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 24 h, but not at 72 h, after patch removal in the dermal irritation experiments and conjunctival redness and blood vessel hyperemia at 1 h, but not at 24 h, 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.     

茂名市某钓鱼竿厂苯、甲苯、二甲苯危害的调查分析

目的了解茂名市某钓鱼竿厂苯、甲苯、二甲苯（“三苯”）对作业人员健康的危害程度。方法对茂名市某钓鱼竿厂工作场所进行“三苯”浓度测定,并对50例“三苯”作业工人（接触组）和68例不接触“三苯”工人（对照组）进行体检。结果该厂工作场所空气中的苯、甲苯、二甲苯时间加权平均容许浓度（PC—TWA）、短时间接触容许浓度（STEL）均未超标。接触组头痛、头晕、失眠、记忆力减退的发生率高于对照组,差异有显著性（P〈0．05或P〈0．01）。血常规检查结果显示,接触组白细胞减少的检出率高于对照组（P〈0．01）。结论该厂“三苯”对作业工人的健康存在一定的潜在危害,应做好“三苯”危害的预防工作。     

Effect of methyl substitution of benzene on the percutaneous absorption and skin irritation in hairless rats

The permeation rate and skin retention of benzene and methylbenzenes were assessed in vitro using hairless rat skin. The effects of unocclusive dermal exposures of these chemicals (15 microl every 2h for 8h a day for 4 days) on the transepidermal water loss (TEWL), erythema and skin histopathology were measured in CD hairless rats. The expression of IL-1 alpha and TNF-alpha in the skin and blood were measured at the end of dermal exposures. The flux of benzene was about 1.5-, 2.5- and 80-fold higher than toluene, xylene and tetramethyl benzene isomers (TMB), respectively, and the values were inversely correlated with molecular weight (r(2)=0.7455) and logoctanol-water partition coefficient (r(2)=0.7831). The retention of chemicals in stratum corneum (SC) was in the order of TMB>xylene>toluene approximately benzene. The TEWL and erythema data demonstrated that the irritation was in the following order: TMB>xylene>benzene. The histo-pathological examination showed that xylene and TMB induced granulocyte infiltration, swelling of the epidermis, and extensive disruption and damage of stratum corneum. Likewise, the expression of IL-1 alpha in the blood and TNF-alpha in the skin after dermal exposures was higher for TMB followed by xylene and benzene compared to control. In conclusion, the aromatic hydrocarbon chemicals induced cumulative irritation upon low-level repeat exposures for a 4-day period and the irritation increased with the number of methyl groups of benzene. The affinity of the chemical to SC and their gradual accumulation in the skin in the present study is the reason for the differences in the skin irritation profiles of different aromatic chemicals. Our ultimate goal is to develop a biologically based model that connects skin retention of chemical to the skin irritation response. The findings of the present study will be helpful in understanding the role of these chemicals in the jet fuel and various petroleum based fuels in inducing skin irritation response.     

舒阴洗液皮肤毒性及黏膜刺激性实验研究

目的对舒阴洗液进行了部分动物实验研究,包括皮肤急性毒性实验、皮肤刺激性实验、皮肤过敏性实验及黏膜刺激性实验,以了解其安全性。方法将不同剂量的舒阴洗液用于大耳白兔背部完整或破损去毛区皮肤,观察其产生急性毒性和皮肤刺激性情况;采用致敏与激发接触舒阴洗液,观察豚鼠过敏情况;将舒阴洗液注入大耳白兔的阴道中,观察大耳白兔阴道黏膜的情况。结果舒阴洗液未产生皮肤急性毒性和刺激性;也未产生阴道黏膜刺激性;反复致敏后无皮肤与全身过敏反应。结论舒阴洗液是安全的新型外用洗剂。     

Human exposure to acrolein: Time-dependence and individual variation in eye irritation

The aim of the study was to examine the time dependence on sensory irritation detection following exposure to threshold levels of acrolein, in humans. The exposures occurred in an exposure chamber and the subjects were breathing fresh air through a mask that covered the nose and mouth. All participants participated in four exposure conditions, of which three consisted of a mixture of acrolein and heptane and one of only heptane. Exposure to acrolein at a concentration half of the TLV-C lead to sensory irritation. The perceived sensory irritation resulted in both increased detectability and sensory irritation after about 6.8 min of exposure in 58% of the participants. The study confirm the previously suggested LOAEL of about 0.34 mg/m³ for eye irritation due to acrolein exposure. The sensory irritation was still significant 10 min after exposure. These results have implications for risk assessment and limit setting in occupational hygiene.     

