Evaluating the in vitro developmental toxicity potency of a series of petroleum substance extracts using new approach methodologies (NAMs).

The present study evaluates the in vitro developmental toxicity and the possible underlying mode of action of DMSO extracts of a series of highly complex petroleum substances in the mouse embryonic stem cell test (mEST), the zebrafish embryotoxicity test (ZET) and the aryl hydrocarbon receptor reporter gene assay (AhR CALUX assay). Results show that two out of sixteen samples tested, both being poorly refined products that may contain a substantial amount of 3- to 7-ring polycyclic aromatic compounds (PACs), induced sustained AhR activation in the AhR CALUX assay, and concentration-dependent developmental toxicity in both mEST and ZET. The other samples tested, representing highly refined petroleum substances and petroleum-derived waxes (containing typically a very low amount or no PACs at all), were negative in all assays applied, pointing to their inability to induce developmental toxicity in vitro. The refining processes applied during the production of highly refined petroleum products, such as solvent extraction and hydrotreatment which focus on the removal of undesired constituents, including 3- to 7-ring PACs, abolish the in vitro developmental toxicity. In conclusion, the obtained results support the hypothesis that 3- to 7-ring PACs are the primary inducers of the developmental toxicity induced by some (i.e., poorly refined) petroleum substances and that the observed effect is partially AhR-mediated.

Metabolism of alcohol ethoxylates (AEs) in rat, hamster, and human hepatocytes and liver S9: a pilot study for metabolic stability, metabolic pathway, and metabolites identification in vitro and in silico.

Alcohol ethoxylates (AEs) are a well-known class of non-ionic surfactants widely used by the personal care market. The aim of this study was to evaluate and characterize the in vitro metabolism of AEs and identify metabolites. Five selected individual homologue AEs (C8EO4, C10EO5, C12EO4, C16EO8, and C18EO3) were incubated using human, rat, and hamster liver S9 fraction and cryopreserved hepatocytes. LC-MS was used to identify metabolites following the incubation of AEs by liver S9 and hepatocytes of all three species. All AEs were metabolized in these systems with a half-life ranging from 2 to 139 min. In general, incubation of AE with human liver S9 showed a shorter half-life compared to rat liver S9. While rat hepatocytes metabolized AEs faster than human hepatocytes. Both hydrophobic alkyl chain and hydrophilic EO head group groups of AEs were found to be target sites of metabolism. Metabolites were identified that show primary hydroxylation and dehydrogenation, followed by O-dealkylation (shortening of EO head groups) and glucuronidation. Additionally, the detection of whole EO groups indicates the cleavage of the ether bond between the alkyl chain and the EO groups as a minor metabolic pathway in the current testing system. Furthermore, no difference in metabolic patterns of each individual homologue AE investigated was observed, regardless of alkyl chain length or the number of EO groups. Moreover, there is an excellent agreement between the in vitro experimental data and the metabolite profile simulations using in silico approaches (OECD QSAR Toolbox). Altogether, these data indicate fast metabolism of all AEs with a qualitatively similar metabolic pathway with some quantitative differences observed in the metabolite profiles. These metabolic studies using different species can provide important reference values for further safety evaluation.

The role of receptor-mediated activities of 4- and 5-ring unsubstituted and methylated polycyclic aromatic hydrocarbons (PAHs) in developmental toxicity.

