Document vVQbBOY4xBy1rV5Xk4mBy2B0Z

01-May-2002 07:25pm. F rom-CharI as River Labs Argus Div. 90S SiKthf Drive, Btdg.A Horsham. PA 19044 Tdephone: (2IS) 443-8710 ke/efivc (2IS) 443SS87 A Z .Z 'l G _ 22115544443383558877 T-007 P.001 F-437 Io q 6 ARGUS RESEARCH Charles River Laboratories Discovery and Development Services From: Raymond G. York, Ph.D., DABT To: Company: Phone: Fax: John Butenhoff, Ph.D., DABT 3M Corporate Toxicology (651)737-1962 (651)733-1773 Date: 01 May 2002 Total Pages: 22 RE: Protocol 418-020 ( CPPe^r'O30a r 0 ^ 3 I 4T TJo " 4rn prvm l-H 0<; 'm o a Attached is the information on covariant analysis for study 418-020. Four different analyses were done for females and two for males. Females: 1. Sexual maturation w/ gestational age at sexual maturation as covariate 1ecm*o0 2. Sexual maturation w! body weight at maturation as covariate 3. Estrous stages w/ day 64 postweaning body weight as covariate 4. Estrous stages w/ gestational age at start of estrous testing as covariate Males: C~Eo 1. Sexual maturation w/ gestational age at sexual maturation as covariate "Do --Hrn om oo 2. Sexual maturation w/ body weight at maturation as covariate o CO W e are defining "gestational age" in this context as the time in days from evidence of mating in the P generation until evidence of sexual maturation in the F1 generation. The reasoning is rats naturally delivery after 21 to 23 days of gestation. Pups that are delivered after 23 days of gestatioh tend to weigh more and appear to develop faster than pups of the same sex after 21 days of gestation. Both pups would be 1 day old but the first pup would be 23 gestational days old and the second pup only 21 gestation days old. If a test substance shortened pregnancy (reduced length of gestation or if more pups were delivered early (reduced parturition length) in one group, the pups may reach sexual maturation a day or two before pups in a group which had a longer gestation or parturition length. It would appear that the test substance was causing a delay in sexual maturation, when it actually affected the maternal animal by changing the time spent in utero. By using each pup's age from time of conception to sexual maturation as the covariant, the length of the stay in utero is factored out. 01-May-2002 ;0c:27pm, Fram-Charles Rivar labs Argus Div. 2154438587 T-007 P.002/022 F-437 ljic am taiy^id ui v<aiicn vvao u o c u wj cvaiuoi,g pi^p^uau o ^ ^ uiuuwi <C4iiv vv*^uiui patency of F1 generation male and female rats, respectively, using gestational age at sexual maturity and the body weight recorded on the day of preputial separation (male rats) or day of vaginal patency (female rats) as the covariates. The Analysis of Covariance was also used to evaluate estrous cycling of F1 generation female rats, using gestational age at the start of estrous testing and day 64 postweaning body weight as the covariates. Please review the attached information and if you have any questions, please do not hesitate to contact me. 3 Ql-May*2(lQ2 O t: 27pm From-Charlas Rivar Labs Argus DivV DAY OF MATURITY 10:26 Monday, March 4, 2002 ANCOVA (DUNNETT'S TEST) WITH BODY WEIGHT AS COVARIATE SEX GROUP N MEAN SD F 1 60 34.8833 2.07562 2 S8 35.5345 2.26503 3 58 34.0862 2.56347 4 58 34.8103 2.11475 5 54 36.7222 2.43726 * M 1 52 48.4615 2.13708 2 58 49.4655 2.68991 3 57 49.3333 3.42956 4 58 49.7241 3.10513 5 54 52.3333 3.02817 * * Significantly different from Control group 1 (p<0.05) 1 ANCOVA (DUNNETT'S TEST) WITH GESTATION AGE AS COVARIATE (#) SEX GROUP N MEAN SD F 1 60 34.8833 2.07562 2 58 35.5345 2.26503 3 58 34.0862 2.56347 4 54 34.9630 2.08284 5 52 36.7115 2.47605 M 1 52 48.4615 2.13708 2 58 49.4655 2.68991 3 57 49.3333 3.42956 4 54 49.8333 3.19049 5 53 52.2642 3.01376 (#) No statistically significant differences when compared with control group 1 (p>0.05) 2154438587 T-007 P.003/022 F-437 4 T)1-May-ifl02 02:27pm From-Charlss River Labs Argus 0 iv.