退热贴的急性毒性、刺激性及过敏性试验观察

目的:观察退热贴的急性毒性、刺激性及过敏性,为其临床应用安全性提供依据.方法:通过一般情况观察与组织病理学检查综合观察退热贴1次给药经豚鼠完整皮肤及破损皮肤吸收后所产生的急性毒性反应和刺激性反应,及豚鼠皮肤重复接触退热贴后的过敏反应情况.结果:退热贴对实验动物无急性毒性;对豚鼠完整皮肤及破损皮肤均无刺激性作用;豚鼠皮肤重复接触不引起皮肤过敏反应.结论:研究结果提示退热贴在临床应用中有较好的安全性.     

稳定性二氧化氯喷雾剂对家兔皮肤的刺激性实验

目的考察稳定性二氧化氯喷雾剂局部经皮给药的安全性。方法将健康家兔背部两侧对称脱毛3 cm×3 cm后,每次给予二氧化氯喷雾剂0.5 ml,对其完整皮肤和破损皮肤分别进行单次给药及多次给药的皮肤刺激性试验。结果该喷雾剂对于家兔完整皮肤和破损皮肤单次给药及多次给药后于1、24、48、72 h观察均无红肿或斑块出现。结论本试验条件下二氧化氯喷雾剂局部经皮给药无明显不良反应,表明安全性良好。为二氧化氯喷雾剂用于临床经皮给药提供可靠的试验依据和安全保障,为临床经皮给药提供一种新的二氧化氯新剂型。     

复方利多卡因乳膏对豚鼠皮肤刺激性实验观察

目的对复方利多卡因乳膏进行豚鼠皮肤刺激性实验,以了解其安全性。方法以一次给药和多次给药方法,将复方利多卡因乳膏外涂于白色豚鼠背部正常或破损去毛区皮肤,观察其产生豚鼠皮肤刺激性反应。结果复方利多卡因乳膏对豚鼠完整皮肤平均反应分值均〈0.5分,对豚鼠破损皮肤平均反应分值在给药后1h和24h均〉0.5分,但〈2.99分,48h后平均反应分值均〈0.5分。结论复方利多卡因乳膏对豚鼠完整皮肤无刺激性,对破损皮肤有轻度刺激性,但给药后48h刺激性消失。     

HSP27 as a biomarker for predicting skin irritation in human skin and reconstructed organotypic skin model

In vitro alternative tests aiming at replacing the traditional animal test for predicting the irritant potential of chemicals have been developed, but the assessing parameters or endpoints are still not sufficient. To discover novel endpoints for skin irritation responses, 2DE-based proteomics was used to analyze the protein expression in human skin exposed to sodium lauryl sulfate (SLS) following the test protocol of the European Centre for the Validation of Alternative Methods (ECVAM) in the present study. HSP27 was up-regulated most significantly among the eight identified proteins, consistent with our previous reports. Acid and basic chemicals were applied on human skin for further validation and results showed that the up-regulated expression of HSP27 was induced in 24 h after the exposure. Skin-equivalent constructed with fibroblasts, basement membrane and keratinocytes was used to investigate the potential of HSP27 as a biomarker or additional endpoint for the hazard assessment of skin irritation. Our skin-equivalent (Reconstructed Organotypic Skin Model, ROSM) had excellent epidermal differentiation and was suitable for the skin irritation test. HSP27 also displayed an up-regulated expression in the ROSM in 24 h after the irritants exposure for 15 min. All these results suggest that HSP27 may represent a potential marker or additional endpoint for the hazard assessment of skin irritation caused by chemical products.     

