Summary. Introduction

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1 Original Contribution Blackwell Publishing ORIGINAL Inc CONTRIBUTIONS Cleansing lotion containing tamarind fruit pulp extract. II. Study of cumulative irritation effects in human Ratree Maenthaisong, BSc Pharm, 1,2 Jarupa Viyoch, PhD, 3,5 Nathorn Chaiyakunapruk, PharmD, PhD, 1 & Prateep Warnnissorn, MD, PhD 4,5 1 Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand 2 Department of Clinical Pharmacy and Research, Faculty of Pharmacy, Mahasarakham University, Mahasarakham, Thailand 3 Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand 4 Naresuan University Hospital, Naresuan University, Phitsanulok, Thailand 5 Cosmetic and Natural Product Research Center, Naresuan University Hospital, Naresuan University, Phitsanulok, Thailand Summary Background Cleansing lotion containing extract of tamarind fruit pulp was developed to provide skin a lighter effect. Skin irritation may occur due to keratolytic effect of α- hydroxyl acids (AHA) in the tamarind fruit pulp extract. Objective To assess the cumulative irritation effect of cleansing lotion containing tamarind fruit extract with 2% (w/w) tartaric acid on human skin compared with placebo product and de-ionized water. Methods The study design was a single-blinded, randomized side of arm, and controlled study. Three samples, including test product, placebo product, and de-ionized water, were repeatedly applied on the inner forearm of 15 healthy females (aged 28.3 ± 3.1 years) for 30 min daily for 5 days under semi-occlusive patch. Skin irritation was measured by using visual scoring and instruments such as Tewameter and Mexameter. All measurements were done before application of samples every day from day 1 until day 5. Final measurements were done after the last application for 3 days (day 8). Results The results obtained from the visual scoring scale indicated no irritation signs and symptoms of test product. Mean differences of transepidermal water loss and erythema values between test product and de-ionized water and between test and placebo products were not statistically significant (P > 0.05). Conclusions These findings indicate a preliminary safety evidence of our developed cleansing lotion containing the natural AHAs and can be used as cumulative evidence for supporting the future home use study of this product in human. Keywords: α-hydroxyl acids, cleansing lotion, clinical trial, irritation, tamarind Introduction Facial cleansing lotion is a skin care product that is used to remove dirty materials such as oily residues from Correspondence: Jarupa Viyoch, PhD, Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand. jarupav@nu.ac.th, jarupav@hotmail.com Accepted for publication October 28, 2006 cosmetics. Normally, this cleanser is applied by hand to remove oily deposits of colors or pigments from the skin and then wiped off with tissues or cloth. Oil-in-water (o/w) emulsions work satisfactorily for this purpose because the water phase can wash the dirt without leaving any oily feeling. In the last decade, there has been increasing interest in adding ingredients for other cosmetic purposes. For example, α-hydroxyl acids (AHA) are added to the facial skin cleanser formula to provide skin-lightening effects Blackwell Publishing Journal of Cosmetic Dermatology, 6,

2 Tamarind (Tamarindus indica L.) is a common tree grown in Southeast Asia including Thailand. Dry powder of its fruit pulp contains AHAs such as tartaric acid (8 23.8%), lactic acid (2%), citric acid, and malic acid. 1 4 In the past, fruit pulp of tamarind has been used as skin scrubbing materials to provide smoother and lighter skin appearance. 5 The effects of the tamarind fruit pulp on skin were postulated to be due to the presence of AHAs that exfoliate dead skin cells by weakening bonds that hold dead skin cells, thus stimulating skin renewal. In addition, pectin and invert sugar that exhibit as moisturizer were found in the fruit pulp. 6 Because of the keratolytic effect of AHAs, there has been recommendation that the concentration of AHAs contained in cosmetic products should not exceed 10%. 