- E5TR0US stages 10:26 Monday, March 4, 2002 2 ANCOVA (DUNNETT'S TEST) WITH DAY 64 BODY WEIGHT AS COVARIATE SEX GROUP N MEAN SD F 1 30 4.66667 0.88409 2 29 5.03448 0.90565 3 30 5.03333 0.80872 4 30 5.03333 1.06620 5 30 5.40000 0.67466 *(1) ANCOVA (DUNNETT'S TEST) WITH ESTROUS AGE AS COVARIATE SEX GROUP N MEAN SD F 1 30 4.66667 0.88409 2 29 5.03448 0.90565 3 30 . 5.03333 0.80872 4 28 5.03571 1.10494 5 28 5.39286 0.68526 *(1) * significantly different from Control group 1 (p<0.05) (1) Note: Results from Dunnett's test are reported without regard to the significance of the global treatment group effect in the overall a n c o v a (Haseman, Toxicological sciences, June, 2001). In both analyses (covariate = day 64 body weight and covariate = estrous age) the global treatment group effect was not statistically significant in the overall a n c o v a . 2154438587 T-007 P.004/022 F-437 i *01-May-41102 02:28pm From-Char 1es Rivar Labs Argus D i w DAY OF MATURITY 10:26 Monday, March 4, 2002 5 ANCOVA WITH BODY WEIGHT AS COVARIATE The Mixed Procedure Model information Data set Dependent Variable Covariance Structure Estimation Method Residual Variance Method Fixed Effects SE Method Degrees of Freedom Method WORK.AAA DAY_MAT Diagonal REML Profile Model-Based Residual class GROUP class Level information Levels values 5 12345 Dimensions Covariance parameters Columns in X Columns in Z Subjects Max Obs Per Subject Observations Used Observations Not Used Total Observations 1 7 0 1 300 288 12 300 2154438587 T-007 P.005/022 F-437 QI-May->W02 _ 02:28pm From-Charles River Labs Argus D iv v Effect GROUP GROUP GROUP DAY OF MATURITY 10:26 Monday, March 4, 2002 6 ANCOVA WITH BODY WEIGHT AS COVARIATE ---SEX=F------------------------------------------------ The Mixed Procedure covariance Parameter Estimates Cov Parm Estimate Residual 4.3642 Fit Statistics -2 Res Log Likelihood AIC (smaller is better) AICC (smaller is better) Bic (smaller is better) 1246.4 1248.4 1248.4 1252.0 -Type 3 Tests of Fixed Effects Effect Num Den DF DF F Value Pr > F BWT GROUP 1 282 4 282 59.63 9.50 <.0001 <.0001 Least Squares Means GROUP Estimate Standard Error DF t Value Pr > 1t| 1 34.8248 0.2698 282 129.07 <.0001 2 35.7142 0.2753 282 129.73 <.0001 3 34.1967 0.2747 282 124.50 <.0001 2154438587 T-007 P.006/022 P-437 7 01-May-2002 02:28pm From-CharI as River Labs Argus 0 iv :' Effect GROUP GROUP GROUP GROUP DAY o f MATURITY 10:26 Monday, March 4, 2002 7 ANCOVA WITH BODY WEIGHT AS COVARIATE -- ------------- s EX=F------------------------------------------------- --- The Mixed Procedure Least Squares Means Effect GROUP Estimate standard Error DF t value Pr > It | GROUP GROUP 4 5 34.8710 36.4103 0.2744 282 127.07 0.2871 282 126.80 Differences of Least Squares Means <.0001 <.0001 standard GROUP _GROUP Estimate Error OF t Value Pr > |t | Adjustment Adj P 21 31 41 51 0.8893 -0.6281 0.04619 1.5855 0.3859 0.3853 0.3850 0.3932 282 282 282 28 2 2.30 -1.63 0.12 4.03 0.0219 0.1042 0.9046 <.0001 Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu 0.0736 0.3006 0.9999 0.0003 2154438587 T-007 P.007/022 F-437 'Q1-May^?0Q2 02 :28pm From-Charlas Rivsr Labs Argus Div. DAY OF m a t u r i t y 10:26 Monday, March 4, 2002 8 ANCOVA