S-Phenylcysteine formation in hemoglobin as a biological exposure index to benzene

Benzene is metabolized to intermediates that bind to hemoglobin, forming adducts. These hemoglobin adducts may be usable as biomarkers of exposure. In this paper, we describe the development of a gas chromatography/mass spectroscopy assay for quantitating the binding of the benzene metabolite, benzene oxide, to cysteine groups in hemoglobin. We used this assay to study the hemoglobin adduct, S-phenylcysteine (SPC), in the blood of rats and mice exposed to benzene either by inhalation or by gavage. We were able to detect SPC in the hemoglobin of exposed rats and mice, to show the linearity of the exposure dose-response relationship, and to establish the sensitivity limits of this assay. For the same exposure regime, rats showed considerably higher levels of SPC than did mice. As yet, we have not been able to detect SPC in the globin of humans occupationally exposed to benzene. We attempted to determine whether the SPC found in hemoglobin originated from the metabolism of benzene within or outside of the red blood cell. We hypothesized that the greatest red blood cell metabolism would be associated with peripheral reticulocytes, which retain high metabolic capacity. After exposing rats to benzene, we isolated the red blood cells and used discontinuous Percoll gradients to fractionate them into age groups. No differences in SPC levels were found among any of the fractions, suggesting that the SPC found in globin originates from the metabolism of benzene to benzene oxide in a location external to the red blood cell. To our knowledge, this is the first demonstration of the nonenzymatic binding of the benzene metabolite, benzene oxide, to protein. The products of the interaction of benzene oxide with protein are particularly appealing as potential biomarkers. These adducts are stable both in vivo and during isolation, and because high background levels in unexposed individuals are unlikely, the presence of such adducts should be a specific indicator of exposure to benzene. If SPC can be detected in humans, the present assay, with some modification, may be amenable to routine monitoring of worker exposures to benzene.     

重组人皮肤模型在化妆品皮肤刺激性评价中的应用研究

目的 探讨重组人皮肤模型应用于化妆品体外皮肤刺激性评价的可行性.方法 以重组人皮肤模型(EpiSkinTM)为受试模型,在对10个已知刺激性分类的标准化学品进行方法验证的基础上,再根据化妆品的使用特性对23个产品进行皮肤刺激性分类评价,其中6个基础型化妆品和10个美容型化妆品暴露18 h、7个清洁型化妆品分别暴露18、...     

Toxicity and penetration of TiO2 nanoparticles in hairless mice and porcine skin after subchronic dermal exposure

The present study investigated the penetration and potential toxicity of titanium dioxide (TiO₂) nanoparticles following its dermal exposure in vitro and in vivo. In vitro, after exposure to isolated porcine skin for 24 h, titanium dioxide nanoparticles of carious sizes cannot penetrate through stratum corneum. Interestingly, when studied in vivo, quite different results were obtained. After topically applied on pig ear for 30 days, TiO₂ nanomaterials (4 nm and 60 nm) can penetrate through horny layer, and be located in deep layer of epidermis. Furthermore, after 60 days dermal exposure in hairless mice, nano-TiO₂ particles can penetrate through the skin, reach different tissues and induce diverse pathological lesions in several major organs. Notably, P25 (21 nm) TiO₂ nanomaterials shows a wider tissue distribution, and can even be found in the brain without inducing any pathological changes. Among all of the organs examined, the skin and liver displayed the most severe pathological changes that correspond to the significant changes in SOD and MDA levels. These results suggest that the pathological lesions are likely to be mediated through the oxidative stress induced by the deposited nanoparticles. Accordingly, the collagen content expressed as HYP content are also significantly reduced in mouse skin samples, indicating that topically applied nano-TiO₂ in skin for a prolonged time can induce skin aging. Altogether, the present study indicates that nanosize TiO₂ may pose a health risk to human after dermal exposure over a relative long time period.     

