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.

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.

Reproductive and Developmental Toxicity of Amitraz in Sprague-Dawley Rats

Jeong-Hyeon Lim,Sung-Hwan Kim,Kang-Hyeon Kim,Na-Hyeong Park,In-Sik Shin,Changjong Moon,Soo-Hyun Park,Sung-Ho Kim,Jong-Choon Kim 한국독성학회 2010 Toxicological Research Vol.26 No.1

The present study was conducted to obtain information on the effects of amitraz on reproductive and developmental parameters in rats. The test chemical was administered via the drinking water containing 0, 40, 120, and 360 ppm to male rats from 2 weeks before mating to the end of 14-day mating period and to females from 2 weeks before mating, throughout mating, gestation and up to lactational day 4. During the study period, clinical signs, body weights, food intake, organ weights, reproductive and littering findings, necropsy findings, sperm parameters, and histopathology were examined. At 360 ppm, decreases in the body weight gain, food consumption, and the number of live pups and an increase in the post-implantation loss were observed. In addition, decreases in the seminal vesicle weight and sperm motility were found in males. At 120 ppm, a decrease in the food consumption was found transiently in both males and females, but no reproductive and developmental toxicity was observed in both sexes. There were no signs of either general or reproductive and developmental toxicity in the 40 ppm group. Based on these results, it was concluded that the repeated oral administration of amitraz to rats resulted in a decrease in the food consumption at 120 ppm and decreases in the seminal vesicle weight, sperm motility, and the number of live pups and an increase in the post-implantation loss at 360 ppm in rats. Under these experimental conditions, the no-observed-adverse-effect level (NOAEL) of amitraz for general and reproduction/developmental toxicity was believed to be 120 ppm, and the no-observed-effect level (NOEL) of amitraz was believed to be 40 ppm in rats.

Reproductive and developmental toxicity screening test of new TiO₂ GST in Sprague-Dawley rats

Hyung Seon Baek,Myeong Kyu Park,Hye Min Kim,Jae Min Im,Heung Sik Seo,Hee Ju Park,Sung Soon Nah 환경독성보건학회 2022 환경독성보건학회지 Vol.37 No.3

TiO₂ nanoparticles are widely used in paints, plastics, cosmetics, printing ink, rubber, food products, pharmaceuticals and other products (photocatalyst, etc.). However, there is little toxicological information during reproduction and developmental period. This study was performed to obtain safety data for new TiO₂ powder, GST (Green Sludge Titanium) produced through sludge recycling of the sewage treatment plant for Reproduction/developmental toxicity screening test in Sprague-Dawley (SD) rats in according to the OECD test guideline (TG 421). Based on the results of the dose-range finding study (14-day repeated toxicity), GST was orally administered to rats at doses of 0, 500, 1000, and 2000 mg/kg B.W/day. Males were dosed for 35 days beginning 14 days before mating, and females for a maximum of 53 days beginning 14 days before mating to day 13 of lactation, including throughout the mating, gestation and lactation periods. In the reproductive and developmental examinations, there were no marked toxicities in terms of general clinical signs, body weight, food consumption, organ weights, macroscopic / microscopic findings, stages of spermatogenesis in the testis, reproductive finding (estrous cycle, copulation-fertility-gestation index), developmental finding (number of corpora lutea and implantations, pups parameters including live birth and viability index). The NOAEL for reproductive/developmental screening toxicity was concluded to be 2000 mg/kg/day under the present study conditions.

Reproductive and Developmental Toxicity of Amitraz in Sprague-Dawley Rats

Lim, Jeong-Hyeon,Kim, Sung-Hwan,Kim, Kang-Hyeon,Park, Na-Hyeong,Shin, In-Sik,Moon, Chang-Jong,Park, Soo-Hyun,Kim, Sung-Ho,Kim, Jong-Choon Korean Society of ToxicologyKorea Environmental Mu 2010 Toxicological Research Vol.27 No.1

The present study was conducted to obtain information on the effects of amitraz on reproductive and developmental parameters in rats. The test chemical was administered via the drinking water containing 0, 40, 120, and 360 ppm to male rats from 2 weeks before mating to the end of 14-day mating period and to females from 2 weeks before mating, throughout mating, gestation and up to lactational day 4. During the study period, clinical signs, body weights, food intake, organ weights, reproductive and littering findings, necropsy findings, sperm parameters, and histopathology were examined. At 360 ppm, decreases in the body weight gain, food consumption, and the number of live pups and an increase in the post-implantation loss were observed. In addition, decreases in the seminal vesicle weight and sperm motility were found in males. At 120 ppm, a decrease in the food consumption was found transiently in both males and females, but no reproductive and developmental toxicity was observed in both sexes. There were no signs of either general or reproductive and developmental toxicity in the 40 ppm group. Based on these results, it was concluded that the repeated oral administration of amitraz to rats resulted in a decrease in the food consumption at 120 ppm and decreases in the seminal vesicle weight, sperm motility, and the number of live pups and an increase in the post-implantation loss at 360 ppm in rats. Under these experimental conditions, the no-observed-adverse-effect level (NOAEL) of amitraz for general and reproduction/developmental toxicity was believed to be 120 ppm, and the no-observed-effect level (NOEL) of amitraz was believed to be 40 ppm in rats.