The present study evaluated the aryl hydrocarbon receptor (AhR), estrogen receptor-α (ER-α), and retinoic acid receptor (RAR) mediated activities of nine 4- and 5-ring unsubstituted and monomethylated polycyclic aromatic hydrocarbons (PAHs) using a series of Chemical-Activated LUciferase gene eXpression (CALUX) assays. The potential role of these aforementioned receptors in relation to the developmental toxicity of these PAHs was further assessed in the zebrafish embryotoxicity test (ZET). The results show that all nine tested PAHs were AhR agonists, benz[a]anthracene (BaA) and 8-methyl-benz[a]anthracene (8-MeBaA) were ER-α agonists, and none of the tested PAHs induced ER-α antagonistic or RAR (ant)agonistic activities. In the AhR CALUX assay, all the methylated PAHs showed higher potency (lower EC50) in activating the AhR than their respective unsubstituted PAHs, implying that the addition of a methyl substituent on the aromatic ring of PAHs could enhance their AhR-mediated activities. Co-exposure of zebrafish embryos with each individual PAH and an AhR antagonist (CH223191) counteracted the observed developmental retardations and embryo lethality to a certain extent, except for 8-methyl-benzo[a]pyrene (8-MeBaP). Co-exposure of zebrafish embryos with either of the two estrogenic PAHs (i.e., BaA and 8-MeBaA) and an ER-α antagonist (fulvestrant) neutralized embryo lethality induced by 50 µM BaA and the developmental retardations induced by 15 µM 8-MeBaA. Altogether, our findings suggest that the observed developmental retardations in zebrafish embryos by the PAH tested may partially be AhR- and/or ER-α-mediated, whereas the RAR seems not to be relevant for the PAH-induced developmental toxicity in the ZET.

Toxicological Assessment of Higher Olefins in OECD TG 422 Repeated Dose and Reproductive /Developmental Toxicity Screening Tests in Han Wistar Rats.

Higher olefins (HO) are used primarily as intermediates in the production of other chemicals, such as polymers, fatty acids, plasticizer alcohols, surfactants, lubricants, amine oxides, and detergent alcohols. The potential toxicity of five HO (i.e., 1-Octene, Nonene, Decene, Hexadecene, and 1-Octadecene) with carbon ranging from C8 to C18 was examined in a combined repeated dose and reproduction/developmental toxicity screening study (OECD TG 422). These five HO were administered to Han Wistar rats by gavage at 0 (controls), 100, 300, and 1000 mg/kg bw/day. As a group of substances, adaptive changes in the liver (liver weight increase without pathological evidence), as well as increased kidney weight in male rats, were observed in HO with carbon numbers from C8 to C10. The overall systemic no observed adverse effect level (NOAEL) for all HO was determined at 1000 mg/kg bw/day. In the reproductive/developmental toxicity assessment, offspring viability, size, and weights were reduced in litters from females treated with Nonene at 1000 mg/kg bw/day. The overall no observed effects level (NOEL) for reproductive toxicity was considered to be 300 mg/kg bw/day for Nonene and 1000 mg/kg bw/day for the other four HO, respectively. These data significantly enrich the database on the toxicity of linear and branched HO, allowing comparison with similar data published on a range of linear and branched HO. Comparisons between structural class and study outcome provide further supportive data in order to validate the read-across hypothesis as part of an overall holistic testing strategy.

Assessment of the Intestinal Absorption of Higher Olefins by the Everted Gut Sac Model in Combination with In Silico New Approach Methodologies.

To reduce the number of animals and studies needed to fulfill the information requirements as required by Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (EC no. 1907/2006), a read-across approach was used to support approximately 30 higher olefins. This study aimed to assess the absorption potential of higher olefins through the gut wall as the experimentally determined bioavailability which would strengthen the read-across hypothesis and justification, reducing the need for toxicity studies on all of the higher olefins. The absorption potential of a series of higher olefins (carbon range from 6 to 28, with five configurations of the double bond) was determined in the in vitro everted rat small intestinal sac model and subsequently ranked. In addition, in silico approaches were applied to predict the reactivity, lipophilicity, and permeability of higher olefins. In the in vitro model, everted sacs were incubated in "fed-state simulated small intestinal fluid" saturated with individual higher olefins. The sac contents were then collected, extracted, and analyzed for olefin content using gas chromatography with a flame ionization detector. The C6 to C10 molecules were readily absorbed into the intestinal sacs. Marked inter-compound differences were observed, with the amount of absorption generally decreasing with the increase in carbon number. Higher olefins with ≥C14 carbons were either not absorbed or very poorly absorbed. In the reactivity simulation study, the reactivity is well described by the position of the double bond rather than the number of carbon atoms. In the lipophilicity and permeability analysis, both parameter descriptors depend mainly on the number of carbon atoms and less on the position of the double bond. In conclusion, these new approach methodologies provide supporting information on any trends or breakpoints in intestinal uptake and a hazard matrix based on carbon number and position of the double bond. This matrix will further assist in the selection of substances for inclusion in the mammalian toxicity testing programme.

