There are several design issues that should be noted when evaluating the results of this study. First, although there were 2 study years ( 1993 and 1995), the populations were not independent. Sixty eight employees participated in both years. Second, there were 31 fewer employees who participated in the study in 1995, thus reducing the power of the study. There were also very few employees in either year with serum PFOA levels greater than 10 ppm. Third, the crosssectional design of the study does not allow for analysis of temporality of an association. Since the half life of PFOA is at least 1 year, the authors suggest that it is possible that there may be some biological accommodation to the effects of PFOA. Fourth, only one sample was taken for each hormone for each of the study years. In order to get more accurate measurements for some of the hormones, pooled blood taken in a short time period should have been used for each participant. Fifth, some of the associations that were measured in this study were done based on the results of an earlier paper that linked PFOA with increased estradiol and decreased testosterone levels. However, total serum organic fluorine was measured in that study instead of PFOA, making it difficult to compare the results. Finally, there may have been some measurement error of some of the confounding variables.

3.2.5 Study on Episodes of Care ( Morbidity)

In order to gain additional insight into the effects of fluorochemical exposure on workers' health, an " episode of care" analysis was undertaken at the Decatur plant to screen for morbidity outcomes that may be associated with long term, high exposure to fluorochemicals ( Olsen et al., 2001g). An " episode of care" is a series of health care services provided from the start of a particular disease or condition until solution or resolution of that problem. Episodes of care were identified in employees' health claims records using Clinical Care Groups ( CCG) software. All inpatient and outpatient visits to health care providers, procedures, ancillary services and prescription drugs used in the diagnosis, treatment, and management of over 400 diseases or conditions were tracked.

Episodes of care were analyzed for 652 chemical employees and 659 film plant employees who worked at the Decatur plant for at least 1 year between January 1, 1993 and December 31, 1998. Based on work history records, employees were placed into different comparison groups: 41 Group A consisted of all film and chemical plant workers; Group B had employees who only worked in either the film or chemical plant; Group C consisted of employees who worked in jobs with high POSF exposures; and Group D had employees who worked in high exposures in the chemical plant for 10 years or more prior to the onset of the study. Film plant employees were considered to have little or no fluorochemical exposure, while chemical plant employees were assumed to have the highest exposures.

Ratios of observed to expected episodes of care were calculated for each plant. Expected numbers were based on 3M's employee population experience using indirect standardization techniques. A ratio of the chemical plant's observed to expected experience divided by the film plant's observed to expected experience was calculated to provide a relative risk ratio for each episode of care ( RREpC). 95% confidence intervals were calculated for each RREpC. Episodes of care that were of greatest interest were those which had been reported in animal or epidemiologic literature on PFOS and PFOA: liver and bladder cancer, endocrine disorders involving the thyroid gland and lipid metabolism, disorders of the liver and biliary tract, and reproductive disorders.

The only increased risk of episodes for these conditions of a priori interest were for neoplasms of the male reproductive system and for the overall category of cancers and benign growths ( which included cancer of the male reproductive system). There was an increased risk of episodes for the overall cancer category for all 4 comparison groups. The risk ratio was greatest in the group of employees with the highest and longest exposures to fluorochemicals ( RREpC = 1.6, 95% CI = 1.2   2.1). Increased risk of episodes in long time, high exposure employees also was reported for male reproductive cancers ( RREpC = 9.7, 95% CI = 1.1  458). It should be noted that the confidence interval is very wide for male reproductive cancers and the sub category of prostate cancer. Five episodes of care were observed for reproductive cancers in chemical plant employees ( 1.8 expected), of which 4 were prostate cancers. One episode of prostate cancer was observed in film plant employees ( 3.4 expected). This finding should be noted because an excess in prostate cancer mortality was observed in the Cottage Grove plant mortality study when there were only 2 exposure categories ( chemical division employees and non chemical division employees). The update of the study sub divided the chemical plant employees and did not confirm this finding when exposures were divided into definitely exposed and probably exposed employees.

There was an increased risk of episodes for neoplasms of the gastrointestinal tract in the high exposure group ( RREpC = 1.8, 95% CI = 1.2 3.0) and the long term employment, high exposure group ( RREpC = 2.9, 95% CI = 1.7   5.2). Most of the episodes were attributable to benign colonic polyps. Similar numbers of episodes were reported in film and chemical plant employees.

In the entire cohort, only 1 episode of care was reported for liver cancer ( 0.6 expected) and 1 for bladder cancer ( 1.5 expected). Both occurred in film plant employees. Only 2 cases of cirrhosis of the liver were observed ( 0.9 expected), both in the chemical plant. There was a greater risk of lower urinary tract infections in chemical plant employees, but they were mostly due to recurring 42 episodes of care by the same employees. It is difficult to draw any conclusions about these observations, given the small number of episodes reported.

Chemical plant employees in the high exposure, long term employment group were 2 ½ times more likely to seek care for disorders of the biliary tract than their counterparts in the film plant ( RREpC = 2.6, 95% CI = 1.2  5.5). Eighteen episodes of care were observed in chemical plant employees and 14 in film plant workers. The sub categories that influenced this observation were episodes of cholelithiasis with acute cholecystitis and cholelithiasis with chronic or unspecified cholecystitis. Most of the observed cases occurred in chemical plant employees.

Risk ratios of episodes of care for endocrine disorders, which included sub categories of thyroid disease, diabetes, hyperlipidemia, and other endocrine or nutritional disorders, were not elevated in the comparison groups. Conditions which were not identified a priori but which excluded the null hypothesis in the 95% confidence interval for the high exposure, long term employment group included: disorders of the pancreas, cystitis, and lower urinary tract infections.

The results of this study only should be used for hypothesis generation. Although the episode of care design allowed for a direct comparison of workers with similar demographics but different exposures, there are many limitations to this design. The limitations include: 1) episodes of care are reported, not disease incidence, 2) the data are difficult to interpret because a large RREpC may not necessarily indicate high risk of incidence of disease, 3) many of the risk ratios for episodes of care had very wide confidence intervals, thereby providing unstable results, 4) the analysis was limited to 6 years, 5) the utilization of health care services may reflect local medical practice patterns, 6) individuals may be counted more than once in the database because they can be categorized under larger or smaller disease classifications, 7) episodes of care may include the same individual several times, 8) not all employees were included in the database, such as those on long term disability, 9) the analysis may be limited by the software used, which may misclassify episodes of care, 10) the software may assign 2 different diagnoses to the same episode, and 11) certain services, such as lab procedures may not have been reported in the database.

3.3 Acute Toxicity Studies in Animals 3.3.1 Oral Studies The acute oral toxicity of APFO was tested in male and female rats in three studies. Death occurred at concentrations   464 mg/ kg ( Internat'l Res and Dev Corp., 1978). Abnormal findings upon necropsy ( kidney, stomach, uterus) were observed ( Glaza, 1997) at 500 mg/ kg ( higher concentrations were not tested). Clinical signs of toxicity observed in these three studies included the following: red stained face, stained urogenital area, wet urogenital area, hypoactivity, hunched posture, staggered gait, excessive salivation, ptosis, piloerection, decreased limb tone, ataxia, corneal opacity, and hypothermic to touch.

In one study ( Internat'l Res and Dev Corp., 1978), the oral LD50 values for Charles River CD 43 rats were 680 mg/ kg ( 399   1157 mg/ kg 95% confidence limit) for males; 430 mg/ kg ( 295   626 mg/ kg 95% confidence limit) for females; and 540 mg/ kg ( 389   749 mg/ kg 95% confidence limit) for males and females. The remaining two studies provided LD50 values of ( 1) > 500 mg/ kg for male Crl: CD( SD) BR rats, and 250 500 mg/ kg for female Crl: CD( SD) BR rats ( Glaza, 1997); and ( 2) < 1000 mg/ kg for male and female Sherman Wistar rats ( 3M Company, 1976b).

3.3.2 Inhalation Studies The acute inhalation toxicity of APFO was tested in male and female Sprague Dawley rats, at a dose level of 18.6 mg/ L ( nominal concentration), and exposure duration of one hour. Signs of toxicity during and up to 14 days after the exposure period, included the following: excessive salivation, excessive lacrimation, decreased activity, labored breathing, gasping, closed eyes, mucoid nasal discharge, irregular breathing, red nasal discharge, yellow staining of the anogenital fur, dry and moist rales, red material around the eyes, and body tremors. Upon necropsy, lung discoloration was observed in a higher than normal incidence of rats ( 8/ 10). Based on the study results, the test substance was not fatal to rats at a nominal exposure concentration of 18.6 mg/ L and exposure duration of one hour ( Bio/ dynamics, Inc. 1979).

3.3.3 Dermal Studies The acute dermal toxicity of APFO was tested in male and female Hra( NZW) SPF rabbits, at a dose level of 2000 mg/ kg, and a 24 hour exposure period. All animals appeared normal and exhibited body weight gain throughout the study, with the exception of one male that lost weight during the first week. Dermal irritation consisted of slight to moderate erythema, edema, and atonia; slight desquamation; coriaceousness; and fissuring. No visible lesions were observed upon necropsy. The dermal LD50 in rabbits was determined to be greater than 2000 mg/ kg ( Glaza, 1995).

3.3.4 Eye Irritation Studies The eye irritation potential of APFO was tested in albino rabbits, at a dose level of 0.1 gram. In two of three studies, APFO was determined to be a primary ocular irritant. In the studies in which APFO was found to be a primary ocular irritant, APFO was left in contact with the eye for 7 days, then rinsed, or not rinsed. Irritation scores varied during the observation period. Irritation scores of the conjunctivae, iris, and cornea ranged from 2   4 in one study ( Biosearch, Inc. 1976) and from 2   10 in the other study ( 3M Company, 1976a). In both studies, irritation remained evident for the duration of the observation period ( 7 days post exposure). In the study in which APFO was determined to be a non irritant ( Gabriel), the test substance was left in contact with the eye for 5 or 30 seconds, and then the eyes were rinsed. In this study, positive scores were reported for conjunctivae irritation for up to 7 days post exposure, so the author's negative conclusion for ocular irritancy is problematic. 44 3.3.5 Skin Irritation Studies The skin irritation potential of APFO was tested in albino rabbits in two studies, at a dose level of 0.5 grams, under occluded test conditions. In one study ( Riker Laboratories, Inc. 1983), APFO produced irreversible tissue damage in female rabbits, following a 3 minute, 1 hour, and 4 hour contact period. Moderate erythema and edema, as well as chemical burn, eschar, and necrosis, were observed following all three contact periods. An endpoint was not achieved in this study due to extreme irritation following each contact period. In contrast in the second study ( Gabriel), APFO was reported as a non irritant of skin after an exposure period of 24 or 72 hours, based on primary irritation scores of zero.

3.4 Mutagenicity Studies APFO was tested twice ( Lawlor, 1995; 1996) for its ability to induce mutation in the Salmonella   E. coli/ mammalian microsome reverse mutation assay. The tests were performed both with and without metabolic activation. A single positive response seen at one dose level in S. typhimurium TA1537 when tested without metabolic activation was not reproducible. APFO did not induce mutation in either S. typhimurium or E. coli when tested either with or without mammalian activation. APFO did not induce chromosomal aberrations in human lymphocytes when tested with and without metabolic activation up to cytotoxic concentrations ( Murli, 1996c; NOTOX, 2000). Sadhu ( 2002) recently reported that APFO did not induce gene mutation when tested with or without metabolic activation in the K 1 line of Chinese hamster ovary ( CHO) cells in culture.

Murli ( 1996b) tested APFO twice for its ability to induce chromosomal aberrations in CHO cells. In the first assay, APFO induced both chromosomal aberrations and polyploidy in both the presence and absence of metabolic activation. In the second assay, no significant increases in chromosomal aberrations were observed without activation. However, when tested with metabolic activation, APFO induced significant increases in chromosomal aberrations and in polyploidy ( Murli, 1996b).

APFO was tested in a cell transformation and cytotoxicity assay conducted in C3H 10T ½ mouse embryo fibroblasts. The cell transformation was determined as both colony transformation and foci transformation potential. There was no evidence of transformation at any of the dose levels tested in either the colony or foci assay methods ( Garry & Nelson, 1981).

APFO was tested twice in the mouse micronucleus assay. APFO did not induce any significant increases in micronuclei and was considered negative under the conditions of this assay ( Murli, 1996a).

3.5 Subchronic Toxicity Studies in Animals Subchronic toxicity studies have been conducted in rats, mice, rhesus monkeys and cynomolgus monkeys. Two unpublished 28 day feeding studies were performed at Industrial Bio Test 45 Laboratories, Inc. ( Metrick and Marias, 1977 and Christopher and Marias, 1977). In both rats and mice the liver was the target organ. In rats, males had more pronounced hepatotoxicity and histopathologic effects than females. Three 90 day subchronic toxicity studies have been conducted in rats ( Goldenthal, 1978a, Palazzolo, 1993) and rhesus monkeys ( Goldenthal, 1978b). Thomford ( 2001a, b) recently conducted a range finding and a 6 month toxicity study in cynomolgus monkeys. In addition, chronic/ carcinogenicity studies have been conducted which are described in section 3.8. In all species, the liver is the main target organ. In rats, males had more pronounced hepatotoxicity and histopathologic effects than females, presumably because of the gender difference in elimination of APFO.

In a 28 day study of ChR CD albino rats, eight randomly assigned groups of five males and five females were studied ( Metrick and Marias, 1977). After rats were allowed to acclimate for a week in individual cages they then received similar feed containing 0, 30, 100, 300, 1000, 3000, 10,000, or 30,000 ppm APFO for 28 days. The animals were observed daily and body weights and food consumption were recorded weekly. Animals that died during the study were examined for gross pathology, as were surviving animals at 28 days. It is stated that the study included a complete examination of gross pathology and a complete set of tissues and organs were examined, but the specific list is not supplied. Livers were weighed to determine relative organ weight then stained for histopathologic examination.

