Document 1g6KOX0QLB8Y3M9V4okZ8mxjm

HLR 401-85 Subchronic Feeding Study of Ammonium Perfluoranonanoate (C-9) in Mice SUMMARY Croups of 5 male and 5 female mice were fed diets that contained 0, 1, 3, 1U, 30, 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 concentrations 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~0d 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 ppm 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 30 ppm were not possible because there were no concurrent control groups. Work by: }].t. GLrvtu* & T G * / W M / T S ------ CsIviSThi. Wylie----------------- Technician JWS/cgs(5) Acute Investigations 2- Cosncsr anlticcd, Does; rvr c.anisin T C-CA CSi Haskell Laboratory Report No. 401-85 Material Tested: Sponsor: Material Submitted b y : Haskell No. 15,437 Nonanoic acid, 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluoro-, ammonium aalt Polymer Products Department C . I. du Pont de Nemours and Company Wilmington, Delaware D A Polymer Products Apartment L. I. du Pont de Nemours and Company Washington Works Parkersburg, West Virginia Test Facility: Haskell Laboratory for toxicology and Industrial Medicine C. 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 There are 16 pages in this report Distribution: Acknowledgements: Rayanne L. Ferenz, Bruce A. Burgess and Gerald l. Kennedy, Jr. also participated in the conduct of this study. Company Sanitizad Does not contain T S C A CBI - 3- INTRODUCTION HLR 401-85 The purpose of this study was to evaluate the effects of ammonium perfluorononanoate (C-9) on the liver weights of male and female mice after dietary administrat ion of the test material for 14 consecutive days. A. Animal Husbandry MATERIALS AND METHODS Male and female 5 to 6-week old, Crl:CD-UICR)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, and 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 number 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 a"Timer-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 d;d 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 cr injury. Mice of each sex were randomly divided into 10 groups of i male and 10 groups of 5 female mice. The mean body weights for 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 chow (I5QQ2) 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 was test day 1. The mice were weighed and observed daily (weekends included when warranted by signs of toxicity). All mice killed in extremis or by design were euthanized by chloroform asphyxiation and exsanguinated. Liver weights were recorded for all mice killed in extremis well as those found dead or killed by design. Body weigHFs and liver weights were analyzed using a one-way analysis of variance. Pairwise comparisons between control and treatment groups were made using least significant difference and/or Dunnett's test. i jj j I 3! i % i i ,S J ''* $ :1 9 I 1 :.ft J1 -I J I .Jf :J C. Test Material HLR 401-B5 Physical Form: Purity: Synonyms : Other Codes: Stabi 1ity: D. Records Retention White solid Ammonium perFluorononanoate C-9 . rQ Verification of the concentration of the test material in the prepared diets Mas performed by personnel of the Polymer Products Department. The test material Mas assumed to be stable throughout the exposure phase of the study. All raM data and the .'inai report will 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 fron the top, middle and bottom of the mixed diet during preparation of the 100 and 10,000 ppm 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. The diets were refrigerated until used. B. Clinical Signs Clinical signs were similar for male and female mice. Nonlethai Concentrations Mice fed C-9 at concentrtionr of 100 ppm and less exhibited slight to severe sporadic weight loss (i.e., 3 - 165 of body weight). Lethal Concentrations Mice fed C-9 at concentrations of 300 ppm and higher exhibited severe weight loss (i.e., 32 - 455 bf body weight), ruffled fur, ; ,,/: lethargy, low posture and limpness. # nnAiiw n iill'ilAflA ppm group on test day 4. - 5- ...  ! -r-~m c . Body Weight and liver Weight Analysis ppm concent ration groups Tre s i m i l ^ J o X " * ^ 0 " '* m d 30 throughout the feedino studi ih " , ^ ? f the contro1 * * * male and female mice are presented i n j u r e s * ! "and 2?"** ** th J* andmice S d " T s t o ^ u l n i n T c - r a ' t f male -** ' - t o significantly heavier when compared t^the'cnn? f *' 30 BP* "ere r e * a t i v r it i s / w^ l tCnCfntration 9 rouP had e i g n i f i M n t i y ^ e a J S m e ^ ' 2-). ' y a wie" comParea to the control group (fabler 1 and ppm were^not Sposs iblebecause ^ h e r o ^ e r The * * - SE ' * * fed diets aboye .30, CONCLUSIONS ggm is s m a X ' S L r z r j z M f i r f c . ' C-9 at a distsr, c o n c i m t r a t ^ ? 9? ~ Of 1, 3, 10 or 30 DDm had m ,, f f - J PP^* C 9 at dletary concentrations : male or female mice? A d m i n i s t r t bdy "eights of eiI* e r ' a ^ n l s t i t " " V ie t rcSU" ea in s l l ht * w S T S k ' w * ,0 " " administration at concentrations of 300 ppm or higher caused iteith. ! 0'if ; ,| ISll im . . 1:V" Company Sanitized.Does r.otcontainTSCAjdBli FIGURE 1 gr w th c u r v e s o f m a le m ice fe d c -9 A. ./" /> A' cr" if B" ' -- J. jar'' / -a B-- + a. -- ef -X-- - < / /i 4' f 4 .e ' .; -fy er' ' -si V -r - 0-- X - -- - - 1 0 . 0 J-0 1__ L IS. 0 1-- 0 1-- I__ l_ i-- 1__ L 1-1.00 IB. 00 ft|tritfly-IiTTi'ir'i &:!?** 13 3 O o o 3 S' 3 C/> o > 0o AVERAGE BODY WEIGHT IN GRAMS 26.00 25.50 FIGURE 2 G R O W TM GURVES OF FEMALE MICE FED C -9 t -- r-i-T--i-- i-- r r T-r r-r-i -T - r - t - 2 5 .0 0 -- _ 2 4 .5 0 2 4 .0 0 " 2 3 .5 0 * 23.00 22.50 22.00 -- --pr ,V '* / / ^ ur /" / ' la-.- / /^ -*/ ft-- ' a a A B W-. A' o> / f- - (H. '4 Cl `_ A a I .j fi 6 1 4 A -1 21.50 21.00 0.00 a :oow s---0 COfflfO. -4 1ppa " J 4 J6 o J^ * V6 .k0 0x --J-- 0 . 0 0 ' - I - L _ L -J-- 1-- J-- J..-- J__ 1__ i , j__ , 10.00 12.00 14.00 16.00 days on test A 3 w + 10 ppa X 30 ppa ! t HLR 401-85 APPENDIX T Piet Analysis - 9- nol contain TSCACBi SanUiied .Does C o mpanv Polymer Product department 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; P8AL Nos 84-5799 to 5805) Six samples of rodent chow, 3 nominally 100 ppm and 3 nominally 10,000 ppm have been analyzed by the derivatizacion/gas chromatographic method using electron capture detector originally developed for CS 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 CIO region. Samples were run in duplicate with CIO as an Internal standard added to samples and standards. Unfortunately the C9 we had available as a standard has some C8 and possibly CIO 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 In 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 sample 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 to call me. Attachment Keywords: GC Perf1uorononanoate C9 S. R. Lui N. J. VIlone TABLE I PEEFLUOaOMONABOATE IH BQDEMT /no Prai No. _ Designation vg Perfluorononanoate/g.earnsle -- OuPlleate A n a l y ( * ^ 84-5799 0 ppm None detected^) 84-5800 100 ppm - Top 86; 85 84-5801 100 ppm - Middle 97; 90 84-5802 100 ppm - Bottom I 84-5803 10,000 ppm - Top 83; 78 11. 000; 10,500 84-5804 10,000 ppm - Middle 11, 200; 11,400 84-5805 10,000 ppm -- Bottom 11, 700; 11,100 I a) r s * - . . Of 10,000 ppm samples. ^ ' W r n . 0.1 g used In each analyst used for analyzingCioo^^sample? Peak b#erwed uo`ler conditions - 11 - Company Sanitized. Does not contain TSC A CBi table 1 MALE MICE LIVER & b o d y w e i g h t SUMMARY MEAN SOLUTE AND [ELATIVE DATA f i fi 15437 GROUP CONC final w t . 