Human biomonitoring of low-level benzene exposures.

Historically, benzene has been widely used in a large variety of applications. Occupational exposure limits (OELs) were set for benzene as it was found to be acutely toxic, causing central nervous system depression at high exposures. OELs were lowered when it was discovered that chronic exposure to benzene could cause haematotoxicity. After confirmation that benzene is a human carcinogen causing acute myeloid leukaemia and possibly other blood malignancies, OEL were further lowered. The industrial application of benzene as solvent is almost completely discontinued but it is still used as feedstock for the production of other materials, such as styrene. Occupational exposure to benzene may also occur since it is present in crude oil, natural gas condensate and a variety of petroleum products and because benzene can be formed in combustion of organic material. In the past few years, lower OELs for benzene in the range of 0.05-0.25 ppm have been proposed or were already established to protect workers from benzene-induced cancer. The skin is an important potential route of exposure and relatively more important at lower OELs. Consequently, human biomonitoring - which integrates all exposure routes - is routinely applied to control overall exposure to benzene. Several potential biomarkers have been proposed and investigated. For compliance check of the current low OELs, urinary S-phenylmercapturic acid (S-PMA), urinary benzene and blood benzene are feasible biomarkers. S-PMA appears to be the most promising biomarker but proper validation of biomarker levels corresponding to airborne benzene concentrations below 0.25 ppm are needed.

The effect of alkyl substitution on the oxidative metabolism and mutagenicity of phenanthrene.

Alkyl-substituted PAHs may be present in certain petroleum-derived products and in the environment and may eventually end up in consumer products, such as foodstuffs, cosmetics and pharmaceuticals. Safety concerns over possible exposure to alkylated PAHs have emerged. Bioactivation is a prerequisite for the mutagenicity and carcinogenicity of PAHs and has been extensively studied for non-substituted PAHs, while data on the bioactivation of alkyl-substituted PAHs are scarce. The present study investigated the effect of alkyl substitution on the CYP 450-mediated metabolism of phenanthrene and eight of its alkylated congeners by quantifying metabolite formation in rat and human liver microsomal incubations. Furthermore, the mutagenicity of four selected methylated phenanthrenes was compared to that of phenanthrene using the Ames test. The obtained results support the hypothesis that alkyl substitution shifts the oxidative metabolism from the aromatic ring to the alkyl side chain. Increasing the length of the alkyl chain reduced overall metabolism with metabolic conversion for 1-n-dodecyl-phenanthrene (C12) being negligible. 1- and 9-methyl-phenanthrene, in which the methyl group generates an additional bay region-like structural motif, showed mutagenicity toward Salmonella typhimurium TA98 and TA 100, whereas phenanthrene and also 2- and 3-methyl-phenanthrene, without such an additional bay region-like structural motif, tested negative. It is concluded that the position of the alkylation affects the metabolism and resulting mutagenicity of phenanthrene with the mutagenicity increasing in cases where the alkyl substituent creates an additional bay region-like structural motif, in spite of the extra possibilities for side chain oxidation.

RespiraTox - Development of a QSAR model to predict human respiratory irritants.

Respiratory irritation is an important human health endpoint in chemical risk assessment. There are two established modes of action of respiratory irritation, 1) sensory irritation mediated by the interaction with sensory neurons, potentially stimulating trigeminal nerve, and 2) direct tissue irritation. The aim of our research was to, develop a QSAR method to predict human respiratory irritants, and to potentially reduce the reliance on animal testing for the identification of respiratory irritants. Compounds are classified as irritating based on combined evidence from different types of toxicological data, including inhalation studies with acute and repeated exposure. The curated project database comprised 1997 organic substances, 1553 being classified as irritating and 444 as non-irritating. A comparison of machine learning approaches, including Logistic Regression (LR), Random Forests (RFs), and Gradient Boosted Decision Trees (GBTs), showed, the best classification was obtained by GBTs. The LR model resulted in an area under the curve (AUC) of 0.65, while the optimal performance for both RFs and GBTs gives an AUC of 0.71. In addition to the classification and the information on the applicability domain, the web-based tool provides a list of structurally similar analogues together with their experimental data to facilitate expert review for read-across purposes.

