Potassium 1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulphonate

Endpoint summary

Description of key information

Five repeat dose studies were conducted with PFBSK+. The results of the studies were:

 

A 28 day oral gavage study in rats resulted in a NOAEL of 900 mg/kg/day when tested according to OECD 407.

 

A 30 day oral gavage study in rats resulted in a NOAEL of 1000 mg/kg/day when tested according to OECD 407.

 

A 90 day oral gavage study in rats resulted in a NOAEL of 200 mg/kg/day for males and 600 mg/kg/day for females when tested according to OECD 408.

 

A 10 day range finding study via oral gavage in rats resulted in a NOAEL of 1000 mg/kg/day.

 

Dietary PFBSK+ was administered for 6 weeks to male APOE*3, Leiden, CETP mice. At the end of the treatment, serum PFBS concentrations ranged between 32-38 ug/mL. PFBS treatment-related effects were limited to a decrease in serum triglyceride.

Additional information

The subacute oral toxicity of the test article was evaluated in Sprague Dawley rats following 28 consecutive daily doses. This study was performed in compliance with OECD GLP (1981). The study design was based on OECD 407. The test material was prepared in 1% carboxymethyl-cellulose in deionized water (vehicle). Rats (10/sex/dose) received 0 (vehicle), 100, 300, or 900 mg/kg/day of the test article via oral gavage at a dose volume of 25 mL/kg for 28 consecutive days. In addition, recovery groups (5/sex/dose) received 0 or 900 mg/kg/day of the test article in a similar manner. With the exception of the recovery groups, all groups were euthanized 24 hours following the last dose. The recovery groups were euthanized following a 14-day recovery period. Parameters evaluated: clinical observations (pretest and at least weekly thereafter or when a change was noted), body weights (weekly), food consumption (weekly), neurological function observations (Week 4), gross necropsy (termination), hematology (termination), clinical chemistry (termination), organ weights of select organs (termination), and histopathology of select organs and tissues (termination). All animals survived. Localized alopecia and scabbed area on back and neck were observed among all dose groups. At 900 mg/kg, one male exhibited urine-stained abdominal fur, and another male exhibited soft or liquid feces. These clinical observations were considered incidental and unrelated to test material administration. There were no test article-related effects on body weights or food consumption. Absolute and relative male liver (25% to 30%) and female kidney (9% to 11%) weights were significantly increased in animals that received 900 mg/kg/day test article when compared to the corresponding controls. These significant changes were not recorded during the recovery period, and since there does not appear to, be an effect of body weight on organ development, these data suggest the changes are test material-related. However, the biological and toxicological significance is unclear due to the lack of any supportive gross or histopathological findings. The remaining significant findings are considered incidental and unrelated to test material administration. No toxicologically relevant changes were noted in the clinical chemistry and hematology parameters evaluated or tissues subjected to histopathological evaluation. Based on the results of the study, the No Observed Adverse Effect Level (NOAEL) is 900 mg/kg/day.

 

 

The repeat dose toxicity potential of the test article was examined in male and female Wistar rats. The study was conducted in compliance with OECD GLP (1997) regulations. The study was conducted according to OECD 407 (1995). The test article was administered orally by gavage to 5 male and 5 female Wistar (Hsd Cpb:WU) rats per dose group, in daily doses of 0,100, 300 or 1000 mg/kg body weight for a period of 30 days. The animals were regularly observed and a functional observational battery as well as motor activity assessment were performed. Body weights and food intake were determined. In addition, clinical laboratory investigations of blood samples were performed. At necropsy selected organs were weighed and organs and tissues were subjected to gross and histopathological investigations. Clinical observations of the animals revealed no treatment-related findings. Detailed behavioral investigations (functional observational battery, assessment of motor activity) gave no evidence for a neurotoxic potential of the test substance. Survival was not affected by the treatment. Body weight development and food consumption were not affected by the treatment with the test substance up to 1000 mg/kg. The hematological and histopathological investigations gave no evidence for treatment-related effects on red or white blood or blood coagulation. The investigations revealed treatment-related effects on the liver. Absolute and relative liver weights were increased at 1000 mg/kg in males and females. At histopathology diffuse hepatocellular hypertrophy was observed in males and females at 1000 mg/kg, and cytoplasmic change in males beginning at 300 mg/kg and in 1000 mg/kg females. A slight increase in activity of alkaline phosphatase at 1000 mg/kg and decrease in cholesterol concentration at 300 mg/kg and above were observed in males. Gross and histopathological investigations of other organs and tissues gave no indication of test compound-related functional or morphological changes in both sexes. Based on the results of the study, the No Observed Adverse Effect Level of the test article is 1000 mg/kg/day.