Subchronic and Reproductive/Developmental Toxicity Studies of Tetrahydrocurcumin in Rats

Muhammed Majeed,Sankaran Natarajan,Anjali Pandey,Sarang Bani,Lakshmi Mundkur 한국독성학회 2019 Toxicological Research Vol.35 No.1

Tetrahydrocurcumin (THC) is a major metabolite of curcumin, which is obtained from Curcuma longa. THC has various benefits and overcomes the bioavailability issue of curcumin. To establish it as a pharmacologically active molecule, its safety profile has to be determined. Thus, the present study aimed to determine the preclinical safety profile of THC in a 90-day subchronic and reproductive/developmental toxicity study in Wistar rats. THC at oral doses of 100, 200, and 400 mg/kg was administered daily for 90 days. Rats in the recovery group were kept for 14 days after treatment termination. The animals were observed for treatment-related morbidity, mortality, and changes in clinical signs, clinical pathology, and histopathology. In the reproductive/developmental toxicity study, THC at 100, 200, and 400 mg/kg was administered orally to rats and the reproductive/developmental parameters in adult male and female rats and pups were observed. THC at up to 400 mg/kg/day of did not have any significant effect on all parameters in male and female rats in both toxicity studies. Thus, 400 mg/kg/day can be considered as the no-observed-adverse-effect-level of THC in rats.

Subchronic and Reproductive/Developmental Toxicity Studies of Tetrahydrocurcumin in Rats

Majeed, Muhammed,Natarajan, Sankaran,Pandey, Anjali,Bani, Sarang,Mundkur, Lakshmi Korean Society of ToxicologyKorea Environmental Mu 2019 Toxicological Research Vol.35 No.1

Tetrahydrocurcumin (THC) is a major metabolite of curcumin, which is obtained from Curcuma longa. THC has various benefits and overcomes the bioavailability issue of curcumin. To establish it as a pharmacologically active molecule, its safety profile has to be determined. Thus, the present study aimed to determine the preclinical safety profile of THC in a 90-day subchronic and reproductive/developmental toxicity study in Wistar rats. THC at oral doses of 100, 200, and 400 mg/kg was administered daily for 90 days. Rats in the recovery group were kept for 14 days after treatment termination. The animals were observed for treatment-related morbidity, mortality, and changes in clinical signs, clinical pathology, and histopathology. In the reproductive/developmental toxicity study, THC at 100, 200, and 400 mg/kg was administered orally to rats and the reproductive/developmental parameters in adult male and female rats and pups were observed. THC at up to 400 mg/kg/day of did not have any significant effect on all parameters in male and female rats in both toxicity studies. Thus, 400 mg/kg/day can be considered as the no-observed-adverse-effect-level of THC in rats.

Combined repeated-dose toxicity study of silver nanoparticles with the reproduction/developmental toxicity screening test

Hong, Jeong-Sup,Kim, Suhyon,Lee, Sang Hee,Jo, Eunhye,Lee, Byungcheun,Yoon, Junheon,Eom, Ig-Chun,Kim, Hyun-Mi,Kim, Pilje,Choi, Kyunghee,Lee, Moo Yeol,Seo, Yeong-Rok,Kim, Younghun,Lee, Yeonjin,Choi, Jon Informa UK, Ltd. 2014 Nanotoxicology Vol.8 No.4

<P>Combined repeated-dose toxicity study of citrate-capped silver nanoparticles (7.9 ± 0.95 nm) with reproduction/developmental toxicity was investigated in rats orally treated with 62.5, 125 and 250 mg/kg, once a day for 42 days for males and up to 52 days for females. The test was performed based on the Organization for Economic Cooperation and Development test guideline 422 and Good Laboratory Practice principles. No death was observed in any of the groups. Alopecia, salivation and yellow discolouration of the lung were observed in a few rats but the symptoms were not dose-dependent. Haematology, serum biochemical investigation and histopathological analysis revealed no statistically significant differences between control group and the treated groups. Toxicity endpoints of reproduction/developmental screening test including mating, fertility, implantation, delivery and foetus were measured. There was no evidence of toxicity.</P>

시너(thinner)의 물리화학적 특성과 랫트의 생식기에 미치는 영향 연구

김현영 ( Hyeon Yeong Kim ),이성배 ( Sung Bae Lee ),한정희 ( Jeong Hee Han ),김태균 ( Tae Gyun Kim ),이정석 ( Jung Suk Lee ),강민구 ( Min Gu Kang ),이채관 ( Chae Kwan Lee ) 한국산업위생학회 2008 한국산업보건학회지 Vol.18 No.3

