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두 가지 단계적 접근법을 이용한 30종 화학물질의 안자극성 평가
전혜련 ( Hye Lyun Jeon ),조아랑 ( Ah Rang Cho ),고경육 ( Kyung Yuk Ko ),김주환 ( Joohwan Kim ),정미경 ( Mi Kyung Jeong ),박교현 ( Kyo-hyun Park ),김배환 ( Bae-hwan Kim ),오원준 ( Won Jun Oh ),이종권 ( Jong Kwon Lee ),박기숙 ( Ki S 한국동물실험대체법학회 2020 동물실험대체법학회지 Vol.14 No.1
Various alternative test methods are being developed to replace in vivo Draize rabbit test that evaluates eye irritation. However, a single alternative method has difficulty to be applied in safety evaluation on substances, because it cannot fully replace the in vivo test by simulating only part of the in vivo system. For this reason, different studies using several alternative test methods and test results of literatures have been under way. Our previous study suggested effective tiered approaches using three tests among Short Time Exposure (STE), Bovine Corneal Opacity and Permeability (BCOP), Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM), re-constructed human cornea epitheliums (RhCE) tests. This study aimed to evaluate eye irritation potential for 30 new test chemicals using two tiered approaches. According to the data generated by direct test performance and literature survey, the accuracy, sensitivity and specificity of the two tiered approaches were 83.3%∼86.7%, 93.3%, 73.3%∼ 80%, respectively. Furthermore, the accuracy, sensitivity, and specificity of the two tiered approaches by applying combined data against 30 test chemicals in our present study and 47 test chemicals in previous study were 90.9%∼92.2%, 95.9%∼98.0%, 78.6%∼85.7%. Consequently, the two tiered approaches may be used to identify between irritants and non-irritants to replace in vivo test.
연구논문 : 인체구강모델을 이용한 구강점막자극 동물대체시험법 개발
임혜림 ( Hye Rim Lim ),이정선 ( Jung Sun Yi ),김태성 ( Tae Sung Kim ),고경육 ( Kyung Yuk Ko ),안일영 ( Il Young Ahn ),김주환 ( Joo Hwan Kim ),이진하 ( Jin Ha Lee ),양송이 ( Song Yi Yang ),김광만 ( Kwang Mahn Kim ),손수정 ( Soo Jun 한국동물실험대체법학회 2015 동물실험대체법학회지 Vol.9 No.1
Ensuring the biological safety of dental materials is important, because people of all ages use them and they stay inside the mouth of a person for years once they are put into it. However, current oral irritation tests bring pain to animals, and there are no internationally accepted alternative test methods. In this study, we have developed a new method to test for oral irritation using three-dimensional (3D) human oral mucosal models in the light of growing needs for alternatives to animal testing in order to ensure the safety of dental materials. The commercially available 3D human oral mucosal models (EpiOral™, HOM™) were cultured, using normal oral epithelial cells, and histologically and phenotypically similar to native buccal tissues. Two controls and three dental materials that are widely used in dentistry were selected for testing. These included a polyethylene film (negative control), 1% Triton X-100 (positive control), resin, denture base resin and impression materials. These three dental materials were prepared according to manufacturers’ instructions, and were turned into 0.7-cm discs in diameter. And then, we directly placed the controls and the three dental materials on the top of each human oral mucosal model for 24 hours and measured tissue viability via the MTT assay and cytokine secretion. When cell viability is less than 50% or cytokine secretion is more than 250 pg/ml, a test material is evaluated as an irritant. The negative control, resin and denture base resin turned out non-irritants while the positive control and the impression materials irritants. Since the results of the oral mucosal irritation test of dental materials using human oral mucosal models matched those of existing cytotoxicity tests, it seems that the oral mucosal test can be a good alternative method. The results of this study suggest that the oral mucosal irritation test employing the 3D human oral mucosal models can be an alternative test method for dental materials. And further validation studies are required in order to take advantage of this method in the future.
