Grouping of esters of 4-hydroxybenzoic acid for hazard assessment.

Hazardous properties of a large number of esters of 4-hydroxybenzoic acid (parabens) have been proposed by ECHA to be assessed as a group. We recommend to restrict the grouping approach to short chain esters, i.e. methyl, ethyl, propyl and butyl paraben which are very similar in chemical structures, physicochemical properties, toxicokinetics, and hazardous properties. While these parabens show a weak estrogenicity in some in vitro or in vivo screening assays, they do not induce estrogen-receptor-mediated adverse effects in intact animals. Therefore, there is no support regarding classification and labeling of endocrine disruption or reproductive toxicity of these parabens.

Grouping of short alkyl-chain branched carboxylic acids for developmental toxicity.

Read-across (RAx) and grouping of chemicals into categories are well-known concepts in toxicology. Recently, ECHA proposed a grouping approach for branched-chain carboxylic acids (BCAs) including more than 60 branched-chain saturated carboxylic acids for hazard identification. Grouping was based only on structural considerations. Due to developmental effects of two members, ECHA postulated that "all short carbon chain acids … are likely reproductive and developmental toxicants". This work analyzes available data for BCAs. The number of compounds in the group can be significantly reduced by eliminating metal and organic salts of BCAs, compounds of unknown or variable composition, and complex reaction products or biological materials (UVCB compounds). For the resulting reduced number of compounds, grouping is supported by similar physicochemical data and expected similar biotransformation. However, analysis of adverse effects for compounds in the group and mechanistic information show that BCAs, as a class, do not cause developmental effects in rats. Rather, developmental toxicity is limited to selected BCAs with specific structures that share a common mode of action (histone deacetylase inhibition). Thus, the proposed grouping is unreasonably wide and the more detailed analyses show that structural similarity alone is not sufficient for grouping branched-chain carboxylic acids for developmental toxicity.

Mammalian toxicity of trifluoroacetate and assessment of human health risks due to environmental exposures.

While trifluoroacetic acid has limited technical uses, the highly water-soluble trifluoroacetate (TFA) is reported to be present in water bodies at low concentrations. Most of the TFA in the environment is discussed to arise from natural processes, but also with the contribution from decomposition of environmental chemicals. The presence of TFA may result in human exposures. For hazard and risk assessment, the mammalian toxicity of TFA and human exposures are reviewed to assess the margin of exposures (MoE). The potential of TFA to induce acute toxicity is very low and oral repeated dose studies in rats have identified the liver as the target organ with mild liver hypertrophy as the lead effect. Biomarker analyses indicate that TFA is a weak peroxisome proliferator in rats. TFA administered to rats did not induce adverse effects in an extended one-generation study and in a developmental toxicity study or induce genotoxic responses. Based on recent levels of TFA in water and diet, MoEs for human exposures to TFA are well above 100 and do not indicate health risks.

Range of the perfluorooctanoate (PFOA) safe dose for human health: An international collaboration.

Many government agencies and expert groups have estimated a dose-rate of perfluorooctanoate (PFOA) that would protect human health. Most of these evaluations are based on the same studies (whether of humans, laboratory animals, or both), and all note various uncertainties in our existing knowledge. Nonetheless, the values of these various, estimated, safe-doses vary widely, with some being more than 100,000 fold different. This sort of discrepancy invites scrutiny and explanation. Otherwise what is the lay public to make of this disparity? The Steering Committee of the Alliance for Risk Assessment (2022) called for scientists interested in attempting to understand and narrow these disparities. An advisory committee of nine scientists from four countries was selected from nominations received, and a subsequent invitation to scientists internationally led to the formation of three technical teams (for a total of 24 scientists from 8 countries). The teams reviewed relevant information and independently developed ranges for estimated PFOA safe doses. All three teams determined that the available epidemiologic information could not form a reliable basis for a PFOA safe dose-assessment in the absence of mechanistic data that are relevant for humans at serum concentrations seen in the general population. Based instead on dose-response data from five studies of PFOA-exposed laboratory animals, we estimated that PFOA dose-rates 10-70 ng/kg-day are protective of human health.

