Document 20oOD6LdyJy03aqmxygpM9Bg

^(* AR226-2762 FOR DU FONT DSE ONLY Copies to: E. I* du Pone da Neoourc and Coapany HaslMll Laboratory for Toxicology and Industrial Medicine Elkeoa Road, Hswsrk, Oslesere 19711 HASHELL LABORATORY REPORT NO. 205-81 Material Tested Occanolc add, pontndacafluoro-, aaaoalua salt* Haskell Ho. 12,037---- Other Codes/Synonyms C-8 ^ Study Inltisted/Cospleeeg """"""Sylg/go - s^237wr "' Mseerial S-ibaitted by 'Polymer Products Dapartnent Washington Works SOBACPTEIiMHALATIOli TOXICITY OF PEHTADECAFLPOROOCTAMOIC ACIP. AMCNIPM SALT . Introduction- Pentadeeafluorooccanoic acid, nBaaniua salt (C-6if^BR has previously been ahown to be highly toxic Co rats by inhalation f'KUC-9.8 "S^jym^SfU^ aod t eauae^slgniftc<ne aniasgeaent of the li^sr^ An inhalation subacuta etudyQlimiWq prfpnBad at 11 and 83 nig/m' caused ratB^IIHB^ liver degeneration, enlarged liters and increases in liver^ enzymes a^Jbofeh concentrAtiona. A recent ubaciCe deraal gtudy vlth &fc 20, 2007 and-27000 aig/Kg/day for-lB Sryi aaa vtaSSwe yilndliigs at airdoe7 along with elevatad blood fluoride throughout the 56 day test. The purpose of this sueacujt Inhalation study with rats at 0, 1, 8, and 80 eg/a was three fold: : 1. attempt to achieve a ''no observable effect" concentration 2. correlate liver pathology, blood organofluoride and blood serua enzyae changes with do<s and clae 3. hold animals for prolonged observation co determine reversibility of effects. General Protocol Six-week old male Crl:CD ^ac were housed 2/cage in stainless-steel wire oesh cages and provided Purina Certified Rodent Chow ^5002 and water ad libitum. Prior to exposure .-its were quarantined for 10 days to assure they exhibited no overt signs of disease and a noraal race of growth. III, II, Rats were tested at 7-8 weeks of age - weighing 2AO-279 g. Design concentrations of C-8 ware 0, 1, 8, and 80 Bg/n for Groups I, and IV, respeccively. Three groupsiof 24 rats were exposed to dusc acmo&pheres 001 Company San,taed. Does not contain TSCA CBf I f III, of C-B (II, and IV), whila an identical control group (I) was II simultaneously exposed to air only* Group I and were contained in one III high velocity hood. Group and IV in another. Group I wae handled with clean gloves during each loading and unloading. All groups were restrained in wire mesh holders and exposed head-only in SO I glass chaBberB for 6 hr/dey, 5 daye/wk for 2 aks (excluding weekends). During exposure rate were observed and clinical signs notedo Post-exposure, rata were weighed and observed daily for 14 recovery days, then weighed and observed twice/wit through 84 days recovery. By design 5 rats/group (until fewer than 5 remained) ware sacrificed at 0, 14, 28, 42, and 84 days recovery, for a total of 96 test day* Generation Procedure Dust atmospheres of C~8 were generated by peeeicg air through a glass generator (2 or 3 stages, depending on desired concentration). An electric aotor and stael rod with plastic paddles agitated the first 2 stages, if necessary to achieve the desired concentration. : Analytical Procedure Two types of analytical methods were used to Bonitor C8 concentrations during exposures. ! ^^ For the high concentration (80 ag/a ), cheaber atmosphere concentrations were priBarily determined by gravimetric analysis. Known volumes of chamber air were drawn through preweighed Geloan glass fiber filters, Type AE, 47 as ^^ at 2 1/min. Samples were taken once per hr. for 30 to 50 oinuees. Concentrations ware-determined' from weight gain (g) of-the fi-l-ter/1 of chamber air. During each expo sure samples from this chamber were also analyzed by wet chemical anelycis (below) in order to cross-check the methods. For the first six exposures 2 1 samples were taken each hr. Fcr the last 4 exposures two of the filters from the gravimetric analysis were choi-en to be analyzed. The C-8 was eluted from the filter as described below* : For the intermediate and low concentrations, chamber atmosphere samples were collected ice per hr (15' I for ineeEaedilsea 100 1 for low) on Selman glass fiber filtere, but wsig^e gei-n could not ba measured. A wee eaeaieal analytical method was used instead. This consisted of dissolving the C-8 from the filters into acidified aqueous methylene blue solution. A colored conplex forms which is excracCed into a nonpolar solvent (e.g., chloroform or aethylene chloride). Concencristioas of C-8/aethylene blue complex ware spectrophoeoaetrically determined 9 650 MB and chamber concentrations were determined by comparing to standards prepared daily. A Sierra Cascade ifflpactor Hodel ?218K Eight Stage was used to detcrfsine particle size. ^""^"-'"-"o.c.^.^cs, - 2 - 002 Clinical Cheaiatry Protocol Following che tenth axpocora (0 daye recovery) and recovery day 14, 28, 42, and 84, blood was taken frda ehe phosphatase calla (AP), of 5 rats in each group. glutaaie-oxaloacetie transaainase Measureaencs of alkaline (GOT) and glutaBic-pyruvte craneaainaae (GfT)were Bade of the blood aerua. Detail* are in ehe clinical pathology report. Pathology Protocol Following the tench exposure (0 daya recovery) and recovery daya 14, 28, 42, and 84, five rata froa each group were sacrificed for pathologic examined grossly and selected ciesues evaluation. At necropcy the rats ware and organs were aaved for ler^aeople evaluation. Weights of the lungs, heart, thyaiM, spleen, livsr, peetes, and kidncya-were obtained. Details of pathological indices are in the pathology report. Body Weight and Organ/Body Weight Analysis II, III, Mean body weights were ca,lculted and plotted to show growth curves of Groups I, and IV froa 0 through 96 days on test. At each sacrifice aean orjgan eeifhte (listed above) and orgsn te body weight ratios were calculated. Procedure for Analyeis of C-8 in Rat Blood At ascrnpsy ^pproxiaately 8 to 10 01 of blood u&s collected (in heparinlzed tubes) froa each rat by cardiac puncture. The blood wa atored at 4"C until all" samples were collected. II III periodB.loodBlosoadmpsleasmpflreos afrGoraouGpsrouI pssad IVanwd ere awnaelryezeadnaalytzeedachonrleycoavt e0ry and 28 recovery days. byHl^BilHB^ Analysis of the blood was conducted Polymer Products Department, Experimental Station. Details appear in Appendix 4. .Company Sanitized. Does not contain TSCA CB! 0 3- 003 Result*; Exposure TiBe-Weighcad-Avaraga (TWA) eoncencratioM and ranges are tabulated below. Comparison of the two analytical methods at the hig^ level denonatraced that ehe 2 1 samples, taken for wet chemical analysis, i e not representative of ehe 60-100 1 samplae taken for gravimetric analysis. These aamplea tended to be lorr (12 to 45Z) than the gravimetric analysis. Direct wet chemical analysis of filters used for the gravimetric analysis shoved