Female B₆C₃F₁ mice were used in these studies as they are the National Toxicology Program’s preferred strain for evaluating general toxicity and have been used previously to investigate PFAS immunotoxicity (Shane et al., 2020; Weatherly et al., 2021, 2023). All mice were purchased from Jackson Laboratory (Bar Harbor, ME) at 7–8 weeks of age. Upon arrival, the animals were allowed to acclimate for a minimum of 5 days. All animals were randomly assigned to treatment groups, weighed, and individually identified via tail marking using a permanent marker. Dose groups were identified by cage cards. Both the dosing group as well as the animal numbers were identified on each cage. The animals were housed 5 mice/cage in ventilated plastic shoe box cages with hardwood chip bedding, modified NIH-31 6% irradiated rodent diet (Harlan Teklad – item #7913), and sterile tap water from water bottles ad libitum. The temperature in the animal facility was maintained between 65 and 78 °F and the relative humidity between 30 and 70%; a light/dark cycle was maintained at 12-hr intervals. All animal experiments were performed in an AAALAC International-accredited National Institute for Occupational Safety and Health (NIOSH) animal facility in accordance with an animal protocol approved by the CDC-Morgantown Institutional Animal Care and Use Committee. This activity was reviewed by CDC, deemed research not involving human subjects, and was conducted consistent with applicable federal law and CDC policy.

Acetone [CAS #67–64-1] and perfluorobutane sulfonic acid (97%, PFBS) [CAS #375–73-5] were purchased from Sigma-Aldrich. Perfluoropentane sulfonic acid (95%, PFPeS) [CAS #2706–91-4] was purchased from SynQuest Laboratories.

To evaluate systemic toxicity, B₆C₃F₁ mice (5/group) were topically treated on the dorsal surface of each ear (25 μl/ear) with vehicle (acetone), PFBS (C4), or PFPeS (C5) concentrations (1.25, 2.5, 5% v/v) once a day for 28 consecutive days. This yielded an approximate topical daily dose of 226, 113, or 57 mg/kg body weight of PFBS(C4) and 225, 113, and 56 mg/kg body weight of PFPeS. Five mice per group have previously been shown to be sufficient to obtain statistical significance (Weatherly et al., 2024a, 2024b; Weatherly et al., 2021, 2023). These studies were conducted for the purpose of hazard identification, and the concentrations of PFBS and PFPeS were selected based on an initial 7-day range finding study and previous skin exposure studies conducted with various PFAS (Weatherly et al., 2024a, 2024b; Weatherly et al., 2021, 2023). Body weights were measured weekly and before exposure to ensure no overt toxicity was occurring. Animals were euthanized by CO₂ asphyxiation approximately 24 hours after the last exposure.

Following euthanasia, animals were weighed and examined for gross pathology. The liver, spleen, kidneys, and thymus were removed, cleaned of connective tissue, and weighed. Left and right auricular draining lymph nodes (dLNs; draining the site of chemical application), one half of the spleen, and one ear pinna were collected and placed in 4 mL RPMI for immune phenotyping as previously described (Weatherly et al., 2021). Half of one ear pinna and a small lobe of the liver (caudate) were placed in 0.5 ml of RNAlater for subsequent gene expression analysis (see below). The remainder of the liver, spleen, ear pinna (1/2), and one kidney (right) were collected in 10% formalin for histopathology analysis.

Blood samples were collected following euthanasia via cardiac puncture, transferred to serum separation tubes, and separated by centrifugation. The serum was frozen at −20 °C for subsequent analysis. Selected serum chemistries were evaluated using a Catalyst DX Chemistry Analyzer (IDEXX Laboratories, Inc.; Westbrook, ME). Endpoints analyzed included: alkaline phosphatase (ALKP), urea nitrogen (BUN), glucose (GLU), alanine aminotransferase (ALT), and cholesterol (CHOL).

Serum and urine samples were collected and analyzed for PFBS or PFPeS by Vista Analytical Laboratory, following Vista’s standard operating procedures (Vista Analytical Laboratory, El Dorado Hills, CA) of solid phase extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) using BEH C18 columns as previously described (Shoemaker et al., 2008). Vista Analytical follows EPA method 537. An aliquot of serum was collected (as described above) from each animal following euthanasia. To have sufficient volume for analysis, voided urine was pooled following euthanasia and stored at −20 °C. The initial calibration and continuing calibration verifications met the acceptance criteria as previously described (Shoemaker et al., 2008). No analytes were detected in the method blank above the reporting limit (200 ng/mL). The labeled standard recoveries for all quality controls and samples were within the acceptance criteria as previously described (Shoemaker et al., 2008). PFBS concentration was set to zero in samples where PFBS was reported as ‘Not Detected.’