WITH BODY WEIGHT AS COVARIATE The Mixed Procedure Model information Data set Dependent Variable Covariance Structure Estimation Method Residual variance Method Fixed Effects SE Method Degrees of Freedom Method WORK.AAA DAY_MAT Diagonal REML Profile Model-Based Resi dual class Levet Information Class Levels Values GROUP 5 12345 Dimensions Covariance Parameters Columns in X Columns in z Subjects Max obs Per Subject Observations used Observations Not used Total Observations 1 7 0 1 300 279 21 300 2154438587 T-007 P.008/022 F-437 [ Ul-May-2l)02 02:28pm From-Charlas River Labs Argus Div.- DAY OF MATURITY 10:26 Monday, March 4, 2002 9 ANCOVA WITH BODY WEIGHT AS COVARIATE Effect GROUP GROUP GROUP The Mixed Procedure Covariance Parameter Estimates Cov Parm Estimate Residual 7.1683 Fit Statistics -2 Res Log Likelihood AIC (smaller is better) AICC (smaller is better) BIC (smaller is better) 1344.4 1346.4 1346.4 1350.0 Type 3 Tests of Fixed Effects Effect Num Den DF DF F value Pr > F BWT GROUP 1 273 4 273 53.38 17.77 <.0001 <.0001 Least Squares Means GROUP Estimate standard Error DF t Value . Pr > i11 1 48.5554 0.3715 273 130.70 <.0001 2 49.6708 0.3527 273 140.84 <.0001 3 49.0207 0.3572 273 137.24 <.0001 2154438587 T-007 P.009/022 F-437 10 5l-May-2002 02:28pm From-CharI as River Labs Argus Div. Effect GROUP GROUP GROUP GROUP DAY OF MATURITY 10:26 Monday, March 4, 2002 10 ANCOVA WITH BODY WEIGHT AS COVARIATE SEX--M The Mixed Procedure Least squares Means Effect GROUP Estimate Standard Error OF t value Pr > !t 1 GROUP GROUP 4 5 49.5845 52.5024 0.3521 273 140.84 0.3651 273 143.81 <.0001 <.0001 Differences of Least Squares Means Standard GROUP _GROUP Estimate Error OF t Value Pr > 1t 1 Adjustment Adj P 21 31 41 51 1.1154 0.4653 1.0291 3.9469 0.5115 0.5164 O.S123 0.5203 273 273 273 273 2.18 0.90 2.01 7.59 0.0301 0.3684 0.0456 <.0001 Dunnett-Hsu Dunnett-hsu Dunnett-Hsu Dunnett-Hsu 0.0964 0.7726 0.1409 <.0001 2154438587 bl II 0l-MayZ0Q2 02:28pm From-Charles River Labs d a y OF m a t u r i t y 10:26 Monday, March 4, 2002 ANCOVA WITH GESTATIONAL AGE AS COVARIATE ______ SEX=F ------ 11 The Mixed Procedure Model information Data Set Dependent variable covariance structure Estimation Method Residual Variance Method Fixed Effects s e Method Degrees of Freedom Method WORK.AAA DAY_MAT Diagonal REML Profile Model-Based Residual Class Level information class Levels values GROUP 5 12345 Dimensions Covariance Parameters Columns in x Columns in z subjects Max obs Per subject Observations used Observations Not Used Total Observations 1 7 0 1 300 282 18 300 Div.- 2154438587 T-007 P.011/022 F-437 JoZ HI-May-ZUOZ 02:29pm F rom-CharI as River Labs Argus D iv .- Effect GROUP GROUP GROUP DAY OF MATURITY 10:26 Monday, March 4, 2002 12 ANCOVA WITH GESTATIONAL AGE AS COVARIATE SEX=F The Mixed Procedure covariance Parameter Estimates cov Parm Estimate Residual 0.2779 Fit Statistics -2 Res Log Likelihood AIC (smaller is better) AICC (smaller is better) Bic (smaller is better) 457.3 459.3 459.3 462.9 - Type 3 Tests of Fixed Effects Effect Num Den DF DF F value Pr > F GEST_AGE GROUP 1 276 4990.64 <.0001 4 276 3.21 0.0135 Least Squares Means GROUP Estimate standard Error DF t Value Pr > |t| 1 35.1763 0.06819 276 515.88 <.0001 2 35.3426 0.06928 276 510.16 <.0001 3 35.0133 0.07046 276 496.94 <.0001 2154438587 T-007 P .012/022 F-437 /3 1-May-2002 02r29pm F rom-Charies River Labs Argus Div... Effect GROUP GROUP GROUP GROUP DAY OF MATURITY 10:26 Monday, March 4, 2002 13 ANCOVA WITH GESTATIONAL AGE AS COVARIATE SEX=F The Mixed Procedure Least Squares Means Effect GROUP Estimate Standard Error DF t value pr > |t| GROUP GROUP 4 5 35.2004 0.07182 276 490.11 35.3069 0.07576 276 466.01 <.0001 <.0001 Differences of Least Squares Means standard GROUP _GROUP Estimate Error DF t Value Pr > |t[ Adjustment Adj P 21 31 41 51 0.1663 -0.1630 0.02402 0.1306 0.09732 0.09749 0.09889 0.1027 276 276 276 276 1.71 -1.67 0.24 1.27 0.0887 0.0956 0.8083 0.2048 Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu 0.2639 0.2816 0.9978 0.5273 2154438587 T-007 P . 