 

The subchronic toxicity of the test article was evaluated in Sprague Dawley rats following 90 consecutive daily doses. The study was performed in compliance with US EPA GLP 40 CFR 160. The study design was based on OECD 408 and US EPA OPPTS 870.3100 (1998). The test material was prepared in 0.1% carboxymethyl-cellulose in deionized water (vehicle). Rats (10/sex/dose) received 0 (vehicle), 60, 200, or 600 mg/kg/day of test article via oral gavage at a dose volume of 10 mL/kg for 90, 91, 92, or 93 consecutive days. All surviving animals were euthanized on the day following the last dose (Days 91-94). Parameters evaluated: clinical observations (daily), body weights (weekly), food consumption (weekly), functional observation battery (not earlier than Week 11), gross necropsy (termination), hematology (termination), clinical chemistry (termination), organ weights of select organs (termination), and histopathology of select organs and tissues (termination). One male treated at 600 mg/kg/day was found dead on Day 85. Prior to death, the following clinical signs were noted on Days 83 and/or 84: red perioral substance, urine-stained abdominal fur, decreased motor activity, cold to touch, ptosis, dehydration, brown substance around mouth, and ungroomed coat. Weekly body weight gains and food consumption values for this animal were normal. Necropsy revealed thin walls of the stomach in the cardiac region, stomach and intestines distended with gas, red thymus, and small spleen. The death was considered unrelated to test article administration since it was a single event, and the sudden onset of adverse clinical observations indicates a possible injury. All other animals survived until scheduled termination. There were no test material-related abnormal clinical observations noted in females. The incidences of a red perioral substance (slight to extreme in degree) and urine-stained abdominal fur were significantly increased in males treated at 600 mg/kg/day. Chormorhinorrhea occurred in 2 and 3 males in the 200 and 600 mg/kg/day dose groups, respectively. There were no test material-related changes in body weights or food consumption in any group. The absolute weights of the spleen and the ratios of the weight of the spleen to the terminal body weight and brain weight were reduced or significantly reduced in the male rats in the 60, 200 and 600 mg/kg/day dosage groups. Organ weights and the ratios of the organ weights to the terminal body weights and brain weights for the female rats were unaffected by dosages of the test article up to 600 mg/kg/day. Average values for red blood cells (RBC), hemoglobin concentration (HGB) and hematocrit (HCT) were reduced in male rats in the 200 and 600 mg/kg/day dosage groups. All other hematological parameters for the male rats and all hematological parameters for the female rats were comparable among the four dosage groups. The average value for chlorine (Cl) was significantly increased in male rats in the 600 mg/kg/day dosage group. Average total protein and albumin values were significantly reduced in female rats in the 600 mg/kg/day dosage group. All other clinical chemistry parameters were unaffected in either sex by dosages of the test article as high as 600 mg/kg/day. No microscopic changes directly related to treatment were observed in any of the male and female rats given 60 or 200 mg/kg/day of the test article for 90 days. Compound-related microscopic changes were observed in the kidneys and stomach of the male and female rats of the 600 mgkglday dosage group. The primary treatment-related change in the kidneys of males and females dosed with 600 mg/kg were hyperplasia of the epithelial cells of the medullary and papillary tubules and ducts in the inner medullary region. These tubules had a dark tinctorial appearance with increased amounts of small interstitial cells with prominent dark nuclei. Other treatment-related changes included a lower incidence of focal papillary edema and a single incidence of papillary necrosis in both kidneys of one male rat in the 600 mg/kg/day dosage group. The treatment-related effect in the stomach consisted of an increased incidence of necrosis of individual squamous epithelial cells in the limiting ridge of the forestomach in the male and female rats in the 600 mg/kg/day dosage group. This change was characterized by individual squamous epithelial cells with dark pyknotic nuclei surrounded by a clear cytoplasmic halo. This change was seen at a very low incidence in the rats of the other dosage groups, including a control female rat, but the increased incidence of this change, along with minimal or mild thickening of the mucosa of the limiting ridge due to hyperplasia and hyperkeratosis, was considered to be treatmentrelated in the 600 mg/kg/day dosage group. Microscopic examination of the nasal cavity and nasal turbinates revealed a few equivocal microscopic changes that occurred at low and sporadic incidences in rats in the 200 and 600 mg/kg/day dosage groups. These changes occurred primarily in the posterior nasal cavity turbinates and involved the olfactory mucosa. These histomorphologic changes included single or low incidences of multifocal necrosis or atrophy of the olfactory mucosa, focal acute/subacute or chronic inflammation, adhesions of the turbinate to either an adjacent turbinate or to the lateral nasal wall, focal hyperostosis of turbinate bone and/or foci of olfactory epithelial hyperplasia. Foci of inflammation may occur spontaneously in the nasal cavity of rats, but in several of the above-mentioned lesions, the inflammation was associated with these other changes. The lesions in the nasal cavity/turbinates are of uncertain significance and origin mainly because they occurred only in the 200 and 600 mg/kg/day dosage groups at very low and sporadic incidences and were focal or multifocal in distribution. The nasal cavity/turbinates 'of most rats of all groups were histologically unremarkable. The varied and focal histomorphologic characteristics of these lesions in the nasal cavity/turbinates are not typical or consistent with a systemic toxic effect. Although the mechanism is unknown, many of these lesions are more suggestive of a local irritating effect on the nasal mucosal membranes. Based on the results of the study, the No Observed Adverse Effect Level (NOAEL) of the test article is 200 mg/kg/day for males and 600 mg/kg/day for females.

