Time-course analysis of pulmonary inflammation induced by intratracheal instillation of nanosized crystalline silica particles in F344 rats.

Crystalline silica is an important cause of serious pulmonary diseases, and its toxic potential is known to be associated with its surface electrical properties. However, in vivo data clarifying the relevance of silica's toxic potential, especially its long-term effects, remain insufficient. To investigate the contribution of physico-chemical property including surface potential on the hazard of nanocrystalline silica, we performed single intratracheal instillation testing using five different crystalline silicas in a rat model and assessed time-course changes in pulmonary inflammation, lung burden, and thoracic lymph node loads. Silica-nanoparticles were prepared from two commercial products (Min-U-Sil5 [MS5] and SIO07PB [SPB]) using three different pretreatments: centrifugation (C), grinding (G), and surface dissolving (D). The five types of silica particles-MS5, MS5_C, SPB_C, SPB_G, and SPB_D-were intratracheally instilled into male F344 rats at doses of 0 mg/kg (purified water), 0.22 mg/kg (SPB), and 0.67, 2, or 6 mg/kg (MS5). Bronchoalveolar lavage, a lung burden analysis, and histopathological examination were performed at 3, 28, and 91 days after instillation. Granuloma formation was present in MS5 group at 91 days after instillation, although granuloma formation was suppressed in MS5_C group, which had a smaller particle size. SPB_C induced severe and progressive inflammation and kinetic lung overload, whereas SPB_G and SPB_D induced only slight and transient acute inflammation. Our results support that in vivo toxic potential of nanosilica by intratracheal instillation may involve with surface electrical properties leading to prolonged effect and may not be dependent not only on surface properties but also on other physico-chemical properties.

Time-course comparison of pulmonary inflammation induced by intratracheal instillation of four different nickel oxide nanoparticles in male Fischer rats.

Occupational exposure to nickel oxide (NiO) is an important cause of respiratory tract cancer. Toxicity is known to be associated with the dissociated component, i.e. nickel (II) ions. To address the relationship between physicochemical properties, including solubility in artificial lysosomal fluid, of NiO and time-course changes in the pulmonary response, we conducted an intratracheal instillation study in male Fischer rats using four different well-characterized NiO products, US3352 (NiO A), NovaWireNi01 (NiO B), I small particle (NiO C), and 637130 (NiO D). The NiOs were suspended in purified water and instilled once intratracheally into male F344 rats (12 weeks old) at 0 (vehicle control), 0.67, 2, and 6 mg/kg body weight. The animals were euthanized on days 3, 28, or 91 after instillation, and blood analysis, bronchoalveolar lavage fluid (BALF) testing, and histopathological examination were performed. The most soluble product, NiO B, caused the most severe systemic toxicity, leading to a high mortality rate, but the response was transient and surviving animals recovered. The second-most-soluble material, NiO D, and the third, NiO A, caused evident pulmonary inflammation, and the responses persisted for at least 91 days with collagen proliferation. In contrast, NiO C induced barely detectable inflammation in the BALF examination, and no marked changes were noted on histopathology. These results indicate that the early phase toxic potential of NiO products, but not the persistence of pulmonary inflammation, is associated with their solubility.

Combined repeated-dose and reproductive/developmental toxicity screening test of benzene, 1,1'-oxybis-, tetrapropylene derivs. in rats.

We have conducted animal toxicity tests of chemicals for a chemical safety program implemented by the Ministry of Economy, Trade and Industry of Japan. Here we conducted a combined repeated-dose and reproductive/developmental toxicity screening test of benzene, 1,1'-oxybis-, tetrapropylene derivs. (BOTD). BOTD was administered to 9-week-old Crl:CD(SD) male and female rats by gavage at 0, 40, 200, or 1000 mg/kg/day. Males were treated for 42 days including mating period. Females were treated for 42-53 days through the premating, mating, pregnancy, and until Day 4 of lactation periods. Increases in prothrombin time and activated partial thromboplastin time values were observed only in males at 200 and 1000 mg/kg/day. Hypertrophy of centrilobular hepatocytes was observed with increased liver weight in both sexes at 200 and 1000 mg/kg/day, but there was no histologic evidence of hepatotoxicity. Diffuse hypertrophy of follicular cells in thyroid glands was observed in females at 200 mg/kg/day and in both sexes at 1000 mg/kg/day, with an increased blood cholesterol level in females at 1000 mg/kg/day. The conception index was decreased for females at 1000 mg/kg/day; and no abnormalities were detected in the reproductive indices of implantation, delivery, or pups' condition, although a slight increase in the pups' body weight was noted at birth. Our data indicate a no-observed-adverse-effect level of 40 mg/kg/day for repeated-dose toxicity on the basis of the prolongation of blood coagulating time, and of 200 mg/kg/day for reproductive/developmental toxicity on the basis of the decreased conception index.

Combined repeated-dose and reproductive/developmental toxicity screening test of 1-tert-butoxy-4-chlorobenzene in rats.

