Document ID: EPA-HQ-OPPT-2003-0027-0006
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-08-06T04:00Z

Optimization
of
the
Sliced
Testis
Steriodogenesis
Assay
Unaudited
Final
Draft
Report
RTI
International
Laboratory
of
Reproductive
and
Endocrine
Toxicology
Chemistry
and
Life
Sciences
Group
Center
for
Life
Sciences
and
Toxicology
Post
Office
Box
12194
Research
Triangle
Park,
NC
27709­
2194
Battelle
Memorial
Institute
505
King
Avenue
Columbus,
OH
43201­
2693
Optimization
of
the
Sliced
Testis
Steriodogenesis
Assay
Unaudited
Draft
Final
Report
RTI
International
Laboratory
of
Reproductive
and
Endocrine
Toxicology
Chemistry
and
Life
Sciences
Group
Center
for
Life
Sciences
and
Toxicology
Post
Office
Box
12194
Research
Triangle
Park,
NC
27709­
2194
Battelle
Memorial
Institute
505
King
Avenue
Columbus,
OH
43201­
2693
EPA
Contract
No.:
68­
W­
01­
023
(
Battelle
Prime
Contractor,
WA
2­
27)
Contract
No.:
08055.001.020
Study
Code:
Rt02­
OPST
Master
Protocol
No.:
RTI­
870
Table
of
Contents
iii
TABLE
OF
CONTENTS
Section
Page
1.0
INTRODUCTION
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1­
1
1.1
Background
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1­
1
1.2
Objectives
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1­
1
2.0
MATERIALS
AND
CHEMICALS
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2­
1
2.1
Reagents
and
Solutions
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2­
1
2.2
Standards
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2­
1
3.0
METHOD
FOR
PROTOTYPICAL
ASSAY
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3­
1
4.0
METHODS
FOR
PHASE
I
 
PRELIMINARY
EXPERIMENTAL
PHASE
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4­
1
4.1
Testosterone
Radioimmunoassay
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4­
1
4.2
LDH
Spectrophotometric
Assay
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4­
2
4.3
Phase
1
Optimization
Experiments
for
Media
Type,
Gaseous
Atmosphere,
Rat
Age,
and
Storage
Container
Type
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4­
3
4.4
Phase
1
Experimental
Design
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4­
4
4.5
Phase
1
Data
Evaluation
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4­
6
5.0
RESULTS
AND
STATISTICAL
ANALYSES
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5­
1
5.1
Testosterone
RIA
Verification
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5­
1
5.2
LDH
Verification
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5­
2
5.3
Statistical
Analysis
of
the
Phase
I
Assay
Optimization
Experiment
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5­
2
5.4
Statistical
Analysis
Methods
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5­
4
6.0
STATISTICAL
ANALYSIS
OF
THE
PHASE
IB
ASSAY
OPTIMIZATION
EXPERIMENT
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6­
1
6.1
Objectives
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6­
1
6.2
Data
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6­
1
6.3
Statistical
Analysis
Methods
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6­
1
7.0
DISCUSSION
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7­
1
8.0
CONCLUSIONS
FOR
PHASE
I
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8­
1
9.0
INCUBATION
EXPERIMENT
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9­
1
9.1
Statistical
Analysis
of
Phase
II
Incubation
Optimization
Experiment
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9­
2
10
TESTES
PREPARATION
EXPERIMENT
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10­
1
10.1
Statistical
Analysis
of
Phase
II
Testis
Preparation
Optimization
Experiment
10­
1
11
EQUILIBRATION
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11­
1
12
SAMPLING
TIMES
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12­
1
13
INDUCED
PARENCHYMAL
FRAGEMENT
INJURY
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13­
1
Table
of
Contents
iv
LIST
OF
FIGURES
No.
Page
1
Sliced
Testis
Steroidogenesis
Assay
Experimental
Design
Organization
Diagram
.
.
.
1­
2
2
Technical
Flow
Illustration
of
the
Sliced
Testis
Steroidogenesis
Assay
.
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3­
2
LIST
OF
TABLES
No.
Page
1
Summary
of
Experimental
Factors
for
Phase
1
Optimization
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4­
5
2
Factorial
Test
Conditions
for
Phase
1
Optimization
Experiment
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4­
5
3
Values
of
Standards
on
Testosterone
RIA
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5­
1
4
Testosterone
RIA
Intra­
Assay
CV
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5­
2
5
Testosterone
RIA
Percent
Recovery
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5­
2
6
Testosterone
RIA
Parallelism
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5­
2
7
Factor
Levels
in
the
Phase
I
Assay
Optimization
Experiment
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5­
6
8
Data
Listing
for
Samples
Without
hCG
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5­
7
9
Data
Listing
for
Samples
With
hCG
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5­
8
10
Summary
of
Data
 
Original
Scale,
Without
Scale,
Without
hCG
.
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5­
11
11
Summary
of
Data
 
Log
Scale,
Without
hCG
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5­
12
12
Summary
of
Data
 
Original
Scale,
With
hCG
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5­
15
13
Summary
of
Data
 
Log
Scale,
With
hCG
.
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5­
16
14
Summary
of
Statistical
Analysis
of
Baseline
Data
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5­
17
15
Summary
of
ANOCOVA
Results
for
Individual­
Hour
Original­
Scale
Models
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5­
18
16
Adjusted
Mean
Levels
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5­
19
17
Summary
of
ANOCOVA
Results
for
Individual­
Hour
Log­
Scale
Models
.
.
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5­
23
18
Adjusted
Mean
Levels
Based
on
Log­
Scale
Models
.
.
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5­
24
19
Least
Squares
Means
for
Reduced
Log­
scale
Model:
Without
hCG
.
.
.
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.
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5­
28
20
Least
Squares
Means
for
Reduced
Original­
scale
Model:
Without
hCG
.
.
.
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.
.
5­
28
21
Least
Squares
Means
for
Reduced
Log­
scale
Model:
With
hCG
.
.
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5­
29
22
Least
Squares
Means
for
Reduced
Original­
scale
Model:
With
hCG
.
.
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5­
29
23
Analysis
of
Differences
in
Levels
for
With
and
Without
hCG
Stimulation
Original­
scale
Models
.
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5­
30
24
Adjusted
Mean
Differences
(
With­
Without
hCG),
Based
on
Original­
Scale
Models
5­
30
25
Analysis
of
Differences
in
Levels
for
With
and
Without
hCG
Stimulation:
Log­
Scale
Models
.
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5­
32
26
Adjusted
Mean
Differences
(
With­
Without
hCG),
Based
on
Log­
Scale
Models
.
.
.
.
5­
32
27
Phase
IB
Data:
Sample
Without
hCG
.
.
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.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
6­
5
28
Phase
IB
Data:
Samples
With
hCG
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
6­
6
29
Summary
of
Data
 
Original
Scale,
Without
hCG
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
6­
7
30
Summary
of
Data
­
Log
Scale,
Without
hCG
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
6­
8
31
Summary
of
Data
­
Original
Scale,
With
hCG
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
6­
9
Table
of
Contents
v
32
Summary
of
Data
 
Log
Scale,
With
hCG
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
6­
10
33
Summary
of
Results
by
Sample
Condition
 
Original
Scale,
Without
hCG
.
.
.
.
.
.
.
6­
11
34
Summary
of
Results
by
Sample
Condition
 
Log
Scale,
Without
hCG
.
.
.
.
.
.
.
.
.
.
.
6­
12
35
Summary
of
Results
by
Sample
Condition
 
Original
Scale,
With
hCG
.
.
.
.
.
.
.
.
.
.
6­
13
36
Summary
of
Results
by
Sample
Condition
 
Log
Scale,
With
hCG
.
.
.
.
.
.
.
.
.
.
.
.
.
6­
14
37
Incubation
Factor
Settings
and
Coded
Values
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9­
3
38
Data
Listing
for
Incubation
Experiment
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9­
6
39
Data
Listing
of
Coded
Factor
Values
and
Logarithm
of
Testosterone
Concentrations
9­
13
40
ANOVA
Results
for
PROC
RSREG
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9­
19
41
Response
Surface
Regression
Results
for
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9­
20
42
Response
Surface
Regression
Results
for
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9­
21
43
Maximum
Values
for
Each
Time
Point
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9­
23
44
Predicted
Values
for
Specified
Optimum
Factor
Combinations
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9­
24
45
ANOVA
Results
for
PROC
RSREG
With
the
Baseline
Concentration
Removed
from
Model
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9­
25
46
Response
Surface
Regression
Results
for
No
hCG
Stimulation
with
Baseline
Concentration
Removed
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9­
26
47
Response
Surface
Regression
Results
for
hCG
Stimuation
with
Baseline
Concentration
Removed
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9­
28
48
Maximum
Values
for
Models
with
Baseline
Concentration
Removed
.
.
.
.
.
.
.
.
.
.
.
9­
29
49
Predicted
Values
for
Specified
Optimum
Factor
Combinations
for
Models
with
Baseline
Concentration
Removed
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9­
32
50
Testis
Preparation
Factor
Settings
and
Coded
Values
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10­
4
51
Data
Listing
for
Incubation
Experiment
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10­
5
52
Data
Listing
of
Coded
Factor
Values
and
Logarithm
of
Testosterone
Concentrations
10­
7
53
ANOVA
Results
for
PROC
RSREG
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10­
11
54
Response
Surface
Regression
Results
for
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10­
12
55
Response
Surface
Regression
Results
for
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10­
13
56
Maximum
Values
for
Each
Time
Point
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10­
15
57
Predicted
Values
for
Specified
Optimum
Factor
Combinations
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10­
17
58
ANOVA
Results
for
PROC
RSREG
with
the
Results
Concentration
Removed
from
Model
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10­
18
59
Response
Surface
Regression
Results
for
No
hCG
Stimulation
with
Baseline
Concentration
Removed
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10­
19
60
Response
Surface
Regression
Results
for
hCG
Stimulation
with
Baseline
Concentration
Removed
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10­
20
61
Maximum
Values
for
Models
with
Baseline
Concentration
Removed
.
.
.
.
.
.
.
.
.
.
10­
21
62
Predicted
Values
for
Specified
Optimum
Factor
Combinations
for
Models
with
Baseline
Concentration
Removed
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10­
23
63
Equilibration
Data
for
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
2
64
Equilibration
Data
for
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
5
Table
of
Contents
vi
65
Means
of
LDH
Concentration
(
mu/
mg)
by
Equilibration
Time
(
minutes)
HCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
8
66
Standard
Deviations
of
LDH
Concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
8
67
Samples
Sizes
of
LDH
Concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
8
68
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
9
69
Means
of
LDH
Concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
10
70
Standard
Deviations
of
LDH
Concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
11
71
Sample
Sizes
of
LDH
Concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
12
72
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
12
73
Means
of
Testosterone
Concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
12
74
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
13
75
Sample
Sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Equilibration
Time
(
Minutes)
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
13
76
Coeff
of
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
Equilibrarion
Time
(
Minutes)
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
13
77
Means
of
Testosterone
Concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
14
78
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
14
79
Sample
Sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Equilibration
Time
(
Minutes)
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
14
80
Coeff
of
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
15
81
Summary
of
Equilibration
Experiment:
Testosterone
Concentration
(
ng/
mg)
by
Time
Pont
With
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
26
82
Summary
of
Equilibration
Experiment:
Testosterone
Concentration
(
ng/
mg)
by
Time
Point
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
26
83
Summary
of
Equilibration
Experiment:
LDH
Concentration
(
mU/
mg)
by
Time
Point
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
27
84
Summary
of
Equilibration
Experiment:
LDH
Concentration
(
mU/
mg)
by
Time
Point
Without
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11­
27
85
Sampling
Time
Factors
Data
for
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
12­
1
86
Sampling
Time
Factors
Data
for
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
12­
4
87
Means
of
LDH
Concentration
(
mu/
mg)
by
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
12­
6
88
Standard
Deviations
of
LDH
Concentration
(
mU/
mg)
by
hCG
Stimulation
.
.
.
.
.
.
.
12­
6
89
Sample
Sizes
of
LDH
Concentration
(
mU/
mg)
by
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
12­
7
90
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
hCG
Stimulation
.
.
.
.
.
.
.
.
.
12­
7
Table
of
Contents
vii
91
Means
of
Testosterone
Concentration
(
ng/
mg)
by
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
12­
8
92
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
HCG
Stimulation
.
.
12­
8
93
Sample
Sizes
of
Testosterone
Concentration
(
ng/
mg)
by
hCG
Stimulation
.
.
.
.
.
.
.
.
12­
9
94
Coeff
of
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
hCG
Stimulation
.
.
.
.
12­
9
95
Summary
of
Sampling
Times
Experiment:
Testosterone
Concentration
(
ng/
mg)
by
Sampling
Time
Points
over
24
Hours
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
12­
14
96
Summary
of
Sampling
Times
Experiment:
LDH
Concentration
(
mU/
mg)
by
Sampling
Time
Points
over
24
Hours
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
12­
14
97
Induced
Parenchymal
Fragment
Injury
Data
for
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
13­
2
98
Induced
Parenchymal
Fragment
Injury
Data
for
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
7
99
Means
of
LDH
Concentration
(
mU/
mg)
by
Type
of
Innjury
Induced
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
8
100
Standard
Deviations
of
LDH
Concentration
(
mU/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
9
101
Sample
Sizes
of
LDH
Concentration
(
mU/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
9
102
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
9
103
Means
of
LDH
Concentration
(
mU/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
10
104
Standard
Deviations
of
LDH
Concentration
(
mU/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
10
105
Sample
Sizes
of
LDH
Concentration
(
mU/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
10
106
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
11
107
Means
of
Testosterone
Concentrationi
(
ng/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
12
108
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
12
109
Sample
Sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
12
110
Coeff
of
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
13
111
Means
of
Testosterone
Concentration
(
ng/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
13
112
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
13
113
Sample
Sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
14
114
Coeff
with
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
14
115
Summary
of
Injury
Experiment:
Testosterone
Concentrations
(
ng/
mg)
by
Sampling
Time
Points
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
30
116
Summary
of
Injury
Experiment:
Testosterone
Concentrations
(
ng/
mg)
by
Sampling
Time
Points
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
30
Table
of
Contents
viii
117
Summary
of
Injury
Experiment:
LDH
Concentrations
(
mU/
mg)
by
Sampling
Time
Points
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
31
118
Injury
Experiment:
LDH
Concentrations
(
mU/
mg)
by
Sampling
Time
Points
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
31
119
Vehicle
and
Vehicle
Concentrations
Data
for
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
32
120
Vehicle
and
Vehicle
Concentrations
Data
for
hCG
Stimualtion
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
35
121
Means
of
LDH
Concentration
(
mUmg)
by
Vehicle
Type
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
37
122
Standard
Deviations
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Type
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
37
123
Sample
Sizes
by
LDH
Concentration
(
mU/
mg)
by
Vehicle
Type
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
37
124
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Type
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
37
125
Means
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
38
126
Standard
Deviations
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
38
127
Sample
Sizes
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
38
128
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
38
129
Means
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Type
with
hCG
Stimulation
.
.
13­
39
130
Standard
Deviationss
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Type
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
39
131
Sample
Sizes
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Type
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
39
132
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Type
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
39
133
Means
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
40
134
Standard
Deviations
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
40
135
Sample
Sizes
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
40
136
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
40
137
Means
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
41
138
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
41
139
Sample
Sizes
by
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
41
140
Coeff
of
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
41
141
Means
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
42
Table
of
Contents
ix
142
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
42
143
Sample
Sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
42
144
Sample
Sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
no
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
42
145
Means
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
43
146
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
43
147
Sample
Sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
43
148
Coeff
of
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
43
149
Means
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
44
150
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
44
151
Sample
Sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
44
152
Coeff
of
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
44
153
Summary
of
Vehicle:
LDH
Concentrations
(
mU/
mg)
by
Vehicle
Type
.
.
.
.
.
.
.
.
.
13­
108
154
Summary
of
Vehicle
Experiment:
LDH
Concentrations
(
mU/
mg)
by
Vehicle
Type
13­
108
155
Summary
of
Vehicle
Experiment:
LDH
Concentrations
(
mU/
mg)
by
Vehicle
Concentration
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
110
156
Summary
of
Vehicle
Experiment:
LDH
Concentrations
(
mU/
mg)
by
Vehicle
Concentration
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
110
157
Summary
of
Vehicle
Experiment:
Testosterone
Concentrations
(
ng/
mg)
by
Vehicle
Type
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
111
158
Summary
of
Vehicle
Experiment:
Testosterone
Concentrations
(
ng/
mg)
by
Vehicle
Type
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
111
159
Summary
of
Vehicle
Experiment:
Testosterone
Concentrations
(
ng/
mg)
by
Vehicle
Concentration
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
112
160
Summary
of
Vehicle
Experiment:
Testosterone
Concentrations
(
ng/
mg)
by
Vehicle
Concentration
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13­
112
Section
1.0
Introduction
1­
1
1.0
INTRODUCTION
1.1
Background
In
1996,
the
Food
Quality
Protection
Act
(
FQPA)
amendments
were
enacted
by
Congress
to
authorize
the
Environmental
Protection
Agency
(
EPA)
to
implement
an
Endocrine
Disruptor
Screening
Program
(
EDSP)
on
pesticides
and
other
substances
found
in
food
or
water
sources
for
endocrine
effects
in
humans
(
FQPA,
1996).
In
this
program,
comprehensive
toxicological
and
ecotoxicological
screens
and
tests
are
being
developed
for
identifying
and
characterizing
the
endocrine
effects
of
various
environmental
contaminants,
industrial
substances,
and
pesticides.
A
two­
tiered
approach
will
be
utilized.
Tier
1
employs
a
combination
of
in
vivo
and
in
vitro
screens,
and
Tier
2
involves
in
vivo
testing
methods
using
two­
generation
reproductive
studies.
A
steroidogenesis
assay
is
proposed
as
one
of
the
Tier
1
screening
battery
assays.

A
detailed
review
paper
(
DRP)
about
steroidogenesis
was
prepared.
The
DRP:
(
1)
summarized
the
state
of
the
science
of
the
in
vivo,
ex
vivo,
and
in
vitro
methodologies
available
for
measuring
gonadal
steroidogenesis;
(
2)
for
each
methodology,
presented
a
review
of
the
individual
assays
and
representative
data
generated
by
investigators
who
used
the
assay
to
evaluate
a
substance
for
steroidogenic­
altering
activity;
(
3)
provided
an
evaluation
of
the
various
methodologies
and
the
assays
as
tools
for
screening
substances
with
suspected
steroidogenic
activity;
(
4)
recommended
a
particular
screening
method
and
assay
as
a
screening
tool;
and
(
5)
described
the
strengths,
weaknesses,
and
implications
for
further
research
associated
with
the
recommended
screening
assay.

The
in
vitro
sliced
testis
steroidogenesis
assay
was
selected
as
the
most
promising
screening
tool
for
identifying
substances
with
steroidogenic­
altering
activity.
The
sliced
testis
assay
was
recommended
because
it
can
be
conducted
at
a
minimal
cost,
quickly,
and
simply
with
standard
laboratory
equipment
and
basic
laboratory
training;
the
preparation
is
stable,
and
the
parenchyma
remains
viable
over
a
sufficient
time
period
to
measure
changes
in
end­
product
hormone
production;
the
assay
is
relatively
sensitive
and
specific;
the
assay
uses
parenchyma
that
maintains
the
cytoarchitecture
of
the
organ;
the
assay
uses
a
reduced
number
of
animals
(
up
to
quartered
testis
slices);
the
assay
should
be
relatively
easy
to
standardize
(
by
optimization);
and
the
assay
has
a
well­
defined
endpoint
in
testosterone
and,
if
desired,
can
be
modified
to
include
additional
intermediate
hormonal
endpoints.

Although
a
promising
tool,
the
sliced
testis
assay
remains
to
be
fully
tested
as
an
assay
that
can
meet
all
the
demands
of
an
endocrine
disruptor
screening
tool.
Concerns
raised
by
the
EPA
and
Endocrine
Disruptor
Methods
Validation
Subcommittee
(
EDMVS)
suggested
that
experiments
be
conducted
to
ensure
the
optimization
of
the
assay
prior
to
more
rigorous
Section
1.0
Introduction
1­
3
optimization
was
to
describe
in
detail
the
experiments
designed
to
provide
data
for
setting
in
place
the
procedures
and
parameters
that
will
optimize
the
performance
of
this
assay.

1.2
Objectives
The
study
plan
for
testing
the
factors
described
in
the
previous
section
involved
two
phases
and
utilized
single
factor
and
factorial
experimental
designs.
A
diagram
of
the
experimental
design
for
this
study
is
illustrated
in
Figure
1.

The
study
was
divided
into
Phases
I
and
II.
In
Phase
I,
the
Preliminary
Experimental
Phase,
the
analytical
assays
planned
for
use
were
verified,
storage
containers
and
lengths
of
storage
were
selected,
and
three
factors
that
may
affect
the
performance
of
the
assay
were
tested.
The
reasoning
for
including
these
3
factors
in
the
preliminary
phase
was
to
establish
early
whether
a
given
level
of
each
factor
was
going
to
affect
assay
performance.
Although
any
factor
listed
in
the
study
plan
could
have
been
rationalized
to
fit
such
a
criteria,
inclusion
in
the
preliminary
phase
also
required
that
the
factor
be
unlikely
to
have
an
interaction
or,
at
best,
a
minimal
interaction
with
another
experimental
factor.
Although
subjective,
these
factors
were
believed
to
best
fit
these
criteria.
Furthermore,
it
was
believed
essential
to
establish
the
optimal
level
for
each
of
these
factors
before
proceeding
with
the
factorial
experiments
since
an
effect
of
one
of
these
would
require
additional
verification
experiments
after
sensitivity
analysis.
Finally,
by
establishing
the
media
type
early
on
in
the
experiment,
the
analytical
assay
verification
testing
(
Phase
I
)
and
Optimization
of
Sample
Testing
(
Phase
II)
could
be
initiated
earlier
in
the
study
milestone
schedule.
Section
1.0
Introduction
1­
3
The
reasoning
for
including
these
3
factors
in
the
preliminary
phase
was
to
establish
early
whether
a
given
level
of
each
factor
was
going
to
affect
assay
performance.
Although
any
factor
listed
in
the
study
plan
could
have
been
rationalized
to
fit
such
a
criteria,
inclusion
in
the
preliminary
phase
also
required
that
the
factor
be
unlikely
to
have
an
interaction
or,
at
best,
a
minimal
interaction
with
another
experimental
factor.
Although
subjective,
these
factors
were
believed
to
best
fit
these
criteria.
Furthermore,
it
was
believed
essential
to
establish
the
optimal
level
for
each
of
these
factors
before
proceeding
with
the
factorial
experiments
since
an
effect
of
one
of
these
would
require
additional
verification
experiments
after
sensitivity
analysis.
Finally,
by
establishing
the
media
type
early
on
in
the
experiment,
the
analytical
assay
verification
testing
(
Phase
I
)
and
Optimization
of
Sample
Testing
(
Phase
II)
could
be
initiated
earlier
in
the
study
milestone
schedule.
Section
2.0
Materials
and
Chemicals
2­
1
2.0
MATERIALS
AND
CHEMICALS
2.1
Reagents
and
Solutions
No
test
substances
were
used
in
this
study.
The
chemicals
used
in
this
study
were
used
to
prepare
reagents
and
solutions
for
the
assay.
All
reagents
and
solutions
had
appropriate
information
documented,
which
included
the
identity,
concentration,
storage
requirements,
and
expiration
date.
Reagents
and
solutions
were
prepared
according
to
Standard
Operating
Procedures.

2.2
Standards
Verification
of
the
analytical
assays
required
testosterone
for
the
radioimmunoassay
(
RIA)
method
and
lactic
dehydrogenase
(
LDH)
for
the
spectrophotometric
assay.
In
addition,
hCG
was
used
as
a
stimulant
of
the
sliced
testis
bioassay.
These
substances
were
considered
standards.

2.2.1
Testosterone
Chemical
Name:
Testosterone
CAS
No.:
58220
Molecular
Weight:
288.4
Solubility:
Clear
colorless
to
very
faint
yellow
solution
at
100
mg/
mL
in
chloroform
Supplier:
Sigma­
Aldrich
Chemical
Company
Lot
No.:
6384­
70­
8
Purity:
NLT
98%
Storage
Conditions:
2­
year
shelf
life
A
safety
protocol
exists
for
the
use
of
the
radioactive
form
of
testosterone.

