Document n9pLJOpY1kpXNJadwxMBqJoz2
Illl 0_-MaY-_.00Z 0_:ZSPm Fr0m-CharlRssivsrLabsAriusOiv. '
ill I
Ill I
!
9o5
A
PA19o44
;Te_hone: (21 S) 443-87f0
iTcie_Dc (215) 443-8587
2154438537
T-007 P.001 F-437
ARGUS RESEARCH Charleslver Laboratories
Discoveryand Development Services
From: Raymond G. York, Ph.D., D_T ,'_ _ /
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
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 2. Sexual maturation w/body weight at maturation as, covariate 3. Estrous stages w/day 64 postweaning body weight as covadate 4. Estrous stages w/gestational age at start of estrous testing as covariate
Males:
....
1. Sexual maturation w/gestational age at sexual maturation as covariate
2. Sexual maturation w/body weight at maturation as covariate
We 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 gestatiol_ tend to weigh more and appear to develop faster ;than pups of the same sex after21 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 idays old. If a test substance shortened pregaancy (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 _thetime spent in utero. By using each pup's age from time of conception to sexual imaturation as the covadant, the length of the stay in utero is factored out.
01".-Ma-200:O2 Z,:27pm, From-CharlesRiver Labs Arlus DJv.
I i lll:_ #'%1101_1_
UI %._UV_I
i_l I_C V_:IO l_lOl_i.I
iW I_QlUlI
215443858?
T-OOT P.OO2/O2Z F-43T
IJi I=-I-Di-4ll_-_i OWl_Ui
UU_I I i.4i I%_1 ls.l_rl
tL.4i
patency of Pl 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.
DAY OF _TURI1-Y
10:26 Monday, March 4, 2002 1
T
ANCOVA(DUNNETT'S TEST) WITH BODYWEIGHTAS COVARIATE
o
SEX GROUP
N
MEAN
SD
F
1
60 34.8833
2.07562
2
58 35.5345
2.26503
3
$8 34.0862
2.56347
4
58 34.8103
2.11475
5
54 36.7222
2.43726 *
I
M
1
52
48. 4615
2.13708
2
58 49.4655
2.68991
3
57 49.3333
3.42956
4
$8 49.7241
3.10513
5
54 52.3333
3.02817 *
Significantly different from Control group i (p_O.05)
ANCOVA (DUNNE1-F'STEST) 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 I (p>O.05)
N N
T
41
ESTROUSSTAGES
10:26 Monday, March 4, 2002 2
._
I
ANCOVA (DUNNETT'S TEST_ WITH DAY 64 BODY WEIGHT AS COVARIATE
SEX GROUP
N
MEAN
SD
F
i
30 4.66667
0.88409
2
29
5.03448
0.90565
3
30
S.03333
0.80872
4
30
5.03333
1.06620
5
30
5.40000 0.67466 *(1)
.. N
"
ANCOVA (DUNNETT'S TEST) WITH ESTROUS AGE AS COVARZATE
SEX GROUP
N
MEAN
SD
F
i
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 *(I)
significantly different from control group i (p_O.05)
_
(i) Note: Results from Dunnett's test are reported 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 signiflcant in the overall ANCOVA.
............... =_.-_=_ ..,_-._ ::__............................
DAY OF MATURTTY
10:26 Monday, March 4, 2002 5
ANCOVA WITH BODY WEIGHT AS COVARIATE
_.=._.___...... - SEX=p--._._..-,,._,.,_.._...........................................
The Nixed Procedure
Model Information
Data Set
Dependent Variable
Covariance Structure Estimation Method Residual Variance Method Fixed Effects SE l_ethod
Degrees of Freedom l_ethod
WORK.AAA
DAY_MAT
Diagonal REML Profile Model-Based
Residual
class GROUP
Class Level Information
Levels 5
values '1 2 3 4 5
Dimensions
Covariance Parameters
I
columns in x
7
columns in Z
0
subjects
I
Max obs Per subject
300
observations used
288
observations Not used
12
Totai observacions
300
P
-_ .