We have carried out animal toxicity tests of chemicals for a chemical safety program implemented by the Ministry of Economy, Trade, and Industry of Japan. Here, we tested 1-tert-butoxy-4-chlorobenzene in a combined repeat-dose and developmental and reproductive toxicity test. The test chemical was administered daily by gavage to 9-week-old Crl:CD (SD) rats at doses of 0, 20, 100, and 500 mg/kg/d. Males were treated for 42 d beginning 14 d before mating. Females were treated from 14 d before mating to day 4 of lactation. Decreased spontaneous locomotion, decreased respiratory rate, and incomplete eyelid opening were observed at 500 mg/kg/d (both sexes), but resolved within 30 min of administration, suggesting central nervous system depression. No notable changes were observed in body weight, food consumption, functional battery tests, or blood test. Increased liver weight with centrilobular or diffuse hepatocyte hypertrophy was observed at 100 and 500 mg/kg/d (both sexes). There were no biochemical or histopathological changes related to hepatotoxicity. Increased kidney weight with basophilic tubules, tubule dilatation, and increased hyaline droplets were observed in males dosed at 100 and 500 mg/kg/d. Immunohistochemical staining indicated α2u-globulin nephropathy, a male rat-specific toxicity. Although kidney weight was also increased in females dosed at 500 mg/kg/d, it was not considered to be an adverse effect because there were no histopathological changes. Pup weights on postnatal day 0 were decreased at 500 mg/kg/d and still decreased on postnatal day 4. Our data indicated the no-observed-adverse-effect-level for repeated-dose and reproductive/developmental toxicity for 1-tert-butoxy-4-chlorobenzene was 100 mg/kg/d.

Effects of pentobarbital, isoflurane, or medetomidine-midazolam-butorphanol anesthesia on bronchoalveolar lavage fluid and blood chemistry in rats.

Bronchoalveolar lavage fluid (BALF) is commonly examined for pulmonary toxicity in animal studies. Two common means of anesthesia before euthanasia and bronchoalveolar lavage in rats are intraperitoneal injection of pentobarbital and inhalation of isoflurane. Medetomidine-midazolam-butorphanol is an alternative anesthesia to pentobarbital for animal welfare; however, the effect of this combination on BALF and blood chemistry is unknown. Here, we compared the effects of anesthesia by intraperitoneal injection of pentobarbital or one of two combinations of medetomidine-midazolam-butorphanol (dose, 0.375-2.0-2.5 or 0.15-2.0-2.5 mg/kg) or by inhalation of isoflurane on BALF and blood chemistry in rats with or without pulmonary inflammation. In BALF, we determined total protein, albumin, lactate dehydrogenase, total cell count and neutrophil count. In serum, we conducted a general chemistry screen. After anesthesia with pentobarbital or isoflurane, there were no significant differences between any of the BALF or blood chemistry parameters with or without inflammation. After anesthesia with either of the combinations of medetomidine-midazolam-butorphanol, lactate dehydrogenase, total cell count, neutrophil count, and almost all of the blood chemistry parameters were comparable with those observed after pentobarbital or isoflurane; however, BALF albumin and serum glucose were significantly increased in rats without inflammation. After the combination of low-dose medetomidine in rats with inflammation, BALF parameters were comparable with those observed after pentobarbital or isoflurane. Our results show that, of the anesthetics examined, inhalation of isoflurane is the most appropriate means of anesthesia when examining BALF or serum for toxicity studies in rats.

Effects of dose volume and delivery device on bronchoalveolar lavage parameters of intratracheally administered nano-sized TiO2 in rats.

The intratracheal (IT) test is useful for screening the pulmonary toxicity of inhaled materials, including nanomaterials. However, a standard procedure has not yet been authorized internationally, and the effects of different test parameters are unknown. To determine appropriate experimental conditions for the IT test, we intratracheally administered nano-sized TiO2 to male F344 rats at 3.0 mg/kg body weight by using two delivery devices (gavage needle or microaerosolizer) and dose volumes of 0.5-3.0 mL/kg (gavage needle) or 0.5-2.0 mL/kg (microaerosolizer). We evaluated the pulmonary deposition and interlobar distribution of TiO2 at both 30 min and 3 days after administration. In addition, the inflammatory components in bronchoalveolar lavage (BAL) fluid were measured 3 days after administration of TiO2. At dose volumes of 0.5-2.0 mL/kg, the BAL values were comparable regardless of the device used. In addition, pulmonary TiO2 burden and lobar concentration patterns were equivalent at all combinations of dose volume and delivery device. In conclusion, the acute pulmonary toxicity of nanomaterials can be assessed effectively by using an IT test in which the test agent is provided to rats at a dose volume of 0.5-2.0 mL/kg with either a gavage needle or microaerosolizer.

Categorization of nano-structured titanium dioxide according to physicochemical characteristics and pulmonary toxicity.