All animals in the 10,000 and 30,000 ppm groups died before the end of the first week. There were no premature deaths or other clinical signs of toxicity in the other groups. Body weight gains were reduced in the groups receiving 1000 or more ppm. Slight reductions in body weight gain were also observed in males exposed to 300 ppm and males and females fed 100 ppm. Reduced food intake was observed in rats fed 1000 ppm or higher in a dose related manner. Relative liver weights were increased in males fed 30 ppm or more and females fed 300 ppm or more. Gross pathological exam did not reveal treatment related effects in kidneys or other organs besides livers. Focal to multifocal cytoplasmic enlargement of hepatocytes was noted in animals fed 300 ppm, and multifocal to diffuse enlargement of hepatocytes was noted in animals fed 1000 ppm or higher. These effects were more pronounced in males ( Metrick and Marias. 1977).

In a 28 day study of Charles River CD albino mice, eight randomly assigned groups of five males and five females were studied ( Christopher and Marisa, 1977). After mice were allowed to acclimate for 8 days in individual cages they then received similar feed containing 0, 30, 100, 300, 1000, 3000, 10,000, or 30,000 ppm of APFO for 28 days. The animals were observed daily and body weights and food consumption were recorded weekly. Animals that died during the study were examined for gross pathology, as were surviving animals at 28 days. It is stated the study included a complete examination of gross pathology and a representative set of tissues and organs were examined, but the specific list is not supplied. Livers were weighed to determine relative organ weight then stained for histopathologic examination.

All animals in the 1000 ppm and higher groups died before the end of day 9. The entire 300 ppm group died within 26 days except 1 male. One animal in each of the 30 and 100 ppm groups 46 died prematurely. Clinical signs were observed in mice exposed to 100 ppm and higher doses of PFOA. At 100 ppm some animals exhibited cyanosis on days 10 and 11 of testing, but appeared normal throughout the rest of the study. Animals feed 300 ppm exhibited roughed fur and muscular weakness as well as signs of cyanosis after 9 days of treatment. Animals fed 1000 ppm exhibited similar effects after 6 days and those receiving 3000 ppm or greater doses exhibited effects after 4 days.

All mice fed APFO lost weight. Reductions in body weight gain were followed by weight losses in mice fed 30, 100, or 300 ppm. A dose related pattern was seen in the depressed body weights.

Relative and absolute liver weights were increased in mice fed 30 ppm or more APFO. Gross pathological examination of kidneys or other organs besides livers is not discussed. Treatmentrelated changes were observed in the livers among all APFO treated animals including enlargement and/ or discoloration of 1 or more liver lobes. Histopathologic examination of all APFO treated mice revealed diffuse cytoplasmic enlargement of hepatocytes throughout the liver ( pan lobular hypertrophy) accompanied by focal to multifocal cytoplasmic vacuoles. Degeneration and / or necrosis of hepatocytes and focal bile duct proliferation were also noted in mice within all groups ( Christopher and Marias, 1977).

Three 90 day subchronic toxicity studies have been conducted. One was conducted in rats ( Goldenthal, 1978a), one was conducted in rhesus monkeys ( Goldenthal, 1978b) and the third was conducted in male rats ( Palazzolo, 1993).

In the monkey study, Goldenthal ( 1978b) administered rhesus monkeys ( 2/ sex/ group) doses of 0, 3, 10, 30 or 100 mg/ kg/ day perfluorooctanoic acid ( FC 143) in 0.5% Methocel7 by gavage for 7 days/ week for 90 days. All doses were given in a constant volume; individual daily doses were based upon the weekly body weights. Animals were observed twice daily for general physical appearance and behavior and pharmacotoxic signs. General physical examinations were performed during the control period and monthly during the study period. Individual body weights were recorded weekly. Blood and urine samples were collected once during the control period and at 1 and 3 months of the study for hematology, clinical chemistry and urinalysis. Monkeys were fasted overnight prior to the collection of blood and urine samples. Organs and tissues from animals that were sacrificed at the end of the study and from animals that died during the treatment period were weighed, examined for gross pathology and samples taken for histopathology. Histopathology was performed on the following organs from all monkeys in the control and treatment groups: adrenals, aorta, bone, brain, esophagus, eyes, gallbladder, heart ( with coronary vessels), duodenum, ileum, jejunum, cecum, colon, rectum, kidneys, liver, lung, skin, mesenteric lymph node, retropharyngeal lymph node, mammary gland, nerve ( with muscle), spleen, pancreas, prostate/ uterus, rib junction ( bone marrow), salivary gland, lumbar spinal cord, pituitary, stomach, testes/ ovaries, thyroid, parathyroid, thymus, trachea, tonsil, tongue, urinary bladder, vagina, identifying tattoo, and any tissues( s) with lesions.

All monkeys in the 100 mg/ kg/ day groups died during the study. The first death occurred during week 2; all animals were dead by week 5. Signs and symptoms which first appeared during 47 week 1 included anorexia, frothy emesis which was sometimes brown in color, pale face and gums, swollen face and eyes, slight to severe decreased activity, prostration and body trembling. Three monkeys from the 30 mg/ kg/ day group died during the study; one male died during week 7 and the two females died during weeks 12 and 13. Beginning in week 4, all four animals showed slight to moderate and sometimes severe decreased activity. One monkey had emesis and ataxia, swollen face, eyes and vulva, as well as pallor of the face and gums. Beginning in week 6, two monkeys had black stools and one monkey had slight to moderate dehydration and ptosis of the eyelids.

No monkeys in the 3 or 10 mg/ kg/ day groups died during the study. Animals in the 3 mg/ kg/ day dose group occasionally had soft stools or moderate to marked diarrhea; frothy emesis was also occasionally noted in this group. One monkey in the 10 mg/ kg/ day group was anorexic during week 4, had a pale and swollen face in week 7 and had black stools for several days in week 12. The other animals in the 10 mg/ kg/ day groups did not show any unusual signs or symptoms.

Changes in body weight were similar to the controls for animals from the 3 and 10 mg/ kg/ day dose groups. Monkeys from the 30 and 100 mg/ kg/ day groups lost body weight after week 1. At the end of the study, this loss was statistically significant for the one surviving male in the 30 mg/ kg/ day group ( 2.30 kg vs 3.78 kg for the control).

Hematology values at the end of the 1 and 3 months of treatment were similar for the control and the 3 and 10 mg/ kg/ day groups. At 30 mg/ kg/ day, the surviving male had decreased numbers of erythrocytes, decreased hemoglobin, decreased hematocrit, and increased platelets. Prothrombin time and activated prothrombin time were also increased. These increases were apparent at 1 month but were much more marked at three months.

Following one month of treatment, glucose was significantly elevated in the 3 mg/ kg/ day group ( 117 vs 89 mg/ 100 ml in the control). The authors of the report attribute this to a single high value for male # 7366 who had a value of 131. The other three monkeys in the 3 mg/ kg/ day groups had levels of 112, 105, and 120 mg/ 100 ml. Glucose levels in the 10 and 30 mg/ kg/ day groups were 104 and 122 mg/ 100 ml, respectively, after one month of treatment. At three months of treatment, glucose levels were 81, 96, 88, and 66 mg/ 100 ml in the control, 3, 10 and 30 mg/ kg/ day groups respectively.

There was a decrease in alkaline phosphatase levels in the 30 mg/ kg/ day group ( 365 vs 597 IU/ l in the control) at one month, which persisted in the one surviving male ( 360 vs 851 IU/ l in the control) at 3 months. Alkaline phosphatase levels in the 3 and 10 mg/ kg/ day groups at three months were 783 and 743 IU/ l showing a dose related trend toward decreased levels.

SGOT levels were reduced in the 30 mg/ kg/ day groups at one month ( 59 vs 29 IU/ l in the control) and in the one surviving male at 3 months ( 88 vs 45 IU/ l in the control). SGPT was elevated in both the 10 and 30 mg/ kg/ day dose groups at 1 month; the levels were 15, 34, and 44 IU/ l in the control, 10 and 30 mg/ kg/ day groups, respectively. SGOT levels in the 10 mg/ kg/ day 48 group were comparable to the controls at 3 months ( 34 vs 31 IU/ l in the control) but were still elevated in the one surviving male in the 30 mg/ kg/ day dose group ( 46 IU/ l).

Cholesterol in the one surviving male in the 30 mg/ kg/ day group was elevated ( 240 vs 165 mg/ 100ml) and total protein and albumin in this animal were reduced. Total protein was 5.52 vs a control level of 8.21 g/ 100 ml and total albumin was 2.00 vs a control level of 4.82 g/ 100 ml.

There were no treatment related changes in urinalysis studies at any time period studied.

There were no macroscopic lesions noted at gross necropsy of any animals which died during the study or which were sacrificed at the end of the treatment period.

The following changes in absolute and relative organ weight changes were noted: absolute and relative weight of the hearts in females from the 10 mg/ kg/ day group were decreased; absolute brain weight of females from this same group were also decreased and relative group mean weight of the pituitary in males from the 3 mg/ kg/ day group was increased. The significance of these weight changes is difficult to assess, as they were not accompanied by morphologic changes.

One male and two females from the 30 mg/ kg/ day group and all animals from the 100 mg/ kg/ day group had marked diffuse lipid depletion in the adrenals. All males and females from the 30 and 100 mg/ kg/ day groups also had slight to moderate hypocellularity of the bone marrow and moderate atrophy of lymphoid follicles in the spleen. One female from the 30 mg/ kg/ day group and all animals in the 100 mg/ kg/ day group had moderate atrophy of the lymphoid follicles in the lymph nodes. No other compound related lesions were seen in at the 30 and 100 mg/ kg/ day groups. No treatment related lesions were seen in the organs of animals from the 3 and 10 mg/ kg/ day groups.

The levels of PFOA in the serum and liver are presented below in Table 3. Individual values are presented so there are double entries for most dose levels.

Table 3. Levels of PFOA in the Serum and Liver Dose Serum ( ppm) Liver ( ppm) Liver total ( ug)

Males Females Males Females Males Females 0 ND 1 0.05 0.07 3 5 3 53 65 3 7 250 350 3 48 50 ND ND ND ND 10 45 79 9 ND 600 ND 10 71 71 ND 10 ND 750 30 ND ND 125 80 8000 7500 30 145 ND 60 125 4000 9000 100 ND ND 100 325 6000 20000 49 In the first rat study, Goldenthal ( 1978a) administered CD rats ( 5/ sex/ group) dietary levels of 0, 10, 30, 100, 300, and 1000 ppm perfluorooctanoic acid. These dose levels are approximately equivalent to 0.6, 1.7, 5.6, 17.9, and 63.5 mg/ kg/ day in males, and 0.7, 2.3, 7.7, 22.4 and 76.5 mg/ kg/ day in females. Animals were housed individually in wire mesh cages and had free access to food and water. Animals were observed twice daily for signs of toxicity and for mortality. Detailed examinations were performed once a week. Individual body weight and food consumption were recorded weekly during the pretest and treatment periods. Blood and urine samples were collected during the pretest period and at 1 and 3 months of the study for hematology and clinical chemistry and urinalysis. At week 13, the serum samples were frozen and shipped to the sponsor for analysis. Organs and tissues from animals that were sacrificed at the end of the study and from two females that died during the treatment period were weighed, examined for gross pathology and samples taken for histopathology. Histopathology was performed on the following organs from rats from the control, 100, 300, and 1000 ppm dose groups: brain with cervical cord, lumbar spinal cord, peripheral nerve, eyes, pituitary, thyroid with parathyroid, adrenals, lung, heart with coronary vessels, aorta, spleen, mesenteric lymph node, thymus, bone with marrow ( sternum), salivary gland, small intestines ( duodenum, jejunum, ileum) colon, pancreas, liver, kidneys, urinary bladder, testes, ovaries, prostate, uterus, skin ( mammary gland), any tissue( s) with gross lesions. Livers from rats from the 10 and 30 ppm dose groups were also examined microscopically and liver samples from all dose groups were frozen and sent to the sponsor for analysis.

One female in the 100 and one female in the 300 ppm group died during collection of blood. These deaths were not considered to be treatment related. All other animals survived until scheduled sacrifice.

There was a significant reduction in mean body weight in males in the 1000 ppm group ( 362 g vs 466 g in the control group). Food consumption was reduced in males in the 100, 300 and 1000 ppm groups, but the differences were not statistically significant.

Males in the 30, 100, 300 and 1000 ppm groups had significantly reduced numbers of erythrocytes at the end of the treatment period. The values were 7.95, 7.05, 7.16, 6.72, and 6.94 in the control, 30, 100, 300 and 1000 ppm groups, respectively. Males had reduced leukocyte values compared to the controls in all dose groups, but were statistically significant at the 300 ppm group only; leukocyte values were 10.64, 8.88, 9.33, 9.35, 7.63, and 8.06 in the control, 10, 30, 100, 300 and 1000 ppm groups, respectively. A similar phenomenon was seen with hemoglobin values, which were reduced at all dose levels but were significant at the 10 ppm dose level only. Hemoglobin values were 16.2, 14.7, 15.0, 15.4, 14.9, 13.1 in the control, 10, 30, 100, 300 and 1000 ppm groups, respectively. There was no similar effect upon the hematological parameters of female rats in the study.

Males at the 30, 100, 300, and 1000 ppm dose levels had increased glucose levels ( mg/ 100 ml), which were statistically significant at all but the 100 ppm dose level. Reported glucose levels were 121, 120, 136, 134, 143 and 135 mg/ 100 ml for the 0, 10, 30 100, 300 and 1000 ppm 50 groups, respectively. B. U. N. levels were elevated in males at the 100, 300, and 1000 ppm dose levels; mean values at 90 days were 20.4, 23.9 and 35.1 mg/ 100 ml for the three dose groups, respectively, compared to 16.2 mg/ 100 ml for the controls. Alkaline phosphatase was elevated in males in the 100, 300, and 1000 ppm groups; the levels were 147, 204 and 212 IU/ l for the three groups, respectively, compared to 104 IU/ l for the controls. Females showed no similar changes in biochemical measurements.

Neither males nor females showed any treatment related changes in urinalysis parameters although females from all groups showed a higher frequency of occult blood in the urine than did males.

The only gross necropsy observation was noted in males at the 1000 ppm dose level. These animals had enlarged livers that showed varying degrees of surface discoloration. Neither females from the 1000 ppm dose level nor males or females from the lower dose levels showed such effects.

