(Miocamycin) Part 1-2. Acute Toxicity in Rats Masayuki
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July 1984 THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-7 1317(81) TOXICOLOGICAL STUDIES ON A NEW MACROLIDE ANTIBIOTIC , MIDECAMYCIN ACETATE (MIOCAMYCIN) PART 1-2. ACUTE TOXICITY IN RATS MASAYUKIYOKOTA, UETO TAKEDA, MASUZO ODAKI, HITOSHI SASAKI, HARUOKAWAOTO, HIROSHI WATANABE, NOBUYOSHI ISHIWATARI and TAKEMIKOEDA Laboratoryof SafetyEvaluation Central ResearchLaboratories, Meiji Seika Kaisha, Ltd., 760, Morooka-cho,Kohoku-ku, Yokohama, Japan (Receivedfor publicationApril 4, 1984) Miocamycin (MOM) is a derivative of midecamycin (MDM), a macrolide antibiotic isolated from a culture broth of Streptomyces mycarofaciens1,2). Its chemical structure is shown in Fig. 1. It is reported that MOM has the most inte- resting antibacterial properties among the Fig. 1. Chemical structure of MOM known macrolide antibiotics3,4). MOM, non- crystalline solid, did not exhibit any acute toxicity in male and female mice after a single dose by the intraperitoneal (i.p.), subcutaneous (s.c.) and oral (p.o.) routes. The LD50 values were estimated as more than 5,000 mg/kg in each route of administration5). The objective of this study was to know the acute toxicity and estimate LD50 values in male and female rats after single i.p., s.c. and p.o. administration of MOM, non-crystalline Table 1. Acute toxicity of MOM, non- solid. crystalline solid, in rats Materials and Methods Male and female Wistar rats (SPF) were purchased from Japan Laboratory Animals Inc. at the age of 4 weeks. After quarantine for about 1 week, only those animals weighing 100•}10 g and considered to be in good general condition were used. Each group was con- sisting of male or female 10 animals. All the animals were housed at tempera- ture of 23 ±1°C and at relative humidity of 55•}5%. They were allowed free access to *: mg (potency)/kg food (NM, animal chow, Oriental Yeast In- dustry Co.) and water. MOM, non-crystalline solid, was suspended in 1% carboxymethylcellulose solution immediately before use. It was decided arbitrarily to dose the test material at a dose of 5,000 mg/kg as maximum physically applicable dose which might be tolerable in consideration of the results from mouse acute toxicity. Test material was once administered via i.p., s.c. and p.o. routes before feeding in the morning. July 1318(82) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-7 1984 The clinical signs of animals were observed for abnormalities mainly before and after administration. Observations were continued for 1 week after treatment. Body weights were recorded once everyday before feeding for the week of observations. All the animals were sacrificed under anesthesia with nembutal(R)(pentobarbital sodium) after the end of observation for 1 week and the following representa- tive organs and tissues were grossly observed and weighed (asterisk indicates the organ weighed); brain*, spinal cord, thymus*, heart*, lungs*, liver*, spleen*, kidneys*, adrenals*, testes or ovaries*, thyroid, hypophysis*, intestine, pancreas, bone marrow and injection site. In the histopathological studies, following organs and tissues were fixed in 10% phosphate buffered formalin solution, sliced and stained with hematoxylin and eosin (H.E.) for microscopic examination after the observations; brain, spinal cord, thymus, heart, lungs, liver, spleen, kidneys, adrenals, testes or ovaries, thyroid, hypophysis, small intestine, pancreas, bone marrow and injection site. LD50 values were estimated according to LITCHFIELD-WILCOXON'Smethod. Results 1. Mortality LD0 values obtained in the present studies are shown in Table 1. No animals died for the week of observations so that LD, values were estimated to be more than 5,000 mg/kg in all routes of administration. 2. Clinical signs and body weights i.p.-dosed groups: A decrease in spontaneous motility, a decrease in respirations and mild Plate 1. Female rat, MOM, non-crystalline solid (5,000 mg/kg), Acute toxicity, i.p., Lung, H.E. stain, x 60 Thrombus like bodies Plate 2. Female rat, MOM, non-crystalline solid (5,000 mg/kg), Acute toxicity, s.c., Injection site, H.E. stain, „‡60 Inflammatory changes July THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-7 1984 1319(83) stretching behavior were observed at about 5 minutes, and crawling behavior appeared about 7 minutes later, but ceased within 40 to 60 minutes. Afterward, unstable gait considered to be due to MOM administration at high doses was observed with a gradual decrease. A general depression in behavio occurred about 2 hours later, and a decrease in spontaneous motility was also observed on the 2nd day, but returned to normal on the 3rd day. On day 6, there was abdominal swelling in a few animals, which remained up to the end of observation. Weight loss was observed from the 2nd to 3rd day in males and from the 2nd to 4th day in the females, but turned to be favorably increasing afterward. s.c.