! N
,_
N
,_
.. N
E __ .<
qC
--_ --_
I c_
C_ N N
---4
DAY OF _tCTURITY
10:26 Monday, March 4, 2002 6
ANCOVA WITH BODY WEIGI{TAS COVARIATE
.............................. --'--- ==_=='- :'= - - =_'='=-- ....................
SEX=F-- ......................
c_
The Mixed Procedure
covar| ance Parameter Esti mates
Coy Parm
Estimate
o
I
Residual
4.3642
Fit Statistics
-2 Res Log Likelihood AIC (smaller is better)
1246.4 1248.4
AICC (smaller is better)
1248,4
_
BIC (smaller is better)
1252.0
Type 3 Tests of Fixed Et=fects
_=
NUm Den
_:
Effect
DF
DF F Value
Pr > F
':
B_rr GROUP
1
282
4
282
59.63 9.50
<. 0001 <. 0001
Least Squares Means
N
Standard
Effect
GROUP Estimate
Error
DF t Value
Pr > ltl
GROUP 1 GROUP 2 GROUP 3
34. 8248 35. 7142 34. 1967
0. 2698
28Z
129.07
0,2753
282
129.73
0. 2747
282
124.50
<. O001 <. 0001 <. 0001
--I I
c_ N N
,T'
c_
DAY OF MATURITY
10:26 I4onday,14arch4, 2002 7
.,
ANCOVA WITH BODY WEIGHT AS COVARIATE
............... _-'=....................................
-_.......... =-_ _-__".... "..... SEX=F....................................
c_
N
Effect GROUP
The Mixed Procedure
Least Squares Means
standard
Estimate
Error
DF
t Value
Pr > Itl
GROUP
4
34.8710
O.2744
282
127.07
<.0001
=_
GROUP 5
36.4103
0.2871
282
126.80
<,0001
Differences of Least Squares Means
standard
Effect GROUP _GROUP Estimate
Error
OF t Value Pr > ltl Adjustment
Adj P
E
GROUP 2
1
0.8893
O. 3859
282
2.30
0.0219 Dunnett-Hsu
O. 0736
_>
GROUP 3
1
-0.6281
O. 3853 282
-1.63
O. 1042 Dunnett-Hsu
O. 3006
"=
GROUP 4
1
O. 04619
O. 3850 282
O._L2 O. 9046 Dunnet t-Hsu
O. 9999
GROUP 5
1
1, 5855
0. 3932
282
4.03
<. 0001 Dunnet t-Hsu
0,0003
_
N "4
DAY OF MATURITY
20:26 Monday, March 4, 2002 8
T
ANCOVA WITIIBODY WEIGHT AS COVARIATE
...................................;_ .....--".........-'SEX=M................... N
The Mixed Procedure
.. N
Model Information
Data Set
WORK.AAA
Dependent Variable
DAY_MAT
covariance Structure
Diagona]
Estimation Method
REML
E
Residual variance Method
Profile
Fixed Effects SE Method
Model-Based
"_
Degrees of Freedom Method Residual
-C
class Leve_,I,nformation
class Levels values
GROUP
5 1234 5
_=
Dimensions
Covariance Parameters
i
;.
columns in x
7
columns in Z
0
subjects
I
Max obs Per subject
300
observations used
279
observations Not Used
21
Total observations
300
DAY OF MATURITY
10:26 Monday, Harch 4, 2002 9
._
ANCOVAWITH BODY WEIGHT AS COVARIATE
......................................................................................
SEX=M--___........
The Mixed Procedure
c_
u
Covariance Parameter Estimates
Coy Parm
Estimate
r
o
Residual
7,1683
Fit Statistics
-2 Res Log Likelihood
1344.4
AIC (smaller is better)
1346.4
AICC (smaller is better)
1346.4
_-
BIC (smaller is better)
1350.0
Type 3 Tests of Fixed Effects
"_
Num
Den
_
Effect
DF
DF F Value Pr > F
_.
