Personal Cleansing Compositions And Methods Of Stabilizing The Microbiome

Abstract

A personal wash composition which provides an effective antimicrobial benefit against fungi, gram positive and gram negative bacteria, which composition comprises: a) from 0.1% to 10 wt % betaine surfactant, b) from 0.1% to 5.0 wt % lactic acid, c) from 0.1% to 20 wt % polyhydric C.sub.2-C.sub.6 alcohol, d) from 0% to 10 wt % alkyl polyethoxy carboxylate of the formula RO(CH.sub.2CH.sub.2O).sub.kCH.sub.2COO.sup.M.sup.+ wherein R is a C.sub.8-C.sub.22 alkyl, k is an integer from 0 to 20, and M is a soluble salt-forming cation, e) from 0% to 10 wt % alkyl polyethoxy amides of the formula RO(CH.sub.2CH.sub.2O).sub.kCH.sub.2CONH.sub.2 wherein R is a C.sub.8-C.sub.22 alkyl, k is an integer from 0 to 20, f) >8 wt % alkyl ether sulphate, g) water, h) a pH of 3.8-4.5, i) 10-20 wt % total surfactant, j) from 0.1 to 10 wt % metal salt.

Claims

1. A personal wash composition comprising: from 0.1% to 10 wt % betaine surfactant; from 0.1% to 5.0 wt % lactic acid; from 0.1% to 20 wt % polyhydric C.sub.2-C.sub.6 alcohol; from 0% to 10 wt % alkyl polyethoxy carboxylate; from 0% to 10 wt % alkyl polyethoxy amides; greater than 8 wt % alkyl ether sulphate; water; a pH of 3.8-4.5; from 10-20 wt % total surfactant; and from 0.1 to 10 wt % metal salt.

2. The composition according to claim 1, wherein the alkyl polyethoxy carboxylate has the formula RO(CH.sub.2CH.sub.2O).sub.kCH.sub.2COO.sup.M.sup.+; wherein R is a C.sub.8-C.sub.22 alkyl; wherein k is an integer from 0 to 20; wherein M is a soluble salt-forming cation; and wherein the alkyl polyethoxy carboxylate is present in an amount of from 2-7 wt %.

3. The composition according to claim 2, wherein in the alkyl polyethoxy carboxylate, R is a C.sub.12 alkyl, K is 11, and M is Na.

4. The composition according to claim 1, wherein the betaine surfactant is present in an amount of from 1-3 wt %.

5. The composition according to claim 1, wherein the betaine comprises cocoamidopropyl betaine.

6. The composition according to claim 1, wherein the lactic acid is present in an amount of from 0.2-8 wt %.

7. The composition according to claim 1, wherein the polyhydric C.sub.2-C.sub.6 alcohol is present in an amount of from 2-18 wt %.

8. The composition according to claim 1, wherein the polyhydric C.sub.2-C.sub.6 alcohol comprises glycerol.

9. The composition according to claim 1, wherein the alkyl polyethoxy amide comprises PEG 4 rapeseed amide.

10. The composition according to claim 1, wherein the metal salt is present in an amount of from 0.1-5 wt %.

11. The composition according to claim 1, wherein the metal salt comprises one or both of an alkali and an alkaline metal halide.

12. The composition according to claim 1, wherein the alkyl ether sulphate comprises sodium laureth sulphate.

13. A personal wash composition in the form of a foam comprising: from 0.1 wt % to 5 wt % betaine surfactant; from 0.1 wt % to 5 wt % lactic acid; from 0.1 wt % to 20 wt % polyhydric C.sub.2-C.sub.6 alcohol; water; a pH of 3.8-4.5; and from 0.1-5 wt % total surfactant.