Risk assessment of local dermal effects and skin sensitisation under the EU Chemicals Regulation REACH: a proposal for a qualitative, exposure scenario specific, approach

Within the framework of REACH, an assessment regarding local dermal effects and skin sensitisation should be performed for substances. Quantitative hazard information for these effects is often not available. Furthermore, it is difficult to relate the way in which animals are exposed in dermal toxicity studies directly to dermal exposure in practice. In the absence of quantitative information, a qualitative assessment for dermal effects is the most reasonable option. The qualitative approach as proposed in the REACH guidance recommends only general risk management measures (RMM) for three categories with a low, moderate and high identified hazard, without specifying which RMM are needed for a specific exposure scenario. We propose to differentiate frequency of exposure based on differences in activities and to compare measured and estimated local skin exposure levels with rules of thumb for evaluation of control of risks per hazard category. For workers, specific RMM regimes are assigned to each combination of hazard category and process category (PROC). For consumers, a strategy in which RMM are arranged from product-integrated measures to the use of personal protective equipment (PPE) is presented. Our approach may be transferred into automated assessment tools like Chesar and CEFIC GES.     

Testicular atrophy and loss of nerve growth factor-immunoreactive germ cell line in rats exposed to n-hexane and a protective effect of simultaneous exposure to toluene or xylene

Testicular and germ cell line morphology in rats were studied 2 weeks, 10 months and 14 months after cessation of a 61-day inhalation exposure to 1000 ppm n-hexane. Androgen biosynthetic capacity of testis, testosterone blood concentration, vas deferens morphology and noradrenaline (NA) concentration, epididymal sperm morphology, and fertility were also studied. Severe testicular atrophy involving the seminiferous tubules with loss of the nerve growth factor (NGF) immunoreactive germ cell line was found. Total loss of the germ cell line was found in a fraction of animals up to 14 months post-exposure, indicating permanent testicular damage. No impairment of androgen synthesis or androgen dependent accessory organs was observed. Simultaneous administration of 1000 ppm n-hexane and 1000 ppm toluene, or 1000 ppm n-hexane and 1000 ppm xylene, did not cause germ cell line alterations or testicular atrophy. Toluene and xylene were thus found to protect from n-hexane induced testicular atrophy.     

复方咪康唑乳膏豚鼠皮肤刺激性试验观察

目的:探讨复方眯康唑乳膏使用的安全性.方法:以1次给药和多次给药方法,将复方咪康唑乳膏外涂于白色豚鼠背部正常或破损去毛区皮肤,观察豚鼠皮肤刺激性反应.结果:复方咪康唑乳膏对豚鼠正常皮肤平均反应分值均低于0.5分,对豚鼠破损皮肤平均反应分值在给药后1 h和24 h均高于0.5分,但低于2.99分,48 h后平均反应分值均低于0.5分.结论:复方眯康唑乳膏对豚鼠正常皮肤无刺激性,对破损皮肤有轻度刺激性,但给药后48 h,这种刺激性消失.     

Effect of jet fuels on the skin morphology and irritation in hairless rats

Jet A and JP-8 are the major jet fuels used in civilian and military (US Air Force) flights, respectively. JP-8+100 is a new jet fuel recently introduced by US Air Force in some of its locations. The purpose of this study was to investigate the effects of dermal exposure of jet fuels (Jet A, JP-8, and JP-8+100) on the skin morphology, barrier function, moisture content, blood flow, and skin irritation (erythema and edema) in hairless rats. Jet fuels were applied by both occlusive and unocclusive methods. The skin of treated and control (untreated) sites were excised and analyzed by magnetic resonance imaging (MRI) (500 MHz, 11.7 Tesla). Unocclusive application of JP-8, Jet A, and JP-8+100 increased the transepidermal water loss (TEWL) gradually and the values at 120 h were significantly greater than the baseline value (P<0.05). Both occlusive and unocclusive application of jet fuels decreased the skin moisture content significantly (P<0.05). Unocclusive application of JP-8, Jet A, and JP-8+100 increased the skin blood flow, though the values returned to the baseline levels within 24 h. Occlusive application of jet fuels (8 h/day for 2 days) caused a substantial increase in the skin blood flow and the values at 48 h were about 6-fold greater than the baseline value. Occlusive application of jet fuels caused a moderate to severe erythema and a moderate edema. MRI was used to obtain proton images and water self-diffusion maps of hairless rat skin exposed to jet fuel. Exposure to JP-8 showed the largest difference from the control with regards to visual observations of the stratum corneum and hair follicles, while JP-8+100 appeared to affect the hair follicle region. The results of the present study demonstrate that exposure to jet fuels can disrupt the skin barrier function, cause skin irritation, and alter the skin structure (stratum corneum and viable epidermis) and MRI can be used as a tool to investigate the alterations in the skin morphology after exposure to toxic chemicals.     

