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FOR DU PONT USE ONLY AR226-2943 Du Pont HLR 651-89 Study Title Inhalation Approximate Lethal Concentration and Author Rudolph Valentine Study Completed On November 22, 1989 Performing Laboratory E. I. du Pont de Nemours and Company, Inc. Haskell Laboratory for Toxicology and Industrial Elkfton Road, P. 0. Box 50 Newark, Delaware 19714 Medicine Laboratory Project ID Haskell Laboratory Report No. 651-89 Page 1 of 19 Company Sanitized. Does not contain TSCA CBI GENERAL INFORMATION Du Pont HLR 651-89 Material Tested; Medical Research No. Haslcell No.: Physical Form; Composition: Contaminants: Synonym; Other Codes: 17,699 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, Wilmington, Delaware Inc. Z - Company Sanitized. Does not eonSain TSCA CB'i GENERALINFORMATION(Cont'd) Du Font HLR 651*89 Material Submitted By. Study Initiation Pate; In-Life_Pbase Inltfated - Completed; Notebook: olyner Products Department |. I. du Pont de Nemours and Company, Inc. Washington Laboratory Parkersburg, West Virginia 4/26/89 S/3/89 - 6/8/89 There are 19 pages In this report. Distribution: 1^ - 3 - >HpaYiySanitized. Does w\ c&rrtam T8CA CB1 Du Pont HLR 651-89 Inhalation Approximate lethal Concentration and Pulmonary Pathology of"^||B|||H|B|||J|||~Exp^ed^ats SUMMARY The acute Inhalation toxicity and pulmonary pathology of ,^_------^ was assessed In groups of male Crl:CDBR rats. Test atmosphe_re_s were generated by suspension of the paniculate test material in a high-pressure air stream, the atmospheric concentration of aerosol was measured by gravimetric analysis. To determine an Approximate Lethal Concentration (ALC). groups of 6 rats were exposed nose-only for a single, 4-hour period to W------------------hat concentrations of 1100 or 2400 mg/m . After exposure, rats were observed for clinical signs of toxicity to^HMBBM------lll^ during a 14-day recovery period. To assess pulmonary pathology, a separate group of 9 rats was exposed nose-only at a concentration of 980 mg/nr for 4 hours. Three subgroups of 3 rats were killed at 2, 7 and 30 days post exposure; the lungs from these rats were weighed at necropsy, perfuseld with formal in, stained with hematoxylin and eosin, and examined by light microscopy. A procedural control group of 6 male Crl:CDBR rats. was useid for histomorphologic comparison; subgroups of 3 rats from this group were killed at 2. and 30 days. Three of 6 rats died within 24 hours of exposure to 2400 nig/n^------l --------------------thRe highest concentration tested. Clinical signs of toxicity observed immediately after exposure included compound-stained fur. brown oral discharge and red ocular discharge. Other than slight to moderate body weight losses (up to 61 of initi?"; body weight within 24 hours of exposure), no clinical signs of toxicity were observed during the postexposure period. Rats began to regain body weight by day 2 post exposure. IHHHBHHBfHBl* To evaluate pulmonary pathology, rats were expasfidjbo a sublethal concentration (approximately 1000 mg/m3)^ Although no gross changes were found in any animal at necropsy, several compound-related microscopic changes were noted. These changes Included the accumulation of test material within the alveoli and alveolar macrophages, and minute, focal areas of inflammation within the alveoli and alveolar ducts. Although test material was still discernible within alveolar macrophages throughout the study, other microscopic pulmonary changes were transient, of minimal severity and had resolved within 7 days of exposure. tfHHI^is Under the conditions of this study, the ALC for^------^------iR 2400 mg/m3. This material is considered to have very low toxTcity on an acute Inhalation basis. 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. 