The BlueScreen HC assay to predict the genotoxic potential of fragrance materials.

BlueScreen HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemical compounds and mixtures. The BlueScreen HC assay has been utilized at the Research Institute for Fragrance Materials in a safety assessment program as a screening tool to prioritize fragrance materials for higher-tier testing, as supporting evidence when using a read-across approach, and as evidence to adjust the threshold of toxicological concern. Predictive values for the BlueScreen HC assay were evaluated based on the ability of the assay to predict the outcome of in vitro and in vivo mutagenicity and chromosomal damage genotoxicity assays. A set of 371 fragrance materials was assessed in the BlueScreen HC assay along with existing or newly generated in vitro and in vivo genotoxicity data. Based on a weight-of-evidence approach, the majority of materials in the data set were deemed negative and concluded not to have the potential to be genotoxic, while only a small proportion of materials were determined to show genotoxic effects in these assays. Analysis of the data set showed a combination of high positive agreement but low negative agreement between BlueScreen HC results, in vitro regulatory genotoxicity assays, and higher-tier test results. The BlueScreen HC assay did not generate any false negatives, thereby providing robustness when utilizing it as a high-throughput screening tool to evaluate the large inventory of fragrance materials. From the perspective of protecting public health, it is desirable to have no or minimal false negatives, as a false-negative result may incorrectly indicate the lack of a genotoxicity hazard. However, the assay did have a high percentage of false-positive results, resulting in poor positive predictivity of the in vitro genotoxicity test battery outcome. Overall, the assay generated 100% negative predictivity and 3.9% positive predictivity. In addition to the data set of 371 fragrance materials, 30 natural complex substances were evaluated for BlueScreen HC, Ames, and in vitro micronucleus assay, and a good correlation in all three assays was observed. Overall, while a positive result may have to be further investigated, these findings suggest that the BlueScreen HC assay can be a valuable screening tool to detect the genotoxic potential of fragrance materials and mixtures.

Commentary: cumulative risk assessment of perfluoroalkyl carboxylic acids and perfluoralkyl sulfonic acids: what is the scientific support for deriving tolerable exposures by assembling 27 PFAS into 1 common assessment group?

This commentary proposes an approach to risk assessment of mixtures of per- and polyfluorinated alkyl substances (PFAS) as EFSA was tasked to derive a tolerable intake for a group of 27 PFAS. The 27 PFAS to be considered contain different functional groups and have widely variable physicochemical (PC) properties and toxicokinetics and thus should not treated as one group based on regulatory guidance for risk assessment of mixtures. The proposed approach to grouping is to split the 27 PFAS into two groups, perfluoroalkyl carboxylates and perfluoroalkyl sulfonates, and apply a relative potency factor approach (as proposed by RIVM) to obtain two separate group TDIs based on liver toxicity in rodents since liver toxicity is a sensitive response of rodents to PFAS. Short chain PFAS and other PFAS structures should not be included in the groups due to their low potency and rapid elimination. This approach is in better agreement with scientific and regulatory guidance for mixture risk assessment.

Immunomodulation and exposure to per- and polyfluoroalkyl substances: an overview of the current evidence from animal and human studies.

Per- and polyfluoroalkyl substances (PFAS) have been widely used and represent a class of environmental persistent chemicals. An association of a reduction of vaccination efficacy with PFAS serum levels in humans was used by the European Food Safety Authority as a key effect for PFAS risk assessment. The data support for using this association is reviewed by a critical analysis of the respective human epidemiology and the available animal studies on the immunomodulation of PFAS. Based on an analysis of the available human epidemiology, the overall level of evidence regarding associations between PFAS serum levels and reduced antibody response remains weak. Absence of an association between an increase in clinical infections and PFAS serum levels and the limited understanding of the importance of antibody levels as an isolated data point further support this conclusion. Animal toxicity studies with PFAS focusing on immunomodulation also provide only limited support for immunomodulation as an important endpoint in PFAS toxicity. While immunomodulation is observed after PFAS administration, generally at blood concentrations several orders of magnitude above those seen in environmentally exposed humans, the relevance of these observation is hampered by the high doses required to influence immune endpoints, the limited number of endpoints assessed, and inconsistent results. The limitations of the current database on associations of human PFAS exposures outlined here indicate that more evidence is required to select immunomodulation as a critical endpoint for human PFAS risk assessment.

