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.

Grouping of UVCB substances with dose-response transcriptomics data from human cell-based assays.

The application of in vitro biological assays as new approach methodologies (NAMs) to support grouping of UVCB (unknown or variable composition, complex reaction products, and biological materials) substances has recently been demonstrated. In addition to cell-based phenotyping as NAMs, in vitro transcriptomic profiling is used to gain deeper mechanistic understanding of biological responses to chemicals and to support grouping and read-across. However, the value of gene expression profiling for characterizing complex substances like UVCBs has not been explored. Using 141 petroleum substance extracts, we performed dose-response transcriptomic profiling in human induced pluripotent stem cell (iPSC)-derived hepatocytes, cardiomyocytes, neurons, and endothelial cells, as well as cell lines MCF7 and A375. The goal was to determine whether transcriptomic data can be used to group these UVCBs and to further characterize the molecular basis for in vitro biological responses. We found distinct transcriptional responses for petroleum substances by manufacturing class. Pathway enrichment informed interpretation of effects of substances and UVCB petroleum-class. Transcriptional activity was strongly correlated with concentration of polycyclic aromatic compounds (PAC), especially in iPSC-derived hepatocytes. Supervised analysis using transcriptomics, alone or in combination with bioactivity data collected on these same substances/cells, suggest that transcriptomics data provide useful mechanistic information, but only modest additional value for grouping. Overall, these results further demonstrate the value of NAMs for grouping of UVCBs, identify informative cell lines, and provide data that could be used for justifying selection of substances for further testing that may be required for registration.

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.

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.

Grouping of UVCB substances with new approach methodologies (NAMs) data.

One of the most challenging areas in regulatory science is assessment of the substances known as UVCB (unknown or variable composition, complex reaction products and biological materials). Because the inherent complexity and variability of UVCBs present considerable challenges for establishing sufficient substance similarity based on chemical characteristics or other data, we hypothesized that new approach methodologies (NAMs), including in vitro test-derived biological activity signatures to characterize substance similarity, could be used to support grouping of UVCBs. We tested 141 petroleum substances as representative UVCBs in a compendium of 15 human cell types representing a variety of tissues. Petroleum substances were assayed in dilution series to derive point of departure estimates for each cell type and phenotype. Extensive quality control measures were taken to ensure that only high-confidence in vitro data were used to determine whether current groupings of these petroleum substances, based largely on the manufacturing process and physico-chemical properties, are justifiable. We found that bioactivity data-based groupings of petroleum substances were generally consistent with the manufacturing class-based categories. We also showed that these data, especially bioactivity from human induced pluripotent stem cell (iPSC)-derived and primary cells, can be used to rank substances in a manner highly concordant with their expected in vivo hazard potential based on their chemical compositional profile. Overall, this study demonstrates that NAMs can be used to inform groupings of UVCBs, to assist in identification of repre­sentative substances in each group for testing when needed, and to fill data gaps by read-across.

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.

In vitro prenatal developmental toxicity induced by some petroleum substances is mediated by their 3- to 7-ring PAH constituent with a potential role for the aryl hydrocarbon receptor (AhR).

To test the hypothesis that 3-7 ring polycyclic aromatic hydrocarbons (PAHs) are responsible for the prenatal developmental toxicity (PDT) as observed with some petroleum substances (PS), the present study evaluates the PDT potency of DMSO-extracts of 7 heavy fuel oils (HFO), varying in their PAH content, and 1 highly refined base oil (HRBO), containing no aromatics, in the embryonic stem cell test (EST). All DMSO-extracts of HFO inhibit ES-D3 cell differentiation in a concentration-dependent manner and their potency is proportional to the amount of 3-7 ring PAHs they contain. All DMSO-extracts of HFOs also show aryl hydrocarbon receptor (AhR)-mediated activities, as tested in the AhR-CALUX assay. Contrarily, the HRBO-extract tested negative in both assays. Co-exposure of ES-D3 cells with selected DMSO-extracts of PS and the AhR-antagonist trimethoxyflavone, successfully counteracted the PS-induced inhibition of ES-D3 cell differentiation, confirming the role of the AhR in mediating the observed PDT of PS extracts in the EST. A good correlation exists when comparing the in-vitro with the in-vivo PDT potencies of the PS under study. Altogether, our findings corroborate the hypothesis that PS-induced PDT is caused by 3-7 ring PAHs present in these substances and that the observed PDT is partially AhR-mediated.

Prenatal developmental toxicity testing of petroleum substances using the zebrafish embryotoxicity test.

