In vivo mutagenicity assessment of orally treated tert-butyl hydroperoxide in the liver and glandular stomach of MutaMouse.

BACKGROUND: tert-Butyl hydroperoxide (TBHP; CAS 75-91-2), a hydroperoxide, is mainly used as a polymerization initiator to produce polyethylene, polyvinyl chloride, and unsaturated polyester. It is a high-production chemical, widely used in industrial countries, including Japan. TBHP is also used as an additive for the manufacturing of food utensils, containers, and packaging (UCP). Therefore, there could be consumer exposure through oral intake of TBHP eluted from UCPs. TBHP was investigated in various in vitro and in vivo genotoxicity assays. In Ames tests, some positive results were reported with and/or without metabolic activation. As for the mouse lymphoma assay, the positive result was reported, regardless of the presence or absence of metabolic activation enzymes. The results of some chromosomal aberrations test and comet assay in vitro also demonstrated the genotoxic positive results. On the other hand, in in vivo tests, there are negative results in the bone marrow micronucleus test of TBHP-administered mice by single intravenous injection and the bone marrow chromosomal aberration test using rats exposed to TBHP for 5 days by inhalation. Also, about dominant lethal tests, the genotoxic positive results appeared. In contrast, there is little information about in vivo mutagenicity and no information about carcinogenicity by oral exposure. RESULTS: We conducted in vivo gene mutation assay using MutaMice according to the OECD Guidelines for the Testing of Chemicals No. 488 to investigate in vivo mutagenicity of TBHP through oral exposure. After repeated dosing for 28 days, there were no significant differences in the mutant frequencies (MFs) of the liver and glandular stomach up to 300 mg/kg/day (close to the maximum tolerable dose (MTD)). The positive and negative controls produced the expected responses. CONCLUSIONS: These findings show that orally administrated TBHP is not mutagenic in the mouse liver and glandular stomach under these experimental conditions.

Derivation of subacute guidance values for chemical contaminants of drinking water quality standard in Japan.

The concentration of chemicals in drinking water may transiently and accidently exceed the Drinking Water Quality Standard (DWQS). If the level of a contaminant is not expected to cause adverse effects for a limited period of exposure, immediate suspension of the water supply may not be necessary. Assessments should be conducted using subacute guidance values (SGVs). In this study, we assessed 26 chemicals for the DWQS to establish the SGVs. Principally, a key study was selected from subacute studies to derive a Subacute Reference Dose (saRfD). The SGV was calculated from the saRfD for adults (drinking water intakes: 40 mL/kg/day) and children (drinking water intakes: 150 mL/kg/day). No allocation factor was applied to derive the SGV. We established the SGV for 20 chemicals, which were 2-38 times higher than the corresponding DWQS. However, SGVs for six chemicals were the same as the corresponding DWQS. Therefore, immediate action will be required for these six accidental contaminants. Our established SGVs are useful for assessing accidental contamination.

In vivo mutagenicity assessment of styrene in MutaMouse liver and lung.

BACKGROUND: Styrene (CAS 100-42-5) is widely used as polystyrene and acrylonitrile-butadiene-styrene resin such as plastic, rubber, and paint. One of the primary uses of styrene is food utensils and containers, but a small amount of styrene transferred into food can be ingested by eating. Styrene is metabolized into styrene 7,8-oxide (SO). SO is mutagenic in bacteria and mouse lymphoma assays. It is clastogenic in cultured mammalian cells. However, styrene and SO are not clastogenic/aneugenic in rodents, and no rodent in vivo gene mutation studies were identified. METHODS: To investigate the mutagenicity of orally administered styrene, we used the transgenic rodent gene mutation assay to perform an in vivo mutagenicity test (OECD TG488). The transgenic MutaMouse was given styrene orally at doses of 0 (corn oil; negative control), 75, 150, and 300 mg/kg/day for 28 days, and mutant frequencies (MFs) were determined using the lacZ assay in the liver and lung (five male mice/group). RESULTS: There were no significant differences in the MFs of the liver and lung up to 300 mg/kg/day (close to maximum tolerable dose (MTD)), when one animal with extremely high MFs that were attributed to an incidental clonal mutation was omitted. Positive and negative controls produced the expected results. CONCLUSIONS: These findings show that styrene is not mutagenic in the liver and lung of MutaMouse under this experimental condition.

[Application of the Benchmark Dose Method to the Incidence Data for Various Pathological Findings and Its Validation Analysis].