 

 

The objective of this study was to identify a maximum dose of the test article and identify dose levels for a 28 days repeated dose study. Healthy male and female Crl:CD®(SD) IBS BR Stock out-bred albino rats (5/sex/dose) were exposed to a single dose of 0 (vehicle control), 100, 300, and 1000 mg/kg bw/day test article in carboxymethylcellulose (medium viscosity) for 10 consecutive days via oral gavage. Observations were recorded for mortality and morbidity (twice daily), clinical signs (once daily), body weights (study day 1, 7, 10, and 11), feed consumption (study day 1, 7, and 10), hematology (at necropsy), clinical chemistry (at necropsy), necropsy (study day 11), organ weights (at necropsy), and histopathology. Following the exposure period, no mortality occurred. Clinical observations, body weights, body weight changes, feed consumption, gross findings, and histopathology did not reveal any adverse effects associated with the 10 day exposure to ≤1000 mg/kg bw/day test article. Hematological and clinical chemistry changes reported in female rats that received 1000 mg/kg bw/day test article appear to be isolated changes in one gender and had no clear association with other reported findings, including histopathology. In the absence of supportive clinical chemistry and histopathology, the marginal but significant absolute and relative liver weights recorded in both male and female rats that received 1000 mg/kg bw/day are not considered biologically or toxicologically significant. Based on the results of the study, the No Observed Adverse Effect level of the test article is 1000 mg/kg/day.

 

 