A potentially useful means of predicting the pulmonary risk posed by new forms of nano-structured titanium dioxide (nano-TiO2) is to use the associations between the physicochemical properties and pulmonary toxicity of characterized forms of TiO2. In the present study, we conducted intratracheal administration studies in rats to clarify the associations between the physicochemical characteristics of seven characterized forms of TiO2 and their acute or subacute pulmonary inflammatory toxicity. Examination of the associations between the physicochemical characteristics of the TiO2 and the pulmonary inflammatory responses they induced revealed (1) that differences in the crystallinity or shape of the TiO2 particles were not associated with the acute pulmonary inflammatory response; (2) that particle size was associated with the acute pulmonary inflammatory response; and (3) that TiO2 particles coated with Al(OH)3 induced a greater pulmonary inflammatory response than did non-coated particles. We separated the seven TiO2 into two groups: a group containing the six TiO2 with no surface coating and a group containing the one TiO2 with a surface coating. Intratracheal administration to rats of TiO2 from the first group (i.e., non-coated TiO2) induced only acute pulmonary inflammatory responses, and within this group, the acute pulmonary inflammatory response was equivalent when the particle size was the same, regardless of crystallinity or shape. In contrast, intratracheal administration to rats of the TiO2 from the second group (i.e., the coated TiO2) induced a more severe, subacute pulmonary inflammatory response compared with that produced by the non-coated TiO2. Since alteration of the pulmonary inflammatory response by surface treatment may depend on the coating material used, the pulmonary toxicities of coated TiO2 need to be further evaluated. Overall, the present results demonstrate that physicochemical properties may be useful for predicting the pulmonary risk posed by new nano-TiO2 materials.

Combined repeated dose and reproductive/developmental toxicity screening test of 4-methoxy-2-nitroaniline in rats.

4-Methoxy-2-nitroaniline (4M2NA) is widely used as an intermediate for the synthesis of dyes, pigments and other chemical compounds. Since 4M2NA has amino-group and nitro-group on the benzene ring, it was expected that it induced obvious hemolytic anemia. We conducted a combined repeated dose and reproductive/developmental toxicity screening test according to Organisation for Economic Co-operation and Development (OECD) Test Guideline No. 422 (OECD TG 422) to enrich the toxic information and ensure the safety of 4M2NA. 4M2NA was administered to Crl:CD(SD) male and female rats by gavage at 0, 12.5, 75 or 450 mg/kg/day for 42 to maximum of 54 days through pre-mating, mating, pregnancy and lactation periods. An extramedullary hematopoiesis and congestion in spleen, and higher reticulocyte ratio were noted in only females at 450 mg/kg/day without decreased anemic parameters in the hematological examination. Hypertrophy of centrilobular hepatocytes in both sexes was observed with increased relative liver weight at 450 mg/kg/day. Furthermore, the diffuse follicular cell hypertrophy of the thyroid was observed in females at 450 mg/kg/day. No abnormalities were detected in the reproductive indices of copulation, delivery or fetal viability. We concluded the no-observed-adverse-effect level (NOAEL) for repeated-dose toxicity was 75 mg/kg/day based on the trace evidences of hemolytic anemia, and the NOAEL for reproductive/developmental toxicity as 450 mg/kg/day based on no toxicological concerns for reproductive endpoints. The hemolytic anemia was much milder than expected. Thus, we discussed the reason of this much less hemolytic effect from the point of view of the structural characteristics of 4M2NA.

Prediction of the bioconcentration factor in common carp (Cyprinus carpio L.) using data from the dietary exposure bioaccumulation fish test.

An approach to predicting the bioconcentration factor (BCFpre ) from the predicted uptake rate constant (k1 pre ) and the depuration rate constant measured in the dietary exposure bioaccumulation fish test (k2 dietary ) [BCFpre = k1 pre /k2 dietary ] is proposed in test guideline 305 of the Organization for Economic Cooperation and Development Guidelines for Testing of Chemicals. Data were collected on the BCFs of 197 test chemicals from Japan's Chemical Substances Control Law database. To demonstrate how the BCFpre compares with experimentally derived BCF under optimum conditions, 48 of 197 test chemicals, including a number of studies that could be considered problematic, were excluded from the analysis. The k1 pre was calculated by using 22 published prediction methods: the correlations between experimental uptake rate constants (k1 aqueous ) and k1 pre for all prediction methods were very low and were statistically nonsignificant (p > 0.05). Three prediction methods were also selected that gave relatively good values for the geometric mean of k1 pre /k1 aqueous and calculated values of BCFpre for 12 test chemicals. Linear relationships (p < 0.05) are presented between logarithm of experimental and predicted BCF. The correlation coefficients of growth-corrected experimental and predicted BCF tended to be higher than values that were not growth corrected. For some test chemicals, use of predicted BCF led to a bioaccumulation classification different from that of existing regulatory criteria.

Photochromic isomerization of spirobenzopyran in nanoholes of anodic alumina.

A notable change in the photochromic characteristics was observed when the benzene solution of spirobenzopyran was put in nanoholes of anodic alumina. The absorption peak that appeared in the ultraviolet irradiation process shifted to a shorter wavelength, and the decay time of the decoloration process became approximately 200 times longer than that of the original solution. After a preservation period of several days, however, both the absorption wavelength and the decay time recovered to those of the original solution. These experimental results suggest that the photochromic isomerization in the alumina nanoholes is affected by the large surface area of the matrix rather than the limited free volume.