A 104-week pulmonary toxicity assessment of long and short single-wall carbon nanotubes after a single intratracheal instillation in rats.

We compared long-term pulmonary toxicities after a single intratracheal instillation of two types of dispersed single-wall carbon nanotubes (SWCNTs), namely, those with relatively long or short linear shapes with average lengths of 8.6 and 0.55 µm, respectively. Both types of SWCNTs were instilled intratracheally in male F344 rats at 0.2 or 1.0 mg/kg (long SWCNTs) or 1.0 mg/kg (short SWCNTs). Pulmonary responses were characterized at 26, 52 and 104 weeks after a single instillation. Inflammatory changes, test substance deposition, test substance engulfment by macrophages, and alveolar wall fibrosis were observed in the lungs of almost all test rats at 52 and 104 weeks after short nanotube instillation. The incidences of these changes were much lower in the long nanotube-treated groups. In almost all rats of the long nanotube-treated groups, fibrosis and epithelium loss in the terminal bronchiole with test substance deposition were observed. These bronchiolar changes were not observed after administering short nanotubes. Both bronchiolo-alveolar adenoma and carcinoma were found in the negative-control group, the high-dose long-nanotube group, and the short-nanotube group at 104 weeks post-instillation, although the incidences were not statistically different. The genotoxicity of the SWCNTs was also evaluated by performing in vivo comet assays with lung cells obtained 26 weeks post-instillation. No significant changes in the percent tail deoxyribonucleic acid were found in any group. These findings suggested that most long SWCNTs were deposited at the terminal bronchioles and that a considerable amount of short SWCNTs reached the alveolus, resulting in chronic inflammatory responses, but no genotoxicity in the lungs.

Length effects of single-walled carbon nanotubes on pulmonary toxicity after intratracheal instillation in rats.

We aimed to evaluate the effects of the length of single-walled carbon nanotubes (SWCNTs) on pulmonary toxicity in rats. Each rat received a single intratracheal instillation of short (S-) (average length of 0.40 µm) or long (L-) (average length of 2.77 µm) SWCNTs at a dose of 1 mg/kg and was observed for the next 6 months. Neither S- nor L-SWCNTs affected clinical signs, body weight, or autopsy findings. An increase in lung weight was observed after instillation of S- or L-SWCNTs; however, lung weights were slightly higher in the rats that were administered the S-SWCNTs. Distinct differences in bronchoalveolar lavage fluid (BALF) composition were observed between the S- and L-SWCNT-treated rats as early as 7 days after the intratracheal instillations of the SWCNTs. The S-SWCNTs caused persistent lung injury and inflammation during the 6-month observational period. However, the L-SWCNTs induced minimal lung injury and inflammation. Although the S- and L-SWCNTs changed BALF parameters and histopathological features of the lung, the magnitudes of the changes observed after the S-SWCNT treatment were greater than the respective changes observed after the L-SWCNT treatment. These findings indicate that the severity of the pulmonary toxicity caused after intratracheal instillation of SWCNT depends on the length of the SWCNTs. It appears that shorter SWCNTs induce greater pulmonary toxicity than longer SWCNTs do.

Long-term retention of pristine multi-walled carbon nanotubes in rat lungs after intratracheal instillation.

As a result of the growing potential industrial and medical applications of multi-walled carbon nanotubes (MWCNTs), people working in or residing near facilities that manufacture them may be exposed to airborne MWCNTs in the future. Because of concerns regarding their toxicity, quantitative data on the long-term clearance of pristine MWCNTs from the lungs are required. We administered pristine MWCNTs well dispersed in 0.5 mg ml(-1) Triton-X solution to rats at doses of 0.20 or 0.55 mg via intratracheal instillation and investigated clearance over a 12-month observation period. The pristine MWCNTs pulmonary burden was determined 1, 3, 7, 28, 91, 175 and 364 days after instillation using a method involving combustive oxidation and infrared analysis, combined with acid digestion and heat pretreatment. As 0.15- and 0.38-mg MWCNTs were detected 1 day after administration of 0.20 and 0.55 mg MWCNTs, respectively, approximately 30% of administrated MWCNTs may have been cleared by bronchial ciliary motion within 24 h of administration. After that, the pulmonary MWCNT burden did not decrease significantly over time for up to 364 days after instillation, suggesting that MWCNTs were not readily cleared from the lung. Transmission electron microscopy (TEM) showed that alveolar macrophages internalized the MWCNTs and retained in the lung for at least 364 days after instillation. MWCNTs were not detected in the liver or brain within the 364-day study period (<0.04 mg per liver, < 0.006 mg per brain).

