Evaluation of the chronic toxicity and carcinogenicity of perfluorohexanoic acid (PFHxA) in Sprague-Dawley rats.

Perfluorohexanoic acid (PFHxA), a 6-carbon perfluoroalkyl (C6; CAS # 307-24-4), has been proposed as a replacement for the commonly used 8-carbon perfluoroalkyls: perfluorooctanoic acid and perfluorooctane sulfonate. PFHxA is not currently a commercial product but rather the ultimate degradation product of C6 fluorotelomer used to make C6 fluorotelomer acrylate polymers. 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. This article reports on a chronic study (duration 104 weeks) that was performed to evaluate the possible toxicologic and carcinogenic effects of PFHxA in gavage (daily gavage, 7 days per week) treated male and female Sprague-Dawley (SD) rats. In the current study, dosage levels of 0, 2.5, 15, and 100 mg/kg/day of PFHxA (males) and 5, 30, and 200 mg/kg/day of PFHxA (females) were selected based on a previous subchronic investigation. No effects on body weights, food consumption, a functional observational battery, or motor activity were observed after exposure to PFHxA. While no difference in survival rates in males was seen, a dose-dependent decrease in survival in PFHxA-treated female rats was observed. Hematology and serum chemistry were unaffected by PFHxA. PFHxA-related histologic changes were noted in the kidneys of the 200-mg/kg/day group females. Finally, there was no evidence that PFHxA was tumorigenic in male or female SD rats at any of the dosage levels examined.

Safety assessment of heat-sterilized green tea catechin preparation: a 6-month repeat-dose study in rats.

Evidence suggests that the purported health benefits associated with green tea consumption are related to tea catechins. In the present study, potential adverse effects of a standardized heat-sterilized green tea catechin (GTC-H) preparation was investigated following gavage administration to rats at doses of 0, 120, 400, 1200 mg/kg/day for 6 months. A decaffeinated high-dose group (1200 mg/kg/day) (GTC-HDC), was included for comparison. A possibly test article-related clinical finding of intermittent increased activity was noted in the 400 and 1200 mg/kg/day GTC-H groups, but was not considered to be adverse. Lower body weight gains without any decrease in food consumption were noted in the high-dose (1200 mg/kg/day)-treated GTC-H and GTC-HDC females. In the high-dose male GTC-H group, a lower total motor activity count for the 60-min session was noted prior to dosing at the study week 25 evaluations compared to the control group. Similar changes were not observed in the GTC-HDC group. Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for GTC-H was 1200 mg/kg/day for males, the highest dose tested, and 400mg/kg/day for females based on reduced body weight gains. The NOAEL for GTC-HDC was 1200 mg/kg/day for males and could not be determined in females.

A 90-day repeated dose oral (gavage) toxicity study of perfluorohexanoic acid (PFHxA) in rats (with functional observational battery and motor activity determinations).

Possible toxic effects of perfluorohexanoic acid (PFHxA) were evaluated when administered orally by gavage to rats at levels up to 200mg/kg/day for 90 days. Lower body weight gains were noted in the 10, 50 and 200mg/kg/day group males (not dose-responsive) throughout dosing. Other changes included lower red blood cell parameters, higher reticulocyte counts and lower globulin in the 200mg/kg/day group males and females, higher liver enzymes in males at 50 and 200mg/kg/day, lower total protein and higher albumin/globulin ratio, and lower cholesterol, calcium in males at 200mg/kg/day. Minimal centrilobular hepatocellular hypertrophy was present in 200mg/kg/day group males and correlated with higher liver weights and slightly higher peroxisome beta oxidation activity at the end of the dosing period. Based on liver histopathology and liver weight changes, the no-observed-adverse-effect level (NOAEL) for oral administration was 50mg/kg/day for males and 200mg/kg/day for females.

Comparison of the toxicokinetic behavior of perfluorohexanoic acid (PFHxA) and nonafluorobutane-1-sulfonic acid (PFBS) in cynomolgus monkeys and rats.

The toxicokinetics of perfluorohexanoic acid (PFHxA) and nonafluoro-1-butanesulfonic acid (PFBS) were evaluated in Sprague-Dawley rats and cynomolgus monkeys. Systemic exposure to PFHxA was lower than for PFBS following single equivalent intravenous or oral (rat only) doses. Serum clearance was more rapid for PFHxA than for PFBS. In rats, exposure to PFHxA and PFBS was up to 8-fold (intravenous) and 4-fold (oral) higher for males than females and serum clearance of PFHxA and PFBS was more rapid in females than males; however, there was no appreciable difference in the extent or rate of urinary elimination between compounds or genders. There were no apparent differences between genders in the serum half-life for PFHxA following 26 days of repeated oral dosing in rats; exposure decreased upon repeated dosing.