The present study evaluates the applicability of the zebrafish embryotoxicity test (ZET) to assess prenatal developmental toxicity (PDT) potency of the DMSO-extracts of 9 petroleum substances (PS), with variable polycyclic aromatic hydrocarbon (PAH) content, and 2 gas-to-liquid (GTL) products, without any PAHs but otherwise similar properties to PS. The results showed that all PS extracts induced concentration-dependent in vitro PDT, as quantified in the ZET and that this potency is associated with their 3-5 ring PAH content. In contrast and as expected, GTL products did not induce any effect at all. The potencies obtained in the ZET correlated with those previously reported for the embryonic stem cell test (EST) (R2=0.61), while the correlation with potencies reported in in vivo studies were higher for the EST (R2=0.85) than the ZET (R2=0.69). Combining the results of the ZET with those previously reported for the EST (Kamelia et al., 2017), the aryl hydrocarbon (AhR) CALUX assay (Kamelia et al., 2018), and the PAH content, ranked and clustered the test compounds in line with their in vivo potencies and chemical characteristics. To conclude, our findings indicate that the ZET does not outperform the EST as a stand-alone assay for testing PDT of PS, but confirms the hypothesis that PAHs are the major inducers of PDT by some PS, while they also indicate that the ZET is a useful addition to a battery of in vitro tests able to predict the in vivo PDT of PS.

The Role of Endocrine and Dioxin-Like Activity of Extracts of Petroleum Substances in Developmental Toxicity as Detected in a Panel of CALUX Reporter Gene Assays.

Recent evidence suggests that the interaction of polycyclic aromatic hydrocarbons (PAHs), present in some petroleum substances (PS), with particular nuclear-hormone-receptors and/or the dioxin (aryl hydrocarbon receptor [AhR]) receptor, may play a role in the prenatal developmental toxicity (PDT) induced by these substances. To address this hypothesis, we evaluated the possible endocrine and dioxin-like activity of the dimethylsulfoxide (DMSO)-extracts of 9 PS, varying in PAH content, and 2 gas-to-liquid (GTL) products, containing no PAHs but having similar other properties as PS, using a series of Chemical Activated LUciferase gene eXpression (CALUX) assays. The results show that the extracts of PS tested in this study possess various endocrine and dioxin-like activities and these in vitro potencies are associated with the quantity and type of PAHs they contain. All tested DMSO-extracts of PS show a strong AhR agonist activity and rather weak antiprogesterone, antiandrogen, and estrogenic activities. In the assays that evaluate thyroid-related and antiestrogen activity, only minor effects of specific extracts, particularly those with a substantial amount of 4-5 ring PAHs, ie, sample No. 34, 98, and 99, were observed. None of the GTL extracts interacted with the selected receptors. Of all assays, the AhR agonist activity correlates best (R2 = 0.80) with the in vitro PDT of the substances as quantified previously in the embryonic stem cell test, suggesting an important role of the AhR in mediating this effect. Hierarchic clustering of the combined CALUX data clustered the compounds in line with their chemical characteristics, suggesting a PS class-specific effects signature in the various CALUX assays, depending on the PAH profile. To conclude, our findings indicate a high potential for endocrine and dioxin-like activity of some PS extracts which correlates with their in vitro PDT and is driven by the PAHs present in these substances.

Prenatal developmental toxicity testing of petroleum substances: Application of the mouse embryonic stem cell test (EST) to compare in vitro potencies with potencies observed in vivo.

Prenatal developmental toxicity (PDT) as observed with some petroleum substances (PS) has been associated with the presence of 3-7 ring polycyclic aromatic hydrocarbons (PAHs). In the present study, the applicability of ES-D3 cell differentiation assay of the EST to evaluate in vitro embryotoxicity potencies of PS and gas-to-liquid (GTL) products as compared to their in vivo potencies was investigated. DMSO-extracts of a range of PS, containing different amounts of PAHs, and GTL-products, which are devoid of PAHs, were tested in the ES-D3 cell proliferation and differentiation assays of the EST. The results show that PS inhibited the differentiation of ES-D3 cells into cardiomyocytes in a concentration-dependent manner at non-cytotoxic concentrations, and that their potency was proportional to their PAH content. In contrast, as expected, GTL-products did not inhibit ES-D3 cell viability or differentiation at all. The in vitro PDT potencies were compared to published in vivo PDT studies, and a good correlation was found between in vitro and in vivo results (R2=0.97). To conclude, our results support the hypothesis that PAHs are the primary inducers of the PDT in PS.

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.

Assessment of petroleum streams for thyroid toxicity.

