Comparison of MC4PC and MDL-QSAR rodent carcinogenicity predictions and the enhancement of predictive performance by combining QSAR models

This report presents a comparison of the predictive performance of MC4PC and MDL-QSAR software as well as a method for combining the predictions from both programs to increase overall accuracy. The conclusions are based on 10 x 10% leave-many-out internal cross-validation studies using 1540 training set compounds with 2-year rodent carcinogenicity findings. The models were generated using the same weight of evidence scoring method previously developed [Matthews, E.J., Contrera, J.F., 1998. A new highly specific method for predicting the carcinogenic potential of pharmaceuticals in rodents using enhanced MCASE QSAR-ES software. Regul. Toxicol. Pharmacol. 28, 242-264.]. Although MC4PC and MDL-QSAR use different algorithms, their overall predictive performance was remarkably similar. Respectively, the sensitivity of MC4PC and MDL-QSAR was 61 and 63%, specificity was 71 and 75%, and concordance was 66 and 69%. Coverage for both programs was over 95% and receiver operator characteristic (ROC) intercept statistic values were above 2.00. The software programs had complimentary coverage with none of the 1540 compounds being uncovered by both MC4PC and MDL-QSAR. Merging MC4PC and MDL-QSAR predictions improved the overall predictive performance. Consensus sensitivity increased to 67%, specificity to 84%, concordance to 76%, and ROC to 4.31. Consensus rules can be tuned to reflect the priorities of the user, so that greater emphasis may be placed on predictions with high sensitivity/low false negative rates or high specificity/low false positive rates. Sensitivity was optimized to 75% by reclassifying all compounds predicted to be positive in MC4PC or MDL-QSAR as positive, and specificity was optimized to 89% by reclassifying all compounds predicted negative in MC4PC or MDL-QSAR as negative.     

BALB/c 3T3 cell transformation assay for the prediction of carcinogenic potential of chemicals and environmental mixtures

The prediction of the carcinogenic risk for humans is mostly based on animal experiments. For the last 20 years, however, the scientific community has paid great attention to alternative strategies in compliance with common moral and ethical values. The new European chemical regulation REACH (Reg. EC 1907/2006) requires the performance of new studies in vertebrates only as a last resort. REACH asks for the development of validated in vitro protocols that can replace, in the medium to the long term, animal bioassays. An in vitro cell transformation assay (CTA) is proposed as an alternative to in vivo carcinogenicity testing. This assay is reported in the list of accepted methods for REACH (Reg. EC 440/2008).The BALB/c 3T3 model represents one of the most well-known CTAs and is regarded as a useful tool to screen single chemicals or complex mixtures for carcinogenicity prediction. In this study we used a modified protocol to highlight the transforming potential of three single compounds, ethinylestradiol (EE), azathioprine (AZA-T), melphalan, and two polychlorinated biphenyls (PCBs) mixtures, which are known or suspected to be human carcinogens. We also evaluated the activity of the antioxidant alpha-lipoic acid (ALA), a promising tumor chemopreventive. A significant increase in transformation frequency was observed when the BALB/c 3T3 cells were exposed to EE, AZA-T or melphalan as well as after PCBs treatment. On the contrary, ALA did not induce any increase of foci occurrence. Our results confirm the suitability of the improved protocol to discriminate carcinogenic compounds and support the use of BALB/c 3T3 cell transformation assay as a possible alternative to predict carcinogenic risk to humans.     

