Search

The chronic toxicity of short chain perfluoroalkyl sulfonic acids (PFSAs), such as perfluorobutanesulfonic acid (PFBS) and perfluorohexanesulfonic acid (PFHxS), are relatively understudied despite the increasing detection of these compounds in the environment. We investigated the chronic toxicity and bioconcentration of PFBS and PFHxS using northern leopard frog (Rana [Lithobates] pipiens) tadpoles. We exposed Gosner stage (GS) 25 tadpoles to either PFBS or PFHxS at nominal concentrations of 0.1, 1, 10, 100, and 1000 μg/L until metamorphosis (GS42). We then assessed tadpole growth, development, stress, and immune metrics, and measured fatty acid (FA) composition and PFSA concentrations in liver and whole-body tissues. Tadpole growth and development measures were relatively unaffected by PFSA exposure. However, tadpoles exposed to 1000 μg/L PFBS or PFHxS had significantly increased hepatosomatic indexes (HSI) relative to controls. Further, tadpoles from the 1000 μg/L PFHxS treatment had altered FA profiles relative to controls, with increased total FAs, saturated FAs, monounsaturated FAs, and omega-6 polyunsaturated FAs. In addition, tadpoles from the 1000 μg/L PFHxS treatment had a higher probability of waterborne corticosterone detection. These results suggest that PFBS and PFHxS influence the hepatic health of tadpoles, and that PFHxS may alter lipid metabolism in tadpoles. We also observed a higher probability of tadpoles being phenotypically female after exposure to an environmentally relevant concentration (0.1 μg/L) of PFHxS, suggesting that PFHxS may exert endocrine disrupting effects on tadpoles during early development. The measured bioconcentration factors (BCFs) for both compounds were ≤10 L kg -1 wet weight, suggesting low bioconcentration potential for PFBS and PFHxS in tadpoles. Many of the significant effects observed in this study occurred at concentrations several orders of magnitude above those measured in the environment; however, our work shows effects of PFSAs exposure on amphibians and provides essential information for ecological risk assessments of these compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. RIFM staff are employees of the Research Institute for Fragrance Materials, Inc. (RIFM). The Expert Panel receives a small honorarium for time spent reviewing the subject work.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. RIFM staff are employees of the Research Institute for Fragrance Materials, Inc. (RIFM). The Expert Panel receives a small honorarium for time spent reviewing the subject work.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. RIFM staff are employees of the Research Institute for Fragrance Materials, Inc. (RIFM). The Expert Panel receives a small honorarium for time spent reviewing the subject work.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. RIFM staff are employees of the Research Institute for Fragrance Materials, Inc. (RIFM). The Expert Panel receives a small honorarium for time spent reviewing the subject work.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. RIFM staff are employees of the Research Institute for Fragrance Materials, Inc. (RIFM). The Expert Panel receives a small honorarium for time spent reviewing the subject work.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. RIFM staff are employees of the Research Institute for Fragrance Materials, Inc. (RIFM). The Expert Panel receives a small honorarium for time spent reviewing the subject work.