0 ppn 1 ppm 3 ppm 10 ppm 30 ppm TEST - HOMOGENEITY TEST - TREND B A R T L E T T 'S TEST 33.41 0.000) 32.0! 0.204) 34.01 0.580) 33.2( 3.853) 31 -8f 0.149) 0.221 0.411 0.082 Values in paren^he^ec _ n ,... ~~ ABSOLUTE WT. RELATIVE WT. 1 8 1 1 ( 0 .0 0 0 ) 2.162( 0.081) 2. 3651 0.009)# 3.171( 0.000)# 3 793( 0.000)# 5.422( 0.000) 6 . 752( 0.017)+ 6 . 952( 0.007)# 9.528( 0.000)# 11.953( 0.000)# 0.000 0.000 0.015 0.000 0.000 0.082 ====~ ===-==========- _____ * - significantly dif fe r ed s 'gS ec Tsly DIFreS S* .... S S S K S p S l|dd and "" ' P VALUE F r TEST F ~ -- -- EOUA' ` ' E TM ERE POSE-RELATED BARTLETT'S TEST P VALUE OF TEST OF HOMOGENEITY OF VARIANCE - 12 Company Sanitized. Does not contain TSCA CM TABL1? 2 FEMALE MICE LIVER i BODY WEIGHT SUMMARY MEAN ABSOLUTE AND RELATIVE DATA |h 15437 GROUP CONC. FINAL WT. ABSOLUTE WT. RELATIVE WT. 0 PPM 1 PPM 3 PPM 10 PPM 30 PPM 25.0( 0.000) 25.6( 0.580) 25.2( 0.853) 26. (H 0.359) 22.8( 0.052) 1.414( 0.000) 1.663{ 0.107) 1.854( 0.007) 2.493< 0.000)1 3.167( 0.000) 5.664( 0.000) 6.513 0.139) 7.343( 0.006) 9.600( 0.000) 13.896 6.000) TEST - HOMOGENEITY TEST - TREND BARTLETT'S TES'I 0.058 0.109 0.626 0.000 0.000 0.207 0.000 0.000 0.626 IXIIS*BBBB8B||I Values in p a r e n t h e s e s - P VALUE OF STUDENT T TEST COMPARISON iBStfflesss ____________ OF TREATMENT MEAN TO CONTROL MEAN. ! " fi2iiii'ANTLY DIFFERENT (P<0.05) FROM CONTROL GROUP BY LSD # - SIGN F CANTLY DIFFERENT (P<0.05) FROM CONTROL GROUP BY LSD AND DUNNETT'S TEST HOMOGENEITY - P VALUE OF F TEST OF WHETHER GROUP MEANS ARE EQUAL. TREND - P VALUE OF F TEST OF WHETHER THERE IS DOSE-RELATED CHANGE IN GROUP MEANS. BARTLETT'S TEST - P VALUE OF TEST OF HOMOGENEITY OF VARIANCE - 13 - Company Sanitized. Does not contain TSCA CBI HL 401-85 APPENDIX II Individual Body and Liver Weight Data from Animals that were Removed from Tnd Diets diet on tesTdaO r r Mgher mn ^ control death (1/10 mire) in thT?0 ,eier* bd>' "ei9ht loss and "id. from these g r o ^ e r e ' ^ r m r S T ?" ^ ^ 6> th' iving sr r & r s r thr liver weights of these mice are p" s e n t e ' ^ I r e ' i o r ^ - 14 Sanitized. Does not contain TSCA CBI Company APPENDIX I I HU? 401 100 inn3"? nne r ,Weght3 for Mice Fed ---- i00r T,Q0Q 5 .QQQ,and 10.OOP ppm p_Q Concert rat ion (ppm) Mouse No. 10U Males WO females 54716 54717 34718 54719 54720 54779 54780 34 781 54712 34786 500 Males 34721 34722 5472 3 34724 34725 500 Females 1,000 Males 34787 34788 34789 54790 34791 34727 34728 34729 34730 34732 1000 Females 34792 34793 34794 34795 34796 Initial Body Weight (q) 31 28 29 31 29 21 20 23 22 22 29 30 29 30 30 22 22 24 21 23 29 31 29 30 28 22 22 23 20 22 F inal Body Weight (q) 27 22 24 29 27 22 16 20 17 18 17 19 18 18 19 Liver Weight (q) 3.413 2.380 2.658 3.466 3.219 3.021 2.150 . 2.663 1.854 2.205 1.932 2.415 1.885 1.910 2.327 F.D.8J* 15 F.D.[8J 14 F.D.f8] 17 19 17 20 18 1.468 2.138 1.785 1.589 1.B68 1.22 1.984 1.70 1.99 1.34 14 F.D.[6] F.D.[5] F.D.[6J r.D.[6] 1.673 1.34 1.71 1.24 1.390 F.O. s Found Dead; nuifeer in brack*. i. c d to day3 ^ - 15 not contain TSCACBI: Sanitized. 02S Company APPENDIX II (Continued) innBinnar>d! l;Ver Wei9hts for Mice Fed -- 0> 300, 1,QOQ, 3.OQ,and 1Qronp ppm C-9 HL 401-85 Concentration (ppm) 3,000 Males 3,000 Females 10,000 Males 10,000 Females Mouse No. 54735 34734' 34735 34736 54737 54797 34799 34800 34801 34802 34738 . 34739 54740 34741 34742 34803 34804 34805 34806 34807 Initial Body Weight (q) 28 29 31 29 30 20 22 21 20 21 31 30 30 29 29 22 21 22 22 22 Pinal Body Weight (q) r.D.[6] F.D.fJ 17 F.D.[6] 17 F.D.[5J F.D.[6] F.D.[6] r.D.[6] F.D.[5] 17 20 F .0.[ 6 ] 18 17 F.D.[6] F.D.[5] 12 13 F.D.[4] Liver Weight (q) 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 r.D. = Found Dead, nurtier in brackets is equal to days on test. I ifl fl BB not conia.' inTSCACef 16 C O***'8'" * 164-00' teSSi