Evaluation of animal toxicity studies with diisocyanates regarding presence of thresholds for induction and elicitation of respiratory allergy by quantitative weight of evidence.

Animal toxicity studies on diisocyanates were evaluated using quantitative weight of evidence (QWoE) to test the hypothesis that the dose-response curve shows a threshold for the induction and/or elicitation of respiratory sensitization. A literature search identified 59 references that included at least two concentration groups of the diisocyanate and a vehicle-exposed concurrent control in the study design. These studies were subjected to a QWoE-assessment applying scoring criteria for quality and relevance/strength of effects relevant to the selected endpoint of respiratory sensitization. Overall, the studies assessing dose/concentration-response for diisocyanates with the endpoint, respiratory sensitization, were heterogenous regarding study design, animal models used, endpoints assessed, and quality. Only a limited number of the studies subjected to the QWoE-assessment allowed drawing conclusions about possible thresholds for respiratory sensitization. Highest quality and relevance/strength of effects scores were obtained by a series of studies specifically designed to investigate a potential threshold for elicitation of respiratory sensitization in the Brown Norway (BN) rat. These studies applied an elaborate study design to optimize induction of respiratory sensitization and reduce interference by respiratory tract irritation. In summary, the available studies provided moderate to good support for the existence of a threshold for elicitation and limited to moderate support for a threshold regarding induction of respiratory allergy by diisocyanates in experimental animals. However, a quantitative extrapolation of threshold values established in rodents to humans remains complex.

Identifying a reference list of respiratory sensitizers for the evaluation of novel approaches to study respiratory sensitization.

The induction of immunological responses that trigger bio-physiological symptoms in the respiratory tract following repeated exposure to a substance, is known as respiratory sensitization. The inducing compound is known as a respiratory sensitizer. While respiratory sensitization by high molecular weight (HMW) materials is recognized and extensively studied, much less information is available regarding low molecular weight (LMW) materials as respiratory sensitizers. Variability of symptoms presented in humans from such exposures, limited availability of (and access to) documented reports, and the absence of standardized and validated test models, hinders the identification of true respiratory sensitizers. This review aims to sort suspected LMW respiratory sensitizers based on available compelling, reasonable, inadequate, or questionable evidence in humans from occupational exposures and use this information to compose a reference list of reported chemical respiratory sensitizers for scientific research purposes. A list of 97 reported respiratory sensitizers was generated from six sources, and 52 LMW organic chemicals were identified, reviewed, and assigned to the four evidence categories. Less than 10 chemicals were confirmed with compelling evidence for induction of respiratory sensitization in humans from occupational exposures. Here, we propose the reference list for developing novel research on respiratory sensitization.

The EU chemicals strategy for sustainability: in support of the BfR position.

The EU chemicals strategy for sustainability (CSS) asserts that both human health and the environment are presently threatened and that further regulation is necessary. In a recent Guest Editorial, members of the German competent authority for risk assessment, the BfR, raised concerns about the scientific justification for this strategy. The complexity and interdependence of the networks of regulation of chemical substances have ensured that public health and wellbeing in the EU have continuously improved. A continuous process of improvement in consumer protection is clearly desirable but any initiative directed towards this objective must be based on scientific knowledge. It must not confound risk with other factors in determining policy. This conclusion is fully supported in the present Commentary including the request to improve both, data collection and the time-consuming and bureaucratic procedures that delay the publication of regulations.

Evaluation of the carcinogenicity of dichloromethane in rats, mice, hamsters and humans.

