Modern deodorant actives are superior to conventional organohalogen systems.
2-Methyl 5-cyclohexylpentanol has no negative impact on the human axillary microbiome while triclosan disrupts it. Long lasting deodorant efficacy may be achieved without impacting natural human axillary microbiome.
Introduction
The formation of body odor starts with odorless human sweat which is mainly composed of water, however contains small fractions of proteins and natural lipids.
Natural skin microbiota will decompose such lipids and proteins to form molecules with a particularly low odor threshold. These molecules may include short chain fatty acids like 3-hydroxy-3-methyl-hexanoic acid (HMHA) or 3-methyl-2-hexenoic acid (3M2H), as well as organosulfur compounds like 3-mercapto-3-methyl-1-hexanol; and even steroids like androstenol or androstenone are part of the game.[1]
Managing body odor is typically achieved by three individual cosmetic technologies which are used separately or in combination: anti-perspirants, odor masking, or deodorant actives. Deodorant actives are used either to deodorize without the use of aluminum salts (anti-perspirants) or to deodorize with low or no fragrance.
Applying such actives in combination with other technologies typically supports a long lasting effect of odor protection, which is expected from an ideal deodorant product by the consumer.[2]
Deo actives are mostly based on antimicrobial properties. In this regard, the antimicrobial triclosan can be considered a historical benchmark often used for comparison when testing modern actives, representing a conventional organohalogen (organochlorine) system.
Examples for non-organohalogen technologies are cosmetic ingredients like farnesol[3], triethyl citrate[4], or 2-methyl 5-cyclohexylpentanol (SymDeo® B125).[5,6,7]
Considering that malodor prevention by deodorant actives is often based on an antimicrobial effect inhibiting bacterial break-down of natural sweat components, modern antimicrobial technologies often aim for selective activity towards odor causing bacteria.
However, information about the actual impact of these ingredients on the natural axillary microbiome [8,9] has still been missing. Application of a newly developed ex vivo model[10] led to new findings on the influence of deodorant actives on the human axillary microbiome.[11]
This article compares modern and conventional deodorant actives typically used in cosmetics. As a first step we demonstrate a comparison of the readily biodegradable (manometric respiration test: 79%/28d) and patented[7] active 2-methyl 5 cyclohexylpentanol with the conventional system triclosan.
Deodorizing performance of the actives is demonstrated in two clinical sniffing studies. In a second step we are looking in-depth at microbial levels as well as relative abundance of natural microbiota by application of the ex vivo human axillary microbiome model.[10]
Experimental Design
Two in vivo clinical studies on human subjects have been carried out, comparing armpits treated with 2-methyl 5-cyclohexylpentanol and triclosan as well as untreated ones by direct olfactory assessment (sniffing) of the armpit. The actives were applied using a volatile carrier system (pump spray).
A 24 h clinical study of 2-methyl 5-cyclohexylpentanol vs triclosan was conducted at Institute Dr. Schrader in Holzminden, Germany. The direct olfactory assessment of odour
reduction induced by the test products in comparison to each other and to initial value included a randomized, double- blind, halfside-test of the armpits on 20 subjects of 29 –
63 years of age, with distinct perspiration odor in the armpit.
The study was started with a ten days conditioning period, in which the subjects were only allowed to use unscented soap without anti-bacterial ingredients, also excluding the use of anti-perspirants, deodorants or other cosmetics.
The subjects were instructed to only wear clothing not treated with perfumed detergent or softener. Only subjects with an odor score not less than 3 on a 0-5 scale (0 = no odor, 5 = very high odor) were allowed to take part in the study.
After the conditioning period, a sniffing assessment of the armpits six hours after washing and 24 hours after washing (t0 = no product applied) was performed by trained experts.
Half of the volunteers had applied formula A (2-methyl 5-cyclohexylpentanol spray) on the left armpit and formula B (triclosan) on the right; the other volunteers had applied the test products in the other way.
Six and 24 hours after a single application, sniffing assessments were performed (t1 = single application). After the 24 hour assessment, the subjects had to apply the test products in the morning & evening during four additional days ending in one last application in the morning of the fifth day. Six and 24 hours after the last application, sniffing assessments were performed (t11 = 11 applications in total) again.
A 48 h clinical study of 2-methyl 5-cyclohexylpentanol vs untreated was conducted at Kosmoscience Ciência & Tecnologia Cosmética Ltda, Brazil. This included a sensory evaluation of the clinical efficacy of the deodorant effect of the test product at six, 24 and 48 hours after the last application to the underarms of research subjects.
The assessment was carried out, comparing left vs right armpit (product applied on the left, only washing on the right or vice versa) using the test product in form of a simple alcoholic pump spray with 0.3% of 2-methyl 5-cyclohexylpentanol. The evaluation of axillary odor was performed through direct sniffing / olfactory assessment of the armpits by three trained assessors (sensory olfactory method, according to ASTM E1207 – 14 Standard Guide for Sensory Evaluation of Deodorancy).
