Gangliosides in the blood plasma: levels of ganglio-series gangliosides in the plasma after administration of brain gangliosides.

The temporal change in the levels of the gangliotetraose-series gangliosides, i.e., GMla, GDla, GD1b, GT1b, in the blood plasma after intramuscular administration of bovine brain gangliosides (5 mg/kg) to beagle dogs (11.3-12.2 kg) was determined with high sensitivity by a recently developed thin-layer chromatography/enzyme-immunostaining method (Hirabayashi, Y., Koketsu, K., Higashi, H., Suzuki, Y., Matsumoto, M., Sugimoto, M. and Ogawa, T. (1986) Biochim. Biophys. Acta 876, 178-182). The amounts of GMla, GDla, GD1b, GT1b and their combined total in the plasma of beagle dogs before administration of gangliosides were 21 +/- 1, 36 +/- 7, 15 +/- 2, 16 +/- 2 and 88 +/- 6 pmol/ml of blood plasma, respectively. Trapezoidal calculation showed that the times of the maximum levels of GMla, GDla, GDlb, GTlb and the total of the their levels in the plasma were 8.0 +/- 1.2, 8.7 +/- 0.7, 6.3 +/- 2.0, 17.0 +/- 7.0 and 8.7 +/- 0.7 h after the administration of gangliosides, and their maximum concentrations were 517 +/- 37, 654 +/- 53, 160 +/- 5, 184 +/- 20 and 1383 +/- 74 pmol/ml, respectively. The maximum level of each ganglioside decreased gradually, reaching the normal level after 10 days. The half-maximum level of each ganglioside occurred 2-3 days after the administration. Asialo GM1 (GA1) was not detected plasma at any of the test times.

Dog liver glutathione S-transferase and its strong immunoreactivity with rat transferase-P(7-7).

Dog liver cytosolic glutathione S-transferases (GSTs) were investigated to characterize their properties in comparison with rat liver transferases. Dog liver GSTs after the glutathione affinity column chromatography showed three subunit bands on SDS-polyacrylamide gel electrophoresis. These three subunits, designated as Yd1 (mol.wt 26,000), Yd2 (mol.wt 27,000) and Yd3 (mol.wt 28,000), were distinctly different from rat liver GST subunits, i.e. Ya(1) (mol.wt 26,500), Yb1(3)/Yb2(4) (mol.wt 27,500) and Yc(2) (mol.wt 28,500). Western blot analysis revealed that Yd1, Yd2 and Yd3 were immunoreacted with anti-rat GST 7-7, 1-1 and 3-3 antibodies, respectively. Four transferase activity fractions, I (pH greater than 7.63), II (pH 6.92), III (pH 5.80) and IV (pH 5.65), were obtained from affinity purified GSTs by chromatofocusing. Each fraction exhibited a characteristic substrate specificity. GST-II, III and IV were all strongly immunoreacted with anti-rat GST 7-7 antibody by immunoblotting, thus suggesting the occurrence of the heterogeneity of transferases immunologically related to rat GST subunit 7 in dog liver. Immunohistochemical examination showed that transferases immunoreacted with anti-GST 7-7 antibody have diffusely distributed throughout the lobule, while enzymes related to subunit 3 have been localized in a narrow range of cells around the central vein. These data suggest that GSTs immunologically associated with rat transferase subunit 7 may be major forms in dog liver.

Quinoline-induced chromosome aberrations and sister chromatid exchanges in rat liver.

Induction of chromosome aberrations and sister chromatid exchanges (SCEs) was studied in hepatocytes of F344 rats exposed in vivo to the hepatocarcinogen quinoline (Q). Hepatocytes were isolated 4-48 hr after a single dose of 200 mg/kg body weight or 24 hr after 28 repeated doses (once a day) of 25-200 mg/kg body weight/day by gastric intubation, and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 hr. A single dose of Q induced chromosome aberrations in up to 22% of metaphase cells, and SCEs with a frequency of up to 1.27 per chromosome 12 hr after the dose, while the control values were 1% and 0.63 per chromosome, respectively. Treatment with 28 repeated doses of Q induced significant chromosome aberrations and SCEs dose-dependently. Cytogenetic damage induced induced in the liver by repeated doses of Q was greater than induced by a single dose. Furthermore, Q induced replicative DNA synthesis in the liver, but failed to induce micronucleus formation in the bone marrow. The noncarcinogen 8-hydroxyquinoline was also examined and found to be essentially non-genotoxic to rat liver. These results show that Q is a genotoxic carcinogen to rat liver and the present method of in vivo cytogenetic assay should be useful for evaluating the genotoxicity of hepatocarcinogens.

