Field-realistic dose of cefotaxime enhances potential mobility of ß-lactam resistance genes in the gut microbiota of zebrafish (Danio rerio).

With large amounts of cephalosporin end up in natural ecosystems, water has been acknowledged as the large reservoir of ß-lactam resistance over the past decades. However, there is still insufficient knowledge available on the function of the living organisms to the transmission of antibiotic resistance. For this reason, in this study, using adult zebrafish (Danio rerio) as animal model, exposing them to environmentally relevant dose of cefotaxime for 150 days, we asked whether cefotaxime contamination accelerated ß-lactam resistance in gut microbiota as well as its potential transmission. Results showed that some of ß-lactam resistance genes (ßRGs) were intrinsic embedded in intestinal microbiome of zebrafish even without antibiotic stressor. Across cefotaxime treatment, the abundance of most ßRGs in fish gut microbiome decreased apparently in the short term firstly, and then increased with the prolonged exposure, forming distinctly divergent ßRG profiles with antibiotic-untreated zebrafish. Meanwhile, with the rising concentration of cefotaxime, the range of ßRGs' host-taxa expanded and the co-occurrence relationships of mobile genetics elements (MGEs) with ßRGs intensified, indicating the enhancement of ßRGs' mobility in gut microbiome when the fish suffered from cefotaxime contamination. Furthermore, the path of partial least squares path modeling (PLS-PM) gave an integral assessment on the specific causality of cefotaxime treatment to ßRG profiles, showing that cefotaxime-mediated ßRGs variation was most ascribed to the alteration of MGEs under cefotaxime stress, followed by bacterial community, functioning both direct influence as ßRG-hosts and indirect effects via affecting MGEs. Finally, pathogenic bacteria Aeromonas was identified as the critical host for multiple ßRGs in fish guts, and its ß-lactam resistance increased over the duration time of cefotaxime exposure, suggesting the potential spreading risks for the antibiotic-resistant pathogens from environmental ecosystems to clinic. Overall, our finding emphasized cefotaxime contamination in aquatic surroundings could enhance the ß-lactam resistance and its transmission mobility in fish bodies.

In vitro interaction of cefotaxime with calf thymus DNA: Insights from spectroscopic, calorimetric and molecular modelling studies.

Cefotaxime is third generation antibiotic with known therapeutic efficacy against bacterial infections including cerebral abscesses and bacterial meningitis. The ß-lactam group of drugs are considered safest antibiotics. Many antibiotics directly interact with DNA and alter their expression profile. Thus, it is necessary to understand the binding mode and its relevance to drug activity and toxicity. There is considerably a remarkable focus on deciphering the binding mechanism of these therapeutic agents as DNA is one of the major target for wide range of drugs. Cefotaxime has been extensively studied for its pharmacological properties while its binding mode to DNA has not been explicated so far. In this study, we have unveiled the binding mechanism of cefotaxime to DNA by using various biophysical, thermodynamic and in silico techniques. UV-vis spectroscopy confirmed the formation cefotaxime-DNA complex along with a brief idea about the extent of interaction. Fluorescence spectroscopy yielded the values of various binding constants and explained mode of fluorescence quenching to be static. CD spectroscopy, thermal denaturation, KI quenching and viscosity measurement explained that cefotaxime is groove binder. Measuring the effect of ions on cefotaxime-DNA complex ensured that it does not bind to DNA electrostatically. Dye displacement experiments finally confirmed that cefotaxime binds to the minor groove of DNA. ITC gave the thermodynamic profile of this binding in which negative value of Gibb's free energy change revealed that the process is spontaneous. Molecular modelling finally strengthened our experimental results that cefotaxime was located in curved contour of minor groove of DNA. The findings support on safety of drug and may have a little interference on normal biological functions.

Severe hemolysis after plasma transfusion in a neonate with necrotizing enterocolitis, Clostridium perfringens infection, and red blood cell T-polyagglutination.