7,8 Generally, AHAs are available in the form of synthetic compound or naturally occurring compound. The AHAs derived from natural sources are often referred to as fruit acids. The debate about the differences between natural and synthetic AHAs has been reported in the past. It has been found that the AHAs derived from natural sources have lower potential for irritation, as indicated by the larger therapeutic index of the natural AHAs. 9 Therapeutic index is ratio of a dose with stimulation effect to a dose with irritation effect. Recently, we have developed a facial cleansing formula (o/w emulsion) containing tamarind fruit pulp extract. 10 Such formula consisted of 5.9% (w/w) of Tween 60; 1.5% (w/w) of Span 80; 0.2% (w/w) of Carbopol aqua SF; 5.0% (w/w) of glycerin, isopropyl myristate, and propylene glycol; 0.15% (w/w) of disodium EDTA; 0.7% (w/w) of triethanolamine; 1% (w/w) of stearyl alcohol; 1.5% (w/w) of stearic acid; 3.0% (w/w) of liquid paraffin; 0.2% (w/w) of methyl paraben; 0.02% (w/w) of propyl paraben; and the lyophilized powder of the tamarind fruit pulp extract in an amount corresponding to 2.0% (w/w) of tartaric acid. The quantity of tartaric acid, a main AHA found in the extract of tamarind fruit pulp, in the extract powder and the test product determined by high-pressure liquid chromatography averaged 24.7 ± 0.2% (w/w) and 15.9 ± 0.6 mg/g of lotion, respectively. Due to the concern of the possibility of irritation effect of AHA after prolonged application, our developed formula contains the natural AHAs at a concentration lower than 10% (w/w). We believe that this formula should have minimal irritation effect and be capable of exerting cosmetic benefits such as skin lightening. To determine the safety of our product, a preliminary study assessing the irritation effects of the product after cumulative exposure was designed and conducted. The method of repeated application of the products on the skin using closed patch test was selected because it mimics cumulative exposure and has been shown to possess a good predictability of the effects of long-term exposure. 11 Materials and methods Study design The study was a single-blinded, randomized side of arm, controlled study, conducted at the Cosmetics and Natural Product Research Center, located at Naresuan University Hospital, Naresuan University, Thailand. The study was conducted in accordance with the ethics principles of the Declaration of Helsinki and was consistent with Good Clinical Practice guidelines. The protocol was approved by the institutional review board at Naresuan University. Written informed consent was obtained from all participating subjects. A repeated-application closed patch epicutaneous test under semi-occlusion method was used to evaluate the cumulative irritant contact dermatitis of each sample, including (i) cleansing product with tamarind fruit pulp extract (test product), (ii) cleansing product without tamarind fruit extract (placebo product), and (iii) de-ionized water (negative control). The subject s arm side was randomly selected. Three test areas (of each size) on the inner forearm were sequentially selected with a space of 3 cm between one another. All three areas were randomly allocated to receive 0.2 g of one of the products. The allocation assignments of the side of arm areas were generated using a simple randomization. Each material was applied and then covered with a semi-occlusive patch (Tegaderm tape with webril cotton) for 30 min. After that, the patch was removed, and the test material was wiped off from the skin. This procedure was repeatedly performed for five consecutive days. 11 During the study period, subjects were allowed to shower and instructed to avoid application of detergents and any cosmetics on the application areas. This procedure was modified from the test guideline for assessment of skin compatibility of finished products in man by Walker et al. Instead of applying the materials for 24 h on the first day and 6 h for the next five consecutive days, we shortened the time of application because the test product was a rinse-off product. Having a less intensified exposure protocol would better represent how consumers will use the test product. Study population Eligible subjects were Thai healthy females aged 18 to 40 years. Exclusion criteria included exposure to a topical steroid, AHAs, and salicylic acid on inner forearm 1 week before the study. Further exclusion criteria included 2007 Blackwell Publishing Journal of Cosmetic Dermatology, 6,