that the two methods uere comparable (wet method 6Z lower than gravimetric). Bxpoaure Group II Group III No. TOA.ng/n"^RangaTWA.ag/^Range Group IV THA.ng/ttRange 1 0.1 0 - 0.2 6.7 0.5 - 10.5 110.0 17 - 167 2 0.6 0.02 - 1,2 10.7 5.5 - 21.9 83.5 44-148 3 1.5 0.4 - 2.8 9.0 2.4 - 16.5 79.5 23 - 136 4 1.0 0.7 - 1.3 8.9 5.6 - 13.3 67.8 45 - 115 5 1.6 0.7 - 3.0 10.0 2.1- 24.9 95.1 63-158 6 1.4 0.2 - 2.2 7.3 1.4 - 9.3 88.6 61 - 160 7 1.2 0.5 - 1.8 8.1 4.1 - 15.1 78.1 61 - 97 8 0.9 0.5 - 1.4 7.5 1.7 - 15,0- 89.1 62 - 122 9 1.0 0.5 - 2.0 7.6 0.3 - 11.3 77.4 72 - 86 10 0.4 0.1 - 0.7 1.3 0.7 " 3..2 75.1 67 - 94 Overall mean concentrations were: Group 9 Cone. (relative) Coneenteation, Bg/a S.D. 11 Low III latermedlace IV Hl^h 1.6^ 0.5 7.6 + 2.5 83.9 7 12.8 Hany attempts were oade to determine particle Size but the particles did not behave typically. The information obtained from the high concentration chamber suggests the particles w^re 1 to 2 u. Thd intermediate concentration chamber was analyzed during the $Ch exposure. Mass median diameter was 3.8 u. These data indicate most of the particles generated were respirable. Clinical Signs During exposures a high percentage of the rats had slight to mild nasal III and ocular discharge, which appeared to be dose-ralated. After the third exposure one rat in Group IV was sacrificed "in extremis". During the fourth exposure one rat in Group IV died. Both anissle wre necropsied and tissues were saved for microscopic examimation. Bats In Groups I, II, and showed moderate weight loss for 1 to 3 days and sporadic weight loss throughout the remainder of the exposure and recovery periods. Three of 24 rats la Group IV had lung noise and all showed aavere wight loss during ehe 12 day exposure period with sporadic weight loss; throughout recovery. - 4 - Company Sanitized "oDooeas.n,^o.t contain TSCACBI 004 C; ( ) s-- o f: ^ Body Weight Analysis Maan body weighta were evaluated statistically (Appendix 1)* Groups I 6 II were not significantly different. Group ZIZ was not significantly III different froa I and II Ui-'U recovery day 5, when Group weights were higher for 13 day. Group IV weights were significantly lower froa test day 2 through 16 (Figure i). Organ Keight Analvsia Organ/Body Weight Ration Relative organ Co body weight ratios for each g-'oup after each recovery period are outlined in Table 1 and Appendix 3. The dose-related changes include increased liver, lungs:and tes-es weights. Liver/body weight ratios are depicted graphically in Figure 2. Other changes in organ/body weight ratios, were found (Table 1). relationship to compound adainistratioc is unclear. Their Mean Absolute Organ Weight Analysis Mean absolute organ weight analysis (Appendix 2) deaonocrated findings similar to organ/body weight ratios (Table 1). However, this analysis III, III appeared Co be more sensitive, in thae i": showed more dose-related changes is Group but this may be an artifact caused by an increase in Group body weight" (which cannot be esplslnad) accaspssied by a noraal increase in organ weights. J Other changes in mean absolute organ weights were found (Table 1). Their relationship to compound!administration is unclear. Clinical Laboratory Measurements III Elevated alkaline phoapha^aae (AP) activity was found in all groups exposed to C-8 after 10 exposures (0 recovery), but only Groups and IV were significantly different from Group I. After 14 days recovery AP was elevated only in Group IV. After 28, 42, and 84 days recovery no differences were found. Pathology Macroscopic The morphologic changes observed at necropsy were varied. The only change considered to be compound-related was hepatic (i.e., heavy livers). II III This occurred ia 1/24 Group I rats, 0/24 Group rats, 5/24 Group r-ts and 10/24 Group IV rats. This change was dose and recovery-time related. III After 14 days recovary this change vs&e not found in Group and after 28 days was not found in Group IV. CompanySanitized Dn.oesootcon^TSC^ 005 ^P Microscopic ^\ Three compound-related hiatomorphologie changes were observed. All uere hepatic. These include panlobular hepacocellular hypertrophy, centrolobular hepatocellular hypertrophy and hepacocellular necrosis. All three lesions III II. were observed in Groups and IV but not in Group One lesion was observed in Group I. recovery-tine related The presence of hepatic lesions was dose and as outlined below. After 28 daya recovery all changes III are considered to b* reversible in Group and reversible by 42 days in Group IV. Number of Rats with Microscopic Hepatic Lesions Recovery Day 0 14 28 42 84 Group I 0/5 1/5 0/5 0/5 0/4 Group II 0/5 0/5 0/5 0/5 0/4 Group III 5/5 1/5 0/5 1/5 1/4 Group IV 5/5, 4/5 2/5 0/4 0/3 Cause of death could not be determined for the two rats that died or were sacrificed "in exegesis" is Group IV. Blood Fluoride Analysis Blood fluoride ass elevated is all groups (including controls) 0 through ( . 84 days recovery and was dose-related as shown on the table below. However, --' after 42 days recovery, blood fluoride in the controls BBS only 4 S above the ligit-of detactafclllty (0,007-u(>- while Group IV .fluoride was 250 X above. After 84 days recovery the control blood fluoride was only 2 X above background, while Group IV was still 120 X above. The presence of C-8 in control blood was probably caused by cross-contaalnation during handling, exposure or housing. Average Concentration of C-8 in Rat Blood. ugF/g Recovery Day 0 14 28 42 84 Group I 5.3* 0.28 0.10 0.032 0.015 Group II 13 1.2 Group III 47 3.8 Group IV 108 10 . 7.1 1.8 0.84 * 1.4 excluding one high value 006 ^mpanv san.taea.boas oesnnoot, contain TSCAC8(. i^ Summary Pentadecafluorooctanoie eld, ammonium 16 (C-8;^J|^B} was adainlscered co male Crl;CD rats by inhalation 6 hr/day, 5 days/wk for 2 wfcs at concentrations ng/n (Group IV). of 0 (Group I), 1.0 (Group II), 7.6 (Group III), and 83.9 Observations of elinic&l signs during exposures showed only slight signs of dose-related to^city (nasal and ocular discharge). However, at the high concentration after 3-4 days on test one rat died during exposure and one rat uas sacrificed "in extremis", Both of these rats were severely losing weight. Mortality ws probably exposure-related although pathologic evaluation could not deteemiae cauBe of death. The other rats in this group lost a significant amount of weight during the exposure period. III II. I) Body weight analysis deaonstraced no significant differences between controls (Group and Group Group body weights were significantly higher than controls from test days 17 through 33. Group IV body weights were significantly lower than controls from test days 2 through 16. Organ to body weight ratios demonstrated a dose-related significant increase in lung, liver and tastes weights after 0 recovery days. The liver/body weight raeioe osre significanely higher in Group IV through 28 III days recovery. Mean absolute liver weights were significantly higlt.