Blood and urine samples for clinical pathology evaluations (hematology, serum chemistry, and urinalysis) were collected from the 10 animals/sex/group in groups 1 to 3 and from 20 animals/sex/group in group 4 during study weeks 25 and 51 (hematology and cholecystokinin) and during study weeks 26 and 52 (serum chemistry, urinalysis, estradiol, luteinizing hormone [LH], and testosterone). Blood smears were prepared from all animals that were euthanized in extremis and from all animals during study weeks 51 and 76. Blood samples collected during the 12th month of the study were collected after study weeks 51 or 52. Hematology parameters were evaluated from all animals at the scheduled necropsy (study week 104). The animals were fasted overnight prior to blood collection. Blood was collected from the retro-orbital sinus of animals anesthetized by inhalation of isoflurane at the time of necropsy. Urine samples were collected overnight using metabolism cages prior to the day blood samples were collected.

Functional observational battery (FOB) assessment was recorded for the first 12 animals/sex/group during the 52nd week of treatment as previously described ( Chengelis et al. 2009 ). The FOB used was based on previously developed protocols ( Moser, McDaniel, and Phillips 1991 ; Irwin 1968 ; Gad 1982 ; Moser et al. 1988 ; Haggerty 1989 ; O’Donoghue 1989 ). Locomotor activity was assessed for the first available 12 animals/sex/group during the 52nd week of treatment. Locomotor activity was measured automatically using the Kinder Scientific Motor Monitor System (Kinder Scientific, LLC, Poway, CA) in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB immediately following completion of the FOB assessments. The testing of treatment groups was conducted according to replicate sequence with each animal tested separately. Data for ambulatory and total locomotor activity were tabulated. Total locomotor activity was defined as a combination of fine locomotor skills (i.e., grooming; interruption of a single photobeam) and ambulatory locomotor activity (e.g., interruption of 2 or more consecutive photobeams).

Rats were randomly assigned by body weight stratification to 1 of the 4 treatment groups for each gender. Groups 1 to 3 consisted of 60 rats/sex and group 4 consisted of 70 rats/sex ( Table 1 ). Group 1 males and females received deionized water (vehicle control). Male rats in groups 2, 3, and 4 received 2.5-, 15-, and 100-mg PFHxA/kg body weight/day, respectively. Female rats in groups 2, 3, and 4 received 5-, 30-, and 200-mg PFHxA/kg body weight/day, respectively. Dose selection was based on a previous subchronic toxicity study ( Chengelis et al. 2009 ) in which 100-mg PFHxA/kg body weight/day for males and 200-mg PFHxA/kg body weight/day for females was determined to be the maximum tolerated dose (MTD; Chengelis et al. 2009 ). Rats in the PFHxA and vehicle control groups were administered the respective concentration of compound via gavage daily, 7 days per week for up to 104 consecutive weeks. Solutions were not buffered. During the treatment, rats were observed at least twice daily for mortality or morbidity throughout the treatment period. Clinical examinations were performed daily at the time of dosing. Physical examinations including palpable mass observations were conducted and recorded on all animals weekly.

All rodent treatment, toxicity assessments, and data acquisition were performed at WIL Research (Ashland, OH). The protocol was reviewed and approved by the Institutional Animal Care and Use Committees (IACUC), in compliance with the Animal Welfare Act (AWA). Male and female Crl:CD (SD) rats, approximately 26 days old, were obtained from Charles River Laboratories, Inc. (Raleigh, NC). Rats were acclimated for 14 days prior to being randomly assigned to treatment groups. Rats were individually housed during treatment in wire mesh cages suspended above a cage board. All animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals in the animal facilities at WIL Research (American Association for the Accreditation of Laboratory Animal Care [AAALAC] accredited). Rats were provided food and water ad libitum . Diet consisted of a basal rodent meal (PMI Nutrition International, LLC, and Certified Rodent LabDiet ® 5002). Drinking water was delivered by an automatic watering system. Rodent room temperature and humidity controls were maintained at a temperature of 22 ± 3°C and a humidity of 50 ± 20%, respectively. A 12-hr light/12-hr dark photoperiod and a minimum of 10 fresh air changes per hour were maintained in the rodent room.