Dichloromethane (DCM) is a high production volume chemical (>1000 t/a) mainly used as an industrial solvent. Carcinogenicity studies in rats, mice and hamsters have demonstrated a malignant tumor inducing potential of DCM only in the mouse (lung and liver) at 1000-4000 ppm whereas human data do not support a conclusion of cancer risk. Based on this, DCM has been classified as a cat. 2 carcinogen. Dose-dependent toxicokinetics of DCM suggest that DCM is a threshold carcinogen in mice, initiating carcinogenicity via the low affinity/high capacity GSTT1 pathway; a biotransformation pathway that becomes relevant only at high exposure concentrations. Rats and hamsters have very low activities of this DCM-metabolizing GST and humans have even lower activities of this enzyme. Based on the induction of specific tumors selectively in the mouse, the dose- and species-specific toxicokinetics in this species, and the absence of a malignant tumor response by DCM in rats and hamsters having a closer relationship to DCM toxicokinetics in humans and thus being a more relevant animal model, the current classification of DCM as human carcinogen cat. 2 remains appropriate.

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity: how to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity. How to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine-disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine-disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower than S-EDCs. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea, and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

A flow scheme for cumulative assessment of pesticides for adverse liver effects.

The European Food Safety Authority (EFSA) is developing approaches to cumulative risk assessment by assigning pesticides to cumulative assessment groups (CAGs). For assignment to CAGs, EFSA relies on common toxic effects (CTEs) on the target system. The developed flow scheme for assignment to liver CAGs sequentially assesses the consistency of the CTE, its adversity, its potential to be secondary to other toxicities, its human relevance, and the relation of the NOAEL for the CTE to the overall NOAEL. If the responses to all questions are "yes", allocation to a CAG is supported; "no" stops the process.

Tetrahydrofuran-induced tumors in rodents are not relevant to humans: Quantitative weight of evidence analysis of mode of action information does not support classification of tetrahydrofuran as a possible human carcinogen.

Inhalation of tetrahydrofuran (THF) causes a marginal increase in the incidence of renal tumors in male rats and an increase in the incidence of liver tumors in female mice. Quantitative weight of evidence (QWoE) was applied to assess experimental support for biologically plausible modes of action (MoA) of tumor formation by THF and their human relevance. QWoE did not obtain support for a MoA to induce kidney tumors in male rats from THF exposure via α2u -globulin nephropathy, exacerbation of chronic progressive nephropathy (CPN), DNA-damage, or recurrent cytotoxicity but obtained moderate to good support for a constitutive androgen receptor (CAR)-mediated MoA for the induction of liver tumors in female mice. Tumors as a consequence of CAR-activation are not considered relevant to humans. Considering the previous conclusion that the increases in kidney tumors in male rats are unlikely related to THF-exposure and the support for a CAR-mediated MoA in mice obtained here, these tumors should not be used as a basis for THF cancer classification.

Obfuscating transparency?

Several recent and prominent articles in Science and Nature deliberately mischaracterized the nature of genuine scientific evidence. Those articles take issue with the United States Environmental Protection Agency's recent proposal to structure its policies and rules only from studies with transparently published raw data. The articles claim it is an effort to obfuscate with transparency, by eliminating a host of studies not offering raw data. A remarkable declaration by a Science editorial is that properly trained experts can verify the scientific evidence of studies without access to raw data, We assert the Agency's proposal must be sustained. Transparency in reporting is a fundamental ethical imperative of objective scientific research justifying massive official regulations and policies. Putative hazards bereft of independent scientific evidence will continue to stoke public anxieties, calling for precautionary regulations and policies. These should rely not on spurious science but on transparent tradeoffs between the smallest exposures compatible with utility and with social perceptions of affordable precaution.

Approaches for grouping of pesticides into cumulative assessment groups for risk assessment of pesticide residues in food.

The European Food Safety Authority (EFSA) is developing approaches to cumulative risk assessment of pesticides by assigning individual pesticides to cumulative assessment groups (CAGs). For assignment to CAGs, EFSA recommended to rely on adverse effects on the specific target system. Contractors to EFSA have proposed to allocate individual pesticides into CAGs relying on NOAELs for effects on target organs. This manuscript evaluates the assignments by applying EFSAs criteria to the CAGs "Toxicity to the nervous system" and "Toxicity to the thyroid hormone system (gland or hormones)". Assignment to the CAG "Toxicity to the nervous system" based, for example, on neurochemical effects like choline esterase inhibition is well supported, whereas assignment to the CAG "Toxicity to the thyroid hormone system (gland or hormones)" has been based in the examined case studies on non-reproducible effects seen in single studies or on observations that are not adverse. Therefore, a more detailed effects evaluation is required to assign a pesticide to a CAG for a target organ where many confounders regarding effects are present. Relative potency factors in cumulative risk assessment should be based on benchmark doses from studies in one species with identical study design and human relevance of effects on specific target organs should be analyzed to define minimal margins of exposure.