2.2.2
Human
Chorionic
Gonadotropin
(
hCG)

Chemical
Name:
hCG
CAS
No.:
9002­
61­
3
Molecular
Weight:
36,700
Solubility:
H
2
O
Supplier:
Calbiochem
Lot
No.:
B11174,
B51120
Section
2.0
Materials
and
Chemicals
2­
2
Purity:
Approx.
30%
hCG
by
weight
Storage
Conditions:
Freezer
(­
20
°
C).
Following
reconstitution,
aliquot
and
freeze
(­
20
°
C).
Stable
for
2
years
as
supplied.

2.2.3
LDH
Chemical
Name:
Lactate
Dehydrogenase
CAS
No.:
EC
1.1.127
Source:
Rabbit
muscle
Solubility:
Supplier:
Sigma­
Aldrich
Chemical
Company
Lot
No.:
99H7480
Purity:
NLT
%
Storage
Conditions:
2­
8
°
C;
1­
year
shelf
life
Section
3.0
Methods
for
Prototypical
Assay
3­
1
3.0
METHOD
FOR
PROTOTYPICAL
ASSAY
The
prototypical
assay
describes
the
sliced
testis
assay
using
the
conditions
that
are
believed
to
be
the
starting
conditions
of
the
assay.
This
section
does
not
describe
any
experiments
to
be
conducted;
rather,
it
describes
the
settings
of
all
factors,
except
for
the
one
that
is
being
tested
in
order
to
perform
the
optimization
experiments
described
in
the
following
sections.
A
run
is
defined
as
a
single
sample
vessel
with
assay
components.

The
sliced
testis
assay
prototype
uses
a
15­
week­
old
male
Sprague­
Dawley
rat,
which
is
euthanized
and
its
testes
removed.
The
testes
are
decapsulated,
weighed,
and
placed
in
cold
(
4
°
C)
DPBS.
The
media
is
medium­
199
(
Gibco)
that
has
added
0.71
g
sodium
bicarbonate,
2.1
g
HEPES,
1.0
g/
L
BSA,
and
0.025
g/
L
soybean
trypsin
inhibitor,
and
is
adjusted
to
a
pH
of
7.4.
The
time
from
removal
to
the
time
of
slicing
is
held
to
under
1
hour.
Each
testis
is
sliced
along
the
longitudinal
axis
into
approximately
4
slices.
Each
slice
is
placed
in
a
20
mL
borosilicate
scintillation
vial
(
loosely
capped)
that
contains
5
mL
of
media
(
Figure
2).
The
vials
containing
the
testicular
sections
and
media
are
incubated
at
34
°
C
on
a
shaker
(
at
135
rpm)
in
5%
CO
2/
95%
air.
After
the
first
period
of
incubation,
the
media
is
removed
and
discarded.
Fresh
media
(
5
mL)
is
added
to
the
vial
and
an
aliquot
of
media
(
0.5
mL)
is
collected.
The
sample
is
centrifuged
and
the
supernatant
transferred
to
a
labeled
vial
and
stored
at
approximately
­
70
°
C
in
a
siliconized
plastic
container
for
no
more
than
1
month.
This
sample
is
the
baseline
sample.
Next,
1/
2
of
the
vials
are
challenged
with
a
stimulant
(
e.
g.,
hCG)
and
the
other
1/
2
are
not.
The
final
hCG
concentration
is
0.1
IU/
mL.
Additional
media
samples
are
collected
from
the
vials
at
1,
2,
3,
and
4
hours
postchallenge.
These
media
samples
are
also
stored
frozen
for
later
analysis.
Samples
are
analyzed
for
testosterone
using
an
RIA
method.
All
samples
for
a
given
day's
set
of
runs
should
be
analyzed
in
the
same
testosterone
RIA.
Section
3.0
Methods
for
Prototypical
Assay
3­
2
Figure
2.
Technical
Flow
Illustration
of
the
Sliced
Testis
Steroidogenesis
Assay
Section
4.0
Methods
for
Phase
1
 
Preliminary
Experimental
Phase
4­
1
4.0
METHODS
FOR
PHASE
I
 
PRELIMINARY
EXPERIMENTAL
PHASE
Phase
I
is
comprised
of
the
verification
experiments
for
the
2
analytical
assay
methods
(
testosterone
RIA
and
LDH
spectrophotometry),
the
determination
of
the
preferred
storage
container
and
storage
length,
and
the
optimization
experiments
for
the
3
factors
to
be
tested
(
media
type,
incubation
atmosphere,
and
animal
age).
The
testosterone
RIA
method
was
verified
prior
to
conducting
any
optimization
assay
since
it
was
needed
as
an
endpoint
to
determine
optimization
of
the
sliced
testes
assay.
The
storage
container
determination
was
also
performed
before
any
further
optimization
assays
were
run.
The
determination
was
made
using
standards
of
testosterone
in
the
prototypical
media,
modified
M­
199
without
phenol
red.

4.1
Testosterone
Radioimmunoassay
The
objective
of
this
experiment
was
to
verify
that
testosterone
can
be
measured
in
the
sliced
testis
assay
media.
A
RIA
commercial
kit
(
Diagnostic
Products
Corporation,
Los
Angeles,
CA),
that
utilizes
125I­
testosterone
and
a
testosterone­
specific
antibody
affixed
to
polypropylene
culture
tubes,
was
used
to
measure
testosterone.
The
assay
was
verified
in
all
3
of
the
potential
assay
media.

Testosterone
(
Sigma,
St.
Louis,
MO;
T­
1500)
was
used
for
preparing
the
standard
curve
and
was
stored
desiccated
at
room
temperature
in
the
RTI
vault.
A
standard
was
prepared
in
ethanol
(
0.1
mg/
mL).
Up
to
an
8­
point
standard
curve
(
but
not
less
than
a
4­
point
standard
curve)
was
prepared
using
standards
with
concentrations
of
0.07,
0.16,
0.41,
1.02
,
2.56,
6.4,
16,
and
40
ng/
ml
in
PBS
gel
buffer
(
0.1
M
phosphate
buffered
saline
with
0.1%
[
w/
v]
sodium
azide
and
0.1%
[
w/
v]
gelatin,
pH
7.4).
In
addition,
procedural
controls
were
included
in
each
run.
The
standard
curve
points
and
the
procedural
controls
were
prepared
in
quadruplicate;
the
bioassay
unknowns
and
the
internal
standards
(
see
below)
were
prepared
in
duplicate.
The
volume
of
all
standards
and
controls
(
including
bioassay
unknowns)
were
adjusted
to
50
µ
L
by
adding
the
PBS­
Gel
Buffer.
Next,
1
mL
125I­
testosterone
was
added
to
each
antibody­
coated
tube
and
mixed
(
vortexed).
The
tubes
were
incubated
in
a
37
°
C
water
bath
for
3
hours,
during
which
time
testosterone,
whether
labeled
or
unlabeled,
competed
for
testosterone­
specific
antibody
binding
sites.
At
the
end
of
the
incubation
period,
the
free
(
unbound)
testosterone
in
the
supernatant
fluid
of
all
tubes
was
aspirated,
and
tubes
were
wiped
clean
of
fluid.
The
bound
testosterone
was
counted
in
a
gamma
counter
for
1
minute.
The
concentration
of
testosterone
was
estimated
against
the
standard
curve.
Values
were
reported
as
a
mean
concentration
(
ng/
mL)
of
duplicate
analyses,
unless
only
a
single
value
was
available.
Verification
of
the
testosterone
assay
involved
preparation
of
internal
standards
(
at
least
3)
using
spiked
media,
with
concentrations
ranging
from
12.5
to
500
ng/
mL.
Each
concentration
was
run
at
each
of
3
volumes
 
10,
25,
and
50
°
L,
to
check
for
parallelism,
and
each
sample
was
adjusted
to
50
°
L
by
adding
the
PBS
gel
buffer.
The
Section
4.0
Methods
for
Phase
1
 
Preliminary
Experimental
Phase
4­
2
low
and
high
standards
were
analyzed
in
at
least
duplicate.
Verification
was
based
on
results
determined
for
accuracy,
precision,
specificity,
and
linearity.
Accuracy
is
expressed
as
the
relative
error,
which
was
determined
by
comparing
the
measured
to
the
target
concentration.
Relative
errors
within
15%
were
acceptable.
Precision
is
expressed
as
the
relative
standard
deviation
(
RSD)
or
coefficient
of
variance
(
CV),
which
was
determined
by
calculating
the
mean
and
standard
deviation
(
SD)
of
the
low
and
high
standards.
A
RSD
or
CV
within
15%
was
acceptable.
The
sensitivity
was
acceptable
if
the
means
of
the
blanks
and
low
standards
were
significantly
different
at
the
5%
significance
level.
Linear
determinations
of
the
standard
curve
line
were
made
and
a
correlation
coefficient
(
r)
calculated.
An
r
of
0.90
or
greater
was
considered
acceptable.

Inter­
and
intra­
assay
variability
was
determined.
The
intra­
assay
variability
was
determined
from
the
precision
results
calculated
from
the
results
obtained
by
measuring
the
low
and
high
standards
in
triplicate.
The
inter­
assay
variability
was
determined
by
repeated
analyses
of
the
standards
by
generating
a
standard
curve
on
3
different
days.

4.2
LDH
Spectrophotometric
Assay
The
objective
of
this
experiment
was
to
verify
that
LDH
can
be
measured
in
the
sliced
testis
assay
media.
The
LDH
assay
measures
the
rate
at
which
NADH
is
formed
(
when
NAD
is
reduced)
when
it
catalyzes
the
oxidation
of
lactate
to
pyruvate.
NADH
is
measured
at
340
nm
using
a
kinetic­
spectrophotometric
method.
The
assay
and
samples
are
temperature
sensitive.
The
samples
should
not
be
refrigerated
or
frozen.
The
assay
has
been
characterized
for
assay
conditions
at
37oC.
LDH
activity
is
expressed
in
U/
L.

Qualification
of
the
assay,
using
Media
199
without
phenol
red,
consisted
of
determination
of
sensitivity
and
dilutional
linearity.
These
were
the
only
qualification
tests
specific
to
media
use.
Intra­
assay
and
inter­
assay
imprecision
were
based
on
serum
based
controls
normally
used
for
instrument
monitoring
of
quality
control.
Accuracy
was
based
on
the
linear
regression
of
a
media
sample
spiked
with
purified
LDH
and
diluted
with
media.

Intra­
assay
imprecision
was
measured
by
assaying
10
quality
control
samples
within
a
single
run.
The
mean
SD
and
CV
were
calculated.
The
CV
should
be
less
than
10%.
Inter­
assay
imprecision
was
measured
by
assaying
10
quality
control
samples
over
10
separate
runs.
The
mean
SD
and
CV
were
calculated.
The
CV
should
be
less
than
10%.

Sensitivity
of
the
assay
or
limit
of
detection
was
determined
by
analysis
of
20
samples
of
media
with
no
LDH
present
in
the
media.
A
low
level
of
control
was
also
analyzed
to
determine
Section
4.0
Methods
for
Phase
1
 
Preliminary
Experimental
Phase
4­
3
the
lowest
level
of
detection.
Two
standard
deviations
from
the
mean
of
the
media
activity
were
used
as
the
limit
of
detection.

Dilutional
linearity
was
used
to
determine
accuracy
and
linearity
limits.
A
sample
of
media
was
spiked
with
purified
LDH
(
Sigma
#
L1254).
This
was
diluted
with
Media
199
without
phenol
red
to
within
the
linear
limits
of
the
assay.
A
series
of
dilutions
by
serial
dilution
of
the
spiked
sample
were
conducted
in
triplicate.
The
linear
regression
of
the
line
compared
to
the
theoretical
activity
should
be
between
0.990
and
1.100.

All
calculations
were
performed
using
EP
Evaluator,
Release
3.0
statistical
analysis
software
from
David
Rhoads
Associates,
Inc.,
Kennett
Square,
PA.

4.3
Phase
1
Optimization
Experiments
for
Media
Type,
Gaseous
Atmosphere,
Rat
Age,
and
Storage
Container
Type
4.3.1
Media
Type
The
objective
was
to
determine
the
effect
of
different
types
of
media
(
with
specified
components)
on
testosterone
production
using
the
sliced
testis
assay.
The
prototypical
assay
conditions
were
used
except
with
regard
to
the
types
of
media.

The
media
types
tested
were:

?
RPMI­
1640
media
(
without
phenol
red),
10%
FCS,
50
ug/
mL
soybean
trypsin
inhibitor
?
Medium­
199
(
Gibco),
0.71
g
Na
bicarbonate,
2.1
g
HEPES,
1.0
g/
L
BSA,
0.025
g
/
L
soybean
trypsin
inhibitor,
adjusted
to
pH
7.4
?
Eagles
MEM
4.3.2
Gaseous
Atmosphere
The
objective
was
to
determine
the
effect
of
different
types
of
gaseous
atmospheres
on
testosterone
production
using
the
sliced
testis
assay.
The
prototypical
assay
conditions
were
used
except
with
regard
to
the
gaseous
atmosphere.

The
atmospheres
tested
were:

?
5%
CO
2
/
95%
air
?
5%
CO
2
/
95%
O
2
Section
4.0
Methods
for
Phase
1
 
Preliminary
Experimental
Phase
4­
4
?
Air
(
3
gases)

4.3.3
Rat
Age
The
objective
will
be
to
determine
the
effect
of
age
on
testosterone
production
using
the
sliced
testis
assay.
The
prototypical
assay
conditions
will
be
used
except
with
regard
to
the
age
of
the
rat
used
to
obtain
the
testes.

The
ages
to
be
tested
are:

?
11
weeks
of
age
?
15
weeks
of
age
?
22
weeks
of
age
4.3.4
Storage
Container
Type
The
objective
was
to
determine
the
effect
of
storage­
type
containers
on
the
stability
of
testosterone
in
media.
The
types
of
containers
tested
were:
siliconized
plastic
and
nonsiliconized
plastic.

4.4
Phase
1
Experimental
Design
4.4.1
Factorial
Design
Experiments
These
experiments
were
conducted
as
a
33
full
factorial
design
with
1
replicate
per
condition.
The
experimental
factor
levels
are
summarized
in
Table
1.
The
factorial
test
conditions
are
displayed
in
Table
2.
The
27
factor
level
combinations
were
run
in
random
order.
Each
combination
was
run
with
and
without
hCG
stimulation
for
a
total
of
54
test
runs.
Each
test
run
response
(
ng
T/
mL)
were
determined
at
1,
2,
3,
and
4
hours
after
media
refreshment.
Section
4.0
Methods
for
Phase
1
 
Preliminary
Experimental
Phase
4­
5
Table
1.
Summary
of
Experimental
Factors
for
Phase
1
Optimization
Factor
Identification
Units
Experimental
Levels
Coded
Experimental
Levels
1
2
3
1
2
3
Media
Type
NA
RPMI­
1640
Medium­
199
Eagles­
MEM
­
1
0
+
1
Gaseous
Atmosphere
NA
5%
CO
2
?
95%
air
5%
CO
2
?
95%
O
2
air
­
1
0
+
1
Rat
Age
Wks
11
15
22
­
1
0
+
1
NA
=
not
applicable.

Table
2.
Factorial
Test
Conditions
for
Phase
1
Optimization
Experiment
Media
Type
Gaseous
Atmosphere
Rat
Age
­
1
­
1
­
1
­
1
­
1
0
­
1
­
1
+
1
­
1
0
­
1
­
1
0
0
­
1
0
+
1
­
1
+
1
­
1
­
1
+
1
0
­
1
+
1
+
1
0
­
1
­
1
0
­
1
0
0
­
1
+
1
0
0
­
1
0
0
0
0
0
+
1
0
+
1
­
1
0
+
1
0
0
+
1
+
1
+
1
­
1
­
1
+
1
­
1
0
+
1
­
1
+
1
+
1
0
­
1
(
Continued)
Table
2.
Factorial
Test
Conditions
for
Phase
1
Optimization
Experiment
(
Continued)
Section
4.0
Methods
for
Phase
1
 
Preliminary
Experimental
Phase
Media
Type
Gaseous
Atmosphere
Rat
Age
4­
6
+
1
0
0
+
1
0
+
1
+
1
+
1
­
1
+
1
+
1
0
+
1
+
1
+
1
4.4.2
Phase
1
Single
Factor
Experimental
Design
The
objective
of
this
set
of
experiments
was
to
determine
the
effect
of
storage
container
type
on
the
stability
of
testosterone
in
the
media.
This
experimental
series
did
not
use
the
sliced
testis
assay.
Stability
was
assessed
as
a
function
of
sample
handling
factors.
The
incubation
medium
used
will
be
determined
in
the
factorial
experiments
(
Phase
I).
To
conduct
the
stability
experiments,
a
known
amount
of
testosterone
was
added
to
the
media
to
achieve
a
specified
target
concentration.
The
target
concentration
was
determined
from
the
media
type
experiments
in
Phase
I
and
was
in
the
range
of
the
lowest
testosterone
concentrations
measured
in
the
sliced
testis
assay.
Using
this
target
concentration,
the
measured
concentration
was
compared
to
the
target
concentration.
The
stability
was
evaluated
based
on
the
difference
between
the
measured
and
target
concentrations.
If
no
statistical
difference
existed
at
the
5%
level,
then
the
sample
was
determined
to
be
stable
under
the
conditions
tested.

4.5
Phase
I
Data
Evaluation
Upon
completion
of
the
Phase
I
optimization
experiments,
the
results
were
reviewed
for
a
possible
change
in
the
prototype
conditions
with
regard
to
the
4
factors
tested.
A
decision
was
made
as
to
whether
the
Phase
II
optimization
experiments
would
be
conducted
using
the
original
prototype
or
modifications
made
to
the
media
type,
atmosphere,
age
of
rat,
and/
or
storage
container
type
used
for
the
remaining
experiments.
The
endpoint
was
the
amount
of
testosterone
released
into
the
media
during
the
sliced
testis
assay.
The
test
conditions
resulting
in
the
highest
values
for
testosterone
were
used
in
Phase
II.
Section
5.0
Results
and
Statistical
Analyses
5­
1
5.0
RESULTS
AND
STATISTICAL
ANALYSES
5.1
Testosterone
RIA
Verification
The
testosterone
RIA
kit
from
Diagnostic
Products
was
used
to
verify
that
the
M­
199
media
without
phenol
red
could
be
used
for
the
sliced
testis
assay
and
provide
accurate
results
for
the
RIA
assay.

Table
3.
Values
of
Standards
on
Testosterone
RIA
Value
of
Standard
Factor
Reading
8
ng/
ml
1
9.33
8
ng/
ml
1
8.70
8
ng/
ml
1
9.19
8
ng/
ml
1
8.86
8
ng/
ml
1
9.35
8
ng/
ml
2
9.63
8
ng/
ml
2
10.16
8
ng/
ml
2
10.54
8
ng/
ml
2
11.08
8
ng/
ml
2
11.86
8
ng/
ml
5
10.96
8
ng/
ml
5
11.60
8
ng/
ml
5
12.41
8
ng/
ml
5
11.56
8
ng/
ml
5
13.03
2
ng/
ml
1
2.56
2
ng/
ml
1
2.77
2
ng/
ml
1
2.56
2
ng/
ml
1
2.59
2
ng/
ml
1
2.46
0.5
ng/
ml
1
0.63
0.5ng/
ml
1
0.80
0.5ng/
ml
1
0.76
0.5ng/
ml
1
0.74
0.5ng/
ml
1
0.71
Unspiked
Media
Below
Detection
Limits
of
0.04
ng/
mL
Section
5.0
Results
and
Statistical
Analyses
5­
2
Table
4.
Testosterone
RIA
Intra­
assay
CV
Number
50
?
l
25
?
l
10
?
l
Unspiked
M199
2
Blanks
+
8
ng/
ml
10
5.24%
8.64%
7.68%

+
2
ng/
ml
10
6.09%

+
0.5
ng/
ml
10
13.34%

Table
5.
Testosterone
RIA
Percent
Recovery
50
?
l
25
?
l
10
?
l
+
8
ng/
ml
113.8
133.3
149.0
+
2
ng/
ml
129.5
0.5
ng/
ml
146.4
Table
6.
Testosterone
RIA
Parallelism
50
?
l
25
?
l
10
?
l
+
8
ng/
mL*
9.10
ng/
mL
10.66
ng/
mL
11.92
ng/
mL
*
The
Index
between
50
and
10
?
l
was
117.1%,
between
25
and
10
?
l
was
111.8%,
and
between
50
and
10
?
l
was
131.0%.

5.2
LDH
Verification
See
Appendix
A.

5.3
Statistical
Analysis
of
the
Phase
I
Assay
Optimization
Experiment
5.3.1
Objectives
The
assay
optimization
experiment
involves
3
factors
(
media
type,
atmosphere,
and
rat
age)
which
are
run
in
a
33
factorial
arrangement.
Each
of
these
27
trials
is
run
with
and
without
hCG
stimulation,
and
repeated
measurements
are
taken
at
baseline
(
time
0)
and
at
1,
2,
3,
and
4
hours
after
baseline.
The
conditions
are
identified
in
Table
7.
Objectives
of
the
experiment
are:

1.
To
determine
the
set
of
conditions
that
yields
the
highest
estimated
testosterone
level,
and
Section
5.0
Results
and
Statistical
Analyses
5­
3
2.
To
determine
the
set
of
conditions
that
yields
the
largest
with­
versus­
without­
hCG
difference
in
testosterone
levels.

5.3.2
Data
Two
basic
SAS
data
sets
were
constructed
from
the
raw
data,
and
2
fundamental
types
of
dependent
variables
were
used
in
the
analyses
of
each
type:

?
Date
Set
1:
Cases
without
hCG
stimulation
Dependent
variables:
testosterone
concentrations
Dependent
variables:
(
natural)
logarithm
of
testosterone
concentrations
?
Data
Set
2:
Cases
with
hCG
stimulation
Dependent
variables:
testosterone
concentrations
Dependent
variables:
(
natural)
logarithm
of
testosterone
concentrations
Each
data
set
can
be
viewed
as
consisting
of
27
observations
(
rows).
Each
observation
includes
dependent
variable
values
for
4
time
points
and
a
corresponding
baseline
level.
Each
observation
also
includes
data
identifying
the
levels
of
the
pertinent
factors.
Data
are
listed
in
Table
8
for
the
unchallenged
samples
and
in
Table
9
for
the
challenged
cases.

5.4
Statistical
Analysis
Methods
5.4.1
Objective
1
Several
statistical
analysis
methods
were
used
to
address
the
first
objective.
Analysis
of
variance
(
ANOVA)
and
analysis
of
covariance
(
ANOCOVA)
were
used
to
analyze
the
data
for
each
individual
time
point
(
including
the
baseline),
and
a
mixed­
model
ANOCOVA
method
was
used
to
jointly
analyze
the
data
(
across
time
points
1
through
4).
The
ANOCOVA
models
utilized
the
baseline
level
(
or
log
level)
as
a
covariate.
For
each
type
of
analysis,
all
main
effects
and
two­
factor
interactions
(
2fi)
of
the
3
factors
were
initially
included
in
the
models.
Tests
for
interactions
were
conducted
and,
where
they
were
not
detected
as
statistically
significant
(
p=
0.05),
a
reduced
model
was
employed
that
retained
the
main
effects,
the
baseline
covariate
(
where
applicable),
and
only
those
2fi
deemed
to
have
significant
effects.
Additional
details
are
provided
in
the
Results
section.

5.4.2
Objective
2
For
each
of
the
27
trials,
differences
between
the
with­
hCG
and
the
without­
hCG
testosterone
levels
were
computed
for
each
hour
(
including
baseline).
These
differences
were
computed
on
both
the
original
and
log
scales.
ANOVA
was
used
to
analyze
these
differences
for
each
individual
time
point
(
including
the
baseline).
For
each
model
and
type
of
data,
all
main
effects
and
2fi
of
the
3
factors
were
initially
included
in
the
models.
Tests
for
interactions
were
conducted
and
where
they
were
not
detected
as
statistically
significant
(
p=
0.05),
a
reduced
model
Section
5.0
Results
and
Statistical
Analyses
5­
4
was
employed
that
retained
the
main
effects
and
only
those
2fi
deemed
to
have
significant
effects.
Additional
details
are
provided
in
the
Results
section.