4 - Company Sanitized. Does not contain TSCA CB1 SJUMM(ACoRnYt'd) Du Pont HLR 651-89 Kork b^ pu^.v..^ fcrgr"Tu<^^ R. T. Turner Technician Study Directors ^.WRufdt.olp\h. Va'^len_t_in_e._P_h_.D_.--_--_--_--_--_ Research lexicologist Acute and Developmental Toxicology Division Reviewed and Approved for Issuei _ _ r ^ l f t ' n u A ' n <VM^&< f --------------Rudolph Valentine Study Director RV:a1r:125.4 5 company Sanitized. Does not contain TSCA CB1 Du Font HLR 651-89 QUALITY ASSURANCEDOCUMENTATION STUDTt H 17,699 Inhalation Approxluate Lethal Concentration and Pulmonary Pathology Rats AUDITS: Items Audited Audijt Dates Pathology Report #70-89 and Necropsy Records 9/13/89 Audit Huaber Findings Reported to Study Director and Management 9/13/89 Protocol, records and Final Report 11/9(10,13/89 ll/U/89 // [\ i P LL Reported by: (JJ f f i^z^- Joseph - PSS^^Af C. BHaaoinll ill~ _ . Quality Assurance Auditor U/l^/^S 'Date "\ v 6- company Sanitized. Does not contain TSCA CBI Du Pont HLR 651-89 INTRODUCTION The purpose of this stujjy was to determine an Approximate Lethal Concentration (ALC) for ------------------b. The ALC was defined as the lowest concentration tested that caused the death of 1 or wore rats either during the exposure or during a 14-day postexposure observation period. To assess the pulmojnary pathology associated with exposure to the test material, an additional! group of 9 rats was exposed to a sublethal concentration of--------M--------------l Subgroups of these rats were killed at 2, 7 and 30 days post exposure; the lung tissues from these rats were weighed, processed and (examined ?"<' light microscopy. Except as documented In the study records, this study was conducted according to the applicable Good Laboratory Practice Regulations. MATERIALSAND METHODS A. Animal Husbandry Young adult male Cr1:CDBR rats were received from Charles River Breeding Laboratories, Raleigh, North Carolina. Each rat was assigned a unique 6-digit Identification number which corresponded to a numbered card affixed to the cage. Rats were quarantined for approximately one week prior to testing, and were weighed and observed three times during the quarantine period, paring the test, rats were housed in pairs in 8" x 14" x 8" 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 water-insoluble markers so that individual rats could be identified after exposure. Except during exposure* Purina Certified Rodent Chow8 #5002 and water were available ad _1ibitum. 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 23 + 2C and relative humidity of 50 + 101. Excursions outside these^ranges were judged to have been oT insufficient magnitude and/or duration to have adversely affected the validity of the study. B. Exposure Protocol Approximate Lethal Concentration Determination Groups of 6 male Cr1:CDBR rats (approximately 7-8 weeks old and weighing 256-295 g) were restrained in perforated, stainless steel cylinders with conical nose pieces. The restrainers were inserted into a Company Sanitized. Does not contain TSCA CBI 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 were exposed nose-only to aerosol atmospheres of ^|^------|------------R 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 weigheddally for 14 days post exposure, weekends and holidays excluded except when warranted by the rats' condition. A11 surviving rats were killed 14 days after exposure. Assessment of Pulmonary Pathology To assess pulmonary pathology, 1 group of 9 male Cr1:CDBR rats (approximately 7 weeks old and weighing 250-276 g at study Initiation) was exposed tolH|||IB||BI|||l|l^^ AtC determination, a sub!etnaFdesign concentration of 1000 mg/m- was selected to assess lung pathology. Subgroups of 3 rats were killed at 2, 7 and 30 days post exposure by sodium pentobarbital anestht-iia and exsangul