PFHxA-related histologic changes were noted in the kidneys of the high-dose (200 mg/kg/day) treated females. These included minimal to severe papillary necrosis and/or minimal to moderate renal tubular degeneration ( Table 7 ). At intermediate and/or high-dose levels, both sexes exhibited several minor tissue changes that were thought to be secondary to accidental aspiration of PFHxA dosing formulations, having a low pH, and that resulted in compromised pulmonary and hepatic perfusion or mucosal irritation associated with the physical properties of PFHxA. These changes included pulmonary and stomach changes such as acute pulmonary congestion and/or hemorrhage, increased pulmonary alveolar macrophages in males and females, and erosion or ulceration of the glandular or nonglandular stomach in females ( Tables 8 and 9 ). Liver effects were also seen including hepatocellular necrosis and congestion ( Table 10 ). Hepatocellular necrosis was seen throughout the liver (necrosis, hepatocellular) as well as in the centrolobular region of the liver lobule (necrosis, hepatocellular, centrilobular). The necrosis seen in the liver was consistent with ischemia resulting from diminished hepatic blood flow. Most liver cell necrosis was observed in animals that died or were euthanized prior to the scheduled necropsy. While liver necrosis appeared to be greater in the female treated rats, liver congestion was increased significantly in the high-dose treated male rats.

PFHxA-related effects on urinalysis parameters consisted of higher mean urine volume and lower specific gravity in the 200-mg/kg/day group females, and lower pH values in the 100-mg/kg/day group males ( Table 6 ). At study week 26, mean urine volume was slightly, but statistically significantly, higher, and mean specific gravity was slightly, but statistically significantly, lower in the 200-mg/kg/day group females when compared to the control group. At study weeks 26 and 52, slightly lower pH values were recorded for the 100-mg/kg/day group males. In the 200-mg/kg/day group females, alterations in urine quantitative parameters corresponded with renal tubular degeneration and/or papillary necrosis and were therefore considered to be PFHxA related. The low urine pH noted in the 100-mg/kg/day group males was attributed to the acidic nature of the test substance.

There were no PFHxA-related alterations in hormone parameters including the evaluations for estradiol, LH, and testosterone at the sampling performed after 26 or 52 weeks of dosing or cholecystokinin at the sampling performed after 27 or 53 weeks of dosing (data not shown). Although none of the hormone values varied from the control group in a statistically significant manner, testosterone and LH levels were slightly lower in the 2.5-, 15-, and 100-mg/kg/day group males after 26 weeks of dosing. These differences, however, were not dose-dependent, and by study week 52, the values had returned to near control group levels. Hormone levels in all PFHxA-treated females were similar to control group values at all evaluations.

In general, there were no PFHxA-related effects on serum chemistry parameters including albumin (A), globulin (G), A/G ratio, ALP, alanine transaminase (ALT), AST, and free fatty acid ( Table 5 ). However, in study week 52, triglycerides were statistically significantly lower by 47.4% and 42.8% in the 2.5- and 100-mg/kg/day treatment group males, respectively; and free fatty acids were significantly lower by 21.1% and 19.4%, respectively, at study week 52 as compared with controls. In contrast, triglycerides were higher by 66.3% (statistically significant) and 27.2%, in the 200-mg/kg/day group females at study weeks 26 and 52, respectively. Significantly higher inorganic phosphorus levels were noted in the 15- and 100-mg/kg/day group males at study week 52 (8.5% and 6.8%, respectively), and sodium was statistically significantly higher by 0.7% in the 100-mg/kg/day group males. Similar differences were not observed in the females. Additional significant changes included lower LDL and very low-density lipoprotein (VLDL) cholesterol values in the 200-mg/kg/day group females at study week 26 ( Table 5 ). Although these clinical chemistry parameters varied from the control group in a statistically significant manner, most alterations were sporadic in nature, were of a magnitude that would be considered to be toxicologically not important, were not dose-dependent, and/or were not associated with histologic changes. Thus, the mean differences were not considered to be PFHxA related.

