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FOR DU PONT USE ONLY AR226-2943 Ou Pont HLR 651-89 Study Title Inhalation Approximate Lethal Concentration and Pulmonary Pathology of Exposed Rats Author Rudolph Valentine Study Completed On November 22, 1989 Performing Laboratory E. !. du Pont de Nemours and Company, Inc. Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P. 0. Box 50 Newark, Delaware 19714 I Laboratory Project ID Haskell Laboratory Report No. 651-89 Page 1 of 19 Company Sanitized. Does not contain TSCA CBI GENERAI INFORMATION Du Pont HLR 651-89 Material Tested: Medical Research Wo.: Haskell No.; Physical Form: Composition: Contaminants: Synonym: Other Codes: lfr,699 White powder CAS Registry No.: Stability: Sponsor: The test material was assumed to be stable throughout the exposure phase of the study. Polymer Products Department E. I. du Pont de Nemours and Company, Inc. Wilmington, Delaware - 2- Company Sanitized. Does no! contain TSCA CB1 GENERAL INFORMATION (Contadi Du Pont HLR 651*89 Material Submitted By; Study Initiation Date: In-Life Phase Initiated - Completed: Notebook: Polymer Product^epartment |. I. du Pont de ttemdiirs and Company. Inc. Washington Laboratory Parkersburg, West Virginia 4/26/89 .... 5/3/89 - 6/8/89 There are 19 pages In this repbrt. Distribution: - 3Coff.pftYiy Sanitized. Does not contain TSCA CB1 Du Pont HLR 651-89 . . Inhalation Approximate Lethal Concentration and ^ 1l"ona^ ^ SUMMARY T h ^ c u t e Inhalation toxicity and pulmonary pathology ]n 9ruP* of male Crl:CDBR rats. Test atmospheres h1ah-?rJJifdafi s^ P e n s 1 o n o f the particulate test material in a mlfciiErrh! 6 air sir*am* Jhe atmospheric concentration of aerosol was c S ^ t i < j r f S t,l i . " " V i,.` - 1 ,leteTM 1"e *" Approximate " S . " i s m h S ^ j 2 y f f i | y ^ 2 * " 0 ^ x p < > se<l nose-only for a single, S f t l /fter T i X Z Si ^ ? c ? i , 2400 groepof 9 r , u f0r,4 TffS^ro!^^!S rV wAfnhfx n J d 30 d s post exposure; the lungs from these rats were eosln and exam1ned*bveiiShSdm ? ith foriBalfjJ* stained with hematoxylin and male Crl CDBR r i ^ bi J l9h^ I A Procedural co"trol group of 6 rraattss ffrroomm tthhi?s5 gIrfoSulp wereu?k?idll,efdrahtl2staonmdor3p0holdaoygsi.c comparison; sub*groupps of 3 ^ ^ T h r e ^ ^ ^ r a t s dled^wlthin 24 hours of exposure to 2400 mg/m3 ^ ^ ^ the highest concentration tested. Clinical s i a n ^ ^ b?iiSfJia?bdirchirlr!M ately afitfir !? pof ure deluded compound-staged fir, ebxoDXoswuerli?gh* t0n0nocslsJens!9ciuaDl ?0r| t Si96n%s olf ito" 1x*ic1*it'ybwode>re*eo*bA9shethrtveewrditthhdeiunnrin2s4glighthhoeut rstoomf oderate exposure!01"6 perfod* Rats be9an to re9ain bdy weight by day 2 post rnnral* e*aluate pulmonary pathology, rats were exposed tn * sublethal concentration (approximately 1000 mg/nr) of 2 5 :fiIL5Sffl?ian9es '?erV ound in any atfd microscopic changes were noted. These changes Included the aSS f e r i a l within the alveoli and alveola? macJSphages d i e t ? 1^ , c 1 .ar! as of inflammation within the alveoli and alveola? ' z " ts. A ^though test material was still discernible within alveolar t ? m l PiiteSofhI ? i ? ^ Ut the study, other microscopic pulmonary changes were transient, of minimal severity and had resolved within 7 days of exposure. Under the conditions of this study, the ALC . ^ ^ ^ ^ f l s 2400 mg/nr. This material is considered to have very low minima^ tiawilSf ba?1s` ACUte exPsures were associated with material' withfn 1aramatdlry pulmonary changes; the presence of test cler.nce i f J e p o i l S d " . K f ! 1"9" thro"9hout the < " * , ongofng 4 Company Sanitized. Does not contain TSC CB1 SUMMARY (Cont'd) Du Pont HLR 651-89 Work byi R. f. Turner Technician Study Director] \ * u . 