Document VGOr9jJZ5qJ6MryX7rby6d3oj

-'R DU FONT USE ONLY AR226-2903 'Copies to E. X. du Font de Nemours and Company Haskell Laboratory for ToxicpTogy and Industrial Medicine Elkton Road, Newark., Delaware 19711 Study Initiated/Completed 8/8/80 - 9/5/80 tied b5 teals & PTgaents Department Jackson Laboratory f SUBACUTE INHALATION TOXICITY STUDY IM RATS I. Introduction; The purpose of this study was to determine the effects of maj.e rats after repeated inhalation exposure. A previous study determined an LC50 of^p------l|to be 35.3 mg/L.' II. Procedures; A. Animals s Hale Crl:CD^ rats were housed 2/cage in 8" x 8" x 14" stainless steel, wire mesh cagefs and provided Purina Certified Rodent Chow 95Q02 and water ad libitun. Rats were quarantined under these conditions and observed for general suitability for one week prior to testing. B. Exposure Protocol; Groups of 10 rats, 8-weeks old and weighing between 240^and 271 grans were exposed to design concentration of 0, 1.0 and 3.0 mg/L ofUB----lQ Rats were exposed whole body, 6 hrs/day, 5 days/week for two weeks. All i-*.ts were Weighed and observed daily through both the exposure period and a 14-day observation period (except weekends and holidays). 01 'Sws^msi B-BM^nofaonMir TSTO CBli it C. Generation; Prior Co tesc, che sample was llqulfled by heating to approximately 100C. A 50 cc plastic syr.-' ige wrapped with heating tape was loaded with llqulfled sample and placed on a syringe drive* The heated sample was sprayed onto the surface of a heated, round bottom flask. Dilution air swept the resultant vapor/aerosol mixture through heated glass connectors Into the cop of a 17 L glass chamber containing the test rats. D. Analytical: Chamber samples were collected approximately every 1/2 hr by drawing a known volume of chamber atmosphere through 2 tandem midget implngers. The Impingers contained c.;etone (ACS grade) as the trapping solvent. Samples were analyzed using a Hewlett Packard )?5730A gas chromatograph equipped with a flame lonlzatlon detector. A 6' x 1/4 ** glass column, packed with 20Z neopentyl glycol sebacate on 60/80 mesh Chromosorb PAW, was used to chromacograph the samples. Oven temperature was programmed to Increase at 8"C per minute from an initial temperature of 90C, until the third sample peak eluted. Ac this point the program was changed to 32C/min until all other sample peaks eluted and the column was clear. U--P----ttJconcentracions were based on the second largest peak in the G.C. chromacograph. This pea^cis identified asf^--1----^^RMon the basis of the G.C. peak height area and I the reported sanpr^^onpoaffRn. Sample concentrations were determiner by comparison with standards prepared by dilutions of the test compound. Chamber temperatures were monitored throughout each exposur-. E. Clinical Pathology : After the 9th exposure and 13th observation day all test animals were individually housed in stainless steel metabolism cages for overnight (16-hour)iurine collection. Water and ground Purina Certified Rodent Chou ^5002 Were available ad libitum. Urinalysis included a measure of the urine volume, osoolality, pH, and tests for sugar, blood, urobilinogen, bilirubin, protein, and acetone. The urine color and appearance we^e noted and sediment from pooled specimens