With the increasing application of cell culture models as primary tools for predicting chemical safety, the quantitative extrapolation of the effective dose from in vitro to in vivo (QIVIVE) is of increasing importance. For developmental toxicity this requires scaling the in vitro observed dose-response characteristics to in vivo fetal exposure, while integrating maternal in vivo kinetics during pregnancy, in particular transplacental transfer. Here the transfer of substances across the placental barrier, has been studied using the in vitro BeWo cell assay and six embryotoxic compounds of different kinetic complexity. The BeWo assay results were incorporated in an existing generic Physiologically Based Kinetic (PBK) model which for this purpose was extended with rat pregnancy. Finally, as a "proof of principle", the BeWo PBK model was used to perform a QIVIVE based on developmental toxicity as observed in various different in vitro toxicity assays. The BeWo results illustrated different transport profiles of the chemicals across the BeWo monolayer, allocating the substances into two distinct groups: the 'quickly-transported' and the 'slowly-transported'. BeWo PBK exposure simulations during gestation were compared to experimentally measured maternal blood and fetal concentrations and a reverse dosimetry approach was applied to translate in vitro observed embryotoxicity into equivalent in vivo dose-response curves. This approach allowed for a direct comparison of the in vitro dose-response characteristics as observed in the Whole Embryo Culture (WEC), and the Embryonic Stem Cell test (cardiac:ESTc and neural:ESTn) with in vivo rat developmental toxicity data. Overall, the in vitro to in vivo comparisons suggest a promising future for the application of such QIVIVE methodologies for screening and prioritization purposes of developmental toxicants. Nevertheless, the clear need for further improvements is acknowledged for a wider application of the approach in chemical safety assessment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Drosophila sechellia is a dietary specialist endemic to the Seychelles islands that has evolved to consume the fruit of Morinda citrifolia. When ripe, the fruit of M. citrifolia contains octanoic acid and hexanoic acid, two medium-chain fatty acid volatiles that deter and are toxic to generalist insects. Drosophila sechellia has evolved resistance to these volatiles allowing it to feed almost exclusively on this host plant. The genetic basis of octanoic acid resistance has been the focus of multiple recent studies, but the mechanisms that govern hexanoic acid resistance in D. sechellia remain unknown. To understand how D. sechellia has evolved to specialize on M. citrifolia fruit and avoid the toxic effects of hexanoic acid, we exposed adult D. sechellia, D. melanogaster and D. simulans to hexanoic acid and performed RNA sequencing comparing their transcriptional responses to identify D. sechellia specific responses. Our analysis identified many more genes responding transcriptionally to hexanoic acid in the susceptible generalist species than in the specialist D. sechellia. Interrogation of the sets of differentially expressed genes showed that generalists regulated the expression of many genes involved in metabolism and detoxification whereas the specialist primarily downregulated genes involved in the innate immunity. Using these data, we have identified interesting candidate genes that may be critically important in aspects of adaptation to their food source that contains high concentrations of HA. Understanding how gene expression evolves during dietary specialization is crucial for our understanding of how ecological communities are built and how evolution shapes trophic interactions., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Perfluorohexanoic acid (PFHxA), one of the short-chain perfluoroalkyl acids (PFAAs), is considered as a substitute of perfluorooctane sulfonate (PFOS). This emerging organic pollutant is persistent and highly bioavailable to humans, raising concerns about its potential health risks. There are currently few researches on the toxicity of PFHxA. Liver has been suggested to be the main target of PFHxA toxicity, and the mechanism remains unclear. Herein, we investigated the transcriptomic, proteomic, and metabolomic landscape in PFHxA-exposed mice. Using these approaches, we identified several valuable biological processes involved in the process of liver injury, comprising fatty acid biosynthesis and degradation pathways, which might be induced by peroxisome proliferator-activated receptor (PPAR) signaling pathway. These processes further promoted oxidative stress and induced liver injury. Meanwhile, abnormalities in purine metabolism and glutathione metabolism were observed during the liver injury induced by PFHxA, indicating the production of oxidative stress. Finally, our present multi-omics studies provided new insights into the mechanisms involved in PFHxA-induced liver injury., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Perfluoroalkyl acids (PFAAs) are persistent environmental contaminants that are associated with various adverse health outcomes. Perfluorooctanoic acid (PFOA) is one of the most prominently detected PFAAs in the environment, which is now replaced with shorter chain carbon compounds including perfluorohexanoic acid (PFHxA) and perfluorobutyric acid (PFBA). The aim of this study was to compare the toxicity of four PFAAs as a function of chain length and head group (carboxylate versus sulfonate) with in vitro and in vivo zebrafish assessments, which were subsequently compared to other cell and aquatic models. Mortality rate increased with chain length (PFOA > PFHxA ≫ PFBA) in both whole embryo/larvae and embryonic cell models. The sulfonate group enhanced toxicity with perfluorobutane sulfonate (PFBS) showing higher toxicity than PFBA and PFHxA in both larvae and cells. Toxicity trends were similar among different aquatic models, but sensitivities varied. Discrepancies with other zebrafish studies were confirmed to be associated with a lack of neutralization of acidic pH of dosing solutions in these other investigations, demonstrating the need for rigor in reporting pH of exposure solutions in all experiments. The zebrafish embryonic cell line was also found to be similar to most other cell lines regardless of exposure length. Overall, results agree with findings in other cell lines and organisms where longer chain length and sulfonate group increase toxicity, except in investigations not neutralizing the exposure solutions for these acidic compounds.

