Document r2QJYRzvz42Jpp5ZV9R81Drv

AR226-3018 t Study Title Repeated Dose Oral Toxicity: 14-Day Feeding Study with H-19683 in Male Mice Author John W. Sarver Study Completed On April 28, 1995 Performing Laboratory E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.0. Box 50 Newark, Delaware 19714 Laboratory Project ID Haskell Laboratory Report No. 325-95 Page 1 of 23 Com pany Sanitized. D oes not contain T SC A CBI DuPont i l l 325-95 REPEATED DOSE ORAL TOXICITY: 14-DAY FEEDING STUDY WITH H-19683 IN HALE MICE GENERAL INFORMATION Substance Tested; Synonyas/Codes; Purity; Physical Form; Composition; Contaminants; C.A.S. Registry No.; Sponsor; Study Initiated/Completed: In-life Study Initiated/Completed; 10/8/92 - 4/28/95 11/4/92 - 11/18/92 - 2- Com pany Sanitized. Does not contain T SC A CB !  DuPont HLR 325-95 REPEATED DOSE ORAL T O XICITY: 14-DAY FEEDING STUDY WITH H-19683 IN MALE H IC l SUMMT The purpose of this study was to evaluate the repeated dose oral toxicity of H-196P3 when incorporated into nutritionally adequate chow and fed to male Crl:CD-l*BR mice for 14 consecutive days. Mice were fed diets containing 0, 3, 30, 300, or 3000 ppm H-19683. Mice were weighed and observed for clinical signs of toxicity once each week. Food consumption was not measured. At the end of the 2 week feeding period, all mice were euthanatized and necropsied. The livers were weighed, and liver weight/final body weight ratios were calculated. No deaths occurred during the study. Other than a body weight effect ii mice from the 3000 ppm dose group, no adverse clinical signs of toxicity weie observed during the study. Significant decreases in mean body weight were * observed in mice from the 3000 ppm dose group on test days 7 and 14. Statistically significant decreases in mean body veight gain were observed in this same dose group for the intervals 0-7 days on test, and overall. Significant increases in mean absolute and relative liver weights were observed in mice from the 300 and 3000 ppm dose groups killed at the end of the 14-day feeding period. The no observable adverse effect level (N0AEL) was determined to l^e 30 ppm. The N0AEL was based on the increases in mean absolute and relative liver weight observed in the 300 and 3000 ppm dose groups and the decreases in ..ean body weights and body veight gains observed in the 3000 ppm dose group. Tracy A. Fillib^h Toxicology Technician Study Director: JVS/alw - 3- Com pany Sanitized. D oes not contain T S C A CBff DuPont BLR 325-95 REPEATED DOSE ORAL TO XICITY: 14-DAY FEEDING STUDY ---------------------- Wif H H-r9683 IN MALE MICE-------------------- The purpose of this study was to evaluate the repeated dose oral toxicity of H-19683 when incorporated into nutritionally adequate chow and fed to male Crl:CD*BR mice for 14 consecutive days. Mice were fed diets contained 0, 3, 30, 300, or 3000 ppm H-19683. MATERIALS AMD METHODS A. Test Substance The test substance, H-19683. was supplied by DuPont Specialty Chemicals, E. I. du Pont de Nemours and Coipany, Wilmington, Delaware. Purity was estimated to Available information about the identity, purity, composition, stability, and toxicity of the test substance was filed with Haskell Laboratory Information Services by the Sponsor when the substance