1. V Rudolph Valentine, Ph.D. Research Toxicologist Acute and Developmental Toxicology Division Reviewed and Approved for Issue] RV:alr:125.4 n Rudolph Valentine Study Director 5- Company Sanitized. Does not contain TSCA CB1 Du Pont HLR 651-89 QUALITY ASSURANCE DOCUMENTATION STUDY: B* 17,699 Inhalation Approxlnate Lethal Concentration and Pulmonary Pathology of Exposed Rats AUDITS: Items Audited Audit Dates Audit Number Pindings Reported to Study Director and Management Pathology Report #70-89 and Necropsy Records 9/13/89 --- 9/13/89 Protocol, records _____ and Final Report 11/9*10,13/89 O H R ll/U/89 - 6- ompany Sanitized. Does not contain TSCA CBI INTRODUCTION Du Pont HLR 651-89 aJ&SKra'g SaSS SS SS STmrare^lLtP,," Der?o dUT<in9 the efosurf Qr durin9 4 14-day postexposure observation tlst M t e H a rJ5it!!!r'ary Pat0]09y associated with exposure to the aijddltlonalj group of 9 rats was exposed to a sublethal killed t 9 7 Subgroups of these rats were wereweiahfd nrfm.fle* ?xp2s!!re,i,the lun9 tissues from these rats and e*amined r U g h t microscopy. Except as a M l T r h f r L hie K tud* recrds this study was conducted according to the applicable Good Laboratory Practice Rgulations. * MATERIALS AND METHODS A. Animal Husbandry Young adult male Crl:CDBR rats were received from Charles River Breeding Laboratories, Raleigh, North Carolina. Each rat was assigned a caidUflffiiiHiL ii ntif<C4ti0n nunber which corresponded to a numbered week D r i o r ^ t M H n n 89! ^ RatS were quarantined for approximately one Pr 'or. testing, and were weighed and observed three times durlna A ul\in9 the test, rats were housed in pairs in I" +hi4iX 8 suspnded, stainless steel, wire-mesh cages. The rat assigned the lower number in each cage was identified by a slash in the right e!r water-insoiuble'markers t ^ t h a t 3?ndividualrrats6cou?d^b*"ident1fied after SPSS; raiLTM"*cert1,,ei Hoh/ll r0?S were aStained on a timer-controlled, 12 hour/12 hour g t/dark cycle* Environmental conditions of the rooms were targeted for a temperature of 23 t 2`C and relative humidity of SO To*! w "s "sidf these ranges were judged to have been o? insufficient studytUde and/or durat1ort to have adversely affected the validity of the B. Exposure Protocol Approximate Lethal Concentration Determination wAinhiiiPL 2 fo L ma!e Cr1iCDBR rats (approximately 7-8 weeks old and weighing 256-295 g) were restrained 1n perforated, stainless steel cylinders with conical ndse pieces. The restrainers were inserted into a - 7Company Sanitized. Does net contain TSCA CB Du Pont HLR 651-89 face plate on the exposure chamber such that only the nose of each rat protruded into the chamber. The rats ware exposed nose-only to aerosol atmospheres of for a single, 4-hour period. Rats were weighed prior to exposure and were observed for clinical signs during and Immediately after exposure. Surviving rats were observed dally; rats were also weighed dally for 14 days post exposure, weekends and holidays excluded except when warranted by the rats' condition. All surviving rats were killed 14 days after exposure. Assessment of Pulmonary Pathology To assess pulmonary pathology, 1 group of 9 male Crl:CDBR rats (approximately 7 weeks old and weighing 250-276 g at study initiation) was exposed to Based on the.results-of the ALC determination, a subletfiaTdesign concentration of 1000 mg/nr was selected to assess lung pathology. Subgroups of 3 rats were killed at 2, 7 and 30 days post exposure by sodium pentobarbital anesthtsia and exsangulnation. The lungs were weighed and examined for gross lesions at necropsy, perfused with buffered formalin, embedded, sectioned, stained with hematoxylin and eosin and examined by light microscopy. For comparative purposes, an unexposed control group of 6 male Crl:CO*BR rats (approximately 7 weeks old and weighing 232-273 g at study Initiation) was used. Subgroups of 3 rats rats from this control group were killed at 2 and 30 days; the lung tissues from these rats were evaluated in the same manner as the treated group. Rats on the extended recovery period were checked dally for morbldlty/mortality; rats were weighed and observed for clinical signs daily, weekends excluded, for approximately 3 weeks and then once a week for the remainder of the study. C. Atmosphere Generation Particulate atmospheres o f w e r e generated by suspension of the test material in a high-pressure air stream. The test material was metered Into a Fluid Energy Processing and Equipment Company Model 00 Jet-O-Mizgr with a K-Tron Model T-20 twin screw volumetric feeder equipped with an electronic speed control. Test material entering the jet mill was entrained