The existing information supports the use of this material as described in this safety assessment. 4-Methylpentanoic acid was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from read-across analog 2-ethylbutyric acid (CAS # 88-09-5) show that 4-methylpentanoic acid is not expected to be genotoxic and provide a calculated margin of exposure (MOE) > 100 for the repeated dose toxicity and reproductive toxicity endpoints. The skin sensitization endpoint was completed using the dermal sensitization threshold (DST) for non-reactive materials (900 μg/cm 2 ); exposure is below the DST. The phototoxicity/photoallergenicity endpoints were evaluated based on ultraviolet (UV) spectra; 4-methylpentanoic acid is not expected to be phototoxic/photoallergenic. The local respiratory toxicity endpoint was evaluated using the threshold of toxicological concern (TTC) for a Cramer Class I material, and the exposure to 4-methylpentanoic acid is below the TTC (1.4 mg/day). The environmental endpoints were evaluated; 4-methylpentanoic acid was found not to be persistent, bioaccumulative, and toxic (PBT) as per the International Fragrance Association (IFRA) Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., Predicted Environmental Concentration/Predicted No Effect Concentration [PEC/PNEC]), are <1., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The existing information supports the use of this material as described in this safety assessment. Ethyl (E)hex-3-enoate was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from read-across analog methyl 3-hexenoate (CAS # 2396-78-3) show that ethyl (E)hex-3-enoate is not expected to be genotoxic. The repeated dose, reproductive, and local respiratory toxicity endpoints were evaluated using the Threshold of Toxicological Concern (TTC) for a Cramer Class I material, and the exposure to ethyl (E)hex-3-enoate is below the TTC (0.03 mg/kg/day, 0.03 mg/kg/day, and 1.4 mg/day, respectively). The skin sensitization endpoint was completed using Dermal Sensitization Threshold (DST) for non-reactive materials (900 μg/cm 2 ); exposure is below the DST. The phototoxicity/photoallergenicity endpoints were evaluated based on ultraviolet (UV) spectra; ethyl (E)hex-3-enoate is not expected to be phototoxic/photoallergenic. The environmental endpoints were evaluated; ethyl (E)hex-3-enoate was found not to be persistent, bioaccumulative, and toxic (PBT) as per the International Fragrance Association (IFRA) Environmental Standards, and its risk quotients, based on its current Volume of Use in Europe and North America (i.e., Predicted Environmental Concentration/Predicted No Effect Concentration [PEC/PNEC]), are <1., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The existing information supports the use of this material as described in this safety assessment. Hexyl hexanoate was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from read-across analog hexyl isobutyrate (CAS # 2349-07-7) show that hexyl hexanoate is not expected to be genotoxic. The repeated dose, reproductive, and local respiratory toxicity endpoints were evaluated using the threshold of toxicological concern (TTC) for a Cramer Class I material, and the exposure to hexyl hexanoate is below the TTC (0.03 mg/kg/day, 0.03 mg/kg/day, and 1.4 mg/day, respectively). The skin sensitization endpoint was completed using the dermal sensitization threshold (DST) for non-reactive materials (900 μg/cm 2 ); exposure is below the DST. The phototoxicity/photoallergenicity endpoints were evaluated based on ultraviolet (UV) spectra; hexyl hexanoate is not expected to be phototoxic/photoallergenic. The environmental endpoints were evaluated; hexyl hexanoate was found not to be persistent, bioaccumulative, and toxic (PBT) as per the International Fragrance Association (IFRA) Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., Predicted Environmental Concentration/Predicted No Effect Concentration [PEC/PNEC]), are <1., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Contaminated poultry meat is considered to be the main source of human infection with Campylobacter spp., a pathogen that asymptomatically colonizes broiler chickens during fattening and contaminates carcasses during slaughter. To prevent or reduce the colonization of broiler flocks with Campylobacter spp., applying different organic acids, especially in combinations, via feed or drinking water seems to be a promising approach. However, only very few combinations of organic acids have been tested for their antibacterial efficacy against Campylobacter spp. Therefore, the in vitro susceptibility of 30 Campylobacter spp. isolates (20 C. jejuni and ten C. coli) to ten organic acids and ten combinations was determined. The testing of minimum inhibitory concentration (MIC) values was performed at pH 6.0 and 7.3 by using the broth microdilution method and included the following organic acids: Caprylic acid, sorbic acid, caproic acid, benzoic acid, ascorbic acid, propionic acid, acetic acid, formic acid, fumaric acid and tartaric acid and combinations thereof. The lowest MIC values were seen for caprylic acid (MIC range at pH 7.3: 0.5-2 mmol/L) and sorbic acid (MIC range at pH 7.3: 1-4 mmol/L). One to two dilution steps lower MIC values were determined at the lower pH value of 6.0. Furthermore, ten combinations consisting of three to five organic acids were developed. In addition to the tested antibacterial activity, other criteria were included such as approval as feed additives, reported synergistic effects and chemical properties. For nine of ten combinations, the MIC90 values of the organic acids decreased 1.25- to 241.5-fold compared to the MIC90 values for the individual substances. Furthermore, nine of ten combinations exhibited synergistic activities against two or more of the tested C. jejuni and C. coli isolates. A combination of caprylic acid, sorbic acid and caproic acid exhibited synergistic activities against the largest number of Campylobacter spp. isolates (six C. jejuni and four C. coli) with fractional inhibitory concentration (FIC) indices (∑FIC) ranging from 0.33 to 1.42. This study shows in vitro synergistic activities of different organic acids in combinations against the major Campylobacter species and could therefore be a promising basis for reducing Campylobacter spp. in vivo., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. RIFM staff are employees of the Research Institute for Fragrance Materials, Inc. (RIFM). The Expert Panel receives a small honorarium for time spent reviewing the subject work.

Poly- and perfluorinated alkyl substances (PFAS) are environmentally persistent chemicals associated with many adverse health outcomes. The National Toxicology Program evaluated the toxicokinetics (TK) of several PFAS to provide context for toxicologic findings.Plasma TK parameters and tissue (liver, kidney, brain) concentrations are reported for perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA) or perfluorodecanoic acid (PFDA) after single-dose administration in male and female Hsd:Sprague-Dawley ® (SD) rats.Generally, longer T max and elimination half-lives, and slower clearance f, were correlated with longer chain length. Male rats administered PFOA had a prolonged half-life compared to females (215 h vs. 2.75), while females had faster clearance and smaller plasma area under the curve (AUC). Females administered PFHxA had a shorter half-life (2 h vs. 9) than males and faster clearance with a smaller plasma AUC, although this was less pronounced than PFOA. There was no sex difference in PFDA half-life. Female rats administered PFDA had a higher plasma AUC/dose than males, and a slower clearance. PFDA had the highest levels in the liver of the PFAS evaluated.Profiling the toxicokinetics of these PFAS allows for comparison among subclasses, and more direct translation of rodent toxicity to human populations.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. RIFM staff are employees of the Research Institute for Fragrance Materials, Inc. (RIFM). The Expert Panel receives a small honorarium for time spent reviewing the subject work.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. RIFM staff are employees of the Research Institute for Fragrance Materials, Inc. (RIFM). The Expert Panel receives a small honorarium for time spent reviewing the subject work.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. RIFM staff are employees of the Research Institute for Fragrance Materials, Inc. (RIFM). The Expert Panel receives a small honorarium for time spent reviewing the subject work.