Risk Assessment of the Carbon Nanotube Group.

This study assessed the health risks via inhalation and derived the occupational exposure limit (OEL) for the carbon nanotube (CNT) group rather than individual CNT material. We devised two methods: the integration of the intratracheal instillation (IT) data with the inhalation (IH) data, and the "biaxial approach." A four-week IH test and IT test were performed in rats exposed to representative materials to obtain the no observed adverse effect level, based on which the OEL was derived. We used the biaxial approach to conduct a relative toxicity assessment of six types of CNTs. An OEL of 0.03 mg/m(3) was selected as the criterion for the CNT group. We proposed that the OEL be limited to 15 years. We adopted adaptive management, in which the values are reviewed whenever new data are obtained. The toxicity level was found to be correlated with the Brunauer-Emmett-Teller (BET)-specific surface area (BET-SSA) of CNT, suggesting the BET-SSA to have potential for use in toxicity estimation. We used the published exposure data and measurement results of dustiness tests to compute the risk in relation to particle size at the workplace and showed that controlling micron-sized respirable particles was of utmost importance. Our genotoxicity studies indicated that CNT did not directly interact with genetic materials. They supported the concept that, even if CNT is genotoxic, it is secondary genotoxicity mediated via a pathway of genotoxic damage resulting from oxidative DNA attack by free radicals generated during CNT-elicited inflammation. Secondary genotoxicity appears to involve a threshold.

Historical control data on developmental toxicity studies in rodents.

Historical control data on rodent developmental toxicity studies, performed between 1994 and 2010, were obtained from 19 laboratories in Japan, including 10 pharmaceutical and chemical companies and nine contract research organizations. Rats, mice, and hamsters were used for developmental toxicity studies. Data included maternal reproductive findings at terminal cesarean sections and fetal findings including the spontaneous incidences of external, visceral, and skeletal anomalies. No noticeable differences were observed in maternal reproductive data between laboratories. Inter-laboratory variations in the incidences of fetuses with anomalies appeared to be due to differences in the selection of observation parameters, observation criteria, classification of the findings, and terminology of fetal alterations. Historical control data are useful for the appropriate interpretation of experimental results and evaluation of the effects of chemical on reproductive and developmental toxicities.

Lung toxicity assessment using bronchoalveolar lavage fluid and pleural lavage fluid cytology by intratracheal treatment in rats.

Usefulness of bronchoalveolar lavage fluid (BALF) and pleural cavity lavage fluid (PLF) as an experimental material was evaluated for the assessment of pulmonary toxicity of chemicals in rats. From the viewpoint of safety, isoflurane can be used for euthanasia/anesthesia because there was no difference in biological properties of BALF between diethyl ether and isoflurane. Here, we also recognized phosphate buffered saline (PBS) and distilled water equally as a solvent/vehicle for negative control. PLF is also provided as a useful target material as well as BALF for assessing chemical lung toxicity. To evaluate the method, we used zinc chloride as a model chemical and obtained the expected and satisfied results. We may conclude that the intratracheal treatment and combination usage of BALF and PLF as a target material is a good method for assessment of chemical pulmonary (lung and plural cavity) toxicity in rats.

Developmental toxicity of diesel exhaust: a review of studies in experimental animals.

Diesel exhaust (DE) is a complex mixture of combustion products of diesel fuel, including gases and diesel exhaust particles (DEPs), commonly known as soot, that contains many toxic air contaminants. Studies of pre- and postnatal exposure to DE or DEPs have revealed changes in growth, sexual development, hormone levels, spermatogenesis, weights of the reproductive and accessory organs, behavior, monoaminergic system, expression of immune-related genes, histopathology of the testes and brain, susceptibility to allergies, and inflammatory and genotoxic endpoints in rodent offspring. Changes in gene expression for gonadal development were also observed after exposure to DE. As for the causative agent for the developmental toxicity of DE, DEPs and the gaseous phase, conflicting findings were reported. Although this paper provides initial information on the potential developmental toxicity of DE including the gaseous phase and DEPs, further studies using relevant concentrations closely reflecting expected levels of human exposure are needed.

