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AR 246 _ 3277 481 R226. 3297 Duron 15308 ~. `TRADE SECRET 125914: Inhalation ApproximSattuedyLeTtihtalel Concenraton (ALC) in Rats AUTHOR: Michael P. DeLorme, Ph.D. STUDY COMPLETED ON: September 9, 2003 PERFORMING LABORATORY: E.L du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 ~ LABORATORY PROJECTID: DuPont-13308 Company Sanized.Doss otconanTSCACBE Page lof 11 1125914: Ibalion Approximate Lethal Concenrution (ALC)inRats --_ CERTIFICATION Dupont13308 1, the undersigned, declare that this reportprovidesan accurate evaluation ofdata obtained from this study. LoudbyStudyDirector: deCRf eTssTDoewsedt:one 23fc o m ~ Company Sanfized. Dossno conte` TSCA CBI ~ FT 105914: tation Appoint Lael Consnsnion ALC) Rats STUDY INFORMATION Dupo 1338 Synonyms/Codes: + H-25914 a `Submitter's Notebook Number(s) (-- - Haskell Number: 25914 CAS Registry une [ re) a Known Impure - \ m-- | Pa Phusial Characteristics: Anber liquid Stability: The test substance appear0ebde stable unde te | acobnsdeirtvieodn.s of the study; no evidence of instability was | `Sponsor: E.L du Pont de Nemours and Company Wilmington, Delaware 19898 USA. Study Initiated/Completed: June 26, 2003 / (see report cover page) --~ Company Sanitized. Does not contain TSCA CBI = 125914: InhalationApprLeothaxlCoincenmtraationt(ALeC) inRats DuPont13308 --_ STUDY PERSONNEL Study Director: Michael P. DeLorme, Ph.D. PrimarMyaTneacghneimceinatn:: WilliaEm. Ells, Jr. Arthur J. O'Neill, B.S. Toxicology Report Preparation: Maryanne M. Wilford, B.A. Management: Nancy S. Selzer, M.S. Laboratory`VMeatnearigneamreinatn:: Thomas W. Maser, D.V.M,, Diplomate A.C.LA.M. Janice L. Connell, M.S, B.A, C.LH. -- `CompanySantzed.Doss notcontarn TSCA cay -- 125914: Inhalation Approximate Lethal Concentration (ALC) in Rats Dupont13308 SUMMARY Thowuorpgerroiuopdstoof62m5a9le14Criln:aCirD.(HS-D2)5I9G1S4 BisRa rsautsspeenascihor exposed nose-only foar single, 4`Concentrationsof H-d25te9s1t4atwmeorsephdeerteesrmwienreed gbeynegrraavtiemdbetYriGceranoaslOyNsZiast.ioSn.inceAHI-r2oo5m9e14 was adilute sduestpeernmsiinoant,iosnaomfpl"eweftil"tearesrwoesorlecwoenicgehnterdatiimomned(isuastpeelnysifoonl)l,owainndgtahiernsasmubpsleeqcuoelnltelcytidoensifcocrated for determinationofthe "dry" aerosol concentration (polymer). Animals were weighed and observed for clinical signsoftoxicity during a 14-day recovery period. Rats were exposed to mean wet aerosol concentrations of 1200 or 4900 mg/m, which corresponded todryaerosol concentrations of 830 or 1600 mg', respectively. The mass `Nmoedriaatsn daieerdoddyunrianmgicexdpioasmuerteertso measured for the wet aerosol tested ranged H-25914or during the recovery period. from 2.2 10 3.5 pm. "The only clinical signoftoxicity observed during his study was alopeciaofthe front paws in animals exposed to 4900 mg/m? wet aerosol (1600 mg/m? dry). The observation ofalopecia was transient in nature and was observed up to 14 days postexposure. Slight body weight losses were observed in 2 o6f animals exposed to1200 mg/m? wet aerosols (830 mg/m? dry), while all `animals exposetdo 4900 mg/m? wet aerosol (1600 mg/m? dry) demonstrated an average 4.5% --~ decrease in body weight 1 day postexposure. However, by the second day following exposure all animals demonstrated normal weight gain. Under the conditionsofthis study the approximate lethal concentration (ALC) for aerosolized polymer in H-25914 isgreaterthan 1600 mg/m' (dry). Based on the dry aerosol concentration and on an acute inhalation basis, H-25914 is considered to be, at worst, slightly toxic (ALC 800 -- 2000 mg/m?) according to Haskell Laboratory toxicity classifications. CompanySanifized.DoesnotconTtSaCAiCnBI --_ -- F-25914: Inhalation Approximate Lethal Concentration (ALC) in Rats DuPont 13308 --~ INTRODUCTION T(hAeLCo)bjoefctHi-v2eo5f9t1h4isinsmtauldyewraatss. toTdheeteArLmCineisade4f-ihnoeudraisnhtahleatlioownesatppartomxoismpahteerilcetchoanlcceonntcreanttiroantion tested which caused the deathofone atleast 14 days following exposure, or more exposed rats either onthedayofexposure The inhalation routeofexposure was chosen based or within on the expected routeofpotential human exposure. STUDY DESIGN Twewroegerxopuopssedonf6osmea-loenlryatfsoreaacshinwgelre,e4e-xhpoousrepdertiooda.eroFsoolllaotwmionsgpheexproessuoref,H-ra2t5s9w1e4reinreaitra.inReadtfsor a 14-day recovery period. Rats were approximately 8 or9 weeksoldand weighed between 241 and 290 grams at the time ofexposure. Rwaetrsewreermeovobesdefrrvoemd ftohre mroesrttraaliinteyrsa.ndDuclriinnicgatlhseirgenscoovfertoyxpiecirtiyodi,mamleldriaatstewleyraefioebrseexrpvoesducraecahsdtahyey for mortality. Rats were weighed and observed for clinical signsof toxicity on the day following ~~ ealxlproastusrweearnedsaactrliefaisctedt wic by e more carbon during the recovery pe dioxide asphyxiation riod. and d At isca the end rded. of t h e re c o v er y p erio d, MATERIALS AND METHODS A. Test Substance "The test substance, H-25914, was suppliedby the sgonsor as an amber liquid that was a The purityofthe organic polymer was test substance was assumed to be stable throughout the exposure phase of the study; no evidenceof instability was observed. B. Test Species YInoc.u,nRgalaediulgth,,mNaolrethCrCalr:oCliDna(.SDT)hIeGSratBsRwerartes awpeprreoxriemcaeitveeldy f7rwoemeCkhsarolledsoRnivtheer dLaayboorfaatrorriiveasl,. Rats have historically been used in safety evaluation studies for inhalation toxicity testing. The -- Crl:CD(SD)IGS BR extensive experience rat was with the ssetlreacitneadt bHaassekdelolnLcaobnosriasttoernyt.ly acceptable health status and on Company Sanitized. Doesnotcontain TSCA CBI - - F-25914: Inhalation Approximate Lethal Concentration (ALC) in Rats Dupont 13308 --_ C. Animal Husbandry I. Quarantine Rats were quarantined after arrival for6 daysprior o testing. During the quarantine period, rals `were weighed and observed for clinical signsofdisease 1 time. 2. Animal Selection Prior to each exposure, 6 male rats were selectedforuse on the study from the rats that were released from quarantine, had no overt signsofdisease,and were the appropriate ageandbody weight. No attempt was made to randomly group animals. 3. Identification Eachratwas assigned an animal numberthatwas recordedon a card affixed to the cage.Priorto exposure, the tail of each animal and cage card were coded with water-insoluble pens so that ach animal could be identified after exposure and during the recovery period. 4. Housing Except during exposure, rats were housed singly in stainless steel,wire.mesh cages suspended above cage boards. ~ 5. Animal Room Environment Rats were housed in proximity to the inhalation chambers. Theanimal room was maintained ata temperature of 18-26C (targeted to 22-24C) and a relative humidityof 30-70% (targeted to 4060%). Animal rooms were artificially illuminated (fluorescent light) on an approximate 12 hour light/dark cycle. Excursions outsideofthese ranges wereofinsufficient magnitude and/or duration to have adversely affected the validityofthe study. 