Italian researchers have found that trifluoroacetic acid (TFA), an atmospheric byproduct of certain f-gases, reduced antibody production in human-relevant in vitro (outside the body) tests at levels similar to those caused by perfluorooctanesulfonic acid (PFOS), which has well-documented immunotoxic effects.
Their study – “Decoding PFAS immunotoxicity: a NAMs-based comparison of short vs. long chains” – took place at the Laboratory of Toxicology and Risk Assessment, Department of Pharmacological and Biomolecular Sciences (Rodolfo Paoletti), University of Milan. The researchers included Emanuela Corsini, Professor of Toxicology at the School of Pharmacy; Valentina Galbiati, Researcher; Martina Iulini, Postdoctoral Researcher; and Marina Marinovich, Professor.
The study is one of the first in the world to assess the potential toxicity of TFA in humans. It was supported by the European Food Safety Authority (EFSA), which is engaged in risk assessments of TFA.
Scientists consider TFA to be an example of PFAS (per- and polyfluorinated substances), known as “forever chemicals” for their persistence in nature. Because they are resistant to stains, grease and water PFAS are widely used in consumer products and also include many refrigerants, but they are considered chemical pollutants when they infiltrate surface water and soil.
TFA is formed from the complete breakdown over two weeks of HFO-1234yf that leaks into the atmosphere, and the partial breakdown of other fugitive HFOs and HFC-134a. Other sources of TFA include the decomposition of certain pesticides and pharmaceuticals, as well as direct releases of industrial waste. The study noted that TFA is currently considered the most abundant PFAS in the global environment.
Long-chain PFAS such as PFOS and PFOA (eight carbons) are linked to reproductive, developmental, liver, kidney, thyroid and immunological effects. In human epidemiological studies, higher blood levels of PFOS and PFOA have been associated with reduced vaccine efficacy and an increased risk of infection in children. By contrast, TFA is an ultrashort-chain PFAS (two carbons) but its prevalence in the environment, drinking water and food and presence in human blood serum has raised concerns about its potential toxicity, which have been largely unexplored.
Concerns about TFA have often been dismissed because of its small chain length and the presumption of low bioaccumulation potential. However, because of the similar effects of TFA and PFOS on antibody production in the University of Milan study, the researchers concluded that “chain length alone is not a reliable predictor of immunotoxic potential.”
“Ultrashort TFA can still exert potent suppressive effects.”
University of Milan study
The researchers examined the immunotoxicity of a number PFAS in addition to TFA, including long-chain (such as PFOS and PFOA) and short-chain (such as PFHxS and PFBS), as well as a fluoropolymer. They employed an in vitro technique using what are called “human-relevant new approach methodologies” (NAMs).
In their analysis, they employed peripheral blood mononuclear cells (PBMCs) – specialized white blood cells that are essential to the human immune system – from healthy donors to assess their production of infection-fighting antibodies IgG and IgM when exposed to TFA and PFOS. The concentrations of the PFAS, none of which harmed the cells, included 0.001, 0.1 and 10mcg/mL. The selection of these concentrations was made in consultation with experts from an EFSA-sponsored project and in accordance with EFSA’s current recommendations for immunotoxicity testing. Studies have found TFA in human blood serum, including a recent one done at North Carolina State University that reported a median level of 17ng/mL (0.017mcg/mL).
The results “aligned with a growing body of evidence from in vivo [in the body] animal studies and human epidemiology and reinforce the importance of integrating NAMs into immunotoxicology risk assessments,” the study said. “In vitro NAMs provided mechanistic, human-relevant insights and reinforce their integration into regulatory frameworks.” NAMs overcome many limitations of animal models, such as differences in immune system development and chemical kinetics between species, the study added.
Several tests on rabbits and rats have pointed to toxic effects of TFA. For example an industry-supported study found that pregnant rabbits exposed to 750mg/kg bw of TFA per day for 23 days produced multiple folded retina in 32% of 28 litters. Based on this and other studies, the German government last year proposed to the European Chemicals Agency (ECHA) that TFA and its trifluoroacetate salts should be classified and labelled as reproductively toxic.
‘An unexpected similarity’
The University of Milan study confirmed that PFOS consistently suppresses antibody production (both IgG and IgM) in a dose-dependent manner in both male and female donors, with statistically significant reductions observed starting at 0.1mcg/mL. The suppression of IgM and IgG antibody release in PBMCs cultures is linked to decreased vaccine responsiveness in epidemiological studies, the study said.
When the effects of TFA were compared to those of PFOS, “an unexpected similarity” emerged for both male and female donors, the study said. “TFA induces a comparable reduction in the release of both IgG and IgM [antibodies], with significant inhibition occurring at higher concentrations.” For example, the production of IgG in males underwent a drop in the stimulation index (SI) from a baseline of 1 to about 0.25 at 0.001mcg/mL of both TFA and PFOS and to about 0.4 and 0.5, respectively, at 10mcg/mL.
These findings suggested that “extremely short-chain PFAS can produce immunosuppressive effects similar in magnitude to those observed with long-chain analogues despite their difference in the carbon-chain and presumed lower bioaccumulation potential,” the study said.