Perfluoroalkyl acids (PFAAs) are a type of persistent organic pollutants that are widely distributed in multiple environmental media and organisms and have a teratogenic effect on and toxicity to animals and humans. The residual levels of seventeen PFAAs in the tissues of two regular consumption fish species, Culter erythropterus and Aristichthys nobilis in Lake Chaohu were measured by a high-performance liquid chromatograph - mass spectrometer (HPLC-MS). The distributions of PFAAs and the effect of the lipid contents were analyzed, and the health risks of typical PFAAs were evaluated. The results showed that perfluorohexanoic acid (PFHxA) was the predominant contaminant (80.50 ± 58.31 ng/g and 19.17 ± 12.57 ng/g wet weight, ww), followed by perfluorooctanesulfonic acid (PFOS) (55.02 ± 34.82 and 14.79 ± 6.24 ng/g, ww) in both fish. The level of total PFAAs was the highest in the liver tissues of Culter erythropterus (359.87 ng/g, ww) and the lowest in the kidney tissues in A. nobilis (10.06 ng/g, ww). Due to the higher trophic level of C. erythropteru, the total PFAA concentrations were significantly higher in all tissues than those in A. nobilis. Liver muscle ratio of C. erythropteru was the highest, indicating the most accumulation in the liver. The concentrations of PFAAs in fish tissues were influenced by the lipid content, resulting in a difference between the lipid-normalized concentrations and the wet weight concentrations of the PFAAs. The non-carcinogenic risks of PFOS were higher than those of PFOA through the ingestion of C. erythropterus and A. nobilis. Both the carcinogenic and non-carcinogenic risks of C. erythropterus were greater than those of A. nobilis, and fish tissue intake could cause an increasing of risks up to 60%, indicating that long-term and large amount ingestion of carnivorous fish and related tissues with higher trophic level, such as C. erythropterus should be avoided., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Poly and perfluoroalkyl substances (PFAS) are a large group of emerging organic pollutants that can persist in the environment and bioaccumulate in biota. They are found in complex mixtures, and although the exact number of PFAS is unknown, it has been estimated to be in the thousands. The objective of this study was two-fold. First, we examined the cytotoxicity of PFAS singly and in binary mixtures using an amphibian fibroblast cell line. Second, we used this experimental data to develop quantitative structure-activity relationship (QSAR) models for single and binary mixtures. We tested the cytotoxicity of four common PFAS: perfluorooctane sulfonate (PFOS); perfluorooctanoic acid (PFOA); perfluorohexane sulfonate (PFHxS); and perfluorohexanoic acid (PFHxA). PFOS was the most toxic and PFHxA the least cytotoxic. Binary mixtures allowed for the construction of isobolograms to test for additivity, synergism, or antagonism. Using this data, QSAR modeling was used for predicting the toxicity of 24 single and 1380 binary mixtures (theoretically generated). Overall, our experimental and modeling results showed that mixtures were approximately additive, with the exception of PFOS and PFOA, which were found to be weakly synergistic. This data shows that certain mixtures of PFAS may have increased toxicity potential above what the simple sum of PFAS concentrations would suggest. More studies are needed that test the toxicity of PFAS mixtures., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This article presents a supplemental data analysis and evaluation of the findings from an oral (gavage) combined developmental and perinatal/postnatal reproduction toxicity study of the ammonium salt of perfluorohexanoic acid (CASRN: 21615-47-4) in Crl: CD-1(ICR) mice. The original study has been cited as supporting a lowest-observed-adverse-effects level of 175 mg/kg/d and no-observed-adverse-effects level of 35 mg/kg/d for developmental effects from perfluorohexanoic acid (PFHxA, CASRN: 307-24-4) in mice. The statistical analysis reported in 2014 was accurate in terms of quantifying statistical significance within phase 2 of the study. However, given the low incidence of findings, the purpose of this article is to extend the analysis and interpretation of findings by pooling the control group information from both phases of the same study, comparing the study findings to the incidence rates for stillbirths and postpartum viability for this species and strain of mouse observed for similar studies conducted by the same laboratory, and evaluating data on the incidence and range of spontaneous eye abnormalities reported in the literature. Based on this supplemental evaluation, the original study supports a NOAEL of 175 mg/kg/d for PFHxA in mice, which is a factor of 5-fold higher than previously reported. Furthermore, to the extent that this study may be considered in the selection of a point of departure for PFHxA in mice, it is noted that 175 mg/kg/d for maternal exposure is an unbounded NOAEL for developmental effects, meaning that the study did not establish a dose at which developmental effects may occur.