In an air stream (approximately 52 L/m1n) and was pulverized by impaction with other particles within the mill's reduction chamber. The test material exited the jet mill and was swept through a short, Tygon transfer tube directly into a cylindrical, borosillcate glass, 38-L exposure chamber. Particles entering the chamber were dispersed with a 4 cm circular baffle/lmpactlon plate located at the outlet of the transfer tube; this baffle acted like an Impaction plate to remove larger particles and promote uniform distribution. Chamber atmospheres were exhausted through a high-capacity flberpac dust filter, dry-fee cold trap and a MSA cartridge filter prior to discharge Into a fume hood. 8- Company Sanitized. Does not contain TSCA CBi % Du Pont HLR 651-89 D. Analytical The atmospheric concentration o f was determined at approximately 30-minute Intervals during each exposure by gravimetric analysis, knonfn volumes of chamber atmospheres were ara*m through preweighed, Gelman glass fiber (Type A/E) filters. Filters were weighed on a Cahn Moael 26 or 28 Automatic Electrobalance. The atmospheric concentration <jf was calculated from the difference In the pre- and post-sampling filter weights. i Particle size (mass median aerodynamic diameter and percent less than 3 and 10 um) was determined with a Sierra Series 210 cascade Impactor during each exposure.1 D u H n g each exposure, chamber temperature was measured with a mercury thermometer, relative humidlty was measured wltn a Bendlx Model 566 psychrometer,-and .chamber oxygen concentration was measured with a Biosystems Model 3100R oxygen analyzer. E. Statistical Analysis At termination, lung ahd final body weights were obtained. Data were analyzed by one-way analysis of variance. Statistical differences were declared at the p = 0.05 probability level. F. Records Retention All raw data and the final report will be stored in the archives of Haskell Laboratory for Toxicology and Industrial Medicine, Newark, Delaware, or in the Du Pont Records Management Center,' E . 1. du Pont ae Nemours and Company, Inc., Wilmington, Delaware. iI i - s- Company Sanitized. Doss not contain TSCA CB! DuPont HLR 651-89 RESULTS A. Exposure Conditions and Associated Mortality Aerosols of were readily observed during the exposures. Chambe^emperatures ranged from 25-26C, relative humidity varied from 48-548, and chamber j jxyoen concentration was 21.0%. Atmospheric concentrations o f a n d rat mortality data for each exposure are summarized 1n the following table. Characterization of ^ M Atmospheres ana Rat Mortality Aerosol Concentration (mg/m3)* Mean S.D. Range n Percent Perce;,t MMADb GSDC < 3 umd < 10 urn- Mortalityf 980 1100 2400 210 630 - 1200 9 330 580 - 1500 8 300 2000 - 2800 8 1.4 2.4 1.5 2.3 1.8 2.5 80 79 73 98 98 98 0/99 0/6 3/6 a Values shown represent the mean, standard deviation (S.D.), range and number of observations (n) for each exposure. Aerosol concentrations were based on filter weights obtained Immediately after sampling. Mars median aerodynamic diameter in urn. 5 Geometric standard deviation. Percent by weight of particles with aerodynamic diameter (AD) less than e 3 urn. Percent by weight of particles with aerodynamic diameter (AD) less than f 10 um. Mortality Is expressed as the number dead/number exposed. 9 Exposure used for assessment of pulmonary pathology. B. Clinical Observations Clinical signs of toxicity could not be taken during the exposures since the dense aerosol prevented visual observation of the rats. Upon release from the restrainers immediately after exposure, rats exhibited compound-stained fur, red ocular discharge, and brown oral discharge. No deaths occurred either during or Immediately after exposure. Deaths occurred within 24 hours of exposure to \______ ___ _ at an aerosol concentration of 2400 mg/rn3, with the exception of slight to moderate weight losses (up to 6% of Initial body weight) in 10 rom narv; O n , not contain TRC.A Cffl Du Pont HLR 651-89 some rats within 1 day of exposure, no clinical signs were noted in rats during the 14-day postexpoisure recovery period. All surviving rats began to regain weight by day 2 post exposure. C. Body and Organ Weight