Flow cytometry was conducted as previously described (Weatherly et al., 2021). For staining, single cell suspensions were resuspended in staining buffer containing anti-mouse CD16/32 antibody (Fc Block; BD Biosciences), then incubated with a cocktail of fluorochrome-conjugated antibodies specific for mouse cell surface antigens. For dLN and spleen cells: B220-V500 (RA3–6B2), CD11b-PerCP-Cy5.5 (M1/70), CD8-PE-CF594 (53–6.7), Siglec-F-PE (E50–2440), Ly6G-FITC (1A8) (BD Biosciences), CD11c-eFluor 450 (N418), CD86-APC (GL1) (eBioscience), CD45-Superbright 780 (30-F11), F4/80-PE-Cy7 (BM8), MHCII-APC-eF780 (M5/114.15.2) (Invitrogen), CD4-BV711 (RM4–5), Ly6C-AF700 (HK1.4) (BioLegend). For ear pinna cells: CD4-BV711 (RM4–5), NKp46-BV605 (29A1.4) (BioLegend), CD8-PE-CF594 (53–6.7), CD3-V500 (500A2), Ly6G-FITC (1A8), CD11b-PerCP-Cy5.5 (M1/70), CD11c-AF700 (HL3), Siglec-F-PE (E50–2440) (BD Biosciences), F4/80-PE-Cy7 (BM8), MHCII-APC-eF780 (M5/114.15.2), CD45-Superbright 780 (30-F11), FcεRI-APC (MAR-1) (Invitrogen), CD117-eFluor 450 (2B8) (eBioscience). Ear total cellularity was determined using a Cellometer with AO/PI (Nexcelom), and dLN and spleen total cellularity was determined using a Z2 Coulter Particle Count and Size Analyzer (Beckman Coulter). Data was acquired on a LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo v10 software (TreeStar Inc., Ashland, OR). Cellular populations were defined using the gating strategies outlined in Supplemental Table 1; Fluorescence minus ones (FMOs) were used as gating controls. An example of gating strategies for dLN, spleen, and ear is outlined in (Weatherly et al., 2024b).

Ear (half an ear pinna/mouse) and liver (caudate) were homogenized on a TissueLyser II in Buffer RLT (Qiagen). Total RNA was isolated using Qiagen’s RNeasy mini spin column kits with DNase treatment on a QIAcube automated RNA isolation machine. RNA concentrations and purity were analyzed on a NanoDrop spectrophotometer (Thermo Fisher Scientific). The cDNA was prepared from 1–2 μg of RNA on an Eppendorf Mastercycler using Applied Biosystems’ High-Capacity Reverse Transcription kit. The cDNA was used as a template for real-time PCR reactions containing TaqMan PCR Master Mix with gene-specific primers (Applied Biosystems) on a 7500 Real-Time PCR System. Relative fold gene expression changes (2^−ΔΔCT) were determined compared to vehicle controls and normalized for expression of reference gene β-actin (Taqman). Evaluated genes are identified in Supplemental Table 2, and those with greater than a two-fold statistically significant change at any exposure were included in the results section.

Each tissue sample stored in 10% formalin was embedded in paraffin, sectioned at 5 microns, stained with hematoxylin and eosin (H&E), and evaluated by a veterinary pathologist at StageBio (Mason, Ohio) using The Society of Toxicologic Pathology Guideline (Crissman et al., 2004). Provantis^™ pathology software v10.2.3.1 was utilized for data capture and table generation. Histopathology grades were assigned as grade 1 (minimal), grade 2 (mild), grade 3 (moderate), grade 4 (marked), or grade 5 (severe) based on an increasing extent of change. The criteria used for skin grading were previously described (Weatherly et al., 2021).