was submitted for testing. Upon receipt, the test substance was assigned a unique Haskell Number (H-19683). B. Test Species On 10/27/92, 30 male Crl:CD-I(ICR)BR mice, born approximately 9/21/92, were received from Charles River Laboratories, Inc., Montreal, Canada. On 11/4/92, the body weights ranged from 24.4 to 29.6 grams. The Crl:CD-lBR mouse was selected on the basis of extensive experience with this strain and its suitability with respect to hardiness, longevity, sensitivity, and low incidence of spontaneous diseases. C. Animal Husbandry All mice were housed singly in suspended stainless steel, wire-mesh cages throughout the study. Animal rooms were maintained on a 12-hour light/dark cycle and targeted at a temperature of 23 2C and a relative humidity of 50 10X. Water was provided ad libitum from an automatic watering system. During the quarantine period (see Section D), all mice were fed (ground) Purina** Certified Rodent Chow #5002. During the test period, all mice were fed the diet of their respective treatment group (see Section F). - 4- Com pany Sanitized. D oes not contain TSCA CBI DuPont BLR 325-95 Haskell Laboratory has an animal health monitoring program. This program is monitored and administered by the Laboratory Veterinarian. Water samples are periodically analyzed for total bacterial counts and for the presence of coliforms, lead, and other contaminants. Additionally, samples from freshly ashed " e s and cage racks are periodically analyzed to assure adequate sanitation by the cagevashers. Data from this program are maintained separately trom1 ^ r e c o r d s . Animal feed is certified by the manufacturer m licet snecified nutritional requirements and to be free of a list of spe'-ified^coiitaminants. On the basis of these analyses, there is no evidence suggesting that contaminants were present in the feed or water in amounts wh" h may have interfered with the results of this study. D. Quarantine Period Upon arrival at Haskell Laboratory, the mice were *'e^ ve^ " ^."quarantine period and observed daily for any gross signs of disease or injury. On the basis of body weights and clinical observations, all mice were released from quarantine on 11/3/92 by the laboratory veterinarian. E. Assignment to Groups and Study Start After the quarantine period, 25 mice were selected for study use on the ba^s of adequate body weight gain and freedom from any clinical signs of disease or injury. The selected mice were divided by computerized, stratified randomization into 5 groups of 5 mice each, so there were no statistically significant differences among 8JouP ^ y^ f order of mice within each group was randomized. Each group of mice a designated as either a control, low-, low-intermediate-, or high-concentration group. After assignment to dosing groups, each mouse was assigned a 5"diS** ,,,`u L w ..b. Each number a printed on a card* * , nl,.,ide of the cage. Each mouse was also assigned a 3-digit luentirication nucber that was marked on the mouse's tail with water insoluble ink. The mice were approximately 44 days of age at study start. Mice not assigned to a test group were sacrificed by C02 asphyxiation. F. concentration Selection and Diet Administration h 19683 was mixed in the diet at concentrations of 0 (control), 