Document 82rr8K2jMB3KnX7NwovNgG2Yk

Toxicological Summary PFOS Dietary Chronic Definitive Reproductive Study: Northern Bobwhite Test Substance: Perfluorooctanesulfonate (PFOS) Structure: 1-Octanesulfonic acid, 1,1,2,2,3,3,4.4,5,5.6.6,7.7,8.8.8-heptadecafluoro, potassium salt. CAS No. 2795-39-3. Test Remarks: The test FC-95. substance is The sample a white powder was stored under (3M Lot ambient #217), also conditions known as and purity was determined to analysis techniques. be 86.9% by LC/MS, "H-NMR, and clemental METHODS 4Method: ASTM Standard E1062-86, OECD 206, and FIFRA Subdivision E., Section 71- Type: Dietary Reproduction Year: 2001 in-lifc phase, 2002-2003 analytical phase, Final report in 2003 Species: Northern bobwhite quail (Colinus virginianus) cEoxnpceernitmraetnitoanls Doefs0i,gn1:0,Ad5u0l,tonror1t5h0erpnpbmobiwnhitthee dqiucatilovweerreaepxeproisoeddotfoaPpFprOoSxiamtantoemliyn2a1l woetehkesr.paiErascohftqrueaaitlmewnetrgeromuapinctoanitnaeidnepder16tpraeiartsmoenftqauanld,ownereepauisredpefrorpebn.looIdn acdoldlietcitoion4n, during the test. The pens that were used for blood collection during the test were not ubsyeWde1e0kas3s,estsheretprreoadtucmteinotn.levDeulewatosorveedrutcseidgntso o2f0topxpimc.ityTnhoete1d5i0n/2t0hep1p5m0 dpopsme twraesattmheennt stiegrnmsionfattoexdicatitWy.eekAll5.reTmhaein5i0ngpapdumltdoqsuaeilwawserteerombisneartveeddadtaiWleyefkor7,siaglnssoodfuteoxtiociotvyerotr abnormal behavior. Body weights were measured at Weeks 2, 4, 6, 8 and at study termination. extra pair of Feed adult cqounasilumapttifoinvew-waesekmeiansteurrvealds.weekAlty.timBeloofodadwualts collected from the termination, blood sfarmopmletshewe1r5e0/c2o0llpepctmedtrferaotmmenatl.l surviving quail ~All bobwhite for chemical quail, except analysis, excluding quail for those in the SO and 150/20 ppm treatments, were euthanized and subjected to gross necropsy. necropsy, tissues were collected for histopathological examination At the time of and chemical analyses. Adult Once quail were brought cgg production was into their initiated, reproductive phase by eggs were set weekly photostimulation for incubation. at Week 7. Eges were 1 cnorry candied so that the developmental stage and any abnormalities could be recorded for each egg set. Reproductive endpoints evaluated during this study included: egg production, embryo viability, hatchability, and hatchling health and survival. After hatching, quail chicks were fed an untreated dit for 14 days. At the end of the study, chick body weights were determined. Prior to euthanasia of the last group of offspring (Lot J), blood samples were collected from 10 chicks from both the control and 10 ppm treatment groups for chemical analysis. In addition, tissues were collected from these chicks for histopathological examination and analysis. Liver weights were recorded for all necropsied quail, and the liver was sampled for chemical analysis. Samples were also taken of the liver, brain, kidney, gonad, proventriculus, gall bladder, adipose, and Bursa of Fabricius for histopathological examination from both adult and offspring bobwhite quails. Test Bird Age: Adult quail were 24 weeks old at test initiation. Number of Replicates: There were 16 pairs of adult quail per treatment with one male and one female adult bird per pen. In addition, 4 pairs of quail were maintained per treatment as parofthe blood monitoring study. Feed and Water: Food and water were provided ad libitum during all phases of the study to both adults and offspring. Feed consumption was measured on a per pen basis, oncea week. Analytical Monitoring: PFOS concentrations in feed, red blood cell, liver tissue, sera and egg components were determined