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.-.,, , -.-/-'sya.-f^nWff AR226-2793 FOR OU PONT USE ONLY HASKELL LABORAl-ORY REPORT NO. 401-85 | HIHUHIf I Subchronic reeding Study of Ammonium Perfluorononanoate (C-9) in Mice Haskell t.. I. du Pont de Nemours and Company Laboratory for Toxicology and -Industrial P. 0. Box 50, Elktbn Road Newark, Delaware 19714 Medicine Date Issued: August 7, 1985 ia,.ai^^^3ia-g^^agg^iiiB^t^-aga^ Company Sanitized. Does not contain TSCA CBI HLR 401-85 Subchronic Feeding Study of Ammonium Perfluorononanosite (C-9) in Mice SUMMARY Groups of 5 male and 5 female mice were fed diets thafc contained 0, 1, 5, 1U, 50, 100, 300, 1,000, 3,000, or 10,000 ppm ammonium perfluorononanoate (greater than 99% pure). Food consumption was not monitored. Clinical signs of toxicity were observed at lethal and nonlethal concentrations. Deaths were observed at concent rat ions of 300 ppm and higher. All mice fed C-9 at dietary concentrations of 1,000 ppm or higher were killed in extremis on test day 6 and the liver weights were recorded. Mice from the 100 and 300 ppm groups were similarly killed on test day 8. Mice from the remaining test groups and the control group were killed after 14 consecutive days of dietary administration of the test material. Administration of C-9 at concentration of 100 ppro or higher in the diet resulted in slight to severe weight loss; administration of dietary concentrations of 300 ppm or higher caused death. Male and female mice fed diets of 3 ppm or higher for 14 consecutive days had significantly increased mean absolute and mean relative liver weights when compared to the controls. The mean relative liver weights of male mice Fed the 1 ppm diet were also significantly heavier than the controls. Mean body weights of male and female mice from the 1, 3, 10, and 30 ppm groups were similar to those of control mice throughout the test. Comparison of mean body and liver weights of mice fed diets containing C-9 at concentrations greater than 50 ppm were not possible because thnre were no concurrent control groups. jS/cga(5) Work by: (U^u^^- ^/.(?. <?AW Carviff'N. Wylie Technician Hf /PS- /]John Peport by;; -JrC-r^ L-J ^5g./uv^i N. Sarver '-' Technologist ^/'? /y^" Approved by: C.&t^ly, hi.b. Study Director Section Supervisor Acute Iwatigetions - 2 - Comt;an- ?ani^"d. Doey W -snia;:- T^CA CSi c Material Tested: Haskell Laboratory Report (to. 401-85 Haskell No. 15,457 Nonanoic acid, 2,2,3,5,4,4,5,5,6.6,7,7, 8,8,9,9-hexadecafluoro-, ammonium a.alt Sponsor; Material Submitted by: Polymer Products Department E. I. du Pont de Nemours and Company Wilmington, Delaware r' Polymer Products Department L. I. du Pont de Nemours and Washington Works Parkersbui'g, West Virginia Company Test Facility; Haskell Laboratory for toxicology and Industrial Medicine E. I. du Pont de Nemours and Company P. 0. Box 50, Elkton Road Newark, Delaware 19714 Study Initiated - Completed; 7/12/84 - 7/26/84 i\ There are 16 pages in this report Distribution! Acknowledgeent8: Rayanne L. Ferenz, Bruee A. Burgoss and Gerald L. Kennedy, Jr. also participated in the conduct of this study. ..;; .iii-,1''rifias.ssjist'i.tise!STMi;t;B;(afii Company SSaann;ti:ts^ad". Does not contain TSCACB 3 - INTRODUCTION HLR 401-85 The purpose of this study was to evaluate the effects of anuaonium perfluorononanoate (C-9) on the liver weights of male and female mice after dietary administration of the test material for 14 consecutive days. MATERIALS AND METHODS A. Animal Husbandry Male and female 5 to 6-week old, Crl;CO-l(ICR)BR mice were received from Charier River Breeding