14. The composition according to claim 13, wherein the foam has a density of less than 500 g/liter.

15. The composition according to claim 13, wherein the betaine surfactant is present in an amount of from 0.1-2 wt %.

16. The composition according to claim 13, wherein the betaine comprises cocoamidopropyl betaine.

17. The composition according to claim 13, wherein the lactic acid is present in an amount of from 0.2-5 wt %.

18. The composition according to claim 13, wherein the polyhydric C.sub.2-C.sub.6 alcohol is present in an amount of from 2-18 wt %.

19. The composition according to claim 13, wherein the polyhydric C.sub.2-C.sub.6 alcohol comprises glycerol.

20. A method for providing a germicidal benefit to a topical surface comprising: contacting a topical surface with the composition according to claim 13; and providing an effective germicidal benefit against fungi, gram-positive and gram-negative bacteria if the topical surface, prior to contact with the composition, had the presence of one or more of fungi, gram-positive and gram-negative bacteria.

21. A method for providing a germicidal benefit to a dermal surface comprising: contacting a dermal surface with the composition according to claim 13; and providing an effective germicidal benefit against undesired pathogens if the dermal surface, prior to contact with the composition, had the presence of one or more undesired pathogens, while not upsetting or disrupting the natural balance of microflora or microbiome on the dermal surface.

22. A method for stabilizing the pH of a surface comprising contacting a surface upon which the presence of one or more undesired pathogens are known or suspected, with the composition according to claim 13.

23. A method for stabilizing microbiome or microflora of a surface comprising contacting a surface upon which the presence of one or more undesired pathogens are known or suspected, with the composition according to claim 13.

24. A method of moisturizing a surface comprising contacting a surface upon which the presence of one or more undesired pathogens are known or suspected, with the composition according to claim 13.

25. The composition according to claim 1, wherein the alkyl polyethoxy amides has the formula RO(CH.sub.2CH.sub.2O).sub.kCH.sub.2CONH.sub.2, wherein R is a C.sub.8-C.sub.22 alkyl, and k is an integer from 0 to 20; wherein the alkyl polyethoxy carboxylate has the formula RO(CH.sub.2CH.sub.2O).sub.kCH.sub.2COO.sup.M.sup.+, wherein R is a C.sub.8-C.sub.22 alkyl, wherein k is an integer from 0 to 20, wherein M is a soluble salt-forming cation, and wherein the alkyl polyethoxy carboxylate is present in an amount of about 4.8 wt %; wherein the betaine surfactant is present in an amount of about 1.3 wt %; wherein the lactic acid is present in an amount of about 2.25 wt %; wherein the polyhydric C.sub.2-C.sub.6 alcohol is present in an amount of from 10-15 wt %; and wherein the metal salt is present in an amount of about 2 wt %.

26. The composition according to claim 13, wherein the foam has a density of less than 200 g/liter; wherein the betaine surfactant is present in an amount of about 0.6 wt %; wherein the lactic acid is present in an amount of about 2.75 wt %; and wherein the polyhydric C.sub.2-C.sub.6 alcohol is present in an amount of from 10-15 wt %.

Description

DETAILED DESCRIPTION

[0047] Surprisingly it has been found that the compositions of the invention achieve satisfactory cleaning and germ kill whilst avoiding issues caused by irritancy from these sulphur containing compounds, notably sulphur containing surfactants such as Sodium Laureth Sulfate (SLES). SLES is an inexpensive and very effective foaming agent. It has also been shown that SLES causes skin irritation. This is particularly significant, given the main preferred intimate use of the present invention. For this preferred use clearly it is surprisingly advantageous that irritation has been avoided.

[0048] In more detail it has been observed that the composition may be characterized in exhibiting at least a 1 log.sub.10 reduction of various microorgalisms/bacteria, such as E Coli when tested according to the standardized test protocols of ASTM E2315-03 Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure.

