Document 6w4bE9nXz0OEEN0mgVV6qxRa3
Ot-May-002 01 :2Spm F rom-CharI as River Labs Argus Div.
90S Shcehy Drive, BJdg.A Horsham. PA 19044 Telephone: (2IS) 443-8710 Tc/efax (2 IS) 442-3587
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ARGUS RESEARCH
Charles River Laboratories
Discovery and D evelopm ent 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
Ii RE: Protocol 418-020 Attached is the information on covariant analysis for study 418-020. Four different analyses were done for females and two for males. i
' Females: i 1. Sexual maturation w/ gestational age at sexual maturation as covariate : 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 I
Males: I 1. Sexual maturation w/ gestational age at sexual maturation as covariate
2. Sexual maturation w/ body weight at maturation as covariate
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 gestation 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.
QI-May-2002 .02:27pm, Prom-Char Ies River Labs Argus Div.
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I 1 It? r\1
U l V ^ U V < 3 I (Cll I U C W O O U O C U i w ^ V Q t u a v c p * c o u n c i l
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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.
Oi-May-2002 02:27pm From-Charles River Labs Araus Div. v
DAY OF m a t u r i t y
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 58 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)
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OlrMay-2002 02:27pm From-Chartes River Labs Argus Oiv-.-
ESTROUS 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 0.90S65 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
F1 2 3 4
5
30 29 30 , 28 : 28
4.66667 5.03448 5.03333 5.03571 5.39286
0.88409 0.90565 0.80872 1.10494 0.68526 *(1)
* Significantly different from Control group 1 (p<0.05) (1) Note: Results from Dunnett's test are reporteH without regard to the significance of the global treatment group effect in the overall ANCOVA (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 ANCOVA.
2154438587
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0!,-May-2002 02:28pm From-Charles River Labs Argus Div -
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 DAYJMAT 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
0!,-May-2002 02:29pm F rom-Char Ias River Labs Arus DiV.- -
DAY OF MATURITY
10:26 Monday, March 4, 2002 6
ANCOVA WITH BODY WEIGHT AS COVARIATE
,
Effect GROUP GROUP GROUP
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
1
34.8248
0.2698 282 129.07
2
35.7142
0.2753 282 129.73
3
34.1967
0.2747 282 124.50
Pr > !t | <.0001 <.0001 <.0001
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QWMay-2002 02:28pm F rom-Char Ies Rivar Labs Argus Div." '
DAY OF MATURITY
10:26 Monday, March 4, 2002 7
ANCOVA WITH BODY WEIGHT AS COVARIATE
Effect GROUP GROUP GROUP GROUP
The Mixed procedure
Least Squares Means
Effect
GROUP
Estimate
standard Error
DF t Value
GROUP GROUP
4 5
34.8710 36.4103
0.2744 282 127.07 0.2871 282 126.80
Differences of Least Squares Means
Standard
GROUP _GROUP Estimate
F.rror
OF t Value Pr > 11 1
21 31 41 51
0.8893 -0.6281 0.04619
1.5855
0.3859 0.3853 0.3850 0.3932
282 282 282 282
2.30 -1.63
0.12 4.03
0.0219 0.1042 0.9046 <.0001
Pr > |t| <0001 <.0001
Adjustment Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu
Adj p
0.0736 0.3006 0.9999 0.0003
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0l-May-2002 02:28pm From-CharIes R i var Labs Argus Div:-
DAY OF MATURITY
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 Residual
class Levet information
Class Levels values
GROUP - 5 1 2 3 4 5
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
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01-May-2002 02:28pm From-Char Ias River Labs Argus D iv 1-
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
Nm Den DF DF F Value Pr > F
BWT GROUP
1 273 4 273
53.38 17.77
<.0001 <.0001
Least Squares Means
GROUP 1 2 3
