Rat 13-Week Repeated Oral Dose Toxicity Test of Palmul-tang - OECD Guideline 408 based KFDA Guideline 2015-082

( Kim Joo-ik ) 대구한의대학교 제한동의학술원 2022 제한동의학술원논문집 Vol.20 No.1

The objective of this study was to obtain 13 weeks repeated oral dose toxicity information of Palmul-tang (PMT), a representative Korean gi-tonifying polyherbal formula, consisted of eight herbs, has been employed in the treatment of qi deficiency and blood problems caused by consumptive diseases, in female and male Sprague-Dawley rats as a process to develop of natural origin medicinal ingredient or food itself, according to Organization for Economic Co-Operation and Development (OECD) Guideline 408 (1998) based Korea Food and Drug Administration (KFDA) Guideline (KFDA2015-082, 2015). In order to investigate the toxicity and identify target organs, PMT were orally administered to female and male Sprague-Dawley rats at dose levels of 2,000, 1,000, 500 and 0 (vehicle control) mg/kg (body weights), in a volume of 10 ml/kg, dissolved in distilled water, once a day for 91 days, and the mortality and changes on the body weight, food and water consumption, fecal and urine excretion, clinical signs were monitored during 91 days of treatment with gross observation, changes on the organ weights, hematology and serum biochemistry, and histopathology of principle organs based on the recommendation of OECD Guideline 408 (1998) based KFDA Guideline (KFDA2015-082, 2015), as compared with those of equal genders of vehicle control rats. Because LD50 and approximate LD of PMT, which were extracted and standardized by Aribio (Seungnam, Korea) in the both female and male Sprague-Dawley rats after single oral dose were considered over 2,000 mg/kg - the limited dosage of rodents with no specific target or clinical sings in our previous single oral dose toxicity test of PMT in Sprague-Dawley rats, the highest dosage used in the present study were also selected as 2,000 mg/kg in a volume of 10 ml of distilled water - the limit dosages of rodents, and 1,000 and 500 mg/kg were selected as middle and lower dosage groups using common ratio 2, according to OECD Guideline 408 (1998) based KFDA Guideline (KFDA2015-082, 2015). As the results of 13 weeks (91 days) repeated oral treatment of PMT, once a day in female and male Sprague-Dawley rats, no treatment related mortalities were observed within 91 days after end of treatment up to 2,000 mg/kg, the limited dosage of rodents in the both genders, and also no PMT treatment related changes on the body and organ weights, clinical signs, food and water consumption, fecal and urine excretion, hematology and serum biochemistrical analysis, necropsy and histopathological findings were observed in all test dose levels - 2,000, 1,000 and 500 mg/kg of both female and male rats, at least in a condition of the current experiment. The results obtained in this study suggest that the PMT, which were extracted and standardized by Aribio (Seungnam, Korea), is non-toxic in rats and is therefore, likely to be safe for clinical use (Table 24). The maximum tolerated dose (MTD) and No-observed-adverse-effect level (NQAEL) in rats after 13 weeks repeated oral dose of PMT were considered over 2,000 mg/kg, the limited dosage of rodents in both female and male Sprague-Dawley rats, respectively. In addition, no specific target or clinical sings were detected, at least in a condition of the present study, as same as previous oral dose toxicity tests of natural products in rats.

보중익기탕의 랫트 단회 경구투여 독성실험

김주익 ( Joo-ik Kim ),송창현 ( Chang-hyun Song ),박수진 ( Soo-jin Park ),최성훈 ( Seong-hun Choi ),구세광 ( Sae-kwang Ku ) 대구한의대학교 제한동의학술원 2021 제한동의학술원논문집 Vol.19 No.1