임혜림 ( Hye Rim Lim ),이정선 ( Jung Sun Yi ),김태성 ( Tae Sung Kim ),고경육 ( Kyung Yuk Ko ),안일영 ( Il Young Ahn ),김주환 ( Joo Hwan Kim ),이진하 ( Jin Ha Lee ),양송이 ( Song Yi Yang ),김광만 ( Kwang Mahn Kim ),손수정 ( Soo Jun 한국동물실험대체법학회 2015 동물실험대체법학회지 Vol.9 No.1
Ensuring the biological safety of dental materials is important, because people of all ages use them and they stay inside the mouth of a person for years once they are put into it. However, current oral irritation tests bring pain to animals, and there are no internationally accepted alternative test methods. In this study, we have developed a new method to test for oral irritation using three-dimensional (3D) human oral mucosal models in the light of growing needs for alternatives to animal testing in order to ensure the safety of dental materials. The commercially available 3D human oral mucosal models (EpiOral™, HOM™) were cultured, using normal oral epithelial cells, and histologically and phenotypically similar to native buccal tissues. Two controls and three dental materials that are widely used in dentistry were selected for testing. These included a polyethylene film (negative control), 1% Triton X-100 (positive control), resin, denture base resin and impression materials. These three dental materials were prepared according to manufacturers’ instructions, and were turned into 0.7-cm discs in diameter. And then, we directly placed the controls and the three dental materials on the top of each human oral mucosal model for 24 hours and measured tissue viability via the MTT assay and cytokine secretion. When cell viability is less than 50% or cytokine secretion is more than 250 pg/ml, a test material is evaluated as an irritant. The negative control, resin and denture base resin turned out non-irritants while the positive control and the impression materials irritants. Since the results of the oral mucosal irritation test of dental materials using human oral mucosal models matched those of existing cytotoxicity tests, it seems that the oral mucosal test can be a good alternative method. The results of this study suggest that the oral mucosal irritation test employing the 3D human oral mucosal models can be an alternative test method for dental materials. And further validation studies are required in order to take advantage of this method in the future.
연구논문 : 수컷 생식줄기세포를 이용한 생식독성 동물대체시험법 개발
전혜련 ( Hye Lyun Jeon ),김태성 ( Tae Sung Kim ),이정선 ( Jung Sun Yi ),안일영 ( Il Young Ahn ),고경육 ( Kyung Yuk Ko ),이진하 ( Jin Ha Lee ),김주환 ( Joo Hwan Kim ),손수정 ( Soo Jung Sohn ) 한국동물실험대체법학회 2015 동물실험대체법학회지 Vol.9 No.1
Currently, alternative test methods are actively being developed as a replacement for animal testing, based on the 3Rs (Replacement, Refinement, Reduction). However, the development of alternative test methods for the evaluation of reproductive and developmental toxicity is in its early stage, and no established test methods exist. This study is aimed at developing an alternative test method to evaluate reproductive toxicity using male germline stem cells (GSC). We selected a negative toxic substance (Dimethyl sufloxide (DMSO)) and two positive toxic substances (NEthyl- N-Nitrosourea (ENU), methyl methanesulfonate (MMS) characterized in OECD TG 489. We also used seven test substances (2,4-diaminotoluene (2,4-DAT), cyclophosphamide (CP), benzo[α]pyrene (BP), cadmium chloride (CdCl2), D-mannitol (MA), n-butyl chloride (NBC) and trimethyl ammonium chloride (TAC)) suggested in a scientific paper published by ECVAM. The endpoints of toxicological evaluation were cell viability (MTT assay) and comet assay which is a method to measure DNA damage. As a result of our study with a 50% inhibitory concentration (IC50) determined using the MTT assay, IC50 values of ENU and MMS were 1.7 mM and 0.4 mM, respectively. Also, IC50 values of 2,4-DAT, CP, BP andCdCl2 were 10.3 mM, 5.5 mM, 0.4 mM and 0.18 mM, respectively. As cell viability wasn’t significantly different from that of the control, IC50 values of MA, NBC and TAC could not be calculated. In the comet assay, Tail DNA%, Tail Length (TL) and Olive Tail Moment (OTM) of the two positive toxic substances (ENU and MMS) and the four test substances (2,4-DAT, CP, BP and CdCl2) significantly grew in comparison with the control. However, Tail DNA%, TL and OTM of the negative toxic substance (DMSO) and the three positive toxic substances (MA, NBC, TAC) were similar to those of the control. In conclusion, this study demonstrated that the comet assay using GSC could be a candidate test method in predicting male reproductive toxicity.