Protection from drug-induced hepatocellular changes by pretreatment with conjugating enzyme inhibitors in rats.

The present paper describes the role of conjugating enzymes in the development of hepatotoxicity after administration of repeated doses of a novel monoamine oxidase type-A (MAO-A) inhibitor, (5R)-3-[2-(( 1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone (E2011). The effects of pretreatment with three kinds of conjugating enzyme inhibitors on hepatic lesions induced by E2011 were evaluated in female Sprague-Dawley rats. The inhibitors used were 2,6-dichloro-4-nitrophenol (DCNP; inhibitor of sulfotransferase (ST)), pentachlorophenol (PCP; inhibitor of both ST and acetyltransferase (AT)) or ranitidine (inhibitor of UDP-glucuronosyltransferase (UDP-GT)). Two weeks treatment of E2011 alone at an oral dosage of 150 mg/kg induced hepatocellular changes characterized by nuclear enlargement. Daily pretreatment with DCNP (10 mg/kg, i.p.) enhanced the E2011-induced hepatocellular changes accompanied by single cell necrosis. On the other hand, the hepatotoxicity was clearly diminished by PCP (5 mg/kg, i.p.). Ranitidine pretreatment had no effect. Protection by PCP was attributed to the inhibitory effects of AT in addition to ST; it was considered that the hepatocellular changes caused by E2011 were largely dependent on the formation of acetyl conjugate(s).

Hepatic drug metabolizing enzymes induced by clofibrate in rasH2 mice.

Hepatic drug metabolizing enzyme activities were determined, after treatment with clofibrate, in transgenic mice carrying human c-Ha-ras (rasH2 mice). Changes in the drug metabolizing enzyme activities in these mice by gene integration were also evaluated. Male and female rasH2 mice (Tg) and the litter mates not carrying the gene (non-Tg) received orally 500 mg/kg of clofibrate or the vehicle for 12 consecutive days. Liver homogenate and microsomes were prepared and the contents and activities of cytochrome P450 (CYP), cytochrome b5 content and enzyme activities related to peroxisome proliferation were determined. Relative liver weights, CYP4A and activities of catalase and carnitine palmitoyl transferase increased to the same extent in Tg and non-Tg mice treated with clofibrate. In Tg and non-Tg groups that received vehicle, contents and activities of CYP and cytchrome b5 contents were comparable. It was concluded that gene integration did not alter drug metabolizing enzymes and responses to clofibrate.

A short-term assessment of tumor-promotion activity in the livers of rats treated with two genotoxic methylating agents: dimethylnitrosamine and methylnitrosourea.

Induction of replicative DNA synthesis (RDS) and mitoinhibitory effects were studied in the hepatocytes of F344 rats exposed in vivo to the methylating agents dimethylnitrosamine (DMN, hepatocarcinogen) and methylnitrosourea (MNU, non-hepatocarcinogen). Cytotoxicity and chromosome aberrations (CA) in rat liver were also investigated to clarify the cause of changes in RDS and mitoinhibitory effects, respectively. The animals were killed at different intervals (up to 14 days) after a single oral dose, or 1 day after 7 or 14 days of repeated oral doses. The hepatocytes were isolated and cultured with Williams' medium E to assess their RDS, mitoinhibitory effects and CA. Mitoinhibitory effects were investigated by monitoring their effect on epidermal growth factor-induced replicative DNA synthesis (EGF-induced RDS) in rat hepatocytes. Hepatotoxic effects were assessed by measuring aspartate transaminase and alanine transaminase in the plasma and by histopathological examination. In the single-dose study, DMN (20 mg/kg body weight (bw)) induced both RDS and hepatotoxicity. MNU (50 mg/kg bw) induced RDS without causing hepatotoxicity, and thus was classified as a mitogen. In the repeated-dose study, DMN (4 mg/kg bw) induced both RDS and hepatotoxicity, but MNU (10 mg/kg bw) induced neither. Both inhibition of EGF-induced RDS and induction of CA were observed in the hepatocytes of rats treated with DMN, but were not observed with MNU in both single and repeated dose studies. The mitoinhibitory effect of DMN persisted for 14 days after the single dose and time dependently increased for 14 days after repeated administration. This mitoinhibitory effect correlated positively with CA. The mitoinhibitory effect was thought to be attributable to the DNA-damaging effect that induces CA. We concluded that the differences which we found in this study between DMN and MNU contribute to the differences in their hepatocarcinogenicity. Our findings suggested that both cell proliferative and mitoinhibitory properties play an important role in tumor promotion, and measuring them may provide an ancillary index that is useful in predicting hepatocarcinogenicity.

