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Lee, Y.S.,Yi, J.S.,Seo, S.J.,Kim, J.H.,Jung, M.S.,Seo, I.K.,Ahn, I.,Ko, K.,Kim, T.S.,Lim, K.M.,Sohn, S. Academic Press 2017 Regulatory toxicology and pharmacology Vol.83 No.-
The local lymph node assay using 5-bromo-2-deoxyuridine (BrdU) with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA that is used to identify skin sensitizers by counting BrdU-incorporated lymph node cells (LNCs) with flow cytometry. Unlike other LLNA methods (OECD TG 429, 442A and 442B) in which the CBA/J mouse strain is used, LLNA: BrdU-FCM was originally designed to be compatible with BALB/c, a mouse strain that is more widely used in many countries. To justify the substitution of CBA/J for BALB/c, the equivalence of the test results between two strains shall be established prior to the official implementation of LLNA: BrdU-FCM. This study aims to compare the test results of LLNA: BrdU-FCM produced in BALB/c mice with those in CBA/J mice for 18 reference substances, including 13 sensitizers and 5 non-sensitizers, listed in OECD Test Guideline 429. Based on the LLNA: BrdU-FCM test procedure, we selected an appropriate solvent and then performed preliminary tests to determine the non-irritating dose ranges for the main study, which revealed the difference in the irritation responses to 8 of the 18 chemicals between the two strains. In the main study, we measured the changes in the number of total LNCs, which indicated differences in the responses to test chemicals between the two strains. However, the stimulation index obtained with the counts of BrdU-incorporated LNCs with 7-AAD using flow cytometry yielded comparable results and 100% concordance between the BALB/c and CBA/J mouse strains was achieved, suggesting that the performance of LLNA: BrdU-FCM using BALB/c mice was equivalent to that with CBA/J mice.
Local Lymph Node Assay를 이용한 삼황사심탕의 피부감작성 평가
김민지 ( Min Ji Kim ),김배환 ( Bae Hwan Kim ) 한국동물실험대체법학회 2014 동물실험대체법학회지 Vol.8 No.1
The purpose of this study is to evaluate the skin sensitization ability of a natural herbal extract, Samhwang-Sasimtang, using Local Lymph Node Assay (LLNA), a alternative method to guinea pig maximization test (GPMT). A non-radioisotopic LLNA employing 5-bromo-2-deoxyuridine (BrdU) with flow cytometry (FACS) is used in this study instead of the traditional LLNA using a radioisotope, 3H-thymidine. Through the LLNA:BrdU-FCM study in Balb/c mice, Samhwang-Sasimtang extract (SSE, 0.1%, 1%, 2%) was confirmed as a non-sensitizer, because it showed lower than 3 of stimulation index (SI) which is calculated relative to the concurrent vehicle control value. 4-Phenylenediamine (PPD), a positive control, showed sensitization potential with high SIs which were greater than 3 in all tested concentrations. From this study, we also confirmed that the modified LLNA(LLNA:BrdU-FCM) in which BrdU is used in place of radioisotope incorporation is a useful alternative to animal testing for skin sensitization.
연구논문 : Local Lymph Node Assay를 이용한 삼황사심탕의 피부감작성 평가
김민지 ( Min Ji Kim ),김배환 ( Bae Hwan Kim ) 한국동물실험대체법학회 2014 동물실험대체법학회지 Vol.8 No.1
The purpose of this study is to evaluate the skin sensitization ability of a natural herbal extract, Samhwang-Sasimtang, using Local Lymph Node Assay (LLNA), a alternative method to guinea pig maximization test (GPMT). A non-radioisotopic LLNA employing 5-bromo-2-deoxyuridine (BrdU) with flow cytometry (FACS) is used in this study instead of the traditional LLNA using a radioisotope, 3H-thymidine. Through the LLNA:BrdU-FCM study in Balb/c mice, Samhwang-Sasimtang extract (SSE, 0.1%, 1%, 2%) was confirmed as a non-sensitizer, because it showed lower than 3 of stimulation index (SI) which is calculated relative to the concurrent vehicle control value. 4-Phenylenediamine (PPD), a positive control, showed sensitization potential with high SIs which were greater than 3 in all tested concentrations. From this study, we also confirmed that the modified LLNA(LLNA:BrdU-FCM) in which BrdU is used in place of radioisotope incorporation is a useful alternative to animal testing for skin sensitization.