Predicting the in vivo developmental toxicity of benzo[a]pyrene (BaP) in rats by an in vitro-in silico approach.

Developmental toxicity testing is an animal-intensive endpoints in toxicity testing and calls for animal-free alternatives. Previous studies showed the applicability of an in vitro-in silico approach for predicting developmental toxicity of a range of compounds, based on data from the mouse embryonic stem cell test (EST) combined with physiologically based kinetic (PBK) modelling facilitated reverse dosimetry. In the current study, the use of this approach for predicting developmental toxicity of polycyclic aromatic hydrocarbons (PAHs) was evaluated, using benzo[a]pyrene (BaP) as a model compound. A rat PBK model of BaP was developed to simulate the kinetics of its main metabolite 3-hydroxybenzo[a]pyrene (3-OHBaP), shown previously to be responsible for the developmental toxicity of BaP. Comparison to in vivo kinetic data showed that the model adequately predicted BaP and 3-OHBaP blood concentrations in the rat. Using this PBK model and reverse dosimetry, a concentration-response curve for 3-OHBaP obtained in the EST was translated into an in vivo dose-response curve for developmental toxicity of BaP in rats upon single or repeated dose exposure. The predicted half maximal effect doses (ED50) amounted to 67 and 45 mg/kg bw being comparable to the ED50 derived from the in vivo dose-response data reported for BaP in the literature, of 29 mg/kg bw. The present study provides a proof of principle of applying this in vitro-in silico approach for evaluating developmental toxicity of BaP and may provide a promising strategy for predicting the developmental toxicity of related PAHs, without the need for extensive animal testing.

The role of metabolism in the developmental toxicity of polycyclic aromatic hydrocarbon-containing extracts of petroleum substances.

In vitro assays presently used for prenatal developmental toxicity (PDT) testing only assess the embryotoxic potential of parent substances and not that of potentially embryotoxic metabolites. Here we combined a biotransformation system, using hamster liver microsomes, with the ES-D3 cell differentiation assay of the embryonic stem cell test (EST) to compare the in vitro PDT potency of two 5-ring polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (BaP) and dibenz[a,h]anthracene (DBA), and dimethyl sulfoxide extracts from five PAH-containing petroleum substances (PS) and a gas-to-liquid base oil (GTLb), with and without bioactivation. In the absence of bioactivation, DBA, but not BaP, inhibited the differentiation of ES-D3 cells into beating cardiomyocytes in a concentration-dependent manner. Upon bioactivation, BaP induced in vitro PDT, while its major metabolite 3-hydroxybenzo[a]pyrene was shown to be active in the EST as well. This means BaP needs biotransformation to exert its embryotoxic effects. GTLb extracts tested negative in the EST, with and without bioactivation. The PS-induced PDT in the EST was not substantially changed following bioactivation, implying that metabolism may not play a crucial role for the PS extracts under study to exert the in vitro PDT effects. Altogether, these results indicate that although some PAH require bioactivation to induce PDT, some do not and this latter appears to hold for the (majority of) the PS constituents responsible for the in vitro PDT of these complex substances.

The selective determination of potentially carcinogenic polycyclic aromatic compounds in lubricant base oils by the DMSO extraction method IP346 and its correlation to mouse skin painting carcinogenicity assays.

