Development, Tolerability and In Vitro Effectiveness of a Natural Cosmetic Formulation for Mosquito Bites

Abstract

Skin irritation and itching, often intense, are common consequences of mosquito bites. Interest in cosmetics formulated with natural ingredients has markedly increased among consumers, perceiving them as healthier, safer and more sustainable. We developed a natural formulation based on botanical ingredients for the treatment of mosquito bites. In this work, we tested the physicochemical and microbiological stability of this novel formulation, as well as its skin tolerance. Moreover, we performed an in vitro test to assess its soothing efficacy in comparison with an ammonia-based product. According to our findings, the natural formulation resulted in a stable oil-in-water emulsion, with an appealing texture and good skin tolerance; no cases of erythema or edema were observed in any of the volunteers with sensitive skin after product application. In addition, an in vitro test performed on a keratinocytes cell line showed that our formulation significantly reduced IL-1α levels, with a displayed efficacy comparable to the ammonia-based product. This study emphasizes the promising potential of botanical ingredients in the field of cosmetic dermatology, offering high-performance, well-tolerated skincare products suitable for the treatment of common skin irritations, such as mosquito bites.

1. Introduction

Mosquito bites are the most common cause of acute itch in humans [1], an unpleasant sensation that stimulates the scratch reflex. The response to mosquito bites includes an immediate reaction and a delayed reaction, along with large local reactions in some individuals [2]. Immediately after a mosquito bite, a round wheal 2–10 mm in diameter forms, with surrounding erythema peaking in 20–30 min [3,4]. The delayed reaction consists of pruritic papules of the same size, which peak in 24–36 h. These gradually disappear over the course of several days.

The exact mechanism of the mosquito bite has not yet been fully elucidated. Nevertheless, it has been demonstrated that the components of mosquito saliva induce an itch response by directly activating the well-recognized pruritic pathway. Histamine is one of the most well-known direct mediators of acute itch [5]. It binds to specific receptors on sensitive nerve endings in the skin. Furthermore, histamine induces local vasodilation and edema, resulting in wheal formation [6].

Common remedies to manage mosquito bites include the application of an ice pack or topical agents to reduce inflammation, pain, swelling and itchiness; examples include medications, such as antihistamines and corticosteroids, in the form of a cream or ointment, preferred when reactions are intense, or other substances, such as ammonium hydroxide, calamine lotion, aloe vera gel or other home remedies [6,7]. Among these, ammonium hydroxide represents the most common over-the-counter after-bite product. Ammonium hydroxide is formed by the solution of ammonia in water. It is present in a large variety of products, including hair dyes, hair bleaching products, shaving cream, hair grooming products, and after-bite remedies. Zhai and colleagues have demonstrated, in a cohort of twenty-five mosquito-bitten subjects, that a diluted form of ammonium solution (3.6%) is effective in relieving symptoms caused by Aedes aegypti bites [8]. However, the mechanism of action is unknown. Being a weak base, it is possible that ammonium neutralizes the mosquito’s acidic saliva, which contains chemicals that trigger the body’s inflammatory response and cause itching [9,10].

Ammonia is generally considered safe, and its use is approved in cosmetic products. However, it is subject to restriction, as reported in Annex III of the EU Cosmetic Regulation. The maximum concentration of ammonia permitted is 6% NH₃. If the concentration is above 2%, the product must be labeled “Contains Ammonia” [11]. Elevated levels of ammonia or direct exposure can cause skin irritation and eye irritation, and inhalation can lead to respiratory issues [12,13,14]. This represents an important concern, especially for babies and children. Aedes mosquitoes are more attracted to the skin of infants and children because it is warmer and has a stronger odor due to its thinness [15]. This, in turn, can trigger more severe irritations.

In this context, cosmetic products containing naturally derived ingredients have received increasing interest from consumers [16,17]. Natural cosmetic ingredients are particularly valued and demanded by consumers, as they perceive sustainable, environmentally friendly, organic, safe and effective. As demand for non-pharmacological treatments grows, itch-relieving cosmetics and other category products are becoming a pivotal part of dermatological care [18]. Currently, the market offers a wide range of after-bite products labeled as “natural”, most of which have a synthetic formulation base. Small and uncertain amounts of botanical extracts are added to these products to make them appear natural and give them a “green claim”. Consequently, the contribution of these natural components to the overall soothing effect is unclear. Furthermore, despite the high demand and growing market for natural products, few have been studied for their mode of action and/or efficacy.

