Document re5Gobrk6m87weKrVwpeQ2NKq

DO PONT OSE ONLY AR226-2903 E. I. du Pont de Nemours and Company Haskell Laboratory fqr Toxicology and Industrial Medicine Elkton Road, Newark, Delaware 19711 HASKELL LABORATORY REPORT NO. 379-81 Haskell No. 13,383 Other >s Study Initiated/Completed 8/8/80 - 9/5^811 toteria^^^mittted by _ PiIigments Department Jackson Laboratory ------------------ SOBACOTE INHALATION TOXICITY STUUf IN RATS f I. Introduction; ; The purpose of this studyiwas to determine the effects of maxe rats after repeated i n h a l ^ lon exposure. A previous study determined an LC50 be 35.3 mg/L. II. Procedures; A Animals! Male Crl:CD rats were housed 2/cage in 8" x 8" x 14 stainless^steflfwire mesh cag^s and provided Purina Certified Rodent Chow #5002 and water ad libitum. Rats were quarantined under these conditions an observed for geniFal suitability for one week prior to testing. a. Exoosure Protocol: Groups of 10 rats, 8-weeks old and weighing between 240^ n d 271 g g S T w e r e exposed to ^ * 8 v " ? r a t i o n Z, rA.3 0 mtt/L o f l f t H I I ^ W l Rats were exposed whole body, 6 hrs/day, 5 y to 'ill r e * l g h . d ..d .b.T ed dally through both the exposure period and a 14-day observation period (except weekends and holidays). ' - 1- 01 C. Generation: Prior to test, the sample was liquified by heating it to approximately 100C. A SO ec plastic syr.''ige wrapped with heating tape was loaded with liquified 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 top 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 impingers. The impingers contained acetone (ACS grade) as the trapping solvent. Samples were analyzed using a Hewlett Packard #5730A gas chromatograph equipped with a flame Ionization detector. A 6' x 1/4" glass column, packed with 202 neopentyl glycol sebhcate on 60/80 mesh Chromosorb PAH, was used to chromatograph the samples. Oven temperature was programmed to Increase at 8C per minute from an initial temperature of 90C, until the third sample peak eluted. Ac this point the program was changed to 32C/mln until all other sample peaks eluted and; the column was clear. ^ J e o n c e a t r a t l o n B ; were based on the second largest peak in the G.C. chromatograph. This peajc Is Identified a s T B H 9 | | ^ M H o n the basis of the G.C. peak height area and: the reported s a m p r ^ ^ O T ^ W f f l o n . Sample concentrations were determine^ by comparison with standards prepared by dilutions of the test compound. Chamber temperatures were monitored throughout each exposure 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. Hater and ground Purina Certified Rodent Chow #5002 were available ad libitum. Urinalysis included a measure of the urine volume, osmolality, pH, and tests for sugar, blood, urobilinogen, bilirubin, protein, and acetone. The urine color and appearance were noted and sediment from pooled specimens examined microscopically. Fltyorlde concentration and total fluoride excreted in the urine were also measured. Blood samples were taken from the rats' tails after the 10th exposure and the 14th observation day. Hematology measurements included: erythrocyte count, hemoglobin, platelet epunt, mean corpuscularvolume, leukocyte count, and relative number of neutrophils, lymphocytes, eosinophils, monocytes, and basophils. Hematocrit, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were calculated from these data. Blood serum measurements included: alkaline phosphatase, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, urea nitrogen, creatinine, and total protein. 