Both absolute and relative liver weights were significantly increased in males in the 30, 300 and 1000 ppm groups and in one female in the 1000 ppm group. Compound related liver lesions occurred in all male rats in the 100, 300 and 1000 ppm groups. These lesions consisted of focal to multifocal, very slight to slight hypertrophy of hepatocytes in centrilobular to midzonal regions of the affected liver lobules. In some instances these lesions were accompanied by an increased amount of yellowish brown pigment resembling lipofuscin in the cytoplasm of hepatocytes and occasionally in sinusoidal lining cells. The incidence and severity of the lesions was more pronounced among male rats at the 1000 ppm dietary level.

A comparison of the serum levels of PFOA is shown below in Table 4. The greater toxicity observed in the males than in the females is due to the gender difference in elimination as demonstrated by the differences in serum PFOA levels.

Table 4. Comparison of Male and Female PFOA Serum Levels Dose PFOA in Serum ( ppm) Males Females 0 0 0 10 21 ND 30 34 0.15 100 36 ND 300 38 0.25 1000 49 0.65 ND = Not Determined.

In the second rat study, Palazzolo ( 1993) administered 45 55 male Sprague Dawley rats per group, doses of 1, 10, 30, or 100 ppm ( approximate mean compound consumption at week 13 of 51 0.05, 0.47, 1.44, and 4.97 mg/ kg/ day) APFO ad libitum in the diet for 13 weeks. Two control groups ( a nonpair fed control group and a control group pair fed to the 100 ppm dose group) were also exposed during that period. Following the 13 week exposure period, 10 animals per group were fed basal diet for an additional 8 weeks post treatment and observed for any signs of recovery. All test diets were assayed and evaluated for test material homogeneity and stability. All animals were observed twice daily for mortality, moribundity, and general clinical signs of toxicity. Body weights were recorded once before exposures began, weekly during the treatment period, and then on the day of necropsy. Food consumption was recorded weekly for all groups, including the nonpair fed control group; daily for the pair fed animals, and then weekly for all of the animals retained for the recovery phase of the study. A total of 15 animals per group were sacrificed following 4, 7, or 13 weeks of treatment; 10 animals per group were sacrificed after 13 weeks of treatment and following 8 weeks of non treatment. Serum samples collected from 10 animals per group at each scheduled sacrifice during treatment and from 5 animals per group during recovery were analyzed for estradiol, total testosterone, luteinizing hormones, and for test material residue. The level of palmitoyl CoA oxidase, an indicator of peroxisome proliferation, was analyzed from a section of liver that was obtained from 5 animals per group at each scheduled sacrifice. The following organs from all animals at each scheduled sacrifice were weighed: brain, liver, lungs, testis ( one), seminal vesicle, prostate, coagulating gland, and urethra. The following tissues in these same animals were preserved in 10% phosphate buffered formalin and examined macroscopically: external surface of the body, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the nasal cavity and paranasal sinuses; the thoracic, abdominal, and pelvic cavities and viscera; and also examined microscopically: any observed lesions, brain, liver, lungs, testes ( one), seminal vesicle, prostate, coagulating gland, and urethra. In addition, the following tissues were preserved in glutaraldehyde for electron microscopic examination: brain, liver, lungs, testes ( one), seminal vesicle, and prostate.

In the analysis of the data, animals in groups exposed to 1, 10, 30, and 100 ppm APFO were compared to the control animals in the nonpair fed group, while the data from the pair fed control animals were compared to animals exposed to100 ppm APFO. All test diets were considered to be homogeneous and stable under the experimental conditions. All animals survived to scheduled sacrifice, with the exception of one animal in the 100 ppm dosed group that was sacrificed on week 4 due to severe neck sores unrelated to treatment. Twice daily examinations of all animals were unremarkable. At 100 ppm, significant reductions in body weights were seen compared to the pair fed control group during week 1 and the nonpair fed control group during weeks 1 13 ( i. e., throughout treatment). During recovery, however, no reductions in body weights were apparent. Body weight data in the other dosed groups were comparable to controls. At 100 ppm, mean body weight gains were significantly higher than the pair fed control group during week 1 and significantly lower than the nonpair fed control group during weeks 1 13. At 10 and 30 ppm, mean body weight gains were significantly lower than the nonpair fed control group at week 2. These differences in body weight gains were not observed during the recovery period. Significant differences in food consumption were observed at 100 ppm during weeks 1 and 2 only, when compared to the nonpair fed control group; no other significant differences in food consumption were noted. There were no significant differences among the groups for any of the hormones evaluated in the serum. Likewise, serum 52 analysis of test material residue showed no increase in serum APFO levels over the course of treatment. Statistically significant higher hepatic palmitoyl CoA oxidase activity was observed at 30 and 100 ppm; however, this effect returned to control levels by the end of the recovery period. At 10 ppm, statistically significant higher levels of hepatic palmitoyl CoA oxidase activity were observed at week 5 only. Mean enzyme activities were highest during week 8 for animals exposed to 10, 30, and 100 ppm. Significant increases in absolute and relative liver weights and hepatocellular hypertrophy were observed at weeks 4, 7, or 13 in the 10, 30 and 1000 ppm groups. The authors suggested that these changes might be associated with peroxisome proliferation, especially since increases in hepatic palmitoyl CoA oxidase activity were also observed at this dose level during treatment. During recovery, however, none of the liver effects were observed, indicating that these treatment related liver effects were reversible.

Therefore, under the conditions of this study, a NOAEL of 1.0 ppm ( 0.05 mg/ kg/ day) and a LOAEL of 10 ppm ( 0.47 mg/ kg/ day) are indicated based on reductions in body weight and body weight gain, and on increases in absolute and relative liver weights with hepatocellular hypertrophy.

Thomford ( 2001a, b) recently conducted a range finding and a 6 month toxicity study in cynomolgus monkeys. In the range finding study, Thomford ( 2001a) administered male cynomolgus monkeys an oral capsule containing 0, 2, or 20 mg/ kg/ day APFO for 4 weeks. There were 3 monkeys in the 2 and 20 mg/ kg/ day groups and one monkey in the control group. The monkeys weighed 2.1 to 3.6 kg at the start of treatment. Animals were observed twice daily for mortality and moribundity and were examined at least once daily for signs of poor health or abnormal behavior. Food consumption was assessed qualitatively. Body weight data were recorded before the start of treatment, on day 1 of treatment and weekly thereafter. The monkeys were fasted overnight and blood samples were collected one week prior to the start of the study and on day 30 for clinical hematology and clinical chemistry analyses, and hormone and PFOA level. Blood for clinical chemistry was also collected from each animal on day 2 ( approximately 24 hours after the first dose). Samples were analyzed for estradiol, estrone, estriol, thyroid stimulating hormone, total and free triiodothyronine, and total and free thyroxin.

At scheduled necropsy, samples of the right lateral lobe of the liver were collected from each animal and analyzed for palmitoyl CoA oxidase activity. Representative samples of liver, right and left testes, and pancreas were collected from each animal for cell proliferation evaluation using proliferation cell nuclear antigen. Bile was collected from each animal for bile acid determination. A sample of liver was collected from each animal for PFOA concentration analysis. The following tissues ( when present) or representative samples were collected and preserved in 10 % neutral buffered formalin, unless otherwise specified, for possible future examination: adrenal ( 2), aorta, brain, cecum, colon, duodenum, epididymis ( 2), esophagus, eyes [ preserved in Davidson's fixative ( 2)], femur with one marrow ( articular surface of the distal end), gallbladder, heart, ileum, jejunum, kidney ( 2), lesions, liver, lung, lymph node ( mesenteric) mammary gland, muscle ( thigh), pancreas, pituitary, prostate, rectum, salivary gland [ mandibular ( 2)], sciatic nerve, seminal vesicle ( 2) skin, spinal cord ( cervical, thoracic, and lumbar), spleen, sternum with bone marrow, stomach, testis ( 2), thymus, thyroid ( 2) with parathyroid, trachea, 53 and urinary bladder. The adrenals, liver, pancreas, spleen, and testes from each animal were embedded in paraffin, sectioned, stained with hematoxylin and eosin and examined microscopically.

All animals survived to scheduled sacrifice. There were no clinical signs of toxicity in the treated groups and there was no effect on body weight. Low or no food consumption was observed for one animal given 20 mg/ kg/ day. No food consumption was noted for this animal on day 12 and low food consumption was noted on days 5, 7, 11, 14 17, and 24, respectively. For this animal, decreased food consumption is in all likelihood related to APFO administration. There were no effects on estradiol, estriol, thyroid stimulating hormone, total and free triiodothyronine, and total and free thyroxin. Estrone levels were notably lower for males given 2 and 20 mg/ kg/ day APFO. There was no evidence of peroxisome proliferation or cell proliferation in the liver, testes or pancreas of treated monkeys. No adverse effects were noted in either gross or clinical pathology studies.

In the 26 week study, male cynomolgus monkeys were administered APFO by oral capsule at doses of 0, 3, 10 or 30 mg/ kg/ day for 26 weeks ( Thomford, 2001b; Butenhoff et al., 2002). At study initiation the monkeys weighed 3.2 to 4.5 kg. There were 4 monkeys in the 3 mg/ kg/ day group and 6 monkeys in each of the other groups. Dosing of animals in the 30 mg/ kg/ day dose group was stopped from days 11   21 because of toxicity. When dosing was resumed on day 22, animals received 20 mg/ kg/ day and this group was designated the 30/ 20 mg/ kg/ day group. At the end of the 26 week treatment period, 2 animals in the control and 10 mg/ kg/ day groups were observed for a 13 week recovery period.

Animals were observed twice daily for mortality and moribundity and were examined at least once daily for signs of poor health or abnormal behavior; food consumption was assessed qualitatively. Ophthalmic examinations were done before initiation of treatment and during weeks 26 and 40. Body weight data were recorded weekly before the start of treatment, on day 1 of treatment and weekly thereafter. Blood and urine samples were collected for clinical hematology, clinical chemistry, and urinalysis before the start of treatment and on days 11, 31, 63, 91, 182, 217, 245 and 275. Blood samples were also taken for hormone determinations; samples were analyzed for estradiol, estrone, estriol, thyroid stimulating hormone, total and free triiodothyronine, total and free thyroxin, and testosterone. Blood, urine and feces were collected during week 2 and every 2 weeks thereafter during treatment and recovery for PFOA concentration analyses. The animals were not fasted before collections.

At scheduled necropsy, liver samples were taken for determination of PFOA levels. The right lateral lobe of the liver was collected from each animal for palmitoyl CoA oxidase activity analyses, and representative samples of liver, right and left testes, and pancreas were collected from each animal for cell proliferation evaluation using proliferation cell nuclear antigen. All available bile was collected for bile acid determination. The following organs were weighed at scheduled and unscheduled sacrifices; paired organs were weighed separately: adrenal ( 2), brain, epididymis ( 2), kidney ( 2), liver, pancreas, testis ( 2), and thyroid ( 2) with parathyroid. Organ to body weight percentages and organ to brain weight ratios were calculated. The following tissues 54 were collected for histopathology: adrenal ( 2), aorta, brain, cecum, colon, duodenum, epididymis ( 2), esophagus, eyes [ preserved in Davidson's fixative ( 2)], femur with bone marrow ( articular surface of the distal end), gallbladder, heart, ileum, jejunum, kidneys ( 2), lesions, liver, lung, mesenteric lymph node, mammary gland, pancreas, pituitary, prostate, rectum, salivary gland [ mandibular ( 2)], sciatic nerve, seminal vesicle ( 2), skeletal muscle ( thigh), skin, spinal cord ( cervical, thoracic, and lumbar), spleen, sternum with bone marrow, stomach, testis [( 2) preserved in Bouin's solution, thymus, thyroid ( 2) with parathyroid, trachea and urinary bladder.

Two animals, one male from the 30/ 20 mg/ kg/ day dose group and one male from the 3 mg/ kg/ day dose group, were sacrificed in moribund condition during the study. The male in the 30/ 20 mg/ kg/ day dose group was sacrificed on day 29. This animal exhibited signs of hypoactivity, weight loss, few or no feces, low or no food consumption and the entire body was cold to the touch before death. Necropsy revealed esophageal and gastric lesions that were indicative of an injury that occurred during dosing and liver lesions that were presumed to be treatment related. The animal from the 3 mg/ kg/ day dose group was sacrificed on day 137. This animal showed clinical signs of limited use and paralysis of the hind limbs, ataxia and hypoactive behavior, few feces and no food consumption. The cause of death was not determined, but APFO treatment could not be ruled out.

Males given 30 mg/ kg/ day from days 1 11 had clinical signs of few feces and low food consumption and they lost weight during week 1 of treatment. Based on these signs, treatment was stopped on day 11 and was not resumed until day 22. When treatment was resumed, the dose was lowered to 20 mg/ kg/ day; this group was then designated the 30/ 20 mg/ kg/ day group. Of the remaining animals in this group, only 2 tolerated this dose level for the remaining 23 weeks of treatment. Treatment of three males given 30/ 20 mg/ kg/ day was halted on days 43 ( week 7), 66 ( week 10), and 81 ( week 12) respectively. Clinical signs in these animals included thin appearance, few or no feces, low or no food consumption, and weight loss. The animals appeared to recover from compound related effects within 3 weeks after cessation of treatment.

Mean body weight changes were notably lower during weeks 1 and 2 for males receiving 30 mg/ kg/ day. During week 2, this change was statistically significant. Treatment was stopped on day 11 and when it was resumed at 20 mg/ kg/ day on day 21, mean body weight changes were significantly lower than controls during weeks 7, 9 and 24. Overall mean body weight changes through week 27 were notably lower for the males in the 30/ 20 mg/ kg/ day group. There was an increased incidence of low or no food consumption for animals in the 30/ 20 mg/ kg/ day group that was considered to be treatment related.

There were no consistent or clearly dose related effects on estrone, estradiol, estriol, thyroid stimulating hormone, or testosterone that were seen in any treatment group over time. In general, thyroid hormones were decreased beginning on day 35 in animals in the 10 or 30/ 20 mg/ kg/ day groups. Triiodothyronine remained depressed through day 183 in the 30/ 20 mg/ kg group. Total thyroxin was decreased beginning on day 35 in animals administered 10 or 30/ 20 mg/ kg APFO. The effect on thyroxin was most pronounced at day 35 in animals administered 10 mg/ kg/ day and day 66 in animals administered 30/ 20 mg/ kg/ day. Thereafter, the effect began 55 to diminish and recovery was observed either in the last 3 months of dosing or during the recovery phase of the experiment. No changes in cholecystokinin concentrations were seen over time in either dose group.