A staining procedure for micronucleus test using new methylene blue and acridine orange: specimens that are supravitally stained with possible long-term storage.

The micronucleus test has been widely used as an in vivo cytogenetic test. It employs two different kinds of supravital staining methods which use either new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravitally stained with possible long-term storage, using both N and AO. This N/AO-staining method involves three steps; (1) combination of the target tissue or target cells with an equivalent volume of 0.5% solution of new methylene blue (N-staining step), (2) immediate smear of the mixture, followed by treatment with methanol for 10 min for fixation and removal of N and drying (referred to as fixed-decolorized specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by washing with Sörensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To examine whether the N/AO-staining method is useful for the micronucleus test, comparisons were made between N-, N/AO-, and AO-stained specimens prepared supravitally from peripheral blood of rats with and without treatment of cyclophosphamide. The results indicate that N/AO-stained specimens can be supravitally observed after long-term storage with the same coloration and comparable frequencies of micronucleated reticulocytes with a positive response as AO-stained specimens, if the staining process is temporarily stopped before AO-staining (as fixed-decolorized specimens), or if the AO-staining step is repeated. The results also showed that separated reticulocyte types are supravitally stained in a similar fashion to N-stained specimens but not to AO-stained specimens, indicative of the preservation of the supravital feature of N-staining. Taken together these results suggest that the N/AO-staining procedure could offer an additional useful staining tool for the micronucleus test.

Mutagenicity of dihydroxybenzenes and dihydroxynaphthalenes for Ames Salmonella tester strains.

The mutagenicity of 3 dihydroxybenzene (DHB) and 9 dihydroxynaphthalene (DHN) isomers was examined by using 5 different Ames Salmonella mutagenicity tester strains in the presence and absence of phenobarbital and 5,6-benzoflavone-treated rat liver S9-mix. Of the 3 DHB isomers, 1,4-DHB (hydroquinone) was mutagenic, and of the 9 DHN isomers, 1,3-DHN (naphthoresorcinol), 1,4-DHN (hydronaphthoquinone), 1,6-DHN and 1,7-DHN were mutagenic. Mutagenicity of all the compounds tested was observed in the absence of S9-mix, while 1,4-DHN and 1,6-DHN were also mutagenic in the presence of S9-mix. The mutagenicity of 1,4-DHB and 1,4-DHN for TA104, which is a strain sensitive to oxidative mutagens, was almost completely or partially inhibited by superoxide dismutase (SOD) and/or catalase, indicating the involvement of activated oxygen species in mutagenesis. Furthermore, from the finding that the 4 DHNs were mutagenic for TA2637, the strain sensitive to frameshift mutagens, it is possible that the mutagenicity of DHNs for S. typhimurium was also attributable to DNA adducts that form with quinones and/or semiquinones through oxidation of DHNs. The mutagenicity of 1,3-DHN, which showed the largest number of revertants in strains TA100, TA98, TA2637 and TA104, was greatly decreased, when their pKM101 plasmid-deficient strains, TA1535, TA1538, TA1537 and TA2659 were used. This observation suggests that an SOS repair system was involved in the mutagenesis of 1,3-DHN for S. typhimurium.

Comparison of the mutational spectra of the lacZ transgene in four organs of the MutaMouse treated with benzo[a]pyrene: target organ specificity.