At terminal sacrifice (104 weeks), no PFHxA-related changes in hematology parameters were observed ( Table 4 ). After 51 weeks of treatment, mean red blood cell (8.1% lower) and hemoglobin (5.2% lower) values were significantly lower in the 200-mg/kg/day treated females. These red blood cell (RBC) parameters returned to control levels by the terminal sacrifice time. Reticulocyte counts were also statistically significantly higher by 26.3% and 56.3% in the high-dose (200 mg/kg/day) treated females at study weeks 25 and 51. Absolute and percentage reticulocyte counts remained slightly higher in the 200-mg/kg/day group females after 104 weeks of treatment; however, the differences were not statistically significant. The absence of correlating histologic changes and the lack of corresponding hematologic alterations in males suggested that the slight changes in female blood parameters may have been secondary to the renal effects of PFHxA or to gastric ulceration that was slightly increased in the 200-mg/kg/day group females.

FOB observations were not affected by PFHxA treatment (data not shown). No statistically significant changes in locomotor activity patterns (total and ambulatory activity counts) or in the pattern of habituation were seen with PFHxA administration. An increase in the number of male rats in the high-dose (100 mg/kg/day) group asleep or lying on their side compared to the control group was noted. In addition, lower mean grip strength was noted in the 5-mg/kg/day treated females. However, there was a lack of a dose response in these effects and no other correlating FOB or locomotor activity effects were seen. These 2 observations were therefore considered a result of normal biological variability. Hence, consistent with the aforementioned clinical/cage-side observations, it was concluded that PFHxA induced no neurobehavioral toxicity in this study.

PFHxA-related clinical observations including rales and yellow material on the ventral trunk, anogenital, and/or urogenital area/areas were seen in the high-dose (100 mg/kg/day) males and high-dose (200 mg/kg/day) females. Very slight increased incidences of struggling during dosing were noted in these same high-dose groups. The struggling may have been related to the acidic nature of the compound since lower dose groups did not exhibit this adverse behavior. Females in the 200-mg/kg/day treatment group appeared to be slightly more sensitive to PFHxA treatment, having a higher incidence of rales and a greater persistence of yellow material at the time of dosing observation periods. This struggling during treatment may have also contributed to the incidental deaths from the gavage and reflux injury noted previously ( Table 2 ) in the high-dose treatments.

At the scheduled necropsy after 104 weeks of PFHxA treatment, no statistically significant difference was seen in survival rates in male rats in any of the 3 groups compared to control ( Figure 1A ). The survival rate of males, excluding the incidental deaths noted previously, at the end of week 104 in the control, 2.5-, 15-, and 100-mg/kg/day group was 31.0%, 43%, 43%, and 47%, respectively ( Table 3 ). In contrast in treated female rats, a significant dose-related decrease in survival rates was seen ( Figure 1B ). In addition, there was a statistically significant decrease in pairwise comparisons between the control group and high-dose group. The survival rate of female rats, excluding the incidental deaths, at the end of week 104 in the control, 5-, 30-, and 200-mg/kg/day group was 36%, 43%, 33%, and 22%, respectively ( Table 3 ).

Survival data for male and female rats over the 104-week treatment period are shown in Figure 1A (males) and B (females) . Several deaths occurred in rats in the highest dose groups (group 4; males [100 mg/kg/day] and females [200 mg/kg/day] prior to week 52 of the study). These early deaths were considered incidental and classified into 3 categories; reflux injury, gavage/mechanical injury, or undetermined ( Table 2 ). Histopathological evaluations concluded that these incidental deaths were not to be related to the systemic exposure to PFHxA but were primarily due to the gavage dosing. Reflux injury was noted in some of the incidental deaths and appeared to be related to the aspiration of the compound after the gavage dosing that produced localized inflammation and/or epithelial necrosis in the larynx or pulmonary airway epithelium due to the acidic nature of the compound. While the incidence of reflux injury and gavage mechanical injury are noted separately in Table 2 , these 2 categories are probably related to the gavage dosing of the animals rather than toxic properties of the compound. Additional support for this conclusion is found in the clustering of the deaths with regard to time of treatment. If the incidental deaths were directly compound related, one would have seen these deaths appear earlier in treatment and continue through the 2-year treatment period at a consistent rate. The numbers of these incidental early deaths from all causes were similar between control, low-, and mid-dose groups for male and female rats.