Analysis Two rats (#459627 and 459579) were omitted from statistical comparisons since their identification numbers could not be accurately established. No statistically significant differences 1n final lung weight or lung to body weight ratios were found when compared to controls for any group. Statistical analyses are shown in Appendix I. D. Pathology No gross abnormalities were detected in any animal at th tim of necropsy. Pathology data from 2 rats (#459627 and 459579) from the 30-day post-treatment groups were not Included in this report since the Identification numbers frdwn these rats could not be accurately established. Microscopically, a granular, yellow-brown material was present 1n the alveoli and alveolar macrophages of all compound-exposed rats 2 days after exposur; this material was observed in alveolar macrophages throughout th 30-day study. Subacute Inflammation, consisting of small focal Interstitial and alveolar Infiltrates of macrophages and neutrophils, associated with alveoli and alveolar ducts was also observed. These focal inflammatory changes were limited in number, of minimal severity and had resolved by 7 days post exposure. Pathology Report No. 70-89 1s attached as Appendix II. DISCUSSION AND CONCLUSION Under the conditions of this study, the ALC for]___ is 2400 mg/m Based on the atmospheric concentration of aerosol, _______ _____________________ [Is considered have very low toxicity on an acute Inhalation basis (ALC greater than 2000 mg/m3). Acute exposures were associated with minimal, transient Inflammatory pulmonary changes; the presence of test material within alveolar macrophages throughout the study Indicates ongoing clearance Of deposited material.* * Calculation described 1n Sierra Instruments, Inc., Bulletin 7-79-219IM, Instruction Manual; Series 210 Ambient Car Impactors and Cyclone Preseparators. - 11 - Company Sanitized. Does not contain TSCA CBI Du Pont HLR 651-89 Inhalation Approximate Lethal Concentration and Pulmonary Pathology of Exposed Rats Appendix I Final Lung and Body Weights - 12 Company Sanitized. Does not contain TSCA CB1 Du Pont HLR 651-89 Inhalation AppitoxIfflateLetha^oncentr^ and Pulmonary Pathology of H H H H M j H H 9 ExPse(1 Rats F1nat Lung and Body Weights Group: Unexposed Controls Days Post Exposure: 2 Rat Number irniai Lung weight (gj Body weight (g) 459574 459575 459576 1.475 1.738 1.580 289 291 294 Mean + (SD) 1.598 (0.132) 291 (3) o Group: 980 mg/ra Days Post Exposure: 2 Rat Number Final Lung weight (g} Body Weight (g) 459618 459619 459620 1.518 * 1.385 275 267 258 Mean + (SD) 1.452 (0.0940) 267 (9) 0 Group: 980 mg/nr Days Post Exposure: 7 Rat Number Final Lung Weight (g) Body Weight (g) 459621 459622 459623 1.635 2.006 2.296 331 296 332 Mean + (SD) 1.979 (0.331) 320 (21) Lung/Body Weight Ratio 0.00510 0.00597 0.00537 0.J0548 (0.00045) Lung/Body Weight Ratio 0.00552 * 0.00537 0.00544 (0.00011) Lung/Body Weight Ratio 0.00494 0.00678 0.00692 0.00621 (0.00110) ONE-WAY ANALYSIS OF VARIANCE P value for lung weight data 0.104; not significant P value for lung/body weight data 0.459; not significant w Invalid lung Weight, data not presented. 13 - Company Sanitized. Does not contain TSCA CB Du Pont HLR 651-89 Inhalation Approximate Lethal Concentration and Pulmonary Pathology pf ^ ats Final Lyng and Body Heights (Cont'd) Group: Control Days Post Exposure: 30 ___ final Rat Nun&er Lung Weight (g| Body Weight (g) 459577 459578 459579 1.581 1.497 ** 452 336 ** Mean + (SD) 1.539 (0.059) 394 (82) Lung/Body Weight Ratio 0.00350 0.00446 ** 0.00398 (0.00068) 3 Group: 980 mg/m Days Post Exposure: 30 Final Rat Number Lung Weight (g) Body Weight (g) Lung/Body Weight Ratio 459624 459626 459627 Mean + (SD) 1.661 2.033 ft* 1.947 (0.122) 465 502 ** 484 (26) 0.00400 0.00405 * 0.00403 (0.00003) ONE-WAY ANALYSIS OF VARIANCE P value for lung weight data * 0.051; not significant P value for lung/body weight data * 0.927; not significant ** Unable to establish animal Identification numbers; data not used for statistical analyses. 