We recently demonstrated that not all organs with a high rate of induction of mutation in the lacZ transgene develop tumors in the lambdalacZ transgenic mice (MutaMouse) used for a long-term carcinogenicity study with benzo[a]pyrene (BP). To better understand the role of chemical-induced in vivo mutations in carcinogenesis, we compared the mutational spectra of the lacZ transgene in four organs of the MutaMouse obtained 2 weeks after five daily consecutive oral treatments with 125 mg/kg/day BP. lacZ transgenes were analyzed in two target organs (forestomach and spleen) and two non-target organs (colon and glandular stomach) for BP-induced carcinogenesis in MutaMouse, and all of these organs were highly mutated in the lacZ transgene. The sequence data showed similar mutational spectra of the lacZ transgene between the two target organs; the predominant mutations were G:C-->T:A transversions (55% and 50% for forestomach and spleen, respectively), followed by deletions (20% and 21% for forestomach and spleen, respectively) mainly at G:C site. The frequent G:C-->T:A transversions are consistent with reports of the mutational spectra produced in the p53 gene in tumors generated in rats and mice exposed to BP. In contrast, the mutational spectra of the lacZ transgene in the two non-target organs are different from those in the target organs, and are also suggested to differ from one another. These findings suggest an organ/tissue-specific mechanism of mutagenesis.

Multiple organ mutation in the lacZ transgenic mouse (Muta mouse) 6 months after oral treatment (5 days) with benzo[a]pyrene.

We have recently demonstrated that not all organs with high rates of mutation in the lacZ transgene develop tumors using the Muta Mouse. To better understand the role of in vivo mutation in carcinogenesis, we examined the mutant frequencies (MF) of the lacZ transgene in tumor-bearing and non tumor-bearing organs. MF, recovered after 2 weeks (the data taken from our previous study) and after 26 weeks following oral doses of 125 mg kg-1 day-1 benzo[a]pyrene (BP) for five days were compared. The organs examined included the target organs (forestomach, spleen, and lung) and non-target organs (colon, glandular stomach, and liver) for BP carcinogenesis. The data indicated that lacZ MF were markedly increased over spontaneous frequencies in the organs examined and that the organ which showed the highest MF was the colon, followed by the forestomach>spleen>glandular stomach, liver, and lung in that order. These findings indicate that the MF of the lacZ transgene in each organ, even 26 weeks after the start of the treatment does not fully correlate with the known target organs of BP. Furthermore, the lacZ MF in a non-papilloma region of a forestomach with a papilloma was equivalent to the two highest MF observed in the healthy colon (non-target organ) of mice at 26 weeks. These observations also indicate that the generation of tumors requires the induction of mutations as well as other factor(s) specific to the target organs. These results clearly suggest that highly mutated organs do not always progress to tumors in the transgenic mouse.

Auto-induction of E5110 metabolism, a novel non-steroidal anti-inflammatory drug, during toxicokinetic studies in beagle dogs.

Following single-dose intravenous and oral studies to determine the absolute bioavailability of E5110 in beagle dogs, repeated dose pharmacokinetic studies were conducted as toxicokinetics in a subacute toxicity test. E5110 was administered orally once a day for 91 days at doses of 0, 1, 10, 50 and 250 mg/kg/day to dogs. On the first day during repeated administration, the Cmax and AUC values of E5110 in females were lower than those for males, and it seems that this may be due to a sex-related difference in the activity of cytochrome P450 (CYP) 3A. During repeated administration of E5110, the plasma levels of E5110 on day 15 and day 91 were markedly lower than those on the first day. On day 91, the AUCs for E5110 were 55.9%, 38.8%, 10.9% and 7.8% in males, and 76.2%, 80.3%, 10.5% and 11.9% in females on the first day, for doses of 1, 10, 50 and 250 mg/kg/day, respectively. Liver microsomes prepared after the last dose of E5110 showed increased activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase, and aminopyrine N-demethylase, at doses of 1 mg/kg/day or more. A dose-dependent increase in P450 content was also observed. Furthermore, the capacity for 5-hydroxylate E5110 was increased, and Western blot analysis indicated an induction of CYP2B and 3A; therefore CYP3A may contribute to a main metabolic pathway of E5110. These results suggested that the decrease in plasma concentrations of E5110 that were observed during repeated administration represents a typical case of auto-induction of the phenobarbital type.

[General toxicity study of gadobenate dimeglumine formulation (E7155) (1)--single dose intravenous and intracisternal toxicity study in rats].

