Changes in disease characteristics of primary biliary cholangitis: An observational retrospective study from 1982 to 2016.

AIM: Disease characteristics of primary biliary cholangitis have changed recently. However, detailed studies on the subject have been limited. Therefore, we aimed to clarify disease characteristics of patients with recent primary biliary cholangitis using the cohort from Niigata University and 21 affiliated hospitals. METHODS: Overall, 508 patients were enrolled in this study from 1982 to 2016, divided into three cohorts according to their year of diagnosis: ≤1999, 2000-2009 and ≥2010. We compared differences in clinical characteristics, response to ursodeoxycholic acid and prognosis. RESULTS: The male-to-female ratio increased incrementally from 1:16.4 (≤1999) to 1:3.8 (≥2010) (P < 0.001). In women, the median age at diagnosis increased incrementally from 54.0 years (≤1999) to 60.5 years (≥2010) (P < 0.001) and serum albumin decreased gradually (P = 0.001), which might have affected the increase in the Fibrosis-4 Index and albumin-bilirubin score. The ursodeoxycholic acid response rate according to the Barcelona criteria increased incrementally from 26.7% (≤1999) to 78.4% (≥2010) (P < 0.010), and those according to other criteria (Paris-I, Rotterdam and Toronto) were approximately ≥80% in all cohorts. Ten-year survival rate in the ≤1999 and 2000-2009 cohorts were 98.6% and 95.6%, respectively. These earlier cohorts were also characterized by a higher rate of asymptomatic state and mild histology (83.5% [≤1999] and 84.7% [2000-2009], and 93.6% [≤1999] and 91.1% [2000-2009]). CONCLUSIONS: Patients with primary biliary cholangitis were characterized by older age at diagnosis and an increase in male to female ratio as well as higher response rates of ursodeoxycholic acid and longer survival, resulting from the early recognition of primary biliary cholangitis.

The Inhibitory Effect of Wood Creosote on the Movement of Anisakis Larvae: An Implication for the Treatment of Acute Anisakiasis.

INTRODUCTION: Anisakiasis is a common disease in countries such as Japan, where raw or undercooked marine fish are frequently consumed. The disease is caused by accidental ingestion of a live larva of Anisakis in raw or undercooked marine fish. In typical cases, it causes abrupt gastrointestinal symptoms, such as epigastric pain, nausea, and vomiting. According to a published report, the disease was alleviated by oral ingestion of an over-the-counter drug containing wood creosote. METHODS: We performed an in vitro experiment to elucidate whether wood creosote can inhibit the motor activity of Anisakis larvae, using infrared locomotion tracking and agarose gel penetration techniques. RESULTS: Our results clearly demonstrate that wood creosote inhibits the motor activity of Anisakis larvae. The concentration of wood creosote used in our experiment is similar to that found in stomach juice when a usual oral dose is taken of the medicine containing wood creosote. DISCUSSION/CONCLUSION: Our results suggest the potential usefulness of the medicine containing wood creosote in the treatment of acute Anisakis infection of the gastrointestinal tract.

Inhibition of Acetylcholinesterase by Wood Creosote and Simple Phenolic Compounds.

Anisakiasis is common in countries where raw or incompletely cooked marine fish are consumed. Currently, effective therapeutic methods to treat anisakiasis are unavailable. A recent study found that wood creosote inactivates the movement of Anisakis species. Essential oil of Origanum compactum containing carvacrol and thymol, which are similar to the constituents of wood creosote, was reported to inactivate Anisakis by inhibiting its acetylcholinesterase. We examined whether wood creosote can also inhibit acetylcholinesterase. We examined the effect of components of wood creosote using the same experimental method. A computer simulation experiment (molecular docking) was also performed. Here, we demonstrate that wood creosote inactivated acetylcholinesterase in a dose-dependent manner with an IC50 of 0.25 mg/mL. Components of wood creosote were also tested individually: 5-methylguaiacol, p-cresol, guaiacol, o-cresol, 2,4-dimethylphenol, m-cresol, phenol and 4-methylguaiacol inactivated the enzyme with an IC50 of 14.0, 5.6, 17.0, 6.3, 3.9, 10.0, 15.2 and 27.2 mM, respectively. The mechanism of acetylcholinesterase inactivation was analyzed using a computer-based molecular docking simulation, which employed a three-dimensional structure of acetylcholinesterase and above phenolic compounds as docking ligands. The simulation indicated that the phenolic compounds bind to the active site of the enzyme, thereby competitively blocking entry of the substrate acetylcholine. These findings suggest that the mechanism for the inactivation of Anisakis movement by wood creosote is due to inhibition of acetylcholinesterase needed for motor neuron activity.