14 - Company Sanitized. Does not contain T S C A C Du Pont HLR 651-89 Inhalation A p p r o x f r a a t ^ e t h a l C o n c ^ and Pulmonary pathology of ^ B H H H H H j ^ H 9 ^ xPosed Rats Appendix II 15 - Company Sanitized. Does not contain TSCA CBl ES 3 6 6 R t v 12 e s r t i u i i t - r c isoa E. I. du Pont de N emours & C ompany IN C O R P O R A T E D H askell Lab o r ato r y for T oxicology an d In d u s tr ial Medicine P.O. B ox SO, E lkto n R oad Newark, Delaware 19714 central research and development department Du Pont HLR 651-89 HASKELL LABORATORY NO. 17699 POLMONARY PATHOLOGYj OF POLYMER PRODUCTS DATE ISSUED: NOVEMBER 21, 1989 -16- Qompany Sanitized. Does not contain TSCA CBi \ PULMONARY PATHOLOGY OF 1)u Pont HLR 651-89 Introduction Nine male Crl :CDBR rats were exposed nose-only to kat a concentration pf 0.98 mg/L for 4 hours. Subgroups of 3 exposed rats were serially-sacrificediat 2,7, and 30 days post-exposure. Six male Crl:CDBR rats, not exposed td the test material, were used as a control group. Subgroups of 3 control rats were sacrificed at 2 and 30 dayB to correspond to the first and l^st post-exposure subgroups. Euthanasia was by Intraperltoneal injection of sodium pentobarbital and exsangulnation. Lungs from these animals were examined grossly, weighed, perfused, and fixed In formalin. Representative sections of processed lung tissue were embedded in paraffin, sectioned at 5 micrometers, stained with hematoxylin and eosln, and examined microscopically. Results All animals survived until their scheduled sacrifice. No gross abnormalities were detected for any animal at the time of necropsy. Two animals were deleted from the study due to an identification problem. One was a 30 day post-exposure rat (#459627) and the second deleted rat (#459579) was a 30 day cage control. Table 1 contains microscopic observations of all rat lungs. The foreign material present In alveoli apd within alveolar macrophages of the compound exposed lungs was yellow-browri, Isotropic, and finely granular. It was assumed to be the test compound. The subacute Inflammation present In the 3 compound-exposed 2 day post-exposure lungs consisted of very small focal interstitial and alveolar infiltrates of macrophages, primarily, and neutro phils. The minute Inflammatory foci per lung section were few in number and associated with alveolar duct# and alveoli. These foci were most likely a response to the presence of the test compound. This minimal Inflammatory ' response was transient and ha# resolved In the 7 and 30 day post-exposure animals. The other microscopic findings consisting of microfocal hemorrhage and Inflammation in one control aijtlmal and perivascular lymphocytes in a treated -17<3onipany Sanitized. Does not contain TSCACB' Du Font HLR 651-89 animal were considered to be incidental findings and not test compound related. : Approved by: GTM/WCK/wfd GTM1 1.17 C-fyiaj Hllllam C. Krauss, D.V.M. Manager, Pathology Division - 18- Corrtpany Sanitized. Does not contain TSCA CB1 Du Pont HLR 651-89 HN-17699 PULMONARY PATHOLOGY OF i TABLE 1 m i c r o s c o p i c Ob s e r v a t i o n s in m a l e r a t lun g s mmmmmmmimmmmmmmmMmmmmmmmma Animal Number Recovery Days 0.0 ns/L aerosol , Microscopic Observations l 459574 459575 459576 459577 459578 2 2 2 30 30 No abnornjalltles detected No abnormalities detected Microfocal hemorrhage and subacute Inflammation, minimal No abnormalities detected No abnormalities detected 0.98 me/L aerosol 459618 2 459619 2 459620 2 459621 459622 459623 459624 459626 7 7 7 30 30 Foreign Material in alveoli and terminal airways, mild Foreign material within alveolar macrophages, minimal Mlcrofoc^l subacute inflammation, minimal Foreign material in alveoli and terminal airways, mild Foreign n^aterlal within alveolar macrophages, minimal Mlcrofoc^l subacute inflammation, minimal Foreign material in alveoli and terminal airways, mild Foreign material within alveolar macrophages, minimal Mlcrofocal subacute inflammation, minimal Foreign Material within alveolar macrophages, mild Foreign Material within alveolar macrophages, mild Lymphocytic infiltrate, perivascular, minimal Foreign material within alveolar macrophages, mild Foreign material within alveolar macrophages, minimal Foreign i^aterlal within alveolar macrophages, minimal -1 9 - ompany Sanitized. Does not contain TSCfcCHi