6. Feedand Water Except during tap water were exposure, PMI Nutrition availableadlibitum. International, LLC Certified Rodent LabDiet 5002 and 7. Animal Health and Environmental Monitoring Program As specified in the Haskell Laboratory animal health and environmental monitoring program, the following procedures are performed periodically to ensure that contaminant levels are below those that would be expected to impact the scientific integrity ofthe study: + aWnadteorthsearmcpolnetsamairenaanntasl.yzed for total bacterial counts, and the presenceofcoliforms, lead, ~~ + Feedsamplesare analyzed for total bacterial, spore, and fungal counts. En `CompanySanitized.Doesnotcontain TSCA Car F-25914: Inhalation Approximate Lethal Concentration (ALC) in Rats DuPont 13308 --_ + Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrationsofkey contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presenceofthese contaminants below the maximum concentration stated by the manufacturer `would not be expected to impact the integrityofthe study. `The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Evaluationofthese data did not indicate any conditions that affected the validityofthe study. D. Inhalation Exposure System 1. Auosphere Generation Chamber atmospheres were generated by nebulizationofthe test substance in air with a Spraying Systems nebulizer. The test substance was metered into the nebulizer with a Harvard Apparatus `model 22 syringe infusion pump. Filtered, high-pressure air, metered into the nebulizer by a Brooks model S851E mass flow controller, carried the resulting atmosphere into the exposure: chamber. Chamber concentrationsoftest substance were controlledbyvarying the syringe --_ infusion pump rateto the nebulizer. Test atmospheres were exhausted through a water-filled scrubber followed by an MSA charcoalHEPA filter cartridge priotro discharge into the fume hood. 2. Chamber Construction and Design `The exposure chamber was constructed ofglass (cylindrical) with a nominal intemal volume of 34 L. A polycarbonate baffle inside the chamber promoted uniform chamber distributionofthe test atmosphere. 3. Exposure Mode During exposure, animals were individually restrained in perforated stainless steel cylinders with conical nose pieces. The restrainers were inserted into a polymethylmethacrylate faceplate that was attached to the exposure chamber so that the nose of each animal extended into the exposure chamber. E. Characterization of Chamber Atmosphere `Company Sanhized. Doss not contain TSCA CBI I. Test Substance Sampling and Analysis The atmospheric concentration of H-25914 was determined by gravimetric analysis at ~ approximately 30-minute intervals during each exposure. Known volumesofchamber = --_-- H.25914: Inhalation Approximate Lethal Concentration (ALC)inRats DuPont 13308 = atmosphere were drawn from the sampling port through a 25 mm filter cassette containinag preweighed Gelman glass fiber (Type A/E) filter. The filters were desiccated fora least 1 day prior 10 air sampling. Immediately following the collection ofthe air sample, the filters were removed pforsotmstahmeplciasnsgetfitletearnwdeiwgehitghweads orneceoirtdheerdaanCdahunsemdotdoedleCt-er3m0ionre Ct-he31totMailcraoerboasloalncceon.cenTthreation (wet aerosol) in the exposure chamber. The filters were then placed in a desiccator for at least 1 day, and reweighed. The desiccated filter mass was then used to determine thedryaerosol concentration. The contentofthe sample on the desiccated filter was not analyzed, but was assumed to be the fluorinated organic polymer. 2. Particle Size Determination A sample to determine particle size distribution (mass median aerodynamic diameter and percent `particles less than 10 pm diameter) was taken during each exposure with a Sierra Series 210 cyclone preseparator/Cascade impactor and Sierra series 110 constant flow air sampler. 