Introduction: A successful outcome of root canal therapy relies on effective disinfection of the root canal system, including the use of intracanal medicaments, which vary in their bactericidal and cytotoxic properties. Assessing the benefits and risks associated with the use of these medicaments is of extreme importance, especially in regenerative endodontic procedures, because residual stem cells may be harmed. In this study, we tested the cytotoxicity and genotoxicity of a novel agent, 2-hydroxyisocaproic acid (HICA), and compared its properties with those of a well-established medicament, calcium hydroxide., Methods: Human periodontal ligament fibroblasts were exposed to varying concentrations of HICA (1, 2.5, 5, 10, 20, and 40 mg/mL) for 24 hours, and a dose-response curve was generated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Immunofluorescence for 2 markers of DNA double-strand breaks, phosphorylated γH2AX and 53BP1, was used to establish the genotoxicity of HICA at various half maximal effective concentration (EC50) fractions. Cytotoxicity and genotoxicity of HICA and calcium hydroxide at 1 mg/mL were compared at 24 and 48 hours using the same methods., Results: At 10 mg/mL and higher, HICA was significantly more cytotoxic and genotoxic than the control (P < .05 and P < .0001, respectively). Calcium hydroxide at 1 mg/mL was more cytotoxic than HICA at 1 mg/mL at 24 and 48 hours (P < .05 for both), whereas no difference in the accumulated DNA damage was observed., Conclusions: HICA is not cytotoxic and genotoxic at concentrations <10 mg/mL. At the concentration of 1 mg/mL, HICA is significantly less cytotoxic than calcium hydroxide., (Copyright © 2019 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Perfluorohexanoic acid (PFHxA) is a short-chain, six-carbon PFAA and is a primary impurity, degradant, and metabolite associated with the short-chain fluorotelomer-based chemistry used in the United States, Europe and Japan today. With the shift towards short-chain PFAA chemistry, uncertainties remain regarding human health risks associated with current exposure levels. Here, we present a critical review and assessment of data relevant to human health risk assessment to today's short-chain PFAA chemistry. Human biomonitoring surveys indicate that PFHxA is infrequently detected in the environment as well as in human serum and urine; however, human health concerns may persist in locations where PFHxA is detected. In a companion paper (Luz et al., 2019) we comprehensively evaluate the available toxicity data for PFHxA, and derive a chronic human health-based reference dose (RfD) for PFHxA of 0.25 mg/kg-day based on benchmark dose modeling of renal papillary necrosis in chronically exposed female rats. In this paper, we apply this RfD in human health-based screening levels calculations, and derive a drinking water lifetime health advisory of 1400 μg/L and a residential groundwater screening level for children of 4000 μg/L. Compared to environmental concentration data, even sites with more elevated concentrations of PFHxA in the environment are at least an order of magnitude lower than these screening levels. Available PFHxA human serum and urine biomonitoring data, used as a biomarker for general population exposure, demonstrates that the general human population exposures to PFHxA are low. Previous estimates of daily intake rates for infants exposed to PFHxA through breast milk, formula, and baby foods (Lorenzo et al., 2016) combined with the most conservative PFHxA peer-reviewed toxicity value (Luz et al., 2019) demonstrate that the margin of safety for PFHxA is high. Therefore, PFHxA and related fluorotelomer precursors currently appear to present negligible human health risk to the general population and are not likely to drive or substantially contribute to risk at sites contaminated with PFAS mixtures. PFHxA may also represent a suitable marker for the safety of fluorotelomer replacement chemistry used today., (Copyright © 2019. Published by Elsevier Inc.)

Perfluorohexanoic acid (PFHxA) is a short-chain, six-carbon perfluoroalkyl acid (PFAA) and is a primary impurity, degradant, and metabolite associated with the short-chain fluorotelomer-based chemistry used globally today. The transition to short-chain fluorotelomer-based products as a cornerstone in replacement fluorochemistry has raised questions regarding potential human health risks associated with exposure to fluorotelomer-based substances and therefore, PFHxA. Here, we present a critical review of data relevant to such a risk assessment, including epidemiological studies and in vivo and in vitro toxicity studies that examined PFHxA acute, subchronic, and chronic toxicity. Key findings from toxicokinetic and mode-of-action studies are also evaluated. Sufficient data exist to conclude that PFHxA is not carcinogenic, is not a selective reproductive or developmental toxicant, and does not disrupt endocrine activity. Collectively, effects caused by PFHxA exposure are largely limited to potential kidney effects, are mild and/or reversible, and occur at much higher doses than observed for perfluorooctanoic acid (PFOA). A chronic human-health-based oral reference dose (RfD) for PFHxA of 0.25 mg/kg-day was calculated using benchmark dose modeling of renal papillary necrosis from a chronic rat bioassay. This RfD is four orders of magnitude greater than the chronic oral RfD calculated by the U.S. Environmental Protection Agency for PFOA. The PFHxA RfD can be used to inform public health decisions related to PFHxA and fluorotelomer precursors for which PFHxA is a terminal degradant. These findings clearly demonstrate that PFHxA is less hazardous to human health than PFOA. The analyses presented support site-specific risk assessments as well as product stewardship initiatives for current and future short-chain fluorotelomer-based products., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

In vitro assays to assess developmental neurotoxicity of chemicals are highly desirable. The murine neural embryonic stem cell test (ESTn) can mimic parts of early differentiation of embryonic brain and may therefore be useful for this purpose. The aim of this study was to investigate whether this test is able to rank the toxic potencies of three valproic acid analogues and to study their mode of action by investigating their individual effects on four cell types: stem cells, neurons, astrocytes and neural crest cells. Using immunocytochemical read-outs and qPCR for cell type-specific genes, the effects of valproic acid (VPA), 2-ethylhexanoic acid (EHA) and 2-ethyl-4-methylpentanoic (EMPA) were assessed. VPA and EHA but not EMPA downregulated cell type-specific differentiation makers and upregulated stem cell related markers (Fut4, Cdh1) at different time points during differentiation. Expression of Gfap, a marker for astrocytes, was dramatically downregulated by VPA and EHA, but not by EMPA. This finding was verified using immunostainings. Based on the number and extent of genes regulated by the three compounds, relative potencies were determined as VPA > EHA > EMPA, which is consistent with in vivo developmental toxicity potency ranking of these compounds. Thus, ESTn using a combination of morphology, gene and protein expression readouts, may provide a medium-throughput system for monitoring the effects of compounds on differentiation of cell types in early brain development., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recent studies of perfluoroalkylated substances (PFASs) have focused on the toxicity of long chain PFASs, such as PFOS or PFOA, which have been demonstrated to cause an array of developmental and behavioral effects. However, less is known about low molecular weight PFASs and alternatives. This study examined the morphometric and behavioral effects in zebrafish following developmental exposures of C6 PFASs: perfluorohexanoic acid, PFHxA, perfluorohexane sulfonate, PFHxS, and 6:2 fluorotelomer alcohol, 6:2 FTOH. Embryos were exposed to 0.02-20 μM concentrations of these compounds from the high stage (˜3 h post fertilization, hpf) until 120 hpf. Morphometric and gene expression endpoints were examined at 120 hpf. Genes selected for analysis were previously shown to be altered in zebrafish developmentally exposed to PFOS and PFOA. Additionally, exposed larvae were transferred to clean water and reared until 14 days post fertilization, dpf, when behavioral assays were completed and morphometric endpoints examined. While PFHxA was found to be the most acutely toxic at 120 hpf, few morphometric effects were observed. Gene expression was the most sensitive endpoint with significant increased tgfb1a, bdnf, and ap1s1 expression observed with PFHxA exposure. PFHxS exposure produced morphometric effects in the larvae, specifically increased length and yolk sac area at 2 and 20 μM. This phenotype persisted to the 14 dpf time point, where these larvae additionally displayed decreased distance traveled and crosses through the center of the arena of the behavioral assay. Exposure to 6:2 FTOH caused no morphometric effects at 120 hpf, and this compound was the least acutely toxic. However, expression of both tgfb1a and bdnf were increased by greater than 2 fold change at this time point. Effects also persisted to 14 dpf where a significant increase in distance traveled and velocity were observed in the behavioral assay. This study demonstrates effects on behavioral, morphometric and gene expression endpoints with developmental PFHxA, PFHxS, and 6:2 FTOH exposures in zebrafish., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This in vitro study aimed to detect leaching components from an epoxy resin- and a methacrylate-based endodontic sealer and correlate them to cytotoxicity induced by material extracts for up to 36 weeks. We qualitatively determined the substances released by aged AH Plus and RealSeal SE specimens at seven intervals between 0 and 36 weeks. Quantification was performed by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). We determined the viability of murine macrophage J774 cells after 24 h exposure to material extracts, at each interval, using a fluorescence staining/microscopy method. The leachables detected were 1-adamantylamine and bisphenol A diglycidyl ether from AH Plus and N-(p-tolyl) diethanolamine and caprolactone-2-(methacryloyloxy) ethyl ester from RealSeal SE. The largest UPLC/MS chromatogram peak areas of the leachables were detected within 72 h. Induction of cytotoxicity after exposure to AH Plus and RealSeal SE extracts coincided with leachant detected within the first 72 and 24 h, respectively. The clinical impact of the cytotoxicity due to resin-based endodontic sealers is unknown.