examined microscopically. Fittoride concentration and total fluorlde excreted in the urine were also measured. Blood samples were taken;from the rats' tails after the 10th exposure and the 14th observation day. Heaatology measurements included: erythrocyte count, hemoglobin, platelet cdunc, mean corpuscular-volume, leukocyte count, and relative number of neutro^hils, lynphocytes, eosinophlls, monocytes, and basophils. Henatocr-'t, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were calculated' from these data. Blood serum measurements included: alkaline phosphatase, glutamic-pyruvic transaminase, glutamic-oxalacefcic transaminase, urea nitrogen, creatinine, and total protein. 2 - 02 ;, ,r-,W SanM^ B> ^ eenteSn TSC& C5,l c 1 F. Pathology ; After the: 10th exposure, S race from each group were selected at random and sacrificed for gross and histopathologic examination. Remaining rats were sacrificed on the l4ch observation day for an identical examination. Organs and tissues examined included the ear pinna, skin, chymust, medlastinal tissue, spleent, bone narrow (sternua), heartt, trachea, lungst, esophagus, stomach, small intestines (duodenum, jejun iai, and ileum), large intestines (cecum and colon), livert, kidneyst, tescest, epididyoides, thyroids, adrenals, brain, eyes 'and any other tissues observed to be abnormal at necropsy. G* Organ and Body Weight Analysis; Three parameters were calculated for each group and test groups Were then compared with centre Is for II). statistical differences: mean body weight (Appendix I), me .in organ weightt, and organ/body weight ratio (Appendix The mean body weights were also plotted and are attached as Figure I. III. Resultsi Chamber temperature was controlled at <_ 30C by placing dry ice on the top of the exposure chamber. At times a cloud was visible in the high ^.evel chamber. During exposure the generation nebulizer plugged repeatedly, presumably because the low boiling components evaporated readily while high boiling ( solids did not. This problem accounts for the high standard deviation and range for these exposure concentrations. A. Exposure Data Exposure No. 9 10 Overalitt Mean i 0.82 0.59 0.49 1.48 0.96 0.87 0.86 1.031 0.89 1.1.1. 0.90 Design Level - 1.0 mg/L S.D. Range 0.46 0.30 0.34 0.41 0.65 0.12 0.45 0.35 0.64 0.59 0.50 0.21 0.26 - 0.14 0.93 - 0.45 0.77 0.20 0.64 0.19 0.60 0.14 - 1.62 1.09 1.09 2.31 2.33 1.18 1.40 1.74 2.07 2.48 2.48 t Organs weighed at aacropsy tt Mean of all samples from 10 exposures 03 ------^.n----.-----------"^6' Exposure No. 1 2 3 4 5 6 7 8 9 1& Overall!t Mean 1.56 2.90 2.13 3.34 3.53 3.01 3.21 3.02 2.78 3.U_ 2.83 Design Level - 3.0 ag/L S.D. Range 0.83 1.18 0.71 1.84 1.73 0.78 1.10 1.53 1.11 1.19 1.32 0.40 1.40 1.14 1.25 - 1.30 1.82 1.71 1.77 1.20 1.90 0.40 - 3.37 5.34 3.43 6.84 5.59 4.74 4.46 6.46 4.08 5.13 6.