by reverse-phase HPLC and mass spectrometry. Egg components were separated using a method characterized by Berardt and Cook (1960) and Stifuni et a. (1990). Statistical Methods: Upon completion of the study, an analysis of variance (ANOVA) was performed 10 evaluate significant differences between treatment groups. Dunnett' multiple comparison procedure was used to compare PFOS treatment groups with the controls (Dunnett, 1955; 1964). The sample units were the individual pens within each treatment group, except for adult body weights where the sample unit was the individual bird. Statistical analyses of body weights were conducted separately for male and female bobwhite quail. Percentage data (reproduction data) were examined using Dunnett's `method following arcsine square root transformation. Statistical analysisofthe data was. performed using Avian Reproduction Data System (ARDS) Software, a validated software package developed by Wildlife Intemational, Lid. Average Daily Intake (ADI) of PFOS for each treatment group was estimated on a pen basis and did not take into account potential differences between the male and female within a pen. Food consumption and adult quail body weight data were averaged over the exposure duration of the study and the ADI was calculated as follows: 2 GOOF ADHmyKghody ihweigh dayay. Aber FondConsumpion eedbgiyndday) Fesdcon om) `Test Diet Preparation: The test dicts were prepared by mixing PFOS into a premix that was used in a weekly preparation of the final diet. Homogeneity of PFOS in the diet was evaluated throughout the study. RESULTS: Measured Diet Concentrations: To verify the concentrations in the PFOS treatments, feed samples were collected and analyzed. Means and standard deviations of the test dicts are given (Table 1). Feed samples from the control treatment showed no indication of containing PFOS. Analyses of the diet confirmed that PFOS was stable and did not degrade during the study. "Table 1. Mean and standard deviations of PFOS concentrations in the diet of northern bobwhite quail A "Nominal `Measured Concentrations. Concentrations of of PFOS cv Percent PFOS (ppm (ppm) %) Nominal Control (0) <LoQ 10 1020.53 544 102 20 2084214 103 104 50 5094204 401 102 Diet conc1e5n0trations were rporcd1on6a1w4e 15we6igh BRE 9.66 108 PLOQ indicates fmt of quantitation (LOQ= 2.00 ppm) Mortalities and Clinical Observations: No treatment-related mortalities were observed in the control or 10 ppm treatments. However, starting at Week 5, clinical signs were observed among quail in the 10 ppm treatment. Signs of toxicity included reduced reaction to extemal stimuli, ruffled appearance, and lethargy. Within the first 5 weeksofthe study, five-treatment related mortalities occurred in the 50 ppm treatment, and three mortalities occurred in the 150/20 ppm treatment. The expiring quail exhibited overt signs of toxicity prior to death. Due to PFOS-related effects, all surviving quail in the 150/20 ppm treatment were euthanized at Week S, and surviving quail in the 0 ppm treatment were euthanized at Week 7. Signs of toxicity at these doses included reduced reaction stimuli, wing droop, loss of coordination, loss of righting reflex, lower limb rigidity, convulsions, shallow and rapid respiration, ruffled appearance, lower limb weakness, lethargy, gaping, prostrate posture and spasms. Based on clinical signs of toxicity, the Lowest Observable Adverse Effect Concentration (LOAEC) was determined tobe 10 ppm. 3 CRO Adult Body Weight: Compared to controls, there were no apparent treatment-related effects on body weight among quail in the 10 ppm treatment (Table 2). However, stating at Week 2, there was a marked and statistically