Benchmark dose (BMD) method have been used in the toxicological assessment of chemical substances so that the point of departure can be derived, as an alternative to the use of no observable adverse effect level (NOAEL), and the method is often applied to the incidence data of histopathological findings in the toxicity studies. In the present study, the BMD method was applied to various patterns of incidence data derived from some toxicity studies as case studies, and the validity of each application was discussed. Five independent applications including toxicity studies of madder color or semicarbazide hydrochloride were prepared and model averaging over the three models with the lowest three AIC (Akaike information criteria) values (MA-3), a recently proposed model averaging method, was employed. The series of case studies indicated, for the better application of the BMD method to histopathological findings, the following points:(i) If there are incidence data with severity grading of pathologically significant lesions, we must discuss whether the BMD method should be applied to the total incidence data or the incidence data above certain grade with or without data aggregation.(ii) If a lesion of interest had higher toxicological significance rather than the secondary lesions with higher severity, the BMD method should be applied to the incidence data of the lesion of interest.(iii) If it is highly necessary to apply the BMD method to obtained incidence data without toxicological and statistical validity, toxicological pathologists are advised to review individual datasets of histopathology and associated data, and provide new incidence data of comprehensive findings (diagnostic name) such as hepatocellular injury or nephropathy, if possible. In all cases, toxicological significance and mechanism of a lesion of interest need to be considered in light of the dose-dependence. In view of both toxicology and statistics, sufficient discussions must be made on the validity of applying BMD method and its estimate.

Initial hazard assessment of ethyl(dimethyl)(tetradecyl)ammonium ethyl sulfate: Genotoxicity tests and combined repeated-dose and reproductive/developmental toxicity screening in rats.

Ethyl(dimethyl)(tetradecyl)ammonium ethyl sulfate, used in laundry detergents, shampoos, and body soaps, is classified by the Japanese Chemical Substances Control Law as a priority assessment chemical substance for environmental effects. However, its toxicity data for human health are insufficient. This study evaluated this chemical under the Safety Examination of Existing Chemicals and Safety Programmes of the Ministry of Health, Labour and Welfare (MHLW). The MHLW conducted bacterial reverse mutation (Ames test), in vitro chromosomal aberration, and combined repeated-dose and reproductive/developmental toxicity screening tests. We performed a screening assessment of ethyl(dimethyl)(tetradecyl)ammonium ethyl sulfate for human health. The chemical showed a negative reaction in the Ames test and a positive reaction in the in vitro chromosomal aberration test with metabolic activation in rats. The combined repeated-dose and reproductive/developmental toxicity screening test showed significantly decreased food consumption at 50 mg/kg body weight/day, but no reproductive and developmental toxicity was observed. The no-observed-effect level of 15 mg/kg/day was obtained as a screening value. Therefore, this chemical was classified as hazard class 3, with a derived-no-effect level of 0.025 mg/kg/day. The results of this study will be useful for risk assessment of groups of structurally similar alkyl quaternary ammonium surfactants.

In silico prediction of chromosome damage: comparison of three (Q)SAR models.

Two major endpoints for genotoxicity tests are gene mutation and chromosome damage (CD), which includes clastogenicity and aneugenicity detected by chromosomal aberration (CA) test or micronucleus (MN) test. Many in silico prediction systems for bacterial mutagenicity (i.e. Ames test results) have been developed and marketed. They show good performance for prediction of Ames mutagenicity. On the other hand, it seems that in silico prediction of CD does not progress as much as Ames prediction. Reasons for this include different mechanisms and detection methods, many false positives and conflicting test results. However, some (quantitative) structure-activity relationship ((Q)SAR) models (e.g. Derek Nexus [Derek], ADMEWorks [AWorks] and CASE Ultra [MCase]) can predict CA test results. Therefore, performances of the three (Q)SAR models were compared using the expanded Carcinogenicity Genotoxicity eXperience (CGX) dataset for understanding current situations and future development. The constructed dataset contained 440 chemicals (325 carcinogens and 115 non-carcinogens). Sensitivity, specificity, accuracy or applicability of each model were 56.0, 86.9, 68.6 or 89.1% in Derek, 67.7, 61.5, 65.2 or 99.3% in AWorks, and 91.0, 64.9, 80.5 or 97.7% in MCase, respectively. The performances (sensitivity and accuracy) of MCase were higher than those of Derek or AWorks. Analysis of predictivity of (Q)SAR models of certain chemical classes revealed no remarkable differences among the models. The tendency of positive prediction by (Q)SAR models was observed in alkylating agents, aromatic amines or amides, aromatic nitro compounds, epoxides, halides and N-nitro or N-nitroso compounds. In an additional investigation, high sensitivity but low specificity was noted in in vivo MN prediction by MCase. Refinement of test data to be used for in silico system (e.g. consideration of cytotoxicity or re-evaluation of conflicting test results) will be needed to improve performance of CD prediction.