Document gb6VyEdNpyXwJ6K4pGY67Lq5V

AR226-3380 Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat P. M. Hinderliter. M. P. DeLorme, and G. W. Jepson The DuPont Company, Haskell Laboratory for Health and Environmental Sciences, Newark, Delaware, USA Abstract A large r |TMnTMmHnMir-database exists describing the behavior of perfluorooctanoic acid (PFOA) following oral exposure- The objective of this study was to quantify plasma PFOA concentrations to rats, following single and repeated inhalation exposures at three airborne concentrations, for the purpose o fbridging the oral and iniialation exposure data sets. The study was comprised of two separate experiments: Pltase I consisted o f a stogie 6-hour nose only exposure while Phase II consisted of repeated exposures (6 hours per day. 5 days per week for three weeks). In both experiments, male and female rats were exposed nose only to an aqueous aerosol o f 0,1,10. or 25 ing/m3PFOA. Levels were selected to produce plasma concentrations similar to those observed to previous oral gavage studies. In Pltase i. blood was drawn via die tail veto pre-exposure, during exposure at 0.5,1,3 and 6 hours, and post-exposure at 1,3,6.12,18 tuid 24 hours. Rats were not removed from the exposure chamber during the exposure period and blood was taken from the tail veto wltile the animals retrained to die nose-only restrainers attached to the exposure chamber. For Phase II, blood was collected immediately before and after the daily inhalation exposure period three days per week. Plasma derived from the whole blood samples was analyzed by liquid chromatography-mass spectrometry (LC-MS). PFOA appears rapidly to the blood of bodi male and female rats d via th? inhalation route. PFOA elimination is sex-dependem, with female rats elii i PFOA from die plasma much more efficiently than male rats. PFOA plasma coi ions are proportional to die attnosplieric exposure concentrations from 1 to 25 ing/m3 ited daily inhalation exposures produce little plasma carryover to female rats, but sig........ ...;..c.a..r.r,yover in male rats. Male rats reach a steady state plasma concentration by three weeks widt, concentrations of 8,21, and 36 pg/mL respectively when exposed to 1,10, and 25 mg/irf PFOA. Female rats readied post exposure plasma concentrations of 1.2, and 4 pg/mL respectively when exposed to 1.10, and 25 mg/m3 PFOA but returned to baseline levels fc die pre-exposure tuns points. This study provides the data necessary to relate external atmospheric concentrations o f PFOA to PFOA lewis to male and female rat plasma This research was sponsored by the Association of Plastics Manufacturers in Europe (APME) Fluoropolymers Committee. IMaterials and Methods jear perfluorooctanoic acid(PFOA) was obtained from Aldrich Chemicals. Since this ly utilized the inhalation route of exposure, no attempt was made to establish the actual e each rat received and all results were therefore reported as a (unction o f d * group osure concentration(s) radier than a function of dose. Male and female Cr!:CD*(SD)lGS BR rats wereobtained fromCharles River Laboratories, Inc., Raleigh, North Carolina At the time o f exposure, rats were approximately 6-8 weeks of age and die weight variation did not exceed 20% of the mean weight by exposure group. All exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal interna! volume of 150 L with a dispersion plate inside the chamber to promote uniform distribution o f die test atmospliere. Animals were individually restrained in polycarbonate (Phase l) or perforated stainless steel cylinders (Piiase II) with conical nose pieces. During exposure, die animals were kept warm with a lieat lamp (Figure 1). A 5% (weight/weight) solution was prepared and adjusted to a pH of 6.0 to 8.0. Cltainber atmospheres were generated by aerosolization of the 5% test substance solution to air with a Spraying Systems nebulizer. Tlie pump and flow controller were controlled and monitored by the Camile Inhalation Toxicology Automated Data System (C1TADS). Chamber concentrations were controlled by varying die syringe infusion rate to die nebulizer or by using dilulional air. The aurasplieric concentration of PFOA was determined by gravimetric analysis at approximately 60-minute intervals during each exposure. In order to determine if the gravimetric analytical method used to