内科、外科、重症监护病房患者鲍氏不动杆菌耐药性分析和同源性研究

目的　调查鲍氏不动杆菌临床分离株的耐药性 ,并通过对鲍氏不动杆菌耐药表型的聚类分析 ,探讨临床分离株的同源性。方法　鲍氏不动杆菌药物敏感性分析采用纸片扩散法 (K B法 ) ,用WHONET5软件分析结果 ;聚类分析使用SPSS10 0统计学分析软件进行 ;耐药率的显著性检验用 χ2 检验。结果　四川大学华西医院 82株鲍氏不动杆菌临床分离株的主要标本来源为痰 ,占 91 5 % ;从内科、外科和重症监护病房患者分离的菌株分别为 3 7株、15株和 2 4株 ,分别占 45 1%、18 3 %和 2 9 3 %。 82株鲍氏不动杆菌对亚胺培南、环丙沙星、头孢他啶和阿米卡星的耐药率分别为 12 5 %、3 7 8%、40 7%和 42 0 %。重症监护病房和外科分离的菌株对头孢噻肟、阿米卡星、庆大霉素、氨曲南和磺胺甲口恶唑 /甲氧苄啶的耐药率明显高于从内科分离的菌株(P <0 0 5 )。聚类分析表明 ,内科分离的菌株 762 3 3 2和 7693 17、5 4744和 2 80 81、3 0 0 73和 3 0 2 0 0 ,重症监护病房的 18681、7872 3 2、73 1161和 785 0 10、781971和 7860 90、785 2 46和 770 0 10、786110和 764 616和外科病房的 766969和 783 14 1,为分离于相同或相邻病房且间隔时间较短的同源菌株 ,其中多数为多重耐药株。结论　重症监护病房和外科分离的菌株对头孢噻     

pH‐Dependence and QSAR analysis of the toxicity of phenols and anilines to Daphnia magna

The toxicity of 13 anilines and 11 phenols to Daphnia magna has been assessed at pH values of 6.0, 7.8, and 9.0. Toxicity of anilines decreases with decreasing pH, whereas the toxicity of phenols decreases with increasing pH. These variations in toxicity, are clearly brought about by the effect of ionization, especially for the phenols. Toxicity decreased with an increasing proportion of ionized molecules. The relative contribution of the ionized and unionized molecules was assessed. It is believed that, at least for the phenols, the ionized form of the compounds does contribute to the toxicity of the molecules. However, the decreased overall toxicity suggests that a lower relative toxicity is attributable to the ionized form. Attempts were also made to develop quantitative structure‐activity relationship (QSAR) models for these toxicity data. Reasonable QSARs were produced for the anilines and phenols, acting as polar narcotics, considered together; these QSARs included a measure of hydrophobicity, pK_a, and hydrogen bonding. When the phenols and anilines were considered separately, better, hydrophobicity‐based, QSARs were obtained. Most compounds, with the exception of 2,4‐dinitrophenol, resorcinol, and pentachlorophenol, were thought to be acting by a polar narcosis mechanism of toxic action. There was some evidence that 2,4‐dichlorophenol and 2,4,6‐trichlorophenol may be acting as weak acid respiratory uncouplers, despite earlier suggestions that they act as polar narcotics. © 2000 John Wiley & Sons, Inc. Environ Toxicol 15: 140–148, 2000     

Molecular docking reveals the potential of phthalate esters to inhibit the enzymes of the glucocorticoid biosynthesis pathway

Glucocorticoids (GCs) are well known to exert broad‐based effects on metabolism, behavior and immunity. Their impaired synthesis and production lead to adverse health effects. Some environmental toxicants, including phthalate esters (PAEs), are associated with endocrine disruption. These endocrine‐disrupting chemicals (EDCs) also cause adrenal toxicity and alteration of GC biosynthesis and their functions. Using in silico tools of Schrodinger Maestro 9.4, we performed a molecular docking study of 32 ligands including PAEs of a known endocrine‐disrupting potential with the selected enzymes of the GC biosynthesis pathway (GBP) such as CYP11A1, CYP11B2, CYP19A1, CYP17A1, CYP21A2 and 3α/20β‐HSD. Binding affinities of the PAEs were compared with known inhibitors of these enzymes. Amongst PAEs, diphenyl benzene‐1, 2 – dicarboxylate (DPhP) showed the lowest docking score of −8.95616 kcal mol⁻¹ against CYP21A1. Besides, benzyl butyl benzene‐1,2‐dicarboxylate (BBzP), bis(7‐methylnonyl) benzene‐1,2 dicarboxylate (DIDP) and bis(2‐ethylhexyl) benzene‐1,2‐dicarboxylate (DEHP) also showed comparable molecular interaction with enzymes of GBP. DPhP showed a significant molecular interaction with different enzymes of GBP such as CYP21A1, CYP11A1 and CYP11B2. These interactions mainly included H‐bonding, hydrophobic, polar and van dar Waals' interactions. Interestingly, this in silico study revealed that certain PAEs have more inhibitory potential against enzymes of GBP than their respective known inhibitors. Such studies become more relevant in the risk assessment of exposure to mixtures of phthalate eaters. Copyright © 2016 John Wiley & Sons, Ltd.     