Objectives : The objective of this study was to obtain acute (single) oral dose toxicity information of Bojungikki-tang (BJIKT), prepared and standardized by Aribio Ltd. (Seungnam, Korea), in female and male Sprague-Dawley rats as a process to develop of natural origin medicinal ingredient or food itself. Materials and methods : In order to investigate the toxicity and identify target organs, BJIKT were once orally administered to female and male Sprague-Dawley rats at dose levels of 2000, 1000, 500 and 0 (vehicle control) mg/kg (body weights) in a volume of 10 ml/kg, dissolved in distilled water, and the mortality and changes on the body weight and clinical signs were monitored during 14 days after treatment with gross observation, changes on the organ weights and histopathology of principle organs based on the recommendation of Organization for Economic Co-Operation and Development (OECD) Guideline 423 based Korea Food and Drug Administration (KFDA) Guideline (2015-082, 2015), as compared with those of equal genders of vehicle control rats. Because there are no available toxicological data after oral treatment in female and male rats of BJIKT, the highest dosage used in the present study were selected as 2,000 mg/kg in a volume of 10 ml of distilled water - the limited dosages of rodents, and 1,000 and 500 mg/kg were selected as middle and lower dosage groups according to OECD Guideline 423 based KFDA Guidelines (Notification No. 2015-082, 2015). In addition each female and male vehicle controls were added in this experiment. Principal organs for weighing : Lung, Heart, Thymus, Liver, Left Kidney, Left Adrenal Gland, Spleen, Left Testis/Ovary, Splenic lobe of Pancreas, Brain, Urinary bladder, Left Epididymis/total Uterus, Prostate and Left Submandibular Lymph node [Total 16 organs] Specific target organs for histopathology : Brain (Cerebrum, Cerebellum and Medulla oblongata), Heart, Thymus, Lung, Testis, Epididymis (head parts), Uterus, Ovary, Left-kidney, Left-adrenal glands, Spleen, Liver-left lateral lobe, Pancreas-splenic part, Digestive tracts (Esophagus, Fundus, Pylorus, Duodenum, Jejunum, Ileum, Cecum, Colon and Rectum), Left-submandibular lymph nodes, Urinary bladder and Prostate [Total 27 organs]. Results : As the results of single oral treatment of BJIKT on the female and male Sprague-Dawley rats, no treatment related mortalities were observed within 14 days after end of treatment up to 2,000 mg/kg, the limited dosage of rodents in the both genders, and also no BJIKT treatment related changes on the body and organ weights, clinical signs, necropsy and histopathological findings were detected, in this experiment. Conclusion : The results obtained in this study suggest that the BJIKT, prepared and standardized by Aribio Ltd. (Seungnam, Korea), is non-toxic in rats and is therefore, likely to be safe for clinical use. The 50% lethal dose (LD50) and approximate LD in rats after single oral dose of BJIKT were considered over 2,000 mg/kg, the limited dosage of rodents in both female and male Sprague-Dawley rats, respectively. In addition, no specific target or clinical sings were detected in the present study.

수컷랫드에 있어서 새로운 안트라사이클린계 항암제 DA-125의 생식독성 연구

김종춘,김갑호,신호철,정문구 한국독성학회 1998 Toxicological Research Vol.14 No.2

The toxicity of DA-125. a new anthracycline anticancer agent, on the male reproductive system was studied in Sprague-Dawley rats. Forty male rats were rando$m\ell$y assigned to Jour groups with ten rats in each group and given single intraveneous doses of DA-125 at dose levels of 0. 12.5. 25. and 50 mg/kg body weight. On day 56 after treatment the animals were allowed to mate. and their male reproductive Junctions and organs were examined in detail. Copulated females were sacrificed on day 20 of gestation for examination of embryo-fetal development. One out of ten rats in the 50 mg/kg group died on day 12 after treatment. Clinical signs such as emaciation. sedation, anorexia. swelling. dark material around eye. alopecia. and diarrhea were observed in the 25 and/or 50 mg/kg groups. Reduction in the body weight gain. decrease in the absolute weights of testes. epididymis and seminal vesicles. and/or decrease in the number of testicular sperm heads were also found. Although histopathological changes such as atrophy of seminiferous tubules. loss or decrease of spermatogenic cells. exfoliation of spermatogenic cells. vacuolization of Sertoli cells. decrease of sperm. and/or increase of necrotic spermatogenic cells in epididymal ducts were observed. no adverse effects on the motility and morphology of epididymal sperm. copulation index. fertility index. and embryo-fetal development were detected in the 25 and 50 mg/kg groups. There were no evidences of male reproductive toxicity in the 12.5 mg/kg group. These results show that single intravenouse doses of DA-125 produce significant dose-related testicular atrophy. histopathological changes. and oligozoospermia in rats and $LD_{10}$ for DA-125 appears to be 50 mg/kg body weight.

