Document gD4Q04qyyGOey1OgKzxDJmvaJ

TRADE SECRET DuPontHL-1997-00598 AR226-3079 Study Title ly^NHALATTOANPPROXIMATE LETHAL CONCENTRATION (ALC) IN RATS LABORATORY PROJECT ID: HL-1997-00598 AUTHOR; David P. Kelly, B.S. STUDY COMPLETED ON: August 29,1997 PERFORMING LABORATORY: E.I. du Font de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 MEDICAL RESEARCH NUMBERj^^HB I ^,3.^.--------------------- Page 1 of 11 lation Approximate Lethal Concentration (ALC) in Rats DuPont HL-1997-00598 Substance Tested: Synonyms/Codes: GENERAL INFORMATION H-22387 H-22632 Physical Characteristics: Stability: water clear liquid The test substance appears to be stable under the conditions of the study; no evidence of instability was observed. Company Sanitized. Does not contain TSCA CBI 2- GENERAL INFORMATION (Continued) Sponsor: E.I. du Pout de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated - Completed: February 13, 1997 - (see report cover page) In-Life Initiated - Completed: February 13, 1997 - July 9, 1997 All original data and the original of this final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain, 200 Todds Lane, Wilmington, Delaware 19802. Company Sanitized. Does not contain TSGA CBf 3- SUMMARY Groups of six male Cri:CDffiD)BRrats eachwere exposed nose-only for a single, four-hour period to a water mixture oiaBI^^BBBMH-2238'Laad H-22632) in air. Test atmospheres QgHUH^ere were generated by aerosolizSBon,and conceBrations measured by gravimetric filter analysis. Aerosol concentrations were reported as wet and dry aerosol. Rats were weighed and observed for clinical signs oftoxicity during a 14- to 15-day recovery period. CHBHHU^ Rats were exposed to atmospheres K^^^^^^^^^Mf wet aeros0^ concentrations of 320, 330, or 570 mg/m3, corresponding to dry aerosol concentrations of 210,270, or 420 mg/m3. The mass tlUBBH^ median aerodynamic diameters.qf the aerogflls tested were 5.0,2.3, and 3.0 jam, respectively. Rats died following exposure concentrations of 320 and 570 mg/m3 (wet), corresponding to 210 and 420 mg/m3 (dry).t)eaths occurred immediately after exposure or within two days. No deaths occurred after exposure at concentrations of 320 mg/m3 (wet), 270 mg/m3 (dry). Jnder the conditions of this study, the approximate lethal concentration (ALC) for aerosolized ^JMMlMriMHB^20mg/m3 (wet aerosol), 210mg/m3 (dry aerosol). On an acute inhalation ^ ^ ^ ^ ^ ^ T basis and based on the dry aerosol concentratioiqk----l------Hs considered to be moderately toxic (ALC 200-800 mg/m3). ew^sW^^^ -4- SIGNATURES Date Reviewed and Approved for Issue: David P^eliy,B.S. ResearctyToxicoIogist Study Director ^f/^7 STUDY PERSONNEL The following individuals were responsible for conduct of the study: Management: Study Director: Primary Technicians: Toxicology Report Preparation: Scott E. Loveless, Ph.D. David P. Kelly, B .S. M. Ahmad Pulliam, B.S. Dwayne A. Lavoie, B.S. Maryanne M. Wilford, B.A. The following individual was responsible for assessing the health status of the animals on study: Laboratory Veterinarian: Charles E. Cover, V.M.D. cmp"i^sn?Mo-----.,,,^,, .5- INTRODUCTION The objective of this studywistodgtemune a four-hour inhalation approximate lethal ojimjfQ concentration (ALC) niale rats. The ALC is defined as the lowest atmospheric concentration tested which caused me death of one or more exposed rats either on the day of exposure or within at least 14 days following exposure. The inhalation route of exposure was chosen based on the expected route of potential human exposure and was requested by the sponsor. Data for this study was obtained using two protocols: an inhalation screen protocol (SOP AI005-T) and an approximate lethal concentration protocol (SOP AI001-T). MATERIALSAND METHODS A. Test Substance The test substanca|pB^^^My[-22387and H-22632)was supplied by the sponsor as a mixture HBflBH|2>-water This was diluted tQ----tHaskell Laboratory to facilitate spraying. Mf B. Animals Young adult, male, Cri:CD(SD)BR rats were obtained from Charles River Breeding Laboratories. The rats were approximately seven to eight weeks old upon arrival. Rats have historically been used in safety evaluation studies for acute inhalation toxicity testing. The Crl:CD(SD)BRrat has been chosen based on consistently acceptable health status and the extensive experience with the strain at this laboratory. C. Animal Husbandry 1. Quarantine and Animal Selection Rats were quarantined after arrival for approximately six days prior to testing. During the quarantine period, rats were weighed and observed for clinical signs of disease three times. Rats were obtained from the general population of stock rats released from quarantine and were selected for use on this study from those rats exhibiting a normal pattern of weight gain and no overt signs of disease. 