Mineral oils are produced by vacuum distillation of crude oil at temperatures from ∼300 °C to ∼600 °C. Subsequent refining processes to eliminate the carcinogenic potential of mineral oils (by extraction and/or hydrotreatment) are based on the principle of removing substances associated with carcinogenic activity; i.e. PAC (polycyclic aromatic compounds), which include PAH and N or S heterocycles. Traditionally, the carcinogenic potential of the refined product was tested in the mouse skin painting assay. This bioassay is considered the gold standard for petroleum derived products since it uses the most sensitive species and route of exposure, and because mice and humans develop the same type of skin tumors it is a relevant model to assess the carcinogenic potential of mineral oils. Mouse skin painting studies have also been important in distinguishing two types of aromatic compounds found in mineral oil. The first type includes the 3-7 ring PAC associated with potential carcinogenic effects found in the 340-535 °C boiling range, which are removed by refinement. The second type includes highly alkylated aromatic compounds (predominantly 1-2 rings) which are not bioactivated and non-carcinogenic, which are typical of a refined oil. Because mouse skin painting studies are time consuming, a DMSO based method was developed that is capable to distinguish these two types of aromatics. Although this industry method, known as the IP346, has been applied for more than 30 years, the background experimental data underlying its development has not yet been published. This paper presents and discusses the chemical and biological features of mineral oil PAC structures assessed by IP346, especially the crucial role of the DMSO extraction step which allows to discriminate between the two types of aromatics. The DMSO selectivity towards the toxicological relevant PAC is discussed by comparing the composition of the DMSO extract of a distillate aromatic extract and mineral oils of varying viscosities and refining conditions. PAC which have >3 rings (naked or partially alkylated) are preferentially encompassed by the DMSO extract, whereas those PAC which have relatively long alkyl side chains are not. Thus, according to the IP346, refined oils will have lower levels of DMSO extractable material compared to less refined oils. DMSO selectivity towards the potentially carcinogenic >3 ring PAC makes the IP346 method therefore highly correlated to the outcome of mouse skin painting studies, using a pass/fail dichotomy. The accuracy, including the false negative results of the IP346 in the prediction of mineral oil carcinogenicity is discussed. The DMSO based IP346 is thus a simple but clear reflection of refinement efficacy. It links manufacturing conditions to carcinogenic potential of an oil, supported by solid physical-chemical and toxicological associations. In Europe it is the only legally binding method to assess, classify and label lubricating base oils and inherently more reliable for hazard assessment than the determination of an arbitrary selection of PAH.

Toxicological and ecotoxicological properties of gas-to-liquid (GTL) products. 2. Ecotoxicology.

Gas-to-liquid (GTL) products are synthetic hydrocarbons produced from natural gas using a catalytic process known as the Fischer-Tropsch process. This process yields a synthetic crude oil that consists of saturated hydrocarbons which can subsequently be refined to a range of products analogous to those obtained from petroleum refining. However, in contrast to their petroleum-derived analogs, GTL products are essentially free of unsaturated or aromatic compounds and do not contain any sulfur-, oxygen-, or nitrogen-containing compounds. Under new chemical substance notification requirements, an extensive testing program covering the entire portfolio of GTL products has been undertaken to assess their hazardous properties to human health and environment. The results of these studies have been summarized in a two-part review. Part 1 provides an overview of the mammalian toxicity hazardous properties of the various GTL products. This second part of the review focuses on the aquatic, sediment, terrestrial, and avian toxicity studies which assess the ecotoxicological hazard profile of the GTL products. Many challenges were encountered during these tests relating to dosing, analysis and interpretation of results. These are discussed with the intent to share experiences to help inform and shape future regulatory mandates for testing of poorly soluble complex substances. As was the case with the mammalian toxicology review, there were a few cases where adverse effects were found, but overall the GTL products were found to exert minimal adverse ecotoxicological effects and these were less severe than effects observed with their conventional, petroleum-derived analogs.

Toxicological and ecotoxicological properties of gas-to-liquid (GTL) products. 1. Mammalian toxicology.

Gas-to-liquid (GTL) products are synthetic hydrocarbons produced from natural gas using a Fischer-Tropsch process. This process yields a synthetic crude oil that consists of saturated hydrocarbons, primarily linear alkanes, with increasing amounts of branched (methyl-groups) alkanes as the chains get longer. In addition, small amounts of cycloalkanes (branched cyclopentanes and cyclohexanes) may be formed as the polymerization reaction prolongs. This synthetic crude can subsequently be refined to a range of products very similar to petroleum refining. However, in contrast to their petroleum-derived analogs, GTL products are essentially free of unsaturated or aromatic constituents and also no sulfur-, oxygen-, or nitrogen-containing constituents are present. From a regulatory perspective, GTL products are new substances which require extensive testing to assess their hazardous properties. As a consequence, a wide range of GTL products, covering the entire portfolio of GTL products, have been tested over the past few years in a wide variety of toxicological studies, including reproductive and prenatal development toxicity studies. This review provides an overview of the hazardous properties of the various GTL products. In general, the data collected on GTL products provide strong proof that they exert minimal health effects. In addition, these data provide supporting evidence for what is known on the mechanisms of mammalian toxicology of their petroleum-derived analogs. In the few cases where adverse effects were found for the GTL substances, these were usually less severe than the adverse effects observed with their petroleum-derived analogs.

