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AR226-2923 FR DU PONT USE ONLY E. I. du Pont de Nemours and Co., Inc. Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P. 0. Box 50, Newark, Delaware 19714 HASKELL LA30RAT0RY REPORT NO. 110-85 MR NO. Material Tested________________ Haskell Nos. SUMMARY [ale Crl :CD(SD)BR rats osed to aerosol atmospheres an aqueous ` sol id ____________________________ _. . .............. r_rtTcle s i ^ ^ ^ ^ ^ u J u n o n s weregenerated to determine the effect of particle'size on the toxicity of these materials. Further, the relation ship between expected pulmonary deposition (based on particle size) and mortality was investigated. 'i H I tmFor bot size^ For land the ALC increased with increasing particle le ACLX in_cnicreased from 42 mg/m at 1.6 urn MMD to 170 mg/rn at 6.. ^ u m M M D T For he ALC increased from 24 mg/m at 1.7 urn MMD to 360 mg/mn at 5.6 urn MM For these materials, the fraction of the total test atmosphere expected to deposit in the alveolar region (particles smaller than 3.1 um) was most closely associated w ^ ^ ^ ^ ^ J i t y . However, this relationship was unequivocal only f o r j ^ ^ | x p o s u r e s ; one exposure to a low concentration of A r t i c l e s smaller than 3.1 um caused deaths which can not be exi^ffiea by expected pulmonary deposition. The atmospheric concentration of total respirable aerosol did not show a clear dose-response. HLR 110-85 Sanitized. Does not contain TSCA CB! Page 1 of 14 INTRODUCTION as extremely toxic by inhalation when tested as a highly respirable aerosol 1ALC of 42 mg/m ; HLR-423-83). The purpose of this study was to determine the ef fe ct if particle size on the inhalation ' Two forms ofj material Concentrations (AL J^ w a ^ ^ ^ W f f l p e o u s suspension For both materials, Approximate Lethal one was ; were determined for various particle size atmospheres, The ALC was defined as the lowest atmospheric concentration tested which caused the death of 1 or more rats either on the day of exposure or within 14 days post exposure Further, the relationship between expected pulmonary deposition (based on particle size) and mortality was investigated. 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 5-digit identification number which corresponded to a numbered card affixed to the cage. Rats' tails and cage cards were color-coded with water-insoluble markers so that rats could be identified after exposure. Rats were housed singly in 5" x II" x 7" suspended, steel-mesh cages in rooms targeted to have temperatures of 25 _+ 2C and 50 _+ 10'S relative humidities on timer-controlled 12 hour/12 hour light/dark cycles. Rats were quarantined for one week prior to testing, and were weighed and observed twice during the quarantine period. Except d u n no exposure, Purina Certified Rodent Chow #5002 and water were available ad libitum. B. Exposure Protocol Groups of 6 rats, 8 to 9 weeks old and weighing between 224 and 297 grams, were restrained in perforated, stainless steel cylinders with conical nose pieces. Each group was exposed n o s e - o a ^ ^ Q j ^ ^ ^ i n a l e , 4-hour period to an aerosol atmosphere of e i t h e r ^ P H | H H I V n air. Rats were weighed prior to exposure and observed"Tbr clinical signs during exposure. Surviving rats were weighed and observed daily for 14 days post exposure, weekends excluded except when deemed necessary by the rats' condition. . Com pany Sanitizer?. Bees nof eonfa>n TSCA CB HLR 110-85 Page 2 of 14 c. Test Material 1. H-15,048) Physical Form: Composition: Purity: Contaminants: Synonyms: Other Codes: Stability: Submitted by: 2. TBCU (H-15,219) Physical Form: Purity: Contaminants: Synonyms: e test material was assumed to be stable ^ t h r o u f l h o u ^ J h ^ ^ ^ ^ y r e phase of the study, ^Chemicals and Pigments Department Jackson Laboratory Waxy s,olid Other Codes: Stability: Submitted by: The test material was assumed to be stable throughout the exposure phase of the study. Based on the supplier's specifications, the