Differences in gene expression profiles in the liver between carcinogenic and non-carcinogenic isomers of compounds given to rats in a 28-day repeat-dose toxicity study

Some compounds have structural isomers of which one is apparently carcinogenic, and the other not. Because of the similarity of their chemical structures, comparisons of their effects can allow gene expression elicited in response to the basic skeletons of the isomers to be disregarded. We compared the gene expression profiles of male Fischer 344 rats administered by daily oral gavage up to 28 days using an in-house oligo microarray. 2-Acetylaminofluorene (2-AAF), 2,4-diaminotoluene (2,4-DAT), 2-nitropropane (2-NP), and 2-nitro-p-phenylenediamine (2-NpP) are hepatocarcinogenic. However, their isomers, 4-acetylaminofluorene (4-AAF), 2,6-diaminotoluene (2,6-DAT), 1-nitropropane (1-NP), and 4-nitro-o-phenylenediamine (4-NoP), are non-hepatocarcinogenic. Because of the limited carcinogenicity of 2-NpP, we attempted to perform two-parametric comparison analyses with (1) a set of 4 isomers: 2-AAF, 2,4-DAT, 2-NP, and 2-NpP as "carcinogenic", and 4-AAF, 2,6-DAT, 1-NP, and 4-NoP as "non-carcinogenic"; and (2) a set of 3 isomers: 2-AAF, 2,4-DAT, and 2-NP, as "carcinogenic", and 4-AAF, 2,6-DAT, and 1-NP as "non-carcinogenic". After ratio filtering and Welch's approximate t-test analysis, 54 and 28 genes were selected from comparisons between the sets of 3 and 4 isomers, respectively, for day 28 data. Using hierarchical clustering analysis with the 54 or 28 genes, 2-AAF, 2,4-DAT, and 2-NP clustered into a "carcinogenic" branch. 2-NpP was in the same cluster as 4-NoP and 4-AAF. This clustering corresponded to the previous finding that 2-NpP is not carcinogenic in male Fischer 344 rats, which indicates that comparing the differences in gene expression elicited by different isomers is an effective method of developing a prediction system for carcinogenicity.     

基于主成分和聚类分析区分牡丹皮药材的差异性

目的：采用主成分和聚类分析对16批不同产地的牡丹皮药材进行分析,比较不同批次之间的差异。方法：选取每批牡丹皮药材色谱图中峰面积＞总面积1%的色谱峰作为变量,用SPSS 13.0软件进行主成分和聚类分析。结果：根据色谱图提取出3个主成分,累积贡献率84.011%,可基本描述30个变量的变异情况,不同产地的牡丹皮实现了良好的区分,共聚成四类。主成分分析和聚类分析的结果与牡丹皮药材产地有较好的一致性。结论：与指纹图谱相比,主成分和聚类分析对药材差异的区分更有效。     

Characterization and validation of an in silico toxicology model to predict the mutagenic potential of drug impurities

Control and minimization of human exposure to potential genotoxic impurities found in drug substances and products is an important part of preclinical safety assessments of new drug products. The FDA's 2008 draft guidance on genotoxic and carcinogenic impurities in drug substances and products allows use of computational quantitative structure-activity relationships (QSAR) to identify structural alerts for known and expected impurities present at levels below qualified thresholds. This study provides the information necessary to establish the practical use of a new in silico toxicology model for predicting Salmonella t. mutagenicity (Ames assay outcome) of drug impurities and other chemicals. We describe the model's chemical content and toxicity fingerprint in terms of compound space, molecular and structural toxicophores, and have rigorously tested its predictive power using both cross-validation and external validation experiments, as well as case studies. Consistent with desired regulatory use, the model performs with high sensitivity (81%) and high negative predictivity (81%) based on external validation with 2368 compounds foreign to the model and having known mutagenicity. A database of drug impurities was created from proprietary FDA submissions and the public literature which found significant overlap between the structural features of drug impurities and training set chemicals in the QSAR model. Overall, the model's predictive performance was found to be acceptable for screening drug impurities for Salmonella mutagenicity.     