3, 30, 30C m 3 0 0 0 ^ . ! T h e concentrations ..re selected so that the results ol this study could be ceaparad to the results obtained iron an earlier study conducted with C-8. - 5- Company Sanitized. D oes not containT SCA Cffl PH DuPont BLR 325-95 During the test period, nice in each group were fed a diet of ground Purina* Certified Rodent Chow* #5002 that contained the following concentrations of H-19683: Group No. Male Dietary Concentration* ppm # Rats/Group I 0 (Control) 5 III 3 (Low) 5 V 30 (Low-Intermediate) 5 VII 300 (Intermediate) 5 IX 3000 (High) 5 Veight/weight concentration of H-19683 G. Diet Preparation and Sampling For each diet concentration, the appropriate amount of H-19683 was first added to a small amount of ground chow and mixed by hand until it appeared homogeneous. The hand-mixed portion was then added to the remainder of the ground chow weighed out for that diet in a Hobart low speed mixer and was mixed for 15 minutes. After mixing, the diet was visually inspected to ensure it was free of clumps. Control diets were aiso mixed for the same period of time. The diets for this study were prepared the day the study started and stored refrigerated until used. Sampies for concentration, homogeneity, and stability were taken for analyses at a later date if deemed necessary by the study director. H. Bodv eights and Clinical Observations All -nice were weighed and observed for clinical signs of toxicity once each week throughout the study. Cageside examinations to detect moribund or dead mice were conducted twice daily. I . Food Consumption, Food Efficiency, and Intake of H-19683 Food consumption was not determined during this study. J. Necropsy At the end of the 2-week feeding period, all mice were necropsied. The mice were euthanatized by chloroform anesthesia and exanguination. The livers were weighed at necropsy, and liver weight/final body weight ratios were calculated. - 6- Company Sanitized. Does not contain TSCA CB1  DuPont HLR 325-95 STATISTICAL ANALYSES Body weights, body veight gains, and organ weights vere analyzed by a one-way analysis of variance. Vhen the statistic for differences aaong test group means (F statistic) vas significant, pairwise comparisons between test and control groups were made with the Dunnett's test. Significance vas judged at alpha - 0.05. RECORDS AD SAMPLE RETENTION All original records will be retained at Haskell Laboratory or at the Records Management Center, E. I. du Pont de Nemours and Company, ViIslington, Delaware. RESULTS A. Diet Sample Analysis y All diets prepared appeared to be thoroughly mixed. No clumps or other signs of non-uniform mixing were observed. Therefore, diet samples taken at the time of diet preparation vere discarded without analysis. B. Body Weights and Body Veight Gains (Tables 1-2} Appendix I) Statistically significant decreases in mean body veight of mice from the 3000 ppm dose group vere observed on test days 7 and 14. Statistically significant decreases in mean body weight gains of this same group vere observed for the interval 0-7 days on test and overall. C. Clinical Observations One mouse in the 3000 ppm dose group exhibited pallor of the ears on test days 7 and 14. No other clinical signs of toxicity vere observed. D. Necropsy (Tables 