-8A B. Clinical Observations: During exposure rats exposed Col^--------Ajac both levels had reduced response Co sound, clear and red discharge from eyes and nose, ooderace salivation; and slight dose-dependent weight loss. During the 14-day observation period no differences were observed between controls and treated rats. C. Clinical Pathology : After 10 exposures all test animals had significant dose-related increases in serum alkaline phosphatase activity and urinary fluorlde excretion. &?ts exposed at 2.8 og/L also had elevated serum gluCaBie-pyruvic transaminase and glutaolc-oxalacetic transaminase activities and excreted more dilute urine;than controls. After the 14-day observation period, remaining test rats had lower erythrocyte counts and higher urinary fluorlde levels than controls. These rats also tended to have significantly less serum creatlnlne and excreted a more alkaline urine. D. Pathology ; No gross findings related to the exposure were noted in any rat at necropsy. Histologle findings showed mild hepatocyte and renal tubular epithelial damage after 10 expo mres toy^--------I^t 2.8 mg/L. .However, the toxic effect was reversible following the 14-day observation period. No compound related lesions were detected in rats exposed at 0.9 mg/L ac either sacrifice. E. Organ and Body Weight, Analysis (Appendix I & 11^: The mean body weight of the high level rats was significantly lower than controls on test days 2 through 16 (Figure 1). After 10 exposures significant dose dependent increases were noted in liver and kidney weights and organ/body weight ratios. Also noted were decreases <n high level heart and lung weights, an increase in high level cestia/body weight ratio, and Increases in low level lung weight and lung/body weight ratio. 04 tt Mean of all samples from 10 exposures - 4 - Can^sny SanHbed, Does no? eWw.n TSCA cw Following the 14-day observation period high level liver and kidney absolute weights and organ/body weight ratios were still significantly increased. Other anomalies noted at the first sacrifice were no longer evident, however, there was an apparent dose-related increase in thynus-body weight ratio of exposed animate. VI. Summary ofBfff-11 Groups of 10 male Crl:CD rats were exposed 6 hours/day ,5days/week for 2 weeks to average concentrations of 0.9 or 2.8 mg/L *iv- A. control group was simultaneously exposed to air only. r During exposure, moderate salivation, clear to red discharges from eyes and nose, reduced response to sound, and slight dose dependent weight loss were noted in all exposed rats.; During the 14-day observation period there were no visible differences in appearance of controls and treated rats. After 10 exposures test raits had significant dose-related increases in serum alkaline phosphatase activity and in urinary fluoride concentrations. Rats exposed at 2.8 og/L also hjadelevated serum glutamic-pyruvic transaminase and glucamic-oxalapetie transaminase activities and excreted more dilute urine than controls;. c Following a 14-day observation period both exposed groups had lower erythrocyte counts and excreted more fluoride than the controls. Treated racs also exhibited significant1 dose-dependent depression of serum creatinine locals and accreted more alkalijne urine than controls. No gross pathologic findings were noted upon necropsy sacrifice. Bistologic finding^ in rats after 10 exposures mild hepatocyte and renal tubular epithelial damage. This determined Co be reversible upon examination of rats after observation period. during either to 2.8 mg/L were effect was a 14-day Mean body weights of high level rats were significantly lower on test days 2-16 than controls. After 10 exposures significant dose