Antimicrobial Test Protocol:

[0049] A testing protocol according to ASTM E2315-03 Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure was used to evaluate antimicrobial efficacy against both Gram positive (Staphylococcus aureus) (ATCC 6538), Gram negative (Escherichia coli) (ATCC 10536) bacteria and the yeast Candida albicans (ATCC 10231). According to this protocol, first, the challenge cultures (18-24 hours) were prepared by suspension in tryptic sodium chloride, equilibrated to 20 C.-22 C. at room temperature. On the day of such testing, 5 ml sample of a composition product was combined with 4 ml ml of standardized hard water (300 ppm CaCO.sub.3) at room temperature (20 C.-22 C.) in a sterile vessel (e.g, test tube). Subsequently 1 ml of the test culture was added, which resulted in a 50% v/v dilution. The test tube was then vortexed for 5 seconds, and allowed to remain in contact for 60+/5 seconds, immediately after which a 1 ml aliquot was withdrawn and added to a further tube containing 9 ml of a neutralizer. The neutralization was allowed to occur for 5 minutes, and thereafter serial tenfold dilutions using tryptic sodium chloride were plated, and incubated for 24-48 hours at 36+1 C. The inocula used were also serially diluted, plated and incubated for 24-48 hours at 36+1 C. Post-incubation the surviving colony-forming units (CFUs) of the challenge organisms were enumerated and log.sub.10 reduction values for each formulation tested were determined from one or more replicate samples, in the case of plurality of replicate samples the average results were reported.

pH

[0050] It is contemplated that certain preferred embodiments of the inventive formulations may also provide an antiseptic or sanitizing benefit which is aided due to the low pH of particularly preferred embodiments of the invention, particularly wherein the compositions are at a pH of 3.8-4.5.

Viscosity

[0051] The compositions of the first aspect of the invention are viscous and exhibit a viscosity of at least 500 cps at room temperature as measured using a Brookfield viscometer, Type 3 spindle at 12 rpm. Preferably the compositions of the first aspect of the invention exhibit viscosities in the range of at least about 500 cps as measured under these conditions. Yet more preferably the topical compositions of the invention exhibit a viscosity in the range of about 1000 to about 10,000 cps, yet more preferably from about 1500 to 7000 cps, and especially preferably from about 1500-4500 cps.

[0052] While the topical compositions disclosed herein find a primary use in application to the skin to provide a cleaning and an antimicrobial benefit and is contemplated as being provided in a dispenser for use in such a treatment, it is to be understood that this is not a limiting definition and that other forms and other uses of the present inventive composition, such as face lotion, milky lotion, cream, face cleansing cream, massage materials, liquid toilet soap, as well as in hair care products such as shampoo, rinse or other hair or scalp treatment are expressly contemplated as being within the scope of the present invention. It is to be further expressly understood that the compositions disclosed herein may be topically applied to the skin on any part of the body, including the skin on the face, neck, chest, back, arms, axilla, hands, legs, and scalp.

Optional Constituents

[0053] A sequesterant may optionally be present. The overall level of sequesterant present (when present) may be within the range of 0.1 to 10 wt %.

[0054] Particularly preferred sequesterants for use with the invention are iminodisuccinic acid or its salts and/or DTPA (diethylene triamine pentaacetic acid) such as Dequest 2066 (Trade Mark; product available from Solutia Inc., St Louis 6366-6760, USA), EDTA, and Dissolvine (GLDA/glutamate diacetate) EDG (Trade Mark; product available from Akzo Nobel, Gillingham, UK), The inventive compositions may optionally include further constituents useful in improving one or more aesthetic characteristics of the compositions or in improving one or more technical characteristics of the compositions. Exemplary further optional constituents include colouring agents, fragrances and fragrance solubilizers, viscosity modifying agents including one or more thickeners, pH adjusting agents and pH buffers including organic and inorganic salts, optical brighteners, opacifying agents, hydrotropes, and preservatives, as well as other optional constituents providing improved technical or aesthetic characteristics known to the relevant art. When present, the total amount of such one or more optional constituents present in the inventive compositions do not exceed about 10% wt., preferably do not exceed 5% wt, and most preferably do not exceed 2.5% wt.