BACKGROUND: Red blood cell (RBC) Thomsen-Friedenreich antigen exposure (T activation) in infants with necrotizing enterocolitis (NEC) has occasionally been associated with posttransfusional intravascular hemolysis thought to be due to anti-T antibodies in the donor plasma. STUDY DESIGN AND METHODS: We describe an infant with NEC and Clostridium perfringens infection complicated by severe hemolysis after plasma transfusion. After this case, infants with confirmed NEC were prospectively evaluated for T activation. We checked for hemolysis in patients with T activation receiving plasma-containing blood products. RESULTS: The infant had received 80 mL of fresh-frozen plasma (FFP). His RBCs displayed strong T activation, and agglutination was observed with four of six ABO-compatible FFP units. A direct antiglobulin test was negative. IgM-class anti-T antibodies were present in small amounts (titer of 8) in the transfused FFP. Anti-T antibodies from the blood donor were not hemolytic in vitro. In the prospective study, T activation was observed in three of 28 infants with NEC (11%). One infant presented moderate T activation and two infants presented very strong T activation but only moderate decreases in sialic acid expression on the RBC membrane. These three infants presented no signs of hemolysis after transfusion with unwashed blood products or FFP. CONCLUSION: Anti-T antibodies are unlikely to be the etiologic factor for the hemolytic reactions observed in infants with NEC and T activation. Massive RBC desialylation and the direct action of bacterial toxins are more probable causes. Strict avoidance of plasma-containing blood products does not seem justified in these infants.

Toxicity of 13 different antibiotics towards freshwater green algae Pseudokirchneriella subcapitata and their modes of action.

Although modes of action (MOAs) play a key role in the understanding of the toxic mechanism of chemicals, the MOAs have not been investigated for antibiotics to green algae. This paper is to discriminate excess toxicity from baseline level and investigate the MOAs of 13 different antibiotics to algae by using the determined toxicity values. Comparison of the toxicities shows that the inhibitors of protein synthesis to bacteria, such as azithromycin, doxycycline, florfenicol and oxytetracycline, exhibit significantly toxic effects to algae. On the other hand, the cell wall synthesis inhibitors, such as cefotaxime and amoxicillin, show relatively low toxic effects to the algae. The concentrations determined by HPLC indicate that quinocetone and amoxicillin can be easily photodegraded or hydrolyzed during the toxic tests. The toxic effects of quinocetone and amoxicillin to the algae are attributed to not only their parent compounds, but also their metabolites. Investigation on the mode of action shows that, except rifampicin, all the tested antibiotics exhibit excess toxicity to Pseudokirchneriella subcapitata (P. subcapitata). These antibiotics can be identified as reactive modes of action to the algae. They act as electrophilic mechanism of action to P. subcapitata. These results are valuable for the understanding of the toxic mechanism to algae.

Evaluation of the toxic potential of cefotaxime in the third instar larvae of transgenic Drosophila melanogaster.

The present study was carried out to evaluate the toxic potential of cefotaxime in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg(9). Cefotaxime at final concentration of 10, 20, 40, 60 and 80 µg/ml was mixed in the diet and the larvae were exposed to the selected doses for 6, 12, 24, 48 h. The hsp70 expression, trypan blue exclusion test, in situ histochemical ß-galactosidase activity, lipid peroxidation, total protein content, glutathione (GSH) content, glutathione-S-transferase (GST) activity, protein carbonyl content, caspase 3 and 9 activity, apoptotic index and comet assay were taken as parameters for the study. The larvae exposed to 40, 60 and 80 µg/ml for 12, 24 and 48 h showed a dose and duration dependent significant increase in the activity of ß-galactosidase and lipid peroxidation but decrease in the total GSH content as compared to unexposed larvae. The decrease in protein content was observed in the larvae exposed to 40, 60 and 80 µg/ml of cefotaxime for 24 and 48 h. The larvae exposed to 40, 60 and 80 µg/ml of cefotaxime for 24 and 48 h showed a dose and duration dependent increase in the tissue damage, GST, caspase 3 and 9 activity, PC content, apoptosis and the DNA tail length (comet assay). The result suggests that the cefotaxime is toxic at 40, 60 and 80 µg/ml of doses for the third instar larvae of transgenic D. melanogaster (hsp70-lacZ)Bg(9). Cefotaxime at 10 and 20 µg/ml was not toxic for any duration of exposure.

Corneal oedema and its medical treatment.

Corneal oedema is a common sign of acute or protracted corneal disease of various aetiologies. In this paper, we review the causes and pathophysiological bases of corneal oedema, as well as discussing the goals and modalities of its medical treatment. Corneal oedema, if adequately understood and appropriately treated, generally shows a good prognosis.

Toward refinement of the colony-forming unit-granulocyte/macrophage clonogenic assay: inclusion of a metabolic system.

