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AR226-2790 FOR DU PONT USE ONLY Approximate Lethal Concentration by Inhalation (ALC) of Ammonium Periluorononanoate (C-9) Haskell Laboratory Report No. 293-85 E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine P. 0. Box 50, Elkton Road Newark, Delaware 19714 Date Issued: June 3, 1985 Company Sanitized. Does notcontain T5CAG3f HLR 293-85 Approximate Lethal Concentration by Inhalation (ALC) of Ammonium Perf1uorononanoate (C-9) Summary To determine an ALC, groups of 6 male Crl:CD(SD)BR rats were exposed to dust atmospheres of C-9 for a single, 4-hour period, followed by a 14-day recovery period. After determining an ALC, 2 groups of 10 male rats were exposed for 4 hours to a marginally lethal and approximately l/10th of a lethal concentration of C-9. For each test group, a control group of 10 male rats was exposed simultaneously to air only. Five rats per group were killed on the 5th- day of recovery;-and 5 rats per group ware killed on the 12th day of recovery for a gross examination of the liver. Under the conditions of this test, the ALC for C-9 was 590 mg/m3. This material is considered moderately toxic by inhalation. 3 Rats exposed to 67 mg/m had significantly elevated mean liver weights and liver-to-body weight-ratios on the 5th and 12th days after exposure. Rats exposed to 590 mg/m had significantly elevated liver-to-body weight ratios on the 12th day after exposure. ' Liver weights for these rats were not significantly different than controls on the 5th day of recovery, and mean liver weights were significantly depressed on the 12th day of recovery. However, these seemingly inconsistent changes were due to severe body weight loss 1n rats exposed to 590 mg/m . Gross pathologic examination of rats exposed to 590 mg/m3 revealed discolored livers with prominent lobular patterns in 4/5 rats killed on the 5th day of recovery, and similar gross liver lesions in 2/5 rats killed on the 12th day of recovery. Work by: J4 T 7 Roobbeerrt T,. Turner Technician n /vr Study Director: . Laura A. Kinney Chemist ,, 6/3 / ? 1 LAK:smk:HLR9.14 Approved by: Section Supervisor Acute Investigations - 2- Haskell Laboratory Report No. 293-85 Material Tested: Sponsor: Haskell No. 15.437 Nonanoic acid, 2,2.3,3,4,4,5,5.6,6,7,7,8.8,9,9hexadecafluoro-, ansnonium salt Polymer Products Department E. I. du.Pont de Nemours and Company Wilmington, Delaware Test Facility: Polymer Products Department E. I. du Pont de Nemours and Company Parkersburg, West Virginia E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine P. 0. Box 50, Elkton Road Newark, Delaware 19714 Study Initiated/Comoleted: 1/3/85 - 2/3/85 r Notebook E-38738, pp. 55-110 There are 15 pages in this report. INTRODUCTION HLR 293-85 P e Purpose of this study was to determine a 4-hour inhalation ALC for C-9 in male rats. The ALC was defined as the lowest atmospheric concentra tion tested that caused the death of 1 or more rats either on the day of exposure or within 14 days post exposure. In addition, following the determination of the ALC,;additional exposures were conducted to monitor the effects o f C - 9 inhalation on the liver. Except as documented in the study SSSla C0"dUCted accord1"s t0 the tood Laboratory MATERIALS AND METHODS A. Animal Husbandry Young adult male Crl:CD(SD)BR rats were received from Charles River Breeding Laboratories, Kingston, New York. Each rat was assigned a unique 6-digit identification number which corresponded to a numbered card affixed to the cage. Rats were quarantined for one week prior to testing, a n d w e r e weighed and observed twice during the quarantine period. During the test, rats were housed in pairs in 8" x 14" x 8U suspended, stainless steel wire-mesh cages. The rat assigned the lower number in each cage was identified by a slash in the right ear. Prior to exposure, rats tails and cage cards were color-coded with waterinsoluble markers so that individual rats could be identified after exposure. Except during exposure, Purina Certified Rodent Chow #5002 and water were available ad libitum. B. Exposure Protocol . * T dS55rn,,!l* ALC* 9rouPs of 6 rats, 8 weeks old and weighing between 234 and 298 grams, were restrained in perforated, stainless steel cylinders with conical nose pieces. Each group was exposed nose-only for a single, 4-hour period to a dust atmosphere of C-9 in air. Rats were weighed prior to exposure, and were observed for clinical signs of toxicity du m g exposure. Surviving rats were weighed and observed daily for 14 days post exposure, weekends