significant reduction in body weights among quail in the 50 and 150/20 ppm treatments. Based on these results, the body weight Lowest Observable Adverse Effect Concentration (LOAEC) was determined to be 50 ppm, while the No Observable Adverse Effect Concentration (NOAEC) was determined (0 be 10 ppm. Feed Consumption: There were no reatment-related effects on feed consumption for qstuaaritlinign attheWe1e0kpp1,matrteraetatmmeenntt.grreoluaptedwhreednucctoimopnairnedfeteod ccoonntsruolmspt(iToanblwea2s). obsHeorwveevderi,n quail from the 50 and 150/20 ppm treatments. While some recovery in feed consumption was observed in the 150/20 ppm treatment group at Week 4, this improvement was likely due 10 a reduction in the PFOS dose (from 150 to 20 ppm in the feed). Due to the reductions in feed consumption observed through Week 7, the LOAEC for reduction in feed consumption was determined to be 50 ppm, while the NOAEC was determined t0 be: 10 ppm. Liver Weight: When compared to controls, there were no treatment-related effects on liver weight among male bobwhite quail in the 10 ppm treatment (Table 2). In contrast, tfheemraelewsasfraomstatthiesti1c0alplypmsigtnriefaitcmanetnt(pwh<e0.n01c)omtpreaartemdentto.rceolnattreodls.incrBeaasseedinonlitvehresweeirgeshutltisn, the LOAEC for adult female quails was determined to be 10 ppm. In males, the NOAEC based on liver weight was determined to be 10 ppm. Texapbolseed2.toFeveadriocuosnscuomncpetnitornatainodnsboofdPyFaOnSd *liver weights of northern bobwhite quail "Treatment ADIT Average Feed Body (pm) (mg/kgiday) Consumption weight Liver weight @ fecdbirdday) Sex (@) 1) Control NA 10:2 M 205216 443%050 Fo 247420 866111 10 07724006 1943 M 208511 448055 Fo249420 109%+160 50 2644024 942 MFo1s79s04e20 NAC 150 7324126 [3] M 187514 NAC F114 Vamelasuureemaernretsg,ivweinthacvontmreoalnsandan1d0 pstapndarrcdamdeenvtiastisoanmspledBoadtyWeacnkd 2I1v,erwhiwle tehe a5i0reagtnderh1m5i0ns2a0l 2p0omfortrceoanttmreonltsanwde1r0pspammplgerdouaptsWsenedkW7 eand6e,aknrdes3pefcotirvSe0ly.andF1e5e0d12c0onpspumm.prieosnpedcatitvaewlyas for Week ANDAI==Naovteraapgepdaliilycinnaotsbakalempe(lm;eg rPeFpOoSrtIekdg body weightdey). + Asterisk indicate atscaly sgnifiant diffrence from the cone treatment tp< 0.01 4 COE 80 GPrFoOsSs Praetlahtoeldogeyf:feNctescraomposyngresquulatisloinnaltlhesur1v0ivpinpgmfetmraelaetsmeinntdiwcahteend tchaotmpthaerreedwetroetnheo controls. However, male quail in the 10 ppm treatment had a greater incidence of reduced tests size (6 out of 16 males) than the controls (1 out of 16 males). All other findings were considered incidental and not related to treatment. Histopathology: There were no lesions in the liver, kidney, brain, proventriculus, adipose tissue, or bursa fabricius in either adult or juvenile bobwhite quails from the 10 ppm treatment. In adult males, an increase in the incidence of small testes size in the 10 ppm treatment was not accompanied by any morphological change in spermatogenesis. This suggested that PFOS might have accelerated post-reproductive regression, a normal physiological process. Based on these results, the LOAEC was determined to be 10 ppm, based on increased incidenceof small tests size. Reproductive Results: When compared to controls, there were no treatment-related effects on egg production in the 10 ppm treatment. However, while not statistically significant, there were slight reductions in the number of viable embryos as a percentage of egg set, hatchlings as a percentage of live 3-week embryos and in 14-day old survivors as a percentage of hatchlings. Slight reductions in fertility, hatchability and offspring survival also resulted in reductions in the number of hatchlings