Spontaneous hibernomas in Sprague-Dawley rats.

Hibernomas are rare neoplasms originating in brown adipose tissue of humans and other animal species, including laboratory animals. Background incidence values for these tumors in all common strains of laboratory rats are generally accepted as being <0.1%. Between April 2000 and April 2007, however, sixty-two hibernomas (an overall prevalence of 3.52%) were observed in a total of 1760 Sprague-Dawley rats assigned to three carcinogenesis bioassays at two separate research laboratories. All rats were obtained from Charles River's breeding facilities in either Portage, Michigan, or Raleigh, North Carolina. Tumors (twenty-nine benign and thirty-three malignant) were randomly distributed among test article-treated and control groups and were considered to be spontaneous. Most tumors originated in the thoracic cavity, and they were usually described as soft, mottled to tan masses with nodular to lobulated profiles. Immunohistochemical procedures for uncoupling protein 1 (UCP1) confirmed brown adipose tissue as the site of origin rather than white fat. The marked increase in hibernomas in our studies suggests that greater numbers of spontaneous hibernomas may be sporadically encountered in future carcinogenesis studies with Sprague-Dawley rats. The increased potential for hibernomas to arise as spontaneous neoplasms has important implications in studies involving peroxisome proliferators-activated receptor (PPAR) drugs, lipophilic environmental chemicals (e.g., polychlorinated biphenyls), and other molecules or physiologic processes (e.g., beta-adrenergic stimulation) that may target brown fat adipocytes.

Safety assessment of heated diacylglycerol oil: subchronic toxicity study in rats.

Diacylglycerol oil is an edible oil with similar taste and usability characteristics as conventional edible oil rich in triacylglycerol oil. The objective of the present study was to evaluate potential adverse effects of heated diacylglycerol and triacylglycerol oil in rats following subchronic administration. The heated diacylglycerol and triacylglycerol oils were prepared separately following deep frying potato slices at 180 degrees C for 8h per day for three days. Sprague Dawley rats were fed diets containing different ratios (concentrations) of heated to unheated diacylglycerol oil. The ratio of heated to unheated diacylglycerol was as follows: 0%/5.5% (control-1; Group 1), 1.0%/4.5% (Group 2), 2.75%/2.75% (Group 3), and 5.5%/0% (Group 4). Two additional groups received the feed containing 5.5% of unheated or 5.5% of heated triacylglycerol oil. Compared to the unheated oils, feeding of heated diacylglycerol or triacylglycerol oil did not reveal any toxicologically significant changes in clinical observation, body weights, body weight gains, feed consumption, ophthalmic examinations, functional observational battery and motor activity, clinical pathology evaluations and organ weights. Similarly, terminal necropsy did not reveal treatment-related gross or histopathology findings. Based on the results of this subchronic study, the no-observed-effect levels (NOELs) of heated diacylglycerol or triacylglycerol oil were 5.5%, the highest levels tested. The mean dietary exposure levels at the highest dose for the heated diacylglycerol and triacylglycerol oil for male and female rats ranged from 3,178 to 4,120 mg/kg/day.

28-Day oral (gavage) toxicity studies of green tea catechins prepared for beverages in rats.

The beneficial health effects associated with drinking green tea are widely considered to be due primarily to tea catechins. Heat treatment of marketed green tea beverages for sterilization causes epimerization and/or polymerization of tea catechins. Safety studies on heat-treated tea catechins are limited. The objective of the present study was to evaluate potential adverse effects, if any, of two standardized green tea catechin (GTC) preparations: one that underwent heat sterilization (GTC-H) and one that was not heat-sterilized (GTC-UH). A decaffeinated preparation of the GTC-H (GTC-HDC) was also evaluated to ascertain if any effects were due to caffeine. The GTC preparations were administered to rats once daily at levels up to 2000 mg/kg/day for 28 days. There were no deaths attributable to the GTC preparations. The clinical condition of the animals, functional observational battery, motor activity, clinical pathology evaluations, organ weights, and gross necropsy findings were unaffected by any of the GTC preparations. GTC-HDC or GTC-UH dosing had no effects on body weights or microscopic findings, whereas lower body weights and food consumption were observed in the 1000 and 2000 mg/kg/day GTC-H group males. The no observed-adverse-effect level (NOAEL) for localized gastric effects for GTC-H was 1000 mg/kg/day. No other target organs were identified. Thus, the NOAEL for systemic toxicity following oral administration was 2000 mg/kg/day for GTC-H, GTC HDC, and GTC-UH under the conditions of this study.

A 24-month dietary carcinogenicity study of DAG in mice.