A one-way analysis of variance (ANOVA) was conducted for analysis of the data generated from the described animal studies. If the ANOVA showed significance at p ≤ 0.05, the Dunnett’s Multiple Range post-test was used to compare treatment groups with the control group. No correction for multiple endpoints has been applied. Statistical analysis was performed using GraphPad Prism version 9.2 (San Diego, CA). Results represent the mean ± standard error (SE) of 4–5 mice per group. Statistical significance is designated by *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

A significant increase in PFBS serum concentration was observed with 1.25, 2.5, and 5% PFBS (Figure 1A); levels increased from 0 μg/ml (control) to 4.3, 6.72, and 13.6 μg/ml, respectively. PFPeS levels in the serum were also significantly increased with 1.25, 2.5, and 5% exposure, increasing from 0.766 μg/ml (control) to 41, 79.8, and 119.2 μg/ml, respectively (Figure 1A), suggesting potential absorption occurring following PFAS dermal exposure. In general, serum PFPeS levels were higher than those for PFBS. Statistical analysis could not be performed on the urine samples as each concentration was from five pooled samples with a single data point. Urine PFBS concentration increased from 0 (control) to 72 μg/ml with 1.25% PFBS, 250 μg/ml with 2.5% PFBS, and 310 μg/ml with 5% PFBS (Figure 1B). Urine PFPeS concentration increased from 1.5 (control) to 220 μg/ml with 1.25% PFPeS, 430 μg/ml with 2.5% PFPeS, and 470 μg/ml with 5% PFPeS (Figure 1B). Notably, the longer-chain PFAS, PFPeS, had higher levels detected in the serum, but not in the urine.

A significant increase in relative liver weights was observed with PFBS and PFPeS, along with a decrease in spleen weight with PFPeS exposure (Figure 2). Relative liver weight significantly increased following exposure to 2.5 and 5% PFBA and PFPeS (11, 14%, and 32, 63%, respectively, vs. values for vehicle-treated mice) (Figure 2A). Relative spleen weights were significantly decreased by 18% with the highest PFPeS (5%) concentration (Figure 2C). No change in relative weight was observed in the thymus or kidneys (Figure 2B, D). No overt toxicity was observed with PFBS or PFPeS exposure, as measured in loss of body weight (Supplemental Figure 1). Absolute organ weights not corrected for total body weight are reported in Supplemental Table 3, with significant increases only in liver mass with 1.25, 2.5, and 5% PFPeS exposure. No significant changes in any absolute organ weights were observed with PFBS.

After 28 days of PFBS or PFPeS exposure, there was a significant increase in serum glucose (5% PFBS; 1.25, 2.5, 5% PFPeS) and alkaline phosphatase (ALKP) (5% PFPeS) (Figure 3A, B). Glucose increased following 5% PFBS exposure by 25% and by 28, 24, and 25% with PFPeS exposure (1.25, 2.5, and 5%, respectively) (Figure 3B). ALKP increased 47% but only following exposure to 5% PFPeS (Figure 3A). No significant changes were observed in cholesterol, ALT, or BUN (Figure 3C–E).

Histopathology examination of the liver revealed PFBS exposure induced minimal hepatocellular hypertrophy in all 5% exposed animals (Table 1). Hepatocellular hypertrophy was characterized by decreased glycogen content, increased cytoplasmic eosinophilia, and increased hepatocyte volume predominantly in centrilobular locations. Similarly, but to a greater degree, PFPeS exposure induced minimal hepatocellular hypertrophy with 1.25% (3/5 mice), mild hypertrophy in 1.25% (1/5) and 2.5% (5/5), and moderate hypertrophy with 5% (5/5) (Table 1). With increasing severity, more of the hepatic plate was affected. Moderately affected individuals also had distortion of the hepatic cords surrounding the central vein. Individual animals dosed with 2.5 and 5% PFPeS also had focal or multifocal randomly distributed necrosis of hepatocytes, which was likely related to hepatocellular hypertrophy. Minimal hepatocyte necrosis occurred in 3/5 mice in 2.5% and 2/5 mice in 5% PFPeS exposure groups. No necrosis was observed with PFBS exposure. No histopathological changes were observed in the liver with 0% PFBS or PFPeS vehicle controls.

At the site of exposure, dose-related epidermal hyperplasia, or an increased number of keratinocyte layers, was observed with PFBS and PFPeS exposure. Minimal hyperplasia was observed at 1.25% (1/5 mice), 2.5% (3/5), and 5% (5/5) PFBS and with 5% PFPeS (5/5) (Table 1). Minimal mixed cell inflammation was also observed in the dermis with 2.5% (1/5) and 5% (2/5) PFBS and with 5% (3/5) PFPeS exposure (Table 1). Mixed cell inflammation was characterized by neutrophils, lymphocytes, and/or macrophages scattered within the dermis subjacent to areas of epidermal hyperplasia. No histopathological changes were observed in the skin within any of the 0% control groups (acetone). No biologically significant changes were identified in the spleen or kidney with exposure.

Select gene expression profiles in the liver were investigated based on gene profiling data generated from previous findings, summarized in Table 4 (Weatherly et al., 2024b). Most of the significant responses demonstrated a dose-dependent trend for both liver and skin. Genes with statistically significant differences in the liver that also exhibited greater than a 2-fold change in expression are shown in Figure 4. Genes with statistically significant differences in the liver but less than a 2-fold change in expression are shown in Supplemental Figure 2. Genes involved in steatosis (Cd36), fatty acid metabolism (Acox1, Cyp4a10, Ehhadh), hepatoxicity (Avpr1a), and PPAR ligand transport (Cd36) were significantly altered with either PFBS or PFPeS exposure (Fig. 4). Increases in Acox1 (2.13 and 2.97-fold) and Cd36 (4.24 and 8.00-fold) were unique to PFBS and observed with 2.5 and 5% exposure respectively (Fig. 4A, B). PFBS (2.5 and 5%) induced increases in Ehhadh (2.79 and 6.59-fold) and Cyp4a10 (5.34 and 9.20-fold), and 5% PFPeS increased Ehhadh 4.05-fold and Cyp4a10 36.87-fold (Fig. 4C, D). A decrease in Avpr1a was demonstrated with 2.5 and 5% PFPeS (0.48 and 0.48-fold) (Fig. 4E); no change was observed following PFBS exposure. PPAR isoforms were also evaluated, with Pparγ expression increasing with 5% PFBS and 2.5 and 5% PFPeS exposure (Fig. 4F). Gene expression of Apoa1, Cyp7a1, Pparδ, and Serpine1 was not altered in the livers with either PFBS or PFPeS exposure (data not shown).

The dermal toxicity was further evaluated with investigations of inflammatory responses and skin barrier integrity. In the skin, the danger signal, Tslp, increased with 5% PFBS (5.30-fold) and PFPeS (3.82-fold) exposure (Fig. 5A). Expression of both danger-associated molecular patterns (DAMPs) S100a7a and S1008a significantly increased in the skin following PFBS exposure, with S100a7a having the largest increase (5%; 11.92-fold) (Fig. 5B, C). Similar changes were observed with 4 skin barrier integrity genes following exposure to PFBS or PFPeS (Fig. 5). PFBS (2.5 and 5%) and PFPeS (5%) exposure increased Flg gene expression (Fig. 5D) while Flg2 increased with 2.5% PFBS and PFPeS (Fig. 5E). Gene expression of Lor and Krt10 increased with 2.5% PFBS, while PFPeS exposure significantly increased Lor with 2.5% and Krt10 with 2.5% and 5% (Fig. 5F, G). Gene expression of Cxcl1, Il-1β, Il-6, Itgbl1, Krt14, Pparα, δ, or γ, and Serpine1 was not altered in the skin with either PFBS or PFPeS exposure (data not shown).

Phenotypic analysis of the ear pinna following 28 days of PFBS and PFPeS exposure resulted in alterations of several cellular sub-groups (Table 2). Interestingly, PFBS demonstrated more phenotypic changes in the ear compared to PFPeS. In the absence of increases in total cellularity, PFBS (2.5 and 5%) exposure significantly increased cell number and frequency of CD45⁺ cells, CD4⁺ cells, natural killer cells (NK), and dendritic cells (DC). PFBS exposure also increased CD8⁺ and eosinophil cell numbers but decreased neutrophil cell frequency (Table 2). PFPeS (5%) only increased CD4⁺, eosinophil, and DCs cell numbers and frequency (Table 2). The increased changes following PFBS were supported, in part, by the gene expression and histological evaluations.

Fewer changes were observed in the dLN and spleen. In the spleen, PFBS increased NK cell frequency (1.25 and 2.5%) while PFPeS decreased CD4⁺, CD8⁺, and NK cell number (5%) (Table 3). Although not reaching statistical significance, there was a decreasing trend in spleen cellularity following PFPeS exposure. In the dLN, PFBS increased CD8⁺ cell numbers (2.5%) and decreased eosinophil cell frequency (2.5 and 5%) while PFPeS increased eosinophil cell number with only the 1.25% exposure (Supplemental Table 4).