Perfluorooctane sulfonic acid (PFOS) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) are persistent organic pollutants of high concern because of their environmental persistence, bioaccumulation and toxic properties. Besides, the amphiphilic properties of fluorinated compounds such as PFOS and perfluorohexanoic acid (PFHxA) suggest a role in increasing cell membrane permeability and solubilizing chemicals. The present study aimed at investigating whether PFOS and PFHxA are capable of modifying the activation of PCB126 toxicity-related pathways. For this purpose, zebrafish embryos were exposed in semi-static conditions to 7.5μg/L of PCB126 alone, in the presence of 25mg/L of PFOS, 15.7mg/L of PFHxA or in the presence of both PFOS and PFHxA. Quantitative PCR was performed on embryos aged from 24h post fertilization (hpf) to 96 hpf to investigate expression changes of genes involved in metabolism of xenobiotics (ahr2, cyp1a), oxidative stress (gpx1a, tp53), lipids metabolism (acaa2, osbpl1a), and epigenetic mechanisms (dnmt1, dnmt3ba). Cyp1a and ahr2 expression were significantly induced by the presence of PCB126. However, after 72 and 78h of exposure, induction of cyp1a expression was significantly lower when embryos were co-exposed to PCB126+PFOS+PFHxA when compared to PCB126-exposed embryos. Significant upregulation of gpx1a occurred after exposure to PCB126+PFHxA and to PCB126+PFOS+PFHxA at 30 and 48 hpf. Besides, embryos appeared more sensitive to PCB126+PFOS+PFHxA at 78 hpf: acaa2 and osbpl1a were significantly downregulated; dnmt1 was significantly upregulated. While presented as environmentally safe, PFHxA demonstrated that it could affect gene expression patterns in zebrafish embryos when combined to PFOS and PCB126, suggesting that such mixture may increase PCB126 toxicity. This is of particular relevance since PFHxA is persistent and still being ejected into the environment. Moreover, it provides additional information as to the importance to integrate mixture effects of chemicals in risk assessment and biomonitoring frameworks., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are synthetic fluorinated compounds that are highly bioaccumulative and persistent organic pollutants. Perfluorooctanoic acid (PFOA), an eight-carbon chain perfluorinated carboxylic acid, was used heavily for the production of fluoropolymers, but concerns have led to its replacement by shorter carbon chain homologues such as perfluorohexanoic acid (PFHxA) and perfluorobutanoic acid (PFBA). However, limited toxicity data exist for these substitutes. We evaluated the toxicity of PFOA, PFHxA and PFBA on a zebrafish liver cell line and investigated the effects of exposure on cell metabolism. Gross toxicity after 96 h of exposure was highest for PFOA and PFO - , while PFHxA and PFBA exhibited lower toxicity. Although the structural similarity of these compounds to fatty acids suggests the possibility of interference with the transport and metabolism of lipids, we could not detect any differential expression of peroxisome proliferator-activated receptor (ppar-α, -β and -γ), fabp3 and crot genes after 96 h exposure to up to 10 ppm of the test compounds. However, we observed localized lipid droplet accumulation only in PFBA-exposed cells. To study the effects of these compounds on cell metabolism, we conducted fluorescence lifetime imaging microscopy using naturally fluorescent biomarkers, NADH and FAD. The fluorescence lifetimes of NADH and FAD and the bound/free ratio of each of these coenzymes decreased in a dose- and carbon length-dependent manner, suggesting disruption of cell metabolism. In sum, our study revealed that PFASs with shorter carbon chains are less toxic than PFOA, and that exposure to sublethal dosage of PFOA, PFHxA or PFBA affects cell metabolism. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