Dermal and ocular irritation and skin sensitization studies of fullerene C60 nanoparticles.

CONTEXT: Widespread production and use of nanomaterials have caused the release of increasing amounts of nanomaterials into the environment. The introduction of novel materials into industry requires safety evaluations as well as an understanding of the impact of the nanomaterials on human health, because the unique properties and size of nanomaterials may also result in unique health risks. Skin and eyes have the highest risk of exposure to nanomaterials, because deposition to the superficial organs has the potential to be a major route of exposure during the manufacturing, use, and disposal of nanomaterials. However, information on the dermal and eye irritation and sensitization of fullerene C(60) nanoparticles is still lacking. OBJECTIVES: This study was performed to examine the potential irritating and sensitizing effects of fullerenes on the skin and eyes. METHODS: The dermal and eye irritation study was performed using rabbits according to the Organisation for Economic Co-operation and Development (OECD) Guidelines 404 and 405, respectively. The skin sensitization study was carried out in accordance to the OECD Guideline 406 using guinea pigs. The concentrations of the fullerenes in the test substances were the maximum allowable for administration. Fullerenes were applied at 50 mg in dermal irritation, 40 mg in skin sensitization, and 100 mg in eye irritation studies. RESULTS: No dermal responses, including erythema/eschar or edema, were found in rabbits treated with fullerenes. No rabbits exhibited corneal opacity, abnormality of the iris, or chemosis eye at any time point after the application of fullerenes. Fullerenes caused conjunctival redness and blood vessel hyperemia at 1 h, but not at 24 h. No erythema or edema was observed after the challenge with fullerenes in the fullerene-treated guinea pigs. CONCLUSION: Reversible minimal potential for acute irritation of the eyes was induced by fullerenes, but neither irritation nor sensitization was caused on the skin. Although the present study provided initial information on the acute irritation and acute sensitization of highly purified C(60) fullerenes, information on the toxicological effects of fullerenes and their derivatives is still limited. Further information is needed to clarify the potential for toxicity given the complex nature of fullerenes and their derivatives.

Genotoxicity evaluation for single-walled carbon nanotubes in a battery of in vitro and in vivo assays.

The genotoxicity of single-walled carbon nanotubes (SWCNTs) was determined using a battery of genotoxicity assays, comprising a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test and a mammalian erythrocytes micronucleus test. SWCNTs had no mutagenicity in S. typhimurium TA98, TA100, TA1535 or TA1537, or in E. coli WP2uvrA, in the absence or presence of metabolic activation. SWCNTs did not increase the number of structural or numerical chromosomal aberrations after short-term or continuous exposure. In the micronucleus test using CD-1 mice, SWCNTs did not affect the proportion of immature erythrocytes, the total proportion of erythrocytes or the number of micronuclei in immature erythrocytes. SWCNTs appear not to pose a genotoxic risk.

In vivo comet assay of multi-walled carbon nanotubes using lung cells of rats intratracheally instilled.

The genotoxicity of multi-walled carbon nanotubes (MWCNTs) was evaluated in vivo with comet assays using the lung cells of rats given MWCNTs. The MWCNTs were intratracheally instilled as a single dose at 0.2 or 1.0 mg kg(-1) or a repeated dose at 0.04 or 0.2 mg kg(-1) , once a week for 5 weeks, to male rats. The rats were sacrificed 3 or 24 h after the single instillation and were sacrificed 3 h after the last instillation in the repeated instillation groups. Histopathological examinations of the lungs revealed that MWCNTs caused inflammatory changes including the infiltration of macrophages and neutrophils after a single instillation and repeated instillation at both doses. In comet assays using rat lung cells, no changes in % Tail DNA were found in any group given MWCNTs. These findings indicate that MWCNTs do not have the potential to cause DNA damage in comet assays using the lung cells of rats given MWCNTs at doses causing inflammatory responses.

Historical control data on prenatal developmental toxicity studies in rabbits.