The Effect of Vitis vinifera L. Juice on Serum Levels of Inhibin B, Sperm Count in Adult Male Rats

Mohammad Reza Afzalzadeh,Akram Ahangarpour,Ashraf Amirzargar,Mohammad Kazemi Varnamkhasti,Hadi Ganjalidarani 대한남성과학회 2015 The World Journal of Men's Health Vol.33 No.2

Purpose: Vitis vinifera is a species of Vitis that is native to the Mediterranean region, central Europe, and southwestern Asia, and has been used as a drug in traditional medicine. Traditional medicinal plants have been used for medical purposes with increasing effectiveness. It is important to identify drugs that inhibit spermatogenesis. Therefore, the present study aimed to investigate the effect of grape juice (GJ) on serum levels of inhibin B and sperm count in normal male rats.Materials and Methods: Thirty-five adult male rats were randomly divided into five groups, each containing seven rats. Rats in the control group received 1 mL of normal saline over the course of the study. The experimental groups received GJ (100, 200, 400, and 1,600 mg/kg, orally, for 35 days consecutively). At the end of the treatment period, fertility indices were measured, including body weight difference, sex organ weight, sperm motility and count, epididymal sperm reserve, daily sperm production (DSP), and serum inhibin B levels.Results: We found that GJ reduces body weight difference, was associated with decreased sperm motility and count in all treatment groups (p≤0.05 and p≤0.001, respectively). Moreover, DSP was significantly decreased in all treatment groups compared to the control group (p≤0.05), except in the group receiving 100 mg/kg of GJ. Inhibin B levels were significantly decreased in all treatment groups (p≤0.05).Conclusions: The results of our study suggest that GJ in all doses, but especially in higher doses, may decrease fertility in male rats.

항안드로겐성 물질이 성 성숙 이전 단계의 정소에서 미치는 영향 연구

홍진,한순영,문현주,강태석,강일현,김태성,김승희,권기성,Hong Jin,Han Soon-Young,Moon Hyun-Ju,Kang Tae-Seok,Kang Il-Hyun,Kim Tae-Sung,Kim Seung-Hee,Kwon Ki-Sung 환경독성보건학회 2006 환경독성보건학회지 Vol.21 No.3

The experiments investigated whether early exposure to testosterone propionate (TP) during prepuberty alters testis development in Sprague-Dawley male rats. We performed Hershberger assay using the stimulated weanling male rats by OECD protocols, cDNA microarray, and Western blot. TP was subcutaneously injected to uncastrated Sprague-Dawley male rat of 22 days old for 10 consecutive days at doses of 0.4, 0.8, 1.0, 1.2, 1.6 mg/kg per day. At necropsy, the following tissues were removed and weighed: combined testes, epididymides (Epi), Cowper's glands (COW), levator am, and bulbocavernosus muscles (LABC), seminal vesicles, together with coagulating gland (SV) and ventral prostate (VP). We found that TP increased the weights of Epi, VP, SV, COW, and LABC, while testis was decreased in a dose-dependent manner. In cDNA microarray analysis of testis, there were significant reductions in the expression of cytochrome P450 11A (CYP11A), the rate-limiting enzyme of steroidogenesis. Taken together these results, TP exposure before puberty in male rats may produce the delay in testis development by inhibiting the CYP11A gene expression.