Ravi Gautam,DaEun Lee,Anju Maharjan,Manju Acharya,JiHun Jo,HyunJi Park,YuBin Kim,Pramod Bahadur KC,Chang Yul Kim,HyoungAh Kim,JiMin Choi,SooMin Lim,YoungWoo Shin,SuBin Yang,Yong Heo 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7
Nanoparticles (NPs), fine particles ranging from 1 to 100 nm, are being used extensively in various fields including industry, agriculture, engineering. Nanoparticles also have become the imperative parts of cosmetic and dermal products owing to the higher benefits over their counter bulk particles. Manganese oxide (Mn2O3) and copper oxide (CuO) are used in cosmetics and dermal products owing to their UV protection and antimicrobial properties and also to make products transparent and aesthetically acceptable. Although Mn2O3 and CuO are used in consumer products that get exposed to the skin, their skin sensitization potential has not been well investigated. In this study we employed, Local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) to predict the skin sensitization (SS) potential of nanoparticles. LLNA: BrdU-FCM is a method for screening of SS potential that addressed the activation and proliferation of T-lymphocytes, key event-4 (KE-4), on adverse outcome pathway (AOP) for SS. Mn2O3 (5%, 10%, and 25%), and CuO (5%, 10%, and 25 %) NPs were suspended in dimethyl sulfoxide (DMSO) for experimentation. AOO (acetone: olive oil = 4:1) and α-hexyl cinnamaldehyde were used as negative and positive control respectively. Mn2O3 and CuO NPs were both classified as non-sensitizer with maximum stimulation indices of 1.4 and 0.99 respectively. Although both NPs were classified as non-sensitizer using LLNA: BrdU-FCM, further assays addressing other KEs of AOP for SS could be carried out as confirmation and cautious use of products containing these nanoparticles is recommended as various studies have revealed their toxicity in vitro . This study was supported by the Korea National Research Foundation (grant number: 2020R1I1A3A0403650911) and the Ministry of Environment-Educational training program for the management of information on the hazards and risk of chemical substances.
( Da Eun Kim ),( Hyeri Yang ),( Won Hee Jang ),( Kyoung Mi Jung ),( Miyoung Park ),( Jin Kyu Choi ),( Mi Sook Jung ),( Eun Young Jeon ),( Yong Heo ),( Kyung Wook Yeo ),( Ji Hoon Jo ),( Jung Eun Park ) 영남대학교 약품개발연구소 2016 영남대학교 약품개발연구소 연구업적집 Vol.26 No.-
In order for a novel test method to be applied for regulatory purposes, its reliability and relevance, I.e., reproducibility and predictive capacity, must be demonstrated, Here, we examine the predictive capacity of a novel non-radioisotopic local lymph node assay, LLNA:BrdU-FCM (5-bromo-2`-deoxyuridine-flow cytome-try), with a cutoff approach and inferential statistics as a prediction model. 22 reference substances in OECDTG429 were tested with a concurrent positive control. Hexylcinnamaldehyde 25%(PC), and the stimulation index(SI) representing the fold increase in lymph node cells over the vehicle control was obtained. The optimal cutoff SI(2.7≤cutoff<3.5), with respect to predictive capacity, was obtained by a receiver operating character-istic curve, which produced 90.9% accuracy for the 22 substances. To address the inter-test variability in respon-siveness, SI values standardized with PC were employed to obtain the optimal percentage cutoff(42.6≤Cutoff<57.3%of PC), which produced 86.4% accuracy. A test subastance may be diagnosed as a sensitizer if a statistically significant increase in Si is elicited. The parametric one-sided t-test and non-parametric Wilcoxon rank-sum test produced 77.3% accuracy. Similarly, a test substance could be defined as a sensitizer if the SI means of the vehicle control, and of the low, middle, and high concentrations were statistically significantly different, which was test-ed using ANOVA or Kruskal-Wallis, with post hoc analysis, Dunnett, or DSCF(Dwass-Steel-Critchlow-Fligner), respectively, depending on the equal variance test, producing 81.8% accuracy. The absolute SI-based cutoff approach produced the best predictive capacity, however the discordant decisions between prediction models need to be examined further.