2- 02 iY S C c EI F. Pathology2 : After the 10th exposure, 5 rats from each group _ selected at random and sacrificed for gross and histopathologic examination. Remaining rats were sacrificed on the 14th observation day for an Identical examination. Organs and tissued examined included the r P1"" ' J t r a c h e a thymust, mediastinal tissue, spleent, bone marrow (sternum , heartt, trachea, lungst, esophagus, stomach, small intestines (duodenum, jejun *, andileua), large intestines (cecum and colon), llvert, kldneyst, testest, epididymides, thyroids, adrenals, brain, eyes and any other tissues observed to be abnormal at necropsy. G. Orean and Bodv Weight Analysis: Three parameters were calculated for each group and test groups were thn compared with contrcls for statistical differences: mean body weight (Appendix I), meja organ weightT, and g a " b o S weght ratio (Appendix II). The mean body weights were also plotted and are attached as Figre 1. III. Results: ' . 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 level chamber. During exposure the generation nebulizer plugged reputedly, presumably because the low boiling components evaporated readily while high boiling S T J n i H ihl. p r o b l ..count, for th. U g k . c ~ U r d d i . t l o . aud range for these exposure concentrations. A. Exposure Data Exposure No. 1 2 3 4 5 6 7 g 9 10 Overalltt Mean i 0.82 0.59 0.49 1.48 0.96 0.97 0.86 1.01 0.89 1.11 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 - 1.62 0.26 - 1.09 0.14 - 1.09 0.93 - 2.31 0.45 - 2.33 0.77 - 1.18 0.20 - 1.40 0.64 - 1.74 0.19 - 2.07 0.60 - 2.48 0.14 - 2.48 t Organs weighed at necropsy * tt " of all samples from 10 exposures - 3- 03 TSCA CBI Sanitized. Does not con.a' Company Exposure No. 1 2 3 4 5 6 7 8 9 10 Overalltt Mean 1.56 2.90 2.13 3.3* 3.53 3.01 3.21 3.02 2.78 3.13 2.83 Design Level - 3.0 mg/L S.P. 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 6v84 I 1 B. Clinical Observations : During exposure rats exposed. both levels had reduced response to sound, clear and red d i s c h a r g e f r o m # and nose, moderate salivation, and slight dose-dependent w i g h t loss, ^ i n g the 14-day observation period ho differences were observed between controls and treated rats. . - C. Clinical Pathology1 ; After 10 exposures all test animals had siotrifleant dose-related increases in serum alkaline phosphatase activity an urinary fluoride excretion. Bats exposed at 2.8 mg/L also had glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase acti ities and excreted more dilute urine than controls. f K After the 14-day observation period, remaining test rats had lower erythrocyte counts and higher urinary fluoride levels than controls. These r 2 s also tended to have significantly less serum creatinine and excreted a more alkaline urine. D. Pathology2 : No gross findings related to the exposure were noted in any rat at necropsy. Histologic findings showgd mild hepatocyte and renal tubular epithelial damage after^lO expo m r e s 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 at either sacrifice. k _ o r m and Body Analysis (Appendix I & III: The mean body weight of the high level rats was significantly lower than conerols on tes. 