Sprague Dawley 수컷 랏트에서 Bisphenol A Diglycidyl Ether의 경구 투여에 의한 급성 독성

임재언,양윤정,이태진,홍연표 대한산업의학회 2006 대한직업환경의학회지 Vol.18 No.4

목적: 본 연구는 BADGE의 경구 투여에 의한 급성 독성학적 연구를 수행하여 기존의 연구 결과를 확인하고 추후 내분비계 장애 연구에 필요한 기초자료를 얻고자 하였다. 방법: 수컷 SD 랏트에 0, 1000, 2000, 4000과 8000 mg/kg/day의 BADGE와 DES 0.37 mg/kg/day의 농도를 단회 경구로 투여한 후 14일간 일반 증상 등을 관찰하였고 14일 후 부검하였다. 결과: BADGE를 투여한 모든 군에서 투여 후 3일째에 설사와 다른 일반 증상들이 관찰되었고 체중도 감소하는 경향을 보였다. 특히 8000 mg/kg/day의BADGE를 투여한 랏트에서 투여 후 3일째에 대조군에 비해 유의한 수준으로 체중이 감소하는 것을 관찰할 수 있었다. 일부 BADGE 투여군에서 대조군에 비해 심장(1000, 2000과 4000 mg/kg/day), 간(1000, 2000, 4000과 8000 mg/kg/day)과 전립샘(4000 mg/kg/day)의 무게의 감소가 관찰되었다. BADGE 투여군의 간(1000과 4000 mg/kg/day)과 전립샘(8000 mg/kg/day) 의 상대 무게가 대조군과 차이를 보였다. 고환을 제외한 모든 장기에서 조직학적인 변화는 관찰되지 않았으며 고환에서는 세 정관에서 정자세포의 감소가 관찰되었다. 고환과 부고환의 sperm head 수를 보면, 고환에서만 감소하는 경향을 보였으며 정자의 운동성과 기형은 대조군과 차이가 없었다. 혈장 에스트로겐과 테스토스테론 농도는 대조군과 모든 투여군 간의 유의한 차이가 없었다. 결론: BADGE를 랏트에 경구 투여 하였을 때 1000 mg/kg/day의 수준에서도 일반 독성 및 생식독성을 유발시킬 수 있음을 보여준다. Objectives: Bisphenol A Di Glycidyl Ether (BADGE) is the major component in commercial liquid epoxy resins, which are manufactured by co-reacting bisphenol A with epichlorohydrin. The authors investigated the acute toxicity of BADGE. Methods: BADGE was administered by a gavage to 8 week old SPF Sprague Dawley rats in a single dose of 0 (negative control), 0.37 (Diethylstilbesterol, DES), 1000, 2000, 4000, and 8000 mg/kg/day of BADGE. Each treatment group contained 7 rats. The general status and weight of the rats were observed for 14 days. The rats were anesthetized by ether at 14 days, and the changes in morphology, organ weight, sperm count and motility, and hormone level were measured. Results: All the rats treated with BADGE had diarrhea on the 1st day. The rats administered BADGE at 1000, 2000, and 4000 mg/kg/day showed a soiled perineal region and soft stools with diarrhea until the 3rd day. The 8000 mg/kg/day BADGE rats had diarrhea for two days followed by emaciation, soiled fur, a soiled perineal region, staining around the mouth and were moribund for three to eight days. No weight gain was observed after the 1st day in the 2000, 4000, and 8000 mg/kg/day BADGE rats and after the 7th day in all the treatment groups compared with the control groups. Some treatment groups were observed to have a decrease in the weight of the heart (BADGE 1000, 2000 and 4000 mg/kg/day), liver (BADGE 1000, 2000, 4000 and 8000 mg/kg/day) and prostate (BADGE 4000 mg/kg/day) compared with control group. The weight of the liver was significantly lower in all treatment groups compared with the control group. The relative weight of the liver (BADGE 1000 and 4000 mg/kg/day) was significant lower than the control. No pathological changes were observed in the brain, liver, thyroid, heart, spleen, kidney, lung and prostate. The number of spermatid in the seminiferous tubule in the testes was lower in all treatment groups than the control. The sperm motility tended to decrease with increasing concentration but the sperm count was similar in all treatment groups. The plasma Estrogen and testosterone level were similar in the control and treatment groups. Conclusions: These results suggest that BADGE induces general, hepatic and reproductive toxicity at 1000 mg/kg/day.

