국내 서식 담수새우 새뱅이(Neocaridina denticulata)를 이용한 3,4-Dichloroaniline의 급성독성 교차시험(Ring test)

신유진,박경화,이재우,김지은,조재구,김자현,강민호,김경태,김필제 한국환경보건학회 2019 한국환경보건학회지 Vol.45 No.3

Objectives: For suitable risk management of the domestic aquatic environment, it is necessary to conduct toxicity tests using species native to Korea. In the present study, we performed toxicity ring tests using endemic freshwater arthropoda Neocaridina denticulata and evaluated its validity and reproducibility as an international standard test species. Methods: To evaluate the sensitivity levels of N. denticulata to hazardous chemicals, toxicity values for several chemicals were compared with other standard test species. Intra- and inter-laboratory acute toxicity tests were performed both within a single laboratory and among four laboratories respectively using 3,4-Dichloroaniline, which is generally used as a reference test substance in fish toxicity tests. In addition, intra- and inter-laboratory coefficient of variations (CVs) were calculated to evaluate reproducibility based on the estimated toxicity values. Results: The sensitivity of N. denticulata to several chemicals was found to be similar with D. manga, indicating that the species is valid as a test species. The CVs of the intra- and inter-laboratory tests were 22.946% with four qualified runs and 8.828% among the four laboratories, respectively. Conclusions: N. denticulata serves in an important role in the food chain of Korean aquatic ecosystems and also inhabits several other Asian countries. Since the validity and reproducibility of the species were confirmed as a toxicity test species in this study, further efforts are needed to establish N. denticulata as the international standard test species for the appropriate risk assessment of aquatic ecosystems at home and abroad.

포제부자(?製附子) 및 포제부자(?製附子) 함유 삼부탕(蔘附湯)의 aconitine 함량 분석과 단회투여 독성시험

배정후,김경철,신순식,황원덕,Bae, Jeong Hu,Kim, Gyeong Cheol,Shin, Soon Shik,Hwang, Won Deuk 대한한의학방제학회 2017 大韓韓醫學方劑學會誌 Vol.25 No.1

Objectives : The contents of aconitine in aconiti radix lateralis preparata, purified hot water extract of Aconiti Radix lateralis preparata, and purified hot water extract of Sambutang-P that contains Aconiti Radix lateralis preparata was analyzed to compare toxicity. Toxicity of Sambutang-P that contains Aconiti Radix lateralis preparata was assessed with a single oral toxicity test on 6-week-old male and female Sprague-Dawley rats. Methods : 1. The contents of aconitine in Aconiti Radix lateralis preparata, purified hot water extract of Aconiti Radix lateralis preparata, and purified hot water extract of Sambutang-P that contains Aconiti Radix lateralis preparata was analyzed using the purity test according to the "Korean Herbal Pharmacopoeia". 2. 2,000mg/kg was injected for the single oral toxicity test of purified hot water extract of Sambutang-P that contains Aconiti Radix lateralis preparata, and the test was done for a test group (injection) and a control group, each with 5 male and 5 female rats. For 14 days after injection, rats were observed for general symptoms and changes in weight. Afterwards, blood biochemical test, autopsy, and histophathological exam of the liver was conducted. Results : 1. The contents of aconitine was 0.0785% for Aconiti Radix lateralis preparata, 0.1510% for purified hot water extract of Aconiti Radix lateralis preparata, and 0.1248% for purified hot water extract of Sambutang-P that contains Aconiti Radix lateralis preparata. 2. There was no death of either male or female rats in both the control group and the test group (injection of 2,000mg/kg). 3. No unusual symptom was observed in both the control group and the test group (injection of 2,000mg/kg). 4. No significant change in weight was observed for both male and female rats in the test group (2,000mg/kg). 5. The histopathological exam of ALT, AST, ALP, GGT and LDH showed no significant changes for both male and female rats in the test group (2,000mg/kg). 6. According to the autopsy results, no visible abnormality of organs or tissues was found in both the control group and the test group (2,000mg/kg). 7. According to the histopathological exam of the liver, the effect of the injected material was not observed for either male or female rats in the test group (2,000mg/kg). Conclusions : The contents of aconitine in Aconiti Radix lateralis preparata was lower for decoction of Sambutang-P with ginseng radix alba than for decoction of only Aconiti Radix lateralis preparata. This suggests that ginseng radix alba can dilute toxicity of Aconiti Radix lateralis preparata. As for a single oral toxicity test of Sambutang-P that contains Aconiti Radix lateralis preparata, no abnormal reaction was observed even when the injection amount far exceeded a toxic dose or a lethal dose. Thus, it is deemed that using Sambutang-P at a clinically prescribed dose would not lead to hepatoxicity.