excluded except when deemed necessary by the rats' condition. * To monitor the effects of C-9 inhalation on the liver, 2 groups of 10 rats, 8 weeks old and weighing between 237 and 277 grams, were restrained and exposed to a marginally lethal and approximately l/10th of a lethal exposure concentration of C-9, respectively. Two groups of 10 rats. 8 weeks old and weighing between 231 and 267 grams, were restrained and exposed to air only. Each control group was exposed concurrently with : 4- Company Sanitized. Does not contain T S C A CBS HLR 293-85 one of the test groups. Five rats per group were killed 5 days after exposure, and 5 rats per group were killed 12 days after exposure for pathologic examination of the liver. C. Test Material Physical Form: Purity: Synonyms : Other Codes: Stability: I CJ `ononanoate The test material was assumed to be stable throughout the test. D. Atmosphere Generation For most exposures, dust atmospheres of C-9 were generated with a K-Tron Bin Feeder equipped with twin feed screws. The feed rate was regulated with a K-Tron*> Volumetric Feed Controller. The bin feeder metered test material into a glass transfer tube. Air introduced at the tube swept the test material through a size-reducing cyclone and into the exposure chamber. The cyclone removed large particles by inertial impaction, while aerodynamic particles passed through the cyclone and into the exposure chamber. The atmospheric concentration of C-9 was controlled by varying the feed rate. For the lowest exposure concentration, the atmosphere was generated by passing pressurized air through a 2-stage glass generator. A round flask at the bottom of the generator served as a dust reservoir. A cyclone elutriator was inserted above the reservior. A notorized stirring rod with plastic paddles agitated dust in the generator. Air introduced at the bottom of the reservoir and at the cyclone elutriator swept dust particles into the exposure chamber. The atmospheric concentration of C-9 was controlled by varying the 2 airflows. E. Analytical The atmospheric concentration of C-9 was determined at approximately 30-minute intervals by drawing calibrated volumes of chamber atmosphere through preweighed, Gelman glass fiber filters. Filters were weighed on a Cahn model 26 Automatic Electrobalance. The atmospheric concentration of particulate was determined from th filter weight differential before and after sampling. Particle size (mass median aerodynamic diameter and percent respirable) was determined with a Sierra model 210 cascade impactor during each exposure. During most exposures, chamber temperature was 5- Company Sanitized. Does not contain T S C A CBS H U 293-85 measured with a mercury thermometer, relative humidity was measured with a Bendlx model 566 psychirometer, and chamber oxygen content was measured with a BioMarlne model 225 oxygen analyzer. F. Pathology Two groups of 10 rats were exposed for 4 hours to 67 or 590 mg/m3 of C-9 in air, and 2 groups of 10 rats were exposed to air only. Five rats per group were arbitrarily selected and killed by chloroform anesthesia and exsanguination 5 days after exposure. The remaining 5 rats/group were killed 12 days after exposure. Rats were necropsied and the livers were weighed. Mean liver weights and liver-to-body weight ratios for test rats were compared to their respective controls by Least Significant Difference and Dunnett's tests. Significance was judged at the 0.05 probability level. G. Records Retention All raw data and the final reports will be stored in the archives of Haskell Laboratory for Toxicology and Industrial Medicine, Newark, Delaware, or in the DuPont Hall of Records, E. I. du Pont de Nemours and Company, Wilmington, Delaware. RESULTS A. Exposure Conditions and Associated Mortality During most exposures, the chamber walls were coated with the test material. Chamber temperatures ranged between 23-27C, relative humidities ranged from 19-45%, and chamber oxygen contents were 21%. Atmospheric characterization and mortality data are summarized in the following table. 