화장품 원료의 피부자극성과 세포독성의 관련성

이은희(Eun Hee Lee),이종권(Jong Kwon Lee),김용규(Yong Kyu Kim),박기숙(Ki Sook Park),안광수(Kwang Soo Ahn),정경미(Kyoung Mi Jung),정해관(Jung Hai Kwan),이선희(Sun Hee Lee),정수연(Soo Youn Chung),홍진태(Jin Tae Hong) 대한약학회 2001 약학회지 Vol.45 No.3

To compare skin irritation and cytotoxicity of anti-wrinkle agents, we examined skin irritation of six anti-wrinkle agents (ascorbic acid, glycolic acid, all trans-retinoic acid, ginseng extract, retinol, EB) in New Zeland white rabbit. Cytotoxicity of these agents was determined by MTT [tetrasolium salt 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide] at multi-time points in cultured HaCaT cell, a human immortalized keratinocyte cell. We then analyzed correlation between skin irritation and cytotoxicity by spearman's rank correlation analysis. All trans- retinoic acid shewed the highest primary irritation index (0.92) in skin irritation test. Being all the six agents not irritant, retinol showed the most cytotoxic agents. The correlation between skin irritation and cytotoxicity IC50 at different time point was 0.814,0.757,0.814 and 0.7 at 3,24, 48 and 72h, respectively. We also found that IC20 and IC80 of these agents showed similar correlation with skin irritation. These results therefore demonstrated that there is close correlation between skin irritation and cytotoxicity IC50 value by MTT in HaCaT cell at early time points by anti-wrinkle agents or IC20value. IC50 at earily time point or IC20 values may be reliable alter- native determinant of skin irritation.

Eye Irritation, Skin Irritation and Skin Sensitization Studies on Surfactin C

Jong-Hwan Lim,Byung-Kwon Park,Youn-Hwan Hwang,Myoung-Seok Kim,Mi-Hyun Hwang,Seong-Chun Park,Man-Hee Rhee,Hyo-In Yun 한국실험동물학회 2008 Laboratory Animal Research Vol.24 No.1

This study was carried out to investigate the skin and eye irritation of surfactin C in rabbits and skin sensitization of surfactin C in guinea pigs. Skin and eye irritation test were performed in New Zealand white rabbits according to Korea Food and Drug Agency (KFDA) guidelines for toxicological test. Buehler method was applied to evaluate the sensitization potential of surfactin C in Hartley guinea pigs. Surfactin C caused slight conjunctival discharge and redness in rabbits by its mechanical irritation. However, it did not show any skin irritation in rabbits and skin sensitization in guinea pigs. The results indicated that surfactin C was a minimal irritant and non-irritant material on eye and skin, respectively. Moreover, it did not cause any skin sensitization in guinea pigs.

한국인에서 피부생물공학적 측정기법을 이용한 피부자극반응의 표준화에 대한 연구

김성진 ( Kim Seong Jin ),전진희 ( Jeon Jin Hui ),이승철 ( Lee Seung Cheol ),원영호 ( Won Yeong Ho ) 대한피부과학회 2003 대한피부과학회지 Vol.41 No.11

N/A Background : It is believed that certain skin types of different age, ethnic group may be more susceptible to the effect of skin irritants than others. However, some previous studies found controversies among intra-, inter-individual variation along with the possibility of ethnic differences in irritation reaction. Objective : Thus, it is mandatory to explore the characteristics of irritant skin reaction in young and old Koreans with objective, standardized instrumental measurement techniques. Method : We have investigated the patterns of skin irritation between adolescent group (19 persons, average age 21.3 ± 2.3) and old group (18 persons, average age 61.0 ± 3.6) after physicochemical stimuli by using tape strippings (15, 30 times) and 24 hours SLS (sodium rauryl sulfate, 1% 0.5%) patch application with skin bioengineering measurements for color change(erythema index), TEWL(transepidermal water loss), capacitance and dermal microcirculation (Laser Doppler Flowmetry flux). Results : The intensity of irritation represented by skin bioengineering measurements showed 30 strippings > 1% SLS > 0.5% SLS > 15 strippings, respectively. The younger the less irritation response at different time-course. TEWL, erythema index and microvascular blood distribution (LDF flux) of old group reached the plateau faster and higher than adolescent group. Occlusive patch containing water induced a potential of irritation regardless of age group that might affect early interpretation of skin bioengineering results. Conclusion : Intensity of irritation reaction and its kinetic suggested the role of age-dependent change of skin. The standardization on skin bioengineering measurement techniques along with irritation methods is mandatory to explore further inter-individual differences of irritation reaction in Korean. (Korean J Dermatol 2003;41(11) : 1440~1447)

