile environment for infectious microorganisms.^1–3

There is evidence to support the theory that an acidic cutaneous pH decreases survival of microorganisms on the skin. In general, higher bacterial counts are retrieved from skin with alkaline pH levels.⁴ Additionally, when gram-negative bacteria are inoculated onto alkaline skin, they persist longer than on acidic skin.⁴ It has also been proposed that the relatively alkaline pH of canine skin may be a reason that skin infections are more common in dogs compared to humans and other animal species.⁵

Although cutaneous pH modification has been evaluated in humans, information regarding cutaneous pH and pH modulation in domestic animals is sparse.^6–8 The purpose of this study was to evaluate the extent and duration of cutaneous acidification achieved after a single application of four acidifying sprays, vinegar (i.e., acetic acid), and water to the skin of eight normal dogs.

Materials and Methods

Eight (three female, five male) healthy, neutered, adult mixed-breed dogs were used for this study. The dogs were fed the same diet and were housed in a climate-controlled room in separate pens. The pens were cleaned two times a day, and the dogs had no contact with chemical disinfectants. Prior to beginning the study, six 4-cm² areas were clipped with a No. 40 blade on the dorsal aspect of the trunk of each dog, including right and left cranial thorax, right and left caudal thorax, and right and left cranial lumbar regions. These sites were chosen to minimize the possibility of the dogs licking the areas. The dogs were then bathed with a cleansing shampoo^a to remove surface debris. Twenty-four hours later, the shaved areas were evaluated for irritation (e.g., erythema, scale, papules, crusts), and baseline pH values were taken using a flat glass electrode pH meter.^b Flat glass electrode pH meters are designed for measuring cutaneous pH, and the techniques used in this study are validated for use in humans and animals.⁹¹⁰ The device was calibrated with pH 4.0 and 6.9 buffers prior to each set of measurements. The tip of the electrode was placed firmly against the skin for 3.0 seconds and was washed with 3 Mol KCl after every test. Triplicate measurements were taken from each area.

Four acidifying sprays (A-D), vinegar,^c and distilled water^d were selected for use in determining their effects on canine cutaneous pH. The pH levels of the sprays were chosen because they were likely to cause skin pH levels that would have antimicrobial activity (e.g., pH<5.0). The desired pH of the four acidifying sprays was achieved by using a combination of buffers encapsulated in Novasome microvesicles.^e Novasome microvesicles are commonly used in products for time-released activity of the ingredients. The acidifying components of spray A (pH, 2.6) were acetic acid and sodium acetate. Spray B (pH, 3.0) contained citric acid and sodium citrate. Sprays C (pH, 2.2) and D (pH, 3.0) contained both citric acid and sodium phosphate in different concentrations. There are no known adverse reactions to the buffers in the four sprays tested. These four sprays were tested to determine which ingredient combination would most effectively acidify the skin for the longest duration. The vinegar^c (pH, 2.7) contained 5% acetic acid. Vinegar was evaluated because it is often used as an adjunctive therapy in the treatment of cutaneous and otic infections. Distilled water^d (pH, 5.4) was used as a control.

The pH values of the sprays, vinegar, and water were measured with a glass electrode pH meter^f and probe designed for pH measurements of liquids. The device was calibrated with pH 4.0 and 6.9 buffers prior to each set of measurements and was washed with 3 Mol KCl after every test. For every dog, each of the six sprays was randomly assigned to a single shaved area by blindly drawing pieces of paper marked with a spray type and consecutively adding them to a prepared list of sites. The investigator taking the pH measurements was blinded to the application of the sprays.

Each spray was applied to the predesignated area on each dog and was allowed to air dry. The sprays were applied until the shaved area was uniformly covered with the liquid. One minute after application of the sprays, another set of pH readings was taken. The pH was then measured every 6 hours for 72 hours, then every 12 hours for an additional 72 hours. Throughout the study, the treated areas continued to be monitored for signs of irritation. At the completion of the study, each dog was again bathed with shampoo.^a

The null hypothesis was that the effects of the six sprays would be the same. Multivariate repeated measures analysis of variance^g was used to determine if the sprays caused different effects on the cutaneous pH levels of the eight dogs. The triplicate measurements of cutaneous pH values were averaged, and the level of significance was chosen as P<0.05.

Results

No cutaneous irritation occurred after clipping the sites or at any time during the study. The standard deviation of the triplicate pH measurements was ±0.3. The mean baseline pH value for all sites tested was 7.55, with a range of 6.3 to 9.0. Analysis of variance showed no significant difference in baseline pH values between the six different sites tested (F=0.4; P>0.8).