Generation and Measurement of Chlorine Dioxide Gas at Extremely Low Concentrations in a Living Room: Implications for Preventing Airborne Microbial Infectious Diseases.

BACKGROUND/AIMS: Preventing respiratory diseases caused by airborne microbes in enclosed spaces is still not satisfactorily controlled. At extremely low concentrations (about 30 parts per billion), chlorine dioxide (ClO2) gas can inactivate airborne microbes and prevent respiratory disease. It has no toxic effect on animals at this level. However, controversies still remain regarding how to measure concentrations of ClO2 gas at such low levels. It is therefore necessary to prove that measured gas concentrations are accurate and reproducible. METHODS: ClO2 gas was released from a gas generator and its concentration was measured by a novel highly sensitive gas analyzer. We compared its data with those from ion chromatography. RESULTS: We demonstrate that the gas concentrations measured in a room using the gas analyzer are accurate and reproducible after comparing the results with those from ion chromatography. However, the temperature dependence of the gas analyzer was found. Therefore, data correction is required for each temperature at which gas concentration is measured. A theoretical analysis of the gas concentrations predicted by the rate of ClO2 gas released from the ClO2 generator was also performed. CONCLUSION: Our results advance progress toward using low concentration ClO2 gas to prevent airborne infectious diseases such as influenza.

Inactivation of Airborne Bacteria and Viruses Using Extremely Low Concentrations of Chlorine Dioxide Gas.

Infectious airborne microbes, including many pathological microbes that cause respiratory infections, are commonly found in medical facilities and constitute a serious threat to human health. Thus, an effective method for reducing the number of microbes floating in the air will aid in the minimization of the incidence of respiratory infectious diseases. Here, we demonstrate that chlorine dioxide (ClO2) gas at extremely low concentrations, which has no detrimental effects on human health, elicits a strong effect to inactivate bacteria and viruses and significantly reduces the number of viable airborne microbes in a hospital operating room. In one set of experiments, a suspension of Staphylococcus aureus, bacteriophage MS2, and bacteriophage ΦX174 were released into an exposure chamber. When ClO2 gas at 0.01 or 0.02 parts per million (ppm, volume/volume) was present in the chamber, the numbers of surviving microbes in the air were markedly reduced after 120 min. The reductions were markedly greater than the natural reductions of the microbes in the chamber. In another experiment, the numbers of viable airborne bacteria in the operating room of a hospital collected over a 24-hour period in the presence or absence of 0.03 ppm ClO2 gas were found to be 10.9 ± 6.7 and 66.8 ± 31.2 colony-forming units/m3 (n = 9, p < 0.001), respectively. Taken together, we conclude that ClO2 gas at extremely low concentrations (≤0.03 ppm) can reduce the number of viable microbes floating in the air in a room. These results strongly support the potential use of ClO2 gas at a non-toxic level to reduce infections caused by the inhalation of pathogenic microbes in nursing homes and medical facilities.

Inactivation of influenza virus haemagglutinin by chlorine dioxide: oxidation of the conserved tryptophan 153 residue in the receptor-binding site.

Airborne influenza virus infection of mice can be prevented by gaseous chlorine dioxide (ClO(2)). This study demonstrated that ClO(2) abolished the function of the haemagglutinin (HA) of influenza A virus (H1N1) in a concentration-, time- and temperature-dependent manner. The IC(50) during a 2 min reaction with ClO(2) at 25 °C was 13.7 µM, and the half-life time of HA with 100 µM ClO(2) at 25 °C was 19.5 s. Peptides generated from a tryptic digest of ClO(2)-treated virus were analysed by mass spectrometry. An HA fragment, (150)NLLWLTGK(157) was identified in which the tryptophan residue (W153) was 32 mass units greater than expected. The W153 residue of this peptide, which is derived from the central region of the receptor-binding site of HA, is highly conserved. It was shown that W153 was oxidized to N-formylkynurenine in ClO(2)-treated virus. It was concluded that the inactivation of influenza virus by ClO(2) is caused by oxidation of W153 in HA, thereby abolishing its receptor-binding ability.

Cumulative risk of developing a new symptom in patients with primary biliary cholangitis and its impact on prognosis.