실험실에서 제조한 피부를 이용한 피부자극도의 평가

김방순 ( Bang Soon Kim ),이동윤 ( Dong Youn Lee ),한지현 ( Ji Hyun Han ),이종희 ( Jong Hee Lee ),조광현 ( Kwang Hyun Cho ) 대한피부과학회 2002 大韓皮膚科學會誌 Vol.40 No.12

N/A Background : There is an increasing need for the development of in vitro models capable of substituting for animals in cutaneous irritancy studies, Until now, various culture models have been developed, including skin organ cultures, conventional and air-exposed cell cultures. The air-exposed culture forms a multilayered epidermis showing an overall structure which resembles that of a native epidermis. The presence of a coherent stratum cerneum layer in these cultures permits the application of potential irritants at the concentrations and formulations which are applied in vivo. Recently, a new human skin recombinant, made of human keratinocytes cultured on de-epidermized dermis with fibroblast-populated collagen matrix, has been developed and appears to represent a better skin equivalent model than previous models. Objective : In the present study, monolayer-cultured human keratinocytes and the new human skin recombinants were evaluated for the test models of various skin irritants. Methods : The extent of skin irritancy induced after application of 3 different irritants (sodium lauryl sulfate, methyl paraben, and polyethylene glycol-400) was evaluated on the basis of (1) MTT assay, (2) neutral red uptake assay, (3) LDH release, and (4) release of IL-1 alpha. In the human skin recombinants, morphological perturbations and changes in the expression of differentiation-specific protein markers (keratin 1, involucrin, filaggrin, and loricrin) were also evaluated. To determine the difference between in vivo and in vitro models for the detection of irritancy, a patch test was performed on 11 normal human volunteers with various concentrations of the different irritants. Results : The results of the present study show that irritant cytotoxicity correlates well with irritant concentration in both monolayer-cultured human keratinocytes and the new human skin recombinant. The new human skin recombinant is superior to monolayer culture as an in vitro model for skin irritancy screening in that the concentrations of test irritants are the same as in vivo. With the human skin recombinant, morphological changes were observed according to the irritant concentration. Conclusion : The new human skin recombinant can be used as an alternative to animals for skin irritancy screening. (Korean J Dermatol 2002;40(12) : 1505∼1517)

Establishment and evaluation of immortalized human epidermal keratinocytes for an alternative skin irritation test

Kim, Cho-Won,Kim, Chang Deok,Choi, Kyung-Chul Elsevier 2017 Journal of pharmacological and toxicological metho Vol.88 No.2

<P><B>Abstract</B></P> <P>Human skin is located at the outermost part of the body, and various cosmetics and chemicals that may come in contact with human skin need to be evaluated for their potential to cause irritation. Until recently, the Draize test was considered the standard method for skin irritation; however, this technique has disadvantages such as the need to sacrifice many rabbits and subjective scoring. Thus, to contribute to the development of an animal-free alternative skin irritation test, we investigated the cytotoxicity and inflammatory response to standard skin irritants in SV40 large T antigen-transformed human epidermal keratinocyte 2 cells (SV-HEK2 cells). In this study, we established an SV-HEK2 cell line immortalized by SV40 large T antigen (SV40 T) and characterized the inherent morphological and cytological properties. We next used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or neutral red uptake (NRU) assays of cell viability to investigate the optimal experimental conditions for determining SV-HEK2 cell viability after exposure to sodium dodecyl sulfate at 6.25×10<SUP>−3</SUP>% to 1×10<SUP>−1</SUP>% as a standard skin irritant. We then examined the viability of SV-HEK2 cells in response to five skin irritants (benzalkonium chloride, isopropanol, sodium dodecyl sulfate, Triton X-100 and Tween20) at 5×10<SUP>−3</SUP>% to 1×10<SUP>−1</SUP>% by MTT or NRU assay. Finally, we estimated the level of cytokines secretion in response to stimulation by skin irritants in SV-HEK2 cells. The results revealed that SV-HEK2 cells responded well to skin irritants in a concentration-dependent manner and that there was good correlation between irritant concentration and cytotoxicity (or cytokine secretion) when cells were exposed to skin irritants for 10min at room temperature (RT) using an MTT assay. Overall, these findings suggest that SV-HEK2 cells could be a good alternative <I>in vitro</I> model for skin irritation tests.</P>