Two males from the 30/ 20 mg/ kg/ day group, the one sacrificed on day 29 and one for whom treatment was stopped because of poor health, had moderate to marked increased serum enzyme concentrations ( i. e. aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, and creatine kinase) and a mildly increased serum bile acid concentration. At 30/ 20 mg/ kg/ day, there were slight increases in triglyceride concentrations and mild to moderate decreases in absolute neutrophil count, total protein concentration and albumin concentration. Two animals in the 30/ 20 mg/ kg/ day group had markedly increased serum enzyme activities and mildly increased serum bile acid concentrations. At 3 mg/ kg/ day or 10 mg/ kg/ day, APFO had no effects on hematology, coagulation, clinical chemistry, or urinalysis. There was no evidence of persistent or delayed toxic effects on clinical pathology test results during the recovery period.

At terminal sacrifice at 26 weeks, there were statistically significant increases in absolute and relative liver weights in all dose groups. The absolute liver weights were increased by 35, 38 and 50% in the 3, 10 and 30/ 20 mg/ kg/ day groups, respectively; relative liver weights were increased by 20, 20 and 60% in the 3, 10 and 30/ 20 mg/ kg/ day groups, respectively. In addition, mean liver to brain weight was significantly increased in the 10 mg/ kg/ day group. The increased liver weights were considered treatment related. There were no treatment related macroscopic or microscopic changes in any organs at the terminal sacrifice, including liver, adrenal, spleen, pancreas, and testis. While there was no evidence of enhanced cell proliferation in the testes or pancreas of the treated monkeys, the findings in the liver were equivocal. There was evidence of mitochondrial proliferation in the livers of treated monkeys ( Butenhoff et al., 2002).

At the recovery sacrifice, there were no treatment related effects on terminal body weights or on absolute or relative organ weights at recovery sacrifice indicating that the liver weight changes seen at terminal sacrifice were reversible over time. There were no treatment related macroscopic or microscopic changes at the recovery sacrifice.

The results of the analyses of PFOA in the serum, liver, urine and feces are presented in Tables 5 8, respectively ( 3M Environmental Laboratory, 2001c). Low levels of PFOA were often detected in the sera, liver, urine and feces of the control animals. The levels in treated animals were significantly higher than those seen in the controls. In general, PFOA levels in the sera of test animals increased with dose but decreased over time during treatment. On day 9 of treatment, the serum levels were 126 ± 36.1 µ g/ mL in the 3 mg/ kg/ day group and 1597 ± 2392 µ g/ mL in the 30/ 20 mg/ kg/ day group, and during weeks 26/ 27, the serum levels were 52.5 ± 9.14 µ g/ mL in the 3 mg/ kg/ day group and 51.5 ± 77.6 µ g/ mL in the 30/ 20 mg/ kg/ day group. PFOA levels in serum decreased over time during recovery until they reached 1.18 ± 0.827 µ g/ mL in the 10 mg/ kg/ day group as compared to 0.0738 ± 0.00256 in the controls during week 40.

A similar trend was seen in the urine. On day 9, the levels in the urine were 73.5 ± 38.1, 221 ± 56 124, and 909 ± 209 µ g/ mL in the 3, 10 and 30/ 20 mg/ kg/ day groups, respectively, and during week 26, the levels were 51.6 ± 13.7, 109 ± 75.2, and 19.2 ± 27.0 µ g/ g, respectively, in these same groups. During week 2, the levels in the feces were less than the limit of quantitation in the control animals, and 7.43 ± 6.54, 15.4 ± 10.2 and 56.6 ± 73.7 ug/ g in the 3, 10, and 30/ 20 mg/ kg/ day groups, respectively; during week 26 the levels were 2.92 ± 1.35, 43.0 ± 36.9 and 10.3 ± 20.8 µ g/ g in the 3, 10, and 30/ 20 mg/ kg/ day groups, respectively. There is no explanation for the high levels of PFOA seen in the feces of the control animals during week 22. During the recovery period, PFOA levels in both urine and feces fell to levels that were comparable to control levels.

Under the conditions of the study, the LOAEL was 3 mg/ kg/ day ( liver toxicity and possibly mortality) and a NOAEL was not established. 57 Table 5 Average PFOA Concentrations in the Serum ( µ g/ mL) of Treated Animals 0 mg/ kg/ day 3 mg/ kg/ day 10 mg/ kg/ day 30/ 20 mg/ kg/ day Time Point Day 9 0.0613 ± 0.0472 126 ± 36.1 189 ± 48.9 1597 ± 2392 Week 4 0.0206 ± 0.0105 98.4 ± 42.7 172 ± 71.9 1084 ± 1839 Week 6 0.103 ± 0.0113 102 ± 33.6 95.8 ± 20.6 145 ± 21.6 Week 8 < LOQ 94.7 ± 27.3 97.6 ± 23.5 166 ± 98.9 Week 10 0.126 ± 0.0348 105 ± 37.7 93.6 ± 13.7 237 ± 158 Week 12 0.123 ± 0.0507 79.6 ± 25.9 90.6 ± 24.5 140 ± 77.5 Week 14 0.162 ± 0.0643 90.0 ± 28.9 92.2 ± 27.6 79.4 ± 28.3 Week 16 0.128 ± 0.0721 68.6 ± 26.0 98.5 ± 42.3 83.9 ± 58.5 Week 18 0.183 ± 0.0637 29.4 ± 25.4 17.4 ± 14.3 36.2 ± 21.2 Week 20 0.224 ± 0.0730 33.0 ± 26.0 96.6 ± 26.6 97.7 ± 129 Week 22 0.232 ± 0.131 77.6 ± 24.9 105 ± 36.3 58.6 ± 45.6 Week 24 < LOQ 72.2 ± 68.8 90.9 ± 21.2 62.7 ± 74.3 Week 26 0.209 ± 0.156 118 ± 27.5 77.4 ± 16.9 77.8 ± 126 Week 26/ 27 0.223 ± 0.105 52.5 ± 9.14 74.1 ± 33.1 51.5 ± 77.6 Week 28 0.181 ± 0.0391 NS 25.9 ± 7.07 NS Week 30 0.144 ± 0.0238 NS 11.3 ± 5.11 NS Week 32 0.110 ± 0.0216 NS 7.60 ± 3.90 NS Week 34 0.0861 ± 0.0256 NS 3.97 ± 2.09 NS Week 36 0.111 ± 0.0445 NS 2.75 ± 1.88 NS Week 38 0.0941 ± 0.0324 NS 1.84 ± 1.40 NS Week 40 0.0738 ± 0.00256 NS 1.18 ± 0.827 NS LOQ = Limit of Quantitation; NS = No Sample; Results are expressed as group averages ± the standard deviation associated with that group. Data are accurate to within one SD of the average fortified sample recovery. The average fortified sample recovery was 94% with an SD of 11%. 58 Table 6 Average PFOA Concentrations in the Liver ( µ g/ g) of Treated Animals 0 mg/ kg/ day 3 mg/ kg/ day 10 mg/ kg/ day 30/ 20 mg/ kg/ day Time Point Week 20 NS 18.3 NS NS Week 27 0.117 ± 0.0730 15.3 ± 3.02 14.0 ± 7.55 42.8 ± 63.3 Week 40 < LOQ NS 0.114 ± 0.0441 NS LOQ = Limit of Quantitation; NS = No Sample Results are expressed as group averages ± the standard deviation associated with that group. Data are accurate to within one SD of the average fortified sample recovery. The average fortified sample recovery was 90% with an SD of 26%. 59 Table 7. Average PFOA Concentrations in the Urine ( µ g/ mL) of Treated Animals 0 mg/ kg/ day 3 mg/ kg/ day 10 mg/ kg/ day 30/ 20 mg/ kg/ day Time Point Week 2 < LOQ 73.5 ± 38.1 221 ± 124 909 ± 269 Week 4 0.152 ± 0.337 54.9 ± 4.62 190 ± 91.6 240 ± 161 Week 6 0.0587 ± 0.0716 65.7 ± 46.9 128 ± 50.0 272 ± 140 Week 8 0.0161 ± 0.00940 47.6 ± 20.6 206 ± 73.1 180 ± 109 Week 10 0.0177 ± 0.00114 39.9 ± 18.7 175 ± 92.3 359 ± 449 Week 12 0.0141 ± 0.00648 48.8 ± 18.8 201 ± 92.7 118 ± 111 Week 14 7.96 ± 19.5 63.1 ± 47.2 139 ± 52.5 72.9 ± 84.0 Week 16 0.0299 ± 0.0339 50.2 ± 21.0 139 ± 57.0 50.2 ± 67.9 Week 18 0.0256 ± 0.0248 37.7 ± 19.3 186 ± 63.9 43.1 84.6 Week 20 0.0211 ± 0.0130 52.1 ± 9.63 144 ± 135 44.0 ± 59.9 Week 22 0.0231 ± 0.00688 95.8 ± 80.8 158 ± 78.4 98.5 ± 134 Week 24 0.0125 ± 0.00749 46.3 ± 8.52 157 ± 63.3 56.0 ± 77.2 Week 26 0.0268 ± 00.0265 51.6 ± 13.7 109 ± 75.2 19.2 ± 27.0 Week 28 0.118 ± 0.142 NS 0.327 ± 0.0182 NS Week 30 < LOQ NS 0.361 ± 0.118 NS Week 32 < LOQ NS 0.114 ± 0.0608 NS Week 34 < LOQ NS 0.121 ± 0.0305 NS Week 36 0.0117 ± 0.00841 NS 0.0502 ± 0.0166 NS Week 38 < LOQ NS 0.0284 ± 0.0102 NS Week 40 < LOQ NS 0.0254 ± 0.0101 NS LOQ = Limit of Quantitation; NS = No Sample; Results are expressed as group averages ± the standard deviation associated with that group. Data are accurate to within one SD of the average fortified sample recovery. The average fortified sample recovery was 88% with an SD of 17%. 60 Table 8 Average PFOA Concentrations in the Feces ( µ g/ g) of Treated Animals 0 mg/ kg/ day 3 mg/ kg/ day 10 mg/ kg/ day 30/ 20 mg/ kg/ day Time Point Week 2 < LOQ 7.43 ± 6.54 15.4 ± 10.2 56.6 ± 73.7 Week 4 0.0214 ± 0.0178 10.4 ± 12.0 23.4 ± 10.6 22.0 ± 6.23 Week 6 0.108 ± 0.00192 12.1 ± 14.1 23.3 ± 8.46 101 ± 86.7 Week 8 0.0782 ± 0.103 9.46 ± 9.21 41.0 ± 25.0 36.7 ± 34.2 Week 10 < LOQ 3.96 ± 3.68 26.0 ± 17.4 48.0 ± 34.0 Week 12 0.0498 ± 0.0894 7.15 ± 5.65 10.3 ± 6.07 32.0 ± 42.9 Week 14 0.139 ± 0.308 7.50 ± 2.43 27.2 ± 29.4 19.2 ± 25.2 Week 16 0.0572 ± 0.0762 6.88 ± 2.62 31.4 ± 23.3 18.2 ± 28.8 Week 18 0.258 ± 0.654 5.72 ± 7.15 17.3 ± 13.3 22.1 ± 31.7 Week 20 0.405 ± 1.08 6.81 ± 4.89 52.4 ± 39.5 37.8 ± 58.1 Week 22 15.5 ± 36.9 13.8 ± 5.22 39.5 ± 21.0 25.2 ± 36.0 Week 24 0.517 ± 1.13 6.22 ± 5.45 40.5 ± 21.8 34.6 ± 47.7 Week 26 0.0172 ± 0.00892 2.92 ± 1.35 43.0 ± 36.9 10.3 ± 20.8 Weeks 28 34 0.279 ± 0.732 NS 0.387 ± 0.372 NS Weeks 36 40 0.0103 ± 0.00684 NS 0.0336 ± 0.0313 NS LOQ = Limit of Quantitation; NS = No Sample Results are expressed as group averages ± the standard deviation associated with that group. Data are accurate to within one SD of the average fortified sample recovery. The average fortified sample recovery was 117% with an SD of 22%.

3.6 Developmental Toxicity Studies in Animals Three prenatal developmental toxicity studies of APFO have been conducted, one inhalation and two oral studies.

The first of these studies was an oral developmental toxicity study in rats ( Gortner, 1981). Based on the results of a range finding study, an upper dose level of 150 mg/ kg/ day was set for the definitive study in which five groups of 22 time mated Sprague Dawley rats were administered 0, 0.05, 1.5, 5, and 150 mg/ kg/ day APFO in distilled water by gavage on gestation days ( GD) 6 15. Doses were adjusted according to body weight. Dams were monitored on GD 3 20 for clinical signs of toxicity. Individual body weights were recorded on GD 3, 6, 9, 12, 15, and 20. 61 Animals were sacrificed on GD 20 by cervical dislocation and the ovaries, uteri, and contents were examined for the number of corpora lutea, number of viable and non viable fetuses, number of resorption sites, and number of implantation sites. Fetuses were weighed and sexed and subjected to external gross necropsy. Approximately one third of the fetuses were fixed in Bouin's solution and examined for visceral abnormalities by free hand sectioning. The remaining fetuses were subjected to skeletal examination using alizarin red.

Signs of maternal toxicity consisted of statistically significant reductions in mean maternal body weights on GD 9, 12, and 15 at the high dose group of 150 mg/ kg/ day. Mean maternal body weight on GD 20 continued to remain lower than controls, although the difference was not statistically significant. Other signs of maternal toxicity that occurred only at the high dose group included ataxia and death in three rat dams. No other effects were reported. Administration of APFO during gestation did not appear to affect the ovaries or reproductive tract of the dams. Under the conditions of the study, a NOAEL of 5 mg/ kg/ day and a LOAEL of 150 mg/ kg/ day for maternal toxicity were indicated.