Laboratories, Kingston, New York. The mice were housed 3 per cage (sexes separate) in suspended, stainless steel, wire-mesh cages with dimensions of 4.5" x 7" x 5" (w x d x h). Mice were quarantined, weighed, add observed for general health for approximately one week prior to testing. Prior to test start, mice were transferred to individual cages and each mouse was assigned, a unique identification nuiiber which was recorded on tape affixed above the cage. Purina Certified Rodent Chow* ^5002 and water were available ad libitum during the pretest period. Animal rooms were maintained on aTimer-controlled, 12 hour/12 hour light/dark cycle. Environmental conditions of the rooms were targeted for a temperature of 22 ^ 3C and relative humidity of 50 +_ 20%. Excursions outside these ranges were of brief duration and dJd not adversely affect the outcome of the study. B. Protocol . After the pretest period, mice were randomly assigned to study groups on the basis of body weight and freedom from any grossly apparent clinical signs of disease r.r injury. Mice of each sex were randomly divided into 10 groups of J male and 10 groups of 5 female mice. The mean body weights fcr each group of mice within a sex were approximately equal. The test mice were fed diets that consisted of ammonium perfluorononanoate blended with ground rodent cho" C5QQ2) and supplied ad libitum during the test phase. Verification of the concentration of the test material in the prepared diets was performed by personnel of the Polymer Products Department. The day that mice were first administered the test diet Mas test day 1. The mice were weighed and observed daily (weekends included when warranted by signs of toxicity). All mice killed in extreals or by design were euthanized by chloroform asphyxia!ion and exsanguinated. Liver weights were recorded for all mice killed in extremis as well as those found dead or killed by design. Body weigTTEsand liver weights ware analyzed using a one-way analysis of variance. Pairwise CDiparisoris between control and treatment groups were Bade using least significant difference and/or Dunnett's teste ;ompany S. an.,^..,.^^, nD:O-.Ss3 ,n,'..',1--^""nTSCACBl g^a--,a..i.i,,-,a-^,Basg HLR ^01-85 c. Test Material Physical Form; Purity: Synonyms; Other Codes: Stab illty; White solid Ammonium perfluorononanoate C-9 ^ C-9 Verification ofche concentration of the test material in the prepared diets was performed by personnel of the Polymer Products Department. The test material was assumed to be stable throughout the exposure phase of the study. D. Records Retention All raw data and the ."inal report Mill be stored in the archives of Haskell Laboratory for Toxicology and Industrial Medicine, C. I. du Pont de Nemours and Company, Newark, Delaware or in the Du Pont Hall of Records, Wilmington; Delaware. RESULTS A. Diet Information Sufficient diet was prepared for each test concentration to last throughout the 2-week feeding period. One sample each was taken froa the top, middle and bottom of the mixed diet during preparation of the 100 and 10,000 pom diets. These samples plus a control diet sample were analyzed for C-9 only by S. R. Laas, Polymer Products Department. The results are presented in Appendix I. used. The diets were refrigerated until B. Clinical Signs Clinical signs were similar for male and female mice. Nonlefchal Concentrations Mice fed C-9 at concentrations" of 100 ppm and less exhibited slight . to severe sporadic weight loss (i.e., 5 - IfiSi of body weight). ;:! Lethal Concentrations :. ^ Mice fed C-9 at concentrations of 500 ppm and higher exhibited C ^ severe weight