基于主成分分析和判别分析对头茬茸和二茬茸化学成分的对比研究

目的:为研究头茬茸和二茬茸化学成分的差异,以多种化学成分为指标,综合评价头茬茸和二茬茸的品质,并通过判别分析进一步探讨其分类方法.方法:采用多种分析技术手段,测定并比较了头茬茸和二茬茸中多糖、粗蛋白、胶原蛋白、硫酸软骨素、氨基酸、脂肪酸、矿质元素、生物胺、核苷9类化学成分的含量差异,并用SPSS进行主成分分析和判别分析...     

多元非线性回归与BP神经网络在香菇多糖提取工艺研究中的应用

目的 探讨多元非线性回归与BP神经网络在香菇多糖提取工艺研究中的应用;方法 使用香菇多糖提取工艺中提取时间、提取温度、料液比、醇析乙醇量和多糖提取率的实验结果,分别建立多元非线性回归方程和BP神经网络;结果 多元非线性回归拟合mse为0.1483,预测误差为1.44%;BP神经网络拟合mse为0.1474,预测误差为1.29%;结论 多元非线性回归与BP神经网络均可用于多因素的非线性模型建立.     

Metabonomic analysis of quercetin against the toxicity of chronic exposure to a mixture of four organophosphate pesticides in rat plasma

1.?A metabonomics approach was performed to investigate the effect of quercetin on the toxicity of chronic exposure to a mixture of four organophosphate pesticides (OPs) at their corresponding no-observed-adverse-effect level (NOAEL). The rats were divided into six groups (n?=?10/group): control, two different doses of quercetin, OPs mixture and different doses of quercetin plus OPs mixture-treated groups.

2.?Nine metabolites, including two quercetin metabolites and seven endogenous metabolites were identified in plasma. The intensities of metabolites significantly changed in the OP mixture-treated group compared with the control group (p?<?0.01), such as lysoPE (16:0/0:0), lysoPC (17:0/0:0), lysoPC (15:0/0:0) and 4-pyridoxic acid, significantly increased; by contrast, the intensities of arachidonic acid and citric acid significantly decreased. Anomalous intensity changes in aforementioned metabolites were alleviated in the OP mixture plus 50?mg/kg?bw/d quercetin-treated group compared with the OP mixture-treated group (p?<?0.05).

3.?The results indicated that quercetin elicited partial protective effects against the toxicity induced by a mixture of OPs, which include regulation of lipid metabolism, improvement of tricarboxylic acid (TCA) cycle disorders, enhancement of antioxidant defence system to protect the liver.     

Madaline网络在复方雷琐辛醇溶液定量分析中的应用

探讨复方雷琐辛溶液中的三组分的含量。方法：采用混合液的紫外光谱数据和ｍａｄａｌｉｎｅ网络,结果：苯酚和雷琐辛及水杨酸的平均回收率分别为９９．８８％,１００．１％和９９．９９％;ＲＳＤ分别为１．９１％,１．７５％和１．８７％,结论可采用Ｍａｄａｌｉｎｅ网络进行复方雷琐辛醇溶液的紫外定量。     

Bioequivalence assessment of diltiazem preparations by means of discriminant analysis of data from solid-phase extraction and liquid chromatography

A solid-phase extraction technique for sample clean-up coupled with a new LC procedure is reported for the assay of diltiazem in plasma. The use of disposable cartridges provides selective extraction and easy automation. A new LC system based on LiChrospher RP 60 Select B columns is described. For routine analysis, the procedure provides a rapid simultaneous clean-up of several samples prior to chromatography and reproducible recoveries over a concentration range of 10-800 ng. The procedure was used to analyse the plasma samples from a bioequivalence study of three commercial diltiazem preparations. The pharmacokinetic parameters in 12 healthy male volunteers were determined and the assessment of bioequivalence was conducted by discriminant analysis.     

Properties of flavonoids influencing the binding to bilitranslocase investigated by neural network modelling

Bilitranslocase is a plasma membrane carrier firstly identified on the sinusoidal (vascular) domain of liver cells and later on also in the gastric epithelium. It transports diverse organic anions, such as bilirubin, some phthaleins and many dietary anthocyanins, suggesting that it could play a role both in the absorption of flavonoids from dietary sources and in their hepatic metabolism. This work was aimed at characterising the interaction of bilitranslocase with flavonols, a flavonoid sub-class. The results obtained show that, contrary to anthocyanins, flavonol glycosides do not interact with the carrier, whereas just some of the corresponding aglycones act as relatively poor ligands to bilitranslocase. These data point to a clear-cut discrimination between anthocyanins and flavonols occurring at the level of the bilitranslocase transport site. A quantitative structure-activity relationship based on counter propagation artificial neural network modelling was undertaken in order to shed light on the nature of flavonoid interaction with bilitranslocase. It was found that binding relies on the ability to establish hydrogen bonds, ruling out the involvement of charge interactions. This requisite might be at the basis of the discrimination between anthocyanins and flavonols by bilitranslocase and could lie behind some aspects of the distinct pharmacokinetic properties of anthocyanins and flavonols in mammals.     