A significantly higher incidence in fetuses with one missing sternebrae was observed at the highdose group of 150 mg/ kg/ day; however this skeletal variation also occurred in the controls and the other three dose groups ( at similar incidence but lower than the high dose group) and therefore was not considered to be treatment related. No significant differences between treated and control groups were noted for other developmental parameters that included the mean number of males and females, total and dead fetuses, the mean number of resorption sites, implantation sites, corpora lutea and mean fetus weights. Likewise, a fetal lens finding initially described as a variety of abnormal morphological changes localized to the area of the embryonal nucleus, was later determined to be an artifact of the free hand sectioning technique and therefore not considered to be treatment related. Under the conditions of the study, a NOAEL for developmental toxicity of 150 mg/ kg/ day ( highest dose group) was indicated.

A second oral prenatal developmental toxicity study was conducted in rabbits ( Gortner, 1982). Based on the results of a range finding study, an upper dose level of 50 mg/ kg/ day was set for the definitive study in which four groups of 18 pregnant New Zealand White rabbits were administered 0, 1.5, 5, and 50 mg/ kg/ day APFO in distilled water by gavage on gestation days ( GD) 6 18. Pregnancy was established in each sexually mature female by i. v. injection of pituitary lutenizing hormone in order to induce ovulation, followed by artificial insemination with 0.5 ml of pooled semen collected from male rabbits; the day of insemination was designated as day 0 of gestation. A constant dose volume of 1 ml/ kg was administered. Individual body weights were measured on GD 3, 6, 9, 12, 15, 18, and 29. The does were observed daily on GD 3 29 for abnormal clinical signs. On GD 29, the does were euthanized and the ovaries, uterus and contents examined for the number of corpora lutea, live and dead fetuses, resorptions and implantation sites. Fetuses were examined for gross abnormalities and placed in a 370 C incubator for a 24 hour survival check. Pups were subsequently euthanized and examined for visceral and skeletal abnormalities. A blood sample was taken from six does prior to dosing and then on GD 18 and 29; a liver sample was taken from the same animals on GD 29. All samples were sent to the sponsor for analysis. This information was unavailable at the time of this 62 review.

Signs of maternal toxicity consisted of statistically significant transient reductions in body weight gain on GD 6 9 when compared to controls; body weight gains returned to control levels on GD 12 29. Administration of APFO during gestation did not appear to affect the ovaries or reproductive tract contents of the does. No clinical or other treatment related signs were reported. Under the conditions of the study, a NOAEL of 50 mg/ kg/ day, the highest dose tested, for maternal toxicity was indicated.

No significant differences were noted between controls and treated groups for the number of males and females, dead or live fetuses, and fetal weights. Likewise, there were no significant differences reported for the number of resorption and implantation sites, corpora lutea, the conception incidence, abortion rate, or the 24 hour mortality incidence of the fetuses. Gross necropsy and skeletal/ visceral examinations were unremarkable. The only sign of developmental toxicity consisted of a dose related increase in a skeletal variation, extra ribs or 13th rib, with statistical significance at the high dose group ( 38% at 50 mg/ kg/ day, 30% at 5 mg/ kg/ day, 20% at 1.5 mg/ kg/ day, and 16 % at 0 mg/ kg/ day). A statistically significant increase in 13th ribs spurred occurred in the mid dose group of 5 mg/ kg/ day; however, the biological significance of this effect is uncertain since in both the high and low dose groups, this effect occurred at the same rate and was not statistically significantly different from controls. Therefore, under the conditions of the study, a LOAEL for developmental toxicity of 50 mg/ kg/ day ( highest dose group) was indicated.

Staples et al. ( 1984) also conducted a developmental toxicity study of APFO. The study design consisted of an inhalation and an oral portion, each with two trials or experiments. The first trial was the teratology portion of the study, in which the dams were sacrificed on GD 21; while in the second trial, the dams were allowed to litter and the pups were sacrificed on day 35 post partum. For the inhalation portion of the study, the two trials consisted of 12 pregnant Sprague Dawley rats per group exposed to 0, 0.1, 1, 10, and 25 mg/ m3 APFO for 6 hours/ day, on GD 6 15. In the oral portion of the study, 25 and 12 Sprague Dawley rats for the first and second trials, respectively, were administered 0 and 100 mg/ kg/ day APFO in corn oil by gavage on GD 6 15. For both routes of administration, females were mated on an as needed basis and when the number of mated females was bred, they were ranked within breeding days by body weight and assigned to groups by rotation in order of rank. Finally, two additional groups ( six dams per group) were added to each trial that was pair fed to the 10 and 25 mg/ m3 groups.

For the teratology portion of the study ( trial one), dams were weighed on GD 1, 6, 9, 13, 16, and 21 and observed daily for abnormal clinical signs. On GD 21, the dams were sacrificed by cervical dislocation and examined for any gross abnormalities, liver weights were recorded and the reproductive status of each animal was evaluated. The ovaries, uterus and contents were examined for the number of corpora lutea, live and dead fetuses, resorptions and implantation sites. Pups ( live and dead) were counted, weighed and sexed and examined for external, visceral, and skeletal alterations. The heads of all control and high dosed group fetuses were examined for visceral alterations as well as macro and microscopic evaluation of the eyes. 63 For trial two, in which the dams were allowed to litter, the procedure was the same as that for trial one up to GD 21. Two days before the expected day of parturition, each dam was housed in an individual cage. The date of parturition was noted and designated Day 1 PP. Dams were weighed and examined for clinical signs on Days 1, 7, 14, and 22 PP. On Day 23 PP all dams were sacrificed. Pups were counted, weighed, and examined for external alterations. Each pup was subsequently weighed and inspected for adverse clinical signs on Days 4, 7, 14, and 22 PP. The eyes of the pups were also examined on Days 15 and 17 PP for the inhalation portion and on Days 27 and 31 PP for the gavage portion of the study. Pups were sacrificed on Day 35 PP and examined for visceral and skeletal alterations.

Inhalation Exposure Trial One:

Treatment related clinical signs of maternal toxicity for trial one ( teratology) occurred at 10 and 25 mg/ m3 and consisted of wet abdomens, chromodacryorrhea, chromorhinorrhea, a general unkempt appearance, and lethargy in four dams at the end of the exposure period ( highconcentration group only). Three out of 12 dams died during treatment at 25 mg/ m3 ( on GD 12, 13, and 17). Food consumption was significantly reduced at both 10 and 25 mg/ m3; however, no significant differences were noted between treated and pair fed groups. Significant reductions in body weight were also observed at these concentrations, with statistical significance at the highconcentration only. Likewise, statistically significant increases in mean liver weights were seen at the high concentration group. Under the conditions of the study, a NOAEL and LOAEL for maternal toxicity of 1 and 10 mg/ m3, respectively, were indicated.

No effects were observed on the maintenance of pregnancy or the incidence of resorptions. Mean fetal body weights were significantly decreased in the 25 mg/ m3 groups and in the control group pair fed 25 mg/ m3. A detailed microscopic visceral and eye examination of the fetuses did not reveal any treatment related effects; however in the control group that was pair fed 25 mg/ m3, a statistically significant increased incidence of fetuses with partially ossified sternebrae was observed. Under the conditions of the study, a NOAEL and LOAEL for developmental toxicity of 10 and 25 mg/ m3, respectively, were indicated.

Trial Two:

Clinical signs of maternal toxicity seen at 10 and 25 mg/ m3 were similar in type and incidence to those described for trial one. Maternal body weight gain during treatment at 25 mg/ m3 was less than controls, although the difference was not statistically significant. In addition, 2 out of 12 dams died during treatment at 25 mg/ m3. No other treatment related effects were reported, nor were any adverse effects noted for any of the measurements of reproductive performance. Under the conditions of the study, a NOAEL and LOAEL for maternal toxicity of 1 and 10 mg/ m3, respectively, were indicated. 64 Signs of developmental toxicity in this group consisted of statistically significant reductions in pup body weight on Day 1 PP ( 6.1 g at 25 mg/ m3 vs. 6.8 g in controls). On Days 4 and 22 PP, pup body weights continued to remain lower than controls, although the difference was not statistically significant ( Day 4 PP: 9.7 g at 25 mg/ m3 vs. 10.3 in controls; Day 22 PP: 49.0 g at 25 mg/ m3 vs. 50.1 in controls). No significant effects were reported following external examination of the pups or with ophthalmoscopic examination of the eyes. Under the conditions of the study, a NOAEL and LOAEL for developmental toxicity of 10 and 25 mg/ m3, respectively, were indicated.

Oral Exposure Trial One:

Three out of 25 dams died during treatment of 100 mg/ kg APFO during gestation ( one death on GD 11; two on GD 12). Clinical signs of maternal toxicity in the dams that died were similar to those seen with inhalation exposure. Food consumption and body weights were reduced in treated animals compared to controls. No adverse signs of toxicity were noted for any of the reproductive parameters such as maintenance of pregnancy or incidence of resorptions. Likewise, no significant differences between treated and control groups were noted for fetal weights, or in the incidences of malformations and variations; nor were there any effects noted following microscopic examination of the eyes.

Trial Two:

Similar observations for clinical signs were noted for the dams as in trial one. Likewise, no adverse effects on reproductive performance or in any of the fetal observations were noted.

3.7 Reproductive Toxicity Studies in Animals York ( 2002) conducted a two generation reproductive toxicity study of APFO. Five groups of 30 Sprague Dawley rats per sex per dose group were administered APFO by gavage at doses of 0, 1, 3, 10, and 30 mg/ kg/ day six weeks prior to and during mating. Treatment of the F0 male rats continued until mating was confirmed, and treatment of the F0 female rats continued throughout gestation, parturition, and lactation.

The F0 animals were examined twice daily for clinical signs, abortions, premature deliveries, and deaths. Body weights of F0 male rats were recorded weekly during the dosage period and then on the day of sacrifice. Body weights of F0 female rats were recorded weekly during the pre and cohabitation periods and then on gestation days ( GD) 0, 7, 10, 14, 18, 21, and 25 ( if necessary) and on lactation days ( LD) 1, 5, 8, 11, 15, and 22 ( terminal body weight). Food consumption values in F0 male rats were recorded weekly during the treatment period, while in F0 female rats, values were recorded weekly during the precohabitation period, on GDs 0, 7, 10, 14, 18, 21, and 25 and on LDs 1, 5, 8, 11, and 15. 65 Estrous cycling was evaluated daily by examination of vaginal cytology beginning 21 days before the scheduled cohabitation period and continuing until confirmation of mating by the presence of sperm in a vaginal smear or confirmation of a copulatory plug. On the day of scheduled sacrifice, the stage of the estrous cycle was assessed.

For mating, one male rat and one female rat per group were cohabitated for a maximum of 14 days. Female rats with evidence of sperm in a vaginal smear or copulatory plug were designated as GD 0 and assigned to individual housing. Parental females were evaluated for length of gestation, fertility index, gestation index, number and sex of offspring per litter, number of implantation sites, general condition of the dam and litter during the postpartum period, litter size and viability, viability index, lactation index, percent survival, and sex ratio. Maternal behavior of the dams was recorded on LDs 1, 5, 8, 15, and 22.

F0 generation animals were sacrificed by carbon dioxide asphyxiation ( day 106 to 110 of the study for male rats, i. e., after completion of the cohabitation period; and LD 22 for female rats), necropsied, and examined for gross lesions. Gross necropsy included examination of external surfaces and orifices, as well as internal examination of tissues and organs. Individual organs were weighed and organ to body weight and organ to brain weight ratios were calculated for the brain, kidneys, spleen, ovaries, testes, thymus, liver, adrenal glands, pituitary, uterus with oviducts and cervix, left epididymis ( whole and cauda), right epididymis, prostate and seminal vesicles, ( with coagulating glands and with and without fluid). Tissues retained in neutral buffered 10% formalin for possible histological evaluation included the pituitary, adrenal glands, vagina, uterus, with oviducts, cervix and ovaries, right testis, seminal vesicles, right epididymis, and prostate. Histological examination was performed on tissues from 10 randomly selected rats per sex from the control and high dosage groups. All gross lesions were examined histologically. All F0 generation rats that died or appeared moribund were also examined.

Histological examination of the reproductive organs in the low and mid dose groups was conducted in rats that exhibited reduced fertility by either failing to mate, conceive, sire, or deliver healthy offspring; or for which estrous cyclicity or sperm number, motility, or morphology were altered. Sperm number, motility, and morphology were evaluated in the left cauda epididymis of F0 generation male rats; testicular spermatid concentrations were evaluated in the left testis. The number and distribution of implantation sites were recorded in F0 generation female rats. Rats that did not deliver a litter were sacrificed on GD 25 and examined for pregnancy status. Uteri of apparently nonpregnant rats were examined to confirm the absence of implantation sites. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Female rats without a confirmed mating date that did not deliver a litter were sacrificed on an estimated day 25 of gestation.

At scheduled sacrifice, after completion of the cohabitation period in F0 male rats and on LD 22 in F0 female rats, blood samples ( 10 males and 10 females each for the 10 and 30 mg/ kg/ day dose groups; 3 males and 3 females for the control group) were collected and frozen for future analysis. The methods section cites that liver samples were also collected, but no other details were provided and the results did not appear to be available at the time of the report. 66 The F1 generation pups in each litter were counted once daily. Physical signs ( including variations from expected lactation behavior and gross external physical anomalies) were recorded for the pups each day. Pup body weights were recorded on LDs 1, 5, 8, 15 and 22. On LD 12, all F1 generation male pups were examined for the presence of nipples. Pups that died before examination of the litter for pup viability on LD 1 were evaluated for vital status at birth. Pups found dead on LDs 2 to 22 were examined for gross lesions and for the cause of death. All F1 generation rats were weaned on LD 22 based on observed growth and viability of these pups.

At weaning ( LD 22), two F1 generation pups per sex per litter per group ( 60 male and 60 female pups per group) were selected for continued evaluation, resulting in 600 total rats ( 300 rats per sex) assigned to the five dosage groups. At least two male pups and two female pups per litter, when possible, were selected. F1 generation pups not selected for continued observation for sexual maturation were sacrificed. Three pups per sex per litter were examined for gross lesions. Necropsy included a single cross section of the head at the level of the frontal parietal suture and examination of the cross sectioned brain for apparent hydrocephaly. The brain, spleen and thymus from one of the three selected pups per sex per litter were weighed and the brain, spleen, and thymus from the three selected pups per sex per litter were retained for possible histological evaluation. All remaining pups were discarded without further examination.