Background and Aim: Symptoms of primary biliary cholangitis (PBC) frequently impair one's quality of life (QOL). Nonetheless, with improved treatment, the prognosis of PBC also improves. QOL plays an important role in patients with PBC. In this study, we aimed to reevaluate the transition of new symptom development in PBC and its predictive factors. Methods: This retrospective multicenter study enrolled 382 patients with PBC for symptom analysis. The impact of a newly developed symptom on PBC prognosis was investigated by Kaplan-Meier analysis with propensity score matching and logistic progression analysis. Results: The cumulative risk of developing a new symptom after 10 and 20 years of follow-up was 7.6 and 28.2%, and specifically that of pruritus, which was the most common symptom, was 6.7 and 23.3%, respectively. In Cox hazard risk analysis, serum Alb level (hazard ratio [HR], 1.097; 95% confidence interval [CI], 1.033-1.165; P = 0.002), the serum D-Bil level (HR, 6.262; 95% CI, 2.522-15.553, P < 0.001), and Paris II criteria (HR, 0.435; 95% CI, 0.183-1.036; P = 0.037) were significant independent predictors of a new symptom. Kaplan-Meier analysis showed that the overall survival and liver-related death were not significant between patients with and without a new symptom. Conclusion: The cumulative risk of new symptom development is roughly 30% 20 years after diagnosis and could be predicted by factors including serum albumin levels, serum D-Bil level, and Paris II criteria.

[Comparison of antibody responses to hepatitis B surface antigen among four recipient groups of hepatitis B vaccines that have been approved in Japan: evaluation using passive hemagglutination assay and chemiluminescent immunoassay].

In hepatitis B virus (HBV) infection-preventing programs, serum or plasma levels of antibody to hepatitis B surface antigen (anti-HBs) are important to determine whether individuals are protective or not. We compared anti-HBs responses using passive hemagglutination assay (Mycell) and chemiluminescent immunoassay (Architect) among four recipient groups of HB vaccines, Meinyu, HBY, Bimmugen and Heptavax II, that have been approved in Japan. Overall, in a total of 1875 vaccinees Mycell results showed recipient groups of Meinyu and HBY acquired higher anti-HBs levels than those of Bimmugen and Heptavax II. Comparison of anti-HBs responses by both Mycell and Architect in recipient groups of Meinyu (n=150), HBY (n=218), Bimmugen (n=260), and Heptavax II (n=47) demonstrated the order of vaccinees' responses, such as geometric mean titers, ratios of acquiring high antibody levels (Mycell titers over 1024, Architect measurements over 1000 mIU/mL), and ratios of having unsuccessful antibody responses (Mycell titers under 8, Architect measurements under 10 mIU/mL), were somewhat different between the two assays. Comparison of Architect measurements at given Mycell titers revealed Bimmugen-recipients showed significantly lower values than HBY- or Heptavax II-recipients. Around critical protective levels, 5 of 22 Bimmugen-recipients with Mycell titers 16 or 32 showed Architect measurements under 10 mIU/mL, while 8 of 11 Heptavax II-recipients with Mycell titers below 8 demonstrated Architect measurements over 10 mIU/mL. Thus, discrepancies in anti-HBs evaluation between Mycell and Architect seemed to partly depend on administered vaccines. These results indicate anti-HBs concentration should be evaluated carefully so that we could completely prevent HBV infection.

Bleeding from a Small-Intestinal Ulcer Associated with Chronic Hepatitis C.

BACKGROUND Hepatitis C virus infection is probably the most common chronic viral infection and affects an estimated 180 million people worldwide. Extrahepatic manifestations are well recognized among patients with chronic HCV infection. CASE REPORT We report a case of melena occurring in a 69-year-old Japanese man who had been diagnosed with CHC and who was treated with antiviral therapy. CONCLUSIONS Finally, he was diagnosed with multiple small intestine ulcers in a short time. We herein report the case of HCV with rapidly developing small intestine ulcers.

Elongation of palindromic repetitive DNA by DNA polymerase from hyperthermophilic archaea: a mechanism of DNA elongation and diversification.