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. A l s o n o t e d w e r e decreases in high level heart and lung weights, an increase in high level testis/body weight ratio, and increases in low level lung weight an lung/body weight ratio. ( tf Mean of all samples from 10 exposures - ` -04 Company Ssniiteed, Does no? contain TSC.d CRf c S S 1" weight ratio of exposed animals. l ~ ~ ` a -- ^ VI. Summary Groups of 10 mol. CrliCD * " " *' L=52^SS=2STAi2 " - .n d ,, ^ : n? . ^ r r : ; p r r " % r ^ : ia r k f 8rtro : L r^ p r e^ rr i g h n o : r ' 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 exposure. t..c s e n slkaline STM"lta.io-p^.lc Pecs exposed et 2.8 ,,ansssilnsse ectlvlcles end excreted transaminase and glutamic oxaiapecie more dilute urine than control. Folloulng e 14-8.7 observe.. g ^ S d i 'S ^ r t ^ S . ' I x S S sere. creecllpe ? " e l f d r e t e d nor. elkelli* urine the. controls. o prose H * ^ 2 ^ . ' iS . ,S L T r tS S S 2 ? ! r d ^ o c ^ r ' e n S l i q . ^ 2* - TM determined to be reversible upon examination of rats atte observation period. 7 Hea,, M r eight, of high level rets ere signlf IcentlF loner on test days 2-16 than controls. After 10 exposure, slg^fleent dose dependent S ^ H o r ' l C t i e d liner end kidney .`" l" " ^ ^ ^ `^" ^ i g h t s ^ s n lncreese In high S L ' S S K J S S h ! 8" ^ ' % Z S " i f U lenel lung ebsoluc. weights and lung/body weight ratios. U ,, r snd kidney - r a ^ i S 's s j r s 'S i i S r t i - * * * - * * - - " tMs - 5- 05 TSCACBI In conclusion, organ/bod weight trends and clinical results are supported by pathologic lesions observed in the livers and kidneys of rats exposed to 2.8 og/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 e * Other Same: O O 06 Company Sanitized. Does not contain TSCA CB? Report by: tk s j Joseph . Haalll Technologist Reviewed by: N.Brute BurgaBS Research'Toxicologist Study Director Approved by: il J. fe-sgeJ Gerald L. Kenned,. Chief, Acute Investigatitlons Section JCH: jrg:WP:4.17 Date Issued: August 25^_ 1981 'Report No. 379-81 C -7- 07 Company Sanitized. Does not contain TSCA CBI SUBACUTE BODY WEIGHT CURVE GRANS Hf 13384 z annoia Sanitized. Does not contain T S C A C B I 6 8 I 12 H 10 10 20 22 24 26 20 30 r s TEST HAYS C GROUP APPENDIX I 2-WK SUBACUTE BODY WEIGHT SUMMARY TABLE H# 13384 INTERVALS IN TEST DAYS 1. 2. 3. 4, CONTROLS LOW LEVEL(0.9 MG/L) HIGH LEVEL(2.8 MG/L> F RATIO(l) LSD(2> DUNNETT(3 > ' WMS(4) 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 267.6000 260.0000 241.0000* 15.110* 10.2276 11.5645 124.2370 275.6000 263.9000+ 238.0000* 23.503* 11.5168 13.0222 157,5296 GROUP CONTROLS LOW LEVEL(0.9 MG/L) ^~T.GH LEVEL(2.8 MG/L) F RATIO(1) LSD(2) LUNNETT <3 > WMS(4) INTrRVALS IN TEST DAYS 8. T 9. 293.9000 286.4000 269.9000* 299.6000 291.0000 270.3000* 10. 305.0000 292.5000 270,5000# 9.013* 11.8674 13.4186 167.2667 12.590*. 14.786* 12.3165 13.1806 13.9264 14.9035 180.1667 . 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 CBI . APPENDIX I (co n t'd ) GROUP INTERVALS IN TEST DAYS 15. 16. 17. CONTROLS LOU LEVEL(0.9 MG/L) HIGH LEVEL<2.8 MG/L) F RAT10(1) LSD(2) DUNNETT(3) WMS(4) 326.4000 304.00004 302.0000# 4.525# 19.6221 22.5148 202.7667 329.0000 307,8000 306.60004 3.287 21.4144 24.5713 241.5000 335.4000 316.0000 316.6000 1.904 24.6323 28.2636 319.5333 18. 