test material was stable at the temperatures nemica!s and Pigments Department Jackson Laboratory D. Atmospheric Generation 1. ^ ^ ^ ^ 'H-15,048) Aerosn^j^ospheres o f ^ H ^ H ^ ^ r e generated by pumping l i q u i d M H H H n t o a Spraying Systems nebulizer. Air introduced at the'Yf&bulizer aerosolized thr; test material, and swept the aerosol stream through a cyclone elutriator and into the exposure chamber. Particle size distributions were shifted toward larger particles by removing the cyclone, using different sized nebulizers Company anisei"?. no TftCA HLR 110-85 Page 3 o f 14 and changing the airflow. During one exposure, the nebulizer and the cyclone were heated to approximately 100C. 2. Aerosol atmospheres offlHft/ere generated by pumping melted test material into a SprayirrfTystems nebulizer. The test material was heated to 60-132C during generation. Air introduced at the nebulizer aerosolized the test material, and swept the aerosol stream into the exposure chamber. The air was preheated in a furnace heated to 120-278C during generation. Particle size distribution was shifted toward larger particles by changing the test material and air temperatures, airflow and nebulizer size. E. Analytical The atmospheric concentration of particulate was determined at approximately 15- to 30-minute intervals by drawing known volumes of chamber atmosphere through pre-weighed, glass fiber filters. Filters were weighed on a Cahn Model 26 Automatic Electrobalance. Atmospheric concentration of particulate was determined from the filter weight differential before and after sampling. ... 0u?n9 each exposure, the particle size distribution was determined witn a Sierra cascade impactor. In addition, for each exposure, the estimated atmospheric concentrations of particles smaller than 3.1 and 3 urn were calculated from the total atmospheric concentration and particle size data. Chamber temperature was monitored with a mercury thermometer during each exposure. F. Records Retention All raw data and th= Final report 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. DuPont de Nemours and Co., Inc., Wilmington, Delaware. RESULTS A * Exposure Conditions and Associated Mortality Chamber temperature ranged between 18-34C ddiuirriinngiLpexposures to ------- J and between 27-35C during exposures t o M H | Wide temperature ranges are not expected to affect.mortality in nose-only exposures. . ^acb test atmosphere contained a distribution of particles of various sizes, including both small (smaller than 1 urn) and large (larger than 10 urn) particles. The average geometric standard deviation for each particle size distribution was approximately 2. Table I shows total atmospheric concentration, particlr size distribution and associated rat mortality for each exposure. Data are grouped from Company Sanitized. Doos not contain TSCA CBi HLR 110-85 Page 4 o f 14 exposures with similar partitle size distributions. The increasing mass median aerodynamic diameters indicate a shift in the particle size distribution from smaller to larger particles. Company Sanitized. Does not contain T SC A CB! HLR 110-85 Page 5 of 14 Table I Atmospheric Characterization Exposures to .Associated Rat Mortality from Total Particulate Concentration __(mg/m ) A. % by Weight of Particles with . Aerodynamic Diameter less than : 13 urn 5.2 urn 3.1 urn 1.1 urn MMDa Deaths 16 + 6.6 99 92 73 21 1.7 urn 0/6 73I42 7 7.8 99 95 79 30 1.6 urn 3/6 25 98 93 77 21 1.9 urn 6/6 330 + 11C 95 75 44 8.1 3.4 urn 6/6 58 + 42 76 45 18 3.0 6.6 urn 0/6 77 7 5.3 86 52 20 4.9 5.8 urn 0/6 170 + 40 80 51 22 2.3 6.0 urn 4/6 9.4 + 1.9 24 + 21 66 + 48 110 + 72 110 + 48 140 + 34 48 + 10 72 + 8.7 n o + 56 190 + 88 ^ 3 2 0 + 56 390 + 100 520 + 140 57 + 18 84 - 31 190 + 33 ^ 3 6 0 + 33 610 + 82 520 + 42 87 76 98 93 98 93 99 96 92 76 98 89 68 81 77 83 53 62 67 50 78 52 73 50 88 60 89 56 77 48 79 54 34 32 29 26 27 22 28 57 29 10 70 43 23 63 39 19 74 46 22 71 41 18 75 42 17 56 1.1 urn 0/6 37 1.7 urn 6/6 26 2.1 urn 6/6 34 1.7 urn 6/6 20 