In this work, we present a novel soothing formulation prepared with natural ingredients and intended for application to mosquito bites. Moreover, we explore its ability to reduce irritation in human keratinocytes in comparison to a product containing ammonium hydroxide.

2. Materials and Methods

2.1. Development of Natural Formulation

2.1.1. Characteristic and Composition of Formulation Tested

The natural formulation (Formula A) investigated in this study was an oil-in-water (O/W) emulsion that had a light texture, was easy to spread, and had no greasy effect. Glyceryl stearate citrate, glyceryl stearate and sodium cetearyl sulfate were selected as emulsifying bases to form a stable emulsion with a light skin feeling; cetearyl alcohol was used as an emulsion stabilizer. Dicaprylyl ether, caprylic/capric triglyceride and Vitis vinifera seed oil were chosen as emollients. The selection of the active ingredients was based on a review of the literature and related efficacy studies. Active ingredients and their properties are summarized in

Table 1. Key ingredients in Formula A and their properties.

Ingredient	Action
Zanthoxylum bungeanum fruit extract (Zanthalene®)	Soothing, antipruritic and anti-irritation activities [19]
Avena sativa kernel extract	It is rich in avenanthramides, which exhibit anti-inflammatory and anti-itch activity [20]
Bisabolol and Chamomilla recutita flower water	Soothing and anti-redness properties [21]
Guaiazulene	Anti-inflammatory and antioxidant activities [22]
Ledum palustre extract	Anti-inflammatory and antirepellent properties [23]
Menthol and menthyl lactate	Refreshing properties [24]
Hamamelis virginiana water	It contains tannins that act as astringents, reducing inflammation and relieving itching [25]

. Lastly, hydroxyacetophenone, 1,2-hexanediol and caprylyl glycol were chosen as the preservative system, substances with antimicrobial properties but not included in Annex V of the Regulation (EC) No. 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products [11].

The complete ingredients of the formula are as follows (INCI denomination): aqua, glycerin, caprylic/capric triglyceride, dicaprylyl ether, chamomilla recutita flower water, hamamelis virginiana leaf water, vitis vinifera seed oil, glyceryl stearate citrate, cetearyl alcohol, ledum palustre extract, zanthoxylum bungeanum fruit extract, avena sativa kernel extract, bisabolol, guaiazulene, menthol, menthyl lactate, xanthan gum, maltodextrin, lecithin, tocopherol, ascorbyl palmitate, glyceryl stearate, oleyl alcohol, disodium phosphate, sodium cetearyl sulfate, citric acid, caprylyl glycol, 1,2-hexanediol, hydroxyacetophenone, limonene, pinene. The commercial reference of this cosmetic cream formulation was “Ledum DS Roll-on Dopo Puntura” (Prodeco Pharma, Castelfranco Veneto, Italy). A comprehensive list of the formula’s ingredients is detailed in Table S1.

2.1.2. Preparation Procedure

Phase A: The aqueous phase, which incorporated ultrapure water (PURELAB^® Chorus 2+, ELGA LabWater, High Wycombe, UK), vegetable glycerin, Hamamelis virginiana leaf water, Chamomilla recutita flower water, xanthan gum, hydroxyacetophenone and disodium phosphate, was heated to 80 °C. Homogenization was performed to completely disperse all the components.

Phase B: The oil phase, composed of dicaprylyl ether, caprylic/capric triglyceride, glyceryl stearate citrate, cetearyl alcohol, sodium cetearyl sulfate and glyceryl stearate, was melted at 80 °C. Vitis vinifera seed oil, lecithin, tocopherol, ascorbyl palmitate and guaiazulene were added, and homogenization of all the ingredients was performed.

Phase B was added to phase A under continuous stirring, using Silverson L5M-A (Silverson Machine Ltd., Waterside, Chestam, UK) (1600 rpm for 10 min). The emulsion obtained from phases A and B was cooled to below 40 °C.

Phase C, containing active ingredients, was added to the cooled emulsion and contained menthyl lactate, menthol, bisabolol, Avena sativa kernel extract, Ledum palustre extract and Zanthoxylum bungeanum fruit extract. The preservatives 1,2-hexanediol and caprylyl glycol were also added. The ingredients were incorporated sequentially, one at a time, under continuous stirring for 3 min to promote their proper dispersion within the system.