341.6000 321.0000 323.0000 2.129 23.9906 27.5273 303.1000 G 23. 371.8000 352.2000 357.2000 1.029 30.9420 35.5035 504.2000 i 1 i GROUP INTERVALS IN TEST DAYS 24, 25. 26. CONTROLS LOW 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 RATIO(l) LSD(2) DUNNETT(3) WMS(4) 0.873 32.1076 36.8409 : 542.9000 0.796 31.3495 35.9711 517.5667 1.191 31.2636 35.8725 514.7333 (. (1) RATIO OF AMONG- TO WITHIN-QROUP VARIATION--ONE-FACTOR ANALYSIS OF VARIANCE. ifv rhoAfI SIGNIFICANT DIFFERENCE-- GIVEN A SIGNIFICANT (ALPHA=0.05> F RATIO ANY TUQ MEANS DIFFERING BY MORE THAN THE LSD ARE SIGNIFICATLY DTFF'FE'P'mt WITH A VARIABLE-WISE FALSE POSITIVr<iCpHArESROR^a e 5f iti5? ERENT ? ^ N!.TT TEST~ ANY TREATMENT MEAN DIFFERING FROM THE CONTROL MEAN BY MORE DUNNETT STATISTIC IS SIGNIFICANTLY DIFFERENT FROM THE CONTROL MEAN WITH A VARIABLE-WISE FALSE POSITIVE <ALPHA) ERROR RATE OFV o s . <4) WITHIN-GROUP MEAN SQUARE. + SIGNIFICANTLY DIFFERENT (P<0,05) FROM CONTROL GROUP BY LSD. * SIGNIFICANTLY DIFFERENT (PC0.05) FROM CONTROL GRGUP BY DUNNETT TEST AND LSD * SIGNIFICANT AT THE 0.05 PROBABILITY LEVEL. 10 Company Sanitized. Does not contain TSCA CBI r C GROUP APPENDIX I I 2^11 SUBACUTE-ORGAN 8 BODY WEIGHT MEAN ABSOLUTE DATA H# 13304 RATS SACRIFICED AFTER 10 EXPOSURES FINAL WGT HEART LUNGS LIVER SPLEEN CONTROLS LOW LEVEL(0.9 MG/L) HIGH LEVEL(2.8 MG/L) F RATIO(l) LSD(2) DUNNETTO) UMS(4) 302.2000 306.4000 202.4000 8.622# 25.4811 29.2375 341.9335 .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 .1538 .0095 GROUP KIDNEY TESTIS THYMUS CONTROLS LOW LEVEL(0.9 MG/L) HIGH LEVEL(2.S riG/L) F RATIO(l) LSD(2) DUNNETTO) ^ WMS(4) 2.3680 2.61L!) 2.7580+ 2,445 .3890 .4465 .0797 2.9480 3.1820 3.1440 1.488 .3172 .3639 .0530 .6620 .7460 .5780 5.209# .1134 .1301 .0068 (1) Ratio of among- to withinHsroup variation-- one-factor analysis of variance. <2) Least significant difference-- given a significant <alpha=0.05) F ratio? ana two means differing ba morel than the LSD are sianificantia different with a variable-wise false positive (alpha) error rate of 0.05. (3) Dunnett test-- Ana treatment mean differing from the control mean ba more than the Dunnett statistic is siignificantia different from the control mean with a variable-wise false positive (alpha) error rate of 0.05. (4) Within-Sroup Mean Sciuare. j + Significantia different (P<Oj*05) from control group ba LSD. # Signif icantia different (P<0i.O5) from control group ba Dunnett and LSD. test * Significant at the 0.05 probabilit level. 11 Sanitized. Does not contain TSC A CBI Company APPENDIX XX (cont'd) r ,2-WK SUBACUTE-ORGAN/BQDY WEIGHT MEAN RELATIVE DATA H H# 13384 RATS SACRIFICED AFTER 10 EXPOSURES GROUP HEART LUNGS LIVER SPLEEN kidnI D CONTROLS LOW LEVEL(0.9 MG/L) HIGH LEVEL(2.8 MG/L) F RATIO(1> LSD(2) DUNNETT<3> WMS(4) .3255 .3304 .3199 .208 .0356 .0409 .0007 .5621 .6600* .5821 19.896* .0357 .0410 .0007 3.4518 4.7067* 5.7825* 94.735# .3693 .4237 .0718 2195 .1982 .2125 .458 .0495 .0568 .0013 7833. .8542 1.0573* 8.090# 1540 .1767 .0125 GROUP TESTIS THYMUS CONTROLS LOW LEVEL(0.9 MG/L) HIGH LEVEL(2.8 MG/L) .9788 1.0391 1.2077* .2197 2434 .2187 F RATIO(l) LSD(2) DUNNETT(3 > WHS(4 > 5.192* .1605' .1841 .013d 2.110 .0296 .0340 .0005 O (1 Ratio of smona- to