The aim of this study is to confirm the physicochemical property and hazard of thinner (012), which is a diluent of enamel paint used for floor coating for waterproofing and oil painting for the outer wall. The literatures of physicochemical property and hazard of thinner were surveyed and its physicochemical property were evaluated. And then, the inhalation toxicity of thinner affecting the central nervous system and reproductive organs in rats were examined by subchronic (6 h./day. 5 days/ week for 13 weeks) inhalation test. 1) According to the 13-week subchronic inhalation test, there were no significant changes in clinical test and body weight. However, a significant evidence of toxicity was observed in the hematological test and organ weight such as heart, kidney, liver and brain (p<0.01) in the 200 ppm and 1,000 ppm exposure groups in a dose response manner. In the histopathology analysis, there were no significant evidence of toxicity. Therefore, thinner was not classified as an organ targeted toxic agent. In case of Harmfulness, it could be classified as a chronic toxic agent 3(500<LC50≤2,500 ppm/4hr, rat). 2) The reproductive toxicity such as extension of the period of estrous cycle, reduction of serum estradiol concentration and increase of frequency of the abnormal sperm was observed in the 1,000 ppm exposed animals. 3) The result of the physicochemical property of the test material showed that the specific gravity was 0.793, boiling point 155.8℃, steam pressure 2.1 kPa, ignition point 34.5℃, and spontaneous ignition point 280℃. The endothermic and exothermic values were 371.4 J/g and 159.1 J/g. respectively. The explosion limit was 214 mg/l. These data showed that thinner could be classified as an explosion agent level 1.2 and ignitive liquid agent 3 (23-60℃) according to the notification No. 2008-1 of the Labor Ministry, "Classifying Standard of Chemical Materials."

랫드를 이용한 Acetanilide의 반복투여 및 생식/발생독성 병행시험

정문구(Moon-Koo Chung),백성수(Sung-Soo Baek),이상희(Sang-Hee Lee),김현미(Hyun-Mi Kim),최경희(Kyung-Hee Choi),한상섭(Sang-Seop Han) 한국독성학회 2007 Toxicological Research Vol.23 No.2

The study was conducted to assess the repeated dose and reproduction and developmental toxicities of acetanilide, an intermediate for drug production, as a part of OECD Screening Information Data Set (SIDS) program. The test agent was administered by gavage at dose levels of 0, 22, 67, 200 and 600 ㎎/㎏ to Sprague-Dawley rats (12/group/sex) during pre-mating and mating period for males(up to 30 days) and females and pregnancy and early lactation period for females (up to 39~50 days). At 22 ㎎/㎏, decreases in HGB, HCT (males) and MCHC (females), hyperplasia of spleen red pulp, hyperplasia of femur bone marrow (both sexes) were observed. At 67 ㎎/㎏, salivation (males), reduced food consumption (both sexes), decreases in RBC, HGB, HCT and MCHC (males), increases in MCV (males) and spleen weight (males), hyperplasia of spleen red pulp and femur bone marrow (both sexes) were observed. At 200 ㎎/㎏, decreases in locomotor activity and salivation (both sexes), reduced food consumption (both sexes), decreases in RBC, HGB, HCT and increases in MCV, MCH, BUN, T-BIL (males), enlargement of spleen (both sexes), increased weight of spleen (males), hyperplasia of spleen red pulp and femur bone marrow and extramedullary hematopoiesis of liver (both sexes), atrophy of thymus and corpus luteum hyperplasia of ovary (females) were observed. At 600 ㎎/㎏, decreases in locomotor activity, cyanosis (both sexes), reddish tear, and salivation (males), mortality (4 out of 12 females), decreased body weight (females), reduced food consumption (both sexes), decreases in RBC, HGB, HCT and MCHC and increases in WBC, MCV, MCH, reticulocyte, neutrophil, lymphocyte, monocyte, AST, ALT, BUN, T-BIL, ALB, Ca and A/G ratio (males), enlargement of spleen, increased weights of spleen (both sexes), liver (males), kidney and ovary, decreased weights of thymus (females), hyperplasia of spleen red pulp, hyperplasia of femur bone marrow and extramedullary hematopoiesis of liver (both sexes), and atrophy of thymus and corpus luteum hyperplasia of ovary (females) were observed. Regarding the reproduction and development toxicities, there were no treatment- related changes in precoital time, mating index, fertility index and pregnancy index at all doses tested. At 22 and 67 ㎎/㎏, there were no adverse effects on all the parameters observed. At 200 ㎎/㎏, decreased body weight of pups (day 4 p.p.) were observed. At 600 ㎎/㎏, decreased body weight of pups (day 0 and 4 p.p.) and viability index (day 4 p.p.), increased incidence of newborns dead or with abnormal clinical signs were observed. The results suggest that the NOAELs for general toxicity are < 22 ㎎/㎏, LOAELs are 22 ㎎/㎏ and the NOAELs for reproductive toxicity are 67 ㎎/㎏.