After complete homogenization, citric acid was used to adjust the pH of the emulsion.

2.1.3. Evaluation of the Physicochemical and Microbiological Characteristics

In accordance with the current legislation (Regulation (EC) No. 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products [11]), a series of tests was carried out: a. stability testing of the formulation; b. organoleptic analysis; c. determination of pH and viscosity and centrifugation test; and d. microbiological evaluation for the assessment of microbiological contamination and preservative efficacy test (challenge test).

Stability Testing

The stability test involved accelerated conditions for three months in climatic conditions at 40 °C ± 75% RH (Climacell EVO, MMM Medcenter Einrichtungen GmbH, Munich, Germany). The stability of the product was also monitored at 4 °C and at room temperature (25 °C).

Quality Control

Organoleptic characteristics (such as appearance, color and odor), pH (SevenCompact S220, Mettler Toledo Spa, Milan, Italy) and viscosity (spindle 4, 20 rpm, 20 °C; Viscometer DV1, Brookfield Ametek, Middleboro, MA, USA) were assessed to ascertain the quality of the product. A centrifugation test was performed by incorporating the emulsion into centrifugation tubes to be centrifuged at 25 °C and 3000 rpm for 30 min (Neya 16, REMI Sales & Engineering Ltd., Mumbai, India).

Microbiological Control and Challenge Test

The preliminary microbial count of the formula was determined in accordance with ISO 21149:2017 (“Enumeration and detection of aerobic mesophilic bacteria”) and ISO 16212:2017 (“Enumeration of yeast and mould”) [26,27]. Assessment of the adequacy of the preservative system chosen (hydroxyacetophenone, 1,2-hexanediol and caprylyl glycol) for the formulation was also performed using the international cosmetics challenge test standard (ISO 11930:2019) [28]. The test was run using the following strains: Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8739, Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404. Inocula were prepared from each strain according to ISO 11930:2019 to obtain between 1 × 10⁵ CFU/mL and 1 × 10⁶ CFU/mL or g of bacteria, and between 1 × 10⁴ CFU/mL and 1 × 10⁵ CFU/mL or g of C. albicans and A. brasiliensis in the formulation (final concentration). Microbial content was assayed at each specified sampling interval: 7 days (T7), 14 days (T14) and 28 days (T28), according to the test strain. Microbiological load reduction (R_x), expressed in logarithmic units, was calculated for each organism using the following formula:

R_x = lg N₀ − lg N_x

where N₀ is the number of microorganisms inoculated at time t₀, and N_x is the number of surviving microorganisms at each sampling time, t_x. The product is considered to pass the preservative efficacy test if the concentrations of the bacteria are reduced by at least 3 log within 7 days, with no increase in numbers thereafter (being tested also after 14 and 28 days), and if the concentrations of the fungi do not increase over the testing period [28].

These analyses were conducted by an external laboratory (Bio Basic Europe S.r.l., Milan, Italy).

Safety Evaluation

In order to assess the irritant potential and the skin tolerance of the product on healthy human skin, a patch test was performed on 27 subjects with sensitive skin. Subjects were selected according to the following inclusion criteria: Caucasian females or males between 18 and 60 years of age, good general health and mental condition, and healthy skin in the test areas. Exclusion criteria included pregnancy or breastfeeding, known allergies to cosmetics or their ingredients, previous adverse reactions to materials used in the study, skin diseases or dermatological disorders. The assessment of sensitive skin was performed at the clinical level by the physician responsible for the trial, taking into account factors such as the capillary fragility and the skin’s tendency to redden, become irritated and peel particularly easily. Subjects provided written informed consent before being enrolled in the study.