-dosed groups: There were no marked changes in behavior. Weight gain took almost favor- able course. A papule was induced by retained MOM at the site of injection, which remained until the end of observation. p.o.-dosed groups: There were no marked changes in behavior. A white stool was discharged 30 to 50 minutes after administration. An increase in body weight in male animals was favorable all through the period of observation. In females, a decrease in body weights was observed on the 2nd day, which returned to the initial level with favorable course of increase afterward. 3. Gross findings and organ weights Adhesion of visceral organs and retention of test material were observed in i.p.-dosed groups. Retention of test material was also found at the site of injection in s.c.-dosed groups. There were no marked changes in p.o.-dosed groups. 4. Histopathological findings i.p.-dosed groups: Inflammatory changes were observed surrounding the visceral organs. Ac- tivation and phagocytosis of KUPFFER'S cells and occasional giant cells were noted as hepatic changes in some animals. Thrombus like bodies were noted in the lungs of 2 female animals (Plate 1). s.c.-dosed groups: Inflammatory changes (granulomatous inflammation) were observed at the injection site in both male and female animals (Plate 2). p.o.-dosed groups: There were no marked changes in both male and female animals. Discussion and Conclusion MOM, a derivative of MDM, discovered by NIIDA et al.1, 2), occurs as tasteless , odorless and white crystalline solid and is insoluble in water . As previously reported in the acute toxicity studies in mice5) , LD0 values were estimated more than 5,000 mg/kg as the maximum physically applicable dose in male and female mice after single i .p., s.c. and p.o. administration. In the present studies with male and female Wistar rats , we selected ad ose of 5,000 mg/kg as the maximum physically applicable dose in consideration of the results obtained from the acute studies with mice. As a result, LA, values were also estimated more than 5,000 mg/kg for rats in all routes of adminis- tration as no animal died during 1 week period of observation . In the i.p. administration, abnormal gait was observed, which seemed due to physical effects from high dose as 5,000 mg/kg but not to local irritation. In the p.o. administration, white stool was noted 30•`50 minutes after the maximum phys- ically applicable dose as high as 5,000 mg/kg. The same abnormality after p .o. administration was not found at doses of 3,000 and 4,000 mg/kg in previous studies°. Inflammatory changes in the visceral organs together with appearance of giant cells and phagocytic KUPFFER'S cells in the liver after i .p. administration and inflammatory changes at the site of injection after s.c. administration might be due to reactions to excessive dose of test material. In conclusion, MOM, non-crystalline solid, did not exhibit any acute toxicity in the present studies 1320(84) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-7 July 1984 with male and female rats after the single i.p., s.c. and p.o. administrations and LD0 values were es- timated more than 5,000 mg/kg in all routes of administration. It is assumed, therefore, that MOM, non-crystalline solid, has weaker acute toxicity than the other macrolide antibiotics6,7) when it was orally administered to rats. Abstract Miocamycin (MOM) is a derivative of midecamycin, a macrolide antibiotic isolated from a cul- ture broth of Streptomyces mycarofaciens1,2). The object of this study was to determine the acute toxicity in male and female rats (Wistar, 5-week-old) after single i.p., s.c. and p.o. administration of MOM, non-crystalline solid, at a dose level of 5,000 mg/kg as the maximum physically applicable dose. Observations were kept for 1 week after administration. In conculusion, no animal died during an observation period for 1 week so that the LD0 values were estimated to be more than 5,000 mg/kg in all routes of administration. References 1) YOSHIDA, T.; T. WATANABE, T. SHOMURA, S. SOMEYA, R. OKAMOTO, S. ISHIHARA, K. MIYAUCHI & Y. KAZUNO: Bacteriological evaluation of midecamycin acetate and its metabolites. Jap. J. Antibiotics 35: 1462-1474, 1982 2) OMOTO, S.; K. IWAMATSU, S. INOUYE & T. Num: Modifications of a macrolide antibiotic midecamycin (SF-837). I. Synthesis and structure of 9,3"-diacetylmidecamycin. J. Antibiotics 29: 536•`548, 1976 3) KAWAHARAJO, K.; Y. SEKIZAWA & M. INOUE: In vitro and in vivo antibacterial activity of 9,3"-di-O- acetylmidecamycin (MOM), a new macrolide antibiotic. J. Antibiotics 34: 436•`442, 1981 4) NAKAMURA, T.; S. FUKATSU, S. SEKI & T. NIIDA : A convenient method for the preparation of the acylated macrolide antibiotic midecamycin using molecular sieves and acylchloride. Chemistry Letters -1978: 1293 #.4296, 1978 5) YOKOTA, M.; U.