Compared with the extensive toxicological database available for the long-chain PFCs, relatively few mammalian toxicity studies have been conducted with the C6 compounds, and all of these have been conducted in rodents. For PFHxA, 90-day  subchronic and 2-year [34••] oral toxicity studies have been conducted in rats with the free acid, at dose ranges of 0, 10, 50, and 200 mg/kg/day for the subchronic study and doses of 0, 2.5, 15, and 100 mg/kg/day (males) and 0, 5, 30, and 200 mg/kg/day (females) in the chronic study. Ninety-day oral studies have also been conducted in rats with sodium PFHxA  and C6-FTOH [36••] at dose ranges of 0, 20, 100, and 500 mg/kg/day for Na PFHxA and 0, 5, 25, 125, and 250 mg/kg/day of C6-FTOH. One-generation reproductive toxicity and teratogenicity studies have been conducted with sodium PFHxA  and C6-FTOH [37••] in rats using the same dose ranges as the subchronic studies cited above, and a one-generation reproductive toxicity study has been conducted with PFHxA ammonium salt in mice [38••] using dose ranges of 0, 100, 350, or 500 mg/kg/day (phase I) or 0, 7, 35, or 175 mg/kg/day (phase II). A 14-day study conducted with the 6–2 methacrylate in rats is also available [24•]. The subchronic [39••], chronic , and developmental  toxicity studies conducted with PFHxA and C6-FTOH were all compliant with their respective guidelines in the FDA’s Redbook. In particular, the subchronic and chronic studies gavaged rats with the test compound for 90 days and 104 weeks, respectively, and measured the following endpoints: bodyweight and feed consumption; biochemical parameters in serum and urine; hematological parameters; organ weights and histopathology; ophthalmology; and neurological function via conduct of a Functional Observational Battery (FOB).
The subchronic [33, 35, 36••] and developmental/reproductive toxicity [35, 37••, 38••] studies demonstrate that the C6 compounds share some similarities in their toxicological profiles with the C8 compounds (see below), except that PFHxA appears to be at least an order of magnitude less potent than perfluorooctanoate (PFOA). Common findings in the above-cited 90-day studies included mortality and/or decreased bodyweights, hepatocellular hypertrophy and increased liver weights, increased kidney weights, and hematological changes indicative of mild anemia at the high doses tested in these studies. However, unlike PFOA, PFHxA did not induce neoplastic effects in any organ in the chronic study [34••].
Studies noted either decreased survival or decreased bodyweights with PFHxA or C6-FTOH administration [33, 35, 36••]. Decreased survival and early mortality was noted at the highest doses tested in the chronic study [34••] with PFHxA of 100 mg/kg/day (males) and 200 mg/kg/day (females), with early mortality noted in both sexes and decreased survival noted in females only. The early mortality was associated with renal papillary necrosis and renal tubular degeneration. The C6-FTOH also induced mortality in both sexes at 250 mg/kg/day in the subchronic study, the highest dose tested, and in one female at 125 mg/kg/day [36••]. Bodyweights were not affected by treatment in either study. In contrast, significantly decreased bodyweights were noted in the subchronic studies conducted with PFHxA in males at doses of ≥50 mg/kg for the free acid and 500 mg/kg for the Na salt [33, 35], with no mortality noted.
Hepatocellular hypertrophy with increased liver weight parameters was one of the most sensitive effects noted in the subchronic studies [33, 35, 36••] and in the 14-day study [24•]; lowest observed effect levels (LOELs) from 90-day studies for this effect were 25 mg/kg/day for C6-FTOH and 100–200 mg/kg/day for PFHxA in males and 125 mg/kg/day for C6-FTOH and 500 mg/kg/day for PFHxA in females. In the chronic study [34••], hepatocellular hypertrophy was not evident at doses of up to 100 mg/kg/day (males) and 200 mg/kg/day (females); however, hepatocellular necrosis and hepatic congestion were noted in high-dose males and females. The hepatocellular necrosis observed after 104 weeks of PFHxA administration is likely the result of the enzyme induction and peroxisomal proliferation that was noted in the 90-day study. The C6-FTOH also induced single-cell hepatocellular necrosis, oval cell/biliary hyperplasia, and periportal inflammation at ≥25 mg/kg/day in males and ≥125 mg/kg/day in females after 90 days of administration [36••]. In neither study were these changes accompanied by elevations in biochemical indicators of liver injury. Interestingly, while Loveless et al.  noted induction of peroxisomal proliferation at the same dose levels that induced hepatocellular hypertrophy, these changes were not accompanied by alteration in serum cholesterol or triglycerides. Indeed, while all of the studies noted hepatocellular hypertrophy with C6 administration, no consistent effects on serum cholesterol profiles were reported. Supporting this finding, recent studies observed that the potency of PFHxA at the human peroxisome-proliferator activated receptor (PPAR)α, activation of which is associated with decreased blood lipid levels, is approximately half the potency of PFOA in the human hepatocellular carcinoma cell line HepG2 [42•] and approximately six-fold less potent than PFOA in both mouse and human PPARα in transiently transfected COS cells [43•]. Additionally, the decreased retention of PFHxA in the liver compared with the longer-chained compounds such as PFOA greatly decreases its potency to induce hepatic peroxisomal proliferation in vivo . Concomitant with these findings, there was also no association of PFHxA serum levels (0.03 ng/ml median) with blood lipids in a Chinese population [45•].