Historical control data on rabbit prenatal developmental toxicity studies, performed between 1994-2010, were obtained from 20 laboratories, including 11 pharmaceutical and chemical companies and nine contract laboratories, in Japan. In this paper, data were incorporated from a laboratory if the information was based on 10 studies or more. Japanese White rabbits and New Zealand White rabbits were used for prenatal developmental toxicity studies. The data included maternal reproductive findings at terminal cesarean sections and fetal findings including spontaneous incidences of morphological alterations. No noticeable differences between strains or laboratories were observed in the maternal reproductive and fetal developmental data. The inter-laboratory variations in the incidences of fetal external, visceral, and skeletal alterations seem to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, and terminology of fetal alterations.

In vivo genotoxicity study of single-wall carbon nanotubes using comet assay following intratracheal instillation in rats.

The genotoxicity of single-wall carbon nanotubes (SWCNTs) was evaluated in vivo using the comet assay after intratracheal instillation in rats. The SWCNTs were instilled at a dosage of 0.2 or 1.0mg/kg body weight (single instillation group) and 0.04 or 0.2mg/kg body weight once a week for 5weeks (repeated instillation group). As a negative control, 1% Tween 80 was instilled in a similar manner. As a positive control, ethyl methanesulfonate (EMS) at 500mg/kg was administered once orally 3h prior to dissection. Histopathologically, inflammation in the lung was observed for all the SWCNTs in both single and repeated groups. In the comet assay, there was no increase in% tail DNA in any of the SWCNT-treated groups. In the EMS-treated groups, there was a significant increase in% tail DNA compared with the negative control group. The present study indicated that a single intratracheal instillation of SWCNTs (1.0mg/kg) or repeated intratracheal instillation (0.2mg/kg) once a week for five weeks induced a clear inflammatory response (hemorrhage in the alveolus, infiltration of alveolar macrophages and neutrophiles), but no DNA damage, in the lungs in rats. Under the conditions of the test, SWCNTs were not genotoxic in the comet assay following intratracheal instillation in rats.

Evaluation of genotoxicity of multi-walled carbon nanotubes in a battery of in vitro and in vivo assays.

The genotoxic potential of two products of multi-walled carbon nanotubes (coded as N-MWCNTs, diameter of 44 nm/BET surface area of 69 m²/g and MWNT-7, diameter of 70 nm/BET surface area of 23 m²/g) was evaluated using a battery of genotoxicity assays, comprising a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and a mammalian erythrocytes micronucleus test. Neither type exerted mutagenicity in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, or in Escherichia coli WP2uvrA, in the absence or presence of metabolic activation. The products of MWCNTs did not increase the number of structural chromosomal aberrations either, regardless of metabolic activation, though they increased the number of numerical chromosomal aberrations, one slightly and the other distinctly, in the absence of metabolic activation. In ICR mice, the two products did not affect the proportion of immature erythrocytes, the total proportion of erythrocytes, or the number of micronuclei in immature erythrocytes.

Genotoxicity evaluation of fullerene C60 nanoparticles in a comet assay using lung cells of intratracheally instilled rats.

The genotoxicity of fullerene C(60) nanoparticles was evaluated in vivo with comet assays using the lung cells of rats given C(60) nanoparticles. The C(60) nanoparticles were intratracheally instilled as a single dose at 0.5 or 2.5mg/kg or repeated dose at 0.1 or 0.5mg/kg, once a week for 5 weeks, to male rats. The lungs were obtained 3 or 24h after a single instillation and 3h after repeated instillation. Inflammatory responses were observed in the lungs obtained 24h after a single instillation at 2.5mg/kg and repeated instillation at 0.5mg/kg. Histopathological examinations revealed that C(60) nanoparticles caused slight changes including hemorrhages in alveoli and the cellular infiltration of macrophages and neutrophils in alveoli. In comet assays using rat lung cells, no increase in % Tail DNA was found in any group given C(60) nanoparticles. These findings indicate that C(60) nanoparticles had no potential for DNA damage in comet assays using the lungs cells of rats given C(60) even at doses causing inflammation.

In vivo genotoxicity study of titanium dioxide nanoparticles using comet assay following intratracheal instillation in rats.