The product (20 µL/mg) was applied under a patch (8 mm diameter Finn Chamber, SmartPractic, Phoenix, AZ, USA) on the skin of the back, after cleaning the test area with a 70% alcohol solution. Physiological water (20 µL) was applied as a negative control to avoid any inaccurate interpretations related to skin irritations. The patch was removed 48 h after the application. A dermatologist evaluated erythema and edema after 15 min, 60 min and 24 h. Clinical safety evaluation was assessed according the following skin reaction parameters: (I) clinical erythema assessment on a five-point severity scale (0 = no erythema; 1 = very mild erythema; 2 = mild and visible erythema; 3 = moderate erythema; 4 = severe erythema, with possible formation of mild scabs), and (II) clinical edema assessment based on a five-point severity scale (0 = no edema; 1 = very mild edema, barely visible; 2 = mild and visible edema; 3 = moderate edema (with borders raised by approximately 1 mm); 4 = severe edema (extended swelling even beyond the application area)) at all evaluation time points. The product was considered “non-irritating” according to the following criteria:

• Absence of erythema and/or edema at any of the follow-up times;
• Appearance of 1. very slight or 2. slight erythema and/or edema at intermediate times and absence of erythema and/or edema at the time of the final observation (T24, 24 h after the patch was removed):
• Appearance of 1. very slight or 2. slight erythema in ≤6 subjects at the time of the final observation (T24, 24 h after the patch was removed).

This study was conducted by an external laboratory (CDC Dermo Clinical Research Institute of Bio Basic Europe S.r.l., Milan, Italy).

2.2. In Vitro Test Efficacy

2.2.1. Testing Material

The natural formulation (Formula A) previously described was compared to a reference formulation (Formula B). Formula B is an ultra-light emulsion containing (INCI denomination) aqua, ammonia, paraffinum liquidum, dimeticone and C12-13 pareth-3 (“After Bite^®”, Tender Corporation, Littleton, NH, USA).

2.2.2. Cell Culture

The HaCaT keratinocyte cell line (CR1017-500, Quimigen, Madrid, Spain) was cultivated in Dulbecco’s modified Eagle’s medium (DMEM) (PromoCell GmbH, Heidelberg, Germany) supplemented with 10% fetal bovine serum (Gibco Inc., Billings, MT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco Inc., Billings, MT, USA). The cells were kept at 37 °C in a 5% CO₂ incubator, and the complete media was replaced every 2 days before reaching 90% confluence.

2.2.3. Determination of Cell Viability

The effects of Formula A and Formula B on keratinocyte cell viability were investigated via an MTT assay [29] performed at the Bionos Biotech facility (Valencia, Spain). HaCaT cells were cultured overnight at a density of 10⁴ cells/well in a 96 well-plate for 24 h. Then, the cell culture medium was replaced by fresh medium with 0.025% sodium laurate (SL; Sigma-Aldrich, Madrid, Spain) for 30 min, followed by another new culture medium containing the tested sample at 8 different concentrations (3%, 1%, 0.3%, 0.1%, 0.03%, 0.01%, 0.003% and 0.001%). After 24 h of incubation, the medium was removed, and MTT solution (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (Invitrogen, Carlsbad, CA, USA) was added to each well. Plates were incubated at 37 °C for 3 h. MTT reactive solution was removed, and 100% DMSO (Sigma-Aldrich, St. Louis, MO, USA) was added to each well to solubilize formazan crystals prior to absorbance measurements at 550 nm and 620 nm as a reference on a scanning multi-well spectrophotometer (Biotek Synergy LX, Agilent, Santa Clara, CA, USA). Data were statistically analyzed.

2.2.4. Soothing Efficacy Assay

Sodium laurate was used to induce irritation in human keratinocytes. HaCaT cells were cultured in a 24-well plate and treated with 0.025% SL for 30 min. After this incubation, 1 mM hydrocortisone (positive control; Sigma-Aldrich, St. Louis, MO, USA), Formula A or Formula B at 0.1% and 0.01% were added for 24 h. Product soothing efficacy was quantified by measuring Interleukin (IL)-1α levels by ELISA assay (Abcam Limited, Cambridge, UK), following the manufacturer’s instructions.

2.2.5. Statistical Analysis

Data were analyzed with GraphPad Prism 8.0 software (GraphPad Software, Boston, MA, USA). The MTT assay was set with 8 replicates per condition. Data outliers were identified using the ROUT method (Q = 5%) and excluded from the analysis if found. Both raw and normalized data were statistically analyzed via Ordinary one-way ANOVA and Dunnett’s post hoc test. For IL-1α quantification, 4 replicates per condition were tested. Data outliers were identified with ROUT (Q = 5%) and excluded from the analysis if found. Both raw and normalized data were statistically analyzed by Ordinary one-way ANOVA and Dunnett’s post hoc test. Statistical significance was declared at p < 0.05, 95% confidence. Data are expressed as mean ± standard error mean (SEM) for each group of values. More details are provided in Data S1.