nation. The lungs were weighed and examined for gross lesions at necropsy, perfused with buffered formalin, embedded, sectioned, stained with hematoxylln and eosin and examined by light microscopy. For comparative purposes, an unexposed control group of 6 male Cr1:CD*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 tn the same manner as the treated group. Rats on the extended recovery period were checked dally for morbidity/mortality; rats were weighed and observed for clinical signs dally, weekends excluded, for approximately 3 weeks and then once a week for the remainder of the study. C. Atmosphere Generation Paniculate atmospheres of^---------------------- were 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-0-Mlzer with a K-Tron Model T-20 twin screw volumetric feeder equipped with an electronic speed control. Test material entering the jet mm was entrained In an air stream (approximately 52 L/min) and was pulverized by Impaction with other particles within the mITl's reduction chamber. The test material exited the jet mill and was swept through a short, Tygon transfer tube directly Into a cylindrical, borosificate glass. 38-L exposure chamber. Particles entering the chamber were dispersed with a 4 cm circular baffle/lmpaction 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 fiberpac 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 CBE Du Pont HLR 651-89 D. Analytical i determined at approximately 30-m1nute Intervals during each gravimetric analysis. Known volumes of chamber atmospheres through prewelghed, Gelman glass fiber (Type A/El filters. weighed on a Cahn Moael 26 or 28 Automatic E1ectroba1ance. atmospheric concentration 4f|--------------I--------was exposure by were dram Filters Were The calculated from the difference In the pre- and post-sampling filter weights. Particle size (mass median aerodynamic diameter and percent less than 3 and 10 urn) was determined with a Sierra Series 210 cascade Impactor during each exposure.1 DuKng each exposure, chamber temperature was measured with a mercury thermometer, relative humidity was measured with a-BendIx Model 566 -psychrometer-,-and-chamber oxygen concentration was measured with a Biosystems Model 3100R oxygen analyzer. E. Statistical Analysis At termination, lung and 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 win be stored In the archives of Haskell Laboratory for Toxicology and Industrial Medicine, Newark. Delaware, or In the Du Pont Records Management Center ,'E. I. du Pont de Memours and Company, Inc., WHmlngton, Delaware. 9 - Company Sanitized. Doss not contain TSCA CB1 Du Font HLR 651-89 RESULTS A. Exposure Conditions and Associated Mortality f----ffH------were Aerosols of readily observed during the exposures. Chamberieniperaturesranged from 25-26C, relative humidity varied from 48-542, and chamber.ogygen concentration was 21.01. Atmospheric concentrations of----Hi--------------I------andrat mortality data for each exposure are summarized In the following table. Characterization of ""am Atmospheres Aerosol Concentration (mg/ro3)a Mean S.D. Range ~"n- 980 1100 2400 210 630 - 1200 9 330 580 - 1500 8 300 2000 - 2800 8 Percent Perce;.t HMAD" GSD1 < 3 urn" < 10 urn-' 1.4 2.4 80 98 1.5 2.3 79 98 1.8 2.5 73 98 Mortality' 0/99 0/6 3/6 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 a 3 urn. Percent by weight of particles with aerodynamic diameter (AD) less than r 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 toxiclty could not be taken during the exposures since the dense aerosol prevented visual observation of the rats. Upon release from the restralners 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------------B |f--------^at an aerosol concentration of 2400 mg/m3. with the exception of slight to moderate weight losses (up to 62 of initial body