Dietary K+PFBS was administered up to 6 weeks to male ApoE*3. Leiden.CETP mice, a humanized transgenic mouse model that displays human-like lipid profile. At age 8-10 weeks, mice were fed a semisynthetic Western-type diet contianing 0.25% (wt/wt) cholesterol, 1% (wt/wt) corn oil, and 14% (wt/wt) bovine fat for 4 weeks in three independent experiments. Upon randomization according to body weight, plasma TC and TG levels, mice received the Western-type diet without or with PFBS (0.03%, approximately 3 mg/kg/day) for 4-6 weeks. Plasma was obtained via tail vein bleeding and assayed for total cholesterol (TC) and triglycerides (TG), using the commercially available enzymatic kits 236691 and 11488872 respectively. Free fatty acids (FA) were measured using NEFA-C kit from Wako Diagnostics (Instruchemie, Delfzijl, the Netherlands) and glycerol was measured using the free glycerol determination kit (Sigma-Aldrich, St Louis, MO). The distribution of lipids over plasma lipoproteins was determined using fast protein liquid chromatography. Plasma was pooled per group, and 50 ul of each pool was injected onto a Superose 6 PC 3.2/30 column and eluted at a constant flow rate of 50 ul/min in PBS, 1 mM EDTA, pH 7.4. Fractions of 50ll were collected and assayed for TC as described above. HDL was isolated by precipitation of apoB-containing lipoproteins from 20ul EDTA plasma by adding 10ul heparin (500 U/ml) and 10ul 0.2MMnCl2. The mixtures were incubated for 20min at room temperature and centrifuged for 15 min at 13,000 revolutions per minute (rpm) at 4_C. HDL-C was measured in the supernatant using enzymatic kit 236691 (Roche Molecular Biochemicals). Plasma cholesteryl ester transfer protein (CETP) mass was analyzed using the CETP ELISA kit from ALPCO Diagnostics (Salem, NH). Plasma apoAI concentrations were determined using a sandwich ELISA (van der Hoornet al., 2008), with diluted mouse plasma (dilution 1:400,000). Purified mouse apoAI from Bio design International (Saco) was used as a standard. Glycerol tri[3H]oleate (triolein [TO])-labeled VLDL-like emulsion particles (80 nm) were obtained by adding 100 μCi of [3H]TO to 100 mg of emulsion lipids before sonication. Mice were fasted for 4 h, sedated with 6.25 mg/kg acepromazine. 6.25 mg/kg midazolam, and 0.3125 mg/kg fentanyl and injected with a large bolus of radiolabeled emulsion particles (1.0 mg TG in 200 μl PBS) via the tail vein. At indicated time points after injection, blood was taken from the tail vein to determine the serum decay of [3H]TO. At 30 min after injection, plasma was collected by orbital puncture and mice were sacrificed by cervical dislocation. Organs (i.e., liver, heart, perigonadal fat, spleen, and skeletal femoralis muscle) were harvested and saponified in 500 μl Solvable to determine [3H]TO uptake. The half-life of VLDL-[3H]TO was calculated from the slope after linear fitting of semi-logarithmic decay curves. Lipolytic activity of both lipoprotein lipase (LPL) and hepatic lipase (HL) was determined. To liberate LPL from endothelium, 4-h fasted mice were injected ip with heparin (0.5 U/g bodyweight; Leo Pharmaceutical Products BV, Weesp, The Netherlands) and blood was collected after 20 min; 10 ul of postheparin plasma was incubated with 0.2 ml of TG substrate mixture containing triolein (4.6 mg/ml) and [3H]TO (2.5 lCi/ml) for 30min at 37[1]C in the presence or absence of 1M NaCl, which completely inhibits LPL activity, to estimate both the HL and LPL activity. The LPL activity was calculated as the fraction of total triacylglycerol hydrolase activity that was inhibited by the presence of 1M NaCl and is expressed as the amount of free FA released per hour per ml of plasma. Mice were fasted for 4 h prior to the start of the experiment. During the experiment, mice were sedated as described above. At t = 0 min, blood was taken via tail bleeding and mice were iv-injected with 100 μl PBS containing 100 μCi Trans35S-label to measure de novo total apoB synthesis. After 30 min, the animals received 500 mg of tyloxapol per kilogram body weight as a 10% (wt/wt) solution in sterile saline, to prevent systemic lipolysis of newly secreted hepatic VLDL-TG. Additional blood samples were taken at t = 15, 30, 60, and 90 min after tyloxapol injection and used for determination of plasma TG concentration. After 90 min, the animals were sacrificed and blood was collected by orbital puncture for isolation of VLDL by density-gradient ultracentrifugation. 