2.9 urn 6/6 13 2.7 urn 6/6 18 5.2 urn 0/6 17 5.1 urn 0/6 13 5.5 urn 0/6 5.8 4.7 urn 0/6 3.9 4.9 urn 5/6 1.6 5.4 urn 4/6 2.9 5.5 urn 4/6 5.0 9.7 urn 0/6 7.4 6.6 urn 0/6 4.8 6.9 urn 0/6 2.5 5.6 urn 3/6 1.1 7.1 urn 6/6 1.3 6.1 urn 6/6 Mass median aerodynamic diameter. Nebulizer and cyclone were heated during generation. Company Sanitized. Does not contain T S C A CBI HLR 110-85 Page 6 o f 14 ? B. Estimated Lung Deposition and Associated Animal Mortality The fractional deposition of particles within the respiratory tract depends in part on the particles' aerodynamic sizes. However, literature sources vary widely in their estimates of-the size-limits of particles able to be inhaled and to be deposited into various regions of the respiratory tract. Further, data indicate that deposition varies widely amoung individuals and amoung species. ` The Environmental Protection Agency has adopted the following criteria to define the approximate size-limits of particles which may deposit into the various regions of the human respiratory tract: particles smaller than 15 urn can be Inspired and deposited throughout the respiratory tract; and particles smaller than 2.5-3.5 urn (nose and mouth breathing, respectively) are expected to deposit predominantly in the alveolar region. Deposition of particles smaller than 3 urn is similar in rats and humans. Deposition data in rats for particles larger than 3 urn are not available. To investigate the relationship b e t w e e n B H o x i c i t y and particle size, the following assumptions have been madF: particles smaller than 3.1 urn will provide predominantly alveolar deposition, particles smaller than 13 urn" (including particles <3.1 urn) represent total respirable particulate, and particles larger than 13 urn will not be inhaled. The 3.1 urn and 13 urn size-limits were chosen because they are the experimental cut-points provided by the cascade impactor used in these tests which most closely approach the EPA criteria. For each exposure, the atmospheric concentrations of particles smaller than 3.1 and 13 urn were estimated by multiplying the total atmospheric concentration by the mass pt'cent of particules smaller than these cut-points. As shown in Table I, within groups of similar particle size atmospheres, mortality generally increased with increasing concentration. Further, as particle size distributions shifted towards larger particles, the concentration needed to cause death increased. The purpose of back-calculating the atmospheric concentration of particles smaller than and 3.1 urn was to investigate whether the japparent decrease in tc-'c can be explained by the inability of a large fraction of these .it ospheres to either be inhaled or be deposited in the alveolar region. F o r B P H l P i x p o s u r e s , the atmospheric concentration of particles smaller tTfan 3.1 urn was most closely associated with animal mortality; regardless of total atmospheric concentration and MMD, as the concentration o ^ j a r y c l e s smaller than 3.1 urn increased, mortality increased. F o r f l f H x p o s u r e s , as the concentration of particles smaller than S.^Jn^ncreased from 32 to 58 mg/m , mortality increased. However, one exposure containing only 19 mg/m3 of particles smaller than 3.1 urn caused 6/6 deaths. The deaths at this concentration were " u n e x o e c t e d ^ n ^ t h e cause of death is difficult to explain. For both H B t o t a l respirable particulate (all particles smaller t n a r w T u m j d i d n o t have a clear dose-response relationship with Company Sanitized. Does not contain T SC A CBS HLR 110-85 Page 7 of 14 mortality; as the concentration of total respirable particulate increased, a corresponding increase in mortality was not observed. Table II presents the atmospheric concentration of particles smaller than 13 and 3.1 urn and associated rat mortality for representa tive exposures. Data for all exposures are presented in Appendix I. V Company Sanitized. Does net contain T SC A CBS HLR 110-85 Page 8 of 