[0055] These aspects and advantages of the invention are discussed in more detail hereinafter, particularly in reference to one or more of the examples set forth below.

EXAMPLES

Example 1Base Formula and Antimicrobial Performance

[0056] This table demonstrates the impact on micro performance by total surfactant, lactic acid and salt concentration changes to the base gel body wash formula (E1). A passing formula is defined as one achieving at least 1 log kill against both s. aureus and e. coli when tested according to the standardized test protocols of ASTM E2315-03 Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure. A 1 log kill against c. albicans is also preferred given its relevance in the intimate area.

TABLE-US-00001 E1 E2 E3 E4 E5 Raw Material % w/w Chemical composition DI Water 53.39 53.64 52.09 63.12 52.09 Tetrasodium Glutamate Diacetate 0.4 0.2 0.4 0.4 0.4 Sodium Laureth Sulfate 12.16 11.55 12.16 12.16 12.16 (70% active) Sodium Laureth 11 Carboxylate 4.85 4.61 4.85 4.85 4.85 PEG 4 Rapeseedamide 1.7 1.62 1.7 1.7 1.7 Cocamidopropyl Betaine 4.25 4.03 4.45 4.25 4.45 (35% active) Glycerin 14.25 10 10 5 10 Fragrance 0.25 0.15 0.15 0.1 0.15 Extract 0 0.1 0.1 0 0.1 Benzoic acid, sorbic acid, 1 0.6 0.6 1 0.6 benzyl alcohol Lactic Acid (88% solution) 2.5 2.5 2.5 1.75 Lactic Acid (50% solution) 4.5 Sodium Hydroxide (30% solution) 0.5 1.25 1.25 0.84 1.25 Sodium Chloride (20% solution) 5 10 0 5 10 Calcium Chloride (20% solution) 0 0 10 0 0 Formula pH 4.18 4.30 4.19 4.20 4.19 Antimicrobial Performance Log Reduction S. aureus 4.01 1.62; 4.3 0.03 4.48 E. coli >5 0.07; 0.59 3.8 >5 4.96 C. albicans 1.04; 0.99 1.82 1.5 K. pneunomiae 3; 3.89 2.0; 1.5 B. Streptococcus 5.39 C. xerosis >4.56 Overall micro performance Pass Fail Pass Fail Pass Comments Base Low With 10% Low lactic 50% lactic formula surfactant CaCl2 acid acid

[0057] The data demonstrates that reduction of the total surfactant system by 5% results in lower e coli performance based on example E2. In example E4, micro efficacy is also negatively impacted by reducing lactic acid to 1.75%. Example E3 demonstrates that positive efficacy is retained by substituting sodium chloride with calcium chloride. Example E5 demonstrates that positive efficacy is retained by substituting 38% lactic acid with 50% lactic acid. In fact, a 0.5 log increase in kill on C. albicans is noted. It is suspected that this benefit may come from the fact that more dilute solutions of lactic acid are richer in the monomer form of lactic acid which is more potent than the polymeric form.

TABLE-US-00002 TABLE A Ingredients with trade name Raw Material Trade Name Tetrasodium Glutamate Diacetate Dissolvine GL 47 S Sodium Laureth Sulfate Texapon N70A Sodium Laureth 11 Carboxylate Akypo Soft 100 BVC PEG 4 Rapeseedamide Amidet N Cocamidopropyl Betaine Mackam 35 Glycerin Glycerin USP Kosher Benzoic acid, sorbic acid, Microcare SBB benzyl alcohol Lactic Acid Purac HiPure 90, Ultrapure 50

[0058] The E1, E3 and E5 formulations as described above have been found to: [0059] 1 be mild to the skin and provide appropriate antimicrobial action providing at least 1 log reduction of certain germs/bacteria. [0060] 1 be mild to the skin and provide stable pH to the intimate skin over 28 days. (Demonstrated as no statistically significant change over 28 days). [0061] 1 provide moisturization to the skin. (Statistically significant (p<0.05) increase in moisturization of intimate area skin over 28 days. Further demonstrated as a statistically significant (p<0.05 compared to baseline) increase in moisturization of keratinized skin after 1 hour). [0062] 1 respect the natural balance of resident/commensal flora in the intimate area.