loss (i.e., 52 - 45S of body weight), ruffled Fur, ;^; ^ lethargy, low posture and limpness. Deaths began to occur in the 1fl,fli30_ai ppm group on test day 4. -nnla'n'TS^-'^ . rtipsr.o10-' -; " Con^53""1" ,& i.jaiAia ^uc/.aaa^^^as . ,.;.qB-- - ' ' r-wpg.l-t.ffiaffe -ipiF' C. Body Weight and Liver Weight Analysis /: ':;.:'- Body weights of the male and female mice from the 1, 3, 10, and 30: ppffl concentration group-s were similar to those of the control mice . , throughout the feeding study. The mean body weight growth curves for the'... male and female mice are presented in Figures 1 and 2. ::^ The mean absolute and mean relative liver weights of male and fenialL^:; mice fed diets containing C-9 at concentrations of 3, 10, and 30' ppiritBil^"' significantly heavier when compared to the controls. In addition, 'malef^ mice from the 1 ppm concentration group had significantly heavier mean ^lt re'ative liver weights when compared fco the control group (Table." 1 and "1 2)- . . Comparison of mean body and liver weights of mice fed diets above IQ^'s II.iQ ppm were not possible because there were no concurrent control groups. ^.|: The body and liver weights of these animals are presented in Appendix CONCLUSIONS Dietary administration of C-9 to male and female mice at I concentrations of 3, 10, or ?0 pp.-n for 14 consecutive days caused significant increases in mean absolute and relative liver weights. Dietary administration of C-9 at a concentration of 1 ppm caused a significant increase in the mean relative, but not absolute, liver tteiglll'T,' of male mice. The mean liver weights of female mice were unaffeclfeetf^)^^ ' C-9 at a dietary concentration of 1 ppm. C-9 at dietary concentrationsll'; ; of 1, 3, 10 or 30 ppm had no effect on the mean body weights of either'.j;: '"', male or female mice. Administration of C-9 at concentrations ojf TOO ppmf or higher in the diet resulted in slight to severe weight loss; ""^i" n administration at concentrations of 300 ppm or higher caused death. :: ! ..rf,.-..,.. ..^.,,.s^. ' ? 1 -Vtf^ . "'' ':"E:'w. ' "^M '^al '. ^KM i^-'E^ Company Sanitized. Does not contain ^SCfs^ P^.BN' - 6 - -...-.Aj :,.;a-asfeSjJg^^g^sMl^^^^gagSL'.gS^^i FIGURE 1 GROWTH CURVES OF MALE MICE FED C-9 "T. ----1. j----I-- --|--|--11--j--I"--"--T--'I'----I--I1----11--1--'------]'----i'----i ---------1--------\------T --r--\--\--- |b>'.' fc. w ^ 33.00- ife 32.00 - z f"t ,. , ' H ; R 0 '' ;. ' r ^ E fe 1 5, '. 0 ^.: o Q ^ 0 1 ' ^ s. ?1. M s fea, Is-. .%" - wQS a o W 0 ^ ^ *^^, 1 '.... - 31.00 : 30.00 -- : - 29.00 ~ 20.00\- - f^^ ^ " ' <r' -^ 0 0 Is 4'.''... ';:;,, F : ! ' ^ t;"';:,; -4 !%'"...'' ty^. y 0 27.00L 0.0 0 1 2.00 :vf'/^"" ... . B.:^.:,, B^ mliy ass^w!- ...a, . -.^i^;;iffi^H:i::;ffiiiBUintiig..... ...-.--...,...;--.;.,i.^.,;,.,-,^^,^.J)a;^n*^.,,-^,,. , J.;^ni^'i^^^^Ss!^&s?ssa:i: , . ' ^ / -^ / > ^ K^' . / CT'" -a- ^- <- y *r +' ^ 9-- c^, .. K / ' / /' .,-"''' ..^' - V . / / / ra-' A,. Of ^' -y----^ /." v"-. /' / / .; 4i -a ,' cr-'' : X' . . .-of ^^ ljL- ... 4.00 6. 00 iaS 8. oo J, 1J, L-,L10-Jj.0.--01--11 --1l--a.o--o--1----11---1--.010-- ON TEST . . . -, ., . . . ,.--.<.--h-- ,--.,-.Aw-. .. a^-ifilffi*ivi-- ^ ...,'-;'j,-",.,^,.'"i3.,' ..... '.:.,. ...fr---^9^W. . ^. ilAlSjt Sj,;Aa^^^.s..^-.^^t^to......LiK^aii.'ii^ ...;, ...... ...... :S,^,.-.. rfffHtl&iHRa"'*1'" .A-:-- FIGURE 2 PR on GROWTH CURVES OF FEMALE MICE FPn n . i i' ir ji ii ii ii ii-ri-r-i~]i ri ";; - rr----r-rr'''i---ri-r-i---i-r--;--Tr---r1-i--;----1-----r---T- ' ' , ""'- 25.50 :- 1.1 W ; 3 3 oS 25.00 -- ' . i 0 1 (H v is 24.50 -- "< : &-e a 24.00 ~ ^ 23.50 ^ .' : -- . W 00 ' -- .