Titanium dioxide (TiO2) is widely used as a white pigment in paints, plastics, inks, paper, creams, cosmetics, drugs and foods. In the present study, the genotoxicity of anatase TiO2 nanoparticles was evaluated in vivo using the comet assay after a single or repeated intratracheal instillation in rats. The nanoparticles were instilled intratracheally at a dosage of 1.0 or 5.0 mg/kg body weight (single instillation group) and 0.2 or 1.0 mg/kg body weight once a week for 5 weeks (repeated instillation group) into male Sprague-Dawley rats. A positive control, ethyl methanesulfonate (EMS) at 500 mg/kg, was administered orally 3 h prior to dissection. Histopathologically, macrophages and neutrophils were detected in the alveolus of the lung in the 1.0 and 5.0 mg/kg TiO2 groups. In the comet assay, there was no increase in % tail DNA in any of the TiO2 groups. In the EMS group, there was a significant increase in % tail DNA compared with the negative control group. TiO2 nanoparticles in the anatase crystal phase are not genotoxic following intratracheal instillation in rats.

Pulmonary and systemic responses of highly pure and well-dispersed single-wall carbon nanotubes after intratracheal instillation in rats.

The present study was conducted to assess the pulmonary and systemic responses in rats after intratracheal instillation of highly pure, well-dispersed, and well-characterized SWCNTs. Exposure to SWCNTs up to 2 mg/kg did not produce mortality, changes in clinical signs, or body weights during the observation period. Dose-dependent changes were observed in the lung weight, BALF inflammatory cells, and biochemical parameters such as LDH value, protein content, IL-1ß and IL-6 activity, and histopathology. In the 0.04 mg/kg SWCNT-exposed group, almost no changes were observed during the observation period. In the 0.2 mg/kg SWCNT-exposed group, pulmonary inflammatory responses were observed after instillation. In the 1 mg/kg and 2 mg/kg SWCNT-exposed group, acute lung inflammation and subsequent granuloma accompanied by increased lung weights were observed. Furthermore, the histopathological findings in the lungs of rats exposed to SWCNTs showed inflammatory responses related with the vital reaction to the foreign substance that was instilled intratracheally, and there were no fibrosis, atypical lesion, or tumor-related findings even at the highest dose (2 mg/kg) of SWCNT-exposed groups up to 6 months after instillation. For all groups, histopathological changes due to the instillation exposure of SWCNTs were observed only in the lungs and lung-associated lymph nodes and not in the other tissues examined (i.e. the liver, kidney, spleen, and cerebrum).

Evaluation of dermal and eye irritation and skin sensitization due to carbon nanotubes.

The present paper summarizes the results of our studies on dermal and eye irritation and skin sensitization due to carbon nanotubes (CNTs), whose potential applications and uses are wide and varied, including CNT-enhanced plastics, electromagnetic interference/radio-frequency (EMI/RFI) shielding, antistatic material, flexible fibers and advanced polymers, medical and health applications, and scanning probe microscopy. Skin and eyes have the highest risk of exposure to nanomaterials, because deposition of nanomaterials to the surficial organs has the potential to be a major route of exposure during the manufacturing, use, and disposal of nanomaterials. Two products composed of single-walled carbon nanotubes (SWCNTs) and two products composed of multi-walled carbon nanotubes (MWCNTs) were tested regarding acute dermal and acute eye irritation using rabbits, and skin sensitization using guinea pigs. The concentrations of the CNTs in the substances were the maximum allowable for administration. The two products of SWCNTs and one of the products of MWCNTs were not irritants to the skin or eyes. The other product of MWCNTs caused very slight erythema at 24h, but not at 72h, after patch removal in the dermal irritation experiments and conjunctival redness and blood vessel hyperemia at 1h, but not at 24h, in eye irritation experiments. These findings showed that one product of MWCNTs was a very weak acute irritant to the skin and eyes. No products of SWCNTs and MWCNTs exhibited skin-sensitization effects. Our knowledge of the toxicological effects of CNTs is still limited. Further information is needed to clarify the potential for irritation and sensitization given the complex nature of CNTs.

Evaluation of the genotoxic potential of single-wall carbon nanotubes by using a battery of in vitro and in vivo genotoxicity assays.