and 14-day old survivors Washipleercselnitghatg,esthoerfebowtahs tahestnatuimstbiecarlloyfseiggngisfisceatntarneddutchteiomnaxinimthuemnunmubmebrerofof14e-gdgasysoetl.d survivors as percentagesof the number of eggs set. Based on this effect, the LOAEC was determined t0 be 10 ppm. lAidvuerltsaBmlpoloeds asnhdowLeidvethratPaFdOulSt qAunaaillysiinst:heRes1u0lptspmofttrheeatcmheenmticgarlouapnaalcycsuimsuolfatbeldooPdFaOnSd during the study (Table 3). At the termination of the study, liver and serum PFOS concentrations measured in males from the 10 ppm treatment group were approximately 16-18 times greater than the concentrations measured in females. While there was a sex specific difference in tissue concentrations, the serum to liver ratio for both sexes was similar with a male ratio of 1.6 and a female ratio of 1.8. Since concentrations of PFOS were measured only in quail from the 10 ppm treatment, a quantitative relationship between PFOS exposure and liver or serum concentrations was not developed. 5 CRON `Table 3. Mean concentrations of PFOS in liver and serumofadult quail at study termination. * Treatment Tiver Cone Serum Cone. Co(nptprmo)l SeTMMx ug) <L0Q" (ug/ml) <10Q F <L0Q <L0Q 10 M 8852285 14130 F 49:10 87426 Omnelayncsoannd tsaarnnddoa0rldpdepvmiastaimopnlsesonwaewreetnwaetiygehetdbafsios.m study. Alvis are reported 5 L40Q0 pg mLlimit ofquantaton: for ver it was 0.0502 gs while or serum twas Measurement of PFOS concentrations in blood collected throughout the reproduction study indicated that PFOS accumulated in quail in both a time and sex dependent manner (Table 4). `Table 4. Concentrations of PFOS ug/ml) in serum collected from adult bobwhite quail Treatment (pm) Sex 5 Samp1l0e time (week) is Control M0024 0.008 <L0Q" oq 10 Fo 00120004 M 9851 1<5L8O24Q2 1<64L+05Q1 F 84:28 S638 89417 Week 5 Week 7 50 M 557 NA F 525 495 PROS concentrations are eponid as means and sandard devitoms "LOQ- Limitofquantitation; The LOQ wes0.01 pgm. BHoyweWveeerk, Sa,t sWeereukm P10F,OsSexcosnpceecnitfriactdiiofnfserdeindcensotindisfefreubmetPwFeOeSn mcoanlceenatnrdatfieomnaslebeqcuaamiel apparent. The concentrations of PFOS in male bobwhile quail were significantly greater than those observed in females. Moreover, this trend continued to Week 15. Female bobwhite quail serum PFOS concentrations were 18-fold less than those observed in males sampled at the same times. The decrease in female serum PFOS concentrations coincided with the onset of the egg-laying phase in the study. This would scem to indicate that female quail transferred PFOS into their cggs during reproduction. In contrast, male serum PFOS concentrations reached an apparent saturation concentration by Week 10, and they did not statistically differ for the remainder of the study. Concentrations of PFOS were also measured in the red blood cells of blood samples collected at each sampling time (Table 3). 6 oyu Table 5. Concentrations of PFOS in red blood cells (ug/L) from adult bobwhite: quail exposed to various concentrations of PFOS * Treatment Sex Sample Period (week) (ppm) 3 10 is Control M <T0Q <10Q <10Q F <L0Q <L0Q <10Q 10 M 2674232 184325 35226 F 280226 74482 20:10 Week 5 Week 7 50 M 165 359 F 106 10 "ReLdLOQb=lLonidmicetllofcoqnucaentnittraattiioonn:sairn edrepbolrotoeddcaelmsetahnesL3OnQdwsansda0.r0d1 dge/vima.tions There was an apparent sex difference in red blood cell mean concentrations at weeks 10 and 15 but not at week , although there is substantial variability in the data. Red blood cell PFOS concentrations decreased in both male and female over time, particularly from weeks 10 10 15. The ratio of serum to red blood cell concentrations were also similar in `males and females at