Grouping of Petroleum Substances as Example UVCBs by Ion Mobility-Mass Spectrometry to Enable Chemical Composition-Based Read-Across.

Substances of Unknown or Variable composition, Complex reaction products, and Biological materials (UVCBs), including many refined petroleum products, present a major challenge in regulatory submissions under the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) and US High Production Volume regulatory regimes. The inherent complexity of these substances, as well as variability in composition obfuscates detailed chemical characterization of each individual substance and their grouping for human and environmental health evaluation through read-across. In this study, we applied ion mobility mass spectrometry in conjunction with cheminformatics-based data integration and visualization to derive substance-specific signatures based on the distribution and abundance of various heteroatom classes. We used petroleum substances from four petroleum substance manufacturing streams and evaluated their chemical composition similarity based on high-dimensional substance-specific quantitative parameters including m/z distribution, drift time, carbon number range, and associated double bond equivalents and hydrogen-to-carbon ratios. Data integration and visualization revealed group-specific similarities for petroleum substances. Observed differences within a product group were indicative of batch- or manufacturer-dependent variation. We demonstrate how high-resolution analytical chemistry approaches can be used effectively to support categorization of UVCBs based on their heteroatom composition and how such data can be used in regulatory decision-making.

Guidance on the selection of cohorts for the extended one-generation reproduction toxicity study (OECD test guideline 443).

The extended one-generation reproduction toxicity study (EOGRTS; OECD test guideline 433) is a new and technically complex design to evaluate the putative effects of chemicals on fertility and development, including effects upon the developing nervous and immune systems. In addition to offering a more comprehensive assessment of developmental toxicity, the EOGRTS offers important improvements in animal welfare through reduction and refinement in a modular study design. The challenge to the practitioner is to know how the modular aspects of the study should be triggered on the basis of prior knowledge of a particular chemical, or on earlier findings in the EOGRTS itself, requirements of specific regulatory frameworks notwithstanding. The purpose of this document is to offer guidance on science-based triggers for these extended evaluations.

An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride.

The framework analysis previously presented for using DNA adduct information in the risk assessment of chemical carcinogens was applied in a series of case studies which place the adduct information into context with the key events in carcinogenesis to determine whether they could be used to support a mutagenic mode of action (MOA) for the examined chemicals. Three data-rich chemicals, aflatoxin B1 (AFB1), tamoxifen (Tam) and vinyl chloride (VCl) were selected for this exercise. These chemicals were selected because they are known human carcinogens and have different characteristics: AFB1 forms a unique adduct and human exposure is through contaminated foods; Tam is a pharmaceutical given to women so that the dose and duration of exposure are known, forms unique adducts in rodents, and has both estrogenic and genotoxic properties; and VCl, to which there is industrial exposure, forms a number of adducts that are identical to endogenous adducts found in unexposed people. All three chemicals produce liver tumors in rats. AFB1 and VCl also produce liver tumors in humans, but Tam induces human uterine tumors, only. To support a mutagenic MOA, the chemical-induced adducts must be characterized, shown to be pro-mutagenic, be present in the tumor target tissue, and produce mutations of the class found in the tumor. The adducts formed by AFB1 and VCl support a mutagenic MOA for their carcinogenicity. However, the data available for Tam shows a mutagenic MOA for liver tumors in rats, but its carcinogenicity in humans is most likely via a different MOA.

The use of biomonitoring data in exposure and human health risk assessment: benzene case study.