랫드에서 고환독성의 정색을 위한 정량적 평가법의 확립: 2-bromopropane의 예

차신우,배주현,손우찬,신진영,신동호,김성호,박승춘,김종춘,Cha Shin-Woo,Bae Joo-Hyun,Son Woo-Chan,Shin Jin-Young,Shin Dong-Ho,Kim Sung-Ho,Park Seung-Chun,Kim Jong-Choon 한국생명과학회 2005 생명과학회지 Vol.15 No.3

The aims of the study were to establish a short-term screening test for detecting testicular toxicity of chemicals in rats and to determine whether a 2-week administration period is sufficient to detect testicular toxicity of 2-bromopropane (2-BP) as an example. Male Sprague-Dawley rats were subcutaneously administered with 1000 mg/kg/day of 2-BP or its vehicle for 2 weeks. Ten male rats each were sacrificed on days 3, 7 and 14 after the initiation of treatment. Parameters of testicular toxicity included genital organ weights, testicular sperm head counts, epididymal sperm counts, motility and morphology, and qualitative and quantitative histopathologic examinations. The early histopathological changes observed on day 3 of treatment included degeneration of spermatogonia and spermatocytes, multinuclear giant cells, mature spermatid retention, vacuolization of Sertoli cells, and decreased number of spermatogonia in stages II and V. On day 7 of treatment, atrophy of seminiferous tubules, exfoliation of germ cells, degeneration of spermatogonia and spermatocytes, multinuclear giant cells, mature spermatid retention, vacuolization of Sertoli cells, decreased number of spermatogonia in stages II and V, and decreased number of spermatocytes in stages VII and XII. On day 14 after treatment, a significant decrease in the weights of testes and seminal vesicles was found. Atrophy of seminiferous tubules, exfoliation of germ cells, degeneration of spermatogonia and spermatocytes, mature spermatid retention, vacuolization of Sertoli cells, decreased number of spermatogonia in stages II and V, and decreased number of spermatocytes in all spermatogenic stages were also observed. In addition, a slight non-significant decrease in testicular sperm head counts, daily sperm production rate and epididymal sperm counts was found. The results showed that 2 weeks of treatment is sufficient to detect the adverse effects of 2-BP on male reproductive organs. It is considered that the short-term testicular toxicity study established in this study can be a useful tool for screening the testicular toxic potential of new drug candidates in rats.

랫드에서 고환독성평가를 위한 단기검색법의 확립

김종춘(Jong-Choon Kim),신진영(Jin-Young Shin),신동호(Dong-Ho Shin),김성호(Sung-Ho Kim),배춘식(Chun-Sik Bae),박승춘(Seung-Chun Park),차신우(Shin-Woo Cha),강부현(Boo-Hyon Kang),정문구(Moon-Koo Chung) 한국실험동물학회 2005 Laboratory Animal Research Vol.21 No.1

The present study was performed to establish a short-term screening test for detecting testicular toxicity of chemicals in rats and to determine whether a 2-week administration period is sufficient to detect testicular toxicity of 2-bromopropane (2-BP) as an example. Male Sprague-Dawley rats were subcutaneously administered with 1000 ㎎/㎏/day of 2-BP or its vehicle (com oil) for 2 weeks. Ten male rats each were sacrificed on days 3, 7 and 14 after the initiation of treatment. Parameters of testicular toxicity included genital organ weights, testicular sperm head counts, epididymal sperm counts, motility and morphology, and histopathologic examinations. The early histopathological changes observed in testes and epididymides included degeneration and decrease of spermatogonia in stages Ⅰ~Ⅵ, degeneration and decrease of spermatocytes and multinuclear giant cells in stages Ⅶ~Ⅹ, mature spermatid retention in stages Ⅸ~Ⅺ, vacuolization of Sertoli cells, and degeneration of spermatogenic cells in epididymal ducts. On day 14 after treatment, a significant decrease in the weights of testes and seminal vesicles was found. Atrophy of seminiferous tubules, exfoliation of spermatogenic cells, degeneration and decrease of spermatogonia, degeneration and decrease of spermatocytes, multinuclear giant cells, mature spermatid retention, vacuolization of Sertoli cells, oligozoospermia, and degeneration of spermatogenic cells in epididymal ducts were also observed. In addition, a slight non-significant decrease in testicular sperm head counts, daily sperm production and epididymal sperm counts was found. The results showed that 2 weeks of treatment is sufficient to detect the adverse effects of 2-BP on male reproductive organs. It is considered that the short-term testicular toxicity study established in this study can be a useful tool for detecting the testicular toxic potential of new drug candidates in rats.