-- in Group TTI through 28 days recovery, but this may be an artifact caused by an unexplainable increase in Group body weights accompanied by a normal increase in liver weights* Clinical laboratory measurements demonstrated an increase in alkaline phosphatase(AP) in allr groupfr exposed to C-8 after 10 exposures, which persisted in Group IV through 14 days recovery. Pathologic findings included a macroscopic liver change (heavy livers) III and three types of microscopic liver lesions in Groups and IV. Both findings showed a dose-response; relationship but were reversible by 28 days III) recovery (Group or 42 days recovery (Group IV). Other charges were found (organ weights and pathologic lesions), but their relations-tip to compound administration is unclear. Blood fluoride analysis clearly demonstrated a dose-related presence of C-8 in all groups including the controls. The amount of C-8 in blood decreased with time, but was detectable after 84 days recovery in both controls and Group IV. Company Sanitized. Does not contain TSCA CBI 7 - 007 Conclusions eitheAr d0a,ln1i.s0t,rac7i.o6n, oorf 8C3-8*9 baygI/na hadlaemtioonnstrfaocred5 cdhaey foa llwoeweikngfo: r cwo weeks at & no-effpc-c concentration of 1 nig/a for this study Exposure-related mortality at 83.9 ag/a An adverse effect on body and liver weight at 83.9 . mg/m Blevaceii serja alkaline phosphatase at 7.6 and 83.9 Big/in Macroscopic and nicroscopic liver Reversibility of toxic effecta by pathology 28 42 at 7*6 and 83.9 , ng/m Persistent dose-related presence of co C-8 in day recovery blood of all groupa including controls Of the paraaeterj exanineri, aosc sensitive and blood fluorlde analysis proved to be the Expoanre to f-8 is persistent iacticacor of C-8 exposure difficult to avoid as indicated by concrols ! presence in * Composition; Contaginanta ;j| Syaesyms s C~8 t Perfluorooceanoic ';cid, ainsoniuB salt ABBonlua perfluorooctanoate 1 Plastics - D e p a -- rtaeat --Metho -- d ,l|^ ^--f^B'u.s*s u e d 10/9/69. 9U\ 2 f B. L. Moore and J. R. Barnes; S-12,037, Clinical "Inhalation Toxicicy off------I^In;ats,"&*( Pathology Report. August 13,^980. - > - f f r i l '' 3 R. M. Everett and U. C. Rrauss:; "Pathology Report No.J^------i" B-12,037, Sept. 10, 1980. - ^'^^^ 8 - C' 008 Work by: ^fe^_ ^. ^g^/ / Stephen D. Raah Technician Supervised by: /WW f\.^^UW^ --2^ ^y ^ .'f^J^/ Mavift R. Brittelli Toxicologlst Study Director: ________Ladd W. Saith P'- ,f;^rch ToxicologiBt c> Aprroved by: ^QA^JLf '3* i^uLjL>______ Gerald L. Kenn^y Chief, Acute Investigations Section ffiB:jrg Pace Isgi.ied; April 15,^981 Report Ho. 205-81 cwv'wfsw*MD^^r^^ 9 - 009 TABLE I a HANGES IN ORGJAN/BODY WEIGHT RATIOS (CHJWmS IS MEAN ABSOLUTE ORGAN NEIG1HTS) R(aeovery Day Organ Group c 3 14 28 t&2 i$4 Liver I II III IV 0 (0) 0 <0) + (+) + (+) 0 (0) 0 (0) 0 (+) + (+) 0 (0) 0 (0) 0 (+) * (-) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Lungs I IT Ijr IV 0 (0) 0 (0) 0 en + (+) 0 (0) 0 (0) o (&) 0 (0) 0 (0) 0 (0) o m 0 (0) 0 (0) 0 (0) 0 W 0 (0) 0 (0) 0 (0) 0 m 0 (0) Kidney I II III IV 0 (0) 0 (0) 0 W 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) + (0) + (*) + (+) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) + CO) + (0) 0 (0) ?estes I II III IV Thyaus I II III IV 0 (0) 0 (+) 0 (+) + (+) 0 (0) - (0-) 0 (0) (0) 0 (0) 0 (0) 0 (0) 0 (+) 0 (0) n (0) 0 (0) , 0 (0) 0 <0) 0 (0) 0 <<?) ^ (8) (T W) 0 <0) 0 (0) 0 <0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Q (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 0 (0) 0 (0) 0 (0) 0 CO) Spleen I II III TV 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (+) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) + (0) + (+) 0 (0) Heart I II HI IV 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (+> 0 () 0 (0) 0 (p) 0 (0) 0 (0) 0 (0) + (0) + (0) 0 (0) 0 no difference froa concrol + increased t significantly diffarent (p < 0*05 from concrol group by - " decreased t Dimnett and/or LSD* 10 - 010 CompanySanitized. Does not contain TSCACBI 0 OMUL aws r\'\ FIGURE 1 FC-143 BODYWEIGH1 GROWTCHU [----JhnzBz LOKLEVB. (Liffi/^ S INT. UEVB. ^ OLMIC/D HICHL|k o.ai6/u i ! 0 i s i S fl u a 71 HATSCN TEST 11 2TO ISO VOSi uiBluoo iou saoa pazniires AueduiOQ LIVER/'BOD- y U-EIGHT RflTIO (PERCENT) 1(fl JH- 0 ts>  tT.-'.-TO.:*" ""VW"V. "-'?5'1f^^"^y^BF^n.:'----.-w)iTMByr-.aitTOw.ypl^.> --"- -... ...., .^^y_..^ --r-"mi*i)ir- .-i'--.-<p)T^ii,*.n.^.i-. ,.^. ., . ^f^y:,.,,-''.,^_ i-.Bffigoyaaa'nfa^ak . : .,^.^..,,..1:,,;.,...A^..^ jj BODY UEIQHT SUMMARY TI------------I H* 12037 9^^B^'- GROUP CONTROLS LOU (.001 MG/L) INT (0.008 MG/L) HIGH (0.08MG/L) F RATIO(l) LSD(2) DUNNETTO) UMS(4) INTERVALS IN TEST DAYS 1. 2. 3. 255.3333 256*2083 259.3333 262.2273+ 2.276 5.7974 6.9919 99.5832 251.0417 248.2500 249.7500 243.7273+ 2.050 6*1575 7.4261 112.3369 252.3750 247.1667 251.3333 243.4545* 3.339 6.2306 7.5143 115.0194 4. 253.0833 251.5000 252.4583 238.0909* 8.656 6,7015 8.0822 133.0623 0 5* 256.2917 254.0833 255*9583 236.72734 12.35l 7.3751 8.8946 161.1569 GROUP CONTROLS LOW (.001 MG/L) INT (0.008 MG/L) HIGH (0.08MO/L) ^/\ F RATIO(l) LSD(2) ^^ DUNNETTO) UMS(4) INTERVALS IN TEST DAYS 6. ! 259.0833 254.8333 257.7500 ; 237.9091*: 12.264* 77290 9.3214 176,9943 j 7. 266*3750 261.9167 267.7500 253.409I* 4.721* 8.2386 9.9360 201.1031 8. 270.0417 267.3833 271.7917 261.5909 3.094 8.5216 10.2773 215.1563 9. 10. 272.1667 270.5417 274.4167 258.7727* 4.690% 8.8888 10.7202 234.0999 273*8333 273.7500 275.666Z 253.954^) 9.724 9.0707 10.9395 243*7791 GROUP CONTROLS LOU (.001 MG/L) INT (0.008 MG/L) HIGH (0.08MO/L) F RATIO(l) LSD(2) DUNNETT(3) UMS(4) 9 INTERVALS IN TEST DAYS 11. : 12, 277,0417 274.6250 279.5833 2548636* 11.849* 9.0440 10.9073 242.3445 275.6250 277.9583 283.5417 256.59094 13.711* 8.7298 10.5284 225.7984 - 14 - 014 ^ ^ .sas^ COt^ ^CBl C1 APPENDIX 1 (cont'd) (U RATIO OF AMONG- TO UZTHIN-OROUP VARIATION--ONb-FACTOR ANALYSIS OF VARIANCE. (2) LEAST SIGNIFICANT DIFFERENCE--GIVEN A SIGNIFICANT (ALPHA0.05> F RATIOr ^Y TUO MEANS DIFFERING BY MORE MAN THE LSD ARE SIGNIFICATLY DIFFERENT BITH A VARIABLE-WISE FALSE POSITIVE (ALPHA) ERROR RftTE OF 0.05. <3? PUNNETT TEST--ANY TREATMENT MEAct DIFFERING FROM THE CuNiffOL MEAN BY MORE THAN THE DUNNETT STATISTIG-IS-SIBNIFICANTLY UITH A VARIABLE-WISE FALSE POSITIVE <Ai-FH^) DIFFERENT FROM THE CONTROL ERROR RATE OF 0.05. MEAN- --- (4) UITHIN-6ROUP MEAN SQUARE. + SIGNIFICANTLY DIFFERENT <P<0.05) FROM CONTROL GROUP BY LSD. * SIGNIFICANTLY DIFFERENT (P<0.05) FROM CONTROL GROUP BY DUNNETT TEST AND LSO. SIGNIFICANT AT THE 0.05 PROBABILITY LEVEL. ,,,,^csl -_ ^^i^^ ois -is- ^M APPENDIX i (cont'd) BODY_UEIGHT SUMMARY H 12037 OROUP CONTROLS LOW (.001 MB/L) INT <0.008 MG/L) HIGH (0.08MG/L) F RATIO(1) LSD(2) DUNNETT<3> UMS(4) INTERVALS IN TEST DAYS 15* 16* 17. 