The sniffing results were rated on a 10-point scale from 0 (no bad odor) to 10 (extremely strong odor). 30 subjects with 29 – 60 years of age (mean age 50 ± 9) took part in the study. The study was started with a seven days conditioning phase in which the subjects were instructed to use only a neutral soap (cleaning agent) with no bactericidal action supplied by the institute.
A control of residual aluminum in the armpits was performed, only allowing subjects without residual aluminum to take part in the study. The study was performed including a controlled application of the investigational product (0.5 g) on one of the armpits by spraying after controlled washing.
After application the subjects were instructed not to wet, wash or pass any underarm product for the next 48 hours, and not to remove the white cotton shirt provided until the final evaluation of the axillary odor was assessed.
Human Axillary Microbiome Model:
A human axillary ex vivo microbiome model was developed and validated by Symrise based on fresh human sweat from eight healthy subjects of 39-64 years of age (mean age 55 ± 9). The pre-sampled sweat was pooled, aliquotted and mixed with the respective test substance (deodorant active). Afterwards, the samples were assayed at 0 h and, 24 h after being incubated under controlled conditions at 37°C over time.
A microbial analysis was carried out in two different ways:
1) Microbial Load – Aerobic and Anaerobic Colony Forming units (CFU). Petri dishes containing plate count agar were aerobically inoculated with 100 μl of diluted sweat samples and incubated for 48 h at 37°C under either aerobic or anaerobic conditions.
The amount of bacterial colonies was assayed and means of at least two technical replicates were calculated.
2) Microbiome Composition – Bacterial Amplicon Sequencing (16S rRNA gene). Sequencing and bioinformatical evaluation was performed at CeMeT GmbH (Tübingen, Germany). DNA was isolated by Qiagen MagAttract PowerSoil DNA Kit and PCR was done on variable regions 3 and 4 of 16S rRNA gene. Sequencing was performed with MiSeq Reagent Kit v3 (600 cycles) and quality of run was 83.05%.
Data were afterwards compared to NCBI Bacterial 16S rRNA database and ordered according to presence of genus information. 20 most abundant genera (adding up to 99.4% of all detected ASVs) were selected based on total abundance in all experiments and visualized by their relative abundance.
According to the testers assessment the product using triclosan showed a significant reduction (p≤ 0.05) in body odor at six and 24 hours after single and multiple applications,
in comparison with the initial values.
The same effect is observed with the test product containing 2-methyl 5 cyclohexylpentanol, except for the 24 hour value after regular application where no significant difference in comparison to the initial value was documented in this study.
In the self-assessment part of the study a significant difference was reported by the subjects for the 6 hour value after multiple applications for both test products in comparison to the initial value and for the 24 hour value of the test product using triclosan in comparison to the initial value.
For the comparison of products with each other no significant differences in body odor could be obtained in this study.
For the tested product a reduction of 51.2% of the axillary malodor was observed six hours after application. (47.9% after 24 hours, and 45.8% after 48 hours respectively) The
investigational product granted a statistically significant reduction (p≤ 0.05) of axillary malodor when compared to untreated armpits at six, 24, and 48 hours.
All participants showed a reduction in axillary malodor for all three time points. The strongest effect was found 6 hours after application.
Human Axillary Microbiome Model
Figure 3: Cell Count Development
In the untreated sweat samples a cell count (CFU/mL) of 107 microorganisms was detected (see figure 3). There was an ven distribution between aerobic and anaerobic microorganisms.
Untreated sweat samples were incubated under controlled conditions at 37°C (body temperature) and a proliferation to a level of 108 CFU/mL in slight favor of the aerobic cells was observed. Sweat samples incubated under the same conditions but treated with 0.1% of triclosan resulted in a significant drop of viable cells compared to the untreated 24 h samples, showing that viability of cells is negatively impacted.
In comparison, when looking at the 24 h cell count for samples treated with 0.1% of 2-methyl 5-cyclohexylpentanol, the resulting cell counts are almost identical to those of the untreated sample.
In summary, we observed that viable cell numbers (without differentiation of strain abundance) is significantly reduced when samples are treated with triclosan (a strong antimicrobial) but remain at comparable levels like untreated samples when treated with 2-methyl 5-cyclohexylpentanol.
Having explored the abundance of viable microorganisms as a first step leads us to the next step: The relative abundance of different strains and in-depth analy sis of the microbiome by bacterial amplicon sequencing on a 16S rRNA gene level (see figure 4).
Figure 4: Microbiome Composition
The untreated sweat samples, containing the initial (0h) untreated microbiome, were analyzed and found to be dominated by gram-positive bacteria, mainly consisting of the three genera Staphylococcus, Anaerococcus, and Corynebacterium.
Subsequent incubation of the untreated sweat samples at 37°C (simulated body temperature) for 24 hours resulted in a slight change in relative abundance. While the amounts of Anaerococcus spp. and Peptoniphilus spp. increased, the proportion of Corynebacterium spp. remained almost unchanged, whereas the relative amount of Staphylocooccus spp. decreased.