The F1 generation rats were given the same dosage level of the test substance and in the same manner as their respective F0 generation sires and dams. Dosages were given once daily, beginning at weaning and continuing until the day before sacrifice. F1 generation female rats were examined for age of vaginal patency, beginning on day 28 postpartum ( LD 28). F1 generation male rats were evaluated for age of preputial separation, beginning on day 39 postpartum ( LD 39). Body weights were recorded when rats reached sexual maturation.

Estrous cycling was evaluated daily by examination of vaginal cytology beginning 21 days before the scheduled cohabitation period and continuing until confirmation of mating by the presence of sperm in a vaginal smear or confirmation of a copulatory plug. On the day of scheduled sacrifice, the stage of the estrous cycle was assessed.

A table of random units was used to assign F1 generation rats to cohabitation, one male rat per female rat. If random assignment to cohabitation resulted in the pairing of F1 generation siblings, an alternate assignment was made. The cohabitation period consisted of a maximum of 14 days.

Body weights of the F1 generation male rats were recorded weekly during the postweaning period and on the day of sacrifice. Body weights of the F1 generation female rats were recorded weekly during the postweaning period to cohabitation, and on DGs 0, 7, 10, 14, 18, 21 and 25 ( if necessary) and on LDs 1, 5, 8, 11, 15 and 22. Food consumption values for the F1 generation male rats were recorded weekly during the dosage period. Food consumption values for the F1 generation female rats were recorded weekly during the postweaning period to cohabitation, on GDs 0, 7, 10, 14, 18, 21 and 25 and on LDs 1, 5, 8, 11 and 15. Because pups begin to consume 67 maternal food on or about LD 15, food consumption values were not tabulated after LD 15.

At scheduled sacrifice, the F1 animals were subjected to gross necropsy, and selected organs were weighed and examined histologically as described above for the F0 animals. Sperm analyses were also conducted as described for the F0 animals.

F2 generation litters were examined after delivery to identify the number and sex of pups, stillbirths, live births and gross alterations. Each litter was evaluated for viability at least twice each day of the 22 day postpartum period. Dead pups observed at these times were removed from the nesting box. Anogenital distance was measured for all live F2 generation pups on LDs 1 and 22.

Parental Males ( F0)

One F0 male rat in the 30 mg/ kg/ day dose group was sacrificed on day 45 of the study due to adverse clinical signs ( emaciation, cold to touch, and decreased motor activity). Necroscopic examination in that animal revealed a pale and tan liver, and red testes. All other F0 generation male rats survived to scheduled sacrifice. Statistically significant increases in clinical signs were also observed in male rats in the high dose group that included dehydration, urine stained abdominal fur, and ungroomed coat.

Significant reductions in body weight and body weight gain were reported for most of the dosage period and continuing until termination of the study in the 3, 10, and 30 mg/ kg/ day dose groups. Absolute food consumption values were also significantly reduced during these periods at the 30 mg/ kg/ day dose group, while significant increases in relative food consumption values were observed in the 3, 10, and 30 mg/ kg/ day within those same periods.

No treatment related effects were reported at any dose level for any of the mating and fertility parameters assessed, including numbers of days to inseminate, numbers of rats that mated, fertility index, numbers of rats with confirmed mating dates during the first and second week of cohabitation, and numbers of pregnant rats per rats in cohabitation. At necropsy, none of the sperm parameters evaluated ( sperm number, motility, or morphology) were affected by treatment at any dose level.

At necropsy, statistically significant reductions in terminal body weights were seen at 3, 10, and 30 mg/ kg/ day. Absolute weights of the left and right epididymides, left cauda epididymis, seminal vesicles ( with and without fluid), prostate, pituitary, left and right adrenals, spleen, and thymus were also significantly reduced at 30 mg/ kg/ day. The absolute weight of the seminal vesicles without fluid was significantly reduced in the 10 mg/ kg/ day dose group. The absolute weight of the liver was significantly increased in all dose groups. Kidney weights were significantly increased in the 1, 3, and 10 mg/ kg/ day dose groups, but significantly decreased in the 30 mg/ kg/ day group. All organ weight to terminal body weight and ratios were significantly increased in all treated groups. Organ weight to brain weight ratios were significantly reduced for some organs at the high dose group, and significantly increased for other organs among all 68 treated groups.

No treatment related effects were seen at necropsy or upon microscopic examination of the reproductive organs, with the exception of increased thickness and prominence of the zona glomerulosa and vacuolation of the cells of the adrenal cortex in the 10 and 30 mg/ kg/ day dose groups.

Serum analysis for the F0 generation males sampled at the end of cohabitation showed that PFOA was present in all samples tested, including controls. Control males had an average concentration of 0.0344+ 0.0148 ug/ ml PFOA. Treated males had 51.1+ 9.30 and 45.3+ 12.6 ug/ ml, respectively for the 10 and 30 mg/ kg/ day dose groups.

Parental Females ( F0)

No treatment related deaths or adverse clinical signs were reported in parental females at any dose level. No treatment related effects were reported for body weights, body weight gains, and absolute and relative food consumption values.

There were no treatment related effects on estrous cyclicity, mating or fertility parameters. None of the natural delivery and litter observations were affected by treatment, that is, the numbers of dams delivering litters, the duration of gestation, the averages for implantation sites per delivered litter, the gestation index ( number of dams with one or more liveborn pups/ number of pregnant rats), the numbers of dams with stillborn pups, dams with all pups dying, liveborn and stillborn pups viability index, pup sex ratios, and mean birth weights were comparable to controls among all treated groups.

Necropsy and histopathological evaluation were also unremarkable. Terminal body weights, organ weights, and organ to terminal body weight ratios were comparable to control values for all treated groups, except for kidney and liver weights. The weights of the left and right kidney, and the ratios of these organ weights to terminal body weights, and of the left kidney weight tobrain weight were significantly reduced at the highest dose of 30 mg/ kg/ day. The ratio of liver weights to terminal body weight was also significantly reduced at 3 and 10 mg/ kg/ day.

Results of the serum analysis in F0 generation females sampled on LD 22 showed that PFOA was present in all samples tested, except in controls where the level was below the limits of quantitation ( 0.00528ugm/ l). Treated females had an average concentration of 0.37+ 0.0805 and 1.02+ 0.425 ug/ ml, respectively for the 10 and 30 mg/ kg/ day dose groups.

F1 Generation  Males No effects were reported at any dose level for the viability and lactation indices. No differences between treated and control groups were noted for the numbers of pups surviving per litter, the percentage of male pups, litter size and average pup body weight per litter at birth. Pup body weight on a per litter basis ( sexes combined) was significantly reduced in the 30 mg/ kg/ day 69 group on days 1, 5, and 8 of lactation. Of the pups necropsied at weaning, no statistically significant, treatment related differences were observed for the weights of the brain, spleen and thymus and the ratios of these organ weights to the terminal body weight and brain weight.

Significant increases in treatment related deaths ( 7 animals total) were reported in F1 males in the high dose group of 30 mg/ kg/ day. One rat was moribund sacrificed on day 39 postweaning and another was found dead on day 107 postweaning, but the majority of the F1 male rats were found dead on days 2 4 postweaning.

Statistically significant increases in clinical signs of toxicity were also observed in F1 males during most of entire postweaning period. These signs included an increased incidence of annular constriction of the tail at all doses, with statistical significance at the 1, 10, and 30 mg/ kg/ day; a significant increase at 10 and 30 mg/ kg/ day in the number of male rats that were emaciated; and a significant increase in the incidence of urine stained abdominal fur, decreased motor activity, and abdominal distention at 30 mg/ kg/ day.

Body weights and body weight gains were statistically significantly reduced prior to and during cohabitation and during the entire dosing period in all treated groups. Statistically significant reductions in body weights were observed at 10 and 30 mg/ kg/ day during days 8 15, 22 29, 29 36, 43 50, and 50 57 postweaning. Body weight gains were also significantly reduced in the 30 mg/ kg/ day group on days 1 8, 15 22, 36 43, 57 64, and 64 70 postweaning. Statistically significant, dose related reductions in body weight gains were observed for the entire dosage period ( days 1 113 postweaning). Absolute food consumption values were significantly reduced at 10 and 30 mg/ kg/ day during the entire precohabitation period ( days 1 70 postweaning), while relative food consumption values were significantly increased.

Statistically significant ( p< 0.01) delays in sexual maturation ( the average day of preputial separation) were observed in high dose animals versus concurrent controls ( 52.2 days of age versus 48.5 days of age, respectively).

No apparent effects were observed on any of the mating or fertility parameters including fertility and pregnancy indices ( number of pregnancies per number of rats that mated and rats in cohabitation, respectively), the number of days to inseminate, the number of rats that mated, and the number of rats with confirmed mating dates during the first week. No statistically significant, treatment related effects were observed on any of the sperm parameters ( motility, concentration, or morphology).

Necroscopic examination revealed statistically significant treatment related effects at 3, 10, and 30 mg/ kg/ day ranging from tan areas in the lateral and median lobes of the liver to moderate to slight dilation of the pelvis of one or both kidneys.

Statistically significant, dose related decreases in terminal body weights of parental F1 males were observed. The absolute weights of the liver were significantly increased and the absolute weights of the spleen were significantly decreased at all treated groups. The absolute weights of 70 the left and/ or right kidneys were significantly increased in the 1 and 3 mg/ kg/ day dose groups and significantly decreased in the 30 mg/ kg/ day dose group. The absolute weight of the thymus was also significantly decreased in the 10 and 30 mg/ kg/ day dose groups. The absolute weight of the prostate, brain and left adrenal gland were significantly decreased in the 30 mg/ kg/ day dosage group. The ratios of the weights of the seminal vesicles, with and without fluid, liver and left and right kidneys to the terminal body weights were significantly increased in all treated groups. The ratios of the weights of the left testis, with and without the tunica albuginea and the right testis to the terminal body weight, were significantly increased at 3 mg/ kg/ day and higher. The ratios of the weights of the left epididymis, left cauda epididymis, right epididymis and brain to the terminal body weight were significantly increased at 10 mg/ kg/ day and higher. The ratios of the weight of the seminal vesicles with fluid to the brain weight were increased at 1 mg/ kg/ day and higher, with statistical significance at 1 and 10 mg/ kg/ day. The ratios of the liver weight to brain weight were significantly increased in the 1 mg/ kg/ day and higher dosage groups, and the ratios of the left and right kidney weights to brain weight were significantly increased in all treated groups. The ratios of the spleen weight to brain weight were significantly decreased at 1 mg/ kg/ day and higher, and the ratios of the thymus weight to brain weight were significantly decreased at 10 and 30 mg/ kg/ day. The ratios of the left and right testes weight tobrain weight were increased in the 3 mg/ kg/ day and higher dosage groups. These ratios were significantly increased at 10 mg/ kg/ day ( right testis only) and 30 mg/ kg/ day.

Histopathologic examination of the reproductive organs was unremarkable; however, treatmentrelated microscopic changes were observed in the adrenal glands of high dose animals ( cytoplasmic hypertrophy and vacuolation of the cells of the adrenal cortex) and in the liver of animals treated with 3, 10, and 30 mg/ kg/ day ( hepatocellular hypertrophy). No other treatmentrelated effects were reported.

F1 Generation   Females No effects were reported at any dose level for the viability and lactation indices. No differences between treated and control groups were noted for the numbers of pups surviving per litter, the percentage male pups, litter size and average pup body weight per litter at birth. Pup body weight on a per litter basis ( sexes combined) was significantly reduced in the 30 mg/ kg/ day group on days 1, 5, and 8 of lactation.

At 30 mg/ kg/ day, one pup from one dam died prior to weaning on lactation day 1 ( LD1). Additionally, on lactation days 6 and 8, statistically significant increases in the numbers of pups found dead were observed at 3 and 30 mg/ kg/ day. According to the study authors, this was not considered to be treatment related because they did not occur in a dose related manner and did not appear to affect any other measures of pup viability that included, numbers of surviving pups per litter and live litter size at weighing. An independent statistical analysis was conducted by US EPA ( 2002b). No significant differences were noted between dose groups and there was no significant trend. 71 Of the pups necropsied at weaning, no statistically significant, treatment related differences were observed for the weights of the brain, spleen and thymus and the ratios of these organ weights to the terminal body weight and brain weight.

An increase in treatment related mortality ( 6 animals total) was observed in F1 females on postweaning days 2 8 at the highest dose of 30 mg/ kg/ day. No adverse clinical signs of treatment related toxicity were reported for any dose level during any time of the study period.

Statistically significant decreases in body weights and body weight gains were observed in highdose animals on days 8, 15, 22, 29, 50, and 57 postweaning, during precohabitation ( recorded on the day cohabitation began, when F1 generation rats were 92 106 days of age), and during gestation and lactation. Decreases in absolute food consumption were observed during days 1 8, 8 15 postweaning during precohabitation and during gestation and lactation in animals treated with 30 mg/ kg/ day. Relative food consumption values were comparable across all treated groups.

Statistically significant ( p< 0.01) delays in sexual maturation ( the average day of vaginal patency) were observed in high dose animals versus concurrent controls ( 36.6 days of age versus 34.9 days of age, respectively).

Prior to mating, the study authors noted a statistically significant increase in the average numbers of estrous stages per 21 days in high dose animals ( 5.4 versus 4.7 in controls). For this calculation, the number of independent occurrences of estrus in the 21 days of observation was determined. This type of calculation can be used as a screen for effects on the estrous cycle, but a more detailed analysis should then be conducted to determine whether there is truly an effect. 3M Company ( 2002) recently completed an analysis that showed there were no effects on the estrous cycle; there were no differences in the number of females with > 3 days of estrus or with > 4 days of diestrus in the control and high dose groups. Analyses conducted by the US EPA ( 2002a) also demonstrated that there were no differences in the estrous cycle among the control and high dose groups. The cycles were evaluated as having either regular 4 5 day cycles, uneven cycling ( defined as brief periods with irregular pattern) or periods of prolonged diestrus ( defined as 4 6 day diestrus periods) extended estrus ( defined as 3 or 4 days of cornified smears), possibly pseudopregnant, ( defined as 6 greater days of leukocytes) or persistent estrus ( defined as 5 or greater days of cornified smears). The two groups were not different in any of the parameters measured. Thus, the increase in the number of estrous stages per 21 days that was noted by the study authors is due to the way in which the calculation was done, and is not biologically meaningful.