quantitate die airborne test substance concentration included the testsubstance plus water, air samples were taken from dumber armosplieres dial ranged from l to 25 mg/m3test substance. The filters were weighed immediately post-sampling, placed in a desiccation chamber for 24 hours and re weighed. The filters lost <0.03% of their mass, indicating dial die filter reflected the total mass of suspended aerosol to die exposure diambers. A sample to determine panicle size distribution (mass median aerodynamic diameter and percent panicles less than 1,3. and iO (tin diameter) was taken fromeadi test exposure chamber at least once per week during the Phase II study with a Sierra Series 210 cyclone preseparator/Cascadc impactor and Sierra series 110 constant flow air sampler. Clumber airflow was set to achieve at least 10 air changes per liour and was monitored continually. Clumber temperature, relative humidity and oxygen concentration were targeted at 22 * 3'C. 40 to 60%, and 19%, respectively. Airflow, temperature, and relative humidity were monitored continuously and recorded at IS-minute intervals. Chamber oxygen concentration was recorded twice during each exposure Plasma samples were processed by protein precipitation (PPT) using Isolule Array protein precipitation columns (Argonaut Tecluiotogies, Foster City. CA). A 0.5 pg/ml solution of perfluorononanoic acid (Aldrich Cltemieals) to ACN was used as an internal standard. Plasma samples were tluwed, and a 20 /tL aliquot ofeach sample was applied to the PPT array. The plasma samples were precipitated by adding appropriate dilution rate volumes of ACN/totemal standard solution. Dilution rates, ranging from 1:4 (60 pL of internal standard solution) to 1:50(980 pL of internal standard solution), were utilized to order to capture rite sample concentrations witliin tlie range of the standard curve concentrations. Die array was slowly eluted under vacuum into a 96-well receiver plate, centrifuged at -3000 rpm for 5 minutes, and tlie extracts were analyzed by LC/MS. \ Experimental Design Phase 1 (Stogie Exposure) To determine tlie proper exposure concentrations for Phase 11, fourgroupsof 3 male and 3 female rats each were exposed nose only for a stogie, 6-hour period to au aerosol o f the test substance. Die rest groups were exposed to an atmospheric concentration of 1 mg/m3, 10 mg/m3, or 25 mg/m5. A control group of rats was exposed to air only. Blood was collected via the toil veto and was drawn pre-exposure, during exposure at 0.5, 1,3 and 6 hours, and post-exposure at 1,3,6.12. 18 and 24 hours. Individual animal exposure times were staggered by five minutes to allow tune for blood collection. Rats were not removed from tlieexposure chamber during tireexposure lime. Blood was taken from tlie tail veto while the animals remained to tlie nose-only restrainers to tlie exposure chamber. Blood was collected in heparinized tubes and stored on wet ice. Plasma wasseparated byeentrifugation. Rats were sacrificed following tire final blood collection Plasma derived from the whole blood samples was analyzed by LC-MS. Die exposure concentrations selected for Phase were levels that produced plasma concentrations similar to plasma concentrations observed in a previous study of oral gavage dosing to rats." 1 Phase II (Repeated Exposure) After the exposure levels were selected, four groups of 5 male and 5 female rats each were exposed (nose only) for a 6-llour period, 5 days per week (weekends excluded), for 3 weeks to an aerosol of the test substance. Die starting time of dieexposure is defined as tlie time wheu die nose only restrained containing die animals are loaded into die exposure chamber. The exposure end lime is defined as die time wlien die nose only restrained are removed from die chamber. After each exposure, rats were returned to their cages. The control group of rats was exposed to air only. Exposure start times were staggered by 10-30 minutes by exposure concentration group to allow time for blood collection. As in Phase I, blood was collected via the tail vein. Blood was collected before and after the daily tohaladon exposure period diree days per week. Rats were sacrificed following the final blood collection. f Results - Chamber Conditions CHAMBERCONCENTRATIONS OFPFOA PHASE 1 (SINGLE EXPOSURE)__________ Design Measured Coni Concentration img/tr ______ (mg/nPi Mean S.D. Phase I (Single Exposure) Bar both sexes, PFOA plasma concentrations rose during tlte 6 hour inhalationexposure- A proportional relationship was produced to both the male and female rat maximumplasma concentrations ( C ,, J between 1and 25 mg/m3(Figure 2). Die male C ^ , values were approximately 2-3 times higlier than the female ClllBl. Die female occurred at the end of the exposure period or 1hour post exposure while the male Cnal occurred at the end of the exposure period up to 6 hours post exposure. Elimination of PFOA from female rat plasma was rapid and complete at all exposure levels to Phase I. Plasma concentrations dropped below the analytical limit ofquaniification(0.1 pg/mL) by 12 hours post exposure. Plasma elimination was much slower in male rats dian female rats. After 24 liours of post exposure recovery, tlie plasma concentrations were approximately 90% of the peak concentrations at all tested exposure levels. Die male and femaleelimination kinetics are consistent with the previously reported elimination kinetics of PFOA following oral dostog.(l> In addition, there did not appear to be a concentration dependence of tlie elimination rate to either male or female rats as tlie plasma curves are parallel on a log scale. Bor the setting of exposure levels for Phase II of the study, male and female C,,,, values were compared to plasma C ,,,, from an oral gavage study of PFOA."' This study examined single oral gavage doses from 0.1 to 25 mg PFOA/kg body weight. The values forthis study ranged from0.598to 160.0 hg/mL (male) and 0.67 to 132.7 (female). Because the values from Phase 1 fell within the middle of the existing range and oral gavage data, the inhalation exposure concentrations were not changed for Phase II. Phase II (Repeated Exposure) Phase II plasma concentrations are shown to Figure 3. Exposures were conducted five days per week, widi plasma samples taken before and after exposure on the first, third and fifth exposure day per week. The male and female concentration curves demnstrate the effects of the differing elimination kinetics on plasma levels following repeated exposure. In the female rats, there was little day-to-day carryover of PFOA toll* plasma. Diere is no significant difference between tlie peak heights of the sampled exposuredays. For the male rats, diere is carryover between test days as expected from Pilase I. Steady state was not achieved until week 3. This steady state value is approximately 2-3 limes as high as the mean single dose CM . As in Phase I, repeated exposures demonstrate a proportional relationsliip between exposure concentrationand plasma concentrations to male and female rats. Male rats read) n steady state plasma concentration by three weeks with plasma concentrations of 8.21. and 36pg/mL respectively for the 1.10, and 25 ing/m5exposures. Female rats reached post-exposure plasma concentrations of 1.2, and 4 pg/mL respectively for the 1,10, and 25 mg/m3exposures, but returned to baseline levels for die pre exposure time points. PHASE II (REPEATEDEXPOSURE) Design Concentrador Itng/mh Design Mass Median Geometric Concentration Aerodynamic Standard (niE/m'l Diameter (ami Deviation CHAMBER ENVIRONMENTALCONDITIONS PHASE1r.SMC Dote RelativeHumidity M,,,, <n Mraii S.D. Retire Men, S.D. Renee n Q i-j 37 1t 36 -4C 28 0025 28 27 21 34 4.5 31 -48 16 70 005 70 28 10 3( 40 17 38 45 31 is '4 DM 31-25 32 34 25 30 28 32 PHASEII(REPEATEDEXPOSURE) 28 0066 2S 0.031 38 }Conclusions PFOA appears rapidly to die blood of both male and female rats exposed via the inhalation route. PFOA elimtoarion is sex-dependem, with female rats eliminating PFOA from tlie plasma much more efficiently than male rats. At tlie tested concentrations from 1 to 25 mg/m3, PFOA plasma concentrations relate proportionally to die iniialation exposure concentration. Repeated daily iniialation exposures produce liule plasma carryover to female rats, but significant carryover in male rats. Male rats reach a steady state plasma concentration by diree weeks with plasma concentrations of 8,21, and 36(tg/mL respectively for die I, 10, and 25 mg/m3 exposures. Female rats readied post-exposure plasma concentrations of l, 2, and 4 /ig/mL respectively for the l, 10, and 25 mg/m3 exposures, but returned to baseline levels for die pre-exposure time points. Reference: innufont it