This work represents a first attempt to refine the colony-forming unit-granulocyte/macrophage (CFU-GM) clonogenic assay by incorporating liver microsomes and co-factors as a metabolic system into the in vitro test system in response to an ECVAM recommendation. From the comparison of results obtained with the CFU-GM clonogenic assay currently used and with the new experimental protocol, different toxicity on granulocyte/macrophage precursors was demonstrated, when drugs with a known metabolism in vivo were tested.

Acute and genotoxic profile of a dimeric impurity of cefotaxime.

The manufacturing and storage of cefotaxime produces different impurities of various concentrations, which may influence the efficacy and safety of the drugs. Because no report of toxicity data is available on the impurities of cefotaxime, the present acute and genotoxicity studies were designed and conducted to provide the information for establishing the safety profile and qualification of the dimeric impurity. Histidine-requiring mutants of Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains, with or without metabolic activation (S-91, were used for point-mutation tests. Neither increase in numbers of revertants, indicative of mutagenic activity, nor inhibition of bacterial growth, indicative of cytotoxicity, was observed when the dimeric impurity of cefotaxime at concentrations of 0.62, 1.85, 5.56, 16.67, and 50 microg/plate was incorporated into plates containing S. typhimurium bacterial strains. Cultures of Chinese hamster ovary (CHO) cells at a cell density of 2 x 10(5) cells per culture were exposed to the dimeric impurity of cefotaxime at the concentration of 11.25, 22.5, and 45 mg per culture, with or without metabolic activation, and harvested at 18 h after exposure. No chromosomal aberrations in the cultured mammalian cells were recorded. Acute intramuscular administration of the dimeric impurity of cefotaxime in Sprague-Dawley rats did not result in any clinical signs and gross pathological changes up to 2000 mg/kg-body weight. The results of these studies indicated that the dimeric impurity of cefotaxime is nonmutagenic in Ames test, nonclastogenic in vitro, and acutely nontoxic in rats.

Antibiotics induce apoptosis of human peritoneal mesothelial cells.

The peritoneal mesothelial cell is a critical component of the peritoneal membrane. The intraperitoneal use of several antibiotics to treat bacterial peritonitis is current clinical practice. Our previous study showed that cephalothin (CPL) and cefotaxime (CFT) have cytotoxic effects on human peritoneal mesothelial cells (HPMC), however, the exact mechanism of cytotoxicity has not been elucidated. In the present study, flow cytometry, TdT-mediated dUTP nick-end labelling (TUNEL) staining and electron microscopy were used to detect the apoptosis of HPMCs. Immunofluorescent staining was used to evaluate the cytochrome c distribution pattern. Western blotting was used to assess apoptotic signalling proteins. We found that CPL (0.5 mg/mL) and CFT (1 mg/mL) induced apoptosis of HPMCs, whereas cefazolin (0.5 mg/mL) and ceftriaxone (0.5 mg/mL) failed to induce apoptosis of HPMCs. While the DNA content of CFT- or CPL-treated cells was reduced, as determined by flow cytometry, cefazolin and ceftriaxone had no such effect. The CFT- or CPL-treated cells displayed the features of apoptosis both under the electron microscope and by using TUNEL staining. However, cefazolin and ceftriaxone produced the same result as the medium controls. Furthermore, CFT and CPL increased the expression of Bax and p53, and caused the translocation of cytochrome c from the mitochondria to the cytoplasm. The HPMC treated by CFT but not by CPL induced the cleavage of procaspase-3 to form active caspase-3. In conclusion, cefotaxime and cephalothin induce apoptosis of HPMCs in vitro. Signal transduction may be through the mitochondrial pathway.

Randomized controlled monocentric comparison of once daily ceftriaxone with tobramycin and cefotaxime three times daily with tobramycin in neutropenic fever.