The thyroid gland, and its associated endocrine hormones, is a growing area of interest in regulatory toxicology due to its important role in metabolism, growth and development. This report presents a review of the toxicology data on chemically complex petroleum streams for thyroid hormone effects. Toxicological summaries and studies from all available published and un-published sources were considered, drawing upon the European REACH regulatory submissions for 19 petroleum streams, with in depth review of 11 individual study reports and 31 published papers on related products or environmental settings. Findings relevant to thyroid pathology or thyroid hormone homeostasis were specifically sought, summarized, and discussed. A total of 349 studies of 28-days or longer duration were considered in the review, including data on mice, rats, rabbits, dogs, humans, and fish. The thyroid was almost invariably not a target organ in these studies. Three rodent studies did find thyroid effects; one on a jet fuel product (JP-8), and two studies on a heavy fuel oil product (F-179). The JP-8 product differs from other fuels due to the presence of additives, and the finding of reduced T4 levels in mice in the study occurred at a dose that is above that expected to occur in environmental settings (e.g. 2000mg/kg). The finding for F-179 involved thyroid inflammation at 10-55mg/kg that co-occurred with liver pathology in rats, indicating a possible secondary effect with questionable relevance to humans. In the few cases where findings did occur, the polycyclic aromatic hydrocarbon (PAH) content was higher than in related substances, and, in support of one possible adverse outcome pathway, one in-vitro study reported reduced thyroid peroxidase (TPO) activity with exposure to some PAH compounds (pyrene, benzo(k)fluoranthene, and benzo(e)pyrene). However, it could not be determined from the data available for this review, whether these specific PAH compounds were substantially higher in the JP-8 or F-179 products than in studies in which thyroid effects were not observed. Thus, a few products may carry a weak potential to affect the thyroid at high doses in rodents, possibly through secondary effects on the rodent liver or possibly through a pathway involving the inhibition of TPO by specific members of the PAH family. Human epidemiology evidence found weak and inconsistent effects on the thyroid but without identification of specific chemicals involved. Two studies in petroleum workers, which found a lower rate of morbidity and mortality overall, reported a statistically significant increase in thyroid cancer, but the small number of cases could not exclude confounding variables as possible explanations for the statistical findings. Overall, the available data indicates a low potential for thyroid hormone effects from exposure to petroleum streams, especially when the aromatic content is low. Because regulatory studies for most chemicals do not include detailed thyroid function or receptor studies, it remains possible that subclinical effects on this system may exist that were not detectable using conventional pathology or hormone measurements.

Dermal uptake of petroleum substances.

Petroleum products are complex substances comprising varying amounts of linear and branched alkanes, alkenes, cycloalkanes, and aromatics which may penetrate the skin at different rates. For proper interpretation of toxic hazard data, understanding their percutaneous absorption is of paramount importance. The extent and significance of dermal absorption of eight petroleum substances, representing different classes of hydrocarbons, was evaluated. Literature data on the steady-state flux and permeability coefficient of these substances were evaluated and compared to those predicted by mathematical models. Reported results spanned over 5-6 orders of magnitude and were largely dependent on experimental conditions in particular on the type of the vehicle used. In general, aromatic hydrocarbons showed higher dermal absorption than more lipophilic aliphatics with similar molecular weight. The results showed high variation and were largely influenced by experimental conditions emphasizing the need of performing the experiments under "in use" scenario. The predictive models overestimated experimental absorption. The overall conclusion is that, based on the observed percutaneous penetration data, dermal exposure to petroleum hydrocarbons, even of aromatics with highest dermal absorption is limited and highly unlikely to be associated with health risks under real use scenarios.

Toxicogenomics in vitro as an alternative tool for safety evaluation of petroleum substances and PAHs with regard to prenatal developmental toxicity.

The REACH legislation requires chemicals - including petroleum substances - that are put on the EU market in quantities greater than 1000 tonnes/year, to be tested for prenatal developmental toxicity. This will require large numbers of animals since prenatal development toxicity testing is animal-intensive. The application of toxicogenomic technologies might reduce the number of animals to study prenatal developmental toxicity of petroleum substances by allowing their grouping into categories with the same toxicological properties. This substance categorization may be supported by similarities in molecular fingerprints. The developmental toxicity effects observed in some oil products are most likely related to polycyclic aromatic hydrocarbons (PAHs) with high-molecular weight. However, the current review indicates that even though the available studies provide clues regarding the HOX, FOX, SHH and PAX family genes, which regulate functions in skeleton development, single individual genes cannot be used as biomarkers of PAHs exposure and subsequent prenatal developmental toxicity. Furthermore, it should be considered that toxicogenomic technologies applied to specific tissues/organs testing might lead to unreliable results regarding developmental toxicity due to induction of tissue-specific pathways. Thus, an approach which applies a battery of in vitro tests including the zebrafish embryo test, embryonic stem cells, and the whole embryo culture is suggested as it would be more relevant for studying developmental effects in the terms of substances categorization.

A consistent and transparent approach for calculation of Derived No-Effect Levels (DNELs) for petroleum substances.

The REACH legislation introduced Derived No-Effect Levels (DNELs) which are defined as 'the levels of exposure above which humans should not be exposed'. DNELs were required for several categories of petroleum substances and CONCAWE developed a consistent approach for their derivation. First, the No-Observed Effect Level from a relevant study was corrected for pattern and route of exposure to obtain a modified Point-of-Departure (POD(modified)). Subsequently, the DNEL was calculated by dividing the POD(modified) by Assessment Factors (AFs) to adjust for inter- and intraspecies differences. If substance-specific information allowed, Informed Assessment Factors (IAFs), developed by CONCAWE were utilised. When little or no substance-specific information on those differences was known, default AFs from the guidance provided by ECHA were used. Some hazard endpoints did not lend themselves to calculation of DNELs (e.g. aspiration, dermal irritation, mutagenicity). DNEL calculation was considered not appropriate if adverse effects were not observed in tests conducted at a limit dose or if meaningful dose-response curves could not be developed. However, DNELs were calculated when hazards were identified, regardless of whether or not risk characterisation was required under REACH. Examples for gasoline, Lubricating Base Oils, gas oils and bitumen are provided to illustrate CONCAWE's approach.