A quantitative weight of evidence assessment of confidence in modes-of-action and their human relevance.

A quantitative weight of evidence (QWoE) methodology was developed to assess confidence in postulated mode(s) of action for adverse effects in animal toxicity studies. The QWoE is appropriate for assessing adverse effects as relevant endpoints for classification and labeling purposes. The methodology involves definition of mode of actions and scoring supporting data for all key steps using predefined criteria for quality and relevance/strength of effects. Scores for all key steps are summarized, and the summary score is compared to the maximal achievable score for the mode of action. The ratio of the summary score to the maximal achievable scores gives an indication of confidence in a specific mode of action in animals. The mode of action in animals with highest confidence is then taken forward to assess appropriateness to humans. If one of the key steps cannot occur in humans, the mode of action is not relevant to humans. The methodology developed is applied to four case studies.

Nephron Toxicity Profiling via Untargeted Metabolome Analysis Employing a High Performance Liquid Chromatography-Mass Spectrometry-based Experimental and Computational Pipeline.

Untargeted metabolomics has the potential to improve the predictivity of in vitro toxicity models and therefore may aid the replacement of expensive and laborious animal models. Here we describe a long term repeat dose nephrotoxicity study conducted on the human renal proximal tubular epithelial cell line, RPTEC/TERT1, treated with 10 and 35 µmol·liter(-1) of chloroacetaldehyde, a metabolite of the anti-cancer drug ifosfamide. Our study outlines the establishment of an automated and easy to use untargeted metabolomics workflow for HPLC-high resolution mass spectrometry data. Automated data analysis workflows based on open source software (OpenMS, KNIME) enabled a comprehensive and reproducible analysis of the complex and voluminous metabolomics data produced by the profiling approach. Time- and concentration-dependent responses were clearly evident in the metabolomic profiles. To obtain a more comprehensive picture of the mode of action, transcriptomics and proteomics data were also integrated. For toxicity profiling of chloroacetaldehyde, 428 and 317 metabolite features were detectable in positive and negative modes, respectively, after stringent removal of chemical noise and unstable signals. Changes upon treatment were explored using principal component analysis, and statistically significant differences were identified using linear models for microarray assays. The analysis revealed toxic effects only for the treatment with 35 µmol·liter(-1) for 3 and 14 days. The most regulated metabolites were glutathione and metabolites related to the oxidative stress response of the cells. These findings are corroborated by proteomics and transcriptomics data, which show, among other things, an activation of the Nrf2 and ATF4 pathways.

Assessment of reproductive and developmental effects of DINP, DnHP and DCHP using quantitative weight of evidence.

Quantitative weight of evidence (QWoE) methodology utilizes detailed scoring sheets to assess the quality/reliability of each publication on toxicity of a chemical and gives numerical scores for quality and observed toxicity. This QWoE-methodology was applied to the reproductive toxicity data on diisononylphthalate (DINP), di-n-hexylphthalate (DnHP), and dicyclohexylphthalate (DCHP) to determine if the scientific evidence for adverse effects meets the requirements for classification as reproductive toxicants. The scores for DINP were compared to those when applying the methodology DCHP and DnHP that have harmonized classifications. Based on the quality/reliability scores, application of the QWoE shows that the three databases are of similar quality; but effect scores differ widely. Application of QWoE to DINP studies resulted in an overall score well below the benchmark required to trigger classification. For DCHP, the QWoE also results in low scores. The high scores from the application of the QWoE methodology to the toxicological data for DnHP represent clear evidence for adverse effects and justify a classification of DnHP as category 1B for both development and fertility. The conclusions on classification based on the QWoE are well supported using a narrative assessment of consistency and biological plausibility.