The genotoxic potential of a high purity sample of single-wall carbon nanotubes (SWCNTs) was evaluated using a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse mutation test (Ames test), an in vitro chromosomal aberration test, and an in vivo mouse bone marrow micronucleus test. The SWCNTs exerted no genotoxicity in Salmonella typhimurium TA97, TA98, TA100, and TA1535, or in Escherichia coli WP2 uvrA/pKM101, whether in the absence or presence of metabolic activation and at concentrations of 12.5-500 µg/plate. In the chromosomal aberration test, at 300-1000 µg/mL, the SWCNTs did not increase the number of structural or numerical chromosomal aberrations, whether the test was conducted with or without metabolic activation. In the in vivo bone marrow micronucleus test, doses of 60 mg/kg and 200mg/kg SWCNTs did not affect the proportions of immature and total erythrocytes, nor did it increase the number of micronuclei in the immature erythrocytes of mice. The results of these studies show that the high purity and well-dispersed sample of SWCNTs are not genotoxic under the conditions of the in vitro bacterial reverse mutation assay, chromosomal aberration assay, or in vivo bone marrow micronucleus test, and thus appear not to pose a genotoxic risk to human health in vivo.

Biological response and morphological assessment of individually dispersed multi-wall carbon nanotubes in the lung after intratracheal instillation in rats.

Biological responses of multi-wall carbon nanotubes (MWCNTs) were assessed after a single intratracheal instillation in rats. The diameter and median length of the MWCNTs used in this study were approximately 60 nm and 1.5 µm, respectively. Groups of male Sprague-Dawley rats were intratracheally instilled with 0.04, 0.2, or 1 mg/kg of the individually dispersed MWCNT suspension. After instillation, the bronchoalveolar lavage fluid was assessed for inflammatory cells and markers, and the lung, liver, kidney, spleen, and cerebrum were histopathologically evaluated at 3-day, 1-week, 1-month, 3-month, and 6-month post-exposure. Transient pulmonary inflammatory responses were observed only in the lungs of the group of rats exposed to 1 mg/kg of MWCNTs. Morphology of the instilled MWCNTs in the lungs of rats was assessed using light microscopy and transmission electron microscopy (TEM). Light microscopy examination revealed that MWCNTs deposited in the lungs of the rats were typically phagocytosed by the alveolar macrophages and these macrophages were consequently accumulated in the alveoli until 6-month post-exposure. The 400 TEM images obtained showed that all MWCNTs were located in the alveolar macrophages or macrophages in the interstitial tissues, and MWCNTs were not located in the cells of the interstitial tissues. There was no evidence of chronic inflammation, such as angiogenesis or fibrosis, induced by MWCNT instillation. These results suggest that MWCNTs were being processed and cleared by alveolar macrophages.

Reproductive and developmental toxicity studies of manufactured nanomaterials.

This paper reviews studies in vivo and in vitro on the reproductive and developmental toxicity of manufactured nanomaterials including metallic and metal oxide-based particles, fullerenes (C(60)), carbon black (CB), and luminescent particles. Studies in vivo showed increased allergic susceptibility in offspring of mouse dams intranasally insufflated with respirable-size titanium dioxide (TiO(2)), adverse effects on spermatogenesis and histopathological changes in the testes and changes in gene expression in the brain of mouse offspring after maternal subcutaneous injection of TiO(2) nanoparticles, transfer to rat fetuses of radiolabeled gold nanoparticles and C(60) after maternal intravenous injection, death and morphological abnormalities in mouse embryos after maternal intraperitoneal injection of C(60), and adverse effects on spermatogenesis in mouse offspring after maternal intratracheal instillation of CB nanoparticles. Studies in vitro revealed that TiO(2) and CB nanoparticles affected the viability of mouse Leydig cells, that gold nanoparticles reduced the motility of human sperm, that silver, aluminum, and molybdenum trioxide were toxic to mouse spermatogonia stem cells, that silica nanoparticles and C(60) inhibited the differentiation of mouse embryonic stem cells and midbrain cells, respectively, and that cadmium selenium-core quantum dots inhibited pre- and postimplantation development of mouse embryos. Although this paper provides initial information on the potential reproductive and developmental toxicity of manufactured nanomaterials, further studies, especially in vivo, using characterized nanoparticles, relevant routes of administration, and doses closely reflecting expected levels of exposure are needed.