weight) in 10 nom^nv SanW^. ^ ^ rnn^.n W.A Cffl Du Pont HLR 651-89 some rats within 1 day of exposure, no clinical signs were noted In pats during the 14-day postexposure recovery period. All surviving rats began to regain weight by day 2 post exposure. C. Body and Organ Weight Analysis Two rats (1459627 and 459579) were omitted from statistical comparisons since their identification numbers could not be accurately established. No statistically significant differences In 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 the time of necropsy. Pathology data from 2 rats (#459627 and 459579) from the 30-day post-treatment groups were not Included In this repirt since the Identification numbers frijmi these rats could not be accurately established. Microscopically, a granular, yellow-brown material was present In the alveoli and! alveolar macrophages of all compound-exposed rats 2 days after exposure; this material was observed In alveolar macrophages throughout the 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 Is attached as Appendix II. DISCUSSION AND CONCLUSION for-Uf-------- Under the conditions of this study, the ALC Bilfc Is 2400 mg/m . Based on the atmospheric concentration of aerosol, ___H^--------------------^s considered have very low toxicity on an acute Inhalation basis (ALC greater than 2000 mg/m-). 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. 1 Calculation described In Sierra Instruments, Inc.. Bulletin 7-79-219IM, Instruction Manual: Series 210 Ambient Ca-- '^ Impactors and Cyclone Preseparators. 11 Company Sanitized. Does not contain TSCA CB1 Du Pont HLR 651-89 o^'dHBUBBI^ Inhalation Approximate Lethal Concentration and Pulmonary Pathology Exposed Rats Appendix I Final Lung and Body Weights " 12 " Company Sanitized. Does not contain TSCA CB1 Du Pont HLR 651-89 Inhalation Approj Pulmonary Pathology or incentratlon and Exposed Rats Final) Lung and Body Heights Rat Number 459574 459575 459576 Mean + (SD) Lung Weight (g) 1.475 1.738 1.580 1.598 (0.132) Body Weight (g) 289 291 294 291 (31 Lung/Body Weight Ratio 0.00510 0.00597 0.00537 O.J0548 (0.00045) Rat Number 459618 459619 459620 Mean + (SD) Lung Height (g} 1.518 * 1.385 1.452 (0.0940) Body Uei'ght (g 275 267 258 267 (9) lung/Body Weight Ratio 0.00552 * 0.00537 0.00544 (0.00011) Rat Number 459621 459622 459623 Mean + (SO) Lung Uelght (g) 1.635 2.006 2.296 1.979 (0.331) Body Uelght (g) 331 296 332 320 (21) Lung/Body Weight Ratio 0.00494 0.00678 0.00692 0.00621 (0.00110) ONE-HAY ANALYSIS OF VARIANCE P value for lung weight data 0.104; not significant P value for lung/body weight data 0.459; not significant * invalid lung weight, data not presented. 13 Company Sanitized. Does not contain TSCA C3' Ou Font HLR 651-89 Inhalation Approximate Lethal Concentration and Final Lyng and Body Heights (Cont'd) Group: Control Days Post Exposure: 30 Rat Number _Lu_ng__W_ei_gh_t_(Fg}inaBlo_dy_W_e_ig_h_t _(g_f 459577 1.581 452 459578 1.497 336 459579 ** Mean + (SD) 1.539 (0.059) 394 (82) Lung/Body Height Ratio 0.00350 0.00446 ** O.H0398 (0.00068) Group: 980 nig/in Days Post Exposure: 30 Rat Number _Lu_ng__U_dg_h_t _(gF)inaBlo_dy_U_e_lg_h_t T_g_T 459624 459626 459627 Mean + (SD) 1.861 2.033 ** 1.947 (0.122) 465 502 ** 484 (26) Lung/Body Weight Ratio 0.00400 0.00405 ** 0.00403 (0.00003) ONE-HAY ANALYSIS OF VARIANCE P value for lung weight data 0.051; not significant P value for lung/body weight data E 0.927; not significant ** Unable to establish animal Identification numbers; data not used for statistical analyses. I) 14 - Company Sanitized. Does not contain TSCA C Du Pont HLR 651-89 Inhalation Approximate lethal Concentration and Pulmona"/ pathology of 4fH------HU|ftxposed Rats Appendix II 15 Company Sanitized. Does not contain TSCA CB1 ES &: icv 12 a; cni t-cciKt E. 1. DU PONT DE NEMOURS