Abstract A framework of "Common Criteria" (i.e. a series of questions) has been developed to inform the use and evaluation of biomonitoring data in the context of human exposure and risk assessment. The data-rich chemical benzene was selected for use in a case study to assess whether refinement of the Common Criteria framework was necessary, and to gain additional perspective on approaches for integrating biomonitoring data into a risk-based context. The available data for benzene satisfied most of the Common Criteria and allowed for a risk-based evaluation of the benzene biomonitoring data. In general, biomarker (blood benzene, urinary benzene and urinary S-phenylmercapturic acid) central tendency (i.e. mean, median and geometric mean) concentrations for non-smokers are at or below the predicted blood or urine concentrations that would correspond to exposure at the US Environmental Protection Agency reference concentration (30 µg/m(3)), but greater than blood or urine concentrations relating to the air concentration at the 1 × 10(-5) excess cancer risk (2.9 µg/m(3)). Smokers clearly have higher levels of benzene exposure, and biomarker levels of benzene for non-smokers are generally consistent with ambient air monitoring results. While some biomarkers of benzene are specific indicators of exposure, the interpretation of benzene biomonitoring levels in a health-risk context are complicated by issues associated with short half-lives and gaps in knowledge regarding the relationship between the biomarkers and subsequent toxic effects.

Guidance on classification for reproductive toxicity under the globally harmonized system of classification and labelling of chemicals (GHS).

The Globally Harmonised System of Classification (GHS) is a framework within which the intrinsic hazards of substances may be determined and communicated. It is not a legislative instrument per se, but is enacted into national legislation with the appropriate legislative instruments. GHS covers many aspects of effects upon health and the environment, including adverse effects upon sexual function and fertility or on development. Classification for these effects is based upon observations in humans or from properly designed experiments in animals, although only the latter is covered herein. The decision to classify a substance based upon experimental data, and the category of classification ascribed, is determined by the level of evidence that is available for an adverse effect on sexual function and fertility or on development that does not arise as a secondary non-specific consequence of other toxic effect. This document offers guidance on the determination of level of concern as a measure of adversity, and the level of evidence to ascribe classification based on data from tests in laboratory animals.

A systematic approach for evaluating and scoring human data.

An approach is described for how the quality of human data can be systematically assessed and categorised. The approach mirrors the animal data quality considerations set out by Klimisch et al., in order that human data quality can be addressed in a complementary manner and to help facilitate transparent (and repeatable) weight of evidence comparisons. Definitions are proposed for the quality and adequacy of data. Quality is differentiated into four categories. A description of how the scheme can be used for evaluating data reliability, especially for use when contributing entries to the IUCLID database, is shown. A discussion of how the criteria might also be used when determining overall data relevance is included. The approach is intended to help harmonise human data evaluation processes worldwide.

Comparative toxicokinetics of low-viscosity mineral oil in Fischer 344 rats, Sprague-Dawley rats, and humans--implications for an Acceptable Daily Intake (ADI).

Oral repeated-dose studies with low-viscosity mineral oils showed distinct species and strain differences, which are hypothesized to be due to differences in bioavailability, with Fischer 344 rats being more susceptible than Sprague-Dawley rats or dogs. Sensitive analytical methodology was developed for accurate measurement of low levels of mineral hydrocarbons and applied in single-dose toxicokinetics studies in rats and humans. Fischer 344 rats showed a 4-fold higher AUC(0-∞) and consistently higher blood and liver concentrations were found than Sprague-Dawley rats. Hepatic mineral hydrocarbon concentration tracked the blood concentration in both strains, indicating that blood concentrations can serve as functional surrogate measure for hepatic concentrations. In human volunteers receiving 1mg/kg body weight of low-viscosity white oil, all blood concentrations of mineral hydrocarbons were below the detection limit. Comparison with threshold blood concentrations associated with NOAELs in both rat strains, indicate that the margin-of-exposure is at least 37-fold. Using an internal dose metric rather than applied dose reduces the uncertainty around the temporary ADI considerably since it intrinsically accounts for intra- and inter-species differences. The current data support replacement of the temporary ADI of 0.01 mg/kg/day by a (permanent) ADI of at least 1.0mg/kg/day for low- and medium-viscosity mineral oils.