DNA polymerase from hyperthermophilic bacteria can elongate tandem repetitive oligoDNA with a complete or incomplete palindromic sequence under isothermal conditions by "hairpin elongation". However, the product of the reaction has not yet been sufficiently characterized. Here, I demonstrate that when palindromic repetitive oligoDNA, e.g., (5'AGATATCT3')(6), was added as a "seed" to the DNA synthesis reaction catalyzed by DNA polymerase from the archaea Thermococcus litoralis (Vent polymerase) at 74 degrees C, the product was (5'AGATATCT3')(n). The product itself was palindromic and repetitive, and its motif (unit) sequence was exactly the same as that of the seed oligoDNA. On the other hand, when a pseudopalindrome, which contains a palindrome-breaking nucleotide (underlined), was present in seed oligoDNA, e.g., (5'GATTC3')(6), the product was (5'GATATC3')(n), which had a different motif sequence from that of the seed oligoDNA. When a pseudopalindrome (5'AGATATCA3')(6) was added to the reaction, the products were 5'TATCA . (AGATATCA)(3) . AGATATCT . (TGATATCT)(5) . TGATA3', etc. When 5'AGATATCA . (AGATATCT3')(5) was added, products were 5'TATCT . (AGATATCT)(2).TGATATCT . AGATATCT . AGATATCA . AGATATCT . AGA3', etc., demonstrating the generation of many "mutations" in the product DNA. I conclude that a tandem repetitive sequence is faithfully elongated (amplified) by hyperthermophilic DNA polymerase if it is completely palindromic, but is elongated with many errors if it is incompletely palindromic (pseudopalindromic) or mixed with a pseudopalindrome. The results suggest a protein-catalyzed elongation/diversification mechanism of short repetitive DNAs on the early earth.

[A case of advanced gastric cancer with obstructive jaundice due to multiple liver metastasis successfully treated with the following combination therapy of CPT-11 and cisplatin after combination therapy of paclitaxel and TS-1].

A 60-year-old man, who had been admitted to another hospital with complaints of constipation, abdominal fullness and appetite loss, was referred to our hospital for further examination and therapy. The patient was diagnosed as advanced gastric cancer (type-3) with multiple liver metastasis and obstructive jaundice. He was treated with combination therapy of paclitaxel and TS-1 (60 mg/m(2)/day of paclitaxel was iv administered on day 1 and 8, and TS-1 of 80 mg/m(2)/day was orally administered for 2 weeks followed by one drug-free week), and showed a remarkable response. However, because of ascites, elevated serum CEA level and resistance in the liver metastasis and gastric region, we attempted two courses of combination therapy with high-dose CPT-11 and cisplatin (70 mg/m(2)/day of CPT-11 was administered iv on day 1 and 15, and 80 mg/m(2)/day of cisplatin on day 1 followed by two drug-free weeks) which showed a remarkable response. Two courses of combination therapy with low-dose CPT-11 and cisplatin (60 mg/m(2)/day of CPT-11 and 30 mg/m(2)/day of cisplatin were administered iv on day 1 and 15 followed by two drug-free weeks) on an outpatient basis. However, the patient showed resistance to the latter combination therapy, increased ascites due to suspicious peritonitis carcinomatosa and obvious re-growth of the metastatic tumors in the liver. He died on May 23, 2006, about ten months after initial diagnosis. We reported a case of successful treatment of combination chemotherapy for advanced gastric cancer with obstructive jaundice due to progressive multiple metastatic tumors in the liver and obtained comparative long-term survival maintaining high quality of life.

[Requirement of standardizing anti-HBs assay methods in Japan for HBV infection-preventing strategy--discrepancy of anti-HBs measurements among three different kits widely used in Japan].

The strategy to eliminate hepatitis B virus (HBV) infection by administrating an HB vaccine is changing worldwide; however, this is not the case in Japan. An important concern about the HBV infection-preventing strategy in Japan may be that the assay methods for the antibody to hepatitis B surface antigen (anti-HBs) are not standardized. The minimum protective anti-HBs titer against HBV infection has been established as 10 mIU/ml by World Health Organization (WHO) -standardized assay methods worldwide, but that is still determined as a "positive" test result by the passive hemagglutination (PHA) method in Japan. We compared anti-HBs measurements in given samples among PHA(Mycell II, Institute of Immunology), chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse, Fujirebio), and chemiluminescent immunoassay (CLIA) (Architect, Abbott), all of which are currently in wide use in Japan. First, anti-HBs measurements in serum from individuals who received a yeast-derived recombinant HB vaccine composed of the major surface protein of either subtype adr or subtype ayw were compared. The results clearly showed that in subtype adr-vaccinees CLIA underestimated the anti-HBs amount compared with CLEIA and PHA, but in ayw-vaccinees, the discordance in the measurements among the three kits was not prominent. Second, anti-HBs measurements in standard or calibration solutions of each assay kit were compared. Surprisingly, CLEIA showed higher measurements in all three kit-associated standard or calibration solutions than CLIA. Thus, the anti-HBs titer of 10 mIU/ml is difficult to introduce in Japan as the minimum protective level against HBV infection. Efforts to standardize anti-HBs assay methods are expected to share international evidence about the HBV infection-preventing strategy.