Effects of morphine and naloxone on behaviour in the hot plate test: an ethopharmacological study in the rat

The objectives of this study were: i) to analyse the effects of morphine and naloxone on the rat's behaviour in the hot plate test using an ethological approach, and ii) to compare the effectiveness of repeated versus single test paradigms. Animals received either morphine (0, 3, 6 or 9 mg/kg SC) or naloxone (0, 0.01, 0.1 or 1 mg/kg SC). For repeated hot plate measures, rats were tested before and 60, 120, 180 and 240 min following morphine treatment, as well as 30, 60, 90 and 120 min after naloxone injection. For the single test schedule, rats were tested only once 60 min after morphine or 30 min after naloxone administration, or at 60, 120, 180, 240 and 300 min after 9 mg/kg morphine treatment. Behaviour was videotaped and analysed by an ethogram and ethological techniques. A cluster analysis revealed that the most frequently displayed patterns could be categorised into exploratory sniffing reactions (walk-sniff, immobile-sniff) and noxious-evoked elements, including primary (paw-licking, stamping), escape (jumping, leaning posture) and independent (hindleg-withdrawal) patterns. During repeated tests, morphine treatment induced: i) a maximum hypoalgesic effect 60 min post-injection (noxious-evoked patterns were significantly reduced), and ii) an unexpected thermal hyperreactivity rebound effect after 120 min (paw-licking and hindleg-withdrawal were enhanced), although changes in hindpaw-licking are more indicative of a hyperalgesic rebound effect. Most changes were quite similar during the single test schedule at 60 and 120 min after morphine injection. With regard to naloxone treatment, jumping latency was significantly decreased during the repeated test schedule, but not on single exposure to the plate. Other elements were facilitated, however, in the single test (stamping, leaning posture, hindleg-withdrawal). The results indicated that both repeated and single tests paradigms are of value for testing the effects of morphine and naloxone on rats. However, under our conditions the single test paradigm gave a better picture of the overall effects of the drug. Learning as well as habituation and sensitization may mask certain effects during repeated tests. In conclusion, an ethological analysis of the rat's behaviour in the hot plate test following administration of morphine and naloxone has been validated in this study.     

Prior exposure to organophosphorus and organochlorine pesticides increases the allergic potential of environmental chemical allergens in a local lymph node assay

The dysregulation of immune functions by some pesticides leads to various immune disorders, including immunodeficiency, tumorigenesis, allergies, and autoimmunity. This study's primary objective was to examine the relationship between immune disorders and the immunosuppression induced by immunosuppressive pesticides. We focused on the modulation of allergic potential by the organophosphorus pesticide parathion, organochlorine pesticide methoxychlor, phenoxyacetic acid herbicide 2,4-d-butyl, and benzoic acid fungicide eugenol, as detected by a local lymph node assay (LLNA), which was developed initially for hazard identification of skin sensitization. Parathion and methoxychlor are immunosuppressive chemicals, and 2,4-d-butyl and eugenol are contact allergens. After the immunosuppressive characteristics of parathion and methoxychlor were confirmed in a pilot study, 4-week-old mice were orally administered parathion (0, 0.4, 1.2mg/kg) or methoxychlor (0, 100, 300 mg/kg). Four weeks after the last administration, an LLNA was conducted using 2,4-d-butyl (0%, 2.5%, 5%, and 10%) and eugenol (0%, 5%, 10%, and 25%). In addition, detailed analysis of their auricular lymph nodes for number of surface antigen expression of T cells and local cytokine production were performed using 5% 2,4-d-butyl and 5% eugenol treatment groups. EC3 values (estimated concentration to yield a stimulation index of 3) of 2,4-d-butyl and eugenol decreased markedly in parathion- and methoxychlor-pretreated groups. Parathion- and methoxychlor-pretreated groups induced marked increase in number of surface antigen expression of T cells and levels of Th1 cytokines (IFN-γ, TNF-α, and IL-17) produced by ex vivo restimulated lymph node cells. According to our results, the allergic potentials of 2,4-d-butyl and eugenol are increased by prior exposure to parathion and methoxychlor.     