2. Housing Rats were housed either singly or in pairs in suspended, stainless steel, wire-mesh cages. Company San^cf. ^ n ^ ^ ^ ^ -6- 3. Animal Room Environment Animal rooms were maintained on a timer-controlled, 12-hour light/I 2-hour dark cycle. Environmental conditions of the rooms were targeted to be within a temperature range of 23 1C and a relative humidity range of 50 10%. Excursions outside these ranges were of insufficient magnitude and/or duration to have adversely affected the validity of the study. 4. Identification Each rat was assigned a unique six-digit identification number which corresponded to a numbered card affixed to the cage. Prior to exposure, the tail of each rat was color-coded or numbered with water-insoluble markers so that individual rats could be identified after exposure. 5. Feed and Water Except during exposure, Purina Certified Rodent Chow #5002 and tap water from United Water Delaware were available ad libitum. 6. Health Monitoring Program Haskell Laboratory has an animal health monitoring program. The following procedures are performed periodically: Water samples are analyzed for total bacterial counts, and the presence ofcoliforms, lead, and other contaminants. Feed samples are analyzed for the presence of bacteria and fungi. Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cage washers. Haskell Laboratory uses certified animal feed. The feed is guaranteed by the manufacturer to meet specified nutritional requirements and to be free of a list of specified contaminants. The animal health monitoring program is administered by the laboratory animal veterinarian. Data are maintained separately from study records and are not included in the final report. Evaluation of these data did not indicate any conditions that affected the validity of the study. D. Study Design ^^^^- Three groups of six male rats each were exposed to atmospheres o^^^^------pn air. Rats were f^^^^^^^^^^^^ f exposed nose-only for a single, four-hour period. Rats were approximately eight to nine weeks old and weighed between 255 to 289 grams at the time of exposure. Rats were observed for mortality and response to alerting stimuli during the exposure and observed for mortality and clinical signs oftoxicity immediately after they were removed from Company SanNfeed. Does rofewf^rr TSCA CB? _^_ the restrainers following exposure. During a 14- to 15-day postexposure period, all surviving rats were observed each day for mortality, and were weighed and observed for clinical signs of toxicity at least twice weekly. At the end of the recovery period, all surviving rats were sacrificed by carbon dioxide asphyxiation and discarded. E. Inhalation Exposure System 1. Atmosphere Generation o^f^U^^^^n^ Chamber atmospheres were generated by aerosolization air with a Spraying Systems nebulizer. The test substance was metered into tne' nebulizer with a Harvard Apparatus model 22 Syringe Infusion Pump. Filtered houseline air, introduced at the nebulizer, atomized the tesLsubstance^nd carried the aerosol into the exposure chamber. Chamber .concentrations en----JBBjivere controlled by varying the test substance feed rate to the atmosphere generator.--^ Test atmospheres were exhausted through a bubbler, a dry-ice cold trap, and an MSA charcoal/HEPA filter cartridge prior to discharge into the fume hood. 2. Chamber Construction and Design The exposure chamber was constructed of glass (cylindrical) with a nominal internal volume of 34 L. A dispersion plate at the chamber inlet promoted uniform chamber distribution of the test atmosphere. 3. Exposure Mode During exposure, rats were individually restrained in perforated stainless steel cylinders with conical nose pieces. The restrainers were inserted into the polymethylmethacrylate faceplate of the exposure chamber so that only the nose of each rat extended into the chamber. F. Characterization of Chamber Atmosphere 1. Test Substance Sampling and Analysis The atmospheric concentration oflllUB^as determined by gravimetric analysis at approximately 30-minute intervals during exposure. Known volumes of chamber atmospheres were drawn from the reference sampling port through a 25 mm filter cassette that contained a pre-weighed Gelman glass fiber (Type A/E) filter. After samples were collected, the filters were immediately weighed to determine the wet aerosol concentration. After overnight drying in a