The study concluded that “chain length alone is not a sufficient predictor of immunotoxic potential.” While shorter-chain PFAS in the study did often have a lesser impact on antibody suppression, “ultrashort TFA can still exert potent suppressive effects.” In contrast, PTFE (polytetrafluoroethylene), a PFAS with a giant chain structure, “appears to be largely inactive or even stimulatory” of antibody formation, it said. Thus the findings “support a nuanced, compound-specific approach to PFAS risk assessment rather than a simple long- vs. short-chain distinction.”
The findings also agree with previous animal studies demonstrating that PFOS and PFOA reduce antibody responses in rodents, often at doses relevant to human exposure. For instance, exposure to PFOS in mice results in substantial reductions in IgM and IgG production following immunization.
The study noted that the potent antibody suppression observed with TFA is “unlikely to be solely explained by cellular accumulation or bioavailability, as its intracellular presence is relatively low and similar to other short-chain compounds.” What could explain it, the study said, is TFA’s high molar concentration (about 88mcgM) compared to other PFAS. Another explanation is that TFA “operates via a distinct mechanism of action compared to both long- and typical short-chain PFAS, beyond differences in cellular uptake or molar concentration.”
The researchers highlighted that only a few immunotoxicological assessments in lab animals (mostly rats) have been conducted for TFA. “In view of the limited immunotoxicity data for TFA and the results obtained in the present study, it is crucial from a regulatory perspective to address this gap by implementing dedicated immunotoxicity testing strategies, including, where feasible, the use of NAMs specifically targeting immune endpoints, especially considering that TFA is both a PFAS and a degradation product of longer-chain PFAS,” the study said.
The study acknowledged that it focused on acute single-compound exposures, which do not fully reflect the chronic, low-dose and mixture scenarios that characterize real-world PFAS exposure. Combined exposures to multiple PFAS and their possible additive or synergistic immunotoxic effects were not evaluated “and should be a priority for future research.”
Furthermore, although PFAS are generally considered resistant to biotransformation, in vitro models do not account for metabolic activation, detoxification or reabsorption. Thus the study recommended coupling NAM-derived potency data with PBK (physiologically based kinetic) modeling that predicts how chemicals are absorbed, distributed, metabolized and excreted in the body.
Expert reaction
Jamie DeWitt, Professor of Environmental and Molecular Toxicology at Oregon State University, who has extensively studied how PFAS disrupt immune function and human health, called Corsini, the leader of the University of Milan study, “one of the foremost experts on immunotoxicity.” Corsini and her group “have been working on PFAS immunotoxicity for close to two decades; they know and understand PFAS and their effects on the immune system,” DeWitt noted.
“Based on what I know about the quality of the work conducted in the Corsini Lab, the development and evaluation of the new approach method and the robustness of the endpoint that is assessed with the new approach method, it appears as if TFA has immunotoxicological concerns,” DeWitt said. “I would be interested to see if it produces similar results in an in vivo rodent model.”
To Hans Peter Arp, a Norwegian environmental chemist who co-authored a study calling TFA a “planetary boundary threat,” the University of Milan study “is the first evidence of immunotoxicity of TFA.”
Moreover, said Arp, an Expert Advisor at the Norwegian Geotechnical Institute and a Professor at the Norwegian University of Science and Technology, the study justifies the work of the Netherlands’ National Institute for Public Health and the Environment (RIVM) in applying “relative potency factors” (RPF) to derive drinking water thresholds for TFA based on immunotoxicity thresholds of PFOA, PFOS and other PFAS. For example, in a study of the effect of TFA on male rat liver weight, RIVM found TFA to be 0.002 times as toxic as PFOA, corresponding to a TFA drinking water threshold of 2.2mcg/L.
In one of the few other studies to investigate human health outcomes related to TFA exposure, Duke University researchers have looked at a potential link between TFA exposure in blood serum and biomarkers of thyroid dysfunction in North Carolina firefighters. While the researchers initially found a correlation with a small number of data points, additional data indicated that the correlation was weakened and no longer significant, said lead researcher Heather Stapleton, Professor of Environmental Health at Duke University and Director of Duke’s Superfund Research Center.
However, the Duke researchers are continuing to collect data “to understand what variables influence exposure, and then continue to see if it’s related to any risks for thyroid dysregulation” and eventually other diseases including cancer and disruption of lipid regulation, added Stapleton. She will discuss her work on June 3 at the ATMOsphere America conference in Tarrytown, New York. (ATMOsphere is the publisher of NaturalRefrigerants.com.)
Angelica Candido, Sector Manager for Cefic, the European Chemical Industry Council, declined to comment on the University of Milan study. In defending the use of TFA-producing refrigerants, the fluorochemical Industry points to the UN Environment Programme (UNEP) and its 2022 Environmental Effects Assessment Panel (EEAP) report. The report says that TFA is not bioaccumulative, is “not expected to pose significant risk to humans or the environment at the present time” and “is unlikely to cause adverse effects out to 2100.”