<--'' . ^ ' l& / " ^ a ^ ' . T A- rt , .. * : > / /' A' A Q0 23.00 - 'la^-,- a \, CQ : . /*- ,,.-.<<--,.. <r- . <tf( M ^" " ' . 22.50- 0 - ^ ^- K - v. 1 ^ r W 22.00 -- , >' A 1 21.50 ^ '' L 51 1- * ' 1 IB1' a r * 0 0 3. 0) \- a. 21.00l:--J--^L 3 0.0 0 2.0L0-.J--L.-.l.4-J.0. 0J....1...1,0..0l.,0..,l....J....I.B..Oi.O. 1. . 1 1 1 10.00-I.,.!...). .1..-^.. ..i. i-..j-.j,. V) 0 12.00 14.00 ivc i&ol UAIO 3> n HurMN TITCT 2 B--'-BOWrdB. - -< t pf . &- &3pp| <- - -+ 10 pp x.-- X30ppB APPENDIX [ Diet Analysis ^^^w^-' HLR 401-85 9 - ^--------no>co"'anTSCACB Vulymm KruuucCu Uepiirl:ionl Research and Development Division Experimental Station ANALYTICAL REPORT August 10, 1984 Rayanne Ferenz CR&DD Haskell ANALYSIS OF PERFLUORONONANOATE (C9) IN RODENT CHOW (Job No. 841-0896; PRAL Nog 84-5799 to 5805) Six samples of rodent chow, 3 nominally 100 ppm and 3 nominally 10,000 ppm have been analyzed by the derlvatizatlon/gas chromatographic method using electron capture detector originally developed for CB la blood (ES-567). No problems were encountered In applying the method to rodent chow. The samples gave very clean chromatograms with no Interfering peaks In the C8 to C10 region. Samples were run In duplicate with C10 as an Internal standard added Co samples and standards. Unfortunately the C9 we had available as a standard has some C8 and possibly C10 contaminants. The C9 you used to spike the rodent chow appears much purer. We have reported results based on peak height only because of Interfering peaks in the integration of the standards. The CiO la the standards does affect the absolute values, but the relative values between the 6 samples should be valid. The results all summarized in the attached table. The precision for the high level samples Is not as good as for the 100 ppm samples as very large dilutions were necessary to bring the samples within the calibration range. In addition, a smaller saaple of rat chow was used. If you need a more accurate analysis, you could send us a sample of your C9 for a standard and we could rerun the samples. If you have any questions, don't hesitate Co call me. Attachment Keywords: GC Prf1uor ononanoat e C9 10 - .nTSCAC^ noicon^' ^san^-0085 Cow?^ -"a? .---- TABLE I PEaFLUOSONONAMOATIEN KODEMT CHOW Pral Ho. 84-5799 84-5800 84-5801 84-5802 84-5803 84-5804 84-5805 Designation 0 ppm 100 ppa - Top 100 ppx - Middle 100 ppm - BoCCom 10,000 ppa - Top 10,000 ppm - Middle 10,000 ppa - Boccoa V8 Prfluorononanoate/g,suple Duplicate Analys*' None detected^ 86; 85 97; 90 83; 78 11.000; 10,500 H.200; 11,400 11,700; 11,100 (a) Analysis of duplicate allquoCs of rac chow; each analysis of 10,000 ppa of 100 ppa saaples; samples. approx. approx. 0*2 g used In 0.1 g used In each analysis (b) Detection Halt not deteralned; no peak observed used for analyzing 100 ppa saaple* under condlclonc 11 Company Sanitized. Does not contain TSCA CBl g.^.iMj^^J^^^^^-gljjg^ffijia'e^^'S TABLE 1 MALE M:CE LIVER & BODY WEIGHT SUMMARY MEAN APSOLUTEANDRELATIVE DATA GROUP CONC. 3 ppn 1 ppm 3 ppni 10 ppm 30 ppm TEST TEST - HOMOGENEITY TREND BARTLETT'S TEST FINAL WT. 33.41 32.0? 34.0( 33.2( 31.8( 0.000} 0.204) 0.580) 0.853) 0.149) 0.221 0.411 0.082 ABSOLUTE WT. 1.811( 0.000) 2.162C 0.08:) 2.365( 0.009)# 3.17K 0.000)# 3.793( 0.000)# 0.000 0.000 0.015 RELATIVE WT. 5.422( 6.752( 6.952( 9.528( 11. 953 ( 0.000) 0.017)* 0.007) 0.000)f O.OOOX? 0.003 0.000 0.082 .a.iues in parentheses - P VALUE OF STUDENT T TEST COMPARISON + - SIGNIFICANTLY OF DIFFERENT TREATMENT MEAN TO CONTROL MEAN. - SIGDNUIFNICNAETNTT'SLY DIFFERTEENSTT ((PP<<00..0055) FFRROOMM CCOONNTTRROOLL GGRROOUUPP BBYY LLSSDD AND HOMOGENEITY - P VALUE OF F TEST OF WHETHER TREND - P VALUE OF F TEST CHANGE IN GROUP OF WHETHER THERE MEANS. GROUP MEANS ARE IS DOSE-RELATED EQUAL, BARTLETT'S TEST - P VALUE OF TEST OF HOMOGENEITY OF VARIANCE Sanitized. Doss not contain TSCA CBl Company TABLl? 