Estimate 48.5554 49.6708 49.0207
standard Error
0.3715 0.3527 0.3572
DF t Value . Pr > f1 1
273 130.70 273 140.84 273 137.24
<.OO01 <.0001 <.0001
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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
Esti mate
standard Error
OF t value Pr > |t|
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
GROUP
2 3 4 5
_GROUP 1 1 1 1
Estimate 1.1154 0.4653 1.0291 3.9469
Standard Error
0.5115 0.5164 0.5123 0.5203
OF t Value Pr > |t| Adjustment
273
2.18
0.0301 Dunnett-Hsu
273
0.90
0.3684 Dunnett-Hsu
273
2.01
0.0456 Dunnett-Hsu
273
7.59
<.0001 Dunnett-Hsu
Adj P 0.0964 0.7726 0.1409 <.0001
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2154430587
01 -May-2002 02:28pm From-Char Ias River Labs Argus D i W - '
DAY OF MATURITY
10:26 Monday, March 4, 2002
ANCOVA WITH GESTATIONAL AGE AS COVARIATE
--- -------------- S E X = F --------------------
11
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 1 2 3 4 5
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
Effect
GROUP GROUP
GROUP
DAY OF MATURITY
10:26 Monday, March 4, 2002 12
ANCOVA WITH GESTATIONAL AGE AS COVARIATE
---------------------------- S E X = 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
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 _GR0UP Estimate
Error
DF t Value Pr > 11 1 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
DAY OF MATURITY
10:26 Monday, March 4, 2002 14
ANCOVA WITH GESTATIONAL AGE AS COVARIATE
----- SEX=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
cl ass Level s 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
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 > |tI
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
DAY OF MATURITY
10:26 Monday, March 4, 2002 16
ANCOVA WITH GESTATIONAL AGE AS COVARIATE
Effect GROUP GROUP
GROUP 4 S
The Mixed procedure
Least Squares Means
Estimate
Standard Error
DF
49.8734 49.9531
0.08396 0.09000
268 268
t Value 594.04 555.05
Pr > |t| <.0001 <.0001
Effect GROUP GROUP GROUP GROUP
GROUP
2 3 4 5
_GR0UP 1 1 1 1
Differences of Least squares Means
Standard
Estimate
Error
DF t Value Pr > Jt|
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
FEMALES - ESTROUS STAGES
10:26 Monday
ANCOVA WITH ESTROUS AGE AS COVARrATE
--------------- 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 1234 S
Dimensions Covariance Parameters columns in x Columns in z subjects Max Obs Per subject Observations Used Observations Not Used Total observations
1 7 0 1 300 145 155 300
f e m a l e s - ESTROUS STAGES
10:26 Monday, March 4, 2002 18
ANCOVA WITH ESTROUS AGE AS COVARIATE
Effect
GROUP GROUP GROUP
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
E5T_AGE GROUP
1 139 4 139
4.04 1.90
Least Squares Means
0.0462 0.1131
GROUP
Estimate
standard Error
DF t Value
1
4.7408
0.1645
139
2S.83
2
4.9751
0.1657
139
30.03
3
5.0098,
0.1607
139
31.17
Pr > |t| <.0001 <.0001 <.0001
FEMALES - ESTROUS s t a g e s
10:26 Monday, March 4, 202 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 > 1tJ
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
females - estrous stages
10:26 Monday
ANCOVA WITH DAY 64 BODY WEIGHT AS COVARIATE '
----- ---------S E X = 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
Dimensions Covariance Parameters Columns in x Columns in Z subjects Max obs Per Subject Observations used observations Not Used Total observations
1 7 0 1 300 149 151 300
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 > |t|
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
Effect GROUP GROUP GROUP GROUP
FEMALES - ESTROUS STAGES
10:26 Monday, March 4, 2002 22
ANCOVA WITH DAY 64 BODY WEIGHT AS COVARIATE
------ -
S E X = F ------ ------------- ------------------------- --
The Mixed Procedure
Least squares Means
Effect
GROUP
Estimate
Standard Error
DF t Value Pr > 1t !
GROUP GROUP
4 5
5.0455 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 > |tl 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