Subchronic studies conducted with both PFHxA and C6-FTOH noted increased kidney weight parameters [33, 35, 36••]. Kidney weight parameters were increased only in males administered PFHxA for 90 days at ≥10 mg/kg , whereas Na PFHxA increased kidney weight parameters in both sexes at ≥100 mg/kg/day ; these increases occurred in the absence of histopathological changes. However, after administration for 104 weeks, PFHxA induced renal tubular degeneration and papillary necrosis accompanied by increased urine volume and decreased specific gravity in females at 200 mg/kg/day, indicating significant adverse functional alterations in renal concentrating ability [34••]. Therefore, it would appear that the increased kidney weights in males noted in the subchronic studies represented adaptive changes to PFHxA administration, whereas the free acid of PFHxA induced renal injury in females only after chronic exposure. The reasons for this gender-specific effect of PFHxA and difference in kidney-weight response in females between the free acid and the Na salt are not apparent. For the C6-FTOH, the increased kidney weights noted in the subchronic study were accompanied by adverse histopathological changes, and the kidney appeared to be as sensitive as the liver to the adverse effects of C6-FTOH in females, with renal tubular degeneration and necrosis evident at ≥125 mg/kg/day C6-FTOH in females and at 250 mg/kg/day in males [36••].
Decreased erythrocyte parameters (erythrocyte number, hematocrit, hemoglobin) and increased reticulocyte counts were noted in both subchronic and chronic toxicity studies conducted with PFHxA at the highest doses tested, indicative of mild anemia with concomitant regenerative responses [33, 35, 36••]. While this was noted in both sexes in the subchronic study , anemia was only noted in females in the chronic study [34••], and the decreased erythrocyte parameters did not persist through the study duration. In contrast, the C6-FTOH induced adverse changes in the same erythrocyte parameters in the same dose range and gender pattern as was observed for hepatotoxicity [36••]. This disparity in dose ranges for this effect between PFHxA and the C6-FTOH may reflect differences in mechanism of action, the additive effect of the adverse hematopoietic effects of the various metabolites of the FTOH, and/or mechanisms secondary to the renal toxicity of the FTOH.
Ameloblast degeneration and altered tooth mineralization were noted with both the C6-FTOH [36••] and the 6–2 methacrylate [24•]; the study authors speculated that fluoride released from metabolism of the test compounds was the causative agent for the observed changes, and increased urinary fluoride levels were noted at the same doses as the adverse effects on the teeth [36••]. These effects were not seen in any of the studies conducted with PFHxA [33, 35, 34••].
No observed adverse effect levels (NOAELs) for systemic toxicity of PFHxA in the 90-day studies were 20 mg/kg/day for the sodium salt  and 50 and 200 mg/kg/day for the free acid in males and females, respectively , which are considerably higher than the 90-day no observed effect level (NOEL) of 0.06 mg/kg/day reported for the ammonium salt of PFOA in rats . Similarly, the NOAEL levels for systemic toxicity in male and female rats for PFHxA in the chronic study [24•] were 15 and 30 mg/kg/day, respectively, whereas the bioassay conducted with ammonium PFOA in rats noted liver damage in treated rats down to the lowest dose tested of 1.5 mg/kg/day . In contrast, both the 90-day study conducted with the C6-FTOH [36••] and an oral 90-day study conducted with the C8 fluorotelomer alcohol (8–2 FTOH)  reported NOAELs of 5 mg/kg/day for systemic toxicity, indicating that decreased perfluorinated chain length of the FTOH did not decrease the toxic potency under the test conditions in short-term studies.
