"Materials and methods

Methods and materials used in this study have been described previously (Ladics et al., 2005; Mylchreest et al., 2005; Stadler et al., 2008). Where methods differ, the details are as follows.

2.1. Test substance and administration

NaPFHx was supplied by DuPont Chemical Solutions Enterprise, Wilmington, DE, as a white solid, 100% purity. Analysis indicated the test substance was stable throughout the course of the studies. NaPFHx was diluted in NANOpure® water and administered by oral gavage to achieve dosage levels of 0, 20, 100, or 500mg/(kg day) for 90-day subchronic, one-generation reproduction, and developmental studies. Dose volumes were 5.0 mL/kg. Male and female control animals were similarly treated with deionized water at the same dose volume as used in the other groups.

2.2. Animals and animal husbandry

Crl:CD(SD) rats were obtained from Charles River Breeding Laboratories, Raleigh, NC, USA. Rats for the subchronic and reproduction studies were approximately 4 weeks old when received and 6–8 weeks old at study start. Time-mated females for the developmental toxicity study were either at 1, 2, or 3 days of gestation when received. Rats were housed and maintained in accordance with the principles described in the Guide to Care and Use of Laboratory Animals (National Research Council, 1996). Tap water and pelleted chow(Certified Rodent LabDiet® 5002: PMI® Nutrition International, LLC) were available ad libitum throughout the study.

2.3. Acute toxicity

A single dose of 175, 550, 1750, or 5000 mg/kgwas administered to fasted female rats,which were observed for up to 14 days for clinical signs of toxicity, body weight effects, and mortality.

2.4. 90-Day subchronic toxicity study

Dosages were set at 20, 100, and 500mg/(kg day), based upon rangefinder studies. Rats were divided into groups with 55 rats/sex in the 500mg/(kg day) dose and control groups, and 45 rats/sex in the 100 mg/(kg day) and 20 mg/(kg day) dose groups (see Fig. 1). Of these, 10 rats/sex/dose were designated for evaluation of subchronic toxicity; the next 20 rats/sex/dose were designated for reproductive assessment; 10 rats/sex/dose (control and high-dose groups only) were designated for 30-day recovery assessment and 10 rats/sex/dose were designated for 90-day recovery assessment. An additional subgroup (5 rats/sex/dose) was received as a separate shipment and was added to each dose group approximately 84 days after study start, for evaluation of hepatic peroxisomal -oxidation analysis following a 10-day exposure. This study conformed to OECD Guideline 408 (Repeated Dose

90-Day Oral Toxicity Study in Rodents). Rats were weighed at regular intervals, cage site examinations were conducted daily, and detailed clinical observations were recorded weekly. Ophthalmological and neurobehavioral evaluations, including functional observations (i.e., gripstrength, sensory motor function) and motor activity assessments, were conducted prior to study start and during the last week of dosing. Clinical pathology evaluations

were conducted on day 45, day 92 (males) or 93 (females), and at 1 month of recovery. Blood for plasma fluoride analysis was collected from the vena cava at sacrifice. Urine fluoridewas determined at the end of treatment and at the end of the 1-month recovery period. Gross examinations were performed at sacrifice. Selected tissues were weighed and/or processed for microscopic examination, as previously described (Ladics et al., 2005).

2.5. Hepatic peroxisomal ˇ-oxidation evaluation

After 10 or approximately 90 days of administration, or following a 30-day recovery period (control and high dose only), -oxidation activity from liver microsomes was measured in 5 overnight-fasted rats from each group. Livers were removed andweighed, and hepatic peroxisomeswere prepared and peroxisomal -oxidation activity determined as described by Biegel et al. (2001).

2.6. One-generation reproduction study

P1 female rats selected for reproductive evaluations were dosed by oral gavage for approximately 70 days prior to cohabitation, through gestation and lactation for a total of approximately 126 days. P1 male rats in this subset were dosed for a total of approximately 110 days. F1 rats were not dosed. Clinical observations, body weight, and food consumption were determined weekly throughout the study. Estrous cycle, sperm parameters, survival, and reproductive performance parameters were assessed. Litter examinations (number of live and dead, individual pup weights, clinical observations)were determined on day 4, and weekly during the lactation period. F1 offspring were given a gross pathological examination at weaning. A subset of F1 generation rats was maintained for 6 weeks after weaning to assess developmental landmarks. The subset was given a gross pathological examination

and selected reproductive organs were weighed. This study was in alignment with OECD Guideline 415.