3 Figure 1 Test areas of test product, placebo, and de-ionized water: (a) baseline, (b) at day 5, and (c) at day 8. treatment with oral systemic steroids, retinoids, or other systemic photosensitizing drugs within 1 month before the study. In addition, subjects who had skin disease, or wound on inner forearm, history of atopic dermatitis, skin hypersensitivity reaction, or history of allergic reaction to cleansing product ingredients or AHA products were excluded. Evaluation Objective assessment of skin parameters using Mexameter (Mexameter MX 18 Courage & Kazaka, Cologne, Germany) and Tewameter [Tewameter TM 210 Courage & Kazaka, express transepidermal water loss (TEWL) values in g/m 2 h] was done to measure skin redness (erythema index) and TEWL, respectively In addition, clinical evaluation by a dermatologist was done using the visual scoring scale of Frosch & Kligman and COLIPA, 11,14 based on three main types of skin lesions including erythema, scaling, and edema domains as follows: Erythema: 0 = no evidence of erythema; 0.5 = minimal or doubtful erythema; 1 = slight redness, spotty and diffuse; 2 = moderate and uniform redness; 3 = intense redness; 4 = fiery redness Scaling: 0 = no evidence of scaling; 0.5 = dry without scaling or appears smooth and taut; 1 = fine or mild scaling; 2 = moderate scaling; 3 = severe scaling with large flakes Edema: 0 = absence of edema; 1 = presence of edema Whenever the visual score was 1 in single domain, the application was discontinued. In this case, scores and values of the TEWL and skin erythema values obtained at the time of discontinuation were carried forward and used for the final calculations. All measurements were made before application every day for 5 days to assess the cumulative effect of sample. In addition, final assessment was done on day 8 to assess the effect of samples after the last application for 3 days. Before the measurements, all subjects had to rest in a room with a certain condition (22 ± 2 C and 55 ± 5% relative humidity) for at least 30 min. 12,14,15 Statistical analysis The differences of skin parameters of test product and placebo product vs. de-ionized water were tested using Wilcoxon signed ranks test (Wilcoxon test for paired data). In addition, all results were reported using descriptive statistics. Statistical significance was set at P < Results Fifteen Thai healthy females aged 25 to 37 years (28.3 ± 3.1 years) were enrolled in this study. There were no dropouts or loss to follow-ups during study period. Clinical evaluation using visual scoring by dermatologist revealed no irritation signs or symptoms at baseline, day 5, and day 8 of study (Fig. 1, Table 1). At baseline assessment, we found no difference of erythema index and TEWL values of both test product and placebo product vs. de-ionized water. At days 5 and 8, we also found no difference of these values of both test and placebo products vs. de-ionized water (Table 1). Discussion To our knowledge, this is the first study assessing the irritation effect of facial cleansing lotion containing tamarind fruit pulp extract. The results showed that there were no clinically and statistically significant differences of cumulative irritant contact dermatitis between test product (cleansing lotions containing tamarind fruit pulp extract) and de-ionized water (negative control) and between test product and placebo product. The absence of skin irritation is most likely due to the low concentration of natural fruit acids in the product. Generally, the irritation effect is dependent on concentration of AHAs. In addition, the soothing effects of natural tartaric Blackwell Publishing Journal of Cosmetic Dermatology, 6,