dependent increases were noted in liver and kidney absolute weights and organ/body weight ratios. Also noted were decreases in high level heart and lung weights, an increase in high level cescis/body weight ratio and increases in low level lung absolute weights and lung/body weight ratios* Liver and kidney absolute weights and organ/body weight ratios were still significantly increased after the 14-day observation period. Further, a dose-related increase in chymua/body weight ratios was observed at this time. 05 Company Sanifice& Does not contain TSCA CB8 In conclusion, organ/body weight trends and clinical results are supported by pathologic lesions observed in the livers and kidneys of rats exposed to 2.8 ng/L. Some of 'these organ/body weight and clinical results were also observed in rats exposed to 0.9 mg/L however, pathologic lesions were not. 6 - 06 Company Sanitized. 0oes not contain TSCA CBS c Report by; \j^i\ C-- CyT^ g^t 7 Joseph C. Haoill ---- Technologist W^/^u?/^^ r Reviewed by: Bru^^BureJBa-------- Besearch^Toxi.calqgist Study Director JCH:jrg:WP:4.17 Date Issuedi August Approved ; : 25^_ 1981; by: jpf/^.sj. Gerald Chief, Acute J. ^--. \. L. Kennedy!l, Ur. IXnavveessttllggaattionas S! ection C 'Report Mo. 379-81 7 - 07 Company Sanitized. Does not contain TSCA CBI i FIGURE I S S Rl S I CM 0 f\( 0 Cosipany Sanitized. Does not contain TSCA CBK C GROUP CONTROLS LOU LEVEL(0.9 MG/L) HIGH LEVEL(2.8 MQ/L> F RATIO(l) LSD(2> DUNNETT(3> MMS<4) APPENDIX I 2-UK i SUBACUTEBOD H* Y UEIG 13384 H T SUMMARY TABLE INTERVALS IN TEST DAYS I* 2. 3. 250.3000 252.2000 247.1000 0*961 7.6295 8.6267 69.1333 254.3000 250.4000 243.4000* 4.853* 7.2744 8.2253 62.8481 260.7000 254.4000 237.9000* 14.322* 9.0276 10*2076 96.7926 4. 267.6000 260*0000 241.0000* 15.110* 10.2276 11.5645 124.2370 5. 275.6000 263.9000+ 238.0000* 23.503 11,5168 13.0222 157.5296 GROUP CONT ROLS LOU LEVEL(0.9 MG/L) /""^GH LEVEL(2.8 MG/L) F RATIO(l) LSD(2) LUNNETTO) UMS<4) INTERVALS IN TEST DAYS 8. 9. 10. -r--------------- 293.9000 286.4000 269.9000* 299.6000 291.0000 270.3000* 305.0000 292.5000 270.5000* 9.013* 11.8674 13.4186 167.2667 12.590. 12.3165 13.9264 180.1667 14.786* 13.1806 14.9035 206.3333 11. 308.2000 296.9000 270.9000* 15.449* 14.1200 15.9657 236.7926 12. 304.5000 297.9000 268.9000* 12.032 15.8424 17*9132 298.0852 09 Company Sanitized. Does not contain TSCA CB8 . APPENDIX I (cont'd) GROUP CONTROLS LOU LEVEL(0.9 MG/L) HIGH LEVEL(2.8 MG/L) F RATIO<1) LSD<2> DUNNETT(3) UMS(4) INTERVALS IN TEST DAYS 15. 16. 17. 326.4000 304.0000+ 302.00004 4.525K? 19.6221 22.5148 202.76A7 329.0000 307.8000 306.6000+ 3.287 21*4144 24.5713 241.5000 335.4000 316.0000 316.6000 1.904 24.6323 28.2636 319.5333 10. 341.6000 321.0000 323.0000 2.129 23.9906 27.5273 303.1000 23. 371.8000 352.2000 357.2000 1.029 30*9420 35.5035 504.2000 GROUP INTERVALS IN TEST DAYS 24, 25. 