Analysis of variance identified a significant difference in the baseline pH values between the individual dogs (F=20.4; P≤0.001). Tukey’s Honestly Significant Difference (HSD) test showed significant differences in baseline pH values among multiple dogs (P<0.05). Overall, it appeared that the dogs fell into two broad groups; one group of five dogs (three male, two female) had a mean pH of ≥7.80; the other group of three dogs (two male, one female) had a mean pH of ≤6.95. The mean pH of each individual dog ranged from 6.57 to 8.58.

There was a decrease in baseline cutaneous pH levels after the application of each spray, including water. After this initial decline in pH, there was an overall trend toward alkalinity [Figure 1]. The mean pH measurement 6 days posttreatment (8.43; range, 6.9 to 9.6) tended to be higher than the mean baseline pH levels (7.55; range, 6.3 to 9.0).

Multivariate repeated measures analysis of variance revealed a significant difference between the acidifying effects of the six sprays (F=15.31; P≤0.001). Linear contrast tests showed that the cutaneous pH after application of the four acidifying sprays was significantly lower than the cutaneous pH after vinegar and water application at all times tested (F=5.96; P≤0.001). Linear contrast tests showed that the overall cutaneous pH levels after vinegar application were significantly lower than water (F=13.83; P≤0.001), although the differences at some times were not significant.

The four acidifying sprays decreased the cutaneous pH to <5.0 for a mean range of 29 to 35 hours, and <6.0 for a mean range of 50 to 65 hours [see Table]. Vinegar maintained a pH of <5.0 for a mean of 3.8 hours, and <6.0 for a mean of 13 hours. Water caused a decrease below baseline for an average of 24 hours (range, 18 to 30 hours), but it never caused the pH to decrease to <6.0.

Discussion

Cutaneous pH likely plays an important role in maintaining the normal bacterial flora of the skin and in preventing cutaneous invasion by infectious microorganisms.²³¹⁰¹¹ Investigators have proposed that the acidity of human skin may also be important in wound healing and may help regulate the processes of keratinization and desquamation through the effects of pH on cutaneous enzyme activity.¹²¹³ Because of these important functions, concern has been expressed that increasing or decreasing cutaneous pH values will cause irritation or alterations in keratinization. Studies in humans show that product ingredients are more likely to play a role in causing irritation than the pH of the product.¹⁴¹⁵ The findings of this study are consistent with this observation, as none of the dogs in this study exhibited signs of irritation after acidification of the skin.

The pH of canine skin has commonly been described as ranging from 5.5 to 7.2.^16–18 In this study, higher pH values were observed, ranging from 6.3 to 9.0. The mean baseline pH level of 7.55 in these dogs is consistent with a recent report that found the mean pH of flank skin in dogs to be 7.48.¹⁹ It appears that canine skin may be more alkaline than previously reported. These discrepancies could be attributed in part to differences in methods of pH measurement, sex, breed, coat color, or anatomical site.¹⁹ Meyer and Neurand have reported that excitement in dogs can lead to elevations in cutaneous pH levels of up to one pH unit in 1 minute.¹⁶ Although the authors consciously tried not to agitate the dogs in this study, it is possible that excitement affected the pH measurements.

Cutaneous pH varies among different anatomical locations in humans, with areas that are occluded (e.g., axillae, genitoanal region, intertriginous and interdigital areas) tending to be more alkaline.¹¹⁰ Site differences in pH have also been demonstrated in dogs and cattle.¹⁹²⁰ In this study, the baseline pH levels of six dorsal sites were not statistically different. This was not surprising, because the sites were similar in location and environmental exposure. The significant difference in mean baseline pH values between dogs could be consistent with a previous report of breed differences.¹⁹ Although the dogs tested in this study were mixed-breed dogs, characteristics of different breeds (e.g., height, coat color, coat length, head and body shapes) appeared to predominate in some of the dogs (e.g., German shepherd-mix, Doberman pinscher-mix).

There was a decrease in baseline cutaneous pH levels after the application of each spray, including water. The water caused a decrease from a mean pH value of 7.38 to a mean of 6.54. Although a decrease in pH after the application of water may not be expected, the pH of the distilled water used was acidic (pH, 5.4). Distilled water was used instead of tap water to ensure consistency and purity. Water also affected cutaneous pH values in a study performed in humans, in which the application of tap water with a pH of 7.89 to 8.2 caused an increase in the pH of infant skin from an average of 6.60 to 6.79.²¹ These results indicate that even water used for bathing may affect cutaneous pH.