Gadobenate dimeglumine formulation (E7155) was evaluated for its general toxicity potential following a single intravenous and intracisternal administration to rats. Dosage levels tested were 3.3, 4.5, 6.0 and 8.0 mmol/kg at the injection rate of 6 ml/min and 7.50, 8.89, 10.54 and 12.50 mmol/kg at 1 ml/min for the intravenous administration route, and 0.15, 0.21, 0.29 and 0.40 mmol/kg for the intracisternal administration route. Parameters measured during the 14-day observation period were mortality, clinical signs and macroscopic examination. After intravenous administration at the injection rate of 6 ml/min, twitches, respiratory blocking and prostration were observed at 6.0 mmol/kg, and dyspnoea and sedation at 3.3 and 4.5 mmol/kg. Deaths occurred within 1 min after administration at 6.0 mmol/kg and above. LD50 values were 7.97 mmol/kg in males and 6.22 mmol/kg in females. After intravenous administration at the injection rate of 1 ml/min, shallow breathing, twitches and sedation were observed at 7.50 mmol/kg and above and respiratory arrest at 8.89 mmol/kg. Deaths occurred within 1 min after administration at 8.89 mmol/kg and above. LD50 values were 9.0 mmol/kg in males and 9.7 mmol/kg in females. After intracisternal administration, symptoms consisted of sedation, staggering gait, dyspnoea, twitches and ataxia at 0.15 mmol/kg and above, prostration, paralysis of forelimbs, and/or hind limbs and chromodacryorrhea at 0.21 mmol/kg, and convulsions at 0.29 mmol/kg and above. Deaths occurred within 7 days after administration at 0.21 mmol/kg and within 5 min at 0.29 mmol/kg and above. LD50 values were 0.42 mmol/kg in males and 0.25 mmol/kg in females.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 17). Testicular toxicity of E7010, a sulfonamide tubulin polymerization inhibitor.

E7010 (N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide), a sulfonamide antitumor agent that inhibits tubulin polymerization, was orally administered to male Slc:SD rats at doses of 50 and 75 mg/kg once a day for 2 weeks. E7010 at a dose of 75 mg/kg induced severe testicular lesions characterized by marked decrease and/or loss of seminiferous epithelial cells, which sometimes resulted in tubules with only Sertoli cells. In the 50 mg/kg group, alteration of germ cells was evident at various stages of spermatogenesis, and apoptotic figures of meiotic spermatocytes at stage XIV were frequently observed. Single dose treatment of 50 mg/kg was also performed and was revealed a decrease of round spermatids in stage VII at necropsy after 1 week. Thus the target cells were considered to be meiotic spermatocytes at stage XIV. The present study indicates that 2-weeks repeated dosing is sufficient to detect the testicular toxicity of E7010.

Toxicological response of rats to a novel monoamine oxidase type-A inhibitor, (5R)-3-[2-((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5- methoxymethyl-2-oxazolidinone (E2011), orally administered for 13 weeks.

(5R)-3-[2-((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5- methoxymethyl-2-oxazolidinone (E2011) is a novel monoamine oxidase type-A (MAO-A) inhibitor. In order to assess toxicological profiles of E2011, doses of 0 (as controls), 30, 100 mg/kg of E2011 were administered to male and female Sprague-Dawley rats once a day for 13 weeks orally by gavage. No mortality or any toxic signs except salivation occurred due to E2011 treatment. Decreased body weight gain and food consumption, increases of alkaline phosphatase and increases of liver weight were the major treatment-related findings observed predominantly in the 100 mg/kg group. Histological examination revealed nuclear enlargement of hepatocytes with appearance of altered cell foci in some cases, and acinar atrophy in Harderian glands in the 100 mg/kg group. Since the histopathological findings in the liver were indicative of an ongoing carcinogenic process, glutathione S-transferase placental form (GST-P) positive hepatic foci were identified immunohistochemically and examined morphometrically. Although GST-P positive hepatic foci were detected in all groups including controls, the number and area of GST-P positive hepatic foci were significantly higher in female rats treated with 100 mg/kg than those in controls. In this paper, possible mechanisms of specific lesions in the liver and Harderian glands will be discussed.

[General toxicity study of gadobenate dimeglumine formulation (E7155) (2)--Single dose intravenous toxicity study in dogs].