Assessment of the Dermal and Ocular Irritation Potential of Lomefloxacin by Using In Vitro Methods

안준호,엄기환,이미가엘 한국독성학회 2010 Toxicological Research Vol.26 No.1

The evaluation of eye and skin irritation potential is essential to ensuring the safety of human in contact with a wide variety of substances. Despite this importance of irritation test, little is known with respect to the irritation potency of lomefloxacin, a fluoroquinolone antibiotic, which has been known to cause phototoxicity with an abnormal reaction of the skin. Thus, to investigate the tendency of lomefloxacin to cause eye and skin irritation, we carried out in vitro eye irritation test using Balb/c 3T3, and in vitro skin irritation test using KeraSkinTM human skin model system. 3T3 neutral red uptake assay has been proposed as a potential replacement alternative for the Draize Eye irritation test. In this study, the IC50 value obtained for lomefloxacin was 375 μg. According to the classification model used for determining in vitro categories,lomefloxacin was classified as moderately irritant. For evaluation of skin irritation, engineered epidermal equivalents (KeraSkinTM) were subjected to 10 and 25 mg of lomefloxacin for 15 minutes. Tissue damage was assessed by tissue viability evaluation, and by the release of a pro-inflammatory mediator, interleukin-1α. Lomefloxacin increased the interleukin-1α release after 15 minutes of exposure and 42 hours of post incubation, although no decrease in viability was observed. Therefore, lomefloxacin is considered to be moderately irritant to skin and eye.

Assessment of the Dermal and Ocular Irritation Potential of Lomefloxacin by Using In Vitro Methods

Ahn, Jun-Ho,Eum, Ki-Hwan,Lee, Mi-Chael Korean Society of ToxicologyKorea Environmental Mu 2010 Toxicological Research Vol.27 No.1

The evaluation of eye and skin irritation potential is essential to ensuring the safety of human in contact with a wide variety of substances. Despite this importance of irritation test, little is known with respect to the irritation potency of lomefloxacin, a fluoroquinolone antibiotic, which has been known to cause phototoxicity with an abnormal reaction of the skin. Thus, to investigate the tendency of lomefloxacin to cause eye and skin irritation, we carried out in vitro eye irritation test using Balb/c 3T3, and in vitro skin irritation test using $KeraSkin^{TM}$ human skin model system. 3T3 neutral red uptake assay has been proposed as a potential replacement alternative for the Draize Eye irritation test. In this study, the $IC_{50}$ value obtained for lomefloxacin was 375 ${\mu}g$. According to the classification model used for determining in vitro categories, lomefloxacin was classified as moderately irritant. For evaluation of skin irritation, engineered epidermal equivalents ($KeraSkin^{TM}$) were subjected to 10 and 25 mg of lomefloxacin for 15 minutes. Tissue damage was assessed by tissue viability evaluation, and by the release of a pro-inflammatory mediator, interleukin- 1${\alpha}$. Lomefloxacin increased the interleukin-1${\alpha}$ release after 15 minutes of exposure and 42 hours of post incubation, although no decrease in viability was observed. Therefore, lomefloxacin is considered to be moderately irritant to skin and eye.