all ime points but the ratio shifted over time as concentrations in red blood cells declined more than serum levels by week 15. While the biological significance of PFOS concentration changes in red blood cells versus serum is unclear, the blood results show that PFOS concentrations declined during the reproductive phase. Concentrations of PFOS in Blood and Liverof Juvenile Quail: The analysisof serum and liver samples from juvenile quail indicated that PFOS was still present in these quail 12 weeks after hatching. The PFOS concentrations in both liver and serum were increased in the 10 ppm treatment group over that measured in the control group, but were less than the concentrations measured in adult quail from the same treatments (Table 6). Unlike adult quail, no sex specific differences were observed in juvenile liver or serum PFOS concentrations. The serum 10 liver ratio for PFOS in male and female juvenile quail averaged 1.92 + 0.46 and 1.97 = 0.80, respectively. The juvenile serum to liver ratio was similar to the ratio observed in adult qual (males = 1.91; females = 1.42). 7 Coed jTuavbelneile6.quCaioln.ceAntrations of PFOS in the liver and serum of2 week old Treament CoTnhceerntPraOtiSon CSoenrcuemntrPaFtOiSon (ppm) Sex Gig) gmt) Control M <L0Q <L0Q 10 MF 57<6L0O.Q67 12<6L4O3Q37 F 549114 12.4+346 #1i0gQ=hLimitofquatation; OQfo iver 20 gis LOQ for serum = 400 nf Egg Component PFOS Concentrations: PFOS concentrations in egg components were mferaosmuWreedekisn c1g9e.s2c0o,ll2e1ctaedndfryoomlktshewecronetrcoolmpaonsdit1e0dpapnmd tarneaaltmyeznetds,forEgcgonscewnetrreatsieolnesctoefd PFOS. Yolk (whole) composites were then analyzed for PFOS (Table 7). In addition, to bpeptmteturnedaetmresnttandwetrhee dailsstorisbeuptaironatoedf PiFntOoSthirneethefreacgtgionyso,lk,veyroylklsowcomdpeonssiittyesliipnoptrhoete1i0n d(eVtLeDrLm)in,edphionsevaitcihn,yolakndfracltiipoonvi(tTealblilne 7)f.ractions. PFOS concentrations were. then Tcagblyeol7k. cCoomnpcoennternattsioAns of PFOS in bobwhite qual egg yolk and Treatment Fraction TFOS Cwonecelntration C1on0tprpoml YoYlokl-owwhhoolee <LO6Q2"s YolYko-lPhko-sVvLiDtLin 308938 Yolk-Lipovitelin 132 RStEaSndTarrdednevonmexanpsreasndesanprdeacris]ioBneovfahtionpsreopnaraWteiornwandbnaays TofHE #Ldu0pliQ-catLeismiftorohfegusaanmieatuniinq;ueLOsaQm=pl0e0.1cpoelmep.osiofseveral ces. Results indicate that at 10 ppm, there was a significant accumulation of PFOS into the aegqguaynotlikfsiaobflenocrotnhceernntrbaotbiwonhiitnetqhueaciolnstarsolcocmgpgayroeldk.toFcuorntthroelrsm.oreN,orPesFulOtSs swhaoswdeedtetchtaetdthien greatest concentration in the yolk was associated with VLDL fraction, followed by lipovitelln. CONCLUSIONS: Naonrdth1e5r0n72b0opbwphmitfeorquuapiltso 2w1erweeeekxsp.oseDdutotoPsFiOgnSs aotfdoiveetratrytocxoincicteyn,traadtuiltonqsuaoifl 0i,n t10h,e 5500 and 150/20 ppm treatments were terminated at the endof 7 and 5 weeks, respectively. 8 [ERD When compared to controls, there were no PFOS-related effects on adult body weight or feed consumption at a dose of 10 ppm in the feed (Table 8). However, minor overt signs of toxicity were observed in adults dosed at 10 ppm. At 10 ppm, there was a statistically significant increase in female liver weight. In adult males, there was an increase in the incidence of small testes size (Table 8). The increase in the numberof adult males in the 10 ppm treatment group with reduced testes size was not accompanied by any morphological change in spermatogenesis. This suggests that PFOS may have accelerated early post-reproductive phase regression, a normal physiological phenomenon. Additionally, there were slight, but not statistically significant ireatmentrelated reductions