Bid]"
1
273
53.38
<.0001
:
GROUP
4
273
17.77
<. 0001
Least Squares Means
N
standard
Effect GROUP Estimate
Error
DF t value . Pr > It I
GROUP 1 GROUP 2 GROUP 3
48.5554 49. 6708 49.0207
0. 3715
273
130.70
O.3527
273
140.84
0.3572
273
137.24
<. O00I <. 0001 <.0002
DAY OF MATURITY
10:26 Monday, March 4, 2002 10
ANCOVAWITH BODY%_EIGHTAS COVARIATE
--=
SEX=_
............
N
The Mixed Procedure
N
Least Squares Means
standard
Effect GROUP Estimate
Error
OF t Value Pr > Itl
I
GROUP 4 GROUP 5
49.5845 52,5024
0.3521 0,3651
273
140.84
273
143.81
<.0001 <.0001
Differences of Least Squares Means
Effect
GROUP _GROUP
Estimate
standard Error
OF t Value Pr Itl Adjustment
Adj P
GROUP 2
1
1.1154
0.5115
273
2.18
0.0301 Dunnett-Hsu
0.0964
_
GROUP 3
1
0.465.3
0,5164
273
0,90
0.3684 Dunnett-Hsu
0.7126
GROUP 4
1
1.0291
0.5123
273
2.01
0.0456 DunneCt-Hsu
0.1409
GROUP 5
1
3. 9469
O. 5203 273
7.59
<. 0001 Dunnett-Hsu
< .0001
_i
t_ z} co
--g m
o c_ c_ c_
T
...........
DAY OF MATURITY
10:26 Monday, t4arch4, 2002 II
.,--__
ANCOVAWITH GESTATIONAL AGE AS COVARIATE
l
SEX=F........
The Mixed Procedure
Model Information
Data Set
WORK.AAA
Dependent variable
DAY_MAT
Covariance Structure
Diagonal
_,
O
Estimation Method Residual Variance Method
REML Profile
Fixed Effects SE Method
Model-Based
Degrees of Freedom Method Residual
class Level Information
_"
c,ass Levels Values
GROUP
5 12 34 5
Dimensions
_
Covariance Parameters
I
..
columns in X
7
Columns in Z
0
subjects
1
14ax obs Per subject
300
Observati ons used
282
__
Observations Not used
18
Total observations
300
..............
EfFect GROUP GROUP GROUP
DAY OF NATURITY
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
O.2779
Fit Statistics
-2 Res Log LikeliBood
ATC (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
Hum
Den
DF
DF
F Value
Pr > F
GEST_AGE GROUP
i
276 4990.64 <.0001
4
276
3.21
0.0135
Least Squares Means
GROUP
i 2 3
Estimate
35.1763 35.3426 35. 0133
standard Error
0.06819 0.06928 O.07046
DF t Value
276
SIS.88
276
510.16
276
496.94
Pr > Itl
<.0001 <.0001 <. 0001
o__ _
..
B
_
"_ _:
DAY OF MATURZTY
10:26 Monday, March 4, 2002 13
ANCOVA WITH GESTATZONAL AGE AS COVARIATE
---
SEX=F
- ....
N
N
Effect
GROUP GROUP
GROUP
4 5
The Mixed Procedure Least squares Means
standard
Estimate
Error
DF
35.2004
0.07182
276
35.3069
0.07576
276
t value
490.11 466,01
Pr > Itl
<.0001 <.0001
Differences of Least Squares Means
Effect GROUP
_GROUP
Estimate
Standard Error
DF t Value Pr > Itl Adjustment
Adj P
GROUP 2
i
0.1663
0.09732
276
1.71
0.0887 Dunnett-Hsu
0.2639
>
GGRROOUUPP 34
11
0-0.0.12643002
00..0099784899
227766
-10..6274
00..08905863 DDuunnnneetttt--HHssuu
00..29891768
GROUP 5
1
0.1306
0.1027
276
1.27
0.2048 Dunnett-Hsu
0.5273
3
.........