유전자변형생명체 안전관리의 제도적 보장

김훈기(Kim Hoon Gi) 원광대학교 법학연구소 2016 의생명과학과 법 Vol.16 No.-

유전자변형생명체(GMO)의 인체 안전성 여부를 판단할 때 가장 중요한 과정은 독성시험이다. 보통 독성시험은 설치류를 대상으로 한 단회투여 시험과 반복투여 시험으로 이뤄진다. 단회투여의 경우 GMO의 외래 유전자를 한번 투여하고 14일간 급성독성 반응을 확인한다. 이에 비해 반복투여 시험은 GMO 사료를 한달 이상 투여하고 이상 상태를 관찰하는 과정을 거친다. GMO는 인간이 평생 먹을 음식이기 때문에 단회투여보다 반복투여를 통해 안전성을 점검하는 것이 적절하다. 그러나 국가별로 실험기준에 차이가 나타나, 미국은 단회투여, 유럽연합은 반복투여를 시행하고 있다. 한국의 경우 단회투여 방식을 심사기준으로 설정하고 있다. 2000년부터 16년간 국내에 수입된 식용 GMO 138건 가운데 일반 GMO의 60% 이상이 단회투여 독성시험을 거친 것으로 확인됐다. 또한 후대교배종 73건은 독성시험이 면제돼 있다. 국산 GMO의 승인이 임박한데다 다양한 기능과 첨단기술이 동원된 차세대 GMO의 등장이 예고된 상황이기 때문에 국민의 안전성 보장 측면에서 심사기준이 반복투여 방향으로 시급히 개선돼야 할 필요가 있다. The most important step in determining GMO s safety is toxicity test. Generally, the toxicity test consists of single dose test and repeated dose test in rodents. In the case of single dose test, the exogenous gene of GMO is treated once and the acute toxicity is confirmed for 14 days. On the other hand, in the repeated dose test, GMO feed is treated for over one month and the abnormality is observed. Since GMO is lifetime food for humans, it is appropriate to check safety through repeated dose test rather than single dose test. However, there are differences in the experimental standards for each country. While the United States adopts single dose test, the European Union follows repeated dose test. In Korea, the single dose test is set as the criteria for safety screening. It has been confirmed that over 60% of 138 imported events for sixteen years from 2000 were subjected to the single dose toxicity test. In addition, 73 gene stacked events were exempted from the toxicity test. Since the approval of domestic GMO is imminent and the appearance of the next generation GMO with various functions and advanced technologies is anticipated, it is necessary to urgently improve the criteria for screening towards repeated dose test in terms of the safety of the people.

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.

생태독성 숙련도 시료의 적절성 평가

신기식 ( Ki Sik Shin ),박우상 ( Woo Sang Park ),이정서 ( Jung Seo Lee ),유순주 ( Soon Ju Yu ),박태진 ( Tae Jin Park ),김종민 ( Jong Min Kim ) 한국물환경학회 2015 한국물환경학회지 Vol.31 No.1

Acute toxicity test with Daphnia magna has a variety of factors such as incubation condition of test species, food quality and proficiency of laboratory workers. Therefore, proper proficiency testing samples were required for evaluating the competence of laboratory workers. This study aims to propose some appropriate proficiency testing samples for laboratory workers of acute toxicity test with Daphnia magna. Proposed four kinds of standard samples (Sample A, B, C, and D) for proficiency testing were selected from reference materials which were recommended from Canada and USA WET(Whole Effluent Toxicity) authorities. Proposed standard samples were tested by trial application of proficiency testing, homogeneity and stability checks using repeatability test. Sample A, C and D were estimated ad the suitable candidates for proficiency test standard samples except for Sample B. In addition, Sample A was proved to be the most suitable sample.

Aquatic Toxicity Assessment of Phosphate Compounds

Kim, Eunju,Yoo, Sunkyoung,Ro, Hee-Young,Han, Hye-Jin,Baek, Yong-Wook,Eom, Ig-Chun,Kim, Hyun-Mi,Kim, Pilje,Choi, Kyunghee The Korean Society of Environmental Toxicology 2013 환경독성보건학회지 Vol.28 No.-