Measurements in international units of antibody to hepatitis B surface antigen(anti-HBs) after immunization with a yeast-derived, subtype adr hepatitis B vaccine are considerably different between chemiluminescent immunoassay (CLIA) and chemiluminescent enzyme immunoassay (CLEIA).

The worldwide consensus of the minimum protective anti-HBs level against HBV infection is 10 mIU/mL on assays standardized by the World Health Organization (WHO) reference preparations. To investigate whether this value could be applied to recipients of yeast-derived recombinant HB vaccine containing the major surface protein of subtype adr (Bimmugen, Astellas Pharmaceutical, Tokyo), we compared anti-HBs measurements between chemiluminescent immunoassay (CLIA) (Architect Ausab, Abbott Japan, Tokyo) and chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse Forte, Fujirebio, Tokyo) in given serum samples obtained from the vaccinees. The vaccine and the two assay methods are currently in a wide use in Japan. The study included 300 medical students who completed a standard vaccination course (0, 1 and 6 months). Serum samples obtained 1 month or 13 months after completing the vaccination were simultaneously tested for anti-HBs by CLIA and CLEIA. In 147 samples with quantifiable values on both CLIA and CLEIA (10 to 1000 mIU/mL) the geometric mean titer on CLEIA (225.0 mIU/mL) was significantly higher than that on CLIA (94.5 mIU/mL) (p < 0.0001). Of 26 subjects with CLIA measurements below 10 mIU/mL, 15 samples (57.7%) showed CLEIA measurements more than 10 mIU/mL. Thus, in the subtype adr-vaccinees CLEIA demonstrated considerably high serum anti-HBs measurements compared to CLIA and discordance in determining critical anti-HBs level of 10 mIU/mL was observed in more than half the samples. This suggests that the minimum HBV-protective anti HBs titer of 10 mIU/mL is difficult to be introduced to Japan where subtype adr-HB vaccines or -HBV infection are prevalent, unless characteristics of assay methods are carefully evaluated.

[A case of exacerbation of ulcerative colitis induced by combination therapy with PEG-interferon alpha-2b and ribavirin for chronic hepatitis C].

A 55-year-old man with chronic hepatitis C had diarrhea with bloody stool in July, 2003 and ulcerative colitis was suspected. However, he quickly improved. He was treated with percutaneous radiofrequency ablation therapy for adenomatous hyperplasia in S5 of the liver in December, 2004. After the ablation therapy, he was treated with combination therapy of PEG-interferon alpha-2b and ribavirin for chronic hepatitis C. Because exacerbation of ulcerative colitis appeared 10 weeks after beginning of the treatment for hepatitis C, the combination therapy of PEG-interferon and ribavirin was discontinued. He was treated with mesalazine and steroid therapy for ulcerative colitis, and improved. We report the first case in Japan of the exacerbation of ulcerative colitis induced by the combination therapy of PEG-interferon and ribavirin for chronic hepatitis C.

Localized Hepatic Tuberculosis with Imaging Changes Caused by the Progression of Tuberculosis.

Localized hepatic tuberculosis (LHTB) is difficult to diagnose preoperatively, and most cases of LHTB are diagnosed based on pathological findings. A relationship between imaging features and the pathological stage of hepatic tuberculosis (TB) has recently been reported, which could aid in the diagnosis of hepatic TB. We herein present a case study of a patient with LHTB diagnosed postoperatively who demonstrated imaging changes due to the progression of TB. An awareness of the presence of LHTB might have permitted a preoperative diagnosis. This is the first report of an LHTB patient who exhibited imaging changes during the course of the disease.

The long-term clinical outcome of 1-year treatment of chronic hepatitis B with lamivudine-5 years observation.