2 FEMALE MICE LIVER 6 BODY WEIGHT SUMMARY MEAN ABSOLUTE AND RELATIVE DATA GROUP CONC. FINAL WT. ABSOLUTE WT. RELATIVE WT. 0 PPM 1 PPM 3 PPM 10 PPM 30 PPM TEST - HOMOGENEITY TEST - TREND BARTLETT'S TES'* 25 .0( 25 .6( 25 .2( 26 .0( 22 .8( 0. 000) 0. 580) 0. 853) 0. 359) 0. 052) 0. 058 0. 109 0.626 1 .414( ^ 663( 1 854( 2 .493( 3 .167( 0.000) 0.107) 0.007)t 0.000) 0.000) 0. 000 0. 000 0. 207 5 664( 6 .513( 7 .343( 9 .600( 13 .896( 0. 000) 0. 139) 0. 006! 0. 600)* 6. OQOltf 0. 000 0. 000 0. 626 Values in parentheses - P VALUE OF STUDENT T TEST COMPARISON OP TREATMENT MEAN TO CONTROL MEAN. ' - SIGNIFICANTLY DIFFERENT (P<0.05) PROM CONTROL GROUP BY LSD - SIGNIFICANTLY DIFFERENT (P<0.05) FROM CONTROL GROUP BY LSO DUNNETT'S TEST AND HOMOGENEITY - P VALUE OF F TEST Of WHETHER GROUP MEANS ARE EQUAL. TREND - P VALUE OP F TEST OP WHETHER THERE IS DOSE-RELATED CHANGE IN GROUP MEANS. BARTLETT'S TEST - P VALUE OP TEST OF HOMOGENEITY OF VARIANCE 13 Company Saniti.ed. Does not contain TSCA CBI --I.yrtfBTOW^S^g*^?'------------^ HL 401-85 APPENDIX II Individual Body and Liver Weight Data from Animals that were Removed From Test Diets Mice Fed diet containing 1,000 ppm or higher were placed on control diet on test day 5 because of moderate to severe body weight loss and death (1/10 mice) in the 10,000 ppm group. On test day 6, the surviving mice From these groups were sacrificed in extremis and liver weights were recorded. Similarly, mice from the 100 and 500 ppm groups were sacrificed on test day 8. The initial and f nai ou^y weights and the liver weights of these mice are presented here for completeness. TSCACB1 ^Cmomnpaannvy Sani.t. neHd.nDoo.s- nnoot contain -^S^^E APPENDIX II Body and i-.ver Weights for Mice Fed 100, WO , 1,000. 3,000,ard 10. 000 ppm C-9 HU 401-85 Concent rat i on (ppm) 100 Males 100 Females 00 Males 10 Females 1 Males Mouse No. 34716 54717 5471 34719 54720 54779 54780 34781 54:j2 34786 34721 54722 54725 34724 54725 34787 34788 34789 34790 34791 34727 34728 34729 34730 34732 Females 54792 34793 34794 34795 34796 Initial Body Weight (g) 31 J7kDO . 29 31 29 21 20 23 22 22 29 xJUn 29 30 30 22 22 24 21 23 29 31 29 30 9tX0f 22 22 23 20 22 Final Body Weight (q) 27 22 24 29 27 22 16 20 17 18 17 19 18 18 19 F.O.[8J 15 F.OJ8J 14 F.D.f8j 17 19 17 20 18 14 F.D.[6J F.D.[5J r.o.[6j F.D.[6] F.O. Found Dead; number in brackets is equal to days on test. Liver Weight (g) 3.413 2.380 2.658 3.466 3.219 3.021 2.150 2.665 1.854 2.205 1.932 2.415 1.885 1.910 2.327 1.468 2.138 1.785 1.589 1.868 1.22 1.984 t.70 1.99 11' U y* 1.673 1.34 1.71 1.24 1.390 15 - S^a.Oo.n.>c.^"TS"CS., Company "'''TM-iw"^c-%3?a^ '-".'ws'xs, APPENDIX II (Continued) Body and Liv<T Weights for Mice Fed 100, 300, 1, 000,, 3,000,and 10,000 ppm C-9 Concentration Cppm) 3,000 Males 5,000 Females ,000 Males 000 Females Mouse Mo. 54735 34734' 5473^ 34736 34737 34797 34799 34800 54801 34802 54738 34739 54740 34741 54742 34803 34804 34805 54806 34807 Initial Body Weiaht (o) 28 29 n 29 w 20 22 21 20 21 31 in 30 70 27Q9 22 21 97 22 22 Final Body Weight (g) F.D.[6] F.D.[6J 17 F.D.(6J 17 F.D.[5j F.0.[6] F.D.I6] F.0.[6] F.D.[5] 17 20 F.D.[6J 18 17 F.D.[6] F.D.[5J 12 13 F.D.[4] Liver Weight (a) 1.834 1.694 1.79 1.634 1.684 1.24 1.243 1.262 1.352 1.27 1.39 1.650 2.080 1.602 1.523 1.530 1.32 1.691 1.20 1.119 F.D. = Found Dead; number in brackets is equal to days on test. " ..---"---"*"