5.4.3
Results
Overall
Characterization
of
the
Data.
Table
10
provides
summary
statistics
characterizing
the
testosterone
levels
in
the
non­
hCG­
stimulated
data
set.
This
summary
ignores
the
particular
experimental
factors.
The
top
portion
of
the
table
gives,
by
hour,
the
sample
size
(
n),
the
mean,
standard
deviation,
sum,
minimum,
and
maximum.
These
variables
are
denoted
as
yJ,
where
J
denotes
the
hour
and
takes
on
values
of
0,
1,
2,
3,
and
4.
The
lower
portion
of
the
table
gives
the
correlations
between
the
hourly
data.
The
following
trends
are
apparent:

?
the
means
continue
to
increase
over
time
?
the
standard
deviations
also
increase
over
time
(
i.
e.,
as
the
mean
level
gets
larger)

?
the
correlations
are
generally
high
and
tend
to
be
largest
for
adjacent
hours
Table
11
provides
a
similar
summary
for
the
log­
scaled
data;
these
variables
are
denoted
as
lyJ,
where
J
denotes
the
hour.
Similar
trends
for
the
means
and
correlations
are
evident,
but
the
standard
deviations
tend
to
be
fairly
stable
across
the
various
time
points.

Table
7.
Factor
Levels
in
the
Phase
I
Assay
Optimization
Experiment
Factor
Identification
Units
Factor
Name
Experimental
Levels
Coded
Experimental
Levels
1
2
3
1
2
3
Media
Type*
Z1
RPMI­
1640
medium­
199
Eagles­
MEM
­
1
0
+
1
Atmosphere*
Z2
5%
CO
2
/
95%
air
5%
CO
2
/
95%
O
2
air
­
1
0
+
1
Rat
Age*
wks
Z3
11
15
22
­
1
0
+
1
*
Treated
as
a
3­
level
discrete
factor.
5­

5
Section
5
Results
and
Statistical
Analysis
Table
8.
Data
Listing
for
Samples
Without
hCG
Animal
Ear
Tag
Set
Number
Whole
Testis
Weight
g
Testis
Section
g
z1
z2
z3
y0
=
Testos.
Conc.
Baseline
y1
=
Testos.
Conc.
Hour
1
y2
=
Testos.
Conc.
Hour
2
y3
=
Testos.
Conc.
Hour
3
y4
=
Testos.
Conc.
Hour
4
3
303
A
RIGHT­
1.5531
0.2331
­
1
­
1
­
1
0.54
3.23
5.21
6.19
6.11
5
308
B
RIGHT­
1.8189
0.2519
­
1
­
1
0
0.45
4.32
5.02
6.81
8.43
16
317
C
LEFT­
2.0203
0.2626
­
1
­
1
1
0.13
1.17
1.96
2.54
2.94
3
303
D
LEFT­
1.5632
0.2688
­
1
0
­
1
0.43
3.60
5.14
6.48
7.11
5
308
E
LEFT­
1.7979
0.2750
­
1
0
0
0.36
5.56
7.85
8.30
10.48
19
316
F
RIGHT­
2.0218
0.2524
­
1
0
1
0.18
2.10
3.77
5.11
6.26
2
304
G
RIGHT­
1.7860
0.2692
­
1
1
­
1
0.58
3.73
4.99
5.72
6.06
7
315
H
RIGHT­
1.7847
0.2449
­
1
1
0
0.57
5.17
7.01
8.39
9.17
16
317
I
RIGHT­
1.9804
0.2603
­
1
1
1
0.17
1.70
2.67
3.64
4.79
3
303
J
RIGHT­
1.5531
0.2742
0
­
1
­
1
0.63
5.35
6.27
7.52
8.64
5
308
K
RIGHT­
1.8189
0.2349
0
­
1
0
0.81
6.60
8.54
10.28
10.97
16
317
L
LEFT­
2.0203
0.2502
0
­
1
1
0.15
1.10
2.04
2.93
4.15
2
304
M
RIGHT­
1.7860
0.2668
0
0
­
1
0.86
6.60
9.81
11.35
13.48
5
308
N
LEFT­
1.7979
0.2504
0
0
0
0.49
5.58
8.02
10.00
11.23
19
316
O
RIGHT­
2.0218
0.2715
0
0
1
0.34
3.50
6.56
8.68
9.65
2
304
P
LEFT­
1.7809
0.2577
0
1
­
1
0.67
3.11
4.85
5.23
5.58
7
315
Q
RIGHT­
1.7847
0.2419
0
1
0
0.78
5.23
6.54
7.29
7.85
16
317
R
RIGHT­
1.9804
0.2563
0
1
1
0.13
1.31
1.82
2.46
2.83
5­

6
Section
5
Results
and
Statistical
Analysis
Table
8.
Data
Listing
for
Samples
Without
hCG
(
Continued)

Animal
Ear
Tag
Set
Number
Whole
Testis
Teight
g
Testis
Section
g
z1
z2
z3
y0
=
Testos.
Conc.
Baseline
y1
=
Testos.
Conc.
Hour
1
y2
=
Testos.
Conc.
Hour
2
y3
=
Testos.
Conc.
Hour
3
y4
=
Testos.
Conc.
Hour
4
3
303
S
LEFT­
1.5632
0.2651
1
­
1
­
1
0.21
3.98
6.10
7.37
10.16
5
308
T
RIGHT­
1.8189
0.2371
1
­
1
0
0.40
5.33
8.05
9.07
12.25
16
317
U
LEFT­
2.0203
0.2586
1
­
1
1
0.08
1.01
1.45
1.97
2.33
2
304
V
RIGHT­
1.7860
0.2741
1
0
­
1
0.48
3.44
5.53
6.98
8.12
5
308
W
LEFT­
1.7979
0.2340
1
0
0
0.69
4.91
7.36
10.05
11.72
19
316
X
RIGHT­
2.0218
0.2686
1
0
1
0.24
2.31
3.98
5.61
6.55
2
304
Y
LEFT­
1.7809
0.2681
1
1
­
1
0.45
4.44
5.49
6.71
6.86
7
315
Z
RIGHT­
1.7847
0.2560
1
1
0
0.74
5.34
6.15
7.87
8.43
16
317
AA
RIGHT­
1.9804
0.2408
1
1
1
0.09
0.84
1.18
1.73
1.74
5­

7
Section
5
Results
and
Statistical
Analysis
Table
9.
Data
Listing
for
Samples
With
hCG
Animal
Ear
Tag
Set
Number
Whole
Testis
Weight
g
Testis
Section
g
z1
z2
z3
yc0
=
Testos.
Conc.
Baseline
yc1
=
Testos.
Conc.
Hour
1
yc2
=
Testos.
Conc.
Hour
2
yc3
=
Testos.
Conc.
Hour
3
yc4
=
Testos.
Conc.
Hour
4
3
303
AC
RIGHT­
1.5531
0.2422
­
1
­
1
­
1
0.54
6.56
16.16
28.30
42.09
5
308
BC
RIGHT­
1.8189
0.2690
­
1
­
1
0
1.11
5.96
9.69
15.19
23.41
16
317
CC
LEFT­
2.0203
0.2680
­
1
­
1
1
0.16
2.03
4.88
8.87
15.93
3
303
DC
LEFT­
1.5632
0.2374
­
1
0
­
1
0.73
5.91
20.96
41.76
51.74
5
308
EC
LEFT­
1.7979
0.2398
­
1
0
0
0.43
4.39
10.84
22.92
33.45
19
316
FC
RIGHT­
2.0218
0.2515
­
1
0
1
0.26
3.80
9.94
18.38
24.77
2
304
GC
RIGHT­
1.7860
0.2494
­
1
1
­
1
1.29
4.71
7.14
10.26
13.39
7
315
HC
RIGHT­
1.7847
0.2401
­
1
1
0
0.62
6.35
8.09
11.90
14.64
16
317
IC
RIGHT­
1.9804
0.2642
­
1
1
1
0.10
1.30
2.74
3.63
5.13
3
303
JC
RIGHT­
1.5531
0.2613
0
­
1
­
1
0.72
8.26
18.51
31.30
39.20
5
308
KC
RIGHT­
1.8189
0.2386
0
­
1
0
0.47
7.84
15.75
28.83
43.60
16
317
LC
LEFT­
2.0203
0.2672
0
­
1
1
0.11
2.19
6.70
11.23
18.38
2
304
MC
RIGHT­
1.7860
0.2631
0
0
­
1
0.86
9.73
31.45
55.27
77.68
5
308
NC
LEFT­
1.7979
0.2461
0
0
0
0.89
7.33
18.38
33.23
59.89
19
316
OC
RIGHT­
2.0218
0.2516
0
0
1
0.42
4.15
10.45
18.93
27.32
2
304
PC
LEFT­
1.7809
0.2461
0
1
­
1
0.61
5.57
9.65
12.75
15.73
5­

8
Section
5
Results
and
Statistical
Analysis
Table
9.
Data
Listing
for
Samples
With
hCG
(
Continued)

Animal
Ear
Tag
Set
Number
Whole
Testis
Weight
g
Testis
Section
g
z1
z2
z3
yc0
=
Testos.
Conc.
Baseline
yc1
=
Testos.
Conc.
Hour
1
yc2
=
Testos.
Conc.
Hour
2
yc3
=
Testos.
Conc.
Hour
3
yc4
=
Testos.
Conc.
Hour
4
7
315
QC
RIGHT­
1.7847
0.2618
0
1
0
1.38
7.03
9.69
12.35
13.47
16
317
RC
RIGHT­
1.9804
0.2607
0
1
1
0.15
1.03
2.14
2.92
4.01
3
303
SC
LEFT­
1.5632
0.2375
1
­
1
­
1
0.34
6.43
18.23
26.69
38.80
5
308
TC
RIGHT­
1.8189
0.2668
1
­
1
0
0.65
8.79
18.07
29.25
46.72
16
317
UC
LEFT­
2.0203
0.2683
1
­
1
1
0.11
1.81
4.44
7.96
12.42
2
304
VC
RIGHT­
1.7860
0.2593
1
0
­
1
0.66
5.91
15.48
24.92
32.07
5
308
WC
LEFT­
1.7979
0.2515
1
0
0
0.90
9.38
25.90
45.21
67.45
19
316
XC
RIGHT­
2.0218
0.2595
1
0
1
0.18
3.98
10.13
18.33
27.01
2
304
YC
LEFT­
1.7809
0.2591
1
1
­
1
0.43
5.17
10.59
16.60
25.20
7
315
ZC
RIGHT­
1.7847
0.2684
1
1
0
0.52
7.09
12.48
20.01
27.96
16
317
AAC
RIGHT­
1.9804
0.2459
1
1
1
0.12
2.44
5.53
9.16
13.76
5­

9
Section
5
Results
and
Statistical
Analysis
Table
10.
Summary
of
Data
­­
Original
Scale,
Without
hCG
Simple
Statistics
Variable
N
Mean
Std
Dev
Sum
Minimum
Maximum
Label
y0
27
0.43148
0.24012
11.65000
0.08000
0.86000
y0
=_
Testos._
Conc._
Baseline
y1
27
3.72444
1.77928
100.56000
0.84000
6.60000
y1
=_
Testos._
Conc._
Hour
1
y2
27
5.30963
2.33770
143.36000
1.18000
9.81000
y2
=_
Testos._
Conc._
Hour
2
y3
27
6.52889
2.67986
176.28000
1.73000
11.35000
y3
=_
Testos._
Conc._
Hour
3
y4
27
7.55148
3.16645
203.89000
1.74000
13.48000
y4
=_
Testos._
Conc._
Hour
4
Pearson
Correlation
Coefficients,
N
=
27
Prob
>
|
r|
under
H0:
Rho=
0
y0
y1
y2
y3
y4
y0
y0
=_
Testos._
Conc._
Baseline
1.00000
0.83359
<.
0001
0.77555
<.
0001
0.75779
<.
0001
0.64894
0.0003
y1
y1
=_
Testos._
Conc._
Hour
1
0.83359
<.
0001
1.00000
0.96006
<.
0001
0.93735
<.
0001
0.89924
<.
0001
y2
y2
=_
Testos._
Conc._
Hour
2
0.77555
<.
0001
0.96006
<.
0001
1.00000
0.98329
<.
0001
0.96280
<.
0001
y3
y3
=_
Testos._
Conc._
Hour
3
0.75779
<.
0001
0.93735
<.
0001
0.98329
<.
0001
1.00000
0.97499
<.
0001
y4
y4
=_
Testos._
Conc._
Hour
4
0.64894
0.0003
0.89924
<.
0001
0.96280
<.
0001
0.97499
<.
0001
1.00000
5­

10
Section
5
Results
and
Statistical
Analysis
Table
11.
Summary
of
Data
­­
Log
Scale,
Without
hCG
Simple
Statistics
Variable
N
Mean
Std
Dev
Sum
Minimum
Maximum
ly0
27
­
1.04291
0.70964
­
28.15853
­
2.52573
­
0.15082
ly1
27
1.15995
0.62490
31.31878
­
0.17435
1.88707
ly2
27
1.53822
0.57738
41.53197
0.16551
2.28340
ly3
27
1.76591
0.52363
47.67970
0.54812
2.42922
ly4
27
1.90916
0.52876
51.54720
0.55389
2.60121
Pearson
Correlation
Coefficients,
N
=
27
Prob
>
|
r|
under
H0:
Rho=
0
ly0
ly1
ly2
ly3
ly4
ly0
1.00000
0.90646
<.
0001
0.88113
<.
0001
0.86395
<.
0001
0.79479
<.
0001
ly1
0.90646
<.
0001
1.00000
0.97732
<.
0001
0.96441
<.
0001
0.93255
<.
0001
ly2
0.88113
<.
0001
0.97732
<.
0001
1.00000
0.99193
<.
0001
0.97266
<.
0001
ly3
0.86395
<.
0001
0.96441
<.
0001
0.99193
<.
0001
1.00000
0.98411
<.
0001
ly4
0.79479
<.
0001
0.93255
<.
0001
0.97266
<.
0001
0.98411
<.
0001
1.00000
Section
5.0
Results
and
Statistical
Analysis
5­
11
Tables
12
and
13
furnish
comparable
information
for
the
hCG­
stimulated
samples.
Similar
trends
are
evident
for
these
data.
Mean
levels
tend
to
be
much
higher
than
for
the
nonstimulated
samples.

Analysis
of
Baseline
Data.
Since
we
intend
to
adjust
for
baseline
(
time
0)
levels
for
subsequent
analyses
of
the
hourly
(
nonbaseline)
data,
it
is
important
to
understand
how
the
experimental
factors
affect
the
baseline
levels.
For
instance,
if
one
of
the
factors
does
impact
the
baseline
levels,
then
adjusting
for
baseline
levels
in
those
subsequent
analyses
may
obscure
the
effect
of
the
experimental
factor.
Table14
presents
the
results
that
summarize
the
ANOVA
results
for
the
baseline
data.
Initially,
we
fit
an
ANOVA
model
that
included
all
mean
effects
(
denoted
as
z1,
z2,
and
z3)
and
all
2­
factor
interactions
(
denoted
as
z1*
z2,
z1*
z3,
and
z2*
z3).
We
examined
the
statistical
significance
of
each
of
the
interactions
and
reduced
the
model
to
contain
only
main
effects
and
the
pertinent
2fi.
For
3
of
the
4
cases
considered,
only
the
main
effects
were
retained;
for
the
log­
scale,
without
hCG
case,
2
of
the
interaction
effects
were
deemed
significant.
Among
the
3
experimental
factors,
it
is
clear
that
the
rat
age
(
z3)
has
the
most
pronounced
effect
on
the
baseline
levels
(
estimated
testosterone
levels
are
0.54,
0.58,
and
0.17
for
11­
week,
15­
week,
and
22­
week
old
animals).
For
the
non­
hCG­
stimulated
case,
media
type
also
impacted
the
baseline
levels,
with
the
highest
level
occurring
for
the
z1=
0
case
(
0.54
versus
0.38
for
the
other
two
media).
The
lower
portion
of
Table
14
furnishes
statistics
characterizing
the
model
fit:

?
R2
=
the
proportion
of
variability
accounted
for
by
the
model,
?
RMSE
=
root
mean
squared
error
=
the
square
root
of
the
residual
variance,
?
C.
V.
=
the
coefficient
of
variation
=
the
RMSE
divided
by
the
mean
testosterone
level
(
times
100%).

Analyses
Directed
at
Objective
1.
Two
fundamental
types
of
statistical
analysis
were
used
to
address
Objective
1
(
assessing
the
effects
of
the
experimental
factors
on
the
testosterone
levels)
 
separate
analyses
for
each
hour
and
a
combined
mixed­
model
approach.

Individual­
hour
analyses.
These
analyses
involved:

?
fitting
the
testosterone
data
for
a
given
hour
as
a
function
of
the
experimental
factors,
their
2­
factor
interactions,
and
the
baseline
level
?
examining
the
significance
of
the
2­
factor
interactions
(
2fi)
?
choosing
(
and
fitting)
a
reduced
model
form
by
eliminating
any
2fi
that
was
not
satistically
significant
in
any
of
the
four
hourly
models.
5­

12
Section
5
Results
and
Statistical
Analysis
Table
12.
Summary
of
Data
­­
Original
Scale,
With
hCG
Simple
Statistics
Variable
N
Mean
Std
Dev
Sum
Minimum
Maximum
Label
yc0
27
0.54667
0.36060
14.76000
0.10000
1.38000
yc0
=_
Testos._
Conc._
Baseline
yc1
27
5.37556
2.48467
145.14000
1.03000
9.73000
yc1
=_
Testos._
Conc._
Hour
1
yc2
27
12.37074
7.01613
334.01000
2.14000
31.45000
yc2
=_
Testos._
Conc._
Hour
2
yc3
27
20.96852
12.82440
566.15000
2.92000
55.27000
yc3
=_
Testos._
Conc._
Hour
3
yc4
27
30.19333
18.74161
815.22000
4.01000
77.68000
yc4
=_
Testos._
Conc._
Hour
4
Pearson
Correlation
Coefficients,
N
=
27
Prob
>
|
r|
under
H0:
Rho=
0
yc0
yc1
yc2
yc3
yc4
yc0
yc0
=_
Testos._
Conc._
Baseline
1.00000
0.66941
0.0001
0.44159
0.0211
0.38125
0.0497
0.34634
0.0768
yc1
yc1
=_
Testos._
Conc._
Hour
1
0.66941
0.0001
1.00000
0.86213
<.
0001
0.80157
<.
0001
0.78474
<.
0001
yc2
yc2
=_
Testos._
Conc._
Hour
2
0.44159
0.0211
0.86213
<.
0001
1.00000
0.98464
<.
0001
0.96305
<.
0001
yc3
yc3
=_
Testos._
Conc._
Hour
3
0.38125
0.0497
0.80157
<.
0001
0.98464
<.
0001
1.00000
0.98016
<.
0001
yc4
yc4
=_
Testos._
Conc._
Hour
4
0.34634
0.0768
0.78474
<.
0001
0.96305
<.
0001
0.98016
<.
0001
1.00000
Section
5.0
Results
and
Statistical
Analysis
5­
13
Table
13.
Summary
of
Data
 
Log
Scale,
With
hCG
Simple
Statistics
Variable
N
Mean
Std
Dev
Sum
Minimum
Maximum
lyc0
27
­
0.86822
0.80586
­
23.44183
­
2.30259
0.32208
lyc1
27
1.53650
0.61019
41.48556
0.02956
2.27521
lyc2
27
2.34072
0.64378
63.19938
0.76081
3.44840
lyc3
27
2.83766
0.70489
76.61684
1.07158
4.01223
lyc4
27
3.19518
0.71702
86.26981
1.38879
4.35260
Pearson
Correlation
Coefficients,
N
=
27
Prob
>
|
r|
under
H0:
Rho=
0
lyc0
lyc1
lyc2
lyc3
lyc4
lyc0
1.00000
0.84699
<.
0001
0.68581
<.
0001
0.61367
0.0007
0.54138
0.0035
lyc1
0.84699
<.
0001
1.00000
0.92139
<.
0001
0.87024
<.
0001
0.82124
<.
0001
lyc2
0.68581
<.
0001
0.92139
<.
0001
1.00000
0.98599
<.
0001
0.95954
<.
0001
lyc3
0.61367
0.0007
0.87024
<.
0001
0.98599
<.
0001
1.00000
0.98703
<.
0001
lyc4
0.54138
0.0035
0.82124
<.
0001
0.95954
<.
0001
0.98703
<.
0001
1.00000
Section
5.0
Results
and
Statistical
Analysis
5­
14
Table
14.
Summary
of
Statistical
Analysis
of
Baseline
Data
Original­
Scale
Models
Log­
Scale
Models
Without
hCG
Stimulation
With
hCG
Stimulation
Without
hCG
Stimulation
With
hCG
Stimulation
Dependent
Variable:
Y0
Y0
log(
Y0)
log(
Y0)

Dependent
Variable
Mean:
0.431
0.547
­
1.043
0.868
Significance
of
Model
Terms:
z1
xx
xxx
z2
xx
z3
xxx
xxx
xxx
xxx
z1*
z2
na
na
xx
na
z1*
z3
na
na
na
na
z2*
z3
na
na
xx
na
R2
0.766
0.628
0.950
0.822
RMSE
0.132
0.251
0.233
0.388
C.
V.
30.7
45.9
­­
­­
x
=
statistically
significant
effect
at
0.10
level
of
significance.
xx
=
statistically
significant
effect
at
0.05
level
of
significance.
xxx
=
statistically
significant
effect
at
0.01
level
of
significance.
na
=
not
applicable
(
effect
not
included
in
the
model).
5­

15
Section
5
Results
and
Statistical
Analysis
Table
15.
Summary
of
ANOCOVA
Results
for
Individual­
Hour
Original­
Scale
Models
Dependent
Variable:
Without
hCG
Stimulation
With
hCG
Stimulation
Y1
Y2
Y3
Y4
Y1
Y2
Y3
Y4
Dependent
Variable
Mean:
3.72
5.31
6.53
7.55
5.38
12.37
20.97
30.19
Significance
of
Model
Terms:
Y0
xx
xx
xx
z1
xx
x
z2
x
xxx
xxx
xxx
xx
xxx
xxx
xxx
z3
xxx
xx
xxx
xx
xxx
xxx
xxx
xxx
z1*
z2
xx
xx
xx
x
na
na
na
na
z1*
z3
na
na
na
na
x
x
R2
0.931
0.913
0.917
0.877
0.904
0.862
0.865
0.847
RMSE
0.62
0.91
1.02
1.46
1.01
3.43
6.21
9.65
C.
V.
16.6
17.1
15.5
19.3
18.9
27.7
29.6
32.0
x
=
statistically
significant
effect
at
0.10
level
of
significance.
xx
=
statistically
significant
effect
at
0.05
level
of
significance.
xxx
=
statistically
significant
effect
at
0.01
level
of
significance.
na
=
not
applicable
(
effect
not
included
in
the
model).
Section
5.0
Results
and
Statistical
Analysis
5­
16
Table
16.
Adjusted
Mean
Levels
(
Based
on
Original­
Scale
Models)

Level
of
Independent
Variable
Mean
Levels
of
Dependent
Variables:
Without
hCG
Stimulation
Mean
Levels
of
Dependent
Variables:
With
hCG
Stimulation
z1
z2
z3
Y1
Mean
Y2
Mean
Y3
Mean
Y4
Mean
Y1
Mean
Y2
Mean
Y3
Mean
Y4
Mean
­
1
3.55
5.05
6.14
6.99
4.52*
10.08*
18.11
25.30
0
3.95
5.64
6.83
7.92
5.83
13.70
23.41
34.01
+
1
3.67
5.24
6.62
7.75
5.77
13.34
21.38
31.26
­
1
3.72
5.16*
6.31**
7.50*
5.62
12.43*
20.40**
30.39**

0
4.12
6.37
7.97
9.33
6.02
17.10
31.25
45.05
+
1
3.34*
4.40**
5.30**
5.82**
4.49**
7.59**
11.25**
15.14**

­
1
3.86**
5.53*
6.59*
7.67*
6.34
16.58
28.33
38.71
0
4.89
6.58
7.99
9.56
6.91
14.50
25.61
38.99
+
1
2.43**
3.83**
5.01**
5.43**
2.88**
6.03**
8.97**
12.88**

­
1
­
1
3.07**
4.28**
5.44**
6.01**

­
1
0
4.06
6.00
7.11*
8.30
­
1
+
1
3.51*
4.86**
5.88**
6.65**

0
­
1
4.07
5.24**
6.48**
7.60*

0
0
4.85
7.63
9.43
11.03
0
+
1
2.94**
4.04**
4.57**
5.11**

+
1
­
1
4.02
5.96
7.02*
8.89
+
1
0
3.44*
5.48*
7.38*
8.67
+
1
+
1
3.55*
4.29**
5.46**
5.69**

­
1
­
1
5.43**
15.00
28.51
38.78
­
1
0
5.40**
9.68**
17.65**
25.55**

­
1
+
1
2.73**
5.55**
8.18**
11.58**

0
­
1
7.68
20.02
34.14
46.02
0
0
7.05
14.90
26.88
42.62
0
+
1
2.76**
6.17**
9.21**
13.40**

+
1
­
1
5.90*
14.71
22.34*
31.33*

+
1
0
8.28
18.93
32.30
48.80
+
1
+
1
3.13**
6.37**
9.50**
13.67**
Shaded
cell
indicates
highest
mean
estimated
level.
*
Indicates
that
the
mean
level
is
significantly
lower
than
the
cell
with
the
maximum
estimated
level,
p=
0.05.
**
Indicates
that
the
mean
level
is
significantly
lower
than
the
cell
with
the
maximum
estimated
level,
p=
0.01.
Section
5.0
Results
and
Statistical
Analysis
5­
17
Results
for
the
original­
scale
data
are
summarized
in
Tables
15
and
16.
Table
15
provides
an
indication
of
which
effects
were
retained
in
the
reduced
model
and
which
of
those
terms
were
statistically
significant.
The
lower
portion
of
the
table
gives
statistics
characterizing
the
fit
of
the
models.