013/022 F-437 IU 0l-May-2002 32:29pm From-Charles River Labs DAY OF MATURITY 10:26 Monday, March 4, 2002 14 ANCOVA WITH GESTATIONAL AGE AS COVARIATE --- ------------ S E X = M -------------------------------------- ---------- The Mixed Procedure Model Information Data Set Dependent Variable Covariance Structure Estimation Method Residual Variance Method Fixed Effects SE Method Degrees of Freedom Method WORK.AAA DAY_MAT Diagonal REML Profile Model-Based Residual Class Level information Class Levels values GROUP 5 12345 Dimensions Covariance Parameters columns in x columns in Z Subjects Max Obs Per Subject Observations used Observations Not Used Total observations 1 7 0 1 300 274 26 300 Div.- 2152438587 T-007 P.014/022 F-437 ir D!-May-?1)02 02:29pm From-Charles River Labs Argus Div.-. Effect GROUP GROUP GROUP DAY OF MATURITY 10:26 Monday, March 4, 2002 15 ANCOVA WITH GESTATIONAL AGE AS COVARIATE ------- SEX=M-------- The Mixed Procedure Covariance Parameter Estimates Cov Parm Estimate Residual 0.3806 Fit Statistics -2 Res Log Likelihood AIC (smaller is better) Aicc (smaller is better) BIC (smaller is better) 529.4 531.4 531.4 535.0 - Type 3 Tests of Fixed Effects Effect Num Den DF DF F Value Pr > F GEST_AGE GROUP 1 268 5816.42 <.0001 4 268 1.80 0.1290 Least Squares Means GROUP Estimate Standard Error DF t Value Pr > |t| 1 49.7916 0.08731 268 570.26 <.0001 2 49.9788 0.08129 268 614.84 <.0001 3 49.7086 0.08186 268 607.21 <.0001 2154438587 T-007 P.015/022 F-437 1C 01-May-2002 02:29pm F rom-CharI as Rivsr Labs Argus 0 iv . - Effect GROUP GROUP OAY OF MATURITY 10:26 Monday, March 4, 2002 16 ANCOVA WITH GESTATIONAL AGE AS COVARIATE ------ SEX=M------ The Mixed Procedure Least Squares Means GROUP Estimate standard Error DF t Value Pr > |t| 4 49.8734 0.08396 268 594.04 <.0001 5 49.9531 0.09000 268 555.05 <.0001 Effect GROUP GROUP GROUP GROUP GROUP 2 3 4 5 _GROUP 1 1 1 1 Differences of Least Squares Means Standard Estimate Error DF t Value Pr > 111 0.1872 -0.08298 0.08185 0.1615 0.1183 0.1190 0.1211 0.1295 268 268 268 268 1.58 -0.70 0.68 1.25 0.1147 0.4861 0.4995 0.2135 Adjustment Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu Adj p 0.3183 0.8903 0.9003 0.5288 2154438587 T-007 P.016/022 F-437 01-May-2002 02:29pm F rom-CharI es River Labs Argus D iv . - f e m a l e s - ESTROUS STAGES 10:26 Monday, March 4, 2002 17 ANCOVA WITH ESTROUS AGE AS COVARIATE The Mixed procedure Model information Data Set Dependent Variable covariance structure Estimation Method Residual Variance Method Fixed Effects SE Method Degrees of Freedom Method WORK.AAA EST Diagonal REML Profile Model-Based Residual Class GROUP class Level information Levels Values 5 12345 Di tensions Covariance Parameters columns in X Columns in z Subjects Max Obs Per subject Observations Used Observations Not Used Total Observations 1 7 0 1 300 145 155 300 2154438587 T-QQ7 P .017/022 F-437 /? 