14 Table II Atmospheric Concentrations of Particles Smaller Than 13 and 3.1 urn and Associated Rat Mortality Atmospheric , . Concentration (mg/nrV Mortality Data Calculated From 1. Particles smaller than 13 urn: 65 0/6 42 3/6 140 4/6 72 6/6 smaller than 3.1 um: 15 0/6 33 3/6 37 4/6 56 6/6 77 mg/m? P 5.8 um MMD 42 mg/m? @ 1.6 um MMD 170 mg/m? P 6.0 um MMD 73 mg/m"1 P 1.9 um MMD 77 mg/m? P 5.8 um MMD 42 mg/m; P 1.6 um MMD 170 mg/m? P 6.0 um MMD 73 mg/mJ P 1.9 um MMD Particles smaller than 13 urn: 80 0/6 120 0/5 170 0/6 24 6/6 65 6/6 100 6/6 Particles smaller than 3.1 ym: 32 0/6 36 0/6 49 0/6 19 6/6 51 6/6 58 ' 6/6 110 mg/m, P 5.5 um MMD 190 mg/m, P 6.9 um MMD 190 mg/m, P 4.7 urn MMd 24 mg/m, P 1.7 urn MMD 66 mg/m, 0 2.1 urn MMD 110 mg/m P 2.9 urn MMD 110 mg/m? P 5.5 urn MMD 190 mg/m? P 6.9 um MMD 190 mg/m? P 4.7 urn MMD 24 mg/m? P 1.7 urn MMD 66 mg/m, P 2.1 urn MMD 110 mg/m0 P 2.9 urn MMD Atmospheric concentrations were estimated by multiplying the total atmospheric concentration by the percent by weight of particles smaller than 3.1 and 13 um, respectively. Company Sanitized. Doss not contain TSCA CE,, HLR 110-85 Page 9 of 14 C. Clinical Observations In general, very,.few clinical ins were observed in rats that survived exposure toi During or immediately following both lethal and non-lethal exposures, some rats 1n several groups had test material on their faces and heads and had a diminished startle response. Most rats exposed to lethal concentrations had labored breathing, and a few rats exposed to lethal concentrations had red nasal and ocular discharges, ruffled fur decreased activity and pallor. A few rats exposed to non-lethal ' concentrations had red nasal and ocular dischanges. 5%)^ ^ D u r i n ^ h e x e c o v e r y period, most rats which survived exposure to ^ ^ ^ ^ ^ ^ g h a d slight weight loss (less than for 1 day after exposure, and had no major clinical signs. However, a few rats had greater than 5* body weight loss, facial discharges, diarrhea, wet perineum, ruffled or discolored fur, hair loss and labored breathing. For|^|||exposures, most deaths occurred during exposure or 1 day post exposure,although a few rats died between 2 and 8 days post exposure. F o r n B n e x p o s u r e s , most deaths occurred from 1 to 2 days post exposure, with tne latest death occurring 6 days post exposure. Rats that died lost approximately 7-15% of initial body weight 1 day after exposure, and continued to lose weight until they died. Clinical signs for rats that died included labored breathing, facial discharges, limpness, ruffled or discolored fur, wet or stained perineum, diarrhea, pallor and lethargy. DISCUSSION Based on total atmospheric concentCfltion, the Approximate Lethal Concentrations for b o t h f t p H H I B M B n increased with increasing particle size: ^ MMD ALC 1.6 urn 6.0 urn 42 mg/m| 170 mg/m 1.7 urn 5.6 urn 24 mg/ml 360 mg/nr Although pu re f a ^ & p p e a r e d to be more toxic t h a n f a M A i n the smaller particle size range, both materials were considered extremely toxic when administered as highly respirable aerosols. When the particle size distribution was shifted toward larger particles, these materials were considered moderately to highly toxic. no contain T S C A C B I & M Q W S Sanitized. Does HLR 110-85 Page 10 of 14 The apparent decrease in toxicity with larger particle sizes is best explained by considering the fraction of the test atmosphere expected to deposit in the alveolar region. Regardless of total atmospheric concentration, the concentration of particles small^than 3.1 urn was most closely associated with mortality. Except for one ^ H ^ x p o s u r e , as the concentration of particles smaller than 3.1 urn iocrSsed, mortality increased. The cause of death in the o u t - l y i n g j B H E x p o s u r e cannot be explained. CONCLUSION _ ^ ^ | ^ h ^ o ^ i t i o n s of this test, the Approximate Lethal Concentrations o f ^ ^ ^ B J i n c r e a s e d as particle size distribtuions shifted from smaller to larger