To evaluate the safety profile of cationic micelles, based on triblock copolymer poly(dimethylaminoethyl methacrylate)-poly(e-caprolactone)-poly(dimethylaminoethyl methacrylate) (PDMAEMA 9 - PCL 70 -PDMAEMA 9 ), the effects of empty (PM) and curcumin loaded micelles (PM-Curc) on nonenzyme induced lipid peroxidation (LPO) in vitro, hemolytic activity and morphological changes in some organs after repeated intraperitoneal administration in vivo were studied. To induce LPO, rat liver microsomes were incubated with a solution of iron sulfate and ascorbinic acid (Fe 2+ /AA). The effect of empty PM (40 and 100 μg/ml), PM-Curc and free curcumin (both at 3.48 and 8.7 μg curcumin/ml) was assessed at 20 min incubation time. In the non-enzyme induced LPO model, the investigated substances at all concentrations significantly decreased the formation of malondialdehyde (MDA), compared to the Fe 2+ /AA induced LPO group. According to the results it can be concluded that curcumin alone and loaded in PM, exert significant antioxidant activity. In the biocompatibility safety studies, the mean hemolytic index for polymeric carrier was less than 2%, indicating it was non-hemolytic. The general appearance of the organ tissues from Wistar rats, treated in vivo with curcumin loaded PM was similar to that of controls, thus showing no apparent toxicity after repeated 14-days treatment.

To examine the kinetics of low doses of perfluoro compounds (PFCs), we administered perfluorohexanoic acid (C6A), perfluorooctanoic acid (C8A), perfluorononanoic acid (C9A) and perfluorooctane sulfonate (C8S) with a single oral dose (50-100 μg/kg BW), and in drinking water at 1, 5, and 25 μg/L for one and three months to male rats; and examined the distribution in the brain, heart, liver, spleen, kidney, whole blood and serum. C6A was very rapidly absorbed, distributed and eliminated from the tissues with nearly the same tissue t 1/2 of 2-3 hr. Considering serum Vd, and the tissue delivery, C6A was mainly in the serum with the lowest delivery to the brain; and no tissue accumulation was observed in the chronic studies as estimated from the single dose study. For the other PFCs, the body seemed to be an assortment of independent one-compartments with a longer elimination t 1/2 for the liver than the serum. The concentration ratio of liver/serum increased gradually from C 0 to a steady state. The high binding capacity of plasma protein may be the reason for the unusual kinetics, with only a very small fraction of free PFCs moving gradually to the liver. Although the tissue specific distribution was time dependent and different among the PFCs, the Vd and k e of each tissue were constant throughout the study. The possibility of extremely high C6A accumulation in the human brain and liver was suggested, by comparing the steady state tissue concentration of this study with the human data reported by Pérez et al. (2013).

Perfluoroalkyl compounds (PFCs) are globally distributed synthetic compounds that are known to adversely affect human health. Developmental toxicity assessment of PFCs is important to facilitate the evaluation of their environmental impact. In the present study, we assessed the developmental toxicity and teratogenicity of PFCs with different numbers of carbon atoms on Xenopus embryogenesis. An initial frog embryo teratogenicity assay-Xenopus (FETAX) assay was performed that identified perfluorohexanoic (PFHxA) and perfluoroheptanoic (PFHpA) acids as potential teratogens and developmental toxicants. The mechanism underlying this teratogenicity was also investigated by measuring the expression of tissue-specific biomarkers such as phosphotyrosine‑binding protein, xPTB (liver); NKX2.5 (heart); and Cyl18 (intestine). Whole‑mount in situ hybridization, reverse transcriptase‑polymerase chain reaction (RT-PCR), and histologic analyses detected severe defects in the liver and heart following exposure to PFHxA or PFHpA. In addition, immunoblotting revealed that PFHpA significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), while PFHxA slightly increased these, as compared with the control. These results suggest that PFHxA and PFHpA are developmental toxicants and teratogens, with PFHpA producing more severe effects on liver and heart development through the induction of ERK and JNK phosphorylation.

The clinical application of amphotericin B (AmB), a broad spectrum antifungal agent, is limited by its poor solubility in aqueous medium and also by its proven renal toxicity. In this work, AmB was encapsulated in micelles obtained from the self-assembly of PDMAEMA-b-PCL-b-PDMAEMA triblock copolymers. The amount of encapsulated AmB depended on the copolymer composition, and short blocks of polycaprolactone (PCL) and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) showed better performance. All the studied formulations exhibited a controlled release of AmB along 150 h. The formulations presented reduced hemotoxicity while maintaining antifungal activities against Candida albicans, Candida krusei, and Candida glabrata comparable with free AmB. A reduction on the hemotoxicity was found to be due to the slow release and subsequent low aggregation achieved with the use of polymer micelle nanocontainers.

Objectives: Up to now, little research has been focussed on discovering how zeta potential independently affects polymeric nanoparticle (NP) cytotoxicity., Methods: Polymeric nanoparticles of gradient zeta potential ranging from -30 mv to +40 mv were fabricated using the same poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBHHx) biopolymer. Interaction forces between nanoparticles and cells were measured by atomic force microscopy (AFM). Cytotoxicity of the nanoparticles to cells was investigated by using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay., Results: Four kinds of nanoparticle with similar sizes and gradient zeta potentials, were fabricated. Those with positive surface charges were found to be more toxic than those with negative surface charges. Positively charged nanoparticles or nanoparticles with higher 'like' charges, offered higher interaction force with cells., Conclusion: This work proposes a novel approach for investigating interaction between NPs and cells, and discloses the importance of controlling zeta potential in developing NPs-based formulations in the future., (© 2015 John Wiley & Sons Ltd.)