295*4211 294.5789 304.3AS4 283.1765+ 5.756 10*9630 13*2205 280*0308 300.6316 301*0000 311.1053 287.7059+ 5.447 11.3719 13.7149 301.3677 303.5789 305.6842 317.2105* 294.823S 5,240* 11.2223 13.5345 293.4909 18. 302*8947 305*8421 317.2632* 292.2353 6347* 11*3577 13.6978 300*6151 19. 311*7365 314*3158 32&.1053* 304.7647 4.494 11*7205 14*1352 320.1234 GROUP CONTROLS LOW (* 001 MG/L) INT (0 .008 MG/L) HIGH (0.08MG/L) |) F RATIO(1) LSD(2) DUNNETT(3) WMS(4) INTERVALS IN TEST DAYS 22. 23* 24. 9S. 323.7368 327*8421 340*6842* 320.0000 4.094 12.5176 15.0966 365*1474 329.4211 335*0526 346.5789* 326*8235 3,609* 13.0048 15*6841 394*1240 342.5789 345*6316 35a.4737+ 341*8824 2.432 14.0325 16.9236 418.8793 348,0526 351.S947 36S.P105+ 348*8824 2*459 14.4293 17*4021 485*1951 26. 350*7368 355.9474 369*7368* 351.23SCJ 3.017 14*4640 17.4440 487*5339 (1) RATIO OF AMONG- r0 WITHIN-G^OUP VARIATION--ONE-FACTOR ANALYSIS OF VARIANCE. (2) LEAST SIGNIFICANT DIFFERENCE--6IVEN A SIGNIFICANT (ALPHA=0.05) F RATIOr ANY TUO MEANS DIFFERING BY MORE THAN THE LSD ARE SIGNIFICATLY DIFFERENT UITH A VARIABLE-UISE FALSE POSi:tVE (ALPHA) ERROR RATE OF 0.05. (3) THAN UITH DUNNETT TEST--ANY TREATMENT MEAN DIFFERING FROM THE CONTROL MEAN BY MORE THE DUNNETT STATISTIC IS SIGNIFICANTLY DIFFERENT FROM THE CONTROL MEAN A VARIABLE-UISE FALSE POSITIVE (ALPHA) ERROR RATE OF 0.05. (4) MI THIN-GROUP MEAN SQUARE. + SIGNIFICANTLY DIFFERENT <P<0.05) FROM CONTROL GROUP BY LSD. SIGNIFICANTLY DIFFERENT (P<0*05) FROM CONTROL GROUP BY DUNNETT TEST AND LSD. SIGNIFICANT AT THE 0.05 PROBABILITY LEVEL* 016 - 16 - ComP^ Sani^'^ ^ ^ APPENDIX 1 (cont'd) BODY-WEIGHT SUMMARY 11 H 12037 GROUP INTERVALS IN TEST DAYS 30. 33* 37. 40. CONTROLS LOW (.001 MG/L) INT (0.006 MG/L> HIOH (0.08MG/L> 375*0000 376*8572 393.9286 376.6667 388*5000 392*7857 410.3571+ 393.1667 403.2143 401*9286 424.0000 407.6667 414. 42B6 416* 1429 435. 9286 420. 4167 F RATIO(1) LSO(2> DUNNETT(3) UMS(4) 1*687 19.8178 23*9589 653.2662 1.750 311564 25*5772 744.4948 1.736 22*3711 27*0457 832*4391 1.418 23.8561 28.b409 946.6198 (1) RATIO OF AMONG- TO UITHIN-OROUP VARIATION--ONE-FACTOR ANALYSIS OF VARIANCE. (2) LEAST SIGNIFICANT DIFFERENCES-GIVEN A SIGNIFICANT <ALPHA=0.05> F RATIOr ANY TUO MEANS DIFFERING BY MORE TWAN THE LSD ARE SIGNIFICATLY DIFFERENT UITH A VARIABLE-UISE FAL8E POSITIVE (ALPHA) ERROR RATE OF 0.03. /' <3) THAN WITH SUNNETT TEST--ANY TREATMENT WEAN BUFFERING FROM THE CONTROL MEAN SY MORE THE DUNNETT STATISTIC IS SIGNIFICANTLY DIFF@%:NT FROM THE CONTROL MEAN A VARIABLE-WISE FALSE POSITIVE (ALPHA) ERROR RATE OF 0.05. WITHIN-GROUP MEAN SQUARE. SIGNIFICANTLY DIFFERENT (P<O.OS! FROM CONTROL GROUP BY LSD. * SIGNIFICANTLY DIFFERENT (p<0.0^) FRQM CONTROL GROUP BY DUNNETT TEST AND LSD. 17 - 017 TSC^ Does ^ .contain comply Sanded. OROUP CONT ROLS LOW <.00l MO/L) INT <0*008 MG/L) HIGH <0.08MO/L) F RATlO(l) LSO<2) DUNNETT(3) UHS(4) [- - T 'ArPBNDIX 1 (COTSIt'd BODY UEIGI-IT SUMIMARY 12037 INTEIVALS IN TE3T DAY 5 44, 46* 51. 433 .7776 438 9889 452 .9889 423 .1429 1*284 30.9153 37 .8438 962 <4064 440*2222 4450000 459.4444 430*2857 1*250 30*0398 37.7513 957.7069 459 .3333 463 .8889 476 .4444 450 .2857 0.821 34 .1047 41 .7479 1171 .2180 54. 473 .1111 471 .7778 486 .5556 460 .2857 0.664 37.2445 45 ,5914 1396 .8032 > SB. 475,5000 468*7500 485.0000 481*0000 0.114 66*8715 83.8552 1704.1591 (1) RATIO OF AMONG- TO MITHIN-GROUP VARIATION--ONE-FACTOR ANALYSIS OF VARIANCE. (2) LEAST SIGNIFICANT DIFFERENCE--GIVEN A SIGNIFICANT (ALPHA=0.05> F RATIO ANY TUO MEANS DIFFERING BY MORE THAN THE LSD ARE SIGNIFICATLY DIFFERENT hITH A VARIABLE-WISE FALSE POSITIVE (ALPHA) ERROR RATE OF 0.05. (3) THAN WITH 4) DUNNETT TES r--ANY TREATMENT' MEAN DIFFERIN6 FROM THE CONTROL MEAN BY MORE 0 THE DUNNETT STATISTIC IS SIGNIFICANTLY DIFFERENT FROM THE CONTROL MEAN A VARIABLE-WISE FALSE POSITIVE (ALPHA) ERROR RATE OF 0.05. UI THIN-GROUP MEAN SQUARE. + SIGNIFICANTLY DIFFERENT <P<0.05) FROM CONTROL GROUP BY LSD. SIGNIFICANTLY DIFFERENT <P<O.J05) FROM CONTROLGROUP BY DUNNETT TEST AN& LSD. 18 - 018 Company Sanitized. Doe* not contain TSCA CB(\. APPENDIX 1 (cont'd) BODY UEIOHT SUMMARY ' fl H* 12037 GROUP CONTROLS LOW (.001 MG/L) INT (0.008 M8/L> HIGH (0.08MO/L^ F RATIO(1) LSD(2) DUNNETT(3) UMS(4) INTERVALS IN TEST DAYS 61, 65. 68. 483.2500 476.2500 493.0000 491.6667 0.133 67.8778 8S.1171 1755.8333 494*2300 486*0000 505.7500 504.3333 0*174 70*2745 88.1225 1882.0152 503.0000 494,0000 512.0000 513.0000 0.148 72*52^3 90.9500 2004.7273 72. 307.7300 500*2500 519.0000 526.6667 0.220 70.3120 95.6934 2219.2879 75. 514.5000 510.0000 529.5000 535.0000 0.209 80,0436 100.3727 2441.6-364 GROUP CONTROLS QM (.001 MO/L) T (0.009 MG/L) HUGH (0.08MG/L) w F RATIO(l) LSD(2> DUNNETT(3> UMS(4) INTERVALS IN TEST DAYS ,' 7?.. S2. S6. 523.2500 517.2500 538.5000 543.0000 0.224 79.6470 99.8754 2417.5000 528.7500 523.5000 543.5000 554*0000 0.278 82.4204 193.3532 2588,7954 536.7500 528.2500 551.0000 558.3333 0.241 84.8947 l0^.45'39 2746.5606 89. 543.2500 533.7500 557.0000 565.6667 0.231 90.1867 113.0919 3099.6516 93. 549,7500 541.2500 563.2500 576.3333 0.252 92.6716 116,2079 3272.8106 GROUP INTERSrALS IN TEST DAYS 96. CONTROLS LOU (.001 MG/L) INT (0.008 MG/L> 4IGH (0.08MG/L) F RATIO(l) LSD(2) DUNNETTO) WMS(4> .) 554.7500 541.0000 566.0000 578.6667 0.284 91.5977 114.8613 3197.401A f Company SanHfzed.D>oes not contain T!3CACBI - ^19 APPENDIX 1 (cont'd) (1) RATIO OF AMONG- TO UITHIN-GROUP VARIATION--ONE-FACTOR ANALYSIS OF VARIANCE. (3) LEAST SIGNIFICANT DIFFERENCE--GIVEN A 8I8NIFICANT (ALPHA-0.05) F RATIO ANY TUO MEANS DIFFERING BY MORE +HAN THE L8D ARE SIGNIFICATLY DIFFERENT UITH A VARIABLE-WISE FALSE POSITIVE (ALPHA) ERROR RATE OF 0.05. <3) DUNNETT TEST--ANY TREATMENT MEAN DIFFERINO FROM THE CONTROL MEAN BY MORE THAN THE-DUNNETT STATISTIC 18-SIGNIFICANTLY DIFFERENT FROM THE-CONTROL.MEAN WITH A VARIABLE-WISE FALSE POSITIVE (ALPHA) ERROR RATE OF 0.05. (-4) U I THIN-GROUP MEAN SQUARE. + SIGNIFICANTLY DIFFERENT (P<0.05) FROM CONTROL GROUP BY LSD. * SIGNIFICANTLY DIFFERENT (P<O.Q5) FROM CONTROL GROUP BY DUNNETT TEST AND LSD. 0 020 20- Company Sanefeed. Doaioi contain TSCA CBL- 0 ^ APPENDIX 2 21 - r)o^ Company Sanitized. Does not contain TSCA CB| GROUP CONTROLS LOW LEVEL(0.001MG/L) INT. LEVEL(0.008Mfi/L) + HIGH LEVEL(0.08MG/L) P RATIO(l) LSD(2) DUiWETT(3) WMS(4) 266.8000 284.0000+ 281.0000 262.8000 3.96** 15.6916 15.4673 136.9750 .9420 1.0000 .9700 .9000 1.404 .1076 .1335 .0064 1.4340 1.6220 1.5680 1.6720+ 1.761 .2316 .2874 .0298 10.5340 11.5680 15.97^ 16.4300^ 14.424* 2.3757 2.9473 3.1397 .5380 .6480 .6880 .4320 5.736* .1444 .17^1 .0116 GROUP KIDNEY TESTIS THYMUS CONTROLS LOW LEvEL(O.OOlMG/L) INT. LEVEL(0.008MG/L) HIGH LEVEL(0.08MG/L) P RATIO(l) LSD(2) OUNNETTO) WMS(4) 2.3020 2.4860 2.5840+ 2.4200 1.648 .2765 .3430 .0425 2.7580 3.0280+ 3.0140+ 3.0180+ 2.9S2 .2277 .2825 .0288 .6140 .5540 .6640 .4100? 