dessicator, the filter samples were weighed to determine the dry aerosol weight. The filters weis. o f J U m weighed on a Cahn model C-30 Microbalance. The atmospheric concentration was calculated from the difference in the pre- and post-sampling filter weights divided by the volume of chamber atmosphere sampled. Company Saniffzed. DDooees, not, contain TSCACBJ Samples to determine particle size distribution (mass median aerodynamic diameter and percent particles less than 3 jiim diameter) were taken with a Sierra Series 210 Cyclone Preseparator/Cascade Impactor and Sierra Series 110 Constant Flow Air Sampler/^ 2. Environmental Monitoring Chamber airflow was set at the beginning of each exposure to achieve at least 12 air changes per hour. The airflow was monitored continually with a calibrated Brooks model 1355 Sho-Rate Rotometer and recorded one to four times during exposure. Chamber temperature was targeted at 22 2C. The temperature was monitored continually with an Omega model 650 Thermocouple Thermometer and recorded two to five times during exposure. Chamber relative humidity was targeted at 50 10%. The relative humidity was measured with an Omega Digital Psychrometer and recorded two to three times during exposure. Chamber oxygen concentration was targeted to at least 19%. The oxygen concentration was measured with a Biosystems model 3100R Oxygen Analyzer and recorded two to three times during exposure. ^Pany^ DoesRoteont^^ -9- RESULTS A. Exposure Conditions (Table 1) Animals were exposed tIpHHBBp concGatratlons of 210,270, or 420 mg/m. The aerosols generated during the 270 mg/m exposure were considered to be marginally respirable in rats as the mass median aerodynamic diameters (MMAD) was 5.0 pn. TABLE 1 CHARACTERIZATION OF TEST ATMOSPHERES AND ASSOCIATED ANIMAL MORTALITY AEROSOL CONCENTRATION (mg/m3)3 AEROSOL SIZE MORTALITY Wet Filters Dry Filters MMAD Percent Mean S.D. Range Mean S.D. Range n (^m)" GSD0 <3 irn^ 320 98 210 - 480 270 87 160-400 8 5.0 3.1 33 330 35 290 - 390 210 25 190 - 260 8 2.3 2.9 66 570 500 210-1800 420 250 150 - 940 12 3.0 2.3 53 (# deaths/ # exposed) 0/6 5/6 6/6 Represents the mean, standard deviation (S.Dr),-and range for each exposure, based on n samples per exposure. Values are reported to two significant figures. The MMAD (Mass Median Aerodynamic Diameter) is based on one particle size sample taken during each exposure. Geometric Standard Deviation. Percent aerosol mass having aerodynamic equivalent diameters of less than 3 iim. Chamber temperature ranged from 20 to 23 C, chamber relative humidity ranged from 76 to 86%, chamber airflow ranged from 15 to 25 L/min, and the oxygen concentration was 21%. The chamber relative humidity was high because of the aerosolized water content of the test mixture. B. Mortality, Clinical Signs, and Body Weights Deaths occurred ai two days of exposure. ncentrations of 210 mg/m3 or greater. Rats died within A diminished alerting response was observed near the end of the 270 mg/m3 exposure. Following exposures, when the rats were removed from the chambers, the clinical signs of toxicity observed included irregular respiration, lung noise, lethargy, and wet perineum. ^^^--^sc^, 10- alation Approximate Lethal Concentration (ALC) in Rats DuPont HL-1997-00598 Surviving rats showed slight to severe weight loss on the day after exposure. Normal weight gain rates were resumed within one week after exposure. CONCLUSION In these studies, the difference in aerosol concentrations between a lethal level, where 5 of 6 rats died, and a non-lethal level was minimal. It is speculated mat the reason rats died at 330 mg/m3 (wet aerosol), 210 mg/m3 (dry), and none died at 320 mg/m3 (wet), 270 mg/m3 (dry), was that the aerosol particle was larger (5 ^m) in the 270 mg/m3 experiment and smaller in the 210 mg/m3 experiment. Although both aerosol sizes are inhalable, the smaller aerosol would reach deeper in the lung, and possibly cause more acute damage. From an industrial health standpoint, it is recommended that production of small aerosols of this material be minimized. Under the condijjjgns of this study, the approximate lethal concentration (ALC) for aerosolized U U B K j 0 320 mg/m3 (wet aerosol), 21Qjqg/m3 (dry aertjgpl). On an acute inhalation jasis and based wi the dry aerosol ^^^t^tionJMBIHJIHIlHfs considered to be moderately toxic (ALC 200-800 mg/m3). '" * REFERENCES 1. Calculation described in Sierra Instruments, me.. Bulletin 7-79-219IM, Instruction Manual: Series 210 Ambient Cascade mipactors and Cyclone Preseparators. Company Sanitized. Does. not contain TSCA CBj 11-