Table
15
indicates
that
mean
concentration
levels
increase
with
time.
The
models
for
the
without
and
with­
hCG
stimulation
data
are
somewhat
different,
but
both
indicate
statistical
significance
for
z2
(
atmosphere
type)
and
z3
(
rat
age).
For
the
without­
hCG
case,
the
model
also
shows
a
significant
effect
of
the
covariate
and
of
the
z1*
z2
(
media
type
by
atmosphere
type)
interaction.
For
the
with­
hCG
case,
the
covariate
was
not
statistically
significant
and
there
was
some
(
weak)
indication
of
a
z1*
z3
(
media
type
by
rat
age)
interaction.
The
RMSE
values
tend
to
increase
with
increasing
concentration
levels
(
i.
e.,
with
time).
The
C.
V.
s,
on
the
other
hand,
tend
to
be
fairly
stable,
suggesting
that
a
log­
tranform
of
the
concentrations
should
result
in
data
with
approximately
homogeneous
variances
over
the
various
time
points.

Adjusted
means
based
on
the
models
that
were
indicated
in
Table
15
are
presented
in
Table
16.
The
means
are
those
that
are
estimated
to
occur
for
a
given
level
of
a
factor
(
or
given
combination
of
factors)
when
other
effects
in
the
model
(
e.
g.,
the
baseline
level
covariate)
are
fixed
at
their
mean
values.
The
3
first
columns
of
the
table
identify
the
factor
levels
(
see
Table
7).
Within
each
set
of
the
levels
(
e.
g.,
the
three
rows
with
z1=
­
1,
0,
and
+
1),
the
estimated
adjusted
mean
that
is
largest
is
highlighted.
Asterisks
beside
the
other
nonhighlighted
means
indicate
if
that
particular
mean
is
deemed
to
be
statistically
significant
from
the
one
that
is
highlighted.
For
the
nonstimulated
data,
for
instance,
the
table
indicates
no
significant
difference
among
the
z1
levels,
although
the
zero
level
is
consistently
estimated
to
be
the
largest.
For
the
atmosphere
and
rat
age
factors,
the
zero
levels
generally
have
the
highest
estimated
mean
testosterone
concentrations
and
the
other
levels
typically
have
significantly
lower
means.
An
exception
is
the
rat
age
(
z3)
factor
for
the
hCG­
stimulated
case,
where
the
11­
week­
old
and
the
15­
week­
old
rats
had
similar
adjusted
mean
levels.
Even
when
interactions
are
considered
(
lower
portion
of
Table
16),
the
zero
levels
of
all
3
factors
are
either
estimated
to
have
the
highest
adjusted
means
or
to
have
adjusted
means
that
are
not
significantly
different
from
the
factor
combination
having
the
highest
estimated
mean
level.

Tables
17
and
18
show
results
for
the
log­
transformed
data.
These
tables
are
analogous
to
Tables
15
and
16,
respectively.
The
models
are
somewhat
different
than
those
indicated
in
Table
15.
On
the
log
scale,
the
rat
age
factor
does
not
appear
to
be
as
prominent.
Also,
the
Eagle­
MEM
(
z1=
1)
medium
yields
the
highest
mean
levels,
although
the
199
medium
(
z1=
0)
levels
are
not
significantly
smaller.
The
air
atmosphere
consistently
produces
lower
mean
levels.
Section
5.0
Results
and
Statistical
Analysis
5­
18
As
with
the
original­
scale
data,
the
zero
levels
of
all
3
factors
are
either
estimated
to
have
the
highest
adjusted
means
or
to
have
5­

19
Section
5
Results
and
Statistical
Analysis
Table
17.
Summary
of
ANOCOVA
Results
for
Individual­
Hour
Log­
Scale
Models
Dependent
Variable:
Without
hCG
Stimulation
With
hCG
Stimulation
log(
Y1)
log(
Y2)
log(
Y3)
log(
Y4)
log(
Y1)
log(
Y2)
log(
Y3)
log(
Y4)

Dependent
Variable
Mean:
1.160
1.538
1.766
1.909
1.537
2.341
2.838
3.195
Significance
of
Model
Terms:
log(
Y0)
xxx
xxx
xxx
xxx
xx
z1
x
xx
x
x
z2
x
xxx
xxx
xxx
xx
xxx
xxx
xxx
z3
x
x
x
z1*
z2
xx
xx
xx
xx
x
x
z1*
z3
x
x
xx
na
na
na
na
R2
0.963
0.964
0.962
0.951
0.894
0.900
0.917
0.924
RMSE
0.185
0.167
0.156
0.181
0.261
0.268
0.268
0.260
x
=
statistically
significant
effect
at
0.10
level
of
significance.
xx
=
statistically
significant
effect
at
0.05
level
of
significance.
xxx
=
statistically
significant
effect
at
0.01
level
of
significance.
na
=
not
applicable
(
effect
not
included
in
the
model)
Section
5.0
Results
and
Statistical
Analysis
5­
20
Table
18.
Adjusted
Mean
Levels
Based
on
Log­
Scale
Models
Level
of
Independent
Variable
Mean
Levels
of
Dependent
Variables:
Without
hCG
Stimulation
Mean
Levels
of
Dependent
Variables:
With
hCG
Stimulation
z1
z2
z3
log(
Y1)
Mean
log(
Y2)
Mean
log(
Y3)
Mean
log(
Y4)
Mean
log(
Y1)
Mean
log(
Y2)
Mean
log(
Y3)
Mean
log(
Y4)
Mean
­
1
1.162
1.551
1.764
1.903
1.381*
2.147*
2.669*
3.021*

0
1.085
1.456
1.679
1.834
1.527
2.362
2.836
3.190
+
1
1.232
1.608
1.855
1.990
1.702
2.513
3.008
3.374
­
1
1.208
1.591
1.810
1.98
1.628
2.431
2.949*
3.356*

0
1.240
1.690
1.935
2.10
1.644
2.696
3.305
3.687
+
1
1.031*
1.334**
1.552**
1.64**
1.337*
1.895**
2.259**
2.542**

­
1
1.096
1.442
1.640
1.780
1.651
2.610
3.098
3.444
0
1.279
1.550
1.774
1.956
1.698
2.463
2.982
3.408
+
1
1.105
1.623
1.884
1.991
1.261
1.949*
2.433*
2.734*

­
1
­
1
1.021*
1.406**
1.645**
1.752**
1.419
2.192*
2.730*
3.211*

­
1
0
1.371
1.805
1.993
2.157
1.515
2.566
3.251
3.552
­
1
+
1
1.094*
1.440*
1.654*
1.801*
1.208*
1.682**
2.026**
2.302**

0
­
1
1.066*
1.403*
1.659*
1.857*
1.755
2.582
3.124
3.477
0
0
1.292
1.740
1.981
2.144
1.675
2.782
3.379
3.864
0
+
1
0.898**
1.225**
1.396**
1.501**
1.151*
1.723**
2.005**
2.229**

+
1
­
1
1.539
1.963
2.127
2.341
1.711
2.520
2.991
3.381
+
1
0
1.057*
1.524*
1.833
2.001
1.743
2.740
3.285
3.645
+
1
+
1
1.102*
1.338**
1.606**
1.628**
1.651
2.281
2.747*
3.097**

­
1
­
1
0.946*
1.307*
1.512*
1.587**

­
1
0
1.401
1.658
1.855
2.053
­
1
+
1
1.139
1.686
1.925
2.070
0
­
1
0.982*
1.288*
1.470*
1.650*

0
0
1.213
1.470
1.685
1.821
0
+
1
1.061
1.609
1.882
2.031
+
1
­
1
1.359
1.730
1.939
2.104
+
1
0
1.221
1.521
1.781
1.996
+
1
+
1
1.117
1.572
1.846
1.871
Shaded
cell
indicates
highest
mean
estimated
level.
*
Indicates
that
the
mean
level
is
significantly
lower
than
the
cell
with
the
maximum
estimated
level,
p=
0.05.
**
Indicates
that
the
mean
level
is
significantly
lower
than
the
cell
with
the
maximum
estimated
level,
p=
0.01.
Section
5.0
Results
and
Statistical
Analysis
5­
21
adjusted
means
that
are
not
significantly
different
from
the
factor
combination
having
the
highest
estimated
mean
level.

Mixed­
model
analyses.
For
each
of
2
data
sets
and
2
types
of
dependent
variables
indicated
above
(
see
"
Data"),
these
analyses
involved
several
steps.
First,
we
employed
a
mixed
model
that
included:

?
the
main
effects
of
the
experimental
factor,

?
two­
factor
interactions,

?
the
baseline
testosterone
level,

?
a
linear
and
a
quadratic
time
component,

?
cross
products
of
the
linear
and
quadratic
time
components
with
the
main
effects,
and
?
cross
products
of
the
linear
and
quadratic
time
components
with
the
2fi.

For
this
"
full"
model,
we
utilized
the
SAS
PROC
MIXED
procedure
to
determine
a
relevant
covariance
structure
for
the
data
set;
in
particular,
we
examined
10
different
possible
covariance
structures,
using
maximum
likelihood
estimation,
and
selected
one
that
appeared
to
be
optimal
or
near
optimal.
Using
that
structure,
we
estimated
fixed
effects
for
all
of
the
above
model
terms.
We
then
reduced
the
model
by
eliminating
nonsignificant
higher­
order
terms.
We
then
re­
examined
the
covariance
structure
(
again
selecting
from
among
10
possible
structures)
for
this
reduced
model.
Using
the
selected
structure,
we
estimated
the
fixed
effects
in
the
reduced
model
(
using
restricted
maximum
likelihood
estimation)
and
computed
adjusted
means
for
the
various
factor
levels,
along
with
approximate
95%
confidence
intervals
for
the
means.

The
adjusted
means
for
the
various
cases
and
factors
are
given
in
Tables
19
through
22.
These
are
denoted
as
EST.,
where
J
=
1,
2,
3,
or
4
denotes
hour.
Approximate
95%
confidence
limits
are
given
in
the
right
portion
of
the
table.
The
lower
and
upper
limits
are
denoted
as
LOWJ
and
HIJ,
respectively,
where
J
=
1,
2,
3,
or
4
denotes
hour.
Unlike
the
individual­
hour
analyses,
these
estimates
(
and
the
interval
estimates)
rely
on
data
from
all
4
hours
and
also
reflect
a
smoothing
over
time
(
due
to
the
assumed
quadratic
time
dependence).

Analyses
Directed
at
Objective
2.
Differences
in
hCG­
stimulated
and
nonstimulated
testosterone
levels
were
computed
for
the
27
trials
on
an
hour­
by­
hour
basis.
These
differences
were
then
analyzed
by
hour
using
an
initial
ANOVA
model
that
included
all
main
effects
and
2fi.
Section
5.0
Results
and
Statistical
Analysis
5­
22
A
reduced
ANOVA
model
was
then
selected
by
eliminating
those
2fi
that
were
not
statistically
significant.
The
original­
scale
models
are
summarized
in
Table
23;
no
2fi
were
deemed
necessary,
so
that
the
model
only
includes
the
main
effects.
Both
atmosphere
type
and
rat
age
were
judged
to
have
impact
on
the
testosterone
concentration
levels.
Table
24
shows
the
adjusted
means
derived
from
the
model.
The
estimated
adjusted
mean
difference
that
is
largest
is
highlighted.
Asterisks
beside
the
other
nonhighlighted
mean
differences
indicate
if
that
particular
difference
is
deemed
to
be
statistically
significant
from
the
one
that
is
highlighted.
The
media
type
appears
to
have
little
effect,
but
rat
age
and
atmosphere
type
are
significant
factors
affecting
the
with­
minus­
without­
hCG
differences.
Section
5.0
Results
and
Statistical
Analysis
5­
23
Table
19.
Least
Squares
Means
for
Reduced
Log­
scale
Model:
Without
hCG
Mean
covariate
value:
ly0=­
1.043
x1
x2
x3
EST1
EST2
EST3
EST4
LOW1
HI1
LOW2
HI2
LOW3
HI3
LOW4
HI4
­
1
_
_
1.163
1.527
1.773
1.901
1.059
1.267
1.424
1.629
1.670
1.875
1.797
2.005
0
_
_
1.116
1.471
1.708
1.828
0.942
1.290
1.298
1.644
1.536
1.881
1.655
2.002
1
_
_
1.211
1.587
1.846
1.988
1.064
1.357
1.442
1.733
1.701
1.992
1.842
2.134
_
­
1
_
1.184
1.563
1.824
1.968
1.039
1.330
1.418
1.708
1.680
1.969
1.823
2.114
_
0
_
1.278
1.672
1.949
2.109
1.146
1.409
1.542
1.802
1.819
2.080
1.978
2.240
_
1
_
1.028
1.350
1.554
1.640
0.928
1.128
1.251
1.448
1.455
1.652
1.540
1.741
_
_
­
1
1.137
1.472
1.683
1.771
0.901
1.374
1.236
1.708
1.448
1.919
1.534
2.007
_
_
0
1.327
1.606
1.813
1.949
1.031
1.622
1.311
1.901
1.518
2.108
1.654
2.245
_
_
1
1.026
1.507
1.831
1.998
0.523
1.529
1.004
2.010
1.328
2.334
1.494
2.501
Reduced
model
was
fit
using
compound
symmetry
covariance
structure.
Effects
retained
in
the
reduced
model
were
the
following
(
tL
denote
the
linear
time
effect,
tQ
denotes
the
quadratic):
ly0,
z1,
z2,
z3,
z1*
z2,
z1*
z3,
tL,
tL*
z1,
tL*
z2,
tL*
z3,
tL*
z1*
z3,
tQ,
tQ*
z3.

Table
20.
Least
Squares
Means
for
Reduced
Original­
scale
Model:
Without
hCG
Mean
covariate
value:
y0=
0.43
x1
x2
x3
EST1
EST2
EST3
EST4
LOW1
HI1
LOW2
HI2
LOW3
HI3
LOW4
HI4
­
1
_
_
3.70
5.11
6.24
7.09
2.99
4.40
4.41
5.80
5.55
6.94
6.39
7.79
0
_
_
3.74
5.35
6.67
7.72
2.82
4.66
4.43
6.27
5.75
7.59
6.79
8.64
1
_
_
3.76
5.40
6.75
7.82
3.05
4.48
4.69
6.10
6.04
7.46
7.11
8.53
_
­
1
_
3.78
5.30
6.54
7.50
3.07
4.48
4.60
6.00
5.84
7.24
6.80
8.21
_
0
_
4.17
6.18
7.91
9.36
3.54
4.81
5.56
6.81
7.29
8.54
8.73
9.99
_
1
_
3.25
4.37
5.21
5.77
2.59
3.90
3.72
5.01
4.56
5.85
5.12
6.42
_
_
­
1
3.65
5.20
6.47
7.46
2.74
4.57
4.29
6.12
5.56
7.38
6.54
8.38
_
_
0
4.45
6.30
7.87
9.15
3.29
5.62
5.14
7.46
6.70
9.03
7.99
10.32
_
_
1
3.09
4.35
5.32
6.02
1.31
4.87
2.57
6.13
3.55
7.10
4.24
7.80
Reduced
model
was
fit
using
compound
symmetry
covariance
structure.
Effects
retained
in
the
reduced
model
were
the
following
(
tL
denote
the
linear
time
effect,
tQ
denotes
the
quadratic):
y0,
z1,
z2,
z3,
z1*
z2,
z1*
z3,
z2*
z3,
tL,
tL*
z1,
tL*
z2,
tL*
z3,
tL*
z1*
z2,
tL*
z1*
z3,
tL*
z2*
z3,
tQ.
Section
5.0
Results
and
Statistical
Analysis
5­
24
Table
21.
Least
Squares
Means
for
Reduced
Log­
scale
Model:
With
hCG
Mean
covariate
value:
lyc0=­
0.868
x1
x2
x3
EST1
EST2
EST3
EST4
LOW1
HI1
LOW2
HI2
LOW3
HI3
LOW4
HI4
­
1
_
_
1.388
2.153
2.694
3.012
1.199
1.576
1.970
2.335
2.512
2.876
2.823
3.200
0
_
_
1.554
2.314
2.852
3.166
1.360
1.748
2.126
2.503
2.664
3.040
2.972
3.360
1
_
_
1.668
2.471
3.051
3.408
1.469
1.867
2.278
2.665
2.858
3.244
3.209
3.607
_
­
1
_
1.598
2.400
2.996
3.385
1.402
1.795
2.206
2.595
2.801
3.190
3.188
3.581
_
0
_
1.682
2.662
3.319
3.651
1.481
1.884
2.463
2.862
3.119
3.518
3.449
3.852
_
1
_
1.329
1.876
2.283
2.550
1.141
1.518
1.690
2.062
2.097
2.469
2.361
2.738
_
_
­
1
1.726
2.560
3.109
3.372
1.489
1.963
2.325
2.795
2.873
3.344
3.135
3.609
_
_
0
1.793
2.443
2.951
3.317
1.530
2.056
2.182
2.704
2.690
3.212
3.054
3.580
_
_
1
1.091
1.935
2.537
2.896
0.713
1.470
1.558
2.313
2.159
2.914
2.518
3.275
Reduced
model
was
fit
using
first­
order
autoregressive
covariance
structure.
Effects
retained
in
the
reduced
model
were
the
following
(
tL
denote
the
linear
time
effect,
tQ
denotes
the
quadratic):
lyc0,
z1,
z2,
z3,
z1*
z2,
tL,
tL*
z1,
tL*
z2,
tL*
z3,
tL*
z1*
z2,
tQ,
tQ*
z2,
tQ*
z3.

Table
22.
Least
Squares
Means
for
Reduced
Original­
scale
Model:
With
hCG
Mean
covariate
value:
yc0=
0.55
x1
x2
x3
EST
1
EST
2
EST
3
EST
4
LOW
1
HI
1
LOW
2
HI
2
LOW
3
HI
3
LOW
4
HI
4
­
1
_
_
5.11
12.20
20.55
30.17
4.43
5.79
10.49
13.90
17.76
23.34
26.03
34.31
0
_
_
5.52
12.60
20.96
30.57
4.83
6.21
10.90
14.31
18.17
23.75
26.43
34.71
1
_
_
5.59
12.68
21.03
30.65
4.89
6.29
10.97
14.39
18.24
23.82
26.51
34.79
_
­
1
_
5.68
12.69
20.97
30.52
4.71
6.64
9.80
15.59
16.09
25.85
23.51
37.52
_
0
_
6.14
17.64
30.40
44.43
5.18
7.09
14.75
20.53
25.52
35.28
37.43
51.44
_
1
_
4.40
7.15
11.16
16.44
3.45
5.36
4.26
10.04
6.28
16.04
9.43
23.45
_
_
­
1
6.32
16.54
27.02
37.76
5.36
7.29
13.66
19.42
22.24
31.81
30.62
44.90
_
_
0
6.78
14.01
23.91
36.48
5.79
7.78
11.12
16.90
19.12
28.70
29.34
43.63
_
_
1
3.11
6.92
11.60
17.15
2.04
4.17
4.01
9.84
6.80
16.41
9.99
24.30
Reduced
model
was
fit
using
first­
order
autoregressive
covariance
structure
with
heterogeneous
variances.
Effects
retained
in
the
reduced
model
were
the
following
(
tL
denote
the
linear
time
effect,
tQ
denotes
the
quadratic):
y0,
z1,
z2,
z3,
tL,
tL*
z2,
tL*
z3,
tQ,
tQ*
z3.
Section
5.0
Results
and
Statistical
Analysis
5­
25
Table
23.
Analysis
of
Differences
in
Levels
for
With
and
Without
hCG
Stimulation:
Original­
Scale
Models
Dependent
Variable:
diff(
Y0)
diff(
Y1)
diff(
Y2)
diff(
Y3)
diff(
Y4)

Dependent
Variable
Mean:
0.12
1.65
7.06
14.44
22.64
Significance
of
Model
Terms:
z1
z2
xxx
xxx
xxx
z3
xx
xxx
xxx
xxx
R2
0.175
0.457
0.687
0.722
0.712
RMSE
0.25
1.05
3,49
6,57
10.04
x
=
statistically
significant
effect
at
0.10
level
of
significance.
xx
=
statistically
significant
effect
at
0.05
level
of
significance.
xxx
=
statistically
significant
effect
at
0.01
level
of
significance.

Table
24.
Adjusted
Mean
Differences
(
With­
Without
hCG),
Based
on
Original­
Scale
Models
Level
of
Independent
Variable
Mean
Levels
of
Dependent
Variables
z1
z2
z3
Mean
diff
(
Y0)
Mean
diff
(
Y1)
Mean
diff
(
Y2)
Mean
diff
(
Y3)
Mean
diff
(
Y4)

­
1
0.20
1.16
5.20
12.00
18.13
0
0.08
1.64
7.59
15.67
24.99
+
1
0.06
2.16
8.40
15.64
24.80
­
1
0.09
1.98
7.53
14.77*
23.84*

0
0.14
1.89
10.61
22.93
35.20
+
1
0.12
1.09
3.04**
5.62**
8.89**

­
1
0.15
2.31
10.53
20.48
29.31
0
0.19
1.79
7.15
15.65
26.67
+
1
0.01
0.85**
3.50**
7.19**
11.94**

Shaded
cell
indicates
highest
mean
estimated
level.
*
Indicates
that
the
mean
level
is
significantly
lower
than
the
cell
with
the
maximum
estimated
level,
p=
0.05.
**
Indicates
that
the
mean
level
is
significantly
lower
than
the
cell
with
the
maximum
estimated
level,
p=
0.01.
Section
5.0
Results
and
Statistical
Analysis
5­
26
Tables
25
and
26
show
comparable
results
for
the
differences
of
the
log­
transformed
data.
In
this
case,
rat
age
appears
less
important
and
atmosphere
type
is
the
most
dominant
factors.

Table
25.
Analysis
of
Differences
in
Levels
for
With
and
Without
hCG
Stimulation:
Log­
Scale
Models
Dependent
Variable:
diff
(
log(
Y0))
diff
(
log(
Y1))
diff
(
log(
Y2))
diff
(
log(
Y3))
diff
(
log(
Y4))

Dependent
Variable
Mean:
0.175
0.377
0.802
1.072
1.286
Significance
of
Model
Terms:
z1
x
z2
xx
xxx
xxx
z3
R2
0.142
0.305
0.528
0.585
0.503
RMSE
0.393
0.289
0.313
0.335
0.405
x
=
statistically
significant
effect
at
0.10
level
of
significance.
xx
=
statistically
significant
effect
at
0.05
level
of
significance.
xxx
=
statistically
significant
effect
at
0.01
level
of
significance.