10.293* .1027 .1275 .0059 Cl) Rac-'o of among- co wichln-group variation--one-factor analysis of variance. (2) Least significant difference--given s significant (alpha0.05) F ratio, any cwo Beans differing by aore than rhe LSD are significantly different with a variable-wise false positive Calpha) error rate of 0.05. C3) DunnetC test--Any treatment mean differing froa) the control mean by oiore than the Dunnett statistic is significantly different from the control a>ean with a variable-wise false positive (alpha) error rate of 0.05. (4) 'rfithin-group Mean Square. + Significantly different (P<0.05) from control group by LSD. a Significantly different (P<0.05) froa control ^roup by Ounnett test and LSD. * Significant at the 0.05 probability level. Company Sann.zed.Does not contain TSCACBI 022 - 22 GROUP _12_0_37_T_IA_T_S.N../B,ODwY WMB- unji HUH saSSUBW SAW SACRITICBD APTBR 14 DAY -.^^ OBSERVATION niRIOD "HAL WGT CONTROLS LOW LEVEL(0.001MG/L) INT LEVEL(0.008MG/L) HIGH LEVEL(0.08MG/L) F RATIO(l) LSD(2) DOMNETT(3) VMS(4) 337.6000 349.6000 362.2000 354.0000 1.400 25.9962 32.2516 375.9500 1.1820 1.2900 1.2940 1.1860 1.745 .1416 .1757 .0112 1.8620 1.8320 2.0080 .7560 1.363 .2722 .3377 .0412 14.2860 14.8880 16.94201 18.3940^ 7.343* 2.0922 2.595& 2.4351 GROUP KIDNEY COTTROLS LOW LEVEL(0.001MG/L) INT LEVEL(0-008MG/L) HIGH LEVEL(0.08MG/L) F RATIO(.l) LSD(2) OUNNETT(3) WhS(4) 2.7860 2.8920 2.9800 2.9320 .825 .2725 .3331 .0413 3.0120 3.2100 3.0740 3.3620+ .8780 .7980 .9300 .7420 1(1) (2) Satio Least of aaong- to lyithln-group variation--onc-factor any tuo significant difference--given aeans differing by aore than Che a significant LSD aaalysia (alpha-O.OS) of variance. F ratio, with a (3) variable-vise false OunnetC cast-~Any positive (alpha) are signlftcantly different error rate of 0.05. than with the Dunnett statisttirceatims enstiganeiafincadniftfleyridnigffefrroemnt thfreoacontrol ean by aore (^) a varlat '-wise Within-group false positive (alpha) error race of the control 0.05. Bean + Mean Square. * Significantly Significantly different different (P<0.05) (P<0.05) froa control group by LSD. ana LSD. fron control group by Dunnert test * Significant at the 0.05 probability level. 023 - 23 - Company Sanitized. Does not contain TSCA CBI eWM-oAia^^-i:--.' - ----JoneA^Boinr^x.BT-MiHBi-fi^^B-'D^it 12037 RATS SACBIPICBD WCCH 28 (&? ^ DAT OBSERVATION GROUP FINAL WGT HEABT LUNGS LIVER SPLBBH CONTROLS LOW LEVEL(0.001MG/L) IMT LEVEL(0.008MC/L) HIGH LEvEL(0.08MG/L) 408.0000 400.6000 438.8000 441.4000 F RATIO(l) LSD(2) ouNNerro) WKS(4) 2.330 40.9799 50.8407 934.2250 1.2620 1.3780 1.4600+ 1.4600+ 2.557 .1756 .2179 .0172 1.9360 2.1260 1.9360 2.1580 1.367 .3068 .3806 .OS3& 15.6660 15.8460 18.2420419.0520^ 4.940* 2.2972 2.8500 .7400 .8400 .91404.8680 1.907 .1598 .1983 .0142 CROUP KIDNEY rESTIS THYMUS CONTROLS LOW LEVEL(O.OniMG/L) INT LEVEL(0.038MG/L) HIGH LEVEL(0.03XG/L) ? RATIO(l) LSD(2) DUNNETTO) WMS(4) 2.8640 3.0460 3.3580^ 3.4720^ 6.384* .3319 .4117 .0613 3.2320 3.5160 3.4420 3.3720 * QyA.?Q .3721 .4617 .0770 .7040 .8220 .9100 .8140 1.087 .2427 .3011 .0328 |j(l) '(2) i^-eaatisot of among- co wichin-Rroup variation--one-factor any two significant difference--glven aeans differing by a significant analysts of (alpha-0.05) F variance. with (3) a variable-wise false snore than the LSD are positive (alpha) significantly ratio, different Dunnecc test--Any treatment aean er-or rate of differing froa the 0.05. w(t4hi)athn athveaDriuaobnleec-rwissteatfiasltsice pisossitiigvneifi(caalpnhtaly) different froacotnhterocl oanetaronl bayeaanore + Withln-group Mean Square. error rate of 0.05. f Significantly Significantly different (P<0.05) from control group by LSD. and LSD. different (P<0.05) froa control group by Dunnecc * Significant at. the 0.05 probability level. test Company Sanitized. Does not contain TSCA CBf 24 - 024 (1) I (2) Ratio of aaong- to tfiChin-group any Lease two significant dlfference--givveanriaa lon--one-factor Beans differing by significant analysis of variance. with (3) a variable-wise false more than positive che LSD are signifi(caalpnhtalyO.dOiSff)erFenrtatio, than Dunnett test--Any the Ounnect creacacnc (alpha) error Bean differing rate of 0.05. with a statistic variable-wise false is signlflc3ntly fron the control different froa the Bean by acre (4) + Wi chin-group Mean positive Square. (alpha) error rate of control 0.05. mean f Significantly Significantly Jifferenc (P<0.05) froai control and LSO. different (P<0.05) from control group group by by LSD. Dunnett Cast Company Sanitized. Does not contain TSCA CBI 025 12037'SATS SACRIFICED FINAL WGT CONTROLS LOW LEVEL(O.OOIMG/L) INT LEVEL(0.008MG/L) HIGH LEVEL(0.08MG/L) 554.7500 541.0000 566.0000 578.6667 P RATIO(l) LSO(2) DUMMETTO) WMS(4) .284 91.5977 114.8613 3197.4016 1.4850 1.6475 1.6675 1.6067 2.2150 2.1875 2.4175 2.2700 1.345 .2865 .3592 .0313 18.3800 19.4525 20.4775 18.5267 .7775 .9325 1.1100^ .8633 CONTROLS LOW LEVEL(0.001MG/L) INT LEVEL(0.008MG/L) HIGH LEVEL(0.03MG/L) F RATIO(1) LSD(2> OUNNETT(3) .MS(4) 3.3975 3.7675 3.9375 3.6967 1.462 .6042 .7576 .1391 3.4575 3.5950 3.8175 3.6000 .691 .5797 .7270 .1281 .6425 .5925 .6625 .6533 .333 .1734 .2175 .0115 (1) Ratio of among- to within-group variation--one-factor analysis of variance. (2) Least significant difference--givec a significant (alpha-0.05) F ratio, any two aeans differing by aore than the LSD are significantly different with a variable-wise false positive (alpha) error rate of 0.05. (3) Dunnett test--Any treatment mean differing froa the control oean by aore than the Dunnett statistic is significantly different from the control mean with a variable-wise false positive (alpha) error rate of 0.05. (4) Withln-group Mean Square. + Significantly different (P<0.05) froa control group by LSD. f Significantly different (P<0.05) froa control group by Dannett test and LSO. * Significant at the 3.05 probability level. Company Sanitized. Does not contain TSCA CB1 026 - 26 - _'7 - Company Sanitized. Does not contain TSCA CBI 027 GROUP CONTROLS LOW LEVEl.(0.001MG/L) INT. LEVEL(0.008MG/L) HIGH LEVEL(0.08MG/L) F RATIO(l) L3D(2) 3UNNTT(3) WiS(4) LGAN/BODY WEIGHT MEAN RELATIVE DATA Bf 12037 RATS SACRIFICED AFTER 10 EXPOSURES HEART LUNGS LIVER SPLEEN .3534 .3519 34'.6 .3424 .277 .0308 .0382 .0005 .5371 .5698 .5589 .6365? 