Objectives Tricalcium phosphate and calcium hydrogenorthophosphate are high production volume chemicals, mainly used as foodstuff additives, pharmaceuticals, lubricants, synthetic resin, and disinfectants. Phosphate has the potential to cause increased algal growth leading to eutrophication in the aquatic environment. However, there is no adequate information available on risk assessment or acute and chronic toxicity. The aim of this research is to evaluate the toxic potential of phosphate compounds in the aquatic environment. Methods An aquatic toxicity test of phosphate was conducted, and its physico-chemical properties were obtained from a database recommended in the Organization for Economic Cooperation and Development (OECD) guidance manual. An ecotoxicity test using fish, Daphnia, and algae was conducted by the good laboratory practice facility according to the OECD TG guidelines for testing of chemicals, to secure reliable data. Results The results of the ecotoxicity tests of tricalcium phosphate and calcium hydrogenorthophosphate are as follows: In an acute toxicity test with Oryzias latipes, 96 hr 50% lethal concentration ($LC_{50}$) was >100 (measured:>2.14) mg/L and >100 (measured: >13.5) mg/L, respectively. In the Daphnia test, 48 hr 50% effective concentration ($EC_{50}$) was >100 (measured: >5.35) mg/L and >100 (measured: >2.9) mg/L, respectively. In a growth inhibition test with Pseudokirchneriella subcapitata, 72 hr $EC_{50}$ was >100 (measured: >1.56) mg/L and >100 (measured: >4.4) mg/L, respectively. Conclusions Based on the results of the ecotoxicity test of phosphate using fish, Daphnia, and algae, $L(E)C_{50}$ was above 100 mg/L (nominal), indicating no toxicity. In general, the total phosphorus concentration including phosphate in rivers and lakes reaches levels of several ppm, suggesting that phosphate has no toxic effects. However, excessive inflow of phosphate into aquatic ecosystems has the potential to cause eutrophication due to algal growth.

Ceriodaphnia dubia의 먹이섭생 기작과 온도조절에 근거한 급성독성조사법의 비교

박종호,이상일,조영옥,Park, Jong-Ho,Lee, Sang-Ill,Cho, Young-Oak 한국물환경학회 2004 한국물환경학회지 Vol.32 No.2

Two methods, a Ceriodaphnia algal uptake suppression test (CAUST) and a new toxicity test based on temperature control (TTBTC) which are based on feeding behaviour and temperature control, respectively, were developed and compared for the adoption as the better methodology for short-term toxicity screening. As previously published by Lee et aI., (1997), the CAUST method is based on the feeding behaviour of C. dubia and requires as little as 1 hour of contact time between C. dubia neonates and toxicant. However, even though CAUST requires only 1 hour of contact time, this method still take many hours for the preparation and measurement. Before the test starts, neonate digestive tracts were cleared by feeding yeast to the daphnids, Neonates were then exposed to toxicant, followed by addition of Scenedesmus subspiatus into the bioassay vessels. Daphnids were examined under the bright-field microscope with the presence of algae (indicated by a green colored digestive tract) or the absence of algae. Uptake indicated no toxic effect, whereas, absence of uptake indicated toxic inhibition. Unlike CAUST, the newly developed method (TTBTC) is based on just temperature control for the toxicity test of C. dubia. Initially, neonates are exposed to toxicants while the temperature of water bath containing media increased to $35.5^{\circ}C$. After 1.25 hour of contact time, the number of the daphnids, either live (no toxic effect) or dead (toxic effect), is counted without the aid of any instrument. In both methods, median effective concentrations ($EC_{50}$ values) were computed based on the results over a range of dosed toxicant concentrations. It showed that TTBTC was as sensitive as the standard 48-hour acute bioassay and CAUST. TTBTC and CAUST were much more sensitive than the I-hour I.Q. test and 30-minute Microtox. This study indicates that TTBTC is an easier and more rapid toxicity test than the standard 48-hour acute bioassay and even CAUST.

Screening of toxic potential of graphene family nanomaterials using in vitro and alternative in vivo toxicity testing systems

Nivedita Chatterjee,Ji Su Yang,Kwangsik Park,Seung Min Oh,Jeonggue Park,Jinhee Choi 환경독성보건학회 2015 환경독성보건학회지 Vol.30 No.-

Objectives: The widely promising applications of graphene nanomaterials raise considerable concerns regarding their environmental and human health risk assessment. The aim of the current study was to evaluate the toxicity profiling of graphene family nananomaterials (GFNs) in alternative in vitro and in vivo toxicity testing models. Methods: The GFNs used in this study are graphene nanoplatelets ([GNPs]–pristine, carboxylate [COOH] and amide [NH2]) and graphene oxides (single layer [SLGO] and few layers [FLGO]). The human bronchial epithelial cells (Beas2B cells) as in vitro system and the nematode Caenorhabditis elegans as in vivo system were used to profile the toxicity response of GFNs. Cytotoxicity assays, colony formation assay for cellular toxicity and reproduction potentiality in C. elegans were used as end points to evaluate the GFNs’ toxicity. Results: In general, GNPs exhibited higher toxicity than GOs in Beas2B cells, and among the GNPs the order of toxicity was pristine>NH2>COOH. Although the order of toxicity of the GNPs was maintained in C. elegans reproductive toxicity, but GOs were found to be more toxic in the worms than GNPs. In both systems, SLGO exhibited profoundly greater dose dependency than FLGO. The possible reason of their differential toxicity lay in their distinctive physicochemical characteristics and agglomeration behavior in the exposure media. Conclusions: The present study revealed that the toxicity of GFNs is dependent on the graphene nanomaterial’s physical forms, surface functionalizations, number of layers, dose, time of exposure and obviously, on the alternative model systems used for toxicity assessment.