in fertility, hatchability, anda statistically significant reduction in offspring survival (p < 0.05). Based on these effects, the Lowest Observable Adverse Effect Concentration (LOAEC) for PFOS reproductive effects in bobwhite quail was determined to be 10 ppm in feed, based on 21 weeks of exposure. PFOS concentrations associated with the Lowest Observable Adverse Effect Level (LOAEL) in both adult and juvenile tissues are summarized in Table 9. `Table 8. Measurement endpoints and associated dietary NOAEC and LOAEC values for PFOS in a chronic study with northern bobwhite quails and their offspring. `Dietary NOAEC Dietary LOAEC Endpoint (ppm) m) ADULT Mortality 10 50 Body weight 10 50 Feed consumption 10 50 Liver weight Maks = 10 Females = 10 Females <10 Males = 50 Gross pathology Females = 10 Males = 10 Histopathology Males < 10 10 Females = 50 Reproductive 10 OFFSPRING 14-day survivabilit 10 10ATpcpomncgernoturpateixopnossreedpfoorid21owneekwse:r5w0eipgphtmbgarsoiup exposed for 7 weeks 9 6oo is `Table 9. LOAEL values in various matrices in adult and offspring northern bobwhite quails in a chronic dietary study with PFOS. Measures of PFOS Exposure TOAELTM ADULT MALES Dose (ppm) 10 ADI (mg PFOS/kg body weightiday) over 21-week period 0772 `Serum (ug PFOS/mL) at study termination (21-weeks) 141 Liver (1g PFOS/g) at study termination (21-weeks) 885 ADULT FEMALES Dose (ppm) 10 ADI (mg PFOS/kg body weightday) over 21-weck period 0772 Serum (ug PFOS/mL), pre-reproductive phase (5-wecks) 84 Serum (ug PFOS/mL), reproductive phase,a study termination(21-weeks) 87 Liver (ug PFOS/g) at study termination (21-wecks) 49 OFFSPRING Yolk (ug PFOS/mL) 2 Liver (ug PFOS/g) 55 TTOSAeErLumwa(sugbasPeFdOoSna/dmeLc)ree Tn the 14-0ay ld survvabinyofofsping. creased mcidenceofsm1a2ll.5sed resetpeosreindaodnulatmwaeltesw,eiagnhtdabassisa.tsicaly significant increase in adult female ver weight. All concentrations LroepwoerftfeedctasvamleuaessufroedditicstsuaendvaAlDuIesae reportedasdicary concentrations. Scrum, and ivereffect values see LdOaAyEsLurvvailvuaebsilfyorfomralreespraodrubcatsioendonhistopathology, or females on increased liver weight,andon reduced 14- Offspring liver nd serum LOAELvalues ae averages of female and male concentrations. DATA QUALITY: Reliability: Klimish ranking = 1 REFERENCES Berard and Cook. 1960. BBA 44: 86-96. Decker, E.R., Flaherty, JM. 2003. Analysis of perfluorooctane sulfonate in mallard and quail egg yolk. Exygen Report No. 023-070. Dunnett, C.W. 1955. A multiple comparison procedure for comparing several treatments witha control. J. Amer. Satis. Assoc. 50: 1096-1121 Dunnett, CW. 1964. New tables for multiple comparisons with a control. Biometrics 20: 482-491 10 Cos Gallagher, study with SP, Van Hoven, RL, the Northern Bobwhite. Beavers, JB, Final report. Jaber, M. 2003. PFOS: A reproduction Wildlife International, Lid., Project No. 454-108. Gallagher, S. 2003. Extraction of Potassium Perfluoroocatnesulfonate from Red Blood Cells and Serum for Analysis using HPLC-Electrospray/Mass Spectrometry. Exygen Research, Exygen Study Number 023-066. Reagen, WK. 2002. Analysis of Perfluorooctanesulfonate in Mallard and Quail Egg Yolk. Exygen Research. Exygen Report No. 023-070. Stevenson, L.A. 2003. Analytical phase report for PFOS: A reproduction study with the Northern Bobwhite. 3M Environmental Laboratory Study No. EO1-1245. Stifani, S., Nimpf,J, Schneider, W.J. 1990. Vitellogenesis in Xenopus laevis and chicken ~lipcoovgintealtien-lIi.gandBsIaOnLdCooHcEytMe r2e6c5e:pt8o8r2s--8t8h8e.binding site for vitellogenin is located on Wildlife Internationa, Lid. 1994. Avian Reproductive Data System (ARDS), Versio2n, OTHER Last changed: 5/05/04 n coon