Table
26.
Adjusted
Mean
Differences
(
With­
Without
hCG),
Based
on
Log­
Scale
Models
Level
of
Independent
Variable
Mean
Levels
of
Dependent
Variables
z1
z2
z3
Mean
diff
log(
Y0)
Mean
diff
log(
Y1)
Mean
diff
log(
Y2)
Mean
diff
log(
Y3)
Mean
diff
log(
Y4)

­
1
0.282
0.289
0.659**
0.963
1.166
0
0.079
0.307
0.732
0.995
1.197
+
1
0.164
0.533
1.017
1.278
1.496
­
1
0.186
0.493
0.956
1.248
1.486
0
0.242
0.380
0.931
1.298
1.503
+
1
0.096
0.257
0.520**
0.670**
0.869**

­
1
0.236
0.446
0.957
1.259
1.447
0
0.243
0.274
0.637*
0.948
1.170
+
1
0.045
0.410
0.814
1.008
1.241
Shaded
cell
indicates
highest
mean
estimated
level.
*
Indicates
that
the
mean
level
is
significantly
lower
than
the
cell
with
the
maximum
estimated
level,
p=
0.05.
**
Indicates
that
the
mean
level
is
significantly
lower
than
the
cell
with
the
maximum
estimated
level,
p=
0.01.
Section
6.0
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
6­
1
6.0
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
6.1
Objectives
Since
the
gaseous
atmosphere
of
5%
CO
2
/
95%
O
2
was
optimal,
and
it
was
thought
that
most
laboratories
would
not
have
incubators
to
accommodate
this
mixture,
a
comparison
was
made
between
incubated
samples
and
those
in
media
that
had
be
gassed
with
the
mixture.

The
Phase
IB
assay
optimization
experiment
involved
assessing
the
effect
of
a
single
experimental
factor
 
using
gassed
or
incubated
samples.
All
other
factors
were
held
fixed.
Ten
trials
of
each
condition
were
run
both
with
and
without
hCG
stimulation.
For
each
trial,
repeated
measurements
are
taken
at
baseline
(
time
0)
and
at
1,
2,
3,
and
4
hours
after
baseline.
The
objective
of
the
experiment
was
to
assess
whether
the
treatments
differed
in
terms
of
the
resultant
testosterone
levels.

6.2
Data
Two
basic
SAS
data
sets
were
constructed
from
the
raw
data
,
and
2
fundamental
types
of
dependent
variables
were
used
in
the
analyses
of
each
type:

?
Date
Set
1:
Cases
without
hCG
stimulation
Dependent
variables:
testosterone
concentrations
Dependent
variables:
(
natural)
logarithm
of
testosterone
concentrations
?
Data
Set
2:
Cases
with
hCG
stimulation
Dependent
variables:
testosterone
concentrations
Dependent
variables:
(
natural)
logarithm
of
testosterone
concentrations
Each
data
set
can
be
viewed
as
consisting
of
20
observations
(
rows).
Each
observation
includes
dependent
variable
values
for
4
time
points
and
a
corresponding
baseline
level.
Each
observation
also
includes
data
identifying
the
levels
of
the
pertinent
factors.
Data
are
listed
in
Table
27
for
the
unchallenged
samples
and
in
Table
28
for
the
challenged
cases.

6.3
Statistical
Analysis
Methods
ANOVA
was
used
to
analyze
the
data
for
each
individual
time
point
(
including
the
baseline).
ANOVAs
were
performed
for
both
original­
scale
data
and
log­
scaled
data
(
natural
logarithm).
ANOCOVA
models
utilizing
the
baseline
level
(
or
log­
level)
as
a
covariate
were
Section
6.0
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
6­
2
also
employed.
Section
6.0
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
6­
3
6.3.1
Results
Overall
Characterization
of
the
Data.
Table
29
provides
summary
statistics
characterizing
the
testosterone
levels
in
the
non­
hCG­
stimulated
data
set.
This
summary
ignores
the
experimental
factor.
The
top
portion
of
the
table
gives,
by
hour,
the
sample
size
(
n),
the
mean,
standard
deviation,
sum,
minimum,
and
maximum.
These
variables
are
denoted
as
yJ,
where
J
denotes
the
hour
and
takes
on
values
of
0,
1,
2,
3,
and
4.
The
lower
portion
of
the
table
gives
the
correlations
between
the
hourly
data.
The
following
trends
are
apparent:

?
the
means
continue
to
increase
over
time,
?
the
standard
deviations
also
increase
over
time
(
i.
e.,
as
the
mean
level
gets
larger),
and
?
the
correlations
are
generally
high
and
tend
to
be
largest
for
adjacent
hours.

Table
30
provides
a
similar
summary
for
the
log­
scaled
data;
these
variables
are
denoted
as
lyJ,
where
J
denotes
the
hour.
Similar
trends
for
the
means
and
correlations
are
evident,
but
the
standard
deviations
tend
to
be
fairly
stable
across
the
various
time
points.

Tables
31
and
32
furnish
comparable
information
for
the
hCG­
stimulated
samples.
Similar
trends
are
evident
for
these
data.
Mean
levels
tend
to
be
much
higher
than
for
the
nonstimulated
samples.

Analysis
of
Variance
and
Covariance.
Means,
by
hour
and
sample
condition,
are
presented
in
Table
33
for
the
non­
hCG­
stimulated
original­
scale
data.
The
table
gives
the
number
of
observations,
the
approximate
95%
confidence
limits
for
the
mean,
the
estimated
mean,
and
standard
deviation.
The
last
column
gives
the
mean
(
for
hours
1,
2,
3,
and
4)
adjusted
for
the
baseline
level,
as
determined
from
the
ANOCOVA.
Tables
34,
35,
and
36
give
corresponding
results
for
the
log­
scaled
data
and
the
hCG­
stimulated
cases.
The
table
below
summarizes
the
findings
detailed
in
Tables
33
through
36.
For
the
most
part,
the
ANOVAs
and
ANOCOVAs
of
the
hourly
data
did
not
detect
significant
differences
between
the
testosterone
levels
of
the
gassed
and
incubated
samples.
If
the
0.05
significance
level
is
used
to
judge
statistical
significance,
then
only
the
baseline
case
of
Table
33
yielded
a
significant
difference.
Section
6.0
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
6­
4
Source
Type
Data
Analyzed
ANOVA
Results
ANOCOVA
Results
Table
33
Original
scale,
non
hCG
stimulated
Baseline
gassed
samples
have
lower
testosterone
mean
(
p=
0.05);
all
other
hours
not
significantly
different
at
0.10
level.
Hour­
1
and
hour­
4
gassed
samples
have
higher
testosterone
means
(
p=
0.08
and
0.07,
respectively);
other
hours
not
significantly
different
at
0.10
level.

Table
34
Log
scale,
non
hCG
stimulated
Baseline
gassed
samples
have
lower
testosterone
mean
(
p=
0.06);
all
other
hours
not
significantly
different
at
0.10
level.
No
significant
differences
at
0.10
level.

Table
35
Original
scale,
hCG
stimulated
No
significant
differences
at
0.10
level.
No
significant
differences
at
0.10
level.

Table
36
Log
scale,
hCG
stimulated
No
significant
differences
at
0.10
level.
No
significant
differences
at
0.10
level.
6­

5
Section
6
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
Table
27.
Phase
IB
Data:
Sample
Without
hCG
Ear
Tag
Body
Weight
g
Testis
Wt
g
Section
Wt
g
Run
Number
x0
y0
=
Testos.
Conc.
Baseline
y1
=
Testos.
Conc.
Hour
1
y2
=
Testos.
Conc.
Hour
2
y3
=
Testos.
Conc.
Hour
3
y4
=
Testos.
Conc.
Hour
4
320
361.74
RIGHT
­
1.5641
0.2413
1
1
0.35
3.49
5.46
6.66
8.05
321
361.74
LEFT
­
1.5303
0.2657
2
1
0.38
3.92
4.95
6.88
8.27
321
365.84
RIGHT
­
1.8176
0.2623
3
1
0.36
4.66
6.52
7.88
8.53
321
365.84
LEFT
­
1.8273
0.2463
4
1
0.34
3.94
4.94
6.30
7.71
321
365.84
LEFT
­
1.8273
0.2389
5
1
0.32
4.53
6.36
7.56
7.73
322
372.13
RIGHT
­
1.5835
0.2341
6
1
0.14
3.30
4.34
5.65
6.40
322
372.13
LEFT
­
1.5263
0.2436
7
1
0.18
1.67
2.47
3.16
3.63
323
365.46
RIGHT
­
1.7040
0.2722
8
1
0.45
3.87
5.41
6.06
7.90
324
356.94
RIGHT
­
1.4908
0.2626
9
1
0.42
5.32
7.49
9.71
11.01
323
365.46
LEFT
­
1.6760
0.2711
10
1
0.35
5.32
7.07
8.83
10.17
320
361.74
RIGHT
­
1.5641
0.2713
11
2
0.49
5.06
7.37
9.13
10.97
320
361.74
LEFT
­
1.5303
0.2543
12
2
0.38
2.94
4.36
5.44
6.45
321
365.84
RIGHT
­
1.8176
0.2610
13
2
0.38
4.76
6.78
8.51
9.21
321
365.84
LEFT
­
1.8273
0.2408
14
2
0.36
3.64
5.37
6.34
6.87
322
372.13
RIGHT
­
1.5835
0.2472
15
2
0.33
3.38
4.87
5.53
6.41
322
372.13
LEFT
­
1.5263
0.2499
16
2
0.46
4.45
5.56
6.85
8.15
323
365.46
RIGHT
­
1.7040
0.2474
17
2
0.40
4.26
6.30
7.99
9.13
323
365.46
LEFT
­
1.6760
0.2630
18
2
0.39
3.56
5.63
6.78
7.71
324
356.94
RIGHT
­
1.4908
0.2365
19
2
0.37
2.62
3.69
4.82
5.84
324
356.94
LEFT
­
1.4136
0.2684
20
2
0.49
4.56
5.85
6.55
9.02
x0
=
sample
condition:
1
=
gassed,
2
=
incubated
6­

6
Section
6
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
Table
28.
Phase
IB
Data:
Samples
With
hCG
Ear
Tag
Body
Weight
g
Testis
Wt
g
Section
Wt
g
Run
Number
x0
y0
=
Testos.
Conc.
Baseline
y1
=
Testos.
Conc.
Hour
1
y2
=
Testos.
Conc.
Hour
2
y3
=
Testos.
Conc.
Hour
3
y4
=
Testos.
Conc.
Hour
4
320
361.74
RIGHT
­
1.5641
0.2387
1
1
0.36
9.56
20.60
32.84
41.13
320
361.74
LEFT
­
1.5303
0.2727
2
1
0.52
6.31
13.49
22.25
29.13
321
365.84
RIGHT
­
1.8176
0.2518
3
1
0.40
5.12
13.29
18.32
25.33
321
365.84
LEFT
­
1.8273
0.2748
4
1
0.48
7.57
14.92
23.79
30.83
321
365.84
LEFT
­
1.8273
0.2681
5
1
0.43
4.84
9.66
13.47
18.59
322
372.13
RIGHT
­
1.5835
0.2429
6
1
0.28
8.73
18.74
30.71
45.84
322
372.13
LEFT
­
1.5263
0.2523
7
1
0.23
4.18
9.38
12.68
18.38
323
365.46
RIGHT
­
1.7040
0.2550
8
1
0.42
5.95
12.41
21.00
30.28
324
356.94
RIGHT
­
1.4908
0.2489
9
1
0.12
5.09
11.99
17.25
28.43
323
365.46
LEFT
­
1.6760
0.2513
10
1
0.21
3.40
8.04
13.78
19.14
320
361.74
RIGHT
­
1.5641
0.2451
11
2
0.34
6.71
17.75
29.37
39.76
320
361.74
LEFT
­
1.5303
0.2409
12
2
0.38
5.07
12.01
17.63
23.46
321
365.84
RIGHT
­
1.8176
0.2471
13
2
0.24
7.90
17.49
27.99
39.46
321
365.84
LEFT
­
1.8273
0.2695
14
2
0.41
7.52
19.79
30.29
43.69
322
372.13
RIGHT
­
1.5835
0.2452
15
2
0.37
4.24
10.43
14.70
19.41
322
372.13
LEFT
­
1.5263
0.2635
16
2
0.31
5.06
11.70
19.98
29.90
323
365.46
RIGHT
­
1.7040
0.2615
17
2
0.44
5.71
13.87
21.35
30.16
323
365.46
LEFT
­
1.6760
0.2328
18
2
0.72
6.93
21.00
31.05
40.41
324
356.94
RIGHT
­
1.4908
0.2632
19
2
0.81
5.73
13.45
23.01
34.28
324
356.94
LEFT
­
1.4136
0.2491
20
2
0.31
6.14
16.68
26.65
33.43
x0
=
sample
condition:
1
=
gassed,
2
=
incubated
6­

7
Section
6
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
Table
29.
Summary
of
Data
 
Original
Scale,
Without
hCG
Simple
Statistics
Variable
N
Mean
Std
Dev
Sum
Minimum
Maximum
Label
Y0
20
0.36700
0.08652
7.34000
0.14000
0.49000
y0
=_
Testos._
Conc._
Baseline
Y1
20
3.96250
0.92905
79.25000
1.67000
5.32000
y1
=_
Testos._
Conc._
Hour
1
Y2
20
5.53950
1.26145
110.79000
2.47000
7.49000
y2
=_
Testos._
Conc._
Hour
2
Y3
20
6.83150
1.57510
136.63000
3.16000
9.71000
y3
=_
Testos._
Conc._
Hour
3
Y4
20
7.95800
1.76957
159.16000
3.63000
11.01000
y4
=_
Testos._
Conc._
Hour
4
Pearson
Correlation
Coefficients,
N
=
20Prob
>
|
r|
under
H0:
Rho=
0
Y0
Y1
Y2
Y3
Y4
Y0y0
=_
Testos._
Conc._
Baseline
1.00000
0.56839
0.0089
0.57435
0.0081
0.51050
0.0215
0.65502
0.0017
Y1y1
=_
Testos._
Conc._
Hour
1
0.56839
0.0089
1.00000
0.95710
<.
0001
0.94357
<.
0001
0.94257
<.
0001
Y2y2
=_
Testos._
Conc._
Hour
2
0.57435
0.0081
0.95710
<.
0001
1.00000
0.97439
<.
0001
0.94352
<.
0001
Y3y3
=_
Testos._
Conc._
Hour
3
0.51050
0.0215
0.94357
<.
0001
0.97439
<.
0001
1.00000
0.95462
<.
0001
Y4y4
=_
Testos._
Conc._
Hour
4
0.65502
0.0017
0.94257
<.
0001
0.94352
<.
0001
0.95462
<.
0001
1.00000
6­

8
Section
6
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
Table
30.
Summary
of
Data
 
Log
Scale,
Without
hCG
Simple
Statistics
Variable
N
Mean
Std
Dev
Sum
Minimum
Maximum
ly0
20
­
1.03875
0.30294
­
20.77491
­
1.96611
­
0.71335
ly1
20
1.34511
0.27439
26.90217
0.51282
1.67147
ly2
20
1.68265
0.26153
33.65299
0.90422
2.01357
ly3
20
1.89300
0.25530
37.86004
1.15057
2.27316
ly4
20
2.04705
0.25043
40.94100
1.28923
2.39880
Pearson
Correlation
Coefficients,
N
=
20Prob
>
|
r|
under
H0:
Rho=
0
ly0
ly1
ly2
ly3
ly4
ly0
1.00000
0.5842
20.0068
0.60978
0.0043
0.55761
0.0106
0.65998
0.0015
ly1
0.58422
0.0068
1.00000
0.96741
<.
0001
0.95797
<.
0001
0.95831
<.
0001
ly2
0.60978
0.0043
0.96741
<.
0001
1.00000
0.97796
<.
0001
0.95576
<.
0001
ly3
0.55761
0.0106
0.95797
<.
0001
0.97796
<.
0001
1.00000
0.96540
<.
0001
ly4
0.65998
0.0015
0.95831
<.
0001
0.95576
<.
0001
0.96540
<.
0001
1.00000
6­

9
Section
6
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
Table
31.
Summary
of
Data
 
Original
Scale,
With
hCG
Simple
Statistics
Variable
N
Mean
Std
Dev
Sum
Minimum
Maximum
Label
YC0
20
0.38900
0.16189
7.78000
0.12000
0.81000
yc0
=_
Testos._
Conc._
Baseline
YC1
20
6.08800
1.58725
121.76000
3.40000
9.56000
yc1
=_
Testos._
Conc._
Hour
1
YC2
20
14.33450
3.87932
286.69000
8.04000
21.00000
yc2
=_
Testos._
Conc._
Hour
2
YC3
20
22.40550
6.48074
448.11000
12.68000
32.84000
yc3
=_
Testos._
Conc._
Hour
3
YC4
20
31.05200
8.66685
621.04000
18.38000
45.84000
yc4
=_
Testos._
Conc._
Hour
4
Pearson
Correlation
Coefficients,
N
=
20Prob
>
|
r|
under
H0:
Rho=
0
YC0
YC1
YC2
YC3
YC4
YC0yc0
=_
Testos._
Conc._
Baseline
1.00000
0.14785
0.5339
0.24875
0.2903
0.25182
0.2841
0.19761
0.4037
YC1yc1
=_
Testos._
Conc._
Hour
1
0.14785
0.5339
1.00000
0.88953
<.
0001
0.90763
<.
0001
0.87874
<.
0001
YC2yc2
=_
Testos._
Conc._
Hour
2
0.24875
0.2903
0.88953
<.
0001
1.00000
0.97501
<.
0001
0.93343
<.
0001
YC3yc3
=_
Testos._
Conc._
Hour
3
0.25182
0.2841
0.90763
<.
0001
0.97501
<.
0001
1.00000
0.96812
<.
0001
YC4yc4
=_
Testos._
Conc._
Hour
4
0.19761
0.4037
0.87874
<.
0001
0.93343
<.
0001
0.96812
<.
0001
1.00000
6­

10
Section
6
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
Table
32.
Summary
of
Data
 
Log
Scale,
With
hCG
Simple
Statistics
Variable
N
Mean
Std
Dev
Sum
Minimum
Maximum
lyc0
20
­
1.02614
0.42653
­
20.52279
­
2.12026
­
0.21072
lyc1
20
1.77407
0.26184
35.48142
1.22378
2.25759
lyc2
20
2.62709
0.27611
52.54184
2.08443
3.04452
lyc3
20
3.06710
0.30327
61.34207
2.54003
3.49165
lyc4
20
3.39608
0.29444
67.92156
2.91126
3.82516
Pearson
Correlation
Coefficients,
N
=
20Prob
>
|
r|
under
H0:
Rho=
0
lyc0
lyc1
lyc2
lyc3
lyc4
lyc0
1.00000
0.2590
10.2702
0.29545
0.2060
0.3062
40.1891
0.21198
0.3696
lyc1
0.2590
10.2702
1.00000
0.91838
<.
0001
0.91443
<.
0001
0.89007
<.
0001
lyc2
0.29545
0.2060
0.91838
<.
0001
1.00000
0.96817
<.
0001
0.93546
<.
0001
lyc3
0.30624
0.1891
0.91443
<.
0001
0.96817
<.
0001
1.00000
0.97426
<.
0001
lyc4
0.21198
0.3696
0.89007
<.
0001
0.93546
<.
0001
0.97426
<.
0001
1.00000
6­

11
Section
6
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
Table
33.
Summary
of
Results
by
Sample
Condition
 
Original
Scale,
Without
hCG
x0
N
Obs
Variable
Lower
95%
CL
for
Mean
Upper
95%
CL
for
Mean
Mean
Std
Dev
ANOCOVA
Adjusted
Mean
1
10
Y0
Y1
Y2
Y3
Y4
0.26
3.23
4.45
5.57
6.51
0.40
4.77
6.55
8.17
9.37
0.
33
4.00
5.50
6.87
7.94
0.101.08
1.47
1.82
2.00
4.30
5.89
7.32
8.57
2
10
Y0
Y1
Y2
Y3
Y4
0.37
3.34
4.79
5.80
6.82
0.44
4.51
6.36
7.79
9.13
0.41
3.92
5.58
6.79
7.98
0.06
0.81
1.10
1.39
1.62
3.62
5.19
6.34
7.34
x0
=
sample
condition:
1
=
gassed,
2
=
incubated
Bolded
entries
are
statistically
significant
at
the
0.10
level.
Bolded
and
underlined
entries
are
statistically
significant
at
the
0.05
level.
6­

12
Section
6
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
Table
34.
Summary
of
Results
by
Sample
Condition
 
Log
Scale,
Without
hCG
x0
N
Obs
Variable
Lower
95%
CL
for
Mean
Upper
95%
CL
for
Mean
Mean
Std
Dev
ANOCOVA
Adjusted
Mean
1
10
ly0
ly1
ly2
ly3
ly4
­
1.433
1.105
1.437
1.667
1.820
­
0.898
1.583
1.893
2.110
2.252
­
1.165
1.344
1.665
1.889
2.036
0.374
0.334
0.318
0.309
0.302
1.426
1.743
1.961
2.118
2
10
ly0
ly1
ly2
ly3
ly4
­
1.008
1.190
1.553
1.751
1.914
­
0.816
1.502
1.848
2.044
2.202
­
0.912
1.346
1.700
1.897
2.058
0.134
0.218
0.206
0.205
0.202
1.264
1.623
1.825
1.976
x0
=
sample
condition:
1
=
gassed,
2
=
incubated
Bolded
entries
are
statistically
significant
at
the
0.10
level.
Bolded
and
underlined
entries
are
statistically
significant
at
the
0.05
level.
6­

13
Section
6
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
Table
35.
Summary
of
Results
by
Sample
Condition
 
Original
Scale,
With
hCG
x0
N
Obs
Variable
Lower
95%
CL
for
Mean
Upper
95%
CL
for
Mean
Mean
Std
Dev
ANOCOVA
Adjusted
Mean
1
10
YC0
YC1
YC2
YC3
YC4
0.25
4.65
10.39
15.60
22.10
0.44
7.50
16.12
25.62
35.31
0.35
6.08
13.25
20.61
28.71
0.13
1.99
4.00
7.00
9.23
6.14
13.44
20.94
29.02
2
10
YC0
YC1
YC2
YC3
YC4
0.30
5.27
12.82
20.13
27.80
0.57
6.93
18.01
28.28
38.99
0.43
6.10
15.42
24.20
33.40
0.18
1.16
3.62
5.69
7.82
6.03
15.22
23.87
33.09
x0
=
sample
condition:
1
=
gassed,
2
=
incubated
Bolded
entries
are
statistically
significant
at
the
0.10
level.
Bolded
and
underlined
entries
are
statistically
significant
at
the
0.05
level.
6­

14
Section
6
Statistical
Analysis
of
the
Phase
IB
Assay
Optimization
Experiment
Table
36.
Summary
of
Results
by
Sample
Condition
 
Log
Scale,
With
hCG
x0
N
Obs
Variable
Lower
95%
CL
for
Mean
Upper
95%
CL
for
Mean
Mean
Std
Dev
ANOCOVA
Adjusted
Mean
1
10
lyc0
lyc1
lyc2
lyc3
lyc4
­
1.474
1.524
2.331
2.737
3.085
­
0.818
1.989
2.757
3.214
3.538
­
1.146
1.757
2.544
2.975
3.312
0.458
0.325
0.297
0.334
0.317
1.776
2.562
2.995
3.323
2
10
lyc0
lyc1
lyc2
lyc3
lyc4
­
1.176
1.652
2.539
2.978
3.295
­
0.637
1.931
2.881
3.340
3.665
­
0.906
1.792
2.710
3.159
3.480
0.377
0.195
0.239
0.253
0.259
1.772
2.693
3.139
3.469
x0
=
sample
condition:
1
=
gassed,
2
=
incubated
Bolded
entries
are
statistically
significant
at
the
0.10
level.
Bolded
and
underlined
entries
are
statistically
significant
at
the
0.05
level.
Section
7.0
Discussion
7­
1
7.0
DISCUSSION
The
optimization
of
the
sliced
testis
assay
is
necessary
in
order
to
proceed
to
the
prevalidation
and
validation
stages
of
the
testing
of
the
assay
for
use
in
the
Tier
I
tests
for
screening
of
substances
for
potential
as
endocrine
disruptors.
The
Phase
I
studies
have
contributed
to
the
initial
portion
of
this
optimization.
These
factors
will
be
used
for
the
rest
of
the
optimization
in
Part
II.
Without
these
initial
studies,
we
would
not
have
used
the
best
gaseous
atmosphere
for
optimal
testosterone
concentrations
from
the
testicular
tissues.
Section
9.0
Incubation
Experiment
8­
1
8.0
CONCLUSIONS
FOR
PHASE
I
The
testosterone
RIA
and
the
LDH
assay
can
both
be
verified
with
M­
199
without
phenol
red.
Both
were
validated
and
show
the
characteristics
necessary
for
use
to
optimize
the
sliced
testis
assay.