Ceriodaphnia dubia의 먹이섭생 기작과 온도조절에 근거한 급성독성조사법의 비교

박종호 ( Park Jong Ho ),이상일 ( Lee Sang Il ),조영옥 ( Jo Yeong Og ) 한국물환경학회 2004 한국물환경학회지 Vol.20 No.1

Two methods, a Cerioduphniu algal uptake suppression test (CAUST) and a new toxicity test based on temperature control (TTBTC) which are based on feeding behaviour and temperature control, respectively, were developed and compared for the adoption as the better methodology for short-term toxicity screening. As previously published by Lee et al., (1997), the CAUST method is based on the feeding behaviour of C. dubiu and requires as little as 1 hour of contact time between C. dubiu neonates and toxicant. However, even though CAUST requires only 1 hour of contact time, this method still take many hours for the preparation and measurement. Before the test starts, neonate digestive tracts were cleared by feeding yeast to the daphnids. Neonates were then exposed to toxicant, followed by addition of Scenedesrnus subspiatus into the bioassay vessels. Daphnids were examined under the bright-field microscope with the presence of algae (indicated by a green colored digestive tract) or the absence of algae. Uptake indicated no toxic effect, whereas, absence of uptake indicated toxic inhibition. Unlike CAUST, the newly developed method (TTBTC) is based on just temperature control for the toxicity test of C. dubiu. Initially, neonates are exposed to toxicants while the temperature of water bath containing media increased to 35.5 ℃. After 1.25 hour of contact time, the number of the daphnids, either live (no toxic effect) or dead (toxic effect), is counted without the aid of any instrument. In both methods, median effective concentrations (EC_(50) values) were computed based on the results over a range of dosed toxicant concentrations. It showed that TTBTC was as sensitive as the standard 48-hour acute bioassay and CAUST. TTBTC and CAUST were much more sensitive than the I-hour I.Q. test and 30-minute Microtox. This study indicates that TTBTC is an easier and more rapid toxicity test than the standard 48-hour acute bioassay and even CAUST.

Aquatic Toxicity Assessment of Phosphate Compounds

김은주,유선경,노희영,한혜진,백용욱,Ig-Chun Eom,김현미,김필제,최경희 환경독성보건학회 2013 환경독성보건학회지 Vol.28 No.-

Objectives Tricalcium phosphate and calcium hydrogenorthophosphate are high production volume chemicals, mainly used as foodstuff additives, pharmaceuticals, lubricants, synthetic resin, and disinfectants. Phosphate has the potential to cause increased algal growth leading to eutrophication in the aquatic environment. However, there is no adequate information available on risk assessment or acute and chronic toxicity. The aim of this research is to evaluate the toxic potential of phosphate compounds in the aquatic environment. Methods An aquatic toxicity test of phosphate was conducted, and its physico-chemical properties were obtained from a database recommended in the Organization for Economic Cooperation and Development (OECD) guidance manual. An ecotoxicity test using fish,Daphnia, and algae was conducted by the good laboratory practice facility according to the OECD TG guidelines for testing of chemicals, to secure reliable data. Results The results of the ecotoxicity tests of tricalcium phosphate and calcium hydrogenorthophosphate are as follows: In an acute toxicity test with Oryzias latipes, 96 hr 50% lethal concentration (LC50) was >100 (measured:>2.14) mg/L and >100 (measured: >13.5) mg/L, respectively. In the Daphnia test, 48 hr 50% effective concentration (EC50) was >100(measured: >5.35) mg/L and >100 (measured: >2.9) mg/L, respectively. In a growth inhibition test with Pseudokirchneriella subcapitata, 72 hr EC50 was >100 (measured: >1.56)mg/L and >100 (measured: >4.4) mg/L, respectively. Conclusions Based on the results of the ecotoxicity test of phosphate using fish, Daphnia,and algae, L(E)C50 was above 100 mg/L (nominal), indicating no toxicity. In general, the total phosphorus concentration including phosphate in rivers and lakes reaches levels of several ppm, suggesting that phosphate has no toxic effects. However, excessive inflow of phosphate into aquatic ecosystems has the potential to cause eutrophication due to algal growth.