6- - Company Sanitized. Does not contain T S C A CBI Characterization of c-9 Atmnsnhproc and:Associated Rat Mortality1 HLR 293-85 Concentration imo/m3l Mean S.D. Range % Respirable3 620 910 1600 4600 180 490 - 980 760 10 - 1700 420 920 - 1900 600 4100 - 5900 91 94 93 90 59607c 35 290 12 - 120 300 - 1200 95 75 MMDfum)b 3.4 3.2 34 4.2 3.5 3.7 Mortality (# deaths/# exoosed} 0/6 4/6 6/6 6/6 l/5d b Percent by weight of particles with aerodynamic diameter less than in m c Mass median aerodynamic diameter. Iess than 10 um . a?f?surfs f?r subse9 uent pathologic examination, d Although this exposure was conducted for subsequent pathologic B. Clinical Observations nZelad%s*. r9A!l?l; r3atfs"'d'i"e`dshdeudrisntgaretx,peos^uPre t"o 46l0i0d mtge/smt3^. . r f i l on thl" m o /m 3D!iriH9,?h! PstexPsure P ^ o d , 1/5 remaining rats exposed to 590 JJi? d2fd 12 days pos* exposure, 4/6 rats exposed to,,910 mg/m" died 9-11 i n n CP ^ expsure and fi/6 rats exposed to 1600 mg/m3 died 4-8 days post exposure. Rats exposed to 67 mg/m3 lost 1-9% of initial body weiqht 1 h e a t e r t h S 6677 r?ma9//i91'1^rats ?,yosti'0a'pTMpr1oxWi?miagthetly9a6i-n1'5%Atofcin"icteiiatlratbioodiys t h l n ^ h 1 Ja^ PSt exPsure, and continued to lose weight either throughout the recovery period er until they died. Most survivina rats weiahtdwhfn5?h* 620 r 9 1 0 "?/'n|3 weighed only'54-71% of initial body * d,ys P0St eX,,M"re 0r >' rsi^,j,,s?*i?g52r* 7 concentrations included hunched posture, ruffled or discolored fur, red - 7Com pany Sanitized. Does not contain T S C A C B i HLR 293-85 or brown facial discharges, wet or stained perineum, pallor, lung noise or labored breathing, lethargy, limpness and hair loss. Clinical signs were observed throughout the recovery period. C. Pathology Gross pathologic examination of rats exposed to 67 mg/m3 revealed large livers in 5/5 rats killed on the 5th day of recovery. After 12 days3of recovery, none: of the rats' livers were noted as large. At 590 mg/m , -4/5 rats killed'on the 5th day of recovery had discolored livers with prominent lobular patterns or markings. These patterns were probably attributable to diffuse fatty change with a zonal distribution. On the 12th day of recovery, gross liver lesions were noted in 2/5 rats The Pathology report is attached as Appendix I. Statistical analyses showed significantly elevated mean liver, weights and 1iver-to-body weight ratios in rats exposed to 62 mg/m3 on both the 5th and the 12th days of recovery. In the 590 mg/nr exposure group, mean liver weights and liver-to-body weight ratios were not significantly different from controls on the 5th day of recovery. On the 12th day of recovery, mean liver weights were significantly lower than controls; however, liver-to-body weight ratios were significantly elevated. The lack of significant liver weight changes on the 5th day of recovery, and the depressed mean liver weights on the 12th day of recovery were due to a large loss of body weight in rats exposed to 590 mg/m . Mean body weights for rats exposed to 590 mg/m3 were only 70% and 48% of controls on the 5th and 12th days of recovery, respectively. However, mean liver weights were 83% and 72% of controls, respectively. Statistical analyses of mean liver weights and liver-to-body weight ratios are presented in Appendix II. CONCLUSION Under the conditions of this study, the ALC for C-9 was 590 mg/m3. This material^! considered moderately toxic by inhalation (ALC between 200 and 800 gg/m ). However, C-9 caused elevated liver weights in rats exposed to 67 mg/m on the 5th and 12th days after exposure, and gross liver lesions in rats exposed to 590 mg/m . **8 Calculation described in Sierra Instruments, Inc., Bulletin 7-79-219IM, Instruction Manual: Series 210 Ambient Cascade Impactors and Cyclone Preseparators. -- --------- ------------- *----- 8- Company Sanitized. Does not contain TS CA CBI HLR 293-85 9Company Sanitized. Does not contain TSCA CB d I W *CV >t*2 E. I. ou Pont de Nemours S Company Haskeu. L aboratory for Toxicology and Industrial Medicine P.O. Box 30. Elkton Road New ark. Delaware 19711 CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT C-9 APPROXIMATE LETHAL CONCENTRATION (ALC) TOXICITY STUDY ______________ IN MALE Crl:CD*(SD)BR RATS_____________ GROSS PATHOLOGY POLYMER PRODUCTS DEPARTMENT DATE ISSUED: MAY 16, 1985 - TO - Com pany Sanitized. Does not contain T S C A C B ! ALC TOXICITY STUDY IK MALE HATS WITH C-9 Introduction and Results Male Crl:CD(SD)BR rats exposed to C-9 for 4 hours via inhalation were necropsied and livers were weighed and examined at 5 and 12 days post exposure. Method of euthanasia was chloroform anesthesia and exsanguination. Table I identifies the exposure groups and contains the gross observations made at necropsy: The livgr of only a few exposed animals was noted as large at necropsy, however, liver weights (weights and statistics will not be presented in this report) indicate that Che liver o f a l l exposed animals was large..The prdminent lobular pattern noted in 4 of 5 high dose rats examined