DAY OF MATURITY
10:26 Monday, March 4, 2002 14
ANCOVA _ITH GESTATIONAL AGE AS COVARIATE
-_---: --'=-::-= ...... -'_--_--'--= - -'-=_ =- _ _-'== ....... SEX=_
..-..-.-.--_--
The Mixed Procedure
Model Information
Data Set
Dependent Vari able Covariance Structure Estimation Method Residua_ 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 2234 5
Dimensions
Covariance Parameters
I
columns in x
7
columns in Z
0
subjects
I
Max obs Per Subject
300
Observations Used
274
observations Not used
26
Total observations
300
T
"-c I
-
_
_= =< =r-
,._
............
DAY OF MATURITY
10:26 Monday, March 4, 2002 15
_-
ANCOVA WITH GESTATIONAL AGE AS COVARIATE
"_
- ...........
"-- -
SEX=M...........
The Mixed Procedure
Covariance Parameter Estimates
Coy Parm
Estimate
o
Residual
0.3806
Fi t Stati stics
_-
-2 Res Log Likelihood
529.4
_<
AIC (smaller is better}
531.4
AICC (smaller is better)
532.4
_-
BIC (smaller is better)
535.0
Type 3 Tests of Fixed Effects
"=
Num
Den
_'
Effect
DF
DF F Value Pr > F
GEST_AGE
I
268
5816.42
<. 0001
:
GROUP
4
268
1.80
0.1290
Least Squares Means
Effect
GROUP Estimate
standard Error
DF
. t value
Pr > Itl
=
GROUP 1 GROUP 2 GROUP 3
49. 7916
0.08731
268
570.26
49.9788
0.08129
268
614.84
49. 7086
O. 08186
268
607.21
< .O001 <. 0001 <. 0001
DAY OF MATURITY
10:26 Monday, March 4, 2002 16
T
ANCOVAWITH GESTATIONALAGE AS COVARIATE
.'
Effect
GROUP GROUP
GROUP
4 5
The Mixed Procedure
Least Squares Means
standard
Estimate
Error
DF
49.8734
0.08396
268
49.9531
0.09000
268
t Value
594.04 555.05
Pr > Itl
<.0001 <.0001
Differences of LeBst squares Means
Effect
GROUP _GROUP
Estimate
Standard Error
DF C Value Pr > Itl Adjustment
Ad_ P
GROUP 2
1
0.1872
0.1183
268
1.58
0.1147 Ounnett-Hsu
0.3183
GROUP 3
1
-0.08298
0.I190
268
-0.70
O.4861 Dunnett-Hsu
0.8903
GROUP 4
1
0.08185
0.1211
268
0.68
0.4995 Dunnett-Hsu
0.9003
_
GROUP 5
1
0.1615
0.1295
268
1.25
0.2135 Dunnett-Hsu
0.5288
FEMALES - ESTROUS STAGES
10:26 Monday, March 4, 2002 17
_--
ANCOVA WITH ESTROUS AGE AS COVARIATE
"..... ' ....
SEX=F...................................
o_
The Mixed Procedure Model Information
Data Set
Dependent Variable Covariance Structure Estimati on Method Residual Variance Method Fixed Effects SE Method
Degrees of Freedom Method
WORK,AAA
EST Diagonal REML Profile Model-Based
Residual
class Level Information
class Levels Values -=
GROUP
5 1234 5
Di mensi ons
'_
Covariance Parameters
I
columns in X
7
columns in z
0
Subjects
I
Max Obs Per subject
300
observations Used
145
Observations Not' used
155
Total observations
300
o N
'2
FEMALES- ESTROUSSTAGES
10:26 Monday, March 4, 2002 18
"T
ANCOVA WITH ESTROUS AGE AS COVARIATE
N
N
The Hixed 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
Num
Den
<
Effect
DF
DF F value Pr > F
"
EST_AGE GROUP
1
139
4
139
4.04 1.90
0.0462 O.1131
Least Squares Means
N
standard
Effect GROUP GROUP GROUP
GROUP 1 2 ]
Estimate 4.7408 4.9751
5.0098
Error 0.1645 0.1657 0.1607
DF t value Pr Itl
i39
28.83
<.0001
139
30.03
<.0001
139
31.17
<.0001
m
c_
.