2.920 .0752 .0933 .0031 3.9259 4.0695 5.67811' 6.2513^ 24.180* -7078 .S7S1 .2787 .2032 .2269 .2454 .1644 4.059* .0520 .0645 .0315 KIDNEY .8635 .8750 .9188 .9211 1.132 .0835 .1036 .0039 ^ROUP TESTIS THYMUS CON TtIOLS LOW I-EVE I .CO.OOIMG/L) IOT LEVr L(0.008MG/L) HIGi^ LEV]EL(G.9SMG/L) ' ilATIO(l) LSD(2) DUKN27T(3) n'MS(4) 1.0356 1.0561 1.0751 1.1482+ 2-233 .')950 .1179 . .0350 .2300 195CH- .2361 .1563? 10.410* .0342 .0-42'. .0007 (1) (2) Ratio Lease of among- to within-group variation--one-facCor analysis of any two significant. difference--given a significant aeans itffering by more than the LSD (alpha^O.C^) F variance. ratio. with a variable-wise (3) Ounnett false positive (alpha) are significantly different error rate of 0.05. thin the test--Any treatment aean difftirin? DunnetC statistic is significantly ;roai the control mzan by aore wC4i/t'h "a?i vt:^tirni-jgbrloeu-.prisMeeanfalSsqeuapreo.sitive (alpha) edrirfoferrernatte froof a0.t0h5e . control aean af nd SSLiicSg;Orn..liffiiccaarnittllyy ddiiffffeerreenntt ((PP<<^0..0055)) frrrooaa ccoonnttrrooll ggrroouupp bbyy DLSuDn.neci tent * Significant it the 3,05 probableity level. Company Sanitized. Does not contain TSCA CB1 028 - 23 - F GROUP C t ^ APPENDIX 3 (corif'd) --s'.'-'^;i.s;:;.Tl<si.i<;f-.-^5etS? i^wjDRGAN/BODY WEIGHT MEAN RELATIVE DATA MHHT 12037 RRJA! TS SACRIFICED AFTER 14 DAY OBSERVATION PERIOD HEART LUNGS LIVER SPLEEN KIDNEY CONTROLS LOW LEVEL(0.001MG/L) INT LEVEL(O.OOSMG/L) HIGH LEVEL(0.08MG/L) F RATIO(l) LSD(2) DUKNETTO) 1^(5(4) .3504 .3689 .3574 .3.159 1.192 .0379 .0470 .0008 .5574 .5247 .5541 ."965 1.473 .0705 .0874 .0028 4.2285 4.2593 4.6752 5.2236? 5.105* .6170 .7655 .2118 .2155 .2266 .2374 .2000 1.385 .0407 .0505 .0001 .8241 .8278 .8237 .8301 .019 .0671 .0833 .0025 SROUP TESTIS THYMUS CONTROLS LOW LEVEL(0.001MG/L) IN'T LEVEL(0.003MG/L) HIGH LKVEL(0.03>iG/L) F RATIO(l) LSD(2) D[J?METT(3) fM3(4) .8922 .9201 .8505 .9527 1.555 .1041 .1291 .0060 .2598 .2278 .2553 .2104 1.696 .0543 .0574 .0016 (1) Ratio of aaong- to wichin-group trariation--one-f actor analysts of variance. (2) Least significant differen-ce--eiven a significant (alpha=0.05) F ratio, d-iy two aeans differing by more than the L3D are significantly different with a variable-i/iss false positive (alpha) error rate of 0.05. (3) Dunnett test--Any treatment mean differing froa the control aean by aore than the 3jnnett statistic is significant'y different froa the control mean with 3 variable--./ise false positive (alpha) error rate of .').05. (<) ..'i thin-group lean Square. - Significantly -nfferenc (P<0.05) fron contro. troup by LSD. Sisnificintly dliferenc (P<-3.05) fro-n control group by Dunnett test in; L-iD. " SSgnlfi^anc M th3 0.05 probah'. 1ity level. 029 - 29 - [^^ " ----r^Kt>AH/BUt} r WEIGHT ffiIAN RELATIVE DATA 037 RATS SACR'CFICKD AFTIS& 28 DAY OBS1B&VATION fKBLIOO GROUP HEART LOWS t LIVER SPLBRN KIDNEY rUwUtnfTi.DKnUiLcj LOW LEVEL(0.001MC/L) IMT LEVEL(0.008MG/L) HIGH LEVEL(0.08MG/L) F RATIO(l) LSD(2) GEWiIETTO) WMS(4) .3094 .3432+ .3338 .3311 2.060 .0298 .0370 .0005 .4739 .5323 .4439 .4886 2.576 .0687 .0853 .0026 3.8374 3.9509 4.15b2 4.3190i? 3.800* .3295 .4087 .0604 .1812 .2094 .2075 .1969 1.769 .0291 .0361 .0005 .7023 .7618+ .7666+ .78731 3.408* .0593 .0735 .0020 ^ROUP TESTIS THYHUS CONTROLS LOW LEVEL(0.001MG/L) INT LEVEL(0.038MG/L) HIi;H LEVEL(O.OSMG/L) F RATIO(l) LSD(2) DU:'tNTTT(3) WMS(4) .7949 .8799 .7873 .7646 2.540 .0950 .1178 .0050 i X 7 5&A** .2061 .2055 .1840 .981 .0512 .0635 .0015 Cl) (2) Xacio Lease or aaong- to significant yichin-group variaclon--one-faccor analysis rif variance. any EWO aeans difference--given differing by aore than che a significant LSD (alpha-0.05) F ratio, with (3) a variable-wise false positive (alpha) are significantly rflffcreot error rate of 0.05. than Dunnett test--Any treatment aean differing from the the Dunnett statistic is significantly different control aean by more with (^) a variable-wise Wi false positive (alpha) error from the control rate of 0.05. mean chin-group Mean Square. + Significantly different (P<0.05) froa control group by LSD. e Significantly and LSD. different (?<0.05) from control group by Dunnett teat * Significant at the 0.05 probability level. ^Panysan.,^^ ^^tafnTSCAcB/ 030 - 30 - P..j l-- OT ----------j/a.UA 12037 RATS n,OUUT SACRI F W6U> ICED H1 A F P T IBA ER H 4 R 2 f iLAT DAY I VZ OB DATA SERVA T I O N PERIOD GROUP HEART LUNGS LIVER SPLEEN KIDNEY CONTROLS LOW LEVEL(0.001MG/L) IMT LEVEL(0.008MG/L) HIGH LEVEL(0.08MG/L) F RATIO(l) LSD(2) DUNNETT(3) WMS(4) .3067 .3065 .^'993 .3098 .112 .0383 .0476 .0007 .4286 .4464 .4795 .4571 .620 .0817 .1017 .0032 3.6410 3.5886 3.7843 3.7997 .688 .3839 .4779 .0712 .1825 .1829 .1959 .1753 .640 .0309 .0385 .000^ .6442 .7144 .6512 .7127 1.447 .0974 .1213 .0046 GROUP TESTIS THYMUS CONTROLS LOW LEVEL(0.001MC/L) I'n LEVEL(0.008MG/L) HIGH LEvEL(0.08MG/L) F RATIO(i) LSD(2) DUNXETTO) WMS(4) .7257 .7417 .5846 .8095 2.219 .1830 .2278 .0162 .1811 .1719 .1710 .1848 .171 .0492 .0613 .0012 (1) Ratio of among- to wichin-group variation--one-faccor analysts of variance. (2) Lease significant difference--given a significant (alpha0.05) F ratio, any two means differing by more than the LSD are significantly different with a variable-vise false positive (alpha) error rate of 0.05. (3) Dunnett test--Any treatment aea^ differing from the control mean by -Bore than the Dunnett statistic is significantly different fro-n the control nean 'ith n variable-wis" false positive (alpha) error race of 0.05. (4) Within-group Mean Square. + Significantly different (P<0.05) from control group by LSD. s Significantly different (P0.05) froa control group by Dunnett test and LSD. - 31 - 031 ^ ^ ^ff,, " " " ^ '^Oo<^flOf V--------H^--12l0]1^3K7 uf\un CT/ DUUi caujn'i. n&AH RELATIVE I1ATA RATS SACRIFICED AFTER G4 DAY OBSIERVATIOH PER][OD GROUP HEART LUNGS LxVER SPLEEN KIDNEY CONTROLS LOW LEVEL(0.001(1G/L) IKT LEVEL(0.008MG/L) HIGH LEVEL(0.03MG/L) F RATIO(l) LSD(2) DONNETTO) WHS (4) .2668 .3046^ 2946+ .2787 5.485* .0229 .0288 .0002 .40LJ .4054 .4?76 .3965 .487 .0617 .0774 .0015 3.3153 3.5910 3.5955 3.2076 3.609* .3160 .3963 .0331 .1399 .1723+ .1964 .1507 7.572* .0290 .0364 .0001 .6119 .6960? .6955 .6424 7.540* .0485 .0609 .0009 GROUP TESTIS THYMUS CONTROLS LOW LEVEL(0.001MG/L) INT LEVEL(0.003MG/L) HIGH LVEL(O.OSMG/L) F RATIO(i) LSD(2) DUNSETT(3) WMS(4) .6215 .6640 .6779 ^fc^ 1.275 .0774 .0970 .0023 .1153 .1102 .1171 .1155 .OS: .0327 .0411 .0004 (1) Ratio of among- to within-group variation--one-factor analysin of variance. (2) Lease significant difference--given a significant (alphas'0.05) F rado, any two means differing by sore than the LSD are significantly different wi;'i a variable-vise falsa positive (alpha) error race of O.Q5(3) Dunnett tesE--Any tieatmenc fflean differing from the control ncan by aore than the DuaneCt statistic is significantly different froa the control aean with a variable-wise false positive (alpha) error