Development of an in silico evaluation system that quantitatively predicts skin sensitization using OECD Guideline No. 497 ITSv2 defined approach for skin sensitization classification.

The Integrated Testing Strategy version 2 (ITSv2) Defined Approach, which is a reliable skin sensitization hazard and multi-step risk assessment method, does not support quantitative risk assessment such as local lymph node assay EC3 values. In this study, we developed a high-performance in silico evaluation system that quantitatively predicts the EC3 values of chemical substances by combining the ITSv2 Defined Approach for hazard identification (ITSv2 HI) with machine learning models. This system uses in chemico/in vitro test data, molecular descriptors, and distance information based on read-across concepts as explanatory variables. The system achieves an R2 value of 0.617 on external-validation data. Substances misclassified in ITSv2 HI are considered to have properties that do not match the correspondence between tests expressing the adverse outcome pathway assumed in the ITSv2 Defined Approach and skin sensitization. Therefore, ITSv2 HI is assumed to be correct within the applicability domains of this system. When using only substances within the applicability domains to reconstruct CatBoost models, the R2 value reached 0.824 on the external-validation data, representing an improvement in system performance. The results demonstrate the utility of explanatory variables that reflect the read-across concept and the advantages of integrating multiple prediction methods.

Development of an oral mucosal irritation test using a three-dimensional human buccal oral mucosal model.

The oral mucosa can become irritated by oral care products and lip cosmetics. Therefore, it is important to determine the irritation potential of their ingredients and products during safety evaluations. We developed a method for oral mucosal irritation test using EpiOral, which is a three-dimensional cultured model. Exposure of sodium lauryl sulphate (SLS) to EpiOral showed a dose-dependent decrease in cell viability. Under 120 min exposure conditions, SLS irritation was detected when 60% cell viability was set as a criterion. Evaluation of the irritancy of SLS and four other raw materials used in oral products at three laboratories under the above conditions confirmed good transferability of the test. Focused on the similarity of the oral and eye mucous, 32 chemicals categorised by the UN-GHS eye-irritation classification were evaluated to ensure the reliability of our criteria at these laboratories. The concordance rate between the UN-GHS classification and our test results was 100% for irritants and 60% for non-irritants. The good intra-laboratory reproducibility of our test was confirmed from the evaluation results of negative and positive controls, and the good inter-laboratory reproducibility was confirmed from the results of 32 chemicals. These findings showed that oral mucosal irritation can be evaluated using EpiOral.

Evaluation of Poly Lactic-co-Glycolic Acid-Coated ß-Tricalcium Phosphate Bone Substitute as a Graft Material for Ridge Preservation after Tooth Extraction in Dog Mandible: A Comparative Study with Conventional ß-Tricalcium Phosphate Granules.

This study aims to evaluate the safety and efficacy of a poly lactic-co-glycolic acid (PLGA)-coated ß-tricalcium phosphate (ß-TCP) with N-methyl-2-pyrrolidone (NMP) liquid activator (PLGA/ß-TCP) on alveolar ridge preservation after tooth extraction in dog mandible. Thirty-two extraction sites were prepared in eight dog mandibles. A distal root of the mandibular premolar was extracted and randomly grafted with one of the following bone substitutes: (1) PLGA/ß-TCP, (2) ß-TCP, or (3) left empty as a control, and wounds were closed with keratinized mucosa graft. Post-operative wound healing was observed and scored to evaluate safety. After 12 and 24 weeks, the bone regeneration was evaluated with micro-computed tomography (CT) images and histomorphometric analyses. Gingival epithelization progressed over time without complication or infection. Micro-CT images and histological observation revealed that both PLGA/ß-TCP and ß-TCP granules supported sufficient new bone formation. Although bone formation and substrate resorption were delayed slightly with the PLGA and the NMP-containing plasticizer as compared to those treated with conventional ß-TCP, it can be concluded that the PLGA and the NMP-containing plasticizer that facilitated the in situ hardening properties of the material had no negative influence on the biocompatibility of the material.

Confirmation of diabetes-related quantitative trait loci derived from SM/J and A/J mice by using congenic strains fed a high-carbohydrate or high-fat diet.