A trend toward an alkaline pH level after the application of the sprays was expected. This was probably due to the skin’s natural buffering system and the decreasing activity of the ingredients within the sprays. The observation that the mean pH measurement at 144 hours posttreatment was generally higher than the pretreatment levels may also be a reflection of the buffering capacity of the skin. It is possible that this transient “rebound” effect could be associated with negative consequences, such as increased growth of cutaneous bacteria or yeast. Additionally, in humans, increased pH levels are associated with increased transepidermal water loss (TEWL), which indicates decreased cutaneous barrier function.²²²³ Transepidermal water loss was not measured in this study, but an increase in pH and TEWL could make the skin more susceptible to cutaneous infection.

The four acidifying sprays were not statistically different from each other, but it was interesting to note that the extent and duration of acidification were not directly related to the pH of the spray. For example, spray B (pH, 3.0; citric acid and sodium citrate) caused the cutaneous pH to remain <6.0 for a mean of 65.3 hours, while spray D (pH, 3.0; citric acid and sodium phosphate) did so for a mean of 50.3 hours. This indicates that while the product pH may be important, the formulation of the product should also be considered. It is possible that the combination of citric acid with sodium citrate as the buffer in spray B was more stable against the buffering system of the skin. This may also be one of the reasons that vinegar (pH, 2.7; 5% acetic acid), which is more acidic than both sprays B and D but does not contain a buffer, sustained a pH <6.0 for only 12.8 hours. In addition, the absence of Novasome microvesicles (designed for sustained release of the buffers) in vinegar may account for some of the differences between the vinegar and the test sprays.

Although vinegar caused a larger and more sustained decrease in pH than water, the differences were not consistently statistically significant. In fact, the mean pH measurements 6 hours after the application of water and vinegar were not statistically different. In practice, vinegar is often combined with water for therapeutic uses; depending on the source and amount of water used, it may increase the pH of the vinegar solution, possibly resulting in decreased acidification of the skin.

The hair was removed from the test areas in this study to assure accurate cutaneous pH measurements. The presence of hair does not allow for the proper contact of a flat glass electrode probe with the skin surface. These sprays could be used on haired skin but must penetrate through the hair to the skin surface to be effective. Care should be taken to avoid excessive amounts of spray in the hair coat, which may lead to prolonged cutaneous moisture and an increased risk of irritation.

The cutaneous pH required to decrease the survival of cutaneous microorganisms has not been established and probably varies with the target organism. In general, a pH of <5.0 prevents microbial growth.¹ For example, a pH of <4.5 inhibits the growth of most Pseudomonas spp. bacteria.²⁴ Similarly, the growth of Malassezia spp. tends to be inhibited at a pH of <4.0.²⁵

The acidifying effects of the four test sprays and vinegar could potentially decrease cutaneous levels of bacteria; however, the effects of vinegar are not long in duration. The cutaneous effects of the four acidifying sprays and vinegar on Malassezia spp. are more questionable. Although the initial mean pH decreased to <4.0 after two of the sprays (spray B, pH of 3.95; spray C, 3.75), these values were not sustained. Vinegar did not cause a decrease in pH <4.0. The clinical benefits observed from the use of vinegar and other acidifying agents may be due to antimicrobial effects that are unrelated to acidification. Both acetic and lactic acids have demonstrated inhibitory effects on bacteria and yeast independent of pH.²⁶ It is also possible that acetic acid causes an immediate, brief decrease in local pH sufficient to affect microorganisms.

Conclusion

This study shows that canine cutaneous pH can be decreased by topical agents for a substantial amount of time. Additionally, no irritation was caused by a single application of acetic acid or the four acidifying sprays tested. Further studies are necessary to determine if acidification of canine skin for extended periods of time is beneficial in the treatment and prevention of bacterial pyoderma and Malassezia dermatitis.

Acknowledgments

The authors thank Evsco Pharmaceuticals, a division of IGI, Inc., for their financial support of this study. They also thank Nadya Lawrence and Madeline Mertis (Evsco Pharmaceuticals, IGI Inc.) for their technical assistance (i.e., formulation, manufacture, and distribution of experimental spray products).

Addendum

A modification of spray B is now commercially available as Advanced pHormula spray (Evsco Pharmaceuticals, a division of IGI, Inc., Buena, NJ).