4 Table 1 Results of all measurement at baseline, day 5, and day 8. Mean (SD) Parameters De-ionized water Placebo sample Test sample Visual scoring* (n = 15): erythema, scaling, and edema Baseline 0 (0) 0 (0) 0 (0) Day 5 (cumulative effect) 0 (0) 0 (0) 0 (0) Day 8** 0 (0) 0 (0) 0 (0) Erythema index (n = 15) Baseline (8.75) (9.55) (8.88) Day 5 (cumulative effect) (7.24) (9.63) (9.35) Day 8** (9.51) (9.91) (10.20) TEWL values (n = 15) Baseline 8.58 (2.32) 8.24 (1.92) 8.66 (2.04) Day 5 (cumulative effect) 8.92 (1.57) 9.00 (1.48) 9.10 (1.60) Day 8** 7.73 (1.23) 8.14 (1.65) 7.98 (1.24) *Score of visual scoring: 0 = no evidence of erythema, no evidence of scaling, or absence of edema. **The effect after final application for 3 days. acid may reduce its irritation effect as this phenomenon was previously reported in other fruit acids. 9 It is true that having no signs or symptoms of irritation based on this study does not necessarily mean an absolute freedom from irritation. The absence of irritation found for the test product could indicate a potentially low irritancy, or it may simply be a result of using an insensitive test method. The repeated-application closed patch epicutaneous test under semi-occlusion method was modified to better represent how consumers will use the test product in reality. Although it was not possible to be certain for the free-of-irritation effect, based on current evidence, we have not detected signs and symptoms of skin irritations after repeated use of cleansing lotions containing tamarind fruit pulp extract. An adequate number of volunteers should be recruited to satisfy the objective of the test and the ethical requirements. Theoretically, a large number of volunteers, such as 100 to 200 subjects, is required for a study conducted with an intention to claim hypoallergenic property of a product. 16 However, several studies have been conducted to assess the irritation effect of several other products, and only 10 to 20 volunteers were included. Because our study included 15 subjects and showed no signs or symptoms of skin irritation, the findings of this study could be counted as one of cumulative evidence indicating the safety of the product. We believe that repeated-application closed patch test used in the study is the most appropriate method for assessing the irritation effect of our product before home use test. In actual practice, consumers apply this cleansing product twice a day on their face and wait for 2 min before wiping it from the skin. In this study, we applied this product on the inner forearm and occluded this area for 30 min before wiping it from skin. The product had more time to contact the skin than actual use as 15 times and had occlusive condition to increase rate of absorption. The irritation effects of a product are affected by many factors including the composition of product, the concentration and purity of ingredients, the type of application, the susceptibility of the patch site, and the duration of exposure. 23 Cleansing lotions are rinse-off products using short duration of exposure to the skin. Therefore, a singleapplication test or short-duration use test might be viewed as a reasonable test for assessing the irritation effects. However, some chemicals can possibly bind to the skin even after a short exposure time. 11 In addition, daily use or use several times a day of these products may cause ingredients to accumulate in the skin and thus increase risk of irritation. 11,23 Therefore, we decided to use a repeated-application closed patch test to ensure that the irritation effects of the product were properly investigated. Because our findings were based on the experimental condition, several issues should be considered in actual application. First, the product was applied on the inner forearm instead of face, which was an actual target site. Because the face has a thin epidermis and is more sensitive than the inner forearm, some irritation signs and symptoms may occur when applying this product on the face. Second, in our study, all subjects were instructed to avoid application of detergents or any cosmetics on the test sites. In reality, users of the product may apply other 2007 Blackwell Publishing Journal of Cosmetic Dermatology, 6,