Molecular characterization of Salmonella Typhimurium and Salmonella Enteritidis by plasmid analysis and pulsed-field gel electrophoresis

Forty-one paediatric isolates of Salmonella spp. from Cerrahpasa Faculty of Medicine, Istanbul, Turkey, between 2001 and 2004 were examined for susceptibility to various antibiotics and presence of antibiotic resistance genes. Pulsed-field gel electrophoresis and plasmid profiling were used to determine possible genetic relationships among Salmonella enterica subsp. enterica clinical isolates. Plasmids from resistant strains were not transferred by conjugation to recipient Escherichia coli cells. Pulsed-field gel electrophoresis and restriction enzyme digestion analysis of DNA revealed that multidrug-resistant isolates belonged to the same clonal group, characterized by ACSSuT resistotype. Isolates of R-type ACSSuT were positive for the intI gene and possessed a single plasmid of 60 MDa.     

A study of the potential for a herbicide formulation containing 2,4-d and picloram to cause male-mediated developmental toxicity in rats.

Male Vietnam veterans have repeatedly expressed concern that exposure to herbicides in Vietnam may have caused birth defects in their offspring. The second most used herbicide was a mixture of 2,4-D and picloram called Agent White. This study is an investigation into the possible male-mediated reproductive toxicology of this herbicide. Male rats were gavaged for 5 days per week for 9 weeks with a mixture of 2,4-D and picloram called Tordon 75D(R) (the Australian derivative of Agent White). Three doses were tested; the high dose was considered the maximum tolerated dose. Each male was mated with two untreated females during weeks 2 and 3, 4 and 5, and 8 and 9 of treatment, and with four untreated females after an 11-week recovery period. Negative controls were males dosed with distilled water, and positive controls were males dosed with cyclophosphamide at 5.1 mg/kg/day. All mated females were killed on day 20 of gestation, and the fetuses were weighed and examined for either structural malformations or skeletal development. Litter size, fetal weight, and malformation rate were all unaffected by treatment. The cyclophosphamide positive controls showed the expected large increase in postimplantation loss. In general, within the limitations of the power of the study, the results did not show any evidence that exposure to a herbicide formulation containing 2,4-D and picloram is likely to cause male-mediated birth defects or other adverse reproductive outcomes.     

Use of real-time breath analysis and physiologically based pharmacokinetic modeling to evaluate dermal absorption of aqueous toluene in human volunteers.

Toluene is a ubiquitous chemical that is commonly used for its solvent properties in industry and manufacturing, and is a component of many paint products. Although human exposure to toluene is most likely to be through inhalation, toluene is also found in well and surface water. Therefore, an assessment of the dermal contribution to total toluene uptake is useful for understanding human exposures. To evaluate the significance of these exposures, the dermal absorption of toluene was assessed in human volunteers using a combination of real-time exhaled breath analysis and physiologically based pharmacokinetic (PBPK) modeling. Human volunteers wearing swimsuits were submerged in warm tap water to neck level in a stainless steel hydrotherapy tub containing an initial concentration of approximately 500-microg/l toluene. Volunteers were provided purified breathing air to eliminate inhalation exposures, and exhaled breath was continually analyzed before, during, and post exposure to track the absorption and subsequent elimination of the compound in real time. A PBPK model was used to estimate the dermal permeability coefficient (K(p)) to describe each set of exhaled breath data from 4-6 human volunteers. An average K(p) value of 0.012 +/- 0.007 cm/h was found to provide a good fit to all data sets. Volunteers also participated in a second study phase, in which the subject was allowed to breathe the room air during immersion, thus both dermal and inhalation exposures to toluene occurred. Exhaled breath analyses revealed that concurrent inhalation of volatilized toluene resulted in a transient increase in the peak exhaled-breath level by 100 ppb, or an approximate 50% increase over breath levels observed in dermal-only studies. For perspective, the total intake of toluene associated with oral consumption of 2 liters of water containing toluene at bath water concentrations were estimated to be more than 30 times greater than the dermal contribution due to bathing.