26. CONTROLS LOU LEVEL(0.9 MG/L) HIGH LEVEL(2.8 MG/L) 376*0000 357.0000 362.8000 379.0000 361.2000 367.0000 379.8000 357.8000 366.6000 F RATIOd) 0.873 0.796 1.191 LSD(2) 32.1076 31.3495 31.2636 (. DUNNETT(3> 36.8409: 35.9711 35.8725 UMS<4> 542.9000 517.5667 514.7333 (1) RATIO OF AMONG- TO MITHIN-GROUP VARIATION--ONE-FACTOR ANALYSIS OF VARIANCE. <2> LEAST SIGNIFICANT DIFFERENCE---GIVEN A SIGNIFICANT <ALPHA=0.05> F RATIO ANY TUO MEANS DIFFERING BY MORE THAN THE LSD ARE SIGNIFICATLY DIFFERENT Uin A VARIABLE-UISE FALSE POSITIVE (ALPHA) ERROR RATE OF 0.05. (3> DUNNETT TEST--ANY TREATMENT MEAN DIFFERING FROM THE CONTROL MEAN BY MORE THAN THE DUNNETT STATISTIC IS SIGNIFICANTLY DIFFERENT FROM THE CONTROL MEAN UITH A VARIABLE-UISE FALSE POSITIVE (ALPHA) ERROR R^TE OF 0.05. <4) MITHIN-GROUP MEAN SQUARE. + SIGNIFICANTLY DIFFERENT (P<0.05) FROM CONTROL GROUP BY LSD. * SIGNIFICANTLY DIFFERENT <P<0.05) FROM CONTROL GROUP BY DUNNETT TEST AND LSD. * SIGNIFICANT AT THE 0.05 PROBABILITY LEVEL. c:- 10 Ccmpany Sanitized. Does not contain TSCA CBI c GROUP APPENDIX 11 2^W SUBACUTE-OROAN I BODY WEIGHT MEAN ABSOLUTE DATA H* 13384 RATS SACRIFICED AFTER 10 EXPOSURES FINAL WGT. HEART LUNGS LIVER SPLEEN CONTROLS LOU LEVEL<0.9 MG/L) HIGH LEVEL(2.8 MG/L) F RATIOd) LSD(2) DUNNETTO) UMS(4) 302.2000 306.4000 262.4000* 8.622* 25.48U 29.237S 341.93331 .9840 1.0120 ,8360* 7.260* 1082 .1241 .0062 1.6960 2.0220* 1.5260+ 26.709* .1503 .1724 .0119 10.4620 14.4160* 15.1900* 16.309* 1.9349 2.2202 1.9717 *6620 ,6080 .5540 1.542 .1340 .1^38 .0095 GROUP KIDNEY TESTIS THYMUS CONTROLS LOU LEVEL(0. 9 MG/L) HIGH LEUEL(2 S MG/L) F RATIO(l) LSD<2) DUNNETTO) C UMS(4) 2. 3680 2. 61.11 2. 75eo+ 2.443 .389Q ,4463 .0797 2.9480 3.1820 3.1440 1.488 .3172 .3639 .0530 .6620 .7460 .5780 5.209 .1134 .1301 .0068 <l) Ratio of amona- to yithin-lflpoup variation--one-factor analysis of variance. <2) Least significant differerice--aiven a sianificant <alpha=0.05) F patio any two means differina ba morei than the LSD are sianificantly different with a variable-wise false positive (alpha) error rate of 0.05. <3) D'-innett test--Any treatment mean differina froni the control mean by more than the Ounnett statistic is sjidnificantly different from the control mean with a variable-wise false positive (alpha) error rate of 0.05. (4) Uithin-aroup Mean Sauare. j + Significantly different (P<OJ*05) from control aroup ba LSD* * Significantly different (P<d.05) from control Broup by Dunnett test and LSD. * Sianificant at the 0*05 probability level. 11 Company ^Samnittaizeedo. Duuoceas not contain TSCA CBI GROUP APPENDIX II (cont'd) ^|'2-UK SUBACUTE-ORGAN/BODY WEIGHT MEAN H 13384 RATS SACRIFICED AFTER 10 RELATIVE DATA EXPOSURES HEART LUNGS LIVER SPLEEN 0 KIDN! CONTROLS LOU LEVEL<0,9 MG/L ) HIGH LEvEL(2.8 MG/L) F RATIO<1> LSO(2) DUNNETT(3) UMS(4) .3254 .3304 .3199 208 .0356 .0409 .0007 5621 , 66004 .5821 19.896* .0357 .0410 .0007 3 4516 4 * 7067* 5 7825* 94.P.5S* .3693 .4237 .0718 2195 1982 2125 .458 .0495 .0566 .0013 *7833. .6542 1.0573< 8.090* *1540 ,1767 *0125 GROUP TESTIS THYMUS CONTROLS LOU LEVEL(0.9 MG/L) HIGH LEVEL<2.8 HG/L/ .9788 1.0391 1.2077* .2197 2434 .2187 F RATIO(l) LSD(2) 5.192 .1605' 2.110 .0296 DUNNETT(3> .1841 .0340 UMS(4) .0136 .0005 0 (1) Ratio of sinona- to within-dpoup variatior--one-factor snslasis of variance* <2) Least sianificant differenc^--aiven a sianificant <slph3s=005) F rstio any two niesns differina by more than the LSD are significantly different with B variable-wise false positive (alpha) error rate of 0.05. <3) Ounnett test--Any treatment mean differins from the control mean by more than the Dunnett statistic is significantly different froni the control mean with a variable-wise false positive (alpha) error rate of 0.05. (4) Uithin-droup Mean Sauare. + Sianificantly different (P<005) from control aroup by LSD* Sianificantly different (P<065) from control aroup by Dunnett test and LSD* Significant at the 0(/5 probateility level* ..