rate of 0.05. (4) Within-group Mean Square. + Significantly differe.il: (P<0.05) from control group by LSD. ? Significantly different (P<0.05) froa control group by Dunnett test and LSD. * Significant at the 0.05 probability level. - 32 - 032 Gofti ^ r ^ ^San,'^etf. oes tot cs^ ^ , ^0. E. 1. OU PONT OE NEMOURS S COMPANY WlLM(NGTON. DELAWARE 19898 WLYMCR poouera OCTARTMENT CXPCMIMCNTAL tTATION ANALYTICAL REPORT January 25, 1981 G. L. KENNED? CE1TKAL RESEARCH HASKELL LAB AND DEVELOPMENT DEPARTMENT DETEBMIHAriON OF PERELODBOOCIASOIC ACID IN RAT BLOOD SAMPLES (JOB to. 800-598, 802-S477, "800-5562; PRAL Nos. 80-60270-376, 80-67459-485, 80-67751-758, 80-68604-674) &s pare of you^Ufmihalacion subaeuce tesc, & series; of rac blood samples has been analyz&dtor perxluorooceaa&a"s (Cg, acid aad sales). A listing of ehe 107 Samples received 6/80, with identification numbers and current scatus, is given in Table I. As requested, ocly che group of 66 samples (plus blanks) indicated there vas included in these first analyses. The renalntng 26 sanples have been dried for storage under Che same conditions as the others, but not analyzed at this time. The analyses were done using che gas -chroaatographic aetbod developed here last year far specific decenoinafion of perfluorooccanoic acid in blood. As we discussed in our meeting on 1/3/81, this method should give values for cocal Cg cr--arabie to chose of non-specific organic fluorine determination for saaples of ch-. type, as well as providing information on the distribution of 3 isoners which iias not previously been available. A. Comparison of Analytical Methods A eoaparlson of the ..g/GC analysis with the tfickbold Torch analysis for cocal organic fluariae was recently aade wish a series of cest sanoles fron Parkersburg, and cbe results found co agree althin experimental uncertaiaity when corrected _for recovery ^f Cg. Details of the comparison are discussad in II Seven of these rat blood samples vere also sent Col--------Byai: Jackson Lab for Wickbold Torch analysis. Results are given % Table aad Figure 1. As aoced in ehe Cable, recovery of Cg by the ffiLckbold Torch procsdure - 34 - c0^^ ^ 034 Thare's a world of things we're doing something about ^ ^ <es ^ n'o't^f^ !tlrs^ has ben determined co be "^832 la recene work, and resulu have been corrected by this factor for the plot la figure I* Overall agreeoenc seems satisfactory, although based on this lioiead daca the extent to which the 0.8-0.9 ppe F in the blank saople (80-67484} could represent a variable.contribution co che others cannot be determined. For ehe subacoce skin test blood saaples analyzed at Jackson Lab early in 1980, a correcClon for Cg recovery in the 3M Parr Boob method should also be made before comparison with these CC values. According CoftB----f^ the recovery ac chat eliae should have been 92+5Z, as reported in che method publisfied bv.3H (Analytical Biochemistry fl^, 545"'fl9781). 5- Total Concentration of Cg III fiesulcs arcglven in Table for overall Cg c^ncentracion (straight-chain plus four branched Isooers}, calculated as ppm fluorlde co facllitace comparison with non-so_ecl.glc- analyses. As hoced there, none of the six "test blank" samples were found co contain detectable Cg, while all except two of the Group I controls did contain measurable amounts. Average concentrations are plotted against recovery cloe in Figure 1. For comparison with the earlier sistasi seudy an calculation of half-cises, linear least square Hp* have been dr"r> through ehe polaes up eo 42 days (although it Is not clear rhae cds Is che best overall fie). For all four groups, half- cisie (log 2/slope) can be seen eo be about 8 days. i ^ c' Contribution from Branched Cg Zsoners Chromacograins obra^ncd for thgee rat blood samples as veil as for . . . Figure 3. (The^HBFlused here was cbe same as chat used in your inhaiac^n study.) w - r As discussed ia the method, ES response is greaser for the four branched isooers (peaks 1,2,4 and 5) than for the straight-chain material (peak 3), and their concentrations were esclffiacsc' '-ig relative response factors as described there. Since the values ca-1. .-aced for che Individual isoaers are very approximate an attempt has not been sade to csbul6-e chess forHimBBBBIIfMBly separately, but the percentage of cralght-chain Cg calculated for each, sample is shown in Table 17. As noted there, s corresponding value rorA------fjscandard would be ^74Z, and candard T-94Z. -rich a few exceptions, values for sacrifice 1 are lowe- (greater contribution from branched^isoaers} than in the later sacrific' , although never as low as that fbru------1.1 The pattern shown in Figure 3c is also fairly typical for saaples concalain -i high percentage of straight-chain Cg, with the major isoaer contribution coming from peak 95 and a measurable contribution from peak i'1 "^e^ 035 ^Panys.. c0^^ wahicwhej.a1p. pecTrhsuaCofabr,*ffclhB --on--ly --ia--oa--ar MeoR_vhvlcAh a erueeure has been ic0h1eR psuogsgiceisocna chac tBeoeBSr^ -e*.oBpariaon of aaflgned GC gd NMB data)^ i 95, ^^ M^BHMHI^^BjLLong Fluerlxr-lS^1' h&i been ba dct ch chain, buc s:^^^ S. S. Stafford ACtachaaaes fsc Key Wbrda: [ a ^ - j . Blood analysis Perfluorooefiassaee 0 03^ - 36 - '^ c<0^ i0^, ljr^'".if.'i<etf 1C{ t^",^,Vf) ^ u 4 c^ Idendflcaclon of Hat Blood SanpleaTABfrLoEn ilatioc Subacute Test^ :B i 0 Days 2/29/80 Group I (concrol) II (1 ag/as3) I-H (8 as/a?) 0 r -- 14 Days 3/14/80 1tV (BO mg/v?) I n III 17 \ Tubt 1 2 3 4 5 < 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 2 3 4 5 6 7 8 5 10 U 12 13 14 15 f SMt f 270322 270323 270326 270327 270330 PRAL No. , As Beceivd 80-60270 80-60271 80-60272 80-6G273 80-60274 PRAt No., Driad 80-67459 80-67460 80-68604 80-68605 80-68606 270638 270639 270640 270641- 270642 80-60275 80-60276 80-60277 )-6fl2J8 80-60279 80-67461 80-67462 80-68607 oBoA--e-oQo^bAuHo 80-68609 270678 270679 270682 270683 270684 80-60280 80-60281 SO-60282 80-60283 80-60284 80-67463 80-67464 80-68610 80-68611 80-68612 270720 270721 270724 ! 270725 270728 80-60285 80-60286 80-602S7 80-60238 80-60289 80-67465 80-67466 80-68613 80-68614 80-68615 270330 270332 270333 270334 270335 80-60290 80-60291 80-602?^ 80-60293 80-60294 80-67467 80-67468 80-67751 80-68616 80-68617 270643 270644 278645 27bb48 270649 80-60295 80-60296 80-60297 80-60298 80-60299 80-68618 80-68619 80-68620 80-68621 80-68622 270685 270686 270657 270688 270689 80-60300 80-60301 80-60302 80-60303 80-60304 80-68623 80-68624 80-68625 80-68626 80-68627 16 270302 17 270730 80-60305 80-67469 18 270731 80-60306 80-67470 19 270732 80-60307 80-67752 20 270733 80-60306 80-68628 80-60309 80-68629 Scacua(b A A A A A A A A A A A A A A A A A A A L L A A A 0 5 3 D D -- D Q D D D L T A A A H | --...-.----------------.