수컷 생식줄기세포를 이용한 생식독성 동물대체시험법 개발
전혜련 ( Hye Lyun Jeon ),김태성 ( Tae Sung Kim ),이정선 ( Jung Sun Yi ),안일영 ( Il Young Ahn ),고경육 ( Kyung Yuk Ko ),이진하 ( Jin Ha Lee ),김주환 ( Joo Hwan Kim ),손수정 ( Soo Jung Sohn ) 한국동물실험대체법학회 2015 동물실험대체법학회지 Vol.9 No.1
Currently, alternative test methods are actively being developed as a replacement for animal testing, based on the 3Rs (Replacement, Refinement, Reduction). However, the development of alternative test methods for the evaluation of reproductive and developmental toxicity is in its early stage, and no established test methods exist. This study is aimed at developing an alternative test method to evaluate reproductive toxicity using male germline stem cells (GSC). We selected a negative toxic substance (Dimethyl sufloxide (DMSO)) and two positive toxic substances (NEthyl- N-Nitrosourea (ENU), methyl methanesulfonate (MMS) characterized in OECD TG 489. We also used seven test substances (2,4-diaminotoluene (2,4-DAT), cyclophosphamide (CP), benzo[α]pyrene (BP), cadmium chloride (CdCl2), D-mannitol (MA), n-butyl chloride (NBC) and trimethyl ammonium chloride (TAC)) suggested in a scientific paper published by ECVAM. The endpoints of toxicological evaluation were cell viability (MTT assay) and comet assay which is a method to measure DNA damage. As a result of our study with a 50% inhibitory concentration (IC50) determined using the MTT assay, IC50 values of ENU and MMS were 1.7 mM and 0.4 mM, respectively. Also, IC50 values of 2,4-DAT, CP, BP andCdCl2 were 10.3 mM, 5.5 mM, 0.4 mM and 0.18 mM, respectively. As cell viability wasn’t significantly different from that of the control, IC50 values of MA, NBC and TAC could not be calculated. In the comet assay, Tail DNA%, Tail Length (TL) and Olive Tail Moment (OTM) of the two positive toxic substances (ENU and MMS) and the four test substances (2,4-DAT, CP, BP and CdCl2) significantly grew in comparison with the control. However, Tail DNA%, TL and OTM of the negative toxic substance (DMSO) and the three positive toxic substances (MA, NBC, TAC) were similar to those of the control. In conclusion, this study demonstrated that the comet assay using GSC could be a candidate test method in predicting male reproductive toxicity.
국내 인체피부모델을 이용한 광독성 동물대체시험법 개발 연구
이용선 ( Yong-sun Lee ),이정선 ( Jung-sun Yi ),임혜림 ( Hye-rim Lim ),김태성 ( Tae-sung Kim ),안일영 ( Il-young Ahn ),고경육 ( Kyung-yuk Ko ),김주환 ( Joo-hwan Kim ),박혜경 ( Hye-kyung Park ),이종권 ( Jong-kwon Lee ),손수정 ( Soo- 한국동물실험대체법학회 2016 동물실험대체법학회지 Vol.10 No.1
Human reconstituted epidermis models have been widely used in evaluating phototoxicity. However, human reconstituted epidermis models made in Korea have not been employed to assess phototoxicity. In this study, we conducted an in vitro phototoxicity test with Neoderm<sup>®</sup>-E, human reconstituted epidermis model from Korea, in order to see if the model could be used for the evaluation of phototoxicity. Firstly, we performed an UVA sensitivity test and selected 6 J/cm<sup>2</sup> for an UVA irradiation dose. Chlorpromazine as a positive control was then tested to confirm that the substance induces phototoxicity in this model. Afterwards, 3 phototoxins and 2 non-phototoxins were tested using Neoderm<sup>®</sup>-E and Epiderm<sup>TM</sup>, a generally used phototoxicity test model, to compare prediction results for each chemical. The results indicated that all those 5 chemicals were correctly predicted with Neoderm<sup>®</sup>-E, which was accordant with the phototoxic information on them. However, only 3 chemicals were correctly predicted with Epiderm<sup>TM</sup>. Consequently, the predictive capacity of the test with Neoderm<sup>®</sup>-E could be higher than that using Epiderm<sup>TM</sup>. Overall, we reached the conclusion that Neoderm<sup>®</sup>-E could be used in evaluating phototoxicity.