This study evaluated the possible carcinogenic effects of DAG (diacylglycerol) oil when given in the diet at levels up to 6.0% for 24 months to mice. Dietary fat was provided by DAG and/or the control article, TG (triacylglycerol oil). Dietary concentrations (% DAG/% TG) were 0%/6.0% (TG control), 1.5%/4.5%, 3.0%/3.0%, and 6.0%/0%. An additional control group received the standard rodent diet (fat content 4.5%). The clinical condition of the animals, ophthalmic findings, palpable mass occurrence, body weights and gross and histopathologic findings were unaffected by DAG in comparison to TG. The findings in DAG-treated groups were no different than those observed in the TG control group. The standard basal diet had 4.5% fat content. Both TG and/or DAG, when presented separately or together in the diet at a total fat level of 6.0%, resulted in some differences relative to the basal diet control (lower survival, higher body weights, lower food consumption, and higher incidences of macroscopic and microscopic findings), presumably related to the higher dietary fat content and/or the semi-purified diet. However, these parameters were similar in groups fed a diet with 6.0% dietary fat that was either DAG or TG. Thus, DAG at dietary concentrations up to 6.0% for 24 months produced no signs of systemic toxicity and had no effect on the incidence of neoplastic findings.

A chronic dietary toxicity study of DAG (diacylglycerol) in Beagle dogs.

The potential chronic toxic effects of DAG (diacylglycerol) when administered orally for 12 months were evaluated in this dietary study in Beagle dogs. DAG is a cooking oil which contains >80% diglycerides, <20% triglycerides and 5% monoglycerides. For this study, a special diet was prepared with no dietary fat so that all of the dietary fat could be provided by DAG, at various concentrations together with a control oil. The control oil, TG (triacylglycerol), was prepared to contain >85% triglycerides, <10% diglycerides and 5% monoglycerides. The fatty acid composition for DAG and TG was closely matched. Dietary concentrations of 0% DAG/9.5% TG (TG control), 1.5% DAG/8.0% TG, 5.5% DAG/4.0% TG, and 9.5% DAG/0% TG were presented daily, seven days per week, for 52 weeks. A second concurrent control group received the standard basal diet (Certified Canine LabDiet 5007, which has a fat content of 9.5%). The basal diet, control article-treated and DAG-treated groups each consisted of four male and four female dogs. Treatment was initiated in prejuvenile (2.5-month-old) dogs. Statistical evaluations compared the DAG-treated groups both to the basal diet and 9.5% TG control groups. The clinical condition of the animals, body weights, body weight gains and food consumption were unaffected by DAG. Hematology and urinalysis parameters were unaffected. No serum chemistry changes indicative of a toxic effect were observed. There were no effects noted on ECG data. No test article-related gross or histopathologic findings or changes in organ weights were observed. While there were no identifiable differences between the effects of TG and DAG, both caused some differences relative to the basal diet (lower food consumption, higher alkaline phosphatase, cholesterol and triglycerides). These differences were not toxicologically significant and were attributed to the differences in the diet rather than the fat source. Thus, DAG at dietary concentrations up to 9.5% for one year had no effect on normal canine growth and development, in comparison to TG.

A 24-month dietary carcinogenicity study of DAG (diacylglycerol) in rats.

Toxicologic and carcinogenic effects of DAG (diacylglycerol) oil, administered in diet for 24 months to Crl:CD((R))(SD)-IGS BR rats, were evaluated using diet-restricted and ad libitum-fed groups. All dietary fat (consistently 5.5%) was provided by DAG and/or the control article, TG (triacylglycerol) oil. Dietary concentrations (% DAG/% TG) were 0%/5.5%, 1%/4.5%, 2.75%/2.75% and 5.5%/0%. Separate groups were fed the 0%/5.5% and 5.5%/0% diets ad libitum. Another group received the standard rodent diet (fat content 4.5%) on the restricted feeding regimen. Clinical condition, ophthalmic findings, palpable mass occurrence, body composition, clinical pathology parameters and incidence of neoplastic lesions were unaffected by DAG in comparison to TG. Groups fed the 5.5% (DAG and/or TG) fat diet when compared to the 4.5% fat diet group displayed lower survival, higher body weights, organ weights, percent body fat, higher fat-related serum chemistry parameters, incidence of microscopic changes in the heart, kidneys, liver, bone marrow, spleen, and incidences of pituitary and mammary gland neoplasms. Parameters more affected in all the ad libitum groups than in the restricted diet groups (regardless of test article) fed the same diet included survival, body weights, body fat, fat-related serum chemistry parameters, and incidences of heart, kidney and liver microscopic changes. However, the DAG and TG ad libitum-fed groups were not different from one another. Thus, DAG-treated animals had no higher risk of carcinogenic effects than rats fed on similar feeding regimens with a diet in which all dietary fat came from TG.