3,4; Appendix II) Gross observations at necropsy revealed liver discoloration in one mouse from the 300 ppm dose group and in 4 mice from the 3000 ppm dose group. Significant increases in mean absolute and relative liver weights vere observed in mice from the 300 and 3000 ppm dose groups. No other gross observations vere noted. .1 - 7- Company Sanitized. Does not contain T SC A CBS Kale ale* were fed diet* containing target c o M t m t i o M of 0, 30, 300, nr w m b m H-196S3 for 14 d m . Significant body weight effects ooro observed^in aleo froa the 3000 ppo dose p wf. absolute and relative liver weights were observed in nice froa the 300 ana 3000 ppa groups. The no observable adverse effect level observed daring this study was 30 ppa. t ? tin Company Sanitized. Does not contain TSCA CBjl DuPont BLR 325-95 TABLES - 9- Company Sanitized. Does not containT SCA C W iiM'nitowiim11 -rii REPEATED ORAL DOSE TOXTOTTY: 14 DAY FEEDTNf: STUDY WTTH H 196B1 TN MALE MTCE TABLE 1 MEAN BODY WEIGHTS OF MALE MICE MEAN BODY WEIGHTS (g) GROUP: CONCENTRATION (PPM): I 0 III V 3 30 VII IX 300 3000 DAYS ON TEST 0 26.7( 0.5) 26.7( 1.9) 27.2( 1.1) 27.8( 1.6) 27.8( 0.6) 7 29.9( 1.5) 30.6( 1.5) 29.6( 1.2) 29.8( 1.2) 26.3( 2.4)* 14 31.7( 1.3) 31.9( 1.3) 31.5( 1.3) 31.0( 1.3) 28.7( 1.7)* a Standard deviation is reported in parentheses Statistical oethods: One-way AOV and Dunnett's tests were performed on weekly interval data. (See Statistical Analyses) * Statistically significant difference from control at alpha => 0.05. DuPont HLR 325-95 Company Sanitized. Does not contain TS C A CBf REPEATED ORAL DOSE TOXICITY: 14-DAY FEEDING STUDY WITH H-19683 IN HALE MICE TABLE 2 MEAN BODY WEIGHT GAINS OF MALE MICE MEAN BODY WEIGHT GAINS (g) GROUP: CONCENTRATION (PPM): I 0 III 3 DAYS ON TEST 0- 7 7-14 3.2( 1.2) 3.9( 1.5) 1.8( 0.5) 1.4( 0.6) 0-14 5.0( 0.9) 5.3( 1.6) V 30 2.3( 1.1) 1.9( 0.6) 4.2( 1.2) VII IX 300 3000 2.0( 1.1) 1.2( 0.4) 3.2( 0.8) -1.5( 2.6)* 2.4( 1.5) 0.9( 2.0)* * Standard deviation is reported In parentheses Statistical Methods: One-way AOV and Dunnett's tests were performed on both weekly and summary interval data. (See Statistical Analyses) * Statistically significant difference from control at alpha - 0.05. DuPont HLR 325-95 Company Sanitized. Does not contain TS C A CBt DuPont HUt 325-95 REPEATED DOSE ORAL T O XICIT Y: 14-DAY FEEDING STUDY WITH H -19683 IN MALE MICE TABLE 3 MEAN FINAL BODY AND ORGAN WEIGHTS FROM MALE MICE MEAN FINAL BODY WEIGHTS (grams) GROUP CONC. (PPM) MEAN FINAL BODY WEIGHTS I0 III 3 V 30 VII 300 IX 3000 31.7 ( 31.9 ( 31.5 ( 31.0 ( 28.7 ( 1.3) 1.3) 1.3) 1.3) 1.7)# MEAN ABSOLUTE ORGAN WEIGHTS (grams) GROUP CONC. (PPM) LIVER I0 1.811 (0.112) III 3 1.907 (0.154) V 30 2.241 (0.344) VII 300 2.871 (0.336)# IX 3000 4.365 (0.685)# MEAN RELATIVE ORGAN WEIGHTS (X of body weight) GROUP CONC. (PPM) LIVER I 0 5.7043 (.1324) III 3 5.9781 (.5546) V 30 7.0975 (.7963) VII 300 9.2486 (.9889)# IX 3000 15.1789 (1.920)# Standard Deviation in parentheses Significantly different (p<0.05) from control group by Dunnett's Test - 12 - Com pany Sanitized. Does not contain T SC A CBS DuPont BLR 325-95 REPEATED DOSE ORAL TOXICITY: 14-DAY FEEDING STUDY WITH H -19683 IN MALE MICE TABLE 3 (continued) MEAN FINAL BODY AND ORGAN WEIGHTS FROM MALE MICE MEAN