There
were
certain
factors
in
the
initial
Phase
I
experiments
that
definitely
were
not
beneficial
to
use
in
the
assay.
For
instance,
22­
week­
old
rats
do
not
show
the
responsiveness
in
their
testicular
tissue
that
is
necessary
for
an
optimal
assay.
The
air
atmosphere
was
also
not
a
favorable
condition
for
the
assay.
The
prototypical
assay
media,
Media
199
without
phenol
red,
was
equal
to
any
of
the
others
tested.
Statistical
analysis
was
necessary
to
show
that
the
atmosphere
of
5%
CO
2/
95%
O
2
was
optimal,
and
that
rats
of
the
11­
to
15­
week­
old
range
could
be
used
for
the
assay.
From
these
conclusions,
we
were
ready
to
advance
to
the
Phase
II
experiments.

Media
199
without
phenol
red
will
be
used
after
it
is
gassed
with
the
5
%
CO
2/
95%
O
2
mixture
and
pH
adjusted
to
7.4
for
testicular
tissues
from
11­
to
15­
week­
old
rats
for
the
Phase
II
experimental
studies.
Section
9.0
Incubation
Experiment
9­
1
PHASE
II
Phase
II
Experiments
consisted
of
several
groups
of
studies.
The
multifactorial
studies
were
for
the
incubation
conditions
of
the
assay
and
the
testes
slice
preparation
factors
of
the
assay.
After
the
optimal
factors
were
determined,
the
LDH
experiments
were
performed
to
determine
LDH
and
testosterone
concentrations
under
various
conditions.

9.0
INCUBATION
EXPERIMENT
9.1
Statistical
Analysis
of
Phase
II
Incubation
Optimization
Experiment
9.1.1
Objectives
The
objectives
of
the
incubation
optimization
experiment
were
to
determine
the
effect
of
various
incubation
factor
settings
on
testosterone
production
using
the
sliced
testis
assay
and
to
identify
the
incubation
factor
settings
that
maximize
the
amount
of
testosterone
production.
These
experiments
tested
the
effect(
s)
of
six
factors:
incubation
temperature,
incubation
vessel
type,
incubation
shaker
speed,
incubation
media
volume,
hCG
concentration,
and
testicular
fragment
size.
Table
37
presents
the
incubation
factor
settings
used
in
this
experiment.
Each
trial
in
the
experiment
was
run
with
and
without
hCG
stimulation,
repeated
measurements
were
taken
at
baseline
(
time
0)
and
at
1,
2,
3,
and
4
hours
after
baseline.

9.1.2
Data
A
SAS
data
set
was
constructed
from
the
raw
Excel
data
files,
and
2
fundamental
types
of
dependent
variables
were
used
in
the
analyses:
the
original
testosterone
concentrations
and
the
(
natural)
logarithm
of
the
testosterone
concentrations.
Each
observation
includes
dependent
variable
values
for
4
time
points
and
a
corresponding
baseline
level.
Each
observation
also
includes
data
identifying
the
levels
of
the
pertinent
factors.
The
data
used
in
the
analysis
are
displayed
in
Tables
38
and
39.

9.1.3
Statistical
Analysis
Methods
In
order
to
assess
the
effects
of
each
incubation
factor
on
the
amount
of
testosterone
production,
ANOVA
models
were
fit
to
the
data
separately
for
the
trials
with
and
without
hCG
stimulation.
For
each
hCG
stimulation
type,
the
ANOVA
models
were
fit
to
each
individual
time
point.
All
main
effects
and
2­
factor
interactions
(
2fi)
of
the
6
factors
were
initially
included
in
the
models.
Tests
for
interactions
were
conducted,
and
where
they
were
not
detected
as
statistically
significant
(
p=
0.10),
a
reduced
model
was
employed
that
retained
the
main
effects,
Section
9.0
Incubation
Experiment
9­
2
the
baseline
covariate,
and
only
those
2fi's
deemed
to
have
significant
effects.
Once
the
final
ANOVA
model
was
determined,
the
data
were
fit
using
a
response
surface
regression
analysis
to
determine
the
maximum
predicted
value
of
testosterone
and
the
incubation
factor
levels
associated
with
the
maximum.
Additional
details
are
provided
in
the
Results
portion
of
this
section.

9.1.4
Results
To
assess
the
effects
of
each
incubation
factor
on
testosterone
production,
an
ANOVA
model
was
fit
separately
to
the
set
of
data
for
each
hCG
stimulation
type
and
each
time
point
beyond
the
baseline.

Table
37.
Incubation
Factor
Settings
and
Coded
Values
Factor
Identification
Units
Variable
Name
Factor
Name
Experimental
Levels
Coded
Experimental
Levels
1
2
3
4
5
1
2
3
4
5
Incubation
Temperature
oC
Temp
X1
­­
34*
­­
37
­­
­­
­
1
­­
+
1
­­

Incubation
Vessel
Type
NAa
Vessel
X2
scintillation
vial*
test
tube
­­
­­
­­
­
1
+
1
­­
­­
­­

Incubation
Shaker
Speed
NA
Speed
X3
­­
none
low
high*
­­
­­
­
1
0
+
1
­­

Incubation
Media
Volume
ml
Inc_
vol
X4
­­
2.5
5*
10
­­
­­
­
1
0
+
1
­­

hCG
Concentration
IU/
ml
hCG_
co
nc
X5
0.001
0.01
0.1*
1
10
­
2
­
1
0
+
1
+
2
Fragment
Size
mg
mg_
tiss
ue
X6
25
50
125
250*
­­
­
0.8
­
0.6
0
+
1
­­

*
Prototypical
value.
a
NA
­
not
applicable.
Note:
Shaker
speed
was
treated
as
a
continuous
factor.

The
ANOVA
models
containing
all
main
effects
and
2fi's
were
fit
to
the
original
testosterone
concentrations
and
the
logarithms
of
testosterone
concentrations
using
PROC
GLM.
The
logarithm
of
the
baseline
testosterone
concentration
(
T0)
was
also
added
to
the
model
as
a
dependent
variable.
Upon
examining
the
residuals
from
fitting
the
full
ANOVA
models,
the
residuals
for
the
original
testosterone
concentrations
showed
signs
of
heteroskedasticity.
The
residuals
for
the
logarithms
of
testosterone
concentrations
appear
homoskedastic
and
randomly
distributed
around
zero,
thereby
satisfying
the
assumptions
necessary
for
valid
ANOVA
modeling
results.
The
logarithms
of
the
testosterone
concentrations
were
therefore
chosen
as
the
more
appropriate
dependent
variables
for
the
analysis
of
the
incubation
experiment
data.
Section
9.0
Incubation
Experiment
9­
3
For
the
full
ANOVA
models
using
the
logarithm
of
testosterone
concentration
as
the
dependent
variable,
we
examined
the
t­
tests
for
the
significance
of
each
factor
in
the
model.
In
the
first
round
of
model
evaluation,
we
examined
the
t­
test
results
for
the
2fis
to
see
if
any
of
those
terms
could
be
removed
from
the
models.
Any
2fis
with
p­
values
greater
than
0.1
were
removed
from
the
models.
The
reduced
models,
one
model
for
the
data
without
hCG
stimulation
and
one
model
for
the
data
with
hCG
stimulation,
were
fit
to
the
logarithms
of
testosterone
concentration
again
and
the
t­
tests
of
the
model
terms
were
re­
evaluated
for
significance.
After
2
rounds
of
removing
insignificant
2fis,
we
arrived
at
the
final
reduced
models
containing
only
significant
2fis
and
first
order
factors.

To
obtain
the
optimal
combination
of
factors
for
each
time
point,
the
data
were
fit
using
response
surface
regression
models
and
the
SAS
procedure
RSREG.
(
The
PROC
GLM
modeling
results
were
used
to
determine
which
factors
and
2fis
needed
to
remain
in
the
models.
Any
factors
which
did
not
have
any
associated
2fis
in
the
final
models
were
treated
as
covariates
in
the
response
surface
regression
models.)
The
ANOVA
results
for
the
response
surface
regressions
are
summarized
in
Table
40.
The
parameter
estimates
from
the
response
surface
regression
models
for
the
data
without
hCG
stimulation
are
presented
in
Table
41,
and
the
parameter
estimates
for
the
data
with
hCG
stimulation
are
presented
in
Table
42.
Because
the
vessel
type
is
a
categorical
variable,
we
needed
to
run
separate
models
for
each
vessel
type
in
PROC
RSREG.
Differentiation
of
the
prediction
equation
is
used
to
determine
the
location
of
the
stationary
point;
in
most
cases
the
stationary
point
was
not
a
point
of
maximal
response.
Under
these
circumstances,
the
RIDGE
technique
in
SAS
is
used
to
find
a
point
of
maximal
response
within
the
experimental
region.
Starting
at
the
center
of
the
experimental
region,
a
hypersphere
having
a
given
radius
is
considered,
and
the
point
on
the
hypersphere
with
the
maximal
response
is
determined.
The
radius
is
then
incremented
and
the
maximum
on
that
hypersphere
is
determined.
This
process,
when
repeated,
traces
out
a
"
ridge
of
maximal
response."
The
iterations
are
terminated
when
one
of
the
factors
reaches
the
extremity
of
its
range,
as
defined
by
the
levels
used
in
the
experiment.
We
determined
the
factor
combination
that
produced
the
maximum
predicted
value
of
logarithm
of
testosterone
concentration
for
each
time
point
at
each
combination
of
with/
without
hCG
stimulation
and
for
each
vessel
type.
The
optimal
value
factor
combinations
were
plugged
back
into
the
response
surface
regression
models
to
obtain
predicted
values
for
the
optimal
factor
combinations
at
all
time
points.
For
example,
we
used
the
RIDGE
regression
results
to
determine
the
optimal
factor
combinations
for
the
first
time
point
(
T1)
for
the
scintillation
vial
without
hCG
stimulation.
We
then
used
the
optimal
T1
combination
to
obtain
the
predicted
values
for
logarithm
of
testosterone
concentration
and
their
standard
errors
for
the
scintillation
vial
without
hCG
stimulation
for
that
time
point
and
for
all
the
other
time
points
(
T2,
T3
and
T4).
The
optimal
factor
results,
presented
in
Table
43,
show
the
effects
of
the
optimal
factors
for
1
time
point
on
the
other
time
points
in
evaluating
the
best
combination
of
factors
to
use
for
further
experimentation.
Section
9.0
Incubation
Experiment
1
This
suggests
that
the
baseline
levels
are
affected
by
(
some
of)
the
factors
and
that
their
resultant
effect
on
later
times
is
diminished
if
adjustments
for
baseline
levels
are
made.

2
This
indicates
that
baseline
adjustments
for
the
latter
cases
were
unnecessary,
as
was
also
apparent
from
Table
4
(
since
baseline
effect
was
not
significant).

9­
4
After
examining
the
possible
optimal
factors
for
each
time
point,
vessel
type,
and
hCG
stimulation,
the
chosen
optimal
values
and
their
predicted
values
and
standard
errors
are
presented
in
Table
44.

In
order
to
assess
the
effects
of
the
baseline
testosterone
concentrations
on
the
effects
of
factors,
the
same
analysis
steps
that
were
performed
above
were
also
repeated
on
the
logarithms
of
testosterone
concentration
but
with
the
baseline
concentration
removed
from
the
models.
The
ANOVA
results
for
the
final
reduced
model
from
PROC
RSREG
are
shown
in
Table
45.

Comparing
the
results
in
Table
45
with
those
in
Table
40
(
results
for
models
with
baseline
concentration
in
the
models)
indicates
that
there
are
many
more
significant
terms
for
the
models
without
the
baseline
concentrations.
1
The
root
mean
square
errors
(
RMSEs)
in
Table
45
are
higher
than
Table
40
for
the
cases
with
no
hCG
stimulation,
whereas
the
RMSEs
in
Table
45
are
lower
than
Table
40
for
the
cases
with
hCG
stimulation.
2
The
response
surface
regression
results
from
PROC
RSREG
are
presented
in
Tables
46
and
47.
The
optimal
values
for
each
time
point
are
shown
in
Table
48.
The
factor
combinations
that
produced
the
optimal
values
for
each
time
point
in
Table
48
are
very
similar
to
the
combinations
in
Table
43.
The
standard
errors
of
the
predicted
values
for
Table
48
are
higher
for
the
data
without
hCG
stimulation
than
Table
43,
whereas
the
standard
errors
for
Table
48
are
lower
than
Table
43
for
the
data
with
hCG
stimulation.
The
differences
in
the
standard
errors
between
the
two
types
of
models
(
with
and
without
baseline
concentrations)
are
consistent
with
the
results
of
the
RMSEs
in
Tables
40
and
45.
The
predicted
values
for
the
chosen
optimal
combination
of
factors
are
presented
in
Table
49.
The
predicted
values
in
Table
49
are
very
similar
to
the
predicted
values
in
Table
44.
The
standard
errors
for
the
cases
without
hCG
stimulation
are
similar
between
the
two
model
types;
however
the
standard
errors
for
the
cases
with
hCG
are
lower
in
Table
13
than
those
in
Table
8.
9­

5
Section
9
Phase
II
Table
38.
Data
Listing
for
Incubation
Experiment
Run
Temp
Vessel
Speed
Inc
Vol
hCG
Conc
mg
Tissue
Weight
Test
0
Test
1
Test
2
Test
3
Test
4
M
37
test
tube
high
2.5
0.010
250
0.2606
7.061
24.751
30.890
65.349
87.145
MC
37
test
tube
high
2.5
0.010
250
0.2666
7.877
65.791
272.168
N
37
test
tube
high
2.5
1.000
50
0.0569
5.800
22.671
25.308
30.404
33.919
NC
37
test
tube
high
2.5
1.000
50
0.0494
7.490
18.016
44.939
59.717
78.138
O
37
test
tube
high
10.0
0.010
50
0.0554
3.971
12.274
14.079
18.412
18.051
OC
37
test
tube
high
10.0
0.010
50
0.0480
16.667
35.833
57.500
75.000
P
37
test
tube
high
10.0
1.000
250
0.2736
0.365
9.064
11.367
13.852
16.228
PC
37
test
tube
high
10.0
1.000
250
0.2654
0.414
24.265
65.825
93.331
109.570
13
37
vial
high
2.5
0.010
50
0.0509
30.452
69.745
59.332
55.010
50.098
13C
37
vial
high
2.5
0.010
50
0.0506
59.486
103.557
206.522
175.692
13*
37
vial
high
2.5
0.010
50
0.0488
38.934
45.082
51.639
47.746
50.205
13C*
37
vial
high
2.5
0.010
50
0.0492
19.715
24.390
26.829
14
37
vial
high
2.5
1.000
250
0.2653
35.733
46.702
41.425
40.709
39.540
14C
37
vial
high
2.5
1.000
250
0.2569
24.251
240.132
378.786
407.513
635.072
14*
37
vial
high
2.5
1.000
250
0.2621
27.890
90.729
70.927
59.634
51.354
14C*
37
vial
high
2.5
1.000
250
0.2512
39.411
238.774
245.900
502.906
458.718
15
37
vial
high
10.0
0.010
250
0.2580
27.209
28.605
28.488
31.085
15C
37
vial
high
10.0
0.010
250
0.2411
4.977
42.555
110.162
134.343
216.715
15*
37
vial
high
10.0
0.010
250
0.2606
8.711
8.941
15.349
15.503
15C*
37
vial
high
10.0
0.010
250
0.2569
34.761
88.945
134.877
156.637
16
37
vial
high
10.0
1.000
50
0.0490
3.265
3.469
2.245
2.041
1.837
16C
37
vial
high
10.0
1.000
50
0.0535
1.682
2.617
4.486
5.421
6.729
16*
37
vial
high
10.0
1.000
50
0.0506
2.767
5.138
5.336
5.138
5.336
16C*
37
vial
high
10.0
1.000
50
0.0566
2.473
6.184
13.428
17.668
24.205
S
37
test
tube
low
5.0
0.100
125
0.1236
1.375
6.068
9.709
14.968
16.100
SC
37
test
tube
low
5.0
0.100
125
0.1282
4.134
7.098
12.637
15.679
19
37
vial
low
5.0
0.100
125
0.1296
6.096
44.830
59.182
72.068
75.540
9­

6
Table
38.
Data
Listing
for
Incubation
Experiment
(
Continued)

Run
Temp
Vessel
Speed
Inc
Vol
hCG
Conc
mg
Tissue
Weight
Test
0
Test
1
Test
2
Test
3
Test
4
19C
37
vial
low
5.0
0.100
125
0.1158
5.534
43.178
61.658
74.180
94.732
I
37
test
tube
none
2.5
0.010
50
0.0503
4.374
11.332
16.700
21.272
23.658
IC
37
test
tube
none
2.5
0.010
50
0.0544
4.044
19.669
39.338
40.625
57.169
J
37
test
tube
none
2.5
1.000
250
0.2430
2.469
5.802
7.284
10.329
13.951
JC
37
test
tube
none
2.5
1.000
250
0.2592
2.160
8.642
12.539
13.927
18.519
K
37
test
tube
none
10.0
0.010
250
0.2405
2.204
7.318
9.813
11.268
10.894
KC
37
test
tube
none
10.0
0.010
250
0.2496
1.482
4.888
7.973
7.652
9.014
L
37
test
tube
none
10.0
1.000
50
0.0473
3.383
7.400
9.091
8.457
LC
37
test
tube
none
10.0
1.000
50
0.0569
1.933
3.163
4.042
4.394
9
37
vial
none
2.5
0.010
250
0.2510
3.705
14.980
20.478
24.462
27.410
9C
37
vial
none
2.5
0.010
250
0.2499
3.601
15.846
23.609
35.214
10
37
vial
none
2.5
1.000
50
0.0504
8.929
55.556
58.532
70.437
77.579
10C
37
vial
none
2.5
1.000
50
0.0554
14.801
73.466
96.570
129.422
172.924
11
37
vial
none
10.0
0.010
50
0.0474
3.797
13.502
16.245
19.198
21.730
11C
37
vial
none
10.0
0.010
50
0.0515
2.330
1.553
1.748
12
37
vial
none
10.0
1.000
250
0.2467
0.365
4.945
7.053
9.360
12.282
12C
37
vial
none
10.0
1.000
250
0.2341
5.083
6.835
10.423
17.044
E
34
test
tube
high
2.5
0.010
50
0.0499
9.619
9.018
10.621
9.018
10.220
EC
34
test
tube
high
2.5
0.010
50
0.0514
12.257
41.829
57.004
82.101
97.082
F
34
test
tube
high
2.5
1.000
250
0.2463
9.013
18.270
23.752
30.694
46.041
FC
34
test
tube
high
2.5
1.000
250
0.2440
8.893
17.254
22.131
30.574
36.352
G
34
test
tube
high
10.0
0.010
250
0.2534
8.011
13.062
15.746
16.338
17.285
GC
34
test
tube
high
10.0
0.010
250
0.2503
2.797
5.473
8.670
10.228
10.907
H
34
test
tube
high
10.0
1.000
50
0.0506
3.755
7.708
12.451
13.636
16.206
HC
34
test
tube
high
10.0
1.000
50
0.0493
6.085
19.270
21.704
19.675
20.081
U
34
test
tube
high
5.0
0.100
125
0.1225
8.000
13.551
16.653
20.898
22.286
UC
34
test
tube
high
5.0
0.100
125
0.1269
4.649
13.002
14.106
20.016
24.665
U*
34
test
tube
high
5.0
0.100
125
0.1254
6.380
16.667
21.372
27.193
22.488
UC*
34
test
tube
high
5.0
0.100
125
0.1282
6.786
12.871
18.487
20.827
7.878
9­

7
Section
9
Phase
II
Table
38.
Data
Listing
for
Incubation
Experiment
(
Continued)

Run
Temp
Vessel
Speed
Inc
Vol
hCG
Conc
mg
Tissue
Weight
Test
0
Test
1
Test
2
Test
3
Test
4
5
34
vial
high
2.5
0.010
50
0.0546
11.172
11.538
11.722
12.271
9.890
5C
34
vial
high
2.5
0.010
50
0.0457
13.786
10.284
15.536
15.536
12.473
5*
34
vial
high
2.5
0.010
50
0.0520
4.615
4.615
3.654
4.808
6.154
5C*
34
vial
high
2.5
0.010
50
0.0505
7.723
11.485
11.683
8.713
7.921
6
34
vial
high
2.5
1.000
50
0.0481
15.800
12.266
12.474
13.721
16.632
6C
34
vial
high
2.5
1.000
50
0.0508
34.449
91.142
153.346
206.693
197.441
6*
34
vial
high
2.5
1.000
50
0.0485
14.639
32.371
36.907
42.474
50.928
6C*
34
vial
high
2.5
1.000
50
0.0454
16.520
14.978
20.044
20.264
26.432
7
34
vial
high
10.0
0.010
50
0.0498
3.614
7.028
9.438
10.241
15.462
7C
34
vial
high
10.0
0.010
50
0.0541
2.218
2.403
1.664
1.848
7*
34
vial
high
10.0
0.010
50
0.0530
2.642
3.396
4.340
4.717
5.094
7C*
34
vial
high
10.0
0.010
50
0.0472
2.331
2.542
1.907
8
34
vial
high
10.0
1.000
250
0.2520
2.341
12.143
13.373
13.651
14.921
8C
34
vial
high
10.0
1.000
250
0.2685
4.469
22.868
37.877
54.562
70.689
8*
34
vial
high
10.0
1.000
250
0.2441
6.145
7.948
8.726
9.586
10.651
8C*
34
vial
high
10.0
1.000
250
0.2455
5.662
31.772
53.931
98.737
142.729
21
34
vial
high
5.0
0.100
125
0.1196
8.528
8.612
9.448
6.773
9.532
21C
34
vial
high
5.0
0.100
125
0.1289
4.888
40.962
86.656
209.930
324.438
21*
34
vial
high
5.0
0.100
125
0.1227
7.742
8.883
7.661
7.579
9.128
21C*
34
vial
high
5.0
0.100
125
0.1266
8.610
24.329
38.468
48.578
68.325
Q
34
test
tube
low
5.0
0.100
125
0.1256
0.717
4.697
8.599
9.475
11.226
QC
34
test
tube
low
5.0
0.100
125
0.1214
6.096
6.260
9.967
9.720
10.626
Q*
34
test
tube
low
5.0
0.100
125
0.1288
8.463
9.084
8.929
11.102
10.093
QC*
34
test
tube
low
5.0
0.100
125
0.1320
5.909
13.712
15.833
18.561
21.136
R
34
test
tube
low
5.0
0.100
125
0.1302
2.995
4.378
9.908
15.131
14.132
RC
34
test
tube
low
5.0
0.100
125
0.1256
5.494
6.210
11.306
13.217
12.341
V
34
test
tube
low
2.5
0.100
125
0.1232
1.948
7.143
13.555
16.883
19.643
VC
34
test
tube
low
2.5
0.100
125
0.1269
2.837
11.348
20.331
31.678
49.724
W
34
test
tube
low
10.0
0.100
125
0.1237
7.842
10.024
12.369
16.006
18.432
9­

8
Table
38.
Data
Listing
for
Incubation
Experiment
(
Continued)

Run
Temp
Vessel
Speed
Inc
Vol
hCG
Conc
mg
Tissue
Weight
Test
0
Test
1
Test
2
Test
3
Test
4
WC
34
test
tube
low
10.0
0.100
125
0.1270
2.441
3.858
5.669
7.559
8.583
X
34
test
tube
low
5.0
0.001
125
0.1262
7.369
11.094
14.659
16.640
18.463
XC
34
test
tube
low
5.0
0.001
125
0.1258
6.200
12.480
13.752
17.647
19.873
Y
34
test
tube
low
5.0
10.000
125
0.1263
2.692
6.809
8.947
11.718
12.827
YC
34
test
tube
low
5.0
10.000
125
0.1247
2.165
8.340
14.354
17.562
21.010
Z
34
test
tube
low
5.0
0.100
25
0.0223
3.587
ZC
34
test
tube
low
5.0
0.100
25
0.0235
12.766
23.404
31.064
49.787
AA
34
test
tube
low
5.0
0.100
250
0.2450
1.143
4.082
6.898
8.286
10.490
AAC
34
test
tube
low
5.0
0.100
250
0.2530
1.502
3.162
4.862
7.747
8.814
17
34
vial
low
5.0
0.100
125
0.1254
20.335
24.402
21.132
50.718
53.907
17C
34
vial
low
5.0
0.100
125
0.1275
45.333
28.941
40.314
17*
34
vial
low
5.0
0.100
125
0.1223
20.687
28.373
21.096
50.286
54.538
17C*
34
vial
low
5.0
0.100
125
0.1323
11.413
13.983
12.472
34.618
46.712
18
34
vial
low
5.0
0.100
125
0.1294
27.821
36.012
23.416
56.260
62.906
18C
34
vial
low
5.0
0.100
125
0.1290
17.674
20.388
18.527
48.372
63.256
22
34
vial
low
2.5
0.100
125
0.1262
2.853
22.979
30.349
40.254
49.366
22C
34
vial
low
2.5
0.100
125
0.1288
4.581
37.112
55.745
86.568
125.000
23
34
vial
low
10.0
0.100
125
0.1298
2.234
9.014
11.325
13.482
14.946
23C
34
vial
low
10.0
0.100
125
0.1255
0.637
9.004
14.821
19.602
26.056
24
34
vial
low
5.0
0.001
125
0.1226
3.507
23.002
31.403
37.847
43.312
24C
34
vial
low
5.0
0.001
125
0.1238
1.858
12.278
15.670
20.679
24.717
25
34
vial
low
5.0
10.000
125
0.1285
0.623
12.918
19.377
22.957
24.358
25C
34
vial
low
5.0
10.000
125
0.1235
2.429
18.462
21.862
31.741
50.526
26
34
vial
low
5.0
0.100
25
0.0274
7.664
6.204
7.299
7.299
26C
34
vial
low
5.0
0.100
25
0.0272
27
34
vial
low
5.0
0.100
250
0.2647
3.060
12.694
17.265
22.289
22.818
27C
34
vial
low
5.0
0.100
250
0.2537
2.877
25.778
46.078
90.579
121.167
A
34
test
tube
none
2.5
0.010
250
0.2435
7.885
18.070
22.300
27.556
36.468
AC
34
test
tube
none
2.5
0.010
250
0.2606
7.866
14.275
19.916
22.909
26.592
9­