The interaction between causative genes and diet is known to influence the onset of obesity and diabetes in humans, although it has remained difficult to identify diabetogenic gene(s) because humans are genetically and environmentally heterogeneous. Mouse SMXA recombinant inbred (RI) strains are established from parental inbred strains (SM/J and A/J) and have been shown to be beneficial tools for analyzing polygenic traits. We previously mapped a significant quantitative trait locus (QTL, T2dm1sa) on Chromosome (Chr.) 10 and suggestive QTLs on Chr. 2, 6, and 18 for diabetes-related traits by using SMXA RI strains fed a high-carbohydrate diet. As a first step in identifying the responsible gene among QTLs for glucose tolerance mapped on Chr. 10 and 18, we established new strains of A.SM-T2dm1sa and SM.A-D18Mit19-D18Mit7 congenic mice. Each congenic strain bears the diabetogenic allele of an introgressed chromosomal region on a genetic background strain carrying the non-diabetogenic allele. The diabetogenic effect of T2dm1sa mapped on Chr. 10 was not supported by studies of A.SM-T2dm1sa congenic mice when the mice were fed a high-carbohydrate or high-fat diet. SM.A-D18Mit19-D18Mit7 congenic mice showed impaired glucose tolerance not only when they were fed a high-carbohydrate diet, but also when they were fed a high-fat diet. Thus, it appears that gene(s) affecting diabetes-related traits under either dietary condition may be present on Chr. 18.

Carbon tetrachloride-induced hepatotoxicity in pregnant and lactating rats.

Carbon tetrachloride (CCl4) is well known to induce hepatotoxicity after being metabolized to trichloromethyl free radical ((.)CCl3) by CYP2E1. In the present study, the hepatotoxicity induced by a single oral dose (2,000 mg/kg) of CCl4 was compared between pregnant (gestation days (GD) 13 and 19) or postpartum (postpartum days (PPD) 1, 13 and 27) and non-pregnant rats. Hepatotoxicity in CCl4-treated pregnant rats evaluated by blood chemistry (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities) and histopathological finding (area of damaged hepatocytes) was minimal on GD19, being weaker than that in non-pregnant rats. CYP2E1 expression in non-treated pregnant rats decreased as pregnancy progressed and reached minimum level on GD19. Thus, the degree of CCl4-induced hepatotoxicity roughly corresponded to CYP2E1 levels during pregnancy. After delivery, hepatotoxicity in CCl4-treated lactating rats was maximal on PPD13, being stronger than that in non-pregnant rats, and then it decreased slightly on PPD27. The CYP2E1 level in the non-treated lactating rats tended to increase but remained at lower levels until PPD13 compared with that in non-pregnant rats. Thus, the degree of CCl4-induced hepatotoxicity did not correspond to CYP2E1 levels during lactation. This suggests that during lactation, there may be certain factors other than CYP2E1 expression responsible for the degree of CCl4-induced hepatotoxicity.

Blood coagulation-related parameter changes in Sprague-Dawley (SD) rats treated with phenobarbital (PB) and PB plus vitamin K.

Effects of dose and duration of phenobarbital (PB) administration and those of co-administration of PB and vitamin K on blood coagulation-related parameters were examined in specific pathogen-free (SPF) rats of Sprague-Dawley strain kept on an ordinary diet. In Experiment 1, oral administration of PB (0, 25, 50, 100 or 150 mg/kg/day) for 2 weeks induced increases in hepatic cytochrome P450 content and CYP2B expression, prolongation of coagulation time (activated partial thromboplastin time (APTT) and Thrombotest (TBT)) and an increase in anti-thrombin III (AT III) concentration in a dose-dependent manner. In Experiment 2, PB administration (100 mg/kg/day) for up to 14 days produced time-dependent increases in hepatic cytochrome P450 content and CYP2B (CYP2B1 and CYP2B2) expression. APTT was prolonged from day 1 and AT III concentration was increased from day 2, whereas the coagulation time (TBT) was prolonged from day 7. In Experiment 3, APTT prolonged by PB (100 mg/kg/day) was shortened after vitamin K(2) (30 mg/kg/day) co-administration, although AT III concentration was still increased. This suggests that not AT III but PB-induced vitamin K deficiency may play an important role in PB-induced prolongation of coagulation time in SPF rats kept on an ordinary diet.