Discussion

PFHxA (C6) is an important compound because it has been identified by regulatory agencies as the most likely long-term environmental degrading of a new class of chemicals (C6 chemistry) that have been proposed as alternatives to traditional oil and water repellents often used on outerwear and carpets (USEPA 2013). PFHxA is currently not a commercial product and is not likely to become one in the near future. It is, rather, the ultimate degradation product of C6 fluorotelomer acrylate polymers, and the C6 acrylate monomers and C6 alcohols that are the intermediates used to make C6 fluorotelomer acrylate polymers. These are chemicals that are already being widely marketed as substitutes for C8 fluorotelomer acrylates for which there is a concern for degradation to PFOA. They have both water and oil repellent properties and are very durable. Unlike PFOA, which itself was used as an emulsifier for the polymerization of fluoropolymers, there is no current literature suggesting that PFHxA will be so used. 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. The traditional chemistry of these products was based on C8, linear chains of 8 carbons, with 2 fluorines attached to each of the carbons and additional fluorine attached to a carbon at one end of the chain. The C8 compound, PFOA, is the primary environmental degradant of C8-based chemistry. PFOA has been shown to be highly persistent in the environment, and it has a high retention time in mammals, including humans. It is suspected of being related to various toxicity and ecotoxicity end points and has been shown to be carcinogenic in rodents. Although not generally used in the manufacture of the new class of C6-based chemicals, PFHxA is a nearly unavoidable low-volume impurity in their manufacture, and, probably, the most significant terminal environmental degradant of these chemicals. If C6 chemistry successfully replaces C8 chemistry, it must be expected that PFHxA will migrate to the environment through multiple pathways (Martin et al. 2003a, 2003b; Russell, Nilsson, and Buck 2013). As an example, apparel and carpet applications for C6 chemistry would be expected to lead to environmental releases of PFHxA through laundering and landfill disposal scenarios. So it is of considerable importance to correctly characterize the toxicology of this compound.

This report describes the findings of the 2-year study performed to evaluate the possible toxicologic and carcinogenic effects of the PFHxA when administered orally to SD rats. The dosage levels of 2.5, 15, and 100 mg/kg/day of PFHxA (males) and 5, 30, and 200 mg/kg/day of PFHxA (females) selected for this 2-year bioassay were based on a previous 13-week range-finding study (Chengelis et al. 2009). There was no evidence of carcinogenicity in either male or female rats treated with PFHxA when administered orally daily for 7 days per week for 104 weeks. Pathologic changes related to PFHxA administration were restricted to the kidneys of the high-dose, 200-mg/kg/day treatment group females and were characterized by papillary necrosis and tubular degeneration. These renal effects, however, were sporadic, and the majority of female rats from the 200-mg/kg/day group females survived to the 24-month sampling without evidence of PFHxA-related renal damage. It is unclear from this study what the mechanism was for the selective kidney effects in the female rat. Further investigation will be needed to understand this effect. Minor hematological alterations (lower red blood cell counts and higher reticulocyte counts) were also observed in the 200-mg/kg/day group females. These changes were attributed to slight blood loss from gastric erosion and ulceration, and possibly from renal hemorrhage associated with papillary necrosis, although distinct hemorrhage was not observed microscopically in the renal pelvises. Toxicologically meaningful alterations in serum chemistry parameters were not observed. Changes in urine parameters in male and female rats were likely related to the test substance possibly through the reduced ability of the high-dose (200 mg/kg/day) group females to concentrate urine and as the result of the lower urine pH seen in the high-dose (100 mg/kg/day) group males.

In summary, PFHxA when administered orally daily for 104 weeks at multiple doses was not carcinogenic to male and female rats at dosages that included maximally tolerated levels. Nonneoplastic changes of note including renal changes, papillary necrosis, and tubular degeneration in female rats and changes in urine parameters in male and female rats were likely related to PFHxA treatment. After 104 weeks of PFHxA treatment, no significant difference in survival rates in male rats was seen while the survival rate of female rats at the highest dose studied was significantly decreased. In this regard, PFHxA differed from PFOA (at doses of 300 ppm in the diet), which has been shown to be carcinogenic in SD rats, inducing the tumor triad pattern (liver adenomas, Leydig cell tumors, and pancreatic acinar cell tumors) similar to several PPARα agonists (Biegel et al. 2001). It is important to note that none of the tumor triad organ sites usually seen with PPARα activators (liver, pancreas, and Leydig cells) were modified by PFHxA chronic treatment. Similarly, no preneoplastic changes (i.e., hyperplasia) in these 3 organs were noted following PFHxA treatment. Therefore, under the treatment conditions in this chronic bioassay (daily gavage treatment with PFHxA), PFHxA was not carcinogenic.