3 1-May-2'002 02:29pm From-Char Ias River Labs Argus Div Effect GROUP GROUP GROUP FEMALES - ESTROUS STAGES 10:26 Monday, March 4, 2002 18 ANCOVA WITH ESTROUS AGE AS COVARIATE --------------- SEX=F--------- ---------------- The Mixed Procedure covariance Parameter Estimates cov Parm Estimate Residual 0.7707 Fit Statistics -2 Res Log Likelihood Aic (smaller is better) AICC (smaller is better) BIC (smaller is better) 381.0 383.0 383.0 385.9 . Type 3 Tests of Fixed Effects Effect Num Den DF DF F value Pr > F EST_AGE GROUP 1 139 4 139 4.04 1.90 0.0462 0.1131 Least Squares Means GROUP Estimate Standard Error OF t value Pr > |t| 1 4.7408 0.1645 139 28.83 <0001 2 4.9751 0.1657 139 30.03 <.0001 3 5.0098 0.1607 139 31.17 <.0001 2154438587 T-007 P.018/022 F-437 >1 01-May-2002 02:30pm From-Charles River Labs Argus Div.-- FEMALES - ESTROUS STAGES 10:26 Monday, March 4, 2002 19 ANCOVA WITH ESTROUS AGE AS COVARIATE Effect GROUP GROUP GROUP 4 5 The Mixed Procedure Least Squares Means Estimate Standard Error DF 5.0590 5.3769 0.1663 0.1661 139 139 t value 30.42 32.37 Pr > |t| <.0001 <.0001 Effect GROUP GROUP GROUP GROUP GROUP 2 3 4 5 _GROUP 1 1 1 1 Differences of Least Squares Means Standard Estimate Error DF t Value Pr > |t| 0.2343 0.2690 0.3182 0.6361 0.2381 0.2318 0.2321 0.2350 139 139 139 139 0.98 1.16 1.37 2.71 0.3266 0.2479 0.1725 0.0076 Adj ustment Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu Adj P 0.7202 0.5956 0.4501 0.0267 2154438587 T-007 P O I 9/022 F-437 o iO Q1-May-2002 02 :30pm F rom-CharI es R i var Labs Argus D i v - - FEMALES - ESTROUS STAGES 10:26 Monday, March 4, 2002 20 ANCOVA WITH DAY 64 BODY WEIGHT AS COVARIATE ------ SEX=F ------ The Mixed Procedure Model information Data Set Dependent Variable Covariance Structure Estimation Method Residual Variance Method Fixed Effects SE Method Degrees of Freedom Method WORK.AAA EST Diagonal REML Profile Model-Based Residual Class GROUP class Level Information Levels values 5 12345 Di mensions Covariance Parameters Columns in X Columns in Z subjects Max obs Per subject observations used Observations Not Used Total observations 1 7 0 1 300 149 151 300 2154438587 T-007 P 020/022 F-437 JLl 3!-May-iOQ2 02:30pm . From-Charles River Labs Argus 0 i v Effect GROUP GROUP GROUP FEMALES - ESTROUS STAGES 10:26 Monday, March 4, 2002 21 ANCOVA WITH DAY 64 BODY WEIGHT AS COVARIATE -- -------------- SEX=F-------------------------------------------- The Mixed Procedure Covariance Parameter Estimates cov parm Estimate Residual 0.7394 Fit statistics -2 Res Log Likelihood AIC (smaller is better) AICC (smaller is better) Bic (smaller is better) 390.6 392.6 392.6 395.6 - Type 3 Tests of Fixed Effects Effect Num Den DF DF F Value Pr > F BWT_64 GROUP 1 143 4 143 6.80 2.11 0.0101 0.0829 Least squares Means GROUP Estimate Standard Error DF t value Pr > |tj 1 4.6807 0.1571 143 29.80 <.0001 2 5.0123 0.1599 143 31.35 <.0001 3 5.1143 0.1600 143 31.96 <.0001 2154438587 T-007 P.021/022 F-437 J L iL wHWajr?tl02 02:30pm From-Char Ias R ivar Labs Argus Div... Effect GROUP GROUP GROUP GROUP FEMALES - ESTROUS STAGES 10:26 Monday, March 4, 2002 22 ANCOVA WITH DAY 64 BODY WEIGHT AS COVARTATE ------ --------- S E X = F ----------------------------------------------- The Mixed procedure Least Squares Means Effect GROUP Estimate standard Error DF t value Pr > jt| GROUP GROUP 4 5 5.045S 5.3143 0.1571 0.1604 143 143 32.12 33.13 <.0001 <.0001 Differences of Least Squares Means Standard GROUP _GROUP Estimate Error DF t value Pr > it| Adjustment Adj P 21 31 41 51 0.3316 0.4337 0.3648 0.6336 0.2244 0.2235 0.2220 0.2253 143 143 143 143 1.48 1.94 1.64 2.81 0.1416 0.0543 0.1025 0.0056 Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu 0.3873 0.1682 0.2948 0.0201 2154438587 T-007 P. 022/022 F-437 -3