Deep sequencing reveals microRNA signature is altered in the rat epididymis following bilateral castration

Yanling Liu,Haiyan Wang,Yangmei Qin,Juan Liu,Ning Li,Zhiliang Ji,Jianyuan Li 한국유전학회 2019 Genes & Genomics Vol.41 No.7

Background In the epididymis of bilateral castrated male rat, gene expression profile changed significantly. However, up to date, no study has investigated how these genes were regulated by microRNAs (miRNAs). Objective We investigated the alterations in the miRNA signature of the epididymis from sham-operated and bilaterally castrated rats. Methods By employing deep sequencing technology and qPCR, the global alterations of epididymal miRNA signature between sham-operated (Con-EP library) and bilaterally castrated rats (Cas-EP library) were explored. MiRNA-target interaction networks were annotated by GO and KEGG enrichment. Results We identified 313 and 306 known miRNAs as well as 152 and 114 novel miRNAs in the Con-EP and Cas-EP libraries, respectively. 59 miRNAs were differentially expressed, including 24 up-regulated and 35 down-regulated miRNAs, among which two up-regulated and three down-regulated ones were validated using qPCR. The expression of these miRNAs in the epididymides of rats at different postnatal ages showed regular changes from birth to adult, suggesting they were androgen-regulated. GO analysis showed that many of the miRNA targets were enriched in metabolic processes. KEGG analysis demonstrated that the targets mainly participated in the mitogen-activated protein kinase (MAPK) pathway. Moreover, 3 and 6 functional modules were detected among the up- and down-regulated miRNA target interaction networks, respectively, and these modules were involved in various biological processes. Conclusion This study represents the first systematic investigation of alterations in the miRNA signature of the epididymis from bilaterally castrated rats and will provide useful resources for functional studies of the miRNAs in the male reproductive system.

Effects of Postnatal Administration of Diethylstilbestrol on Puberty and Thyroid Function in Male Rats

SHIN, Jae-Ho,KIM, Tae Sung,KANG, Il Hyun,KANG, Tae Seok,MOON, Hyun Ju,HAN, Soon-Young Society for Reproduction and Development 2009 Journal of Reproduction and Development Vol.55 No.5

<P>To examine the effects of diethylstilbestrol (DES) on male pubertal development and thyroid function, juvenile male Sprague-Dawley rats were given DES daily by oral intubation at doses of 10, 20 and 40 μg/kg/day from postnatal day 33 for 20 days. Prepuce separation was significantly delayed at the dose of 20 μg/kg/day and above in the DES-treated rats. DES treatment induced a significant reduction in the weights of testes, epididymides, the ventral prostate, seminal vesicles plus coagulating glands and fluid, levator ani bulbocavernosus muscles, Cowper's glands and the glans penis. The weights of the liver and adrenals increased in the DES-treated animals. DES caused a dose-dependent reduction in germ cells; in particular the spermatids were mainly affected. The serum levels of testosterone and luteinizing hormone were significantly reduced in the DES-treated groups, but that of estradiol decreased. No differences were observed in the serum thyroxine levels of the control and DES-treated groups. In microscopic observation of the DES-treated animals, degeneration of germ cells and tubular atrophy in the testis were noted, but there were no microscopic changes in the thyroid. These results indicate that DES affected the pubertal development of juvenile male rats and that its mode of action may be related to alterations in hormone levels.</P>