3. Results

3.1. Formulation Characteristics

The investigated soothing cream formulated in this study was characterized as an oil-in-water emulsion, with a light texture and rapid absorption. It appeared to be light blue in color due to the presence of guaiazulene, a dark blue crystalline constituent of chamomile oil (Figure 1). Guaiazulene is a cosmetic ingredient, known for its soothing properties, permitted under European Regulation (EC) No. 1223/2009 [11]; additionally, it is approved by the U.S. FDA as a cosmetic color additive.

At the olfactory level, the formulation had a characteristic menthol scent, without being pungent.

Throughout the stability study, the cream formulation exhibited no visible changes in color, homogeneity, pH, and viscosity nor phase separation after 90 days at ambient temperature (25 °C) and storage under accelerated (40 ± 2 °C/75 ± 5% RH) or refrigerated (4 °C) conditions, indicating excellent physical stability. In particular, pH values remained within the set range, even under extreme conditions (40 ± 2 °C/75 ± 5% RH), 5.20–5.80. Viscosity analysis did not show notable changes over time, although it tended to decrease with high temperatures (

Table 2. Physicochemical stability of Formula A at accelerated storage conditions.

Parameter	Specification/ Limits	T0	T30	T60	T90
Aspect	Light blue cream	Conform	Conform	Conform	Conform
Odor	Menthol, delicate	Conform	Conform	Conform	Conform
pH	5.20–5.80	5.23	5.31	5.33	5.39
Viscosity	3000–7000 mPas	6590 mPas	6280 mPas	5920 mPas	5390 mPas
Centrifugation	Homogeneous	Conform	Conform	Conform	Conform

). The formulation was stable under rotational stress. The consistent appearance under ambient, refrigerated and stress conditions suggested that the formulation successfully preserved the structural integrity of its components.

3.2. Microbiological Assessment

The performed challenge test has confirmed the effectiveness of the antimicrobial protection of the cosmetic formulation. All bacterial counts were reduced by more than 3 log from day 7, with no increase at the other time points. In addition, the fungal load was also lowered to zero after 28 days.

The results of the challenge test in

Table 3. Inoculum load and logarithmic reduction in microbial load (R_x) obtained in challenge test.

	S. aureus ATCC 6538		P. aeruginosa ATCC 9027		E. coli ATCC 8739		C. albicans ATCC 10231		A. brasiliensis ATCC 16404
	CFU/mL	Rx	CFU/mL	Rx	CFU/mL	Rx	CFU/mL	Rx	CFU/mL	Rx
Inoculum	1.7 × 105	-	6.0 × 105	-	9.0 × 105	-	6.1 × 104	-	2.0 × 104	-
7 days	<10	4.23	<10	4.78	<10	4.95	<10	3.79	-	-
14 days	<10	4.23	<10	4.78	<10	4.95	<10	3.79	<10	3.30
28 days	<10	4.23	<10	4.78	<10	4.95	<10	3.79	<10	3.30

showed compliance with the accepted standard.

3.3. Dermatological Test

Patch testing on sensitive skin showed that none of the subjects experienced erythema or edema/irritation at any of the observation times (15 min (T15′), 1 h (T1) and 24 h (T24)) after application of Formula A (

Table 4. The absolute frequencies of the assessments of edema and erythema at the observation times.

	T0	T15′	T1	T24
Erythema score
Absent	27	27	27	27
Very slight	0	0	0	0
Slight	0	0	0	0
Moderate	0	0	0	0
Marked	0	0	0	0
Edema score
Absent	27	27	27	27
Very slight	0	0	0	0
Slight	0	0	0	0
Moderate	0	0	0	0
Marked	0	0	0	0

).

3.4. Viability Assay

The MTT assay was performed to evaluate the impact of the formulations on HaCaT cell viability after SL treatment. The results from the MTT assays demonstrated that when HaCaT cells were treated with varying concentrations of Formula A or Formula B, the percentage of cell viability exhibited a concentration-dependent response, providing a preliminary indication for the assessment of consumer exposure levels and risk characterization.