Example 2Dermal Irritation or Sensitisation Study

[0063] The gel formulae E5 was placed in a clinical study to determine the dermal irritation and sensitisation potential following external contact with the skin on human subjects.

[0064] The study was performed on 104 healthy subjects, using application of semi-occlusive patches bearing the test products (diluted to 1%) to marked skin sites on the upper back (between the scapulae) for nine exposure periods of 24 hours during a 3-week induction phase. Skin sites were assessed for erythema, edema and other signs of irritancy approximately 48 hours following each application. Following approximately a 2 week rest period after the induction phase, semi-occlusive product challenge patches were applied for 24 hours to a previously untreated site on the lower back. Challenge sites were assessed for erythema, edema and other signs of irritancy 24, 48, and 72 hours after patch application for determination for sensitization potential.

[0065] No visible skin reactions were observed.

[0066] The study concluded that based on a test population of 104 subjects and under conditions of the study as described, the formula identified above did not demonstrate a potential for eliciting dermal irritation or sensitization, thus supporting that the formula is mild and gentle.

Example 3Comparison of Inventive Feminine Gel Vs. Competitor Product

[0067] Kora is an intimate hygiene gel product containing a lactic acid/chamomile antimicrobial complex. However, when tested the low dosage of lactic acid in the Kora product does not provide sufficient anti-microbial efficacy to promote adequate hygiene of the intimate feminine area. See results below.

TABLE-US-00003 Lactic acid % Glycerin %, Sample information active basis active basis pH Kora 0.08 1.81 5.0-6.0 Inventive 2.25 10 4.19 formulation E5

TABLE-US-00004 S. aureus E. coli Sample Log10 Reduction Log10 Reduction information 60 sec 60 sec Kora 0, 0, 0.02 0, 0, 0 (0) (0) Inventive (4.3) (3.8) formulation E3

[0068] In addition, it was found that the Kora product (like many commercial intimate hygiene products) has a very low level of glycerine, which may not provide adequate moisturization benefits as compared to a gel according to the invention.

[0069] The inventive formulation demonstrated that the key to an effective antimicrobial benefit is the three surfactant systems with high amount of lactic acid synergy.

Example 4Intimate Foam Cleanser

[0070] Intimate Foam wash is another format of well received feminine products. Lactic acid was used again as an antimicrobial active in the foam formulas. The formulations contain a lower amount of total surfactant versus the prior gel examples and further contain a higher level of lactic acid to help balance the micro efficacy of the formulation. The formulations are water thin to allow for use with either a foam pump package or a squeeze foam dispenser.

[0071] A passing formula for foam is also defined as one achieving at least 1 log kill against both s. aureus and e. coli when tested according to the standardized test protocols of ASTM E2315-03 Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure. A 1 log kill against c. albicans is also preferred given its relevance in the intimate area.

TABLE-US-00005 Intimate Foam E6 E11 E12 E13 Raw Material % w/w Chemical composition DI Water 87.97 87.47 93.07 85.9 Tetrasodium 0.4 0.4 0.4 0.4 Glutamate Diacetate Sodium Laureth 11 0 0 0 2 Carboxylate Cocamidopropyl 2 2 0.5 2 Betaine (30%) Glycerin 5 5 1 5 Benzoic acid, 1 1 1 1 sorbic acid, benzyl alcohol Lactic Acid (based 2.25 2.75 2.75 2.25 on 100% active) 50% Sodium 1.38 1.38 1.28 1.45 Hydroxide pH 4.2 4.2 4.18 4.2 Germ kill efficacy Log reduction S. aureus 0.65 1.32, 1.48 2.12, 1.50 0 E. coli >5.59, >5.35 5.36, 4.73 1.91, 2.47 C. albicans 0.04 Over micro Fail Pass Pass Fail performance Summary Low High lactic High lactic Low surfactant acid level level, low surfactant, betaine low lactic