Gadobenate dimeglumine formulation (E7155) was given by single intravenous injection to 4-5 month-old beagle dogs at doses of 2 or 6 mmol/kg. Treatment was followed by a 14-day observation period in order to evaluate the test article's toxicity. The male and female dogs at 6 mmol/kg vomited and showed reddened gums and ears as clinical signs. One male dog at 6 mmol/kg was euthanized approximately 23 hr after administration due to its very poor clinical condition, which included an unwillingness to move, pale gums and weak pulse. Body weight was decreased at 6 mmol/kg, and also slightly at 2 mmol/kg. Decreased food consumption was noted both at 2 and 6 mmol/kg. Hematology for the euthanized male at 6 mmol/kg showed increases in the total white blood cell count, packed cell volume, hemoglobin and red cell count and a decrease in the platelet count. Biochemistry showed a dose-related increase in alkaline phosphatase, GPT and GOT at 2 and 6 mmol/kg. Males and females at 6 mmol/kg showed increases in bilirubin, calcium and urea, and a reduction in glucose. Females at 6 mmol/kg also showed a reduction in total protein. Urinalysis showed an increase in pH at 2 mmol/kg and above. For females at 6 mmol/kg, an increase in urine volume and a decrease in specific gravity and osmolality were noted. An increase in relative liver and kidney weights was recorded for males and females dosed at 6 mmol/kg. For the euthanized male at 6 mmol/kg, postmortem examination revealed a pale liver with rounded edges and an accentuated lobular pattern, and dark material on the gastro-intestinal mucosal surface. In macroscopic pathology, the male at 6 mmol/kg revealed single liver cell necrosis, minimal early hyperplasia in small biliary ductules, inflammatory cells in the sinusoidal and portal tracts, centrilobular inflammatory cells, diffuse vacuolation of the hepatocytes and sinusoidal dilatation in the liver, and cortical tubular vacuolation in the kidneys. In the female dog treated at 6 mmol/kg, hyperplasia in the small biliary ductules, inflammatory cells in the portal tracts, diffuse vacuolation of hepatocytes and sinusoidal dilatation were seen in the liver, and increases in the severity of cortical tubular basophilia, cortical tubular dilatation and cortical tubular casts were detected in the kidney. Based on these results, the lethal dose of E7155 was set at 6 mmol/kg. It is also concluded that a dose of 2 mmol/kg was tolerated in the beagle dog after a single injection followed by a 14-day observation period.

[General toxicity study of gadobenate dimeglumine formulation (E7155) (3)--4-week repeated dose intravenous toxicity study followed by 4-week recovery period in rats].

A 4-week repeated dose toxicity study of gadobenate dimeglumine formulation (E7155) was conducted in Sprague-Dawley rats to assess its non-clinical safety. E7155 was administered intravenously at doses of 0.3, 1.0 and 3.0 mmol/kg/day to male and female rats once a day during 4 weeks. The reversibility of toxicity was evaluated during a 4-week recovery period at 3.0 mmol/kg/day. At 0.3 mmol/kg/day and higher, vacuolation of the cortical epithelium was seen in the kidneys and an increase in the incidence of local damage at the injection sites. In the 1.0 and 3.0 mmol/kg/day male and female groups, scabbing/ulceration of the tail at the injection sites, macroscopic pale/thickened fundic mucosa in the stomachs, vacuolation of the urinary bladder, and mucosal mineralization with epithelial hyperplasia of the glandular stomach were found. In the 1.0 and 3.0 mmol/kg/day male group and 3.0 mmol/kg/day female group, increases of water consumption and urinary potassium excretion, increased kidney weight and enlargement of the kidneys were observed. In the 3.0 mmol/kg/day male and female group, hepatocyte necrosis with inflammatory cells in the liver and epithelial degranulation in the interlobular ducts of the salivary glands were found. In addition, in the 3.0 mmol/kg/day male group, increases in plasma sodium and decreases of urinary sodium and chloride excretion, and degenerative changes in the testes and epididymides were observed. After the 4-week recovery period, except for an increase in urinary potassium excretion, increased kidney weights and changes in the testes and epididymides, all of the above findings had complete or partial recovery. Vacuolation of renal tubular cells was common, expected, and known as an adaptive change of treatment with hypertonic solutions, and an increase in the incidence of local damage at the injection sites was due to irritation by repeated intravenous dosing with hypertonic solutions. Therefore, these changes were not toxic changes. In conclusion, the dose level of 0.3 mmol/kg/day should be regarded as the No Observed Adverse Effect Level (NOAEL) after repeated administration of E7155 in rats.

[General toxicity study of gadobenate dimeglumine formulation (E7155) (4)--4-week repeated dose intravenous toxicity study followed by 4-week recovery period in dogs].