A prospective, randomized, controlled monocentric trial was performed to evaluate the efficacy and safety of once daily ceftriaxone 2 g plus tobramycin 5 mg/kg in comparison to cefotaxime 2 g t.i.d. plus tobramycin 5 mg/kg qd in the treatment of neutropenic fever. In cases of fever > or = 38.5 degrees C and a neutrophil count below 1000/microliter, patients with hematological malignancies were assigned to ceftriaxone or cefotaxime, each with tobramycin. The primary endpoint was defined as defervescence < 37.5 degrees C on day 4-6 followed by at least 7 afebrile days. Secondary endpoints were overall response, defined as defervescence on day 25 and toxicity. There were 160 episodes of 114 patients included. Fever of unknown origin accounted for 79 episodes (51%), microbiologically defined infection for 36 (23%), clinically defined infection for 27 (17%), and both clinically and microbiologically defined infection for 14 episodes (9%). On an intent-to-treat basis 156 episodes could be evaluated for the primary endpoint. Ceftriaxone plus tobramycin and cefotaxime plus tobramycin resulted in a primary response in 46.9% and 45.3%, respectively. Overall response was achieved on study day 25 in 87.7% and 80%, respectively. No significant difference in toxicity was observed. Once-daily ceftriaxone plus tobramycin was not inferior to cefotaxime t.i.d. plus tobramycin qd in the empirical treatment of neutropenic fever.

Cefodizime, a new 2-aminothiazolyl cephalosporin: physicochemical properties, toxicology and structure-activity relationships.

Cefodizime is a 2-aminothiazolyl cephalosporin for parenteral use. Cefodizime has a bisubstituted thiothiazole moiety in position 3 of the cephem nucleus. The presence of this moiety does not alter the in-vitro antibacterial activity, or safety in animal studies, which are similar to those of cefotaxime, but results in an apparent long elimination half-life in rodents and dogs, and in novel immunological properties.

Biochemical aspects of the renal tolerance for cefpirome and other cephalosporins.

Effects of cefpirome (CFP, HR 810; CAS 84957-29-9) and other cephalosporins such as cefotaxime (CFX), cephaloridine (CPH) and ceftazidime (CFZ) on the renal biochemical processes such as peroxidation of lipids, organic cation transport or gluconeogenesis were investigated in vitro or after i.v.-administration of cephalosporins to 200 g male Wistar rats. In a series of in vitro experiments renal cortical slices were incubated for 60 min in a cephalosporin free medium or in a cephalosporin containing medium (1.25, 2.5, 5.0 and 10 mg/ml) at 37 degrees C under a 100% O2 atmosphere. Subsequently, peroxidation of lipids (LPO), measured as malondialdehyde (MDA) production, tissue accumulation of the organic cation tetraethylammonium (TEA) and gluconeogenesis were determined. In one series of in vivo experiments, 2 h after i.p.-administration of saline, CFP, CFX, CPH and CFZ (0, 500, 1000 and 2000 mg/kg), rats were killed and the amount of the reduced glutathione (GSH) in the renal cortex was measured. In another series of experiments, CFP, CFX, CPH and CFZ were administered (1200 mg/kg/d, i.v.) for 5 days. Subsequently, the effects of these cephalosporins on MDA production, cytosolic lactate dehydrogenase (LDH) activity, TEA accumulation and gluconeogenesis in the renal cortex were investigated. Results of the in vitro experiments show a significant concentration-dependent increase in MDA production only after incubation of renal cortical slices with CPH. CFZ and CPH caused a dose-dependent decrease in gluconeogenesis and except CFX, all other investigated cephalosporins induced a dose-dependent decrease in TEA accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

[General toxic and organotropic properties of cefotaxime in acute and chronic experiments]. / Izuchenie obshchetoksicheskikh i organotropnykh svoistv tsefotaksima v ostrykh i khronicheskikh éksperimentakh.

The action of cefotaxime on the functions of the liver and kidneys, the peripheral blood count, growth and development of young animals, blood circulation, respiration and the central nervous system was studied in acute and chronic experiments on mice and rats. Allergenic, immunomodulating, embryotoxic and teratogenic properties of the antibiotic were also studied. Cefotaxime was shown to be low toxic. After intravenous administration to mice, its LD50 amounted to 7000 (6295-7805) mg/kg. In the chronic experiments on rats with intramuscular and intravenous administration of the antibiotic in doses equivalent by the body surface to the course doses for humans there were no significant shifts in the function of the liver and kidneys, the count of the blood formed elements and the histologic pattern of the viscera. In the therapeutic doses the antibiotic had no action on hemopoiesis, respiration and the central nervous system. The allergenic properties of cefotaxime were slightly pronounced and similar to those of klaforan. The antibiotic had no action on the host immunity and showed no embryotoxic and teratogenic properties. After intravenous and intramuscular administration, cefotaxime had a slight irritating action on the tissues which was similar to that of klaforan.

Safety evaluation of meropenem in animals: studies on the kidney.