2.7. Developmental toxicity study

Twenty-two female rats per group were dosed once daily on days 6–20 of gestation according to OECD Guideline 414. In-life observations were recorded and rats sacrificed on gestation day (GD) 21. All dams underwent a gross pathological examination and the fetuses were removed from the uteri by Cesarean section. Fetuses were weighed and sexed and examined for morphological alterations. All fetuses were examined for external and skeletal alterations, and approximately 50% of the fetuses were examined for soft tissue and visceral head examinations.

2.8. Genotoxicity testing

NaPFHx was evaluated for mutagenicity in the bacterial reverse mutation (Ames) assay using the plate incorporation method, according to OECD Guideline 471. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were tested in the presence and absence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). All tester strains and supplies, as well as liver S9 fraction prepared from male Sprague–Dawley rats induced with Aroclor 1254, and positive controls of benzo[a]pyrene, 4-nitroquinoline N-oxide, acridine mutagen ICR-191, sodium azide, 2-aminoanthracene, and 2-nitrofluorene, were purchased fromMoltox Inc. (Boone,NC). The testwas performed in two phases.

The first phase was the toxicity-mutation test which established the dose range for the mutagenicity test. The second phase was the mutagenicity test which evaluated and confirmed the mutagenic potential of NaPFHx. NaPFHx was soluble in sterile water at 50mg/mL, the highest concentration that was tested. The dose levels used were 333, 667, 1000, 3333, and 5000g/plate for all tester strains (TA98, TA100, TA1535, TA1537, and WP2uvrA) in the presence and absence of S9 metabolic activation. The plate incorporation method was employed. NaPFHx was evaluated for its ability to induce structural and numerical chromosome aberrations in vitro using human peripheral blood lymphocytes (HPBL) in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9) according to OECD Guideline 473. Human blood was collected from a healthy volunteer donor. The positive controls, mitomycin C and cyclophosphamide (CP), were purchased from Sigma–Aldrich (St. Louis, MO). Cell culture media and buffers were obtained from Mediatech (Manassas, VA). To establish a concentration range for the chromosome aberration assay, a preliminary toxicity test was conducted. NaPFHx was soluble in water at 38.6mg/mL (100 mM). The cells were treated for 4 or 22 h in the non-activated test condition and for 4 h in the S9-activated test condition. All cells were harvested 22 h after treatment initiation. The maximum concentration tested in the preliminary toxicity assay based on the formula weight of NaPFHx was 10mM (3860g/mL), the recommended limit dose for this test system (i.e., 5000g/mL or 10mM, whichever is lower – OECD 473 testing guideline). The cells were exposed to nine concentrations ranging from 5 to 3860g/mL,

as well as vehicle controls. No visible precipitate was observed in the treatment medium at the beginning or end of the treatment period at any concentration tested. Based on the results from the toxicity test, the chromosome aberration assay was conducted with cytogenetic evaluations conducted at 2000, 3000, and 3860g/mL (10mM) for the 4-h non-activated test condition and at 250, 500, and 1000g/mL (2.59mM) for the 4-h activated and 22-h non-activated test conditions.

2.9. Statistical analyses

A variety of statistical tests were used, as appropriate, for each study. Preliminary tests were conducted for homogeneity of variance (Levene, 1960) and

normality (Shapiro and Wilk, 1965). A one-way analysis of variance (Snedecor and Cochran, 1967) followed by Dunnett’s test (Dunnett, 1964) were conducted if data were normally distributed and had homogeneity of variance. For data that did not show homogeneity of variances, a robust version of Dunnett’s test (Dunnett, 1980) was used. Non-normally distributed data were analyzed using the non-parametric Kruskal–Wallis test (Kruskal and Wallis, 1952) followed by Dunn’s test (Dunn, 1964). For all statistical analyses, significance was judged at p < 0.05. Comparisons were made between the vehicle control group and the dosed or positive control groups. Statistical methods applied in the subchronic, reproductive, and developmental toxicity

studies were as follows. Monotonal endpoints were evaluated with Levene’s test for homogeneity and Shapiro–Wilk test for normality. If the Shapiro–Wilk test was not significant, but Levene’s test was significant, a one-way analysis of variance followed by Dunnett’s test (Dunnett, 1980) was used. If the Shapiro–Wilk test was significant, Kruskal–Wallis testwas followed by Dunn’s test.No statistical evaluation was conducted to determine the significance of the nasal lesions."

F1 Combined Crl:CD(SD) Rats