-_i
Q N N
T
FEMALES - ESTROUS STAGES
10:26 Monday, March 4, 2002 19
ANCOVA WITH ESTROUS AGE AS COVARIATE
"_
I
_
.............SEX=F........
_-_
The Mixed Procedure
Least Squares Means 3
standard
Effect GROUP Estimate
Error
DF t value Pr > itl
!
GROUP
4
5.0590
0.1663
139
30.42
<.0001
GROUP 5
5.3769
0.1661
139
]2.37
<.0001
DifFerences of Least Squares Means
_
Effect
GROUP
_GROUP
Estimate
Standard Error
DF t value Pr > Itl Adjustment
Adj P
GROUP 2
1
0.2343
0.2381
139
0.98
0.3266 Dunnett-Hsu
0.7202
GROUP 3
1
0.2690
0.2318
139
1.16
0.2479 Dunnett-Hsu
0.5956
GROUP 4
1
0.3182
0.2321
139
1.37
0.1725 Dunnett-Hsu
0.4501
_
GROUP 5
1
0.6361
0.2350
139
2.71
0.0076 Dunnett-Hsu
0.0267
N
.................
FE&IALES- ESTROUS STAGES
10:26 Monday, March 4, 2002 20
-T
ANCOVA WZTH DAY 64 BODY WEIGHT AS COVARIATE
I
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 _4ethod
WORK.A_
EST Diagonal REML Profile Model-Based Residual
class Level Information
class GROUP
Levels 5
values 12345
-c
oimensions
"
Covariance Parameters
1
Columns in x
7
Columns in Z
0
Subjects
i
._
Max obs Per subject
300
Oobbsseerrvvaatitoinosns uNsoetdUsed
149 151
Total observations
300
o_
.....................
FB4ALES - 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
o
I
O
Resi dual
O. 7394
E
Fit Statistics
-2 Res Log Likelihood
390.6
_
AIC (smaller is better)
392.6
AICC (smaller is better)
392.6
BIC (smaller is better)
395.6
Type 3 Tests of Fixed Effects
Effect
Num
Den
DF
DF F Value Pr > F
BWT_64
I " 143
6.80 O.0101
"
GROUP
4
143
2.11
0. 0829
EfFect
GROUP GROUP GROUP
GROUP
i 2 3
Least squares Means
Standard
Estimate
Error
DF
4. 6807 5.0123 5.1143
O.i.57i
143
O,1599
143
O, 1600
143
t Value
29.80 31, 35 31.96
Pr > Itl
<. 000i <, 0001 <. 0001
!
-1_
FEMALES - ESTROUS STAGES
10:26 Monday, March 4, 2002 22
ANCOVA WZTH DAY 64 BODY WEIGHT AS COVARTATE
.....
"
_-
"" -_ ....... _---'--_
SEX=F ....
Effect
GROUP GROUP GROUP GROUP
EFfect
GROUP GROUP
GROUP
4 5
The Mixed Procedure
Least Squares Means
s_andard
Estimate
Error
DF
5.0455 5.3143
0.1571
143
0.1604
143
t Value
32.12 33.13
GROUP
2 3 4 5
_GROUP
1 1 1 1
Differences of Least Squares Means
Estimate
standard Error
DF t Value Pr > Ltl
0.3316 0.4337 0.3648 0.6336
0.2244
143
0.2235
143
0.2220
143
0.2253
143
1.48 1.94 1.64 2.81
0.1416 0.0543 0.1025 0.0056
Pr > It_ <.0001 <.0001
Adjustment Dunnett-HSU Dunnett-Hsu Dunnett-Hsu Dunnett-Hsu
Adj P
0.3873 0.1682 0.2948 0.0201
.-
I
__ -_
---I