9
Section
9
Phase
II
Table
38.
Data
Listing
for
Incubation
Experiment
(
Continued)

Run
Temp
Vessel
Speed
Inc
Vol
hCG
Conc
mg
Tissue
Weight
Test
0
Test
1
Test
2
Test
3
Test
4
B
34
test
tube
none
2.5
1.000
50
0.0523
13.767
15.488
17.400
19.503
BC
34
test
tube
none
2.5
1.000
50
0.0533
18.949
30.769
32.645
35.835
37.148
C
34
test
tube
none
10.0
0.010
50
0.0452
9.513
15.708
18.142
16.372
19.027
CC
34
test
tube
none
10.0
0.010
50
0.0516
12.984
24.225
34.884
45.543
48.062
D
34
test
tube
none
10.0
1.000
250
0.2408
7.766
7.226
9.053
9.925
12.209
DC
34
test
tube
none
10.0
1.000
250
0.2406
6.151
7.606
11.887
14.630
16.417
T
34
test
tube
none
5.0
0.100
125
0.1254
7.257
16.188
18.979
16.108
27.193
TC
34
test
tube
none
5.0
0.100
125
0.1287
4.895
15.540
28.594
36.752
T*
34
test
tube
none
5.0
0.100
125
0.1253
6.305
11.333
19.154
24.900
18.037
TC*
34
test
tube
none
5.0
0.100
125
0.1296
3.858
11.960
17.515
19.444
23.843
1
34
vial
none
2.5
0.010
50
0.0476
15.336
86.134
120.588
135.294
150.420
1C
34
vial
none
2.5
0.010
50
0.0448
18.750
41.071
55.580
62.500
78.125
2
34
vial
none
2.5
1.000
250
0.2439
12.423
30.217
53.424
61.337
71.628
2C
34
vial
none
2.5
1.000
250
0.2345
19.446
49.595
88.913
134.371
3
34
vial
none
10.0
0.010
250
0.2314
1.642
6.785
14.866
11.841
16.854
3C
34
vial
none
10.0
0.010
250
0.2462
3.249
7.433
13.891
11.779
16.288
4
34
vial
none
10.0
1.000
50
0.0521
2.495
7.294
9.405
10.749
10.940
4C
34
vial
none
10.0
1.000
50
0.0528
4.167
10.795
16.288
20.644
24.621
20
34
vial
none
5.0
0.100
125
0.1239
4.358
18.402
27.603
32.849
37.530
20C
34
vial
none
5.0
0.100
125
0.1263
1.504
30.404
37.609
42.439
47.348
20*
34
vial
none
5.0
0.100
125
0.1242
18.438
23.108
31.481
33.253
20C*
34
vial
none
5.0
0.100
125
0.1224
26.389
34.967
42.157
44.444
9­

10
Table
39.
Data
Listing
of
Coded
Factor
Values
and
Logarithm
of
Testosterone
Concentrations
Run
hCG
Stimulation
Temp
Code
Vessel
Code
Speed
Code
Inc
Vol
Code
hCG
Conc
Code
mg
Tissue
Code
L
Test
0
L
Test
1
L
Test
2
L
Test
3
L
Test
4
M
0
1
1
1
­
1
­
1
1.0
1.95
3.21
3.43
4.18
4.47
MC
1
1
1
1
­
1
­
1
1.0
2.06
4.19
5.61
N
0
1
1
1
­
1
1
­
0.6
1.76
3.12
3.23
3.41
3.52
NC
1
1
1
1
­
1
1
­
0.6
2.01
2.89
3.81
4.09
4.36
O
0
1
1
1
1
­
1
­
0.6
1.38
2.51
2.64
2.91
2.89
OC
1
1
1
1
1
­
1
­
0.6
2.81
3.58
4.05
4.32
P
0
1
1
1
1
1
1.0
­
1.01
2.20
2.43
2.63
2.79
PC
1
1
1
1
1
1
1.0
­
0.88
3.19
4.19
4.54
4.70
13
0
1
­
1
1
­
1
­
1
­
0.6
3.42
4.24
4.08
4.01
3.91
13C
1
1
­
1
1
­
1
­
1
­
0.6
4.09
4.64
5.33
5.17
13*
0
1
­
1
1
­
1
­
1
­
0.6
3.66
3.81
3.94
3.87
3.92
13C*
1
1
­
1
1
­
1
­
1
­
0.6
2.98
3.19
3.29
14
0
1
­
1
1
­
1
1
1.0
3.58
3.84
3.72
3.71
3.68
14C
1
1
­
1
1
­
1
1
1.0
3.19
5.48
5.94
6.01
6.45
14*
0
1
­
1
1
­
1
1
1.0
3.33
4.51
4.26
4.09
3.94
14C*
1
1
­
1
1
­
1
1
1.0
3.67
5.48
5.50
6.22
6.13
15
0
1
­
1
1
1
­
1
1.0
3.30
3.35
3.35
3.44
15C
1
1
­
1
1
1
­
1
1.0
1.60
3.75
4.70
4.90
5.38
15*
0
1
­
1
1
1
­
1
1.0
2.16
2.19
2.73
2.74
15C*
1
1
­
1
1
1
­
1
1.0
3.55
4.49
4.90
5.05
16
0
1
­
1
1
1
1
­
0.6
1.18
1.24
0.81
0.71
0.61
16C
1
1
­
1
1
1
1
­
0.6
0.52
0.96
1.50
1.69
1.91
16*
0
1
­
1
1
1
1
­
0.6
1.02
1.64
1.67
1.64
1.67
16C*
1
1
­
1
1
1
1
­
0.6
0.91
1.82
2.60
2.87
3.19
S
0
1
1
0
0
0
0.0
0.32
1.80
2.27
2.71
2.78
SC
1
1
1
0
0
0
0.0
1.42
1.96
2.54
2.75
19
0
1
­
1
0
0
0
0.0
1.81
3.80
4.08
4.28
4.32
19C
1
1
­
1
0
0
0
0.0
1.71
3.77
4.12
4.31
4.55
I
0
1
1
­
1
­
1
­
1
­
0.6
1.48
2.43
2.82
3.06
3.16
Table
39.
Data
Listing
of
Coded
Factor
Values
and
Logarithm
of
Testosterone
Concentrations
(
Continued)

Run
hCG
Stimulation
Temp
Code
Vessel
Code
Speed
Code
Inc
Vol
Code
hCG
Conc
Code
mg
Tissue
Code
L
Test
0
L
Test
1
L
Test
2
L
Test
3
L
Test
4
IC
1
1
1
­
1
­
1
­
1
­
0.6
1.40
2.98
3.67
3.70
4.05
J
0
1
1
­
1
­
1
1
1.0
0.90
1.76
1.99
2.33
2.64
JC
1
1
1
­
1
­
1
1
1.0
0.77
2.16
2.53
2.63
2.92
K
0
1
1
­
1
1
­
1
1.0
0.79
1.99
2.28
2.42
2.39
KC
1
1
1
­
1
1
­
1
1.0
0.39
1.59
2.08
2.03
2.20
L
0
1
1
­
1
1
1
­
0.6
1.22
2.00
2.21
2.13
LC
1
1
1
­
1
1
1
­
0.6
0.66
1.15
1.40
1.48
9
0
1
­
1
­
1
­
1
­
1
1.0
1.31
2.71
3.02
3.20
3.31
9C
1
1
­
1
­
1
­
1
­
1
1.0
1.28
2.76
3.16
3.56
10
0
1
­
1
­
1
­
1
1
­
0.6
2.19
4.02
4.07
4.25
4.35
10C
1
1
­
1
­
1
­
1
1
­
0.6
2.69
4.30
4.57
4.86
5.15
11
0
1
­
1
­
1
1
­
1
­
0.6
1.33
2.60
2.79
2.95
3.08
11C
1
1
­
1
­
1
1
­
1
­
0.6
0.85
0.44
0.56
12
0
1
­
1
­
1
1
1
1.0
­
1.01
1.60
1.95
2.24
2.51
12C
1
1
­
1
­
1
1
1
1.0
1.63
1.92
2.34
2.84
E
0
­
1
1
1
­
1
­
1
­
0.6
2.26
2.20
2.36
2.20
2.32
EC
1
­
1
1
1
­
1
­
1
­
0.6
2.51
3.73
4.04
4.41
4.58
F
0
­
1
1
1
­
1
1
1.0
2.20
2.91
3.17
3.42
3.83
FC
1
­
1
1
1
­
1
1
1.0
2.19
2.85
3.10
3.42
3.59
G
0
­
1
1
1
1
­
1
1.0
2.08
2.57
2.76
2.79
2.85
GC
1
­
1
1
1
1
­
1
1.0
1.03
1.70
2.16
2.33
2.39
H
0
­
1
1
1
1
1
­
0.6
1.32
2.04
2.52
2.61
2.79
HC
1
­
1
1
1
1
1
­
0.6
1.81
2.96
3.08
2.98
3.00
U
0
­
1
1
1
0
0
0.0
2.08
2.61
2.81
3.04
3.10
UC
1
­
1
1
1
0
0
0.0
1.54
2.57
2.65
3.00
3.21
U*
0
­
1
1
1
0
0
0.0
1.85
2.81
3.06
3.30
3.11
UC*
1
­
1
1
1
0
0
0.0
1.91
2.55
2.92
3.04
2.06
5
0
­
1
­
1
1
­
1
­
1
­
0.6
2.41
2.45
2.46
2.51
2.29
5C
1
­
1
­
1
1
­
1
­
1
­
0.6
2.62
2.33
2.74
2.74
2.52
5*
0
­
1
­
1
1
­
1
­
1
­
0.6
1.53
1.53
1.30
1.57
1.82
5C*
1
­
1
­
1
1
­
1
­
1
­
0.6
2.04
2.44
2.46
2.16
2.07
6
0
­
1
­
1
1
­
1
1
­
0.6
2.76
2.51
2.52
2.62
2.81
6C
1
­
1
­
1
1
­
1
1
­
0.6
3.54
4.51
5.03
5.33
5.29
9­

12
Table
39.
Data
Listing
of
Coded
Factor
Values
and
Logarithm
of
Testosterone
Concentrations
(
Continued)

Run
hCG
Stimulation
Temp
Code
Vessel
Code
Speed
Code
Inc
Vol
Code
hCG
Conc
Code
mg
Tissue
Code
L
Test
0
L
Test
1
L
Test
2
L
Test
3
L
Test
4
6*
0
­
1
­
1
1
­
1
1
­
0.6
2.68
3.48
3.61
3.75
3.93
6C*
1
­
1
­
1
1
­
1
1
­
0.6
2.80
2.71
3.00
3.01
3.27
7
0
­
1
­
1
1
1
­
1
­
0.6
1.28
1.95
2.24
2.33
2.74
7C
1
­
1
­
1
1
1
­
1
­
0.6
0.80
0.88
0.51
0.61
7*
0
­
1
­
1
1
1
­
1
­
0.6
0.97
1.22
1.47
1.55
1.63
7C*
1
­
1
­
1
1
1
­
1
­
0.6
0.85
0.93
0.65
8
0
­
1
­
1
1
1
1
1.0
0.85
2.50
2.59
2.61
2.70
8C
1
­
1
­
1
1
1
1
1.0
1.50
3.13
3.63
4.00
4.26
8*
0
­
1
­
1
1
1
1
1.0
1.82
2.07
2.17
2.26
2.37
8C*
1
­
1
­
1
1
1
1
1.0
1.73
3.46
3.99
4.59
4.96
21
0
­
1
­
1
1
0
0
0.0
2.14
2.15
2.25
1.91
2.25
21C
1
­
1
­
1
1
0
0
0.0
1.59
3.71
4.46
5.35
5.78
21*
0
­
1
­
1
1
0
0
0.0
2.05
2.18
2.04
2.03
2.21
21C*
1
­
1
­
1
1
0
0
0.0
2.15
3.19
3.65
3.88
4.22
Q
0
­
1
1
0
0
0
0.0
­
0.33
1.55
2.15
2.25
2.42
QC
1
­
1
1
0
0
0
0.0
1.81
1.83
2.30
2.27
2.36
Q*
0
­
1
1
0
0
0
0.0
2.14
2.21
2.19
2.41
2.31
QC*
1
­
1
1
0
0
0
0.0
1.78
2.62
2.76
2.92
3.05
R
0
­
1
1
0
0
0
0.0
1.10
1.48
2.29
2.72
2.65
RC
1
­
1
1
0
0
0
0.0
1.70
1.83
2.43
2.58
2.51
V
0
­
1
1
0
­
1
0
0.0
0.67
1.97
2.61
2.83
2.98
VC
1
­
1
1
0
­
1
0
0.0
1.04
2.43
3.01
3.46
3.91
W
0
­
1
1
0
1
0
0.0
2.06
2.31
2.52
2.77
2.91
WC
1
­
1
1
0
1
0
0.0
0.89
1.35
1.74
2.02
2.15
X
0
­
1
1
0
0
­
2
0.0
2.00
2.41
2.69
2.81
2.92
XC
1
­
1
1
0
0
­
2
0.0
1.82
2.52
2.62
2.87
2.99
Y
0
­
1
1
0
0
2
0.0
0.99
1.92
2.19
2.46
2.55
YC
1
­
1
1
0
0
2
0.0
0.77
2.12
2.66
2.87
3.05
Z
0
­
1
1
0
0
0
­
0.8
1.28
ZC
1
­
1
1
0
0
0
­
0.8
2.55
3.15
3.44
3.91
AA
0
­
1
1
0
0
0
1.0
0.13
1.41
1.93
2.11
2.35
Table
39.
Data
Listing
of
Coded
Factor
Values
and
Logarithm
of
Testosterone
Concentrations
(
Continued)

Run
hCG
Stimulation
Temp
Code
Vessel
Code
Speed
Code
Inc
Vol
Code
hCG
Conc
Code
mg
Tissue
Code
L
Test
0
L
Test
1
L
Test
2
L
Test
3
L
Test
4
AAC
1
­
1
1
0
0
0
1.0
0.41
1.15
1.58
2.05
2.18
17
0
­
1
­
1
0
0
0
0.0
3.01
3.19
3.05
3.93
3.99
17C
1
­
1
­
1
0
0
0
0.0
3.81
3.37
3.70
17*
0
­
1
­
1
0
0
0
0.0
3.03
3.35
3.05
3.92
4.00
17C*
1
­
1
­
1
0
0
0
0.0
2.43
2.64
2.52
3.54
3.84
18
0
­
1
­
1
0
0
0
0.0
3.33
3.58
3.15
4.03
4.14
18C
1
­
1
­
1
0
0
0
0.0
2.87
3.01
2.92
3.88
4.15
22
0
­
1
­
1
0
­
1
0
0.0
1.05
3.13
3.41
3.70
3.90
22C
1
­
1
­
1
0
­
1
0
0.0
1.52
3.61
4.02
4.46
4.83
23
0
­
1
­
1
0
1
0
0.0
0.80
2.20
2.43
2.60
2.70
23C
1
­
1
­
1
0
1
0
0.0
­
0.45
2.20
2.70
2.98
3.26
24
0
­
1
­
1
0
0
­
2
0.0
1.25
3.14
3.45
3.63
3.77
24C
1
­
1
­
1
0
0
­
2
0.0
0.62
2.51
2.75
3.03
3.21
25
0
­
1
­
1
0
0
2
0.0
­
0.47
2.56
2.96
3.13
3.19
25C
1
­
1
­
1
0
0
2
0.0
0.89
2.92
3.08
3.46
3.92
26
0
­
1
­
1
0
0
0
­
0.8
2.04
1.83
1.99
1.99
26C
1
­
1
­
1
0
0
0
­
0.8
27
0
­
1
­
1
0
0
0
1.0
1.12
2.54
2.85
3.10
3.13
27C
1
­
1
­
1
0
0
0
1.0
1.06
3.25
3.83
4.51
4.80
A
0
­
1
1
­
1
­
1
­
1
1.0
2.06
2.89
3.10
3.32
3.60
AC
1
­
1
1
­
1
­
1
­
1
1.0
2.06
2.66
2.99
3.13
3.28
B
0
­
1
1
­
1
­
1
1
­
0.6
2.62
2.74
2.86
2.97
BC
1
­
1
1
­
1
­
1
1
­
0.6
2.94
3.43
3.49
3.58
3.61
C
0
­
1
1
­
1
1
­
1
­
0.6
2.25
2.75
2.90
2.80
2.95
CC
1
­
1
1
­
1
1
­
1
­
0.6
2.56
3.19
3.55
3.82
3.87
D
0
­
1
1
­
1
1
1
1.0
2.05
1.98
2.20
2.30
2.50
DC
1
­
1
1
­
1
1
1
1.0
1.82
2.03
2.48
2.68
2.80
T
0
­
1
1
­
1
0
0
0.0
1.98
2.78
2.94
2.78
3.30
TC
1
­
1
1
­
1
0
0
0.0
1.59
2.74
3.35
3.60
T*
0
­
1
1
­
1
0
0
0.0
1.84
2.43
2.95
3.21
2.89
TC*
1
­
1
1
­
1
0
0
0.0
1.35
2.48
2.86
2.97
3.17
9­

14
Table
39.
Data
Listing
of
Coded
Factor
Values
and
Logarithm
of
Testosterone
Concentrations
(
Continued)

Run
hCG
Stimulation
Temp
Code
Vessel
Code
Speed
Code
Inc
Vol
Code
hCG
Conc
Code
mg
Tissue
Code
L
Test
0
L
Test
1
L
Test
2
L
Test
3
L
Test
4
1
0
­
1
­
1
­
1
­
1
­
1
­
0.6
2.73
4.46
4.79
4.91
5.01
1C
1
­
1
­
1
­
1
­
1
­
1
­
0.6
2.93
3.72
4.02
4.14
4.36
2
0
­
1
­
1
­
1
­
1
1
1.0
2.52
3.41
3.98
4.12
4.27
2C
1
­
1
­
1
­
1
­
1
1
1.0
2.97
3.90
4.49
4.90
3
0
­
1
­
1
­
1
1
­
1
1.0
0.50
1.91
2.70
2.47
2.82
3C
1
­
1
­
1
­
1
1
­
1
1.0
1.18
2.01
2.63
2.47
2.79
4
0
­
1
­
1
­
1
1
1
­
0.6
0.91
1.99
2.24
2.37
2.39
4C
1
­
1
­
1
­
1
1
1
­
0.6
1.43
2.38
2.79
3.03
3.20
20
0
­
1
­
1
­
1
0
0
0.0
1.47
2.91
3.32
3.49
3.63
20C
1
­
1
­
1
­
1
0
0
0.0
0.41
3.41
3.63
3.75
3.86
20*
0
­
1
­
1
­
1
0
0
0.0
2.91
3.14
3.45
3.50
20C*
1
­
1
­
1
­
1
0
0
0.0
3.27
3.55
3.74
3.79
Section
9.0
Incubation
Experiment
9­
15
Table
40.
ANOVA
results
for
PROC
RSREG
hCG
Stimulatio
n
Vessel
Type
Error
df
Time
R2
hCG
Conc.
Incub.
Vol.
Shaker
Speed
Frag.
Size
Incub.
Temp.
Baseline
RMSE
No
scintillation
vial
19
T1
87
NA
**
***
*
***
0.443
T2
77
NA
*
**
0.585
T3
83
NA
*
***
***
0.530
T4
80
NA
***
**
0.570
test
tube
12
T1
86
NA
***
*
**
0.267
T2
82
NA
***
*
0.250
T3
81
NA
**
*
*
0.296
T4
86
NA
**
**
**
*
0.278
Yes
scintillation
vial
10
T1
91
**
***
0.506
T2
85
**
**
0.649
T3
83
*
**
0.740
T4
85
*
**
0.727
test
tube
8
T1
91
**
**
**
*
0.334
T2
91
**
**
**
*
0.339
T3
93
*
***
***
***
**
0.308
T4
87
***
*
*
0.482
*
Statistically
significant
F
test
at
0.10
level
of
significance.
**
Statistically
significant
F
test
at
0.05
level
of
significance.
***
Statistically
significant
F
test
at
0.01
level
of
significance.

Note:
R2
is
the
percentage
of
variation
accounted
for
by
the
model.
RMSE
=
square
root
of
the
residual
(
error)
mean
square
NA
=
not
applicable
Section
9.0
Incubation
Experiment
9­
16
Table
41.
Response
Surface
Regression
Results
for
No
hCG
Stimulation
Vessel
Type
Model
Terms
L
Test
1
L
Test
2
L
Test
3
L
Test
4
Beta
P
Value
Beta
P
Value
Beta
P
Value
Beta
P
Value
Scintillatio
n
Vial
Increasing
volume
code
­
0.47
0.003
­
0.56
0.007
­
0.43
0.020
­
0.40
0.038
Speed
code
­
0.29
0.014
­
0.42
0.007
­
0.55
0.000
­
0.59
0.000
mg
tissue
code
0.05
0.863
0.07
0.848
­
0.02
0.962
0.10
0.786
Temperature
code
0.21
0.035
0.12
0.334
0.11
0.337
0.07
0.557
Increasing
volume
code*
Increasing
volume
code
0.02
0.958
0.06
0.886
0.03
0.942
0.07
0.856
Speed
code*
Increasing
volume
code
0.09
0.436
0.07
0.621
0.12
0.378
0.12
0.414
Speed
code*
Speed
code
­
0.46
0.093
­
0.37
0.299
­
0.85
0.014
­
0.74
0.041
mg
tissue
code*
Increasing
volume
code
0.27
0.102
0.25
0.237
0.31
0.116
0.32
0.124
mg
tissue
code*
Speed
code
0.36
0.017
0.29
0.131
0.25
0.155
0.18
0.326
mg
tissue
code*
mg
tissue
code
­
0.06
0.908
­
0.04
0.954
0.19
0.773
­
0.03
0.967
Temperature
code*
Increasing
volume
code
­
0.09
0.382
­
0.17
0.245
­
0.11
0.411
­
0.11
0.419
Temperature
code*
Speed
code
0.16
0.151
0.19
0.194
0.12
0.381
0.07
0.631
Temperature
code*
mg
tissue
code
0.06
0.683
­
0.01
0.979
0.12
0.537
0.11
0.582
L
test
0
0.36
0.005
0.22
0.158
0.39
0.010
0.39
0.015
Test
Tube
Increasing
volume
code
­
0.07
0.426
­
0.10
0.223
­
0.12
0.214
­
0.17
0.062
Speed
code
0.11
0.147
0.07
0.320
0.13
0.136
0.12
0.155
mg
tissue
code
0.23
0.255
0.24
0.196
0.48
0.040
0.50
0.026
Temperature
code
0.12
0.153
0.06
0.472
0.19
0.047
0.14
0.108
Increasing
volume
code*
Increasing
volume
code
0.14
0.438
0.20
0.253
0.22
0.289
0.32
0.107
Speed
code*
Increasing
volume
code
­
0.01
0.919
­
0.05
0.544
­
0.06
0.588
­
0.08
0.396
Speed
code*
Speed
code
0.50
0.016
0.53
0.008
0.44
0.045
0.45
0.031
mg
tissue
code*
Increasing
volume
code
­
0.06
0.587
­
0.11
0.268
­
0.22
0.075
­
0.30
0.017
mg
tissue
code*
Speed
code
0.21
0.047
0.22
0.028
0.23
0.052
0.25
0.028
mg
tissue
code*
mg
tissue
code
­
0.52
0.177
­
0.69
0.066
­
0.97
0.034
­
0.88
0.038
Temperature
code*
Increasing
volume
code
0.04
0.595
0.01
0.899
­
0.04
0.675
­
0.07
0.388
Temperature
code*
Speed
code
0.16
0.041
0.12
0.105
0.13
0.143
0.14
0.081
Temperature
code*
mg
tissue
code
­
0.06
0.564
­
0.13
0.229
­
0.15
0.226
­
0.17
0.157
LTest_
0
0.31
0.013
0.11
0.282
0.13
0.283
0.07
0.543
Note:
L
Test
J
=
log
testosterone
concentration
at
time
J
Beta
=
regression
coefficient,
as
applied
to
coded
factors
P
Value
=
significance
level
for
t­
test
of
hypothesis
that
the
true
Beta
=
0
Section
9.0
Incubation
Experiment
9­
17
Table
42.
Response
Surface
Regression
Results
for
hCG
Stimulation
Vessel
Type
Model
Terms
L
Test
1
L
Test
2
L
Test
3
L
Test
4
Beta
P
Value
Beta
P
Value
Beta
P
Value
Beta
P
Value
Scintillation
Vial
hCG
concentration
code
0.08
0.718
­
0.02
0.942
0.12
0.710
0.11
0.729
Speed
code
­
0.28
0.321
­
0.16
0.660
0.15
0.717
0.22
0.593
mg
tissue
code
1.05
0.012
1.00
0.045
1.11
0.050
1.23
0.031
Temperature
code
0.29
0.230
0.33
0.294
0.07
0.832
0.06
0.852
hCG
concentration
code*
hCGconcentration
code
­
0.07
0.502
­
0.11
0.428
­
0.16
0.324
­
0.16
0.327
Speed
code*
hCG
concentration
code
0.26
0.255
0.34
0.247
0.23
0.481
0.27
0.399
Speed
code*
Speed
code
0.34
0.331
0.49
0.277
0.18
0.712
0.19
0.704
mg
tissue
code*
hCG
concentration
code
­
0.22
0.492
­
0.46
0.284
­
0.69
0.162
­
0.88
0.079
mg
tissue
code*
Speed
code
0.65
0.086
0.81
0.095
1.21
0.037
1.41
0.017
mg
tissue
code*
mg
tissue
code
­
0.72
0.206
­
0.52
0.466
­
0.98
0.239
­
1.21
0.146
Temperature
code*
hCG
concentration
code
­
0.07
0.773
­
0.13
0.676
0.00
0.998
­
0.03
0.931
Temperature
code*
Speed
code
­
0.30
0.348
­
0.31
0.436
­
0.09
0.834
­
0.05
0.914
Temp
code*
mg
tissue
code
0.36
0.397
0.31
0.568
­
0.03
0.964
­
0.16
0.791
L
test
0
0.04
0.853
­
0.12
0.624
0.11
0.714
0.08
0.765
Increasing
volume
code
­
0.74
0.027
­
0.82
0.048
­
0.79
0.087
­
0.86
0.063
Test
Tube
hCG
concentration
code
­
0.22
0.195
­
0.27
0.122
­
0.33
0.049
­
0.34
0.166
Speed
code
0.33
0.071
0.44
0.026
0.55
0.006
0.48
0.075
mg
tissue
code
­
0.35
0.224
­
0.43
0.150
­
0.50
0.076
­
0.77
0.081
Temperature
code
­
0.16
0.359
0.04
0.825
­
0.04
0.806
­
0.07
0.784
hCG
concentration
code*
hCG
concentration
code
0.10
0.156
0.08
0.250
0.08
0.231
0.10
0.333
Speed
code*
hCG
concentration
code
0.09
0.451
0.07
0.565
0.05
0.650
0.06
0.734
Speed
code*
Speed
code
0.67
0.018
0.62
0.026
0.59
0.023
0.47
0.190
mg
tissue
code*
hCG
concentration
code
0.27
0.066
0.32
0.037
0.42
0.007
0.45
0.039
mg
tissue
code*
Speed
code
0.07
0.609
0.06
0.657
0.05
0.714
0.02
0.923
mg
tissue
code*
mg
tissue
code
0.28
0.448
0.30
0.419
0.44
0.211
0.80
0.153
Temperature
code*
hCG
concentration
code
­
0.14
0.371
­
0.20
0.232
­
0.21
0.178
­
0.20
0.391
Temperature
code*
Speed
code
0.34
0.054
0.50
0.012
0.59
0.003
0.61
0.024
Temperature
code*
mg
tissue
code
0.42
0.021
0.32
0.063
0.31
0.053
0.26
0.250
L
test
0
0.04
0.818
­
0.07
0.714
­
0.18
0.322
­
0.35
0.220
Increasing
volume
code
­
0.38
0.012
­
0.38
0.012
­
0.46
0.003
­
0.57
0.01
Note:
L
Test
J
=
log
testosterone
concentration
at
time
J
Beta
=
regression
coefficient,
as
applied
to
coded
factors
P
Value
=
significance
level
for
t­
test
of
hypothesis
that
the
true
Beta
=
0
9­

18
Section
9
Phase
II
Table
43.
Maximum
Values
for
Each
Time
Point
hCG
Stimulation
Vessel
Type
Time
Predicted
Log(
Testosterone
Concentration)*
Media
Volume
hCG
Conc
Fragment
Size
Temp
Shaker
Speed
Code
Shaker
Speeda
log
(
T0)
T1
T2
T3
T4
No
Scintillation
vial
T1
opt
3.992
(
0.289)
4.139
(
0.382)
4.446
(
0.346)
4.503
(
0.372)
2.5
98.1
36.1
­
0.251
low
1.8108
No
Scintillation
vial
T2
opt
3.945
(
0.293)
4.105
(
0.387)
4.403
(
0.351)
4.484
(
0.377)
2.5
108.6
35.9
­
0.310
low
1.8108
No
Scintillation
vial
T3
opt
4.064
(
0.348)
4.212
(
0.460)
4.671
(
0.417)
4.605
(
0.449)
2.5
=
98
42.0
35.7
­
0.339
low
1.8108
No
Scintillation
vial
T4
opt
3.945
(
0.282)
4.110
(
0.373)
4.418
(
0.338)
4.495
(
0.363)
2.5
102.3
35.8
­
0.343
low
1.8108
No
Test
tube
T1
opt
2.870
(
0.175)
3.121
(
0.163)
3.536
(
0.193)
3.543
(
0.182)
4.7
162.1
35.8
0.998
high
1.4189
No
Test
tube
T2
opt
2.856
(
0.175)
3.135
(
0.164)
3.542
(
0.194)
3.568
(
0.182)
4.3
159.5
35.7
0.998
high
1.4189
No
Test
tube
T3
opt
2.913
(
0.180)
3.190
(
0.168)
3.634
(
0.199)
3.685
(
0.187)
3.9
164.8
35.9
0.991
high
1.4189
No
Test
tube
T4
opt
2.990
(
0.204)
3.317
(
0.191)
3.794
(
0.226)
3.927
(
0.213)
3.1
173.5
35.8
0.993
high
1.4189
Yes
Scintillation
vial
T1
opt
5.327
(
0.864)
5.703
(
1.108)
4.876
(
1.263)
5.048
(
1.240)
2.5
0.053
172.5
36.6
­
0.994
none
1.8142
Yes
Scintillation
vial
T2
opt
5.087
(
0.543)
5.919
(
0.696)
6.421
(
0.794)
6.919
(
0.779)
2.5
0.085
234.8
35.8
0.990
high
1.8142
Yes
Scintillation
vial
T3
opt
4.961
(
0.574)
5.766
(
0.736)
6.377
(
0.840)
6.904
(
0.824)
2.5
0.077
223.3
35.5
0.994
high
1.8142
Yes
Scintillation
vial
T4
opt
4.937
(
0.588)
5.743
(
0.754)
6.374
(
0.860)
6.909
(
0.844)
2.5
0.071
220.7
35.4
0.999
high
1.8142
Yes
Test
tube
T1
opt
3.169
(
0.355)
3.876
(
0.360)
4.163
(
0.328)
4.101
(
0.513)
2.5
0.083
135.2
35.7
0.991
high
1.4847
Yes
Test
tube
T2
opt
3.242
(
0.403)
4.025
(
0.409)
4.323
(
0.372)
4.267
(
0.583)
2.5
0.071
130.3
36.0
0.999
high
1.4847
Yes
Test
tube
T3
opt
3.276
(
0.398)
4.075
(
0.404)
4.392
(
0.368)
4.375
(
0.576)
2.5
0.061
115.6
36.0
0.998
high
1.4847
Yes
Test
tube
T4
opt
2.945
(
0.403)
3.693
(
0.409)
4.140
(
0.372)
4.711
(
0.582)
2.5
0.046
25.3
35.5
0.350
low
1.4847
*
Values
in
parentheses
are
standard
errors
a
If
the
coded
value
for
shaker
speed
was
between
­
1
and
­
0.5
then
the
shaker
speed
was
defined
as
"
none".
If
the
coded
value
for
shaker
speed
was
between
­
0.5
and
0.5
then
the
shaker
speed
was
defined
as
"
low".
If
the
coded
value
for
shaker
speed
was
between
0.5
and
1
then
the
shaker
speed
was
defined
as
"
high".
9­

19
Section
9
Phase
II
Table
44.
Predicted
values
for
Specified
Optimum
Factor
Combinations
hCG
Stimulation
Vessel
Type
Time
Predicted
Log(
Testosterone
Concentration)*
Media
Volume
hCG
Concent
Fragment
Size
Temp
Shaker
Speed
Code
Shaker
Speeda
log
(
T0)
T1
T2
T3
T4
No
Scintillation
vial
Optimal
3.368
(
0.204)
3.333
(
0.270)
3.800
(
0.245)
3.861
(
0.263)
5.0
175.0
36.0
0.0
low
1.8108
No
Scintillation
vial
Optimal
2.820
(
0.305)
2.728
(
0.403)
2.538
(
0.365)
2.624
(
0.392)
5.0
175.0
36.0
1.0
high
1.8108
Yes
Scintillation
vial
Optimal
4.496
(
0.358)
4.810
(
0.459)
5.120
(
0.524)
5.448
(
0.515)
2.5
0.100
175.0
36.0
0.0
low
1.8142
Yes
Scintillation
vial
Optimal
4.715
(
0.590)
5.363
(
0.757)
5.906
(
0.864)
6.398
(
0.848)
2.5
0.100
175.0
36.0
1.0
high
1.8142
*
Values
in
parentheses
are
standard
errors
a
If
the
coded
value
for
shaker
speed
was
between
­
1
and
­
0.5
then
the
shaker
speed
was
defined
as
"
none".
If
the
coded
value
for
shaker
speed
was
between
­
0.5
and
0.5
then
the
shaker
speed
was
defined
as
"
low".
If
the
coded
value
for
shaker
speed
was
between
0.5
and
1
then
the
shaker
speed
was
defined
as
"
high".
9­

20
Section
9
Phase
II
Table
45.
ANOVA
results
for
PROC
RSREG
with
the
Baseline
Concentration
Removed
from
Model
hCG
Stimulation
Vessel
Type
Error
df
Time
R2
hCG
Conc.
Incub.
Vol.
Shaker
Speed
Frag.
Size
Incub.
Temp.
RMSE
No
scintillation
vial
24
T1
77
NA
***
***
**
**
0.532
T2
72
NA
***
**
*
*
0.600
T3
72
NA
***
***
*
0.625
T4
69
NA
***
**
*
0.653
test
tube
14
T1
71
NA
***
*
0.390
T2
76
NA
*
***
*
0.279
T3
76
NA
**
***
**
*
0.316
T4
82
NA
***
***
**
**
0.300
Yes
scintillation
vial
16
T1
91
***
***
***
0.455
T2
88
*
***
***
***
*
0.552
T3
85
***
*
***
*
0.681
T4
85
***
*
***
*
0.693
test
tube
12
T1
93
***
***
***
***
***
0.278
T2
92
**
***
***
***
***
0.315
T3
94
***
***
***
***
***
0.288
T4
87
*
***
***
**
***
0.447
*
Statistically
significant
F
test
at
0.10
level
of
significance.
**
Statistically
significant
F
test
at
0.05
level
of
significance.
***
Statistically
significant
F
test
at
0.01
level
of
significance.

Note:
R2
is
the
percentage
of
variation
accounted
for
by
the
model.
RMSE
=
square
root
of
the
residual
(
error)
mean
square
NA
=
not
applicable
Section
9.0
Incubation
Experiment
9­
21
Table
46.
Response
Surface
Regression
Results
for
No
hCG
Stimulation
with
Baseline
Concentration
Removed
Vessel
Type
Model
Terms
L
Test
1
L
Test
2
L
Test
3
L
Test
4
Beta
P
Value
Beta
P
Value
Beta
P
Value
Beta
P
Value
Scintillation
Vial
Inc_
vol_
code
­
0.78
<.
0001
­
0.75
<.
0001
­
0.75
<.
0001
­
0.72
<.
0001
Speed_
code
­
0.14
0.206
­
0.30
0.022
­
0.38
0.008
­
0.41
0.006
mg_
tissue_
code
0.18
0.409
0.32
0.194
0.36
0.163
0.44
0.106
Temp_
code
0.22
0.047
0.15
0.218
0.15
0.239
0.12
0.378
Inc_
vol_
code*
Inc_
vol_
code
0.10
0.734
0.26
0.430
0.26
0.447
0.29
0.414
Speed_
code*
Inc_
vol_
code
0.04
0.722
0.06
0.675
0.10
0.491
0.11
0.477
Speed_
code*
Speed_
code
­
0.23
0.385
­
0.17
0.573
­
0.46
0.144
­
0.39
0.229
mg_
tissue_
code*
Inc_
vol_
code
0.18
0.243
0.22
0.208
0.27
0.145
0.30
0.126
mg_
tissue_
code*
Speed_
code
0.48
0.005
0.37
0.043
0.40
0.038
0.34
0.086
mg_
tissue_
code*
mg_
tissue_
code
­
0.52
0.226
­
0.62
0.196
­
0.73
0.151
­
0.86
0.107
Temp_
code*
Inc_
vol_
code
­
0.15
0.196
­
0.18
0.179
­
0.13
0.343
­
0.13
0.361
Temp_
code*
Speed_
code
0.22
0.062
0.23
0.076
0.21
0.124
0.16
0.244
Temp_
code*
mg_
tissue_
code
­
0.09
0.555
­
0.10
0.542
­
0.02
0.888
­
0.01
0.947
Test
Tube
Inc_
vol_
code
­
0.19
0.079
­
0.18
0.031
­
0.19
0.037
­
0.25
0.006
Speed_
code
0.21
0.051
0.13
0.078
0.19
0.032
0.18
0.029
mg_
tissue_
code
0.41
0.151
0.33
0.107
0.58
0.020
0.58
0.014
Temp_
code
­
0.06
0.542
­
0.04
0.596
0.10
0.225
0.06
0.451
Inc_
vol_
code*
Inc_
vol_
code
0.29
0.264
0.25
0.185
0.28
0.188
0.35
0.089
Speed_
code*
Inc_
vol_
code
­
0.03
0.762
­
0.03
0.668
­
0.05
0.584
­
0.04
0.610
Speed_
code*
Speed_
code
0.79
0.002
0.63
0.001
0.56
0.005
0.51
0.007
mg_
tissue_
code*
Inc_
vol_
code
­
0.04
0.783
­
0.07
0.458
­
0.19
0.087
­
0.24
0.031
mg_
tissue_
code*
Speed_
code
0.09
0.487
0.15
0.125
0.16
0.151
0.17
0.099
mg_
tissue_
code*
mg_
tissue_
code
­
0.89
0.095
­
0.81
0.038
­
1.12
0.015
­
0.95
0.025
Temp_
code*
Inc_
vol_
code
­
0.12
0.226
­
0.07
0.312
­
0.12
0.145
­
0.15
0.070
Temp_
code*
Speed_
code
0.23
0.025
0.17
0.022
0.17
0.039
0.20
0.013
Temp_
code*
mg_
tissue_
code
­
0.07
0.572
­
0.10
0.310
­
0.13
0.219
­
0.12
0.261
Note:
Ltest_
J
=
log
testosterone
concentration
at
time
J
Beta
=
regression
coefficient,
as
applied
to
coded
factors
Pvalue
=
significance
level
for
t­
test
of
hypothesis
that
the
true
Beta
=
0
9­

22
Section
9
Phase
II
Table
47.
Response
Surface
Regression
Results
for
hCG
Stimulation
with
Baseline
Concentration
Removed
Vessel
Type
Model
Terms
L
Test
1
L
Test
2
L
Test
3
L
Test
4
Beta
P
Value
Beta
P
Value
Beta
P
Value
Beta
P
Value
Scintillation
Vial
hCG_
conc_
code
­
0.09
0.534
­
0.17
0.333
­
0.11
0.609
­
0.07
0.734
Speed_
code
­
0.09
0.545
­
0.03
0.870
0.02
0.933
­
0.04
0.869
mg_
tissue_
code
0.80
0.007
0.87
0.013
1.05
0.016
1.18
0.008
Temp_
code
0.33
0.035
0.40
0.034
0.46
0.048
0.50
0.034
hCG_
conc_
code*
hCG_
conc_
code
­
0.06
0.485
­
0.07
0.509
­
0.13
0.334
­
0.13
0.372
Speed_
code*
hCG_
conc_
code
0.20
0.266
0.23
0.281
0.19
0.473
0.24
0.363
Speed_
code*
Speed_
code
0.41
0.136
0.57
0.091
0.35
0.380
0.30
0.467
mg_
tissue_
code*
hCG_
conc_
code
­
0.09
0.606
­
0.25
0.228
­
0.20
0.428
­
0.27
0.293
mg_
tissue_
code*
Speed_
code
0.70
0.001
0.71
0.003
0.78
0.006
0.82
0.005
mg_
tissue_
code*
mg_
tissue_
code
­
0.71
0.098
­
0.67
0.192
­
0.89
0.163
­
1.03
0.116
Temp_
code*
hCG_
conc_
code
­
0.28
0.043
­
0.35
0.038
­
0.41
0.048
­
0.41
0.055
Temp_
code*
Speed_
code
­
0.03
0.825
­
0.04
0.806
­
0.02
0.927
­
0.10
0.656
Temp_
code*
mg_
tissue_
code
­
0.06
0.679
­
0.14
0.457
­
0.26
0.277
­
0.23
0.327
Inc_
vol_
code
­
1.00
<.
0001
­
0.96
<.
0001
­
1.02
<.
0001
­
0.95
0.000
Test
Tube
hCG_
conc_
code
­
0.20
0.011
­
0.19
0.028
­
0.21
0.011
­
0.23
0.055
Speed_
code
0.31
0.001
0.38
0.001
0.46
<.
000
mg_
tissue_
code
­
0.36
0.043
­
0.34
0.083
­
0.36
0.053
­
0.47
0.093
Temp_
code
­
0.24
0.004
­
0.05
0.499
0.00
0.978
0.07
0.507
hCG_
conc_
code*
hCG_
conc_
code
0.11
0.074
0.09
0.159
0.08
0.194
0.09
0.300
Speed_
code*
hCG_
conc_
code
0.13
0.117
0.16
0.088
0.12
0.176
0.12
0.372
Speed_
code*
Speed_
code
0.70
0.001
0.62
0.006
0.50
0.013
0.28
0.301
mg_
tissue_
code*
hCG_
conc_
code
0.27
0.017
0.30
0.018
0.37
0.003
0.40
0.023
mg_
tissue_
code*
Speed_
code
0.07
0.509
0.09
0.413
0.12
0.248
0.12
0.463
mg_
tissue_
code*
mg_
tissue_
code
0.31
0.295
0.32
0.329
0.37
0.229
0.66
0.169
Temp_
code*
hCG_
conc_
code
­
0.12
0.096
­
0.13
0.122
­
0.11
0.156
­
0.13
0.268
Temp_
code*
Speed_
code
0.33
0.001
0.42
0.000
0.48
<.
0001
0.53
0.001
Temp_
code*
mg_
tissue_
code
0.44
0.001
0.38
0.004
0.33
0.006
0.32
0.057
Inc_
vol_
code
­
0.41
0.000
­
0.39
0.000
­
0.41
0.000
­
0.47
0.002
Note:
Ltest_
J
=
log
testosterone
concentration
at
time
J
Beta
=
regression
coefficient,
as
applied
to
coded
factors
Pvalue
=
significance
level
for
t­
test
of
hypothesis
that
the
true
Beta
=
0
9­

23
Section
9
Phase
II
Table
48.
Maximum
Values
for
Models
with
Baseline
Concentration
Removed
hCG
Stimulation
Vessel
Type
Predicted
Log(
Testosterone
Concentration)
Incubation
Media
Volume
hCG
Conc
Fragment
Size
Temp
Shaker
Speed
Code
Shaker
Speed
T1
T2
T3
T4
No
Scintillation
vial
T1
opt
4.201
(
0.347)
4.322
(
0.391)
4.735
(
0.407)
4.802
(
0.425)
2.5
127.3
36.0
­
0.069
low
No
Scintillation
vial
T2
opt
4.151
(
0.341)
4.302
(
0.384)
4.726
(
0.400)
4.805
(
0.418)
2.5
132.2
35.8
­
0.177
low
No
Scintillation
vial
T3
opt
4.146
(
0.341)
4.301
(
0.385)
4.726
(
0.400)
4.807
(
0.418)
2.5
131.6
35.8
­
0.188
low
No
Scintillation
vial
T4
opt
4.135
(
0.339)
4.298
(
0.383)
4.724
(
0.398)
4.809
(
0.416)
2.5
133.4
35.7
­
0.221
low
No
Test
tube
T1
opt
2.959
(
0.247)
3.164
(
0.177)
3.570
(
0.201)
3.567
(
0.190)
4.4
155.1
35.6
0.996
high
No
Test
tube
T2
opt
2.976
(
0.248)
3.180
(
0.178)
3.590
(
0.201)
3.594
(
0.190)
4.2
156.7
35.6
0.996
high
No
Test
tube
T3
opt
3.062
(
0.256)
3.254
(
0.184)
3.696
(
0.208)
3.720
(
0.197)
3.9
161.5
35.8
1.000
high
No
Test
tube
T4
opt
3.209
(
0.274)
3.387
(
0.196)
3.872
(
0.222)
3.946
(
0.210)
3.2
169.3
35.9
0.996
high
Yes
Scintillation
vial
T1
opt
5.334
(
0.284)
5.883
(
0.345)
6.458
(
0.426)
6.654
(
0.433)
2.5
0.098
207.4
35.8
0.999
high
Yes
Scintillation
vial
T2
opt
5.328
(
0.291)
5.891
(
0.353)
6.462
(
0.436)
6.654
(
0.444)
2.5
0.079
206.8
35.8
1.000
high
Yes
Scintillation
vial
T3
opt
5.355
(
0.288)
5.923
(
0.350)
6.492
(
0.432)
6.683
(
0.439)
2.5
0.078
209.4
35.9
1.000
high
Yes
Scintillation
vial
T4
opt
5.355
(
0.283)
5.920
(
0.343)
6.491
(
0.423)
6.684
(
0.431)
2.5
0.080
211.7
35.9
0.990
high
Yes
Test
tube
T1
opt
3.105
(
0.203)
3.662
(
0.230)
3.967
(
0.210)
3.971
(
0.326)
2.5
0.091
132.7
35.6
0.997
high
Yes
Test
tube
T2
opt
3.112
(
0.221)
3.726
(
0.251)
4.049
(
0.229)
4.069
(
0.355)
2.5
0.099
146.8
35.9
0.998
high
9­

24
Section
9
Phase
II
Table
48.
Maximum
Values
for
Models
with
Baseline
Concentration
Removed
(
Continued)

hCG
Stimulation
Vessel
Type
Predicted
Log(
Testosterone
Concentration)
Incubation
Media
Volume
hCG
Conc
Fragment
Size
Temp
Shaker
Speed
Code
Shaker
Speed
T1
T2
T3
T4
Yes
Test
tube
T3
opt
3.119
(
0.229)
3.756
(