FINAL BODY WEIGHTS (grans) GROUP I III V VII IX CQNC. (PPM) 0 3 30 300 3000 MEAN FINAL BODY WEIGHTS 31.7 (0.000) 31.9 (0.803) 31.5 (0.768) 31.0 (0.444) 28.7 (0.002)# TEST - HOMOGENEITY TEST - TREND BARTLETT'S TEST 0.008 0.002 0.972 MEAN ABSOLUTE ORGAN WEIGHTS (grams) GROUP CONC. (PFM) LIVER I0 1.811 (0.000) III 3 1.907 (0.697) V 30 2.241 (0.092) VII 30C 2.871 (0.000)# IX 3000 4.365 (0.000)# TEST - HOMOGENEITY TEST - TREND BARTLETT'S TEST 0.000 0.000 0.011 Values in parentheses - P value of Student T Test comparison of treatment mean to control mean. # - Significantly different (p<0.05) from control group by Dunnett's Test Homogeneity - P value of F Test of whether group means are equal. Trend - P value of F test of whether there is a dose-related change in group means. . .,, . Bartlett's Test - P value of Test of Homogeneity of Variance. com pany Sanitized. D oes not contain T S C A C H DuPont BLR 325-95 REPEATED DOSE ORAL TO XICITY: 14-BAY FEEDING STUDY WITH H-19683 IN MALE MICE TABLE 3 (continued) MEAN FINAL BODY AND ORGAN WEIGHTS FROM MALE MICE MEAN FINAL BODY WEIGHTS (grans) GROUP I III V VII IX CONC. (PPM) 0 3 30 300 3000 MEAN FINAL BODY WEIGHTS 31.7 (0.000) 31.9 (0.803) 31.5 (0.768) 31.0 (0.444) 28.7 (0.002)# TEST - HOMOGENEITY TEST - TREND BARTLETT'S TEST 0.008 0.002 0.972 MEAN RELATIVE ORGAN WEIGHTS (X of body weight) GROUP CONC. (P?"> LIVER I 0 5.7043 (0.000) III 3 5.9781 (0.688) V 30 7.0975 (0.051) VII 30C 9.2486 (0.000)# IX 300? 15.1789 (0.000)# TEST - HOMOGENEITY TEST - TREND BARTLETT'S TEST 0.000 0.000 0.001 Values in parentheses - P value of Student T Test comparison of treatment mean to control mean. | _ Significantly different (p<0.05) from control group by Dunnett's Test Homogeneity - F value of F Test of whether group means are equal. Trend - P value of F test of whether there is a dose-related change in group means. Bartlett's Test - P value of Test of Homogeneity of Variance. - 14 - Com pany Sanitized. D oes noi contain T S C A C B | S I T E / LE S 1ON : liv er " LARGE DISCOLORATION REPLATED ORAL DOSE T O XICITY: 14-DAY FEEDING STUDY WITH H-19683 IN HALE NICE TABLE 4 INCIDENCES OF GROSS OBSERVATIONS IN MALE HICE GROUP DESIGNATION; CONCENTRATION (PPM ): NUMBER IN GROUP: 1 0 5 III V 3 30 55 VII 300 5 0 0 NOTE: 0 INCIDENCES REFLECT THE NUMBER OF ANIMALS WITH A GIVEN LESION. IX 3000 5 I Ul I DuPont HLR 325-95 C o m p a n y Sanitized. Does n o t contain T S C A C B 1 APPENDIX I INDIVIDUAL BODY WEIGHTS T! I - 16 Com pany Sanitized. D oes not contain T SC A CB1 m DuPont BLR 325-95 REPEATED DOSE ORAL TOXICITY: 14-DAY FEEDING STUDY WITH H-19683 IN MALE NICE APPENDIX I INDIVIDUAL BODY WEIGHTS (g) OF MALE MICE GROUP I - O PPH DAYS ON TEST: 0 7 ANIMAL * 90620 90621 90622 90623 90624 26.4 27.3 27.1 26.7 26.2 30.4 31.0 31.5 28.5 28.3 14 31.5 32.6 33.4 30.8 30.3 GROUP III - 3 PPM DAYS ON TEST: 0 7 14 ANIMAL # 90625 90626 90627 90628 90629 28.2 26.4 29.0 25.3 24.4 30.7 31.6 32.1 28.3 30.2 31.6 32.2 33.6 30.1 32.2 GROUP V - 30 PPM DAYS ON TEST: 07 ANIMAL # 90630 90631 90632 90633 90634 26.3 26.0 28.7 27.3 27.8 30.2 28.6 31.3 28.7 29.0 14 31.7 30.6 33.5 31.4 30.1 17 - Com pany Sanitized. Does not contain T SC A CBI ..... ' '. DuPont HLR 325-95 REPEATED DOSE ORAL TOXICITY: 14-DAY FEEDING STUDY WITH H-19683 IN MALE MICE APPENDIX I (Continued) INDIVIDUAL BODY WEIGHTS (g) OF MALE MICE GROUP VII - 300 PPM DAYS ON TEST: 0 7 14 ANIMAL # 90635 90636 90637 90638 90639 28.6 28.1 29.6 25.2 27.6 29.9 31.4 30.4 28.2 29.2 31.0 32.3 32.1 29.1 30.7 GROUP IX - 3000 PPM DAYS ON TEST: 0 7 14 ANIMAL * 90640 90641 90642 90643 90644 28.1 28.7 27.9 2'. 