The effect of meropenem on animal kidneys has been assessed in rats (5 of each sex/group), rabbits (3 of each sex/group) and monkeys (3 of each sex/group) in comparative iv studies with ceftazidime, cefotaxime, cephaloridine and imipenem (without cilastatin). Diarrhoea occurred in rabbits and monkeys dosed with imipenem or meropenem. Emesis occurred only after the administration of imipenem to monkeys. After 14 days administration to rats evidence of nephrotoxicity was seen only in males dosed with cephaloridine (850 mg/kg); no changes were seen with ceftazidime, cefotaxime or meropenem (all at 1000 mg/kg). Four days after a single dose to rabbits renal tubular necrosis was seen in all animals receiving imipenem (150 mg/kg) and cephaloridine (250 mg/kg). Minimal histopathological changes to the kidneys were seen with cefotaxime, ceftazidime and meropenem (all at 400 mg/kg). After seven days' administration to cynomolgus monkeys imipenem (180 mg/kg) caused moderate to severe tubular necrosis. No tubular damage was seen with meropenem at 180 mg/kg or with cefotaxime or ceftazidime (both at 500 mg/kg). At 500 mg/kg meropenem caused mild tubular regeneration and/or fat accumulation in 3/6 animals, with mild tubular necrosis in one of these. The data from these three species indicate that meropenem has a low nephrotoxic potential in these animal models.

Seizure propensity with imipenem.

Five patients with seizures related to imipenem administration are described. The potential of imipenem therapy to cause seizure was further studied in a mouse model and compared with the potential for seizure with penicillin and cefotaxime therapy. Penicillin caused ataxia and seizure at a mean mouse serum level of 5800 microns/mL, cefotaxime at 3400 microns/mL, and imipenem at a much lower serum concentration of 1900 microns/mL. The potent activity of imipenem therapy against bacteria, allowing for a clinical dose of only 2 g/d, is unfortunately offset by its higher propensity to induce neurologic symptoms in humans and mice at much smaller doses than would therapy with penicillin G or the cephalosporins, such as cefotaxime.

[Acute toxicity study of cefodizime sodium].

Acute toxicity of cefodizime sodium (THR-221) was examined in mice of both sexes, rats of both sexes (including 5-day-old young), and male dogs. The LD50 values of THR-221 (mg/kg) were as follows: (1) mice: intravenous, 7200 for males and 5000 for females; intraperitoneal, 10500 for males and 11000 for females; subcutaneous, 17500 for males and 16500 for females; and oral, 28000 for males and 29000 for females. (2) rats (adult): intravenous, 7000 for males and 8200 for females; intraperitoneal, 9500 for males and 8800 for females; subcutaneous, 17000 for males and 15500 for females; oral, more than 20000 for both sexes; and intramuscular, more than 3200 for both sexes. (3) 5-day-old rats: subcutaneous, 5278 for males and 5314 for females. (4) male dogs: intravenous, more than 5000. Major changes in general conditions observed in mice and rats were decreased spontaneous activity, lying prone, respiratory changes, staggering gait, clonic or clonic-tonic convulsions, and cyanosis, and in the animals dosed orally, diarrhea or salivation was also noted. The changes in 5-day-old rats were respiratory changes, agony, loss of reflex to an external stimulus, and congestion at the injection site, and those in dogs were vomiting, dryness of the nose, and soft or mucous stools. Autopsies on the mice and rats which died revealed hemorrhage on the brain surface. In addition, the following were seen: intraperitoneal retention of fluid and dark red spots on the abdominal wall (i.p.), subcutaneous retention of fluid or jellylike material and hemorrhage at the injection site (s.c.), and retention of fluid and dark red spots on the mucosa in the digestive tract (mice p.o.). In 5-day-old rats which died, the subcutaneous tissue at the injection site showed hemorrhage macroscopically and inflammatory changes microscopically. Hematological and blood chemical tests performed in dogs showed an increase in white blood cells and changes suggesting anemia, increases in GOT, LDH and ALP activities, and slight changes in urea nitrogen and inorganic phosphorus. In one animal given a low dose of 2500 mg/kg, an increase in GPT activity was also seen. However, these changes were all transient. Microscopic findings in dogs were slight inflammatory changes in the subcutaneous tissue around the injection site.

[Six-month chronic intravenous toxicity study of cefodizime sodium in dogs].