Gadobenate dimeglumine formulation (E7155), at doses of 0 (physiological saline), 0.25, 0.5, 1 and 2 mmol/kg/day of body weight, was administered intravenously to male and female beagle dogs once daily for 4 consecutive weeks in order to evaluate the subacute toxicity of the test article. Reversibility of toxicity was evaluated during a 4-week recovery period at 1 and 2 mmol/kg/day. No toxicologically significant changes were observed at 0.25 and 0.5 mmol/kg/day. In animals receiving 1 or 2 mmol/kg/day, transient swelling and redness of the facial and eye areas, lethargy, decreased activity, emesis, retching, watery or unformed stool, decreased body weight or body weight gain, decreased food consumption, decreased hematocrit and hemoglobin concentration, increased APTT, increases in plasma ALP, GPT or gamma-GT, decreased plasma inorganic phosphorus, total protein or albumin, increased liver or kidney weight, subacute inflammatory infiltrates, loss of centrilobular hepatocytes or hepatocellular cytoplamic vacuolation in the liver, vacuoles in the epithelial cells of the renal tubles and/or hypocellularity in the bone marrow were seen. The results of toxicokinetic analysis showed that systemic exposure was similar in males and females, and there was no accumulation of the test material over the treatment period, although AUC tended to be enhanced by slightly more than the proportionate dose increase. These effects were recovered or tended to be reversed after a post-dosing period for 4 weeks. In conclusion, the No Observed Adverse Effect Level (NOAEL) was 0.5 mmol/kg/day.

[Reproductive and developmental toxicity study of gadobenate dimeglumine formulation (E7155) (1)--Fertility study in male rats by intravenous administration].

The influence of gadobenate dimeglumine formulation (E7155) on general reproductive performance and fertility in male rats of the Sprague-Dawley strain was assessed in this study. E7155 was administered by intravenous injection at a dosage of 0.3, 1.0, or 2.0 mmol/kg/day to groups of 22 male rats for 13 weeks. Control animals received 0.9% sterile physiological saline throughout the same period. After four weeks of treatment, each male was paired with an untreated female of the same strain. Each male was paired again after 10 weeks of treatment with another untreated female of the same strain. All females were killed on Day 14 of gestation for examination of pregnancy status. No significant toxicological signs associated with systemic exposure to E7155 were observed. There were no effects of treatment with E7155 on body weight gain, food consumption, macroscopic findings, reproductive organ weights and sperm count or sperm motility in male rats. Mating performance after pairing at Weeks 4 and 10 of treatment as well as litter size and number of survival embryos on Day 14 of gestation were not affected by paternal treatment with E7155. From these results, the No Observed Adverse Effect Level (NOAEL) of E7155 was 2.0 mmol/kg/day for general and reproductive toxicity parameters in male rats treated with E7155 and for development in their embryos.

[Reproductive and developmental toxicity study of gadobenate dimeglumine formulation (E7155) (2)--Combined study of effects on fertility and embryo-fetal toxicity in female rats by intravenous administration].

The influence of gadobenate dimeglumine formulation (E7155) on fertility and general reproductive performance and embryo-fetal development was assessed in female Sprague-Dawley rats. E7155 was administered by intravenous injection at a dose of 0.3, 1.0 or 2.0 mmol/kg/day to groups of 22 female rats for 15 days before pairing. Treatment was continued throughout mating and up to Day 17 of gestation. Control animals received 0.9% sterile physiological saline throughout the same period. All females were killed on Day 20 of gestation for examination of their uterine contents. There were no toxic clinical signs of treatment. The body weight and food consumption of females before pairing and during gestation were not affected by treatment. Estrous cycles, mating performance, litter size and fetal weight, survival and development were also not affected by treatment. Based on the above results, the No Observed Adverse Effect Level (NOAEL) of E7155 was 2.0 mmol/kg/day for general toxicologic effects and reproduction of female rats and the development of their fetuses.

[Mutagenicity study of gadobenate dimeglumine formulation (E7155) (1)--Reverse mutation assays in S. typhimurium and E. coli tester strains].

The ability of gadobenate dimeglumine formulation (E7155) to cause gene mutations was assessed in five strains of Salmonella typhimurium (TA100, TA1535, TA98, TA1538, and TA1537) and a strain of Escherichia coli (CM891; WP2, uvrA-, pKM101) using the Ames test (agar plate assay). The results suggest that E7155 is non-mutagenic towards these bacterial tester strains.