Example 5Feminine Hygiene Wash In-Use Tolerance and Microbiome Study

[0072] Following a 7 day pre-study conditioning period with a pH 5.6 Shower Gel product, 34 healthy female subjects used the test product (Formula E5) to wash the external intimate area at least once per day for 28 days, with assessments at baseline (Day 0) and Days 14 and 28. Tolerance assessments of skin dryness, erythema, oedema, desquamation, itching (verbal questioning of subjects, visual assessment for scratch marks), burning (verbal questioning of subjects), stinging (verbal questioning of subjects) and other clinical signs of irritancy of the intimate area were carried out by a gynaecologist. Subjects provided subjective tolerance assessments at baseline (Day 0) and Days 14 and 28.

[0073] Skin pH measurements of the mid-labium majus were taken with a Multi Probe Adaptor System (MPA System) (Courage & Khazaka GmbH) using the skin-pH meter PH 905 probe at baseline (Day 0) and Days 14 and 28. Skin hydration and moisturization measures were taken with a Corneometer CM 825 (Courage & Khazaka GmbH) using a Multi-Probe Adaptor (MPA) 6 with Corneometer probe after the subject had remained in climate controlled conditions (acclimatized 222 C. and 35-55% RH) for 20 minutes. Skin hydration of the groin area was measured using a Corneometer at baseline (Day 0) and Days 14 and 28 to assess longer lasting benefits. Skin moisturization of the upper inner leg area before and at two time points after application of test product by the study technician were measured using a Corneometer at baseline (T-0 h), T-0 post wash (immediately after test product wash) and T-1 h after application of test product wash to assess single use/short term benefits.

[0074] Microbiological samples were taken from the mid-labium majus area via a liquid cup scrub method at baseline (Day 0) and Days 14 and 28. A glass cylinder was pressed (approx. 4 cm.sup.2 in area) to firmly make contact with the skin exerting adequate pressure to prevent leaking of liquid during collection. 1 ml of 0.1% detergent solution (75 mM phosphate buffer containing 0.1% (v/v) Triton X-100 (full strength wash fluid) buffered to a pH between 7.5-8.0) was pipetted into the sterile glass cylinder circumscribing an area of about 3.0 cm.sup.2 and the skin surface was carefully rubbed as evenly as possible with a blunted sterile glass rod (scrubbing device), applying moderate constant pressure for 1 min. Samples were boiled at 96 C for 10 minutes to sterilize, packaged in dry ice and shipped for microbial diversity analysis.

[0075] Thirty six (36) subjects were enrolled on the study. Two (2) subjects withdrew leaving thirty-four (34) subjects completing the study, and their microbiome data were included in the analysis. Subjects were aged between 19 and 55 years (mean 36.61, SD 11.28). Twelve subjects (33.3%) were aged between 18-29; 13 (36.1%) subjects were aged between 30-44 and 11 (30.6%) subjects were aged between 45-55.

[0076] Dermal irritancy scores (mean total scores) based on a 5-point scale 0=none, 0.5=very slight, 1=slight, 2=moderate, 3=severe, increased from 0.01 at Day 0 to 0.12 at Day 14 and 0.13 at Day 28. The mean within subject change from baseline total irritancy scores at Day 14 and 28 were 0.10 and 0.12 respectively meeting acceptable tolerance criteria. The Overall Tolerance Rating made by the Gynaecologist after 28 days of use was: the test product was well tolerated by most subjects.