3. Environmental Monitoring Chamber airflow was set at the beginningofeach exposure to achieve at least 10 air changes per hour. The airflow was monitored continually with a calibrated Brooks model 5851E mass flow controller and recorded initially and whenever changes were made during each exposure. Chamber temperature was targeted at 22 + 2C. The temperature was monitored continually with a NIST digital traceable thermometer and recorded 3 times during each exposure. Chamber ~~ relative humidity was targeted at 50 + 20%. The relative humidity was measured with an Omega `model RH5100C digital psychrometer and recorded 3 times during each exposure. Chamber oxygen concentration was targeted to be at least 19%. The oxygen concentration was measured with a Biosystems model 3100R oxygenanalyzerand recorded 3 times during each exposure. `Company Sanitized. Dossnotcontain TSCA CBI -_-- 1:25914: Inhalation Approsimat Lethal Concentration (ALC)in Rats --_ RESULTS AND DISCUSSION DuPont13308 A. Exposure Conditions (Table 1) Animals were exposed to H-25914 aerosols at concentrations of 1200 or 4900 mg/m (wet), which corresponded to mean dry aerosol concentrationsof 830 or 1600 mg/m', respectively. The atmospheres generated in this study were considered to be respirable in rats, with mass median aerodynamic diameters (MMAD) ranging from 2.2 10 3.5 pm. TABLE | CHARACTERIZATION OF TEST ATMOSPHERES AND ASSOCIATED ANIMAL MORTALITY "AEROSOL CONCENTRATION (mg/n)" MORTALITY Wet Aerosol MMAD Percent| (# deaths' --~ M1e20a0n _1S3D.0 100R0an-g1e400|R Meana SDn . ge| 49001500 3100-7000| 1600 360 1100-2100 2im)2' 2G4SD <190%um'| #exp0o7s6ed) 35 3s wm 0/6 a eRxepporseusreen.tsVtahleumeesaanr,esrteapnodratredd dteov2isaitginoinfi(c5a.Dn.t),fiagnurdesr.anCgaelcfuolactaicohnsexeproseurpee,rfboarsmeeddopnrniosraomprloeusnpdeirng values. b eTxhpeosMuMreAD (Mass Median Aerodynamic Diameter) s based on 1 particle size sample taken dring cach 4 PGeerocmeenttriaecrSosaonldmaarsdsDhevaivaitnigons.erodymamic equivalent diametersof ess than 10 ym. `7C2h%a,mcbehramtbeempraeirraftluorwewraansge1d8fLriommin2,0atnod2t2heC,oxcyhgaemnbecornrceelnattriavteihounmwiadist2y0r.a8n%g.edAflrtohmou7g0hto tcohahmavbeeradrveleartsievleyhaufmfiedctietdytwhaesvaoluitdsiitdyeoftthehitsarsgteutdeyd.parameters, this variation was considered not B. Mortality, Clinical Signs, and Body Weights No deaths occurred during the study. 2Nodacylsinfioclalloswiignngsoefxtpooxsiucrietytow4e9r0e0obmsge/rmv?edwienttHh-e2a5n9i1ma4laseiromsmoeldi(a1t6e0l0ymagft/emr?exdpryo)s,ur1eo.f H6aonwievmearl,s demonstrated alopecia ofthe front paws that went unresolved at 14 days postexposure. At7 and f1r4ondtapysawfso.llNowoinagloepxepcoisauwraestoob4s9e0r0vemdg/inm?anwiemtalasereoxspolo,se4datnoim1a2l0s0dmegm/omns?twreatteHd-a2l5o9pe1c4iaaoerfotshoel ~~ (830 mg/m? dry). Weight losses were observed in all animals exposed to 4900 mg/m? wet TTT Comp SmtiDurss eorCdoTa:t TOA CBI H-25914: Inhalation Approximate Lethal Concentration (ALC) inRats DuPont13308 A aerosol (1600mg/m? dry) 1 day postexposure. The weight loss ranged from 1.8 ~ 7.6% initial body mass and by 2 days postexposure, all animals demonstrated normal weight gains. Following the 1212 mg/m' wet H-25914 aerosol (830 mg/m? dry) exposure 2 rats lost <I and 4.3%oftheir initial body weighat 1 day postexposure. By 2 days following the 1212 mg/m* wet H-25914 acrosol (830 mg/m? dry) exposure all animals demonstrated normal weight gains. CONCLUSIONS `Under the conditionsofthis study, the approximate lethal concentration (ALC) for the organic polymerin H-25914 is greater than 1600 mg/m'dryaerosol. Based on thedryaerosol concentration and on an acute inhalation basis, H-25914 is considered to be, at worst, lightly toxic (ALC 800 -- 2000 mg/m') accorditnog Haskell Laboratory toxicity classifications. RECORDS AND SAMPLE STORAGE Specimens(if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. --~ REFERENCES 1. Calculation described in Sierra Instruments, Inc., Bulletin 7-79-2191M, Instruction Manual: Series 210 Ambient Cascade Impactors and Cyclone Preseparators. CompanySanitized.DoesnotcontainTSCA CBI -_----mn