Reproductive toxicity studies were conducted with sodium PFHxA  and C6-FTOH [37••] in rats and ammonium PFHxA in mice [38••]. The studies conducted with PFHxA salt and C6-FTOH gavaged male and female CD rats for ~70 days prior to mating, and pregnant females through to lactation day (LD) 22. Separate teratology studies conducted with the sodium PFHxA and C6-FTOH gavaged pregnant rats on gestation days (GD) 6–20, with terminal necropsy on GD 21. The PFHxA study in mice gavaged pregnant ICR dams from GD 6 through LD 22.
There were no effects of sodium PFHxA  or C6-FTOH [37••] on any reproductive indices in rats at doses of up to 500 mg/kg/day and 250 mg/kg/day, respectively; decreased maternal bodyweights and bodyweight gains were noted in the teratology , but not the reproductive, cohort at 500 mg/kg/day PFHxA and in both cohorts at 250 mg/kg/day C6-FTOH [37••]. There were no effects of sodium PFHxA on reproductive organ weights or histopathology in the P0 generation. The only developmental effects of sodium PFHxA noted were a 17–18 % decrease in mean pup weight throughout the lactation period in the F1 generation in the one-generation study and 10 % decreased fetal weight in the teratology study at 500 mg/kg/day, the highest dose tested in both studies. For the C6-FTOH [37••], the reproductive study noted increased pup mortality and decreased pup weights at ≥125 mg/kg/day; increased incidences of delayed ossification and wavy ribs were noted in the teratology study at these doses. The NOELs for prenatal and postnatal toxicity in rats from these studies were 300 mg/kg/day PFHxA and 25 mg/kg/day C6-FTOH. In contrast, mice were far more sensitive to the effects of PFHxA. Decreased bodyweight gains during postnatal days (PNDs) 0–4 (≥350 mg/kg/d) and the entire lactation period (500 mg/kg/d) were noted in dams [38••]. Significant litter observations (mostly at ≥ 350 mg/kg/d) included increased incidences of stillbirths, increased whole litter loss on PNDs 0–3, decreased pup survival during lactation, decreased pup bodyweights, delayed eye opening, and reduced terminal bodyweights in F1 females and terminal bodyweight : liver weight ratios in F1 males. At 175 mg/kg/day, significant findings included increased numbers of stillborn pups and pups dying on PND 1 and decreased pup weight at PND 1. The NOEL for developmental toxicity of PFHxA salt in mice from the study is 35 mg/kg/day. The adverse effects noted on pup bodyweight, postnatal survival, and attainment of developmental landmarks are consistent with effects noted in mice after PFOA administration; however, the LOELs for these effects in the study conducted with PFHxA are at least two orders of magnitude greater than the respective LOELs for PFOA in mice, the most sensitive species , of 0.6 mg/kg/day and 1 mg/kg/day, respectively, emphasizing the decreased potency of the C6 compound compared with PFOA.
In summary, subchronic and chronic oral toxicity studies conducted with PFHxA (free acid and sodium salt) reported an array of toxicological effects that are broadly similar to those noted with PFOA: decreased bodyweights, hepatocellular hypertrophy and peroxisomal proliferation, and anemia. Kidney effects were more pronounced with PFHxA versus PFOA; but the data overall demonstrate that PFHxA is much less toxic than PFOA, with LOELs at least an order of magnitude higher for PFHxA than PFOA. Moreover, PFHxA was non-carcinogenic in rats and did not display the potent postnatal toxicity noted with PFOA in either rats or mice.
In contrast, the toxicological profile for the C6-FTOH is not as well characterized. Subchronic studies conducted with the C6-FTOH identify similar toxic endpoints to those identified for the 8–2 FTOH, with adverse effects on the teeth, the kidneys, the liver, and red blood cell homeostasis. However, the mortality noted during the C6-FTOH study was not seen with the 8–2 FTOH, and the adverse effects on the kidney were more severe in the C6-FTOH study. As such, while the toxicological profile for PFHxA itself appears less concerning than that for long-chain PFCAs, the toxicological profile and potency for the C6-FTOH may be similar to the long-chained FTOHs. Future studies are needed to confirm whether this is the case.