^ i^ 12 S^feed. no. co^l. TSCA CBi c GROUP APPENDIX XI (cont'd) 2-UK SUBACUTE-ORGAN S BODY UEIGHT MEAN ABSOLUTE DATA H 13384 RATS SACRIFICED AFTER 14 DAY OBSERVATION PERIOD FINAL MGT. HEART LUNGS LIVER SPLEEN CONTROLS LOM LEVEL(0*9 MG/L) HIGH LEVEL<2. 8 MG/L) F RATIO<i) LSD<2) DUNNETT<3> UMS(4) 379*8000 357.8000 3666000 1*191 31.2636 35.8725 514*7333 1. 1660 1. 1700 1. 1940 116 .1371 .1573 .0099 2 3280 2 1160 2 1760 1.252 .3009 .3453 .0477 14 .5760 14 .5280 18 .2100* 6798 2.4960 2.8639 3*2809 .7420 .7320 .8020 .657 .1439 .1651 .0109 GROUP KIDNEY TESTIS THYMUS CONTROLS LOW LEVEL(0.9 MG/L) HIGH LEVEL(2.8 M6/L) 2.7040 ' 2.640C 329404J 3.2840 3,2880 3.2460 .6800 .7560 .8300 F RATIO(l) LSD(2) 14986 .2870 .099 .2273 2.190 .1562 DUjNNETTO) *3293 , .2608 .1792 ( UMS<4) .0434 .0272 .0128 (1) Ratio of amona- to within-arou? variation--one-factor analysis of variance* (2) Least sianificant difference--diven a sianificant (alpha^O.OS) F ratio? ana two means differing by more ^than the LSD are significantly different with a variable-wise false positive (alpha) error rate of 0.05. (3) Injnnett test--Any treataentj mean diffepinfl from the control mean by more than the Dunnett statistic is significantly different from the control mean with a variable-wise false positive (alpha) error rate of 0.05. (4) Uithin-aroup Mean Souare. ; + Significantly different <P<005) froffi control arou? ba LSD. * Sianificantly different <P<0;05) from control aroup by Dunnett test and LSD. ' Significant at the 0.05 probability level* 13 Company Sanitized. Does nol contain TSCA CBI U , ^i--^*1 APPENDIX XZ (cont'd) j -WK SUBACUTrE-ORGAN/BODY WEIGHT MEAN RbLAIIvE DAT A 3384 RATS ?SACRIFICED AFT ER 14 DAY OE.S ERVATION PE RIOD GROUPi HEART LUNGS LIVER SPLEEN gf^ KIDN&> CONTROLS LOU LEVEL( 0.9 MG/L) HIGH LEVEL <2.9 MG/L> F RATIO(I) LSD(2) DUNNETT(3) UMS<4) .3072 3269 .3265 1.077 .0333 .0383 .0006 .6119 .5918 .5934 .396 .0547 .0627 .0016 3.8263 4.0587 4.9587* 20.982* .4023 .4617 .0852 1954 .2040 .2194 1.109 .0356 .0409 .0007 .7140 .7398 .9008* 10.39K .0968 .1110 .0049 GROUP TESTIS THYMUS CONTROLS .8660 .1793 LOU LEVEL< 0.9 MG/L) HIGH LEVEL (2.8 MG/L) . 920': ,8910 .2112+ 2248* F RATIO(l) LSD(2) DUNNETTO) UMS(4) .710 .0998 .1145 .0052 5.144* .0318 .0365 .0005 0 1 (1) Ratio of amona- to within-droup variation--one-factor analysis of variance. (2) Least siai-iificsnt difference-- aiven a sidnificarit (alph3=0*05) F rstior any two means differina by more ithan the LSD are significantly different with a variable-wise false positive (alpha) error rate of 0.05. (3) Dunnett test--Any treatment mean differing from the control mean by more than the Dunnett statistic is significantly different from the control mean with a variable-wise false positive (alpha) error rate of 0.05. <4) Uithin-aroup Mean Sauare. ! + Sianificantly different (P<0+05) from control aroup by LSD. ft Significantly different <P<005) from control aroup by Dunnett test and LSD. # Sianifleant at the 0.05 probability level. c 14 Company Sanitized. Does not contain TSCA CBI