For Formula A, cell viability remained relatively high up to concentrations below 0.3%, with values ranging from 74.20% to 100% (Figure 2A). However, as Formula A’s concentration increased to 1%, cell viability declined to 51% compared to the control (Figure 2A). The same trend was observed for Formula B, with a drastic reduction in cell viability with concentrations above 1% (Figure 2B).

3.5. Soothing Efficacy

In this model, the irritated control (SL) elicited an increase in IL-1α protein levels compared to the non-treated condition (C), confirming successful induction of inflammation in keratinocytes (Figure 3). As expected, the anti-inflammatory control hydrocortisone (HC) showed a reduction in IL-1α relative to the SL group (Figure 3). Importantly, both Formula A and Formula B also reduced IL-1α levels versus the SL control at all tested concentrations (Figure 3), supporting a measurable soothing/anti-inflammatory effect under these experimental conditions.

4. Discussion

The present study presents a novel composition with botanical active ingredients indicated for mosquito bites and explores its soothing potential. Reactions to mosquito bites can be very painful and very itchy, especially among babies and children. Compared to adults, children’s immune system is still developing and may react more strongly to mosquito saliva [30]. Itch-relieving remedies are highly requested, as itching is an unpleasant sensation. In this context, the current trend in the cosmetics industry is to formulate novel skin care products that incorporate natural extracts as active ingredients. These ingredients show great promise in terms of their efficacy and safety profile, meeting high market acceptance [31]. On the other hand, ammonia, the best-known non-pharmacological substance for the treatment of mosquito bites, presents health hazards. Ammonia exposure can irritate the eyes, nose and throat, and repeated skin contact can cause dryness, itching and redness. Generally, products with ammonia are not recommended for children under 2 years of age or subjects with sensitive skin. It should also be considered that ammonia is also present in human sweat, and it has been demonstrated that it is an attractive component of host odor for Aedes aegypti mosquitoes [32].

In the current study, we presented a novel formulation with natural active ingredients specifically selected for their soothing effect and/or for their established traditional use. It contains Zanthalene^®, a patented ingredient obtained from CO₂ extraction of the fruit husks of Zanthoxylum bungeanum [33], also known as Sichuan pepper or false pepper. It contains a mixture of lipophilic alkylamides (including hydroxy α- and β-sanshools), which are responsible for the soothing and anti-itching properties of the plant. In particular, hydroxy-α-sanshool is able to interact with the TRPV1 and TRPA1 ion channels [34], receptors that play a significant role in the itching pathway. In a pilot study of forty healthy volunteers with mosquito bites, 67.5% of subjects reported a reduction in itch intensity after application of spray lotion containing Zanthalene^® compared to the blank sample [35]. Oat has been used for centuries as a soothing agent to relieve itching and irritation associated with various xerotic dermatoses. Avenanthramides, polyphenols from oat, have been demonstrated to have a potent anti-inflammatory action due to the inhibition of IkB-α degradation in keratinocytes [20]. The actives bisabolol and azulene, both derived from chamomile essential oil, are also known to soothe sensitive skin [36,37]. Our formulation also contains Ledum palustre extract, which was among the components inside a homeopathic after-bite gel. A pilot study that examined the efficacy of this remedy on sixty-eight healthy volunteers bitten by Aedes aegypti mosquitoes showed that it was significantly superior in reducing erythema compared to no treatment and the placebo gel [38]. Finally, it should be highlighted that Witch hazel (Hamamelis virginiana) water, menthol and menthyl lactate work together to reduce itching. Hamamelis water has vasoconstricting properties that help reduce redness and relieve itching and discomfort [25]. The active menthol, on the other hand, was selected for its cooling properties. When topically applied, menthol produces a cooling sensation, which soothes the affected area by reducing heat and swelling. Menthol works by activating TRPM8 receptors in the skin [24], a part of the body’s temperature-sensing system. The combination with menthyl lactate, obtained via the esterification reaction between natural menthol and lactic acid, resulted in a mild, long-lasting cooling effect. Menthyl lactate exhibits slow evaporation due to its higher molecular weight and intermolecular forces compared to menthol, which allows it to be released gradually onto the skin, providing a prolonged effect compared to the immediate, but potentially short-lived, cooling sensation of menthol [39]. It should be noted that the choice of a light oil-in-water emulsion as the formulation base resulted in a cooling effect due to the water content in the external phase, creating a cooling sensation when it evaporates. Overall, the developed formulation showed stable organoleptic/characteristics, pH and viscosity values, as confirmed by the results obtained from stability testing and quality control. The selection of the preservation system merits consideration, for instance, avoiding chemical preservatives in favor of multifunctional ingredients such as humectants, skin conditioners (1,2-hexanediol), emollients (caprylyl glycol) and antioxidants (hydroxyacetophenone) [40]. The challenge test confirmed the efficacy of the preservative system used, and the stability test confirmed the compatibility of these substances with the other ingredients in the formulation. Finally, it should be noted that it has been previously demonstrated that the combination of these multifunctional ingredients (1,2-hexanediol, caprylyl glycol, hydroxyacetophenone) did not negatively affect the natural flora of the skin. These results were obtained through skin microbiome analysis using 16S rRNA gene sequencing and bioinformatics of volunteers who applied an oil-in-water emulsion with 1,2-hexanediol, caprylyl glycol and hydroxyacetophenone on the skin twice a day for 2 weeks [41]. Lastly, the final consideration is that the dermatological test results showed that none of the subjects, even those with sensitive skin, developed irritative reactions, confirming good skin tolerability. Additionally, the toxicological evaluation and the Cosmetic Product Safety Report have declared the formulation suitable for children of all ages.