No effects on any of the mating and fertility parameters ( numbers of days in cohabitation, numbers of rats that mated, fertility index, rats with confirmed mating dates during the first week of cohabitation and number of rats pregnant per rats in cohabitation).

All natural delivery observations were unaffected by treatment at any dose level. Numbers of dams delivering litters, the duration of gestation, averages for implantation sites per delivered 72 litter, the gestation index ( number of dams with one or more liveborn pups/ number of pregnant rats), the numbers of dams with stillborn pups, dams with all pups dying and liveborn and stillborn pups were comparable among treated and control groups.

No treatment related effects were observed on terminal body weights. The absolute weight of the pituitary and the ratios of the pituitary weight to terminal body weight and to the brain weight were significantly decreased at 3 mg/ kg/ day and higher, but did not show a doseresponse No other differences were reported for the absolute weights or ratios for other organs evaluated. No treatment related effects were reported following necroscopic and histopathologic examinations.

F2 Generation Offspring No treatment related adverse clinical signs were observed at any dose level. Likewise, no treatment related effects were reported following necroscopic examination, with the exception of no milk in stomach in pups that were found dead. The numbers of pups found either dead or stillborn did not show a dose response ( 3/ 28, 6/ 28, 10/ 28, 10/ 28, and 6/ 28 in 0, 1, 3, 10, and 30 mg/ kg/ day dose groups, respectively) and therefore were unlikely related to treatment.

No effects were reported at any dose level for the viability and lactation indices. No differences between treated and control groups were noted for the numbers of pups surviving per litter, the percentage of male pups, litter size and average pup body weight per litter when measured on LDs 1, 5, 8, 15, or 22. Anogenital distances measured for F2 male and female pups on LDs 1 and 22 were also comparable among the five dosage groups and did not differ significantly.

Statistically significant increases ( p< 0.01) in the number of pups found dead were observed on lactation day 1 at the 3 and 10 mg/ kg/ day dosage groups. According to the study authors, this was not considered to be treatment related because they did not occur in a dose related manner and did not appear to affect any other measures of pup viability that included, numbers of surviving pups per litter and live litter size at weighing. An independent statistical analysis was conducted by US EPA ( 2002b). No significant differences were observed between dose groups and there was no significant trend.

Terminal body weights in F2 pups were not significantly different from controls. Absolute weights of the brain, spleen and thymus and the ratios of these organ weights to terminal body weight and to brain weight were also comparable among treated and control groups.

Conclusions Dosing with APFO at 30 mg/ kg/ day appeared to delay the onset of sexual maturation in both male and female F1 offspring. The authors of the study contend that the delays in sexual maturation ( preputial separation or vaginal patency) observed in high dose animals are due to the fact that these animals have a decreased gestational age, a variable which they have defined as the time in days from evidence of mating in the F0 generation until evidence of sexual 73 maturation in the F1 generation. The authors state that gestational age appeared to be decreased in high dose animals at the time of acquisition ( the time when sexual maturation was reached), which they believe meant the animals in that group were younger and more immature than the control group, in which there was no significant difference in sexual maturation.

In order to test this hypothesis, the authors covaried separately the decreases in body weight and in gestational age with the delays in sexual maturation in order to determine whether or not body weights and gestational age were a contributing factor. When the body weight was covaried with the time to sexual maturation, the time to sexual maturation showed a dose related delay that was statistically significant at the p< 0.05. This suggests that the delay in sexual maturation was partly related to body weight, but not entirely. When gestational age was covaried with the time to sexual maturation, there was no significant difference in the time of onset of sexual maturation between controls and high dose animals. This indicates that the effect of delayed sexual maturation could possibly be attributed to decreased gestational age.

While it is known and commonly accepted that changes in the body weights of offspring can affect the time to sexual maturation, whether or not gestational age, as defined by the authors, also affects the time of acquisition is purely speculative, especially since there was no data provided by the authors to support this relationship. Additionally, covarying gestational age with time to sexual maturation is problematic from a statistical standpoint. Since there was no significant change in the length of gestation at 30 mg/ kg/ day, based on the authors' definition of ` gestational age', the decreases in gestational age would have to be due mostly to changes in time to sexual maturation. Therefore, sexual maturation is essentially being covaried with itself. Still, even if a relationship between gestational age and time to sexual maturation were shown, it merely offers an explanation for the observed delays in sexual maturation in high dose animals, but does not diminish its significance.

Therefore, under the conditions of the study, the LOAEL for F0 parental males is considered to be 1 mg/ kg/ day, the lowest dose tested, based on significant increases in the liver and kidney weights to terminal body weight and to brain weight ratios. A NOAEL for the F0 parental males could not be determined since treatment related effects were seen at all doses tested.

The NOAEL and LOAEL for F0 parental females are considered to be 10 and 30 mg/ kg/ day, respectively, based on significant reductions in kidney weight and kidney weight to terminal body weight and to brain weight ratios observed at the highest dose.

The LOAEL for F1 generation males is considered to be 1 mg/ kg/ day, based on significant decreases in body weights and body weight gains, and in terminal body weights; and significant changes in absolute liver and spleen weights and in the ratios of liver, kidney, and spleen weights to brain weights; and based on significant, dose related reductions in body weights and body weight gains observed prior to and during cohabitation and during the entire dosing period. A NOAEL for the F1 males could not be determined since treatment related effects were seen at all doses tested. 74 The NOAEL and LOAEL for F1 generation females are considered to be 10 and 30 mg/ kg/ day, respectively, based on statistically significant increases in postweaning mortality, delays in sexual maturation ( time to vaginal patency), decreases in body weight and body weight gains, and decreases in absolute food consumption, all observed at the highest dose tested.

The NOAEL for the F2 generation offspring was considered to be 30 mg/ kg/ day. No treatmentrelated effects were observed at any doses tested in the study. However, it should be noted that the F2 pups were sacrificed at weaning, and thus it was not possible to ascertain the potential post weaning effects that were noted in the F1 generation.

3.8 Carcinogenicity Studies in Animals 3.8.1 Cancer Bioassays The carcinogenic potential of APFO has been investigated in a two year feeding study in rats ( 3M, 1987). In this study, groups of 50 male and 50 female Sprague Dawley ( Crl: CD BR) rats were fed diets containing 0, 30 or 300 ppm APFO for two years. Groups of 15 additional rats per sex were fed 0, or 300 ppm APFO and evaluated at the one year interim sacrifice. The mean actual test article consumption was: males, 1.3 and 14.2 mg/ kg/ day; females, 1.6 and 16.1 mg/ kg/ day for the low and high dose groups, respectively.

There was a dose related decrease in body weight gain in the male rats and to a lesser extent, in the female rats as compared to the controls; the decreases were statistically significant in the high dose groups of both sexes. The body weight changes are treatment related since feed consumption was actually increased ( rather than decreased). There were no differences in mortality between the treated and untreated groups; the survival rates at the end of 104 weeks for the control, low, and high dose groups were: male, 70%, 72% and 88%; females, 50%, 48% and 58%. The only clinical sign observed was a dose related increase in ataxia in the female rats; the incidences in the control, low and high dose groups were: 4%, 18% and 30%. Significant decreases in red blood cell counts, hemoglobin concentrations and hematocrit values were observed in the high dose male and female rats as compared to control values. Clinical chemistry changes indicative of liver toxicity included increases in alanine aminotransferase ( ALT), aspartate aminotransferase ( AST) and alkaline phosphatase ( AP) in both treated male groups from 3 18 months, but only in the high dose males at 24 months. Increases in relative liver and kidney weights were noted in both high dose male and female rats. Significant nonneoplastic lesions were seen primarily in the liver and testis; there were increases in the incidence of liver masses, hyperplastic nodules and foci, and in testicular masses in the highdose male group. Other liver toxic effects include dose related increases in the incidence of diffuse hepatomegalocytosis, cystoid degeneration, and portal mononuclear cell infiltration in both male and female treated groups; these increases were statistically significant in the highdose males. A statistically significant, dose related increase in the incidence of ovarian tubular hyperplasia was found in female rats; the incidence of this lesion in the control, low, and highdose groups was 0%, 14%, and 32%, respectively. Based on these toxic effects, the high dose selected in this study appears to have reached the Maximum Tolerated Dose ( MTD). Based on 75 decreased body weight gain, increased liver and kidney weights and toxicity in the hematological and hepatic systems, the LOAEL for male and female rats is 300 ppm. [ Based on increases in the incidence of ataxia and ovarian tubular hyperplasia, the LOAEL for female rats is 30 ppm.]

At the termination of the study, a slight increase in the incidence of various neoplasms ( tumors of the liver, testis, thyroid, adrenal and mammary glands, etc.) was seen in the treated animals. Among them, the increased incidences of testicular ( Leydig) cell adenomas in the high dose male rats, and of mammary fibroadenoma in both groups of female rats were statistically significant ( P< 0.05) as compared to the concurrent controls. The incidence of the Leydig cell tumors ( LCT) in the control, low and high dose males was 0/ 50 ( 0%), 2/ 50 ( 4%) and 7/ 50 ( 14%), respectively; the respective incidences of mammary fibroadenoma in the female groups were 11/ 50 ( 22%), 21/ 50 ( 42%) and 24/ 50 ( 48%). The increases are also statistically significant as compared to the historical control incidences ( LCT, 0.82%; mammary fibroadenoma, 19.0%) observed in 1,340 male and 1,329 female Sprague Dawley control rats used in 17 carcinogenicity studies ( Chandra et al., 1992). The spontaneous incidence of LCT in 2 year old Sprague Dawley rats in other studies was reported to be approximately 5% ( cited in: Clegg et al., 1997). Therefore, under the conditions of this study, APFO is carcinogenic in Sprague Dawley rats, inducing Leydig cell tumors in the male rats and mammary fibroadenomas in the female rats.

The induction of Leydig cell tumors was confirmed in a follow up 2 year mechanism study of PFOA in male Sprague Dawley ( CD) rats at a dietary level of 300 ppm ( Cook et al., 1994; Biegel et al. 2001). A significantly increased LCT incidence was observed in the treated rats ( 8/ 76, 11%) as compared to the controls ( 0/ 80, 0%). In addition, PFOA also caused significantly increased incidences of liver tumors and pancreatic acinar cell tumors. The incidences of liver adenomas in the control and treated groups were 2/ 80 ( 3%) and 10/ 76 ( 13%), respectively, whereas those for the pancreatic acinar cell adenomas were 0/ 80 ( 0%) and 7/ 76 ( 9%). There was one pancreatic acinar cell caricinoma in 76 of the treated rats and none in 80 controls. The incidence of combined pancreatic acinar cell adenoma/ carcinoma in the treated rats ( 8/ 76, 11%) was also significantly increased as compared to the controls ( 0/ 80, 0%).

PFOA has also been shown to promote liver carcinogenesis in rodents ( Abdellatif et al., 1991; Nilsson et al., 1991).

3.8.2 Mode of Action Studies The mechanism( s) of toxicological/ carcinogenic action of PFOA is not clearly understood. PFOA was not mutagenic in the Ames test using five strains of Salmonella typhimurium, or in an assay with Saccharomyces cerevisiae ( Griffith and Long, 1980). Short term genotoxicity assays appear to suggest that PFOA is not a DNA reactive compound. However, when tested with metabolic activation, PFOA induced significant increases in chromosomal aberrations and in polyploidy in CHO cells ( Murli, 1996). The significance of these genotoxic effects is unclear. Available data appear to indicate that the induction of tumors by PFOA is due to a non genotoxic mechanism, involving activation of receptors and perturbations of the endocrine system. The 76 Agency is currently examining these postulated modes of action in detail. The following summaries are not meant to be a detailed review of the literature, but simply summarize the current scientific evidence.

<IP_ADDRESS> Liver Tumors It has been well documented that APFO is a potent peroxisome proliferator, inducing peroxisome proliferation in the liver of rats and mice ( e. g., Ikeda et al., 1985; Pastoor et al., 1987; Sohlenius et al., 1992). A sex related difference in the induction of liver peroxisome proliferation exists in rats ( Kawashima et al., 1989), but not in mice ( Sohlenius et al., 1992). The higher induction of liver peroxisome proliferation in male rats was shown to be strongly dependent on the sex hormone testosterone ( Kawashima et al., 1989). Like many other peroxisome proliferators, APFO has also been shown to cause hepatomegaly ( an early biomarker of peroxisome proliferator hepatocarcinogenesis) in rats ( Takagi, et al., 1992; Cook, 1994) and mice ( Kennedy, 1987), and induce oxidative DNA damage in liver of rats ( Takagi et al., 1991). The totality of these data appears to suggest that the liver toxicity and carcinogenicity of APFO may be related to induction of peroxisome proliferation. Meanwhile, estrogen has been shown to promote hepatocarcinogenesis in rats ( Yager and Yager, 1980; Cameron et al., 1982); an increase in estrogen levels after APFO exposure ( discussed below) may also play a role in hepatocarcinogenesis in rats. Recently, IARC ( 1995) concluded that the liver tumors induced in rodents by PPAR alpha agonists are unlikely to be operative in humans based on our current understanding of the animal mode of action.

<IP_ADDRESS> Leydig Cell Tumors A large number of non genotoxic compounds of diverse chemical structures have been reported to induce Leydig cell tumors ( LCT) in rats, mice, or dogs. A review of the available information on LCT induction in animals led a workshop panel to classify these compounds into seven groups based on their modes of action ( Clegg et al., 1997). The common theme in the mode of action for most compounds is that these compounds affect the hormonal control of Leydig cell growth by disrupting the hypothalamic pituitary testicular axis at various points that result in increasing the serum levels of luteinizing hormone ( LH). It has been postulated that in addition to stimulating the production of testosterone, LH may also play a mitogenic role in the Leydig cells; a sustained increase in circulating LH levels and chronic stimulation of Leydig cells by growth stimulating mediators such as IGF 1, TGF  , leukotrienes and various free radicals can lead to LCT development ( rev. in: Clegg et al., 1997).