1 21.3 27.5 26.7 22.1 27.0 28.1 28.6 28.3 26.7 28.4 31.4 I - 18 - Com pany Sanitized. D oes not contain T S C A CEp *4 .1 DuPont BLE 32S-95 APPENDIX II INDIVIDUAL ANIMAL FINAL BODY AND ORGAN WEIGHTS Explanatory Notes STD. DEV. Standard Deviation - 19 - Company Snftfzecf. Does nof contain T SC .C B I REPEATED DOSE ORAL TOXICITY; 14-DAY FEEDING STUDY VITH H-1%83 IN MALE MICE APPENDIX II INDIVIDUAL ANIMAL FINAL BODY AND ORGAN EIGHTS FINAL BODY AW) ABSOLUTE ORGAN WEIGHTS (grans) FROM MALE MICE GROUP I - 0 PPM ANIMAL NUMBER FINAL BODY LIVER 90620 90621 90622 90623 90624 31.5 32.6 33.4 30.8 30.3 1.783 1.903 1.951 1.705 1.711 GROUP MEAN STD. DEV. 31.7 1.3 1.811 0.112 GROUP III - 3 PPM ANIMAL NUMBER FINAL BODY LIVER 90625 90626 90627 90628 90629 31.6 32.2 33.6 30.1 32.2 2.098 1.914 1.946 1.906 1.669 GROUP MEAN STD. DEV. 31.9 1.3 1.907 0.154 GROUP V - 30 PPM ANIMAL NUMBER FINAL BODY LIVER 90630 90631 90632 90633 90634 31.7 30.6 33.5 31.4 30.1 2.370 1.909 2.736 2.265 1.924 GROUP MEAN STD. DEV. 31.5 1.3 2.241 0.344 - 20 Com pany Sanitized. Does not contain T SC A CBI DuPont F.LR 325-95 REPEATED DOSE ORAL TO XICITY! 14-DAY PEEDING STUDY WITH H-19683 IN HALE MICE APPENDIX II (Continued) INDIVIDUAL ANIMAL FINAL BODY AND ORGAN WEIGHTS FINAL BODY AND ABSOLUTE ORGAN WEIGHTS (grams) FROM MALE MICE GROUP VII - 300 PPM ANIMAL NUMBER FINAL BODY LIVER 90635 90636 90637 90638 90639 31.0 32.3 32.1 29.1 30.7 2.886 2.541 3.306 2.540 3.082 GROUP MEAN STD. DEV. 31.0 1.3 2.871 0.336 GROUP IX - 3000 PPM ANIMAL NUMBER 90640 90641 90642 90643 90644 GROUP MEAN STD. DEV. FINAL BODY LIVER 28.6 28.3 26.7 28.4 31.4 4.343 5.014 3.294 4.266 4.909 28.7 1.7 4.365 0.685 - 21 - Company Sanitized. Does not contain T SC A CBI DuPont BLR 325-95 REPEATED DOSE ORAL TOXICITYs 14-DAY FEEDING STUDY WITH H-19613 IN MALE MICE APPENDIX II (continued) TWDTVIDUAL ANIMAL FINAL BODY AND ORGAN WEIGHTS RRI.ATIVE ORGftN WEIGHTS (X of body weight) FROM MALE MICE GROUP I - 0 PPM ANIMAL NUMBER LIVER 90620 90621 90622 90623 9062*+ 5.6603 5.8374 5.8413 5.5357 5.6469 GROUP MEAN STD. DEV. 5.7043 0.1324 GROUP III - 3 PPM ANIMAL NUMBER LIVER 90625 90626 90627 90628 90629 6.6392 5.9441 5.7917 6.3322 5.1832 GROUP MEAN STD. DEV. 5.9781 0.5546 GROUP V - 30 PPM ANIMAL NUMBER LIVER 90630 90631 90632 90633 90634 7.4763 6.2386 8.1672 7.2134 6.3920 GROUP MEAN STD. DEV. 7.0975 0.7963 - 22 Com pany Sanitized. D oes not contain T SC A CBS DuPont BLR 325-95 REPEATED DOSE ORAL TO XICITY: 14-DAY FEEDING STUDY VITH H -19683 IN HALE NICE APPENDIX II (continued) INDIVIDUAL ANIMAL FINAL BODY AND ORGAN WEIGHTS RELATIVE ORGAN WEIGHTS (X of body weight) FROM HALE MICE GROUP VII - 300 PPM ANIMAL NUMBER LIVER 90635 90636 90637 90638 90639 9.3097 7.8669 10.2991 8.7285 10.0391 GROUP MEAN STD. DEV. 9.2486 0.9889 GROUP IX - 3000 PPM ANIMAL NUMBER LIVER 90640 90641 90642 90643 90644 15.1853 17.7173 12.3371 15.0211 15.6338 G??UP MEAN STD. DEV. 15.1789 1.9204 - 23 - Com pany Sanitized. D oes not contain T SC A CB1