-- ----..... ^ ^ . 37 ... ,, . . -37- .,,.-,..,..... ^^-oe^""olcoa--^ -'?;. Sacrifice | 3 I y 28 Days 3/28/80 II III IV 4 I 42 Days 4/11/80 II III IV 5 I 14 Days 723/80 TABLE I (coa'c) Grom i Tube 1 2 3 4 5 6 7 8 9 10 11 12 L3 14 15 16 17 18 19 20 1 2 3 4 5 6 7 i 10 11 12 13 14 15 16 17 18 19 1 2 3 4 f Rac / PRAL No.. As PRAL Ho. Racaivad Dried 270336 270337 270356 270357 270358 80-60310 80-60311 80-60312 80-60313 80-60314 80-6 7A 71 60-67472 80-67753 80-66630 80-68631 270652 270653 270656 270657 270658 80-60315 80-60316 80-60317 80-60318 80-60319 80-67473 80-67474 80-67754 80-68632 80-68633 270690 270691 270692 . 270693 270694 80-60320 80-60321 80-6032^ 80-60323 80-60324 80-67075 80-67476 80-67755 80-68634 80-68635 270734 270735 270738 270739 276744 80-60325 80-60326 80-60327 80-60328 wfln~--vu^jm--^yo 80-67477 80-67478 80-67756 80-68636 80-68637 270359 270384 270385 270414 270415 80-60330 80-60331 80-60332 80-60333 30-60334 80-67479 80-67480 80-68638 80-68639 80-6S640 270659 270662 270663 270668 270669 80-60335 80-60336 80-60337 80-60338 80-60339 80-68641 80-68642 80-68643 80-68644 80-68645 270695 270698 270699 270702 270703 80-60340 80-60341 80-60342 80-60343 80-60344 80-68646 80-68647 SO-6864S 80-68649 80-68650 270745 2/0296 270297 270310 80-60345 80-60346 80-60347 80-50348 80-67481 80-67482 80-68651 0M0M--6QB6C52^ 270444 270445 270474 270475 80-50349 80-60350 80-60351 80-60352 80-68653 80-6865'* 80-67757 80-68655 L L A A A L L A i A A A A A A 0 D D 0 0 N 0 0 y 0 A A A A A A A A /- 038 - 38 - Company Sanitized. Does not contain TSCA CBJ TABLE I (con't) III) (a) As cha samples were no --. ^ o each entire sample was longer homogeneous lyophlllzed and at the cine of analysis (due pirtlons eo analyze. assigned a second PRAL CO clotting), No. before taking <fbb.J) CCiuirryr^e"n*"t S?*t--ac--u-s- ; A, Samples 0, Samples analyzed 12/80-1/81, lyophlllzed resales reported here iC chls das for further storage, buC noc analyzed N, L, No blo 'SSaafafppllee o s s d _ l c,,ol.l.e.,,c.t.c.'Md*t, ost eapecy tubes rceeceeilved lIo/,s*.s- o--ef dur*inag lyophlillzaaattioa" vacuum la the /(;o"t--is g- roup of 13. due analyzed for apparatus^. sacrifices 2 and 3 For chls reason, the to groups c) Blanks Included withh the __ - uu j contain only 3 rats each--(-T*ab* le I, group of samples during samples submolitted preparation and A6//8an0. 'O<n- e of- these was included analysis as a procedural blank. with each 039 - 39 - Company Sanitized. Does not contain TSCA CBI I I TABLE Comparison of Cg/GC and Wiekbold Torch Analyses of Sac Blood Samples^ PRAL No. 80-67461 80-67462 80-67752 80-67756 80-67481 80-67460 80-67484 Sacrifice-Tube 1-6 1-7 2-18 3-18 4-16 1-2 tesc blank <3C Analysis, [Cal. UR F/B blood 7.7 13.1 11.4 8.6 1.3 1.1 <0.007 Cooae deeecfced above reagent background) Wiekbold Torch Analyaie, Tocal Fluoride, ug F/g blood Gacorrected x 1/0.83^ 6.9 8.3 10.5 12.7 ' 9.5 11.4 9.0 10.8 1.8 2.2 1.1 1.3 0.76 0.92 (a) Analyses on doplieaee portions of the lyoptbUlzed blood samples. GC analyses as described la Table HI, U/l/80-12/8/80. Wiekbold Torch analyses by ^ (b) Becovery of U)ln the Wiekbold Torch, procedure is currently 'v.8i33Z2,. is determined in refcceint work bylki--------1 0.10 Company San.fcA Does not contain TSCA C91 - 40 - IABLE in (eon't) (a) Pxocediira as descrloed in the oatood "DeternriLnacioB. in Blood Gas^ghronacpgraphic fjfechod", ^/2/81, ualag ochf&|^'c_ap_il_la_ry_ coloan GC analysis aodpBMBBBHHMBllllBMr^s c&libraeloa scandardc Lower limic for quanticaSon 0.007 ppa F. , Esclinacad uocercalnlty ^102 relative scandard deviation over vose of the range, aomewoac higher ac the louest coneestrations. (b) For the six "test blank" saapJf.es listed as analyzed in Table I, [Csj <6.007 ppn F (none deceeced above rasgenci^adtground). (PRaL Nos. 80-60368-69, 80-60372-75)- (c) Values of 0.005 (sample 5-2) and 0.003 (saapie 5-4) were used to calculate itthe average, but quaatitation cannot really be done at these levels, and is questionable whether 5-4 contained any Cg above backgrouod. ^rs^ 042 - 42 - Percent Scraighc-ChainI fin Rat Blood Sflrpia(a) _(conerol) Percenc Group n .^J^ ^/^ ^ Percent III Group Tuba 9 (8 wsfvft) Pereeac 11 89 12 92 13 90 14 85 15 86 as/^ Group IV (80 Tube S Percent 16 88 17 88 18 87 19 84 20 86 .noc ; 5 eat- 9 eulaced(b) 10 18 . 93 19 92 .' 20 91 i 13 96 14 95 15 96 18 94 19 92 20 93 16 94 17 96 18 95 19 94 not cal culated (b) 13 . 'V97 , 14 - 94 15 93 (a) pCeaalkcsulsaeteedn ians dFeigsucrreibe3db. in che method, for peak ff3 in che group of five Cg isomer The saaa cslculaelon for spiked blood standards (M.O ppm F) gave average values (b) A calculation of branched iaomer wfrioeiha c[hCegJr<e0e.g1enppcms., due eo che- smaclol nscreisnecroaef iocahea. saefeaooncdasrdccyempepackesd afnodr isnacspejr./<fiaorence 'i- i-,. ^ <- V- 043 - 43 . ^ttl,'Oany ^zetf.^oe,^oof ^aro rsc4 C6f t--j Figure 1 - Comparison of Cg/CC and Wickbold Torch Analyses of Bat Blood Samples Daca from Table lit using Torch values corrected for 83Z recovery Linear Lease Squares Ftt; Torch 0.90 + 0.96 CGC) ^ ;:.-(. :;{== * "nt::: :-i= :. ::: :-' ^ 'i' i i r ::" : ::;; -- .1- -i-; :::;(:: ;::.<: ^ 7" ""f " ( .4- .:..(: r --- *.' "'. i - - ;:--i:: ^ i-'t:.-,, '..!' :..: ' . ;.,:: ::: i. i l - - - :.. '. ::.' '::: :" ! . - < ^ !' .... - \:' . ; - .;; .,,; ~Ht- ) : : .;i. . :.! ::; . ''. ? *'.. : ;:: :. i : :;.. .. ::. '^1 ' ::::: :': ^ ' ..: ::- ::.:i:.:' .. . .. :!::: ': :'!: i ;;:'..::;: .:'.1:" . .-.'i;- ^i^. ^ jp-^ -M" :.:-i:: - . -i;. ^ . (f :::^;::t;=:::: ::": ..(. ..1: -:!: :: -T---- " ; * ;. ....: I: . ;-. i :;;;],,; .' / 7-- 1 . 1 y /"\ ; i -.i-.-.l':".: ^ :.. :: :.: : : / "" ; . ';'"!': 4 ^ :. ;" :!' : y ' " . i i ^ \'-/\^ i-'- : r . 0 ^ /i:. ? ^ -- i ---- i " i , . . 7-r--- * ! : j --^ <3r ! -T-- ! I .... ... . , --i--.- - ) I ..!. '!' \~/r^v> r -T-1 ; f. ^--- . ^r'------ 1 i :.i::: ...,'.. .r. i . , i i . S. ^ ^ y 1 | / ! i j^ ^ . -i ^ r /o 7^ ^ 6. . ---- 1 l / .- .... .- ---- -- . ! "? ! ! -- -- 1 . .;. ..... -T-- r-; -f " . . i . . ; i 1 \ : . i ' : 1 1 (0 u ^ GC, ppm 5 as F 044 - 44 - < ^ c. ^a^ 0^ r^t ^tQif, e IT Cocmteion of 53 a Bend septs. mcovary re csfron Table TT, demcagn Tatum 17 0 oo sow A:=iGorowup IIv EEILI- ou. lleoggcC==11,79-o0,l03s9 (to 42 days only) X= Grom I EEA ree pa 108 = 0.078 - 0.038 (to 42 days only, sample 3-20excluded) E N S = SS Se--E e--F e ee e e aa e eefPa e r = CE ee e LE PE E rr = e == e See Ee = P= O; = se ee ee E == or =T T ==E E =--a-N _ Se == e=E EEo = | Ze S a e ee rr s e e ee N eae ] ea == ww, E 2 S r a eEe r --E = E = t1r | E Em S ne i e Ee me emee ee n n S eE e Ee E E : e= eF: = ErrE Ra=a == ree wa" er To = ==F => == = Tecovery Tien, Days = = Company Sanitized. Does not contain TSCA CB o 015 -as- -- EE ----