The biochemical and virological outcomes of 19 patients with chronic hepatitis B who had been treated with 100 mg per day of lamivudine (LMV) for 1 year from 1995 to 1996 were evaluated. Fourteen patients were followed for 4.5-5 years since the end of the treatment without any further active antiviral treatment. During the treatment, DNA levels of hepatitis B virus (HBV) were under the detection limit of a hybridization assay in all the 19 patients. However, YMDD mutants appeared in 5 (26%) patients during the course of treatment and were accompanied in all five by the elevation of serum alanine aminotransferase (ALT). Mutated HBV DNA was not detected at 1 year after the end of treatment in any of the 5 patients. Of the patients who were followed for 4.5-5 years, the rate of seroconversion to anti-HBe and negativity for HBV DNA fluctuated during the course. Four of 11 patients who initially had been positive for hepatitis B virus e antigen (HBeAg) became positive for anti-HBe and all of them remained positive for HBV DNA by a transcription-mediated amplification test at the end of the follow-up. Thus, a 1-year treatment with LMV for chronic hepatitis B resulted in the relapse of HBV viraemia in most of the patients who had been positive for HBeAg, although the clinical course ameliorated in some patients. In addition, HBV DNA remained positive and ALT values were elevated at the end of the follow-up in the three patients who had been treated with interferon, with or without LMV, during the follow-up.

Development of peptide nucleic acid mediated polymerase chain reaction clamping (PMPC)--direct sequencing method for detecting lamivudine-resistant hepatitis B virus (HBV) variants with high sensitivity and specificity.

Reduced sensitivity of HBV to lamivudine, which causes a viral breakthrough during treatment, is attributed to mutations within the tyrosine-methionine-aspartate(YMDD) locus in the reverse transcriptase(rt) domain of HBV polymerase, mainly a methionine(rtM204) substitution. The sensitive detection of such mutations before or early in treatment could assist in optimizing antiviral treatment. For this purpose, we developed peptide nucleic acid(PNA) mediated polymerase chain reaction(PCR) clamping(PMPC) with a PNA probe targeting the YMDD locus. We first tested this method for its sensitivity and specificity in detecting a mutant on HBV DNA standards consisting of serial copy number ratios of a known lamivudine-resistant, mutant clone with rtM204I(ATT) to a wild-type clone with rtM204(ATG). The sensitivity was 0.1 to 0.01% in the coexistence of wild-type clones and the specificity was guaranteed by direct sequencing of the products. We next applied this method to HBV DNA specimens extracted from serum from 4 chronic hepatitis B patients treated with lamivudine. Two of these exhibited a break-through of the HBV mutant with rtM204I(ATT), while the other 2 did not. Before treatment, all 4 patients showed HBV with rtM204I encoded by ATA. During treatment, HBV with the rtM204I(ATT) emerged in the 2 breakthrough patients more than 3 months before the breakthrough, whereas this and other known lamivudine-resistant viruses did not appear in the 2 non-breakthrough patients. Thus, our PMPC-direct sequencing method is highly sensitive and reliable for the early identification of lamivudine-resistant HBV that causes a viral breakthrough.

Six-month low level chlorine dioxide gas inhalation toxicity study with two-week recovery period in rats.

BACKGROUND: Chlorine dioxide (CD) gas has a potent antimicrobial activity at extremely low concentration and may serve as a new tool for infection control occupationally as well as publicly. However, it remains unknown whether the chronic exposure of CD gas concentration effective against microbes is safe. Therefore, long-term, low concentration CD gas inhalation toxicity was studied in rats as a six-month continuous whole-body exposure followed by a two-week recovery period, so as to prove that the CD gas exposed up to 0.1 ppm (volume ratio) is judged as safe on the basis of a battery of toxicological examinations. METHODS: CD gas at 0.05 ppm or 0.1 ppm for 24 hours/day and 7 days/week was exposed to rats for 6 months under an unrestrained condition with free access to chow and water in a chamber so as to simulate the ordinary lifestyle in human. The control animals were exposed to air only. During the study period, the body weight as well as the food and water consumptions were recorded. After the 6-month exposure and the 2-week recovery period, animals were sacrificed and a battery of toxicological examinations, including biochemistry, hematology, necropsy, organ weights and histopathology, were performed. RESULTS: Well regulated levels of CD gas were exposed throughout the chamber over the entire study period. No CD gas-related toxicity sign was observed during the whole study period. No significant difference was observed in body weight gain, food and water consumptions, and relative organ weight. In biochemistry and hematology examinations, changes did not appear to be related to CD gas toxicity. In necropsy and histopathology, no CD gas-related toxicity was observed even in expected target respiratory organs. CONCLUSIONS: CD gas up to 0.1 ppm, exceeding the level effective against microbes, exposed to whole body in rats continuously for six months was not toxic, under a condition simulating the conventional lifestyle in human.