Due to their oleophobic and hydrophobic properties and stability, perfluorinated compounds (PFCs) are used in many applications, particularly as greaseproofing agents for food contact. However, PFCs 8-carbons in length or greater (C8-PFCs) have raised concerns regarding environmental biopersistence, bioaccumulation in humans, and potent toxicity that have resulted in their gradual phase-out for food contact use. Industry has replaced C8-PFCs with shorter-chained C6-based greaseproofing agents, which are intended to have the same favorable physicochemical properties without the problematic toxicological effects in humans and wildlife. Compared with the large body of data available for C8 compounds, however, the available database on toxicity and exposure to the C6 compounds is fairly limited. This article summarizes the information in this database, focusing on aspects of human exposure and potential health risks associated with two types of C6 PFCs found in food packaging: perfluorohexanoic acid (PFHxA) and 6-2 fluorotelomer alcohol (C6-FTOH).

Perfluorohexanoic acid (PFHxA), a 6-carbon perfluoroalkyl (C6; CAS # 307-24-4), has been proposed as a replacement for the commonly used 8-carbon perfluoroalkyls: perfluorooctanoic acid and perfluorooctane sulfonate. PFHxA is not currently a commercial product but rather the ultimate degradation product of C6 fluorotelomer used to make C6 fluorotelomer acrylate polymers. It can be expected that, to a greater or lesser extent, the environmental loading of PFHxA will increase, as C6 fluorotelomer acrylate treatments are used and waste is generated. This article reports on a chronic study (duration 104 weeks) that was performed to evaluate the possible toxicologic and carcinogenic effects of PFHxA in gavage (daily gavage, 7 days per week) treated male and female Sprague-Dawley (SD) rats. In the current study, dosage levels of 0, 2.5, 15, and 100 mg/kg/day of PFHxA (males) and 5, 30, and 200 mg/kg/day of PFHxA (females) were selected based on a previous subchronic investigation. No effects on body weights, food consumption, a functional observational battery, or motor activity were observed after exposure to PFHxA. While no difference in survival rates in males was seen, a dose-dependent decrease in survival in PFHxA-treated female rats was observed. Hematology and serum chemistry were unaffected by PFHxA. PFHxA-related histologic changes were noted in the kidneys of the 200-mg/kg/day group females. Finally, there was no evidence that PFHxA was tumorigenic in male or female SD rats at any of the dosage levels examined., (© 2014 by The Author(s).)

The mechanical properties of highly porous (90% porosity) poly(l-lactide) (PLLA), poly(ε-caprolactone) (PCL) and poly(l-lactide/ε-caprolactone) (PLCL) were investigated. Young's modulus of non-porous PLLA, PCL and PLCL dropped from 2263.4, 183.7 and 5.7 MPa to 16.8, 1.0 and 1.0 MPa, respectively, for their ~90% porous counterparts. Elongation at break of PCL decreased noticeably with porosity fraction while PLCL maintained a highly elastomeric character and strain recovery capacity even in the presence of pores. Inorganic bioactive particles (hydroxyapatite or bioglass) were added to confer bioactivity to the aforementioned synthetic bioresorbable polymers, and their effect on the mechanical properties was also investigated. Addition of 15 vol.% of inorganic bioactive particles increased the Young's modulus of highly porous PLLA from 16.2 to ~30 MPa. On the contrary, the difference between Young's modulus of filled and unfilled PCL and PLCL scaffolds was not statistically significant. Finally, an in vitro study of the cytocompatibility and adhesion of adipose derived stem cells (ADSCs) was conducted. The observed viability and excellent adhesion of these cells to both porous and non-porous templates indicate that the employed materials can be good candidates for application in tissue engineering., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The reproductive toxicity potential of Ammonium Salt of Perfluorinated Hexanoic Acid (PFHxA Ammonium Salt) in pregnant Crl: CD1(ICR) mice was investigated. Twenty females/group were administered the test substance or vehicle once daily from gestation day 6 through 18. Phase 1 doses: 0, 100, 350, and 500 mg/kg/d; phase 2: 0, 7, 35, and 175 mg/kg/d. Parameters evaluated include mortality, viability, body weights, clinical signs, abortions, premature deliveries, pregnancy and fertility, litter observations, maternal behavior, and sexual maturity in the F1 generation. The level of PFHxA Ammonium Salt was measured in the liver of F0 and F1 mice. At doses of 350 and 500 mg/kg/d maternal mortalities, excess salivation and changes in body weight gains occurred. Pup body weights were reduced on postpartum day (PPD) 0 in all the dosage groups, but persisted only in the 350 and 500 mg/kg/d groups. Additional effects at 300 and 500 mg/kg/d included stillbirths, reductions in viability indices, and delays in physical development. Levels of PFHxA Ammonium Salt in the livers of the 100 mg/kg/d dams were all below the lower limit of quantization (0.02 µg/mL); in the 350 mg/kg/d group, 3 of the 8 samples had quantifiable analytical results. In phase 2 no PFHxA Ammonium Salt was found in the liver. Adverse effects occurred only in the 175 mg/kg/d group and consisted of increased stillborn pups, pups dying on PPD 1, and reduced pup weights on PPD 1. Based on these data, the maternal and reproductive no observable adverse effect level of PFHxA Ammonium Salt is 100 mg/kg/d., (© The Author(s) 2014.)