Assessment of the Dermal and Ocular Irritation Potential of Lomefloxacin by Using In Vitro Methods

Jun-Ho Ahn,Ki-Hwan Eum,Michael Lee 한국독성학회 2010 Toxicological Research Vol.26 No.1

The evaluation of eye and skin irritation potential is essential to ensuring the safety of human in contact with a wide variety of substances. Despite this importance of irritation test, little is known with respect to the irritation potency of lomefloxacin, a fluoroquinolone antibiotic, which has been known to cause phototoxicity with an abnormal reaction of the skin. Thus, to investigate the tendency of lomefloxacin to cause eye and skin irritation, we carried out in vitro eye irritation test using Balb/c 3T3, and in vitro skin irritation test using KeraSkin™ human skin model system. 3T3 neutral red uptake assay has been proposed as a potential replacement alternative for the Draize Eye irritation test. In this study, the IC?? value obtained for lomefloxacin was 375 ㎍. According to the classification model used for determining in vitro categories, lomefloxacin was classified as moderately irritant. For evaluation of skin irritation, engineered epidermal equivalents (KeraSkin™) were subjected to 10 and 25 mg of lomefloxacin for 15 minutes. Tissue damage was assessed by tissue viability evaluation, and by the release of a pro-inflammatory mediator, interleukin-1α. Lomefloxacin increased the interleukin-1α release after 15 minutes of exposure and 42 hours of post incubation, although no decrease in viability was observed. Therefore, lomefloxacin is considered to be moderately irritant to skin and eye.

Phenoxyethanol을 이용한 저자극 방부시스템 개발에 관한 연구

안기웅 ( Gi Woong Ahn ),이춘몽 ( Chun Mong Lee ),김형배 ( Heong Bae Kim ),정지헌 ( Ji Hean Jeong ),조병기 ( Byoung Kee Jo ) 대한화장품학회 2005 대한화장품학회지 Vol.31 No.1

최근, 민감성 피부가 증가함에 따라 화장품의 안전성이 매우 중요시되고 있으며, 특히 방부제는 화장품 사용에 따른 부작용을 일으킬 수 있는 주요 자극원의 하나로 알려져 있다. 하지만, 방부제의 세포 독성 피부 투과, 유/수 분배, 항균력 비교 및 이를 통한 피부 자극과의 상관성 분석에 관한 연구는 전무한 실정이다. 본 연구의 목적은 상기의 여러 factor를 고려하여 화장품에서 빈번히 사용되고 있는 방부제의 하나인 phenoxyethanol을 이용한 저자극 방부시스템 개발에 관한 것이다. MTT assay를 통하여 human norm기 fibroblast cell에 대한 독성을 평가해 본 결과, 세포 독성은 propylparaben > butylparaben > ethylparaben > methylparaben > triciosan > phenoxyethanol 순으로 확인되어 phenoxyethanol이 다른 방부제에 비해 낮은 세포 독성을 나타낸 반면, 피부 일차자극을 알아보기 위하여 수행한 인체 첩포시험에서는 triclosan, methylparaben에 비해 높은 피부 자극을 나타내었다. 5~8 주령의 웅성 무모생쥐의 피부를 적출하여 in vitro Franz diffusion cell system을 이용한 방부제의 피부 투과도를 측정하여 본 결과, 피부 투과도는 phenoxyethanol > methylparaben > ethylparaben > propylparaben > butylparaben > triclosan 순으로 확인되어 세포 독성이 낮은 phenoxyethanol의 높은 피부 자극이 높은 피부 투과도와 연관성이 큰 것을 확인하였다. 따라서, 본 연구에서는 비교적 독성이 낮은 phenoxyethanol의 피부 투과도를 감소시킬 수 있는 방법을 찾고자 하였으며, 연구 결과, 제형내 polarity가 낮은 oil을 사용할 경우 phenoxyethanol의 피부 투과가 현격히 감소하며, 피부 자극도 감소함을 알 수 있었다. Oil polarity에 따른 Phenoxyethanol의 유/수 분배 측정 결과, Polarity가 낮은 oil에서는 70% 이상의 Phenoxyethanol이 수상에 존재한 반면, polarity가 높은 oil에서는 약 70~90%의 phenoxyethanol이 유상에 존재하였다. 또한, 미생물에 대한 항균력도 phenoxyethanol이 수상에 많이 존재할수록 증가하는 경향을 나타내었다. 따라서, 제형 내 oil tomposition을 변화시킴으로써 phenoxyethanol의 사용량을 줄일 수 있을 뿐만 아니라, 피부 투과를 감소시켜 보다 피부 자극이 적은 저자극 방부시스템 개발이 가능하리라 보여 진다. Recently, according as people who have sensitive skin increase, we've been giving more importance to the safety of cosmetics. Especially, preservative is known to be one of the main stimuli which cause side-effects of cosmetics. However, there have been few reports describing cell cytotoxicity, skin penetration, oil-aqueous phase partition, anti-microbial activity of preservatives and their correlation with skin irritation. The study is aimed to develop low irritable preservative system with phenoxyethanol, one of the most commonly used preservatives in cosmetics, considering various factors mentioned above. According to our results of cell cytotoxicity against human normal fibroblasts by means of MTT assay, phenoxyethanol showed the lowest cytotoxicity when compared to other preservatives tested (cytotoxicity: pro-pylparaben > butylparaben > ethylparaben > methylparaben > triclosan > phenoxyethanol), but human patch test for assessing shin primary irritation revealed that phenoxyethanol has higher skin irritation than methylparaben and triclosan. We performed in vitro skin penetration test using horizontal Franz diffusion cells with skin membrane prepared from hairless mouse (5~8 weeks, male) to evaluate the rate of skin penetration of preservatives. From the results, we found that the higher irritable property of phenoxyethanol in human skin correlates with its predominant permeability (skin penetration: phenoxyethanol > methylparaben > ethylparaben > propylparaben > butylfaraben > triclosan). Therefore, we made an effort to reduce skin permeability of phenoxyethanol and found that not only the rate of skin penetration of phenoxyethanol but also its skin irritation is dramatically reduced in formulas containing oils with low polarity. In the experiments to investigate the effect of oil polarity on the oil-aqueous phase partition of phenoxyethanol, more than 70% of phenoxyethanol was partitioned in aqueous phase in formulas containing oils with low polarity, while about 70~90% of phenoxyethanol was partitioned in oil phase in formulas containing oils with high polarity. Also, in aqueous phase phenoxyethanol showed greater anti-microbial activity. Conclusively, it appears that we can develop less toxic preservative system with reduced use dosage of phenox-yethanol and its skin penetration by changing oil composition in formulas.