The chronic intravenous toxicity of cefodizime sodium (THR-221) was studied in beagle dogs. Groups of 6 males and 6 females were treated with THR-221 at doses of 0 (saline), 200, 400, 800 and 1600 mg/kg/day for 6 months. The THR-221 related symptoms were vomiting, excessive drinking behavior and salivation. The paleness of the visible mucosa and discoloration of vascular color by funduscopy due to systemic anemia were observed in one animal each of 800 and 1600 mg/kg/day groups. Body weight was depressed transiently or continuously in a few animals of 400-1600 mg/kg/day groups. The hematological, serum chemical and urinalysis findings in a few animals of 400-1600 mg/kg/day groups revealed decreases in RBC count, PCV and hemoglobin, an increase in reticulocyte count, a decrease in WBC count, a decrease in platelet count, slight increase in TP, and albumin, a decrease in AlP, and an increase in urinary Na. Light microscopically, deposition of hemosiderin and increased extramedullary hematopoiesis in the liver and spleen, and deposition of fibroid substance in the white pulp of the spleen and diffuse fibrosis in the bone marrows were detected in a few animals of 800 and 1600 mg/kg/day groups. Electron microscopically, no significant toxic changes were observed. The maximum nontoxic doses of THR-221 are estimated as 200 mg/kg/day in male and less than 200 mg/kg/day in female.

[Fertility study of cefodizime sodium in mice--intravenous administration from pre-conceptional period through early period of gestation].

Cefodizime sodium (THR-221) was intravenously administered to mice of both sexes at dose levels of 300, 1000, 2000 and 3000 mg/kg/day, and its effects on the fertility of male and female mice and on their offspring were examined. Twenty-four males and 24 females were used per dose and as a control group. The males were dosed for 9 weeks prior to mating and through the mating period, and the females were treated from 2 weeks before mating through day 6 of gestation. Cesarean sections were performed on all pregnant mice in each group (16-23) on day 18 of gestation. Of the parental mice, 1, 2 and 1 males died at 0 (control), 2000 and 3000 mg/kg/day, respectively, and 5 females died at 3000 mg/kg/day. Inflammatory changes in the skin at the injection site were observed in the males at 3000 mg/kg/day. In the males, the kidney weight at 3000 mg/kg/day and the spleen weights at 2000 and 3000 mg/kg/day increased, and the thymus weight at 3000 mg/kg/day decreased, as compared with the control values. No differences appeared between any compound-treated group and the control group in the following maternal and fetal parameters: copulation rate, conception rate, and body and placental weights, crown-rump length and sex ratio of the live fetuses. External, visceral and skeletal examinations on the live fetuses revealed no compound-related abnormalities. From the present results, it is considered that the no-effect doses of THR-221 for the parental mice and the fetuses are 1000 and 3000 mg/kg/day, respectively.

[Teratological study of cefodizime sodium in mice--intravenous administration during period of organogenesis].

Cefodizime sodium (THR-221) was intravenously administered at dose levels of 300, 1000 and 3000 mg/kg/day to pregnant mice on days 6-15 of gestation covering the period of organogenesis, and its effects on the dams (F0) and the fetal and postnatal development and fertility of their offspring (F1) were examined. In each group including a control group, 21-24 of the 33-38 F0 dams were submitted to cesarean section on day 18 of gestation, and the remaining 12-14 animals were allowed to litter normally and nurse their offspring until day 21 of lactation. In the F0 dams, no compound effects were seen in general conditions, body weight, food consumption, numbers of corpora lutea and implantations or duration of gestation. The thymus weights of the cesarean sectioned dams at 1000 and 3000 mg/kg/day were less than the control values, but no difference appeared in weights of the other main organs between any treatment group and the control group. No gross visceral abnormalities were noted at any dose. In F1 near-term fetuses, the compound administration exerted no effects on their development at any dose, except the decreased body weight and crown-rump length noted at 3000 mg/kg/day. No difference occurred in incidence of fetuses with external, visceral or skeletal anomalies between any treatment group and the control group. Normally delivered F1 offspring in the treatment groups exhibited no changes from the controls in postnatal development, reflexes, 21-day survival index or behavioral test performance. Skeletal examination on F1 offspring which died during lactation and autopsies on the other animals at the end of study revealed no compound-related abnormalities. The fertility of the F1 mice was normal at all doses, and there were no compound effects on the F1 dams or their near-term fetuses or newborn pups. From the present results, it is considered that the no-effect doses of THR-221 for the maternal mice and their offspring are 300 and 1000 mg/kg/day, respectively.