Timed-pregnant Long-Evans hooded rats (Charles River Laboratory, Raleigh, NC) were obtained (4 shipments over 6 months) on gestational day (GD) 4 and individually housed in ventilated cages (Techniplast, West Chester, PA) with autoclaved, hardwood bedding (PJ Murphy, Montville, NY) within a semi-barrier room (40-60% humidity; 12- hr light/dark cycle:6:00-18:00; 20-24oC). Dams were randomly assigned to exposure group: Two drinking water control groups were maintained on reverse osmosis drinking water (RO-H2O). One group (G1) was maintained on a standard rodent chow (Teklad 2918; Envigo, Madison, WI) and the second (G2) was maintained on a low-fluoride chow (Teklad Custom Diet TD.160173). To examine the effect of fluoride in the drinking water while controlling for dietary fluoride, rats were exposed to the low-fluoride chow and water supplemented with either10ppm F- (G3) or 20ppm F- (G4). Dosing solutions were prepared fresh weekly with sodium fluoride (NaF; lot # X0044851; 99.9%; Materion, Milwaukee, WI). Fluoride levels in drinking water were confirmed [RO-H2O: <0.2ppm; 10ppm and 20ppm F-within <5% of target] (Analytical Method: EPA 300.0; Pace Analytical, Huntersville, NC). The Teklad 2918 diet contained 20.5ppm F- and the TD.160173 custom diet contained 3.24ppm F- (Official Methods of Analysis Methods 944.08 and 978.03; AOAC International, Gaithersburg, MD; Covance, Madison, WI). Food and water were available ad libitum. Exposure to the dams began on GD6 and continued throughout lactation. Pups were allowed free access to drinking water, beginning consumption around PND14, and continued on the same level of exposure after weaning until study termination. All animal procedures were conducted in accordance with protocols approved by the NIEHS Animal Care and Use Committee within AAALAC approved animal facilities.

Timed-pregnant Long-Evans hooded rats (Charles River Laboratory, Raleigh, NC) were obtained (4 shipments over 6 months) on gestational day (GD) 4 and individually housed in ventilated cages (Techniplast, West Chester, PA) with autoclaved, hardwood bedding (PJ Murphy, Montville, NY) within a semi-barrier room (40-60% humidity; 12- hr light/dark cycle:6:00-18:00; 20-24oC). Dams were randomly assigned to exposure group: Two drinking water control groups were maintained on reverse osmosis drinking water (RO-H2O). One group (G1) was maintained on a standard rodent chow (Teklad 2918; Envigo, Madison, WI) and the second (G2) was maintained on a low-fluoride chow (Teklad Custom Diet TD.160173). To examine the effect of fluoride in the drinking water while controlling for dietary fluoride, rats were exposed to the low-fluoride chow and water supplemented with either10ppm F- (G3) or 20ppm F- (G4). Dosing solutions were prepared fresh weekly with sodium fluoride (NaF; lot # X0044851; 99.9%; Materion, Milwaukee, WI). Fluoride levels in drinking water were confirmed [RO-H2O: <0.2ppm; 10ppm and 20ppm F-within <5% of target] (Analytical Method: EPA 300.0; Pace Analytical, Huntersville, NC). The Teklad 2918 diet contained 20.5ppm F- and the TD.160173 custom diet contained 3.24ppm F- (Official Methods of Analysis Methods 944.08 and 978.03; AOAC International, Gaithersburg, MD; Covance, Madison, WI). Food and water were available ad libitum. Exposure to the dams began on GD6 and continued throughout lactation. Pups were allowed free access to drinking water, beginning consumption around PND14, and continued on the same level of exposure after weaning until study termination. All animal procedures were conducted in accordance with protocols approved by the NIEHS Animal Care and Use Committee within AAALAC approved animal facilities.