within-aroue variation-- one-factor analysis of variance <*.) Least sianifleant difference-- aiven a significant (alpha=0.05> F ratio * any two means differina by more than the LSD are sianificantly different with a variable-wise false positive (alpha) error rate of 0.05. lUr treatment!mean differina from the control mean by more than the Dunnett statistic is sianificantly different from the control mean 3 variable-wise false positive (alpha) error rate of 0.05. (4) Within--Sroup Mean Sauare. : + Sianif icantly different (P<0.<i>5) from control aroup by LSD. * Si^"lficantla different (PC0.65) from control aroup by Dunnett test 3HQ LSD* * Sianificant at the 0*05 probability level* * ] 12 e iliteed, Dess n,o0>contain TSCACBi % c GROUP APPENDIX II (cont'd) _ 2-WK SUBACUTE-ORGAN S BODY WEIGHT MEAN ABSOLUTE DATA H# 13384 RATS SACRIFICED AFTER 14 DAY OBSERVATION PERIOD FINAL WGT HEART LUNGS LIVER SPLEEN CONTROLS LOW LEVEL(0.9 MG/L) HIGH LEVEL<2.8 MG/L) F RATIO<1) LSDC2) DUNNETT <3 > WMS<4 ) 379*8000 357.8000 366 *6000 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# 6.798* 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 MG/L) F RATIO(l) LSD<2) DUJNNETT (3 ) WMS <4) ( 2.7040 2.640C : 3.2940$ 14.986* .2870 .3293 , .0434 3.2840 3.2880 3.2460 .099 .2273 .2608 .0272 .6800 .7560 .8300 2.190 .1562 .1792 .0128 (1) Ratio of 3mona- to withm-Sroup variation-- one-factor analysis of variance. <*.) Least sianificant difference-- Siven a significant <alpha=O.OS) F ratio any two means differins by more than the LSD are sisnificantly different r V3r*ak*e"w*se false positive (alpha) error rate of 0.05. (3) Dunnett test-- Any treatment! ean differins from the control mean by more than 3th. eV3Dr ui3nbnleet-tW slsteatii'ssltsiec is sisnificantly positive (alpha) different from the control error rate of 0.05. mean <4; Within--droup Mean Sctuare* ; + Sisnificantly different (PC0.05) from control Sroup by LSD. # Sisnif icantly different <P<0.!05) from control sroup by Dunnett test and LSD. ! * Sianificant at the 0.05 probability level. 13 Company Sanitized. Does net contain TSCA CBI  GROUP APPENDIX XX (cont'd) . __ [2-WK SUBACUTE-ORGAN/BODY WEIGHT MEAN RELATIVE DATA H* 13384 RATS SACRIFICED AFTER 14 DAY OBSERVATION PERIOD HEART LUNGS LIVER SPLEEN KIDNBSS# CONTROLS LOU LEVEL(0.9 MG/L) HIGH LEVEL(2.8 MG/L) F RATIO(l) LSD(2) DUNNETTO) WMS(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# 10.391* .0968 .1110 .0049 GROUP TESTIS THYMUS CONTROLS LOU LEVEL(0.9 MG/L) HIGH LEVEL(2.8 MG/L) .8660 .920'; .8910 .1793 .2112+ .2248# ' ` F RATIQ(l) LSD(2) DUNNETT(3) UMS(4) .710 .0998 .1145 .0052 J 5,144* .0318 .0365 .0005 C ! V (1) Ratio of among- to within-droup variation-- one-factor analasis of variance, (2) Least significant difference-- given a significant (alrha=0.05) F ratior ana two means differing ba more than the LSD are significantla different with s variable-wise false positive (alpha) error rate of 0.05. (3) Dunnett test-- Ana treatment mean differing from the control mean ba 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) Within-group Mean Souare. ' + Significantly different (P<0|05> from control group ba LSD. t Sisnificantla different <P<0;05> from control group ba Dunnett test and LSD. # Significant at the 0.05 probabilit level. C 14 Sanitized. Does not contain TSC A CBI Company