The results obtained from our in vitro experiments show Formula A’s promising soothing activity. However, there are some limitations. In our model, we used sodium laurate to induce irritation on epidermal keratinocytes, the cell type that is primarily exposed to environmental stimuli. Sodium laurate is a known surfactant commonly used in cleansers and soaps, also known for its irritant properties. It causes damage by interrupting barrier function and leading to cell injury. It has been shown that the addition of SL to a human epidermal cell culture increases inflammation [42]. In this model, SL was used as an inflammatory/irritant stimulus at a concentration chosen to preserve keratinocyte viability, thereby enabling evaluation of cytokine-driven irritation (IL-1α induction) without confounding factors derived from the cell death pathways that are unlikely to reflect the localized tissue response to mosquito bites. Despite being a natural product, our results showed that Formula A exhibits a level of IL-1α reduction comparable to that of Formula B. It is important to note, however, that these results are exploratory in nature and limited to IL-1α only. The pathophysiology of itch and inflammation in mosquito bites is complex, involving an intricate crosstalk between keratinocytes, immune cells and sensory nerves. To further elucidate and expand our findings, 3D skin models and/or other advanced skin models (i.e., organ-on-chip technology) that include the immune system are crucial to better reflect human physiology and understand the complex immune cell/skin interactions [43]. Given that tissue models offer a superior representation of the complexity of the three-dimensional structure, they are also instrumental in facilitating a more precise evaluation of the biocompatibility of the product. This, in turn, enables the optimal dosage to be administered to consumers and/or in a clinical setting, given that higher concentrations of the product have shown dose-dependent cytotoxicity, although these findings have been observed primarily in a monolayer model.

Moreover, we used sodium laurate as a skin irritant, which does not completely represent the complexity of mosquitoes’ saliva composition, as it induces not only local skin reactions but also IgE-mediated response and modulations of host immune response [6]; these biological effects remain to be explored. Furthermore, it would be interesting to test the efficacy of the formulation in a real-world setting to verify its effectiveness after mosquito bites, its speed of action in soothing itching and its favorable safety profile, also considering subjects with skin disorders, such as atopic-prone skin or other conditions.

5. Conclusions

The formulation developed in this study, containing 99.2% natural ingredients, embodies the “green cosmetic” trend. This product aligns with consumer preferences, who perceive green products as safer, more sustainable and more ethical than synthetic ones. The formulation has demonstrated desirable chemical stability and microbiological control, and a favorable tolerability profile. Additionally, using an in vitro model, it demonstrated potential soothing efficacy comparable to that of chemical products containing ammonium hydroxide. This is worth further investigation using more complex models, which are needed to better represent the pathophysiology of the skin’s reaction to mosquito bites. All these findings, while promising, need to be confirmed in a real-world setting.

Overall, the investigation of natural ingredients for alleviating skin discomfort remains an ongoing area of research, deserving of further investigation to establish their safety and efficacy, especially in children and the elderly, who could benefit most from the general absence of hazardous substances found in synthetic cosmetics.