연구논문 : 인공피부를 이용한 피부자극성 대체시험법

문지영 ( Ji Young Moon ),맹은호 ( Eun Ho Maeng ),박학수 ( Hak Soo Park ),권민 ( Min Kwon ),장동혁 ( Dong Hyouk Jang ),조영민 ( Young Min Cho ),고은옥 ( Eun Ok Koh ),성하정 ( Ha Jung Sung ),박철범 ( Cheol Beom Park ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 No.1

Reconstructed skin provides advantages to single cell culture testing and leads to promising results as shown by different validation studies. In this study we investigated the efficacy of "MCTT Skin model" compared with "EPI-200" and in vivo test. We conducted endpoint analysis including cell viability (MTT reduction, CCK-8) and IL-1 alpha release. Sodium lauryl sulfate(20%), Potassium hydoxide(5%), Heptanal, Methyl palmitate, 1,1,1-Trichloroethane, 2,4-Xylidine, 3,3`-Dithiodipropionic acid, 4-Amino-1,2,4-triazol and Benzyl chloroformate were selected for this comparison. Potassium hydoxide(5%) and Benzyl chloroformate were excluded from animal tests because of their pH. Sodium lauryl sulfate induced severe irritation, Heptanal induced moderate irritation. Methyl palmitate, 1,1,1-Trichloroethane induced mild irritation. 2,4-Xylidine, 3,3`-Dithiodipropionic acid, 4-Amino-1,2,4-triazole induced no irritation on rabbit skin irritation test. Results of MTT assay and CCK-8 assay were not different between EPI-200 MCTT Skin model but IL-lα levels of some test items different between EPI-200 and MCTT Skin model. We concluded that this MCTT Skin model is useful for alternative skin irritation test, although other tests should be conducted for many other chemicals in international validation study.