The usefulness of toxicogenomics for predicting acute skin irritation on in vitro reconstructed human epidermis

In vitro models aiming at replacing the traditional animal test for determining the skin irritation potential of a test substance have been developed, evaluated in prevalidation studies and recently validated by the European Center for the Validation of Alternative Methods (ECVAM). To investigate the usefulness of toxicogenomic technologies to identify novel mechanistic endpoints for skin irritation responses, the present work challenged the human reconstituted epidermis model validated by ECVAM with four irritant chemicals and four non-classified chemicals tested at subcytotoxic concentrations. Using a specifically designed low-density DNA array, about 50 genes out of 240 were found to be significantly and differentially expressed between tissues exposed to irritant and non-irritant chemicals for at least one test chemical when compared to the seven others. These genes are involved in cell signalling, stress response, cell cycle, protein metabolism and cell structure. Among them, 16 are expressed in the same way whatever the irritant compound applied. The differential gene expressions might represent new or additional endpoints useful for the mechanistic understanding and perhaps also the hazard assessment of the skin irritation potential of chemicals and products.     

Correlation between environmental and biological monitoring of exposure to benzene in petrochemical industry operators

The present work was aimed to study in petrochemical industry operators the correlation, if any, between environmental exposure to low levels of benzene and two biological exposure indexes in end-shift urine, i.e. trans, trans-muconic acid (t,t-MA) and S-phenylmercapturic acid (SPMA). Exposure to benzene was assessed in 133 male subjects employed in outdoor operations in a petrochemical plant, using personal passive-diffusive air samplers worn at the breathing zone; adsorbed benzene was determined by GC-FID analysis. S-PMA was determined by a new HPLCMS/MS method, after (quantitative) acidic hydrolysis of the cysteine conjugate precursor. t,t-MA was measured by an HPLC-UV method. Smoking habits were assessed by means of a self-administered questionnaire.     

Correlations of the components of tea tree oil with its antibacterial effects and skin irritation

Tea tree oil (TTO), the essential oil of Melaleuca alternifolia L., is one of the most popular agents used in cosmetics. This study aimed to explore the correlations of components in TTO with its anti-acne activities and skin irritation. TTOs were isolated from the leaves (TTO-L), twigs, and branches of M. alternifolia by steam distillation, and the components analyzed by gas chromatography–mass spectrometry. Results showed that components of TTO-L satisfied the International Organization for Standardization (ISO) 4730 guidelines. TTO-L and its components, terpinen-4-ol, terpinolene, α-terpinene, and α-terpineol, had strong inhibitory activities against Propionibacterium acnes and Staphylococcus aureus. Moreover, six TTO formulas (DF-TTO) were designed according to ISO 4730 guidelines by adjusting the proportions of these four active components. All DF-TTO formulas showed a positive correlation between terpinen-4-ol concentration and anti-P. acnes activity. In the skin irritation assay, TTO-L, terpinen-4-ol, and 1,8-cineole did not cause significant skin irritation at 2% per site. In conclusion, terpinen-4-ol is the major active component responsible for TTO's antibacterial efficacy, while minor components in TTO also contributed to its efficacy. Moreover, we suggest that a concentration less than 5% is more suitable and safer for treating acne than higher concentrations.     

Hepatic metabolism of heme in rats after exposure to benzene,gasoline and kerosene

-Aminolevulinic acid (ALA) synthetase, -ALA dehydratase and heme oxygenase activities were studied in the liver of albino rats, 3 and 20 h after i.p. administration of benzene, gasoline, and kerosene. -ALA synthetase activity was increased markedly after benzene administration, while gasoline and kerosene treated groups showed decreased enzyme activity. Inhibition of -ALA dehydratase activity was observed in all three groups, but heme oxygenase activity was unchanged.