particles. Regardless of total atmospheric concentration, the atmospheric concentration of particles expected to enter the alveolar region was most closely associated with mortality. However, one exposure to | ^ B r aused death at a much lower concentration than was expected, and the ^ a u s e of death in this exposure can not be explained by this model. Calculation described in Sierra Instruments, Inc., Bulletin 7-79-219IM, Instruction Manual: Series 210 Ambient Cascade Impactors and Cyclone Preseparators. ~~ ------ 2 Air Quality Criteria for Particulate Matter and Sulfur Oxides. External Review Draft No. 2, Office of Research and Development, U. S. Environmental Protection Agency, February, 1981. Company Sanitized. D oss no! contain T SC A CBI HLR 110-85 Page 11 of 14 Acknowledgement: Bruce A. Burgess and Rudolph Valentine also participated in this study. Work by: Robert T. T u n e r Technician Steven C. Carppenter Technician JStudy Director: A .I^ Lauraa A. Kiinney ) i'fi'f 2S Chemist Approved by: C- QAa V/'/^ Nanc/'C. Ch romey, Wi .D. Section Supervisor, Acute Investigations Section LAKrsgl:1.2 Date Issued: April 1, 1985 Study Initiated/Completed: Notebooks : 7/83-2/7/84 Haskell Laboratory Report No. 110-85 Number of pages in this report: 14 Company Sanifized. Doss not confa'n TSCA CBr HLR 110-85 Page 12 of 14 Appendix I Atmospheric Concentrations of Particles Smaller Than 3.1 and 13 urn and Associated Rat Mortality Concentration of particles smaller than 13 urn Atmospheri c , Concentration (mq/m ) Mortality Data Calculated From: 16 0/6 16 mg/m, @ 1.7 um MMD 44 0/6 58 mg/m, @ 6.6 urn MMD 66 0/6 77 mg/m'3 @ 5.8 urn MMD 3 42 3/6 42 mg/m, @ 1.6 urn MMD 140 4/6 170 mg/m'3 @ 6.0 urn MMD 72 6/6 73 mg/nu @ 1.9 urn MMD 310 6/6 330 mg/m13 0 3.4 urn MMD Concentration of particles smaller than 3.1 urn Atmospheric , Concentration (mg/m ) Mortality Data Calculated From: 10 0/6 58 mg/mo 0 6.6 urn MMD 12 0/6 16 mg/m, @ 1.7 urn MMD 15 0/6 77 mg/m'3 @ 5.8 urn MMD 33 3/6 42 mg/riK @ 1.6 urn MMD 37 4/6 170 mg/m"3 0 6.0 urn MMD 56 150 Pa 6/6 73 mg/nu @ 1.9 urn MMD 6/6 330 mg/m0 @ 3.4 urn MMD Concentration of particle1- !smaller than 13 urn Atmospheric , Concentration (mg/m ) Mortality Data Calculated From 8.2 32 32 56 59 80 120 170 . 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 9.4 mg/m~ 0 1.1 urn MMD 48 mg/m, @ 5.2 urn MMD 57 mg/m, @ 9.7 urn MMD 72 mg/m: @ 5.1 urn MMD 84 mg/m, @ 6.6 urn MMD 110 mg/m, 0 5.5 urn MMD 190 mg/m, 0 6.9 urn MMD 190 mg/m'3 0 4.7 urn MMD Company Sanitized. Does not contain T S C CBi HLR 110-85 Page 13 of 14 Appendix I (cont'd) MB. Atmospheric Concentrations of Particles Smaller Than 3.1 and 13 urn and Associated Rat Mortality (cont'd) Concentration of particles smaller than 13 um (cont'd) 24 6/6 24 mg/nu @ 1 . 7 um >MMD 65 6/6 66 mg/mf @ 2 . 1 umiMMD 100 6/6 110 mg/mf @ 2 . 9 um MMD 110 6/6 110 mg/mf @ 1 . 7 urii MMD 140 6/6 140 mg/mf @ 2 . 7 umiMMD 270 3/6 360 mg/mf @ 5 . 6 um'MMD 280 5/6 320 mg/mf @ 4 . 9 um'MMD 300 4/6 390 mg/mf @ 5 . 4 urn*-MMD 390 6/6 400 mg/mf @ 2 . 5 um MMD 410 4/6 520 mg/mf @ 5 . 5 um MMD 430 6/6 610 mg/mf @ 7.1 um MMD 460 6/6 620 mg/mf @ 6.1 um MMD 820 6/6 900 mg/rn @ 2.6 um MMD Concentration of Particles Smaller than 3.1 um Atmospheric 3 Concentration (mq/nr) Mortality Data Calculated From 5.7 6.4 16 19 23 32 36 49 0/6 57 mg/m3 @ 9 . 7 um MMD 0/6 9.4 mg/mf @ 1.1 um, MMD 0/6 48 mg/mf @ 5 . 2 um MMD 0/6 84 mg/mf @ 6 . 6 urn MMD 0/6 72 mg/mf @ 5.1 umiMMD 0/6 110 mg/mf @ 5.5 um; MMD 0/6 190 mg/mf @ 6 . 9 um MMD 0/6 190 mg/nr @ 4 . 7 um: MMD 19 6/6 24 mg/m, @ 1.7 um MMD 51 6/6 66 mg/mf @ 2 . 1 um MMD 58 6/6 110 mg/m3 @ 2 . 9 um MMD 79 3/6 360 mg/mf @ 5.6 um MMD 86 5/6 320 mg/mf @ 4.4 um MMD 86 . 4/6 390 mg/mf @ 5.4 um MMD 87 6/6 140 mg/mf @ 2 . 7 um MMD 91 6/6 110 mg/mf @ 1 . 7 um MMD 110 6/6 610 mg/mf @ 7 . 1 umiMMD 110 6/6 620 mg/mf @ 6 . 1 um MMD 150 4/6 520 mg/mf @ 5.5 um MMD 260 6/6 400 mg/mf @ 2 . 5 um MMD 570 6/6 900 mg/ni @ 2 . 6 um MMD Company Sanitized. Does not contain TSCA CBI HLR 110-85 Page 14 of 14