on day 5 post-exposure is most likely attributable to diffuse fatty change with a zonal distribution (centrllobular or periportal). A portion of the Increase in liver size may be due to fatty change, however, it is more likely that the liver functions as the primary sice of metabolism of the compound and that the Increase in size is due to a proliferation of smooth endoplasmic reticulum. Acknowledgement Joan A. Wolfe was pathology supervisor for this study. Report by: Theodore W. Slone, D.V.M. Diplomate A.C.V.P. Staff Pathologist TWS/WCK/wfd SLONE 3.13 Approved by: William C. Kruass, D.V.M. Manager, Pathology Division - 11 - containCompany Sanitized. Does not TSCA C8I 3 TABLE I GROSS PATHOLOGY C-9 iw a giicaaaagaa--a-iTMALE RATS - INHALATION< Animai Test Recovery Mode of* Number Days Days Death Observations Group IA - Control 386616 6 5 SD Liver - discoloration, dark red area, nan), left lobe 386618 6 5 SD No abnormalities detected 386620 6 5 SD No abnormalities detected 386622 6 5 SD No abnormalities detected 386624 6 5 SD No abnormalities detected 386617 13 12 SD No abnormalities detected 386619 13 12 SD No abnormalities detected 386621 13 12 SD No abnormalities detected 386623 13 12 SD No abnormalities detected 386625 13 12 SD No abnormalities detected Group IB -- Control 386988 6 5 386990 6 5 386992 6 5 '386994 6 5 386996 6 5 386989 13 12 386991 13 12 386993 13 12 SD No abnormalities detected SD No abnormalities detected SD No abnormalities detected SD No abnormalities detected SD No abnormalities detected SD No abnormalities detected SD No abnormalities detected SD No abnormalities detected * SD = S a c r if ic e d by d e s ig n ; FD = Found dead - 12 - Company Sanitized. D oes not contain T S C A C B t - 4- 15437. TABLE I Part 2 i m m GROSS PATHOLOGY : c-9 MALE RATS - INHALATION Animal Test Recovery Mode of Number Days Days Death ______________ Observations 386995 386997 13 13 12 12 SO No abnormalities detected SD No abnormalities detected Group II ~ High Dose (0.59 mg/I) 386545 6 5 SD 386546 6 5 SD 386550 6 5 SD 386552 6 5 SD 386554 6 5 SD 386547 13 12 SD 386549 386551 386553 13 13 13 12 12 12 FD SD SD 386555 13 12 'SD Liver - discoloration, lobular pattern prominent Liver - discoloration, lobular pattern prominent No abnormalities detected Liver - discoloration, lobular pattern prominent Liver - discoloration, lobular markings prominent, tan streaks scattered Liver - foci, white, scattered, (< 2 mm in diameter), all lobes No abnormalities detected No abnormalities detected Liver - discoloration, small, white area, left lobe, (< 2 mm In diameter) Perineum - alopecia, moderate Dorsum - alopecia, right, moderate Group III - Low Pose (0.067 mg/L) 387033 6 5 SD 387035 6 5 SD Liver - large Liver - large - 13 Company Sanitized. Does not contain T S C A CBI TABLE I Animal Number Test Days Recovery Days GROSS PATHOLOGY C-9 HALE RATS - INHALATION Mode oi Death Observations 387037 387039 387041 387034 387036 387038 387040 387042 6 6 6 13 13 13 13 13 5 5 5 12 12 12 12 12 SD Liver - large SD Liver - large SD Liver - large SD No abnormalities detected SD No abnormalities detected SD No abnormalities detected SD No abnormalities detected SD No abnormalities detected Pare 3 - 14 Company Sanitized. Does not contain T S C A CBI % APPENDIX II HLR 293-85 C9 - MEAN LIVER HEIGHTS AND LIVER-TO-BODY WEIGHT RATIOS RATS SACRIFICED ON THE 5TH DAY OF RECOVERY GROUP CONTROL,I 67 MG/MJ CONTROL II 590 MG/M* FINAL HT. 275.6 (O.OOO) 276.6 (0,912) 293.4 (Oi.000) 204.0 (Oi.000)# LIVER 11.306 (0.000) 14.501 (0.002)# 14.511 (0.000) 12.010 (0.164) LIVER-TO-BODY WEIGHT RATIO 4.090 (0.000) 5.243 (0.000)# 4.946 (0.000) 5.803 (0.053) Values in parentheses - P value of Student's t test comparison of treatment mean to control mean. + - Significantly different (p<0.05) from control group by LSD # - Significantly different (p<0.05) from control group by LSD and Dunnett's test C9 - MEAN LIVER WEIGHTS AND LIVER-TO-BODY WEIGHT RATIOS RATS SACRIFICED ON THE 12TH DAY OF RECOVERY GROUP CONTROL-I 67 MG/MJ CONTROL II 590 MG/M3 FINAL WT. 315.4 (0.000) 325.0 (0.374) 335.4 (0.000) 160.0 (0.000)# LIVER 14.806 (0.000) 20.371 (0.001)# 15.557 (0.000) 11.198 (0.002)# LIVER-TO-BODY WEIGHT RATIO 4.678 (0.000) 6.268 (0.000)# 4.649 (0.000) 7.003 (0.000)# Values in parentheses - P value of Student's t test comparison of treatment mean to control mean. + - Significantly different (p<0.05) from control group by LSD # - Significantly different (p<0.05) from control group by LSD and Dunnett's test 15 - ' Company sanitized. Does not contain T S C A C B l