ft COMPANY (NCBMPOHATKD HASKELL LABORATORV FOR TOXICOLOGY AND INDUSTRIAL MEDICINE P.O. BOX 50, ELKTON ROAD NEWARK. DELAWARE (9714 CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT Du Font HLR 651-89 HASKELL LABORATORY NO. 17699 PULMONARY PATHOLOGY OF P' RATS POLYMER PRODUCTS DATE ISSUED: NOVEMBER 21, 19^9 -16Qompany Sanitized. Does not contain TSCA CB! Du Font HLB 651-89 PULMONARY PATHOLOGY OF Introduction Nine male Cri :CDBR rats were exposed nose-only to --------------------. --^----Rat a concentration of 0.98 ng/L for 4 hours. Subgroups of 3 exposed rats were serially-sacrificed :at 2, 7, and 30 days post-exposure* Six male Crl:CDBR rats, not exposed to the test material, were used as a control group. Subgroups of 3 control) rats were sacrificed at 2 and 30 days to correspond to the first and l^st post-exposure subgroups. Euthanasia was by intraperltoneal injection of podium pentobarblcal and ex6angul-atlon. Lungs from these animals were examined grossly, weighed, perfused, and fixed Id formalin. Representative sectfions of processed lung tissue were embedded in paraffin, sectioned at 5 micrometers, stained with hematoxylin and eosin, and examined microscopically* Results ^ ' All animals survived until their scheduled sacrifice. No gross abnonaalitles were detected for any animal at the tine of necropsy. Two animals were deleted from the study due to an identification problem. One was a 30 day post-exposure rat (N59627) 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 a4d within alveolar macrophages of the compound- exposed lungs was yellow-browl, isotropic, and finely granular. It was assumed Co 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 neutrophlls. The minute inflammatory foci per lung section were few in number and associated with alveolar diicti and alveoli. These foci were most likely a response to the presence of the test compound* This minimal inflammatory response was transient and had resolved in the 7 and 30 day post-exposure animals. The other microscopic findings consisting of aicrofocal hemorrhage and Inflammation in one control animal and pefivascular lymphocytes in a treated -17- fionipanySanitized. Does not contain TSCACB? Du Font HLR 651-89 animal were considered to be incidental findings and not test compound related. : Report by: G. Tracy Hakoveci D.V.M. Diplomate^ A.C.V.P. Staff Pathologist Approved by; GTM/WCK/wfd GTM1 1.17 /WWillvllwaawC^.'-Kra, us.s._, _D^.V.-.M._. Manager, Pathology Division -18Coitipany Sanitized. Does not contain 7SCA CB1 Du Font HLR 651-89 HN-17699 PULMONARY PATHOLOOGFY ; TABLE 1 MICROSCOPIC OBSERVATIONS IN MALE RAT LONGS Animal Number Recovery Days 0.0 ng/L aerosol 459574 2 459575 2 459576 2 459577 30 459578 30 Microscopic Observations No abnornkaIlcleB detected No abnornialltles detected Microfocal hemorrhage and subacute Inflammation, minimal No abnormalities detected No abnormalities detected 0.98 mg/L aerosol 459618 2 459619 2 459620 2 459621 7 459622 7 459623 7 459624 30 459626 30 Foreign Material in alveoli and terminal airways, mild Foreign n^aterlal within alveolar macrophages, minimal Mierofocal subacute inflammation, minimal Foreign Material in alveoli and terminal airways, mild Foreign arterial within alveolar macrophages, minimal Mierofocal subacute inflammation, minimal Foreign material in alveoli and terminal airways, mild Foreign material within alveolar macrophages, minimal Mierofocal subacute inflammation, minimal Foreign material within alveolar macrophages, mild Foreign material within alveolar macrophages, mild Lymphocyfic Infiltrate, perlvascular, minimal Foreign Material within alveolar macrophages, mild Foreign material within alveolar macrophages, minimal Foreign niaterlal within alveolar macrophages, minimal -19eompany Sanitized. Does not contain TSCftCW