While the 24 h value for samples treated with 0.1% of 2-methyl 5-cyclohexylpentanol was very similar to those left untreated, the samples treated with 0.1% of triclosan showed a significant enrichment of the gram-negative Pseudomonas genus.
In combination with the results shown for the bacterial cell count (Figure 3) this clearly indicates a selection pressure towards triclosan resistan species, resulting in a severe shirt in the axillary microbiome.
Thus, our analysis of the relative microbiome composition revealed that triclosan has a significant impact on the composition of the microbiome and causes a significant shift
in abundance.
2-Methyl 5-cyclohexylpentanol on the other hand has very low influence and does not significantly imbalance the composition of the microbiome.
Discussion.
Table 3: Summarized Results
Comparison of conventional and contemporary deodorant actives.
The two clinical studies discussed here reveal two findings. Firstly, the 24 hours study shows comparable deodorant efficacy of the two ingredients triclosan and 2-methyl 5-cyclohexylpentanol. Both correspond to a significant reduction of body odor compared to the initial value. Secondly, in the 48 hours study the application of 2-methyl 5-cyclohexylpentanol dramatically reduced body odor compared to untreated armpits at all time points. In terms of clinical efficacy, modern and conventional systems perform equally to reduce malodor, while long lasting performance up to 48 hours was also demonstrated for 2-methyl 5-cyclohexylpentanol.
While modern and conventional systems seem to perform similarly on a sensory level, there are significant differences on the microbiome level. The modern system (2-methyl-5-cyclohexylpentanol) suggests to be advantageous, as it minimally impacts the natural axillary microbiome. As it has been reported in the past[12], it seems unnecessary to indiscriminately kill all bacteria in order to achieve deodorant efficacy.
Our hypothesis is that 2-methyl 5-cyclohexylpentanol causes an inhibitory effect on microorgansims and thus moderates the development of malodors. This effect makes 2-methyl 5-cyclohexylpentanol a microbiome-friendly alternative to conventional organohalogen systems.
Conclusion or Summary
Modern cosmetic formulations claiming long lasting deodorant efficacy can benefit from the excellent performance of modern deodorant actives like 2-methyl 5-cyclohexylpentanol and do not have to utilize strong antimicrobials such as triclosan. This modern alternative allows formulators to create microbiome gentle products, without compromising efficacy.
References
[1] Natsch, A. (2015). What Makes Us Smell: The Biochemistry of Body Odour and the Design of New Deodorant Ingredients, CHIMIA 69 414-420.
[2] Symrise AG: CMI Data Source, Symrise Cosmetic Ingredients consumer data base.
[3] Symrise AG (2019, Nov 11): Farnesol; Nature-identical sesquiterpene alcohol. Available at https://www.symselect.com/deodorants
[4] Niendorf, H. (2012, Sept). Natural deodorising active for modern formulations; Personal Care Magazine 39-42.
[5] Symrise AG (2019, Oct 11): SymDeo® B125; Patented highly effective deodorant active. Available at https://www.symselect.com/deodorants
[6] Pesaro, M., Diesing, B., Schmaus, G., Pillai, R. (2011, Dec). 2-Methyl 5-Cyclohexylpentanol: Development of a Novel Deodorant Agent; SOFW-Journal 137 61-68.
[7] Kuhn, W., Wöhrle, I., Dilk, I., Ewering, Ch., Mampel, J., Krohn, M., Zinke, H. (2009, Apr 28). EP2424829 B1, US8623340B2, BRPI0924661B1…. OMEGA-CYCLOHEXYLALKAN-1-OLES AND USE THEREOF AS ANTIMICROBIAL ACTIVES TO COMBAT BODY ODOR.
[8] US National Institutes of Health, Human Microbiome Project (2019, Oct 11): Available at https://hmpdacc.org/
[9] The Human Microbiome Project Consortium (2012) in Nature 486 (7402) 207-214 and 215-221.
[10] Nordzieke, S., Diesing, B., Singer, M., Wittlake, R., Lanfermann, I., Winkler, S., Schmaus, G., Pesaro, M., Koch, C. (2019). The Good, the Bad, and the Smelly – developing a representative model for the human axillary microbiome. Poster, Annual Conference of the Association for General and Applied Microbiology (Mainz, Germany).
[11] Nordzieke, S., Diesing, B., Singer, M., Wittlake, R., Lanfermann, I., Winkler, S., Schmaus, G., Pesaro, M., Koch, C. (2019). Going ex vivo – Applying a representative model for the human axillary microbiome. Poster, 25th IFSCC Conference on Cosmetic Science and Conscience (Milan, Italy).
[12] Haustein, U.-F., Herrmann, J., Hoppe, U., Engel, W., Sauermann, G. (1993). Growth inhibition of coryneform bacteria by a mixture of three natural products Farnesol, glyceryl monolaurate, and phenoxyethanol: HGQ. J. Soc. Cosmet. Chem. 44 211-220.