A series of studies have been conducted to investigate the mechanism of tumor formation in male Sprague Dawley ( CD) rats exposed to APFO ( Cook et al., 1992; Biegel et al., 1995; Liu et al., 1996). No significant increases in LH were seen in the rats after treatment of APFO at various dose levels for 14 days. However, serum and testicular levels of estradiol were significantly increased and testosterone levels were significantly decreased. It was postulated that the elevated estradiol levels may cause Leydig cell hyperplasia and tumor formation by acting as a mitogen and/ or enhancing growth factor secretion; the transforming growth factor   77 ( TGF  ), which binds to the epidermal growth factor ( EGF) receptor and stimulated cell proliferation, for instance, has been detected in Leydig cells ( Teerds et al., 1990). Subsequent experiments have shown that APFO increased the levels of estradiol by inducing cytochrome P450 XIX ( aromatase), which converts testosterone to estradiol. Peroxisome proliferators are known to induce   oxidation and cytochrome P 450 monooxygenases by binding to the peroxisome proliferation activation receptor   ( PPAR  ; a subfamily of steroid hormone receptors). It is believed that APFO induces cytochrome P450 XIX ( aromatase) by binding to and activating the PPAR .

Although significant increases in LH were not seen in Sprague Dawley rats after treatment of APFO in the 14 day studies, it appears that increase in LH levels cannot be ruled out to be involved ( in addition to increased estradiol level) in the induction of LCT by APFO. In these studies, significant increase in hepatic aromatase ( which converts testosterone to estradiol) activities associated with decreased serum testosterone levels and increased estradiol levels were observed in the treated rats. Testosterone is synthesized and secreted by the Leydig cells, and is regulated by LH; testosterone and LH form a closed loop feedback system in the HPT axis. In order to maintain adequate testosterone plasma levels, reduced testosterone levels ( caused by increased aromatase activity) are expected to lead to increased LH levels through the negative feedback mechanism. It has been pointed out that increases in LH may not always be seen in all studies of chemicals for which the proposed mode of action calls for elevated LH, and that compensation may have occurred to restore homeostasis and inappropriate timing of sampling are some of the explanations for failing to detect changes in LH levels ( Clegg et al., 1997).

<IP_ADDRESS> Mammary Gland Tumors Estradiol has also been shown to stimulate the secretion of TGF   by mammary epithelial cells and the overexpression of TGF   has been suggested as one possible factor in producing sustained cell proliferation of mammary tumor cells and the subsequent development of neoplasia ( Liu et al., 1987). Hence, it is possible that the APFO induced elevation of estradiol levels may also be responsible for the development of mammary fibroadenomas in Sprague Dawley rats in addition to LCT ( discussed above). In fact, this is consistent with the mechanism by which spontaneous mammary neoplasms were developed in aging female Sprague Dawley rats. It has been demonstrated that the early appearance and high spontaneous incidence of mammary gland tumors in untreated, aging female Sprague Dawley rats is due to increased exposure to endogenous estrogen and prolactin as a result of an accelerating effect on normal, age related perturbations of the estrous cycle in this strain of rat ( Cutts and Noble, 1964; Chapin et al., 1996).

<IP_ADDRESS> Pancreatic Tumors The mechanism by which APFO induced pancreatic acinar cell tumors is unknown. A number of other peroxisome proliferators also produce pancreatic acinar cell tumors in rats. Available data suggest that the pancreatic acinar cell tumors are related to an increase in serum cholecystokinin ( CCK) level secondary to hepatic cholestasis ( Cook et al., 1994; Obourn et al., 1997). CCK is a 78 growth factor that has been shown to stimulate normal, adaptive, and neoplastic growth of pancreatic acinar cells in rats ( Longnecker, 1987). However, data on the role of CCK in pancreatic tumor formation are conflicting.

3.9 Immunotoxicology Studies in Animals Four immunotoxicity studies of PFOA have been conducted in mice. The first of these studies was a feeding study in mice ( Yang et al. 2000). For investigation of the effects of perfluorooctanoic acid ( PFOA), and other peroxisome proliferators, on lymphoid organs, 0.02 % PFOA was administered to male C57Bl/ 6 mice in the diet for 2, 5, 7, or 10 days. At the end of the feeding period, mice were sacrificed and the liver, spleen, and thymus were dissected out and weighed. The effect of PFOA administration on the cellularity, cell surface phenotype, and cell cycle of thymocytes and splenocytes was determined. In addition, effects of exposure of thymocytes and splenocytes to PFOA in vitro were examined.

The results showed that administration of 0.02% PFOA for 2, 5, 7, or 10 days resulted in a significant increase, relative to controls, in liver weight, even at the earliest time point. Also, a decrease in body weight caused by PFOA administration was observed. Subsequently, by the day 5 administration period, significant decreases in thymus and spleen weight were detected. After administration of 0.02% PFOA for 7 days, significant decreases ( 85% and 80%, respectively) in the total number of thymocytes and splenocytes were observed. The results also showed that the number of thymocytes expressing both CD4 and CD8 decreased by 95%; the number expressing both CD4 and CD8 decreased by 57%; and the number expressing either CD4 or CD8 decreased by 64% and 72%, respectively. For the splenocytes, both T cells ( CD3) and B cells ( CD19) decreased by 75% and 86%, respectively. Also, significant decreases in both CD4 helper and CD8 cytotoxic splenic T cells were observed. Upon administration of 0.02% PFOA to mice for 7 days, thymocyte proliferation was also inhibited, as detected by cell cycle flow cytometry analyses. In vitro studies showed that there was spontaneous apoptosis occurring in splenocytes and thymocytes after 8 or 24 hours of culturing in the presence of varying concentrations ( 50, 100, or 200 M) of PFOA. However, PFOA did not significantly alter the cell cycle under these conditions.

In order to study mechanism ( Yang et al. 2001) , another mouse feeding study was performed. In order to examine the dose dependency of the effects, C57Bl/ 6 mice received diets consisting of 0.001% 0.05% PFOA ( w/ w) for 10 days. For examining the time course, a diet containing 0.02% PFOA was given for 2, 5, 7 or 10 days. Effects of withdrawal of PFOA were also studied.

The results showed that, at higher doses, a significant decrease, relative to controls, in body weight was observed, although no other apparent signs of toxicity such as sores, lethargy, and poor grooming were noticed. However, a significant decrease in total water intake was observed. Mice receiving dietary PFOA for 10 days experienced significant increases in liver weight and peroxisome proliferation, as measured by induction of acyl CoA oxidase with lauroyl CoA or palmitoyl CoA as substrate. These increases started at the lowest dose and 79 reached their maximal values at a dose of 0.003 0.01%. In contrast, the weight decreases of the spleen and thymus began at a higher dose ( 0.01%) with no maximum reached with the doses given. The time course studies showed that increased liver weights and peroxisome proliferation were evident at the earliest time point examined. In contrast, significant thymus and spleen weight decreases required PFOA administration for a period of at least 5 days, following which the spleen weight remained constant while the thymus weight continued to decrease. However, upon prolonged treatment for one month, no further decreases in thymus and spleen weights were observed. In another set of experiments, animals received 0.02% PFOA for 7 days, and then they received normal chow for a period of 10 days. These recovery experiments showed that the animals rapidly recovered the body weight the second day after withdrawal of PFOA. However, the liver weight did not return to normal even after 10 days of recovery. Thymus recovery started on day 2 and was completed by day 10. The spleen weights returned to normal by day 2 post withdrawal. In addition, the changes in thymus and spleen weight upon PFOA treatment and withdrawal paralleled the changes in total thymocyte and splenocyte counts. Furthermore, flow cytometry cell cycle experiments showed that the decrease in thymocyte number caused by PFOA treatment is due mainly to inhibition of thymocyte proliferation. In contrast, PFOA treatment caused no changes in the cell cycle of splenocytes.

A third feeding study ( Yang et al. 2002a) was designed to examine the possible involvement of the peroxisome proliferator activated receptor alpha ( PPAR  in the immunomodulation exerted by PFOA. This study made use of transgenic PPAR  null mice, which are homozygous with regards to a functional mutation in the PPAR  gene. These mice do not exhibit peroxisome proliferation or hepatomegaly and hepatocarcinogenesis even after exposure to peroxisome proliferators. These mice were fed a diet consisting of 0.02% PFOA ( w/ w) for 7 days. At the end of the feeding period, mice were sacrificed and the liver, spleen, and thymus were removed and weighed. The effect of PFOA on peroxisome proliferation, cell cycle, and lymphoproliferation was ascertained.

The results showed that, in contrast to wild type mice, feeding PPAR  null mice PFOA resulted in no significant decrease in body weight. However, increases in liver weight were still seen in PPAR  null mice, suggesting that this is not a PPAR  dependent process. As expected, peroxisome proliferation, as measured by fatty acid  oxidation, was totally lacking in PPAR  null mice. Also in contrast to wild type mice, feeding PPAR( null mice PFOA resulted in no significant decrease in the weight of the spleen or the number of splenocytes. At the same time, the decrease in weight and cellularity of the thymus was attenuated, but not totally eliminated in the PPAR( null mice. In addition, the decreases in the size of the CD4+ CD8+ population of thymus cells and the number of thymus cells in the S and G2/ M phases of the cell cycle, which reflects inhibition of proliferation, observed in wild type mice administered PFOA were much less extensive in PPAR( null mice. Finally, in contrast to wild type mice, PFOA treatment caused no significant change in splenocyte proliferation in response to mitogens in PPAR( null mice.

A fourth feeding study ( Yang et al. 2002b) was designed to examine the effects of PFOA on specific humoral immune responses in mice. For this study, 0.02 % PFOA was administered to 80 male C57Bl/ 6 mice for 10 days. Then the animals were examined, via plaque forming cell ( PFC) and serum antibody assays, for their ability to generate an immune response to horse red blood cells ( HRBCs). Ex vivo and in vitro splenic lymphocyte proliferation assays were also performed.

The results showed that mice fed normal chow responded to challenge with HRBCs with a strong humoral response, as measured by the PFC assay. In contrast, mice fed with PFOA responded to HRBC immunization with no increase in HRBC specific PFCs, relative to unimmunized controls. However, in experiments where PFOA treated mice received normal chow following HRBC immunization, there was a significant recovery of the numbers of specific PFCs stimulated. The suppression of the humoral immune response by PFOA was confirmed by analysis of the serum anti HRBC response. In ex vivo experiments, splenocytes isolated from control mice responded to both ConA and LPS with lymphocyte proliferation, as measured by thymidine incorporation. However, treating mice with PFOA ( 0.02% for 7 days) attenuated the proliferation. In a set of in vitro experiments, PFOA ( 1 200 M) added to the culture medium of splenocytes cultured from untreated mice did not cause an alteration of lymphocyte proliferation in response to LPS or ConA.

4.0 Hazards to the Environment 4.1 Introduction The aquatic toxicity and hazard of APFO to aquatic organisms was assessed. This task was made more difficult by several problems discussed below. These problems complicated the task of determining if the ecotoxicity tests were valid and could be used in the assessment. Furthermore, these problems limited the confidence that could be placed on the toxicity test values, and thus in turn lowered the confidence of conclusions that could be drawn in assessing the inherent toxicity and hazard of APFO to aquatic organisms.

1) A variety of different APFOs with varying designations and lot numbers were tested. Generally, the ammonium salt or the tetrabutylammonium salt was tested. The exact composition and identification of impurities, which may affect toxicity, in each lot number used is not known.

2) A variety of testing laboratories conducted the APFO toxicity studies over a period of time from approximately 1974 1996. This situation served to increase overall test variability and thus made inter laboratory comparisons more difficult.

3) Purity of the tested material, or percent test material and percent other material( s), was a major concern. Purity was not sufficiently characterized in these tests. In some tests it appeared that 100% test chemical was used; in others a chemical of lesser purity ( approximately 85%) was used. Purity of test material does affect toxicity and should be taken into account when possible, by expressing toxicity on the same purity basis.

4) Water, an isopropanol solvent, or a combination of both were used with the test material in 81 many of the toxicity tests, for no obvious indicated reason. Solvents are mixed with the test material to make it miscible with the test dilution water before the test is begun. Solvents are used in tests where the concentrations of the test material are extremely low and a very small amount of test material must be added to the test chambers. It was not clear from the summaries of these studies why a solvent was used or was even found to be necessary. In fact, 3M summarized each test and stated " Data may not accurately relate toxicity of the test sample with that of the test substance." Thus, in those tests where 100% test material was not used, the toxicity values had to be adjusted to take into account the percent solvent( s), and to express the values on a 100% test chemical basis, so that the tests could be compared.

5) In all these toxicity tests only nominal test chemical concentrations were used. Measured test chemical concentrations are instead always recommended so that one can accurately determine the actual test chemical concentration to which the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values will have to accordingly be adjusted by 50%. Analytical measurements of chemical concentration should have been taken or made available. Then, recovery rates could have been determined, and physicochemical processes ( e. g., hydrolysis, volatility) that might lower the actual concentrations to which the test organisms were exposed could have been taken into account. Nominals may be used when measured concentrations are taken and the relationship of both is known.

In order to proceed with any sort of environmental hazard review it was necessary to ignore these test limitations and to assume that the nominal concentrations were an " adequate" expression of the measured test chemical concentrations. Criteria for assessing degree of acute toxicity are based on well established values ( low is > 100 mg/ L; medium or moderate is > 1< 100 mg/ L; high is < 1 mg/ L).

4.2 Acute Toxicity to Freshwater Species Several species were tested to assess the acute toxicity of APFO; these included the fathead minnow ( Pimephales promelas), bluegill sunfish ( Lepomis machrochirus), water flea ( Daphnia magna), and a green alga ( Selenastrum capricornutum). The toxicity test endpoints have been adjusted to 100% test chemical and test results are presented in Tables 9 ( organized by test substance) and 10 ( organized by test species). Each value is related to a testing facility and reference.

Twelve tests were conducted with fathead minnows; 96 h LC50 values ( based on mortality) ranged from 70 to 843 mg/ L. It is unclear why this range is so wide. Assuming these studies are valid, and due to the limitations discussed above, these toxicity values indicate low toxicity. The two acute values for bluegill sunfish also indicate low toxicity ( 96 h LC50s of > 420, and 569 mg/ L).

Nine acute tests were conducted with daphnids and 48 h EC50 values ( based on immobilization) ranged from 39 to > 1000 mg/ L. The lower values are indicative of moderate toxicity, but the 82 wide range makes interpretation difficult.