5 cosmetics products. This might result in increased risk of skin irritation. Due to these limitations, we cannot be completely certain that the product has no irritation effect when used in real life. It would be prudent to have a study evaluating the safety of products in actual conditions. A larger-scale study using a repeated application closed patch epicutaneous test will be necessary to confirm the validity of its human safety. Conclusion The results of repeated-application closed patch epicutaneous test under semi-occlusion of cleansing lotion containing tamarind fruit pulp extract revealed that there was no difference of the cumulative irritation effects of the test product, water (negative control), and cream base (placebo product). These findings indicate a preliminary safety evidence of our developed cleansing lotion containing the natural AHAs and can be used as cumulative evidence for supporting the future home use study of this product in human. References 1 Lewis YS, Neelakantan S, Bhatin DS. Determination of free and combined tartaric acid in plant products. Food Sci (Mysore) 1961; 10: Ghosh GP, Pathak AK. Studies on complex formation of phosphoric acid with fruits, tartaric and citric acids. Proc Natl Acad Sci Indian Sect A 1981; 51: Ranjan S, Patnaik KK, Laloraya MM. Enzymic conversion of meso-tartrate to dextro-tartrate in tamarind. Naturwissenschaften 1961; 48: Verhaar LG. Tartaric acid and other constituents in the fruits of Tamarindus indica. Chronica Naturae 1948; 104: Silapornpinit P. Natural Cosmetics and Products for Skin Bangkok, Thailand: O.A. Printing House. 6 Batham HN, Nigam LS. Tamarind as a source of alcohol and tartaric acid. Agr Res Inst, Pusa, Bull 1924; 153: Barrows JN. Memorandum to the Administrative File, Guidance for Industry: Labeling for Topically Applied Cosmetic Products Containing Alpha Hydroxy Acids as Ingredients, Office of Cosmetics and Colors, CFSAN, FDA, September 12, Kurtzweil P. Alpha hydroxy acids for skin care. FDA Consumer 1998; 32: 30 5 [revised May 1999]. (retrieved on May 15, 2005). 9 Smith W. Hydroxy acids and skin aging. Cosmet Toiletries 1994; 109: Viyoch J, Klinthong N, Siripaisal W. Development of oil-in water emulsion containing tamarind fruit pulp extract I. Physical characteristics and stability of emulsion. Naresuan Univ J 2003; 11 (3): Walker AP, Basketter DA, Baverel M et al. Test guidelines for assessment of skin compatibility of cosmetic finished products in man. Food Chem Toxicol 1996; 34: Fullerton A, Fischer T, Lahti A, Wilhelm KP, Takiwaki H, Serup J. Guidelines for measurement of skin colour and erythema. A report from the Standardization Group of the European Society of Contact Dermatitis. Contact Dermatitis 1996; 35: Pinnagoda J, Tupker RA, Agner T, Serup J. Guidelines for transepidermal water loss (TEWL) measurement. A report from the Standardisation Group of the European Society of Contact Dermatitis. Contact Dermatitis 1990; 22: Frosch PJ, Kligman AM. The soap chamber test. A new method for assessing the irritancy of soaps. J Am Acad Dermatol 1979; 1: Lee CH, Maibach HI. Study of cumulative irritant contact dermatitis in man utilizing open application on subcuclinically irritated skin. Contact Dermatitis 1994; 30: Engasser PG, Maibach HI. Hypoallergenic cosmetics. In: P Elsner, HF Merk, HI Maibach, eds. Cosmetics: Controlled Efficacy Studies and Regulation. Berlin: Springer-Verlag; 1999: Schliemann-Willers S, Fuchs S, Kleesz P, Grieshaber R, Elsner P. Fruit acids do not enhance sodium lauryl sulphate-induced cumulative irritant contact dermatitis in vivo. Acta Derm Venereol 2005; 85: Lee CH, Maibach HI. Study of cumulative irritant contact dermatitis in man utilizing open application on subclinically irritated skin. Contact Dermatitis 1994; 30: Wilhelm KP, Freitag G, Wolff HH. Surfactant-induced skin irritation and skin repair: evaluation of a cumulative human irritation model by noninvasive techniques. J Am Acad Dermatol 1994; 31: Wigger-Alberti W, Fischer T, Greif C, Maddern P, Elsner P. Effect of various grit-containing cleansers on skin barrier function. Contact Dermatitis 1999; 41: Fluhr JW et al. Fruit acids and sodium hydroxide in the food industry and their combined effect with sodium lauryl sulphate: controlled in vivo tandem irritation study. Br J Dermatol 2004; 151: Wigger-Alberti W, Krebs A, Elsner P. Experimental irritant contact dermatitis due to cumulative epicutaneous exposure to sodium lauryl sulphate and toluene: single and concurrent application. Br J Dermatol 2000; 143: Goossens AE. Allergy and hypoallergenic products. In: JJ Leyden, AV Rawlings, eds. Cosmetic Science and Technology, Vol. 25. New York: Marcel Dekker; 1998: Blackwell Publishing Journal of Cosmetic Dermatology, 6,