[0077] The skin pH (overall population) of the external intimate area at Day 0 was 5.88. At Day 14 the pH decreased very slightly to 5.87 and at Day 28 to 5.85. The mean within subject change from baseline pH was 0.01 at Day 14 and 0.03 at Day 28. Analysis showed no significant differences between the time points; p-value 0.992 between D-0 & D-14, p-value 0.883 between D-0 & D-28 and p-value 0.933 between D-14 & D-28; all much higher than the 0.05 associated with the 95% confidence level. Statistical analysis confirms no significant differences (overall population) in skin pH of external (vulva) area over time with product use. Skin hydration, measured in the groin area, by the mean Corneometer scores was 39.14 at Day 0. At Day 14 this increased to 45.97 and at Day 28 increased to 48.86. Skin hydration increased over the 28 day duration of the study. Analysis showed a statistically significant increase (p<0.05) in skin hydration at D-14 and D-28 (p=<0.0001).

[0078] Skin moisturization, measured in the upper inner leg area by the mean Corneometer scores, for the test product was statistically significantly greater at T-1 hour (p=<0.0001) versus baseline (34.43 vs 29.98).

[0079] To perform the microbial diversity analysis, 16s rRNA sequences were amplified, sequenced and OTUs (operational taxonomic units) were assigned to each sequence based on the NCBI database.

[0080] Diversity was examined from two perspectives. First, overall richness (i.e., number of distinct organisms present within the microbiome), was expressed as the number of operational taxonomic units (OTUs), and was quantified using the Chao1 richness estimator. Secondly, overall diversity (which is determined by both richness and evenness, the distribution of abundance among distinct taxa) was expressed as Shannon Diversity.

[0081] Measures of diversity were screened for group differences using an analysis of variance (ANOVA). Results (shown below) indicates that the gel wash formulation tested has no significant (p>0.05) impact on species richness and diversity at any of the time points assessed for either bacteria or fungi of the external intimate skin microbiome. In fact it was shown to stabilize the intimate skin microbiome.

TABLE-US-00006 TABLE B Bacterial alpha diversity metrics comparing time points: Day 0 (n = 34) Day 14 (n = 34) Day 28 (n = 34) Observed OTUs 222.74 176.47 186.56 Chao1 Richness 246.64 9.64 201.55 10.83 210.2 10.16 Shannon Diversity 2.58 2.37 2.36

TABLE-US-00007 TABLE C Results of the ANOVA, testing for differences in Chao1 Richness among time points: Df Sum Sq Mean Sq F value Pr(>F) Time 2 38932.63 19466.32 2.01 0.1400 Residuals 99 960760.90 9704.66

TABLE-US-00008 TABLE D Results of the ANOVA, testing for differences in Shannon diversity among time. Df Sum Sq Mean Sq F value Pr(>F) Time 2 1.08 0.54 1.19 03097 Residuals 99 44.90 0.45

TABLE-US-00009 TABLE E Fungal alpha diversity metrics comparing time points: Day 0 (n = 16) Day 14 (n = 12) Day 28 (n = 13) Observed OTUs 5.06 4.75 186.56 Chao1 Richness 5.08 0.08 4.75 0.04 6.31 0.05 Shannon Diversity 0.37 0.37 0.48

TABLE-US-00010 TABLE F Results of the ANOVA, testing for differences in Chao1 Richness among time. Df Sum Sq Mean Sq F value Pr(>F) Time 2 17.44 8.72 0.90 0.4169 Residuals 38 369.97 9.74

TABLE-US-00011 TABLE G Results of the ANOVA, testing for differences in Shannon diversity among time. Df Sum Sq Mean Sq F value Pr(>F) Time 2 0.11 0.06 0.44 0.6469 Residuals 38 4.92 0.13

IN CONCLUSION

[0082] The gel wash when used by women on the external genital area at least once per day over a 4 week period (28 days), demonstrated acceptable tolerance, did not significantly change the skin pH in the external vulvar area and provided significant skin hydration and moisturization benefits. Furthermore microbiome analysis of the external vulvar area showed the formula had no significant impact on species richness and diversity for bacteria or fungi, with a stabilizing effect on the microbiome.