35S-apoB was measured in the VLDL fraction after apoB-specific precipitation with isopropanol. Livers were isolated and partly homogenized (30 s at 5000 rpm) in saline (∼10% wet wt/vol) using a mini-bead beater. Lipids were extracted and separated by high-performance thin-layer chromatography. Fecal secretion of neutral sterols and bile acids was determined in feces, collected during a 48- to –72-h time period at two consecutive time points, by gas chromatographic analysis. One mouse of each experimental group was used to obtain autologous HDL that was radiolabeled with [3H]CO. Mice were injected via the tail vein with a trace of autologous radiolabeled HDL (0.1 uCi in 200 ul PBS). At the indicated time points after injection, blood was collected to determine the plasma decay of [3H]CO. The fractional catabolic rate was calculated after curve fitting. Taking into account that plasma levels of HDL-C were changed upon treatment, the fractional catabolic rate (FCR) was also calculated from these data as millimolar HDL-C cleared per hour, based on the actual level of HDL-C in the various groups. Total RNA was extracted from individual livers using RNA-Bee and glass beads according to the manufacturer's instructions. The RNA was further purified using the nucleospin RNA II kit (Machery-Nagel, Düren, Germany) according to the manufacturer's instructions. The integrity of each RNA sample obtained was examined by Agilent Lab-on-a-chip technology using an RNA 6000 Nano LabChip kit and a Bioanalyzer 2100 (Agilent Technologies, Amstelveen, The Netherlands). The Affymetrix 3′ IVT-Express labeling Kit (#901229) and the protocols optimized by Affymetrix were used to synthesize Biotin-labeled coding ribonucleic acid (cRNA) (from 100 ng of total RNA) for microarray hybridization. For the hybridization, 15 μg cRNA was used for further fragmentation and finally 10 μg for the hybridizations. The quality of intermediate products (i.e., biotin-labeled cRNA and fragmented cRNA) was again checked. Microarray analysis was carried out using an Affymetrix technology platform and Affymetrix GeneChip mouse genome 430 2.0 arrays. Briefly, fragmented cRNA was mixed with spiked controls and hybridized with murine GeneChip 430 2.0 arrays. The hybridization, probe array washing and staining, and washing procedures were executed as described in the Affymetrix protocols, and probe arrays were scanned with a Hewlett-Packard Gene Array Scanner (ServiceXS, Leiden, The Netherlands). Quality control of microarray data was performed using BioConductor packages (including simpleaffy and affyplm), through the NuGO pipeline that is available as a Genepattern procedure on http://nbx2.nugo.org (de Groot et al., 2008). All samples passed the QC. Raw signal intensities (from CEL-files) were normalized using the GCRMA algorithm (gc-rma slow). For annotation of probes and summarization of signals from probes representing one gene the custom MNBI CDF-file was used (based on EntrezGene, version 11.0.2. This resulted in expression values for 16,331 genes, represented by unique Entrez gene identifiers. Genes were filtered for expression above five in three or more samples, resulting in a set of 11,587 genes that was used for further analysis. Gene expression data were log-transformed (base 2). Statistical analysis on resulting data was performed using the moderated t-test with correction for multiple testing (Storey and Tibshirani, 2003). Cutoff for statistically significant changes was set at corrected p value (q value) < 0.05. In addition, T-profiler analysis (Boorsma et al., 2005) was performed using expression values corrected for mean expression in the control group. This analysis resulted in scores (t-scores) and significance values for functional gene sets and biological processes (based on gene ontology annotation). Gene sets and biological processes with significant scores (> 4 or < −4) in five or six animals per group were selected. A hierarchical clustering of these pathways and biological processes and their scores in all samples was generated in GenePattern (Broad Institute, MIT; Reich et al., 2006). The data discussed in this publication have been deposited in National Center for Biotechnology Information's Gene Expression Omnibus according to MIAME compliance (GEO series accession number GSE22940). At the end of the treatment where serum PFBS concentration ranged between 32 – 38 mg/mL. PFBS did not affect VLDL-TG production and modestly reduced VLDL-apoB production (-17%). Additionally, it did not affect liver weights, white perigonadal fat pad weight, plasma free fatty acids, plasma glycerol and did not alter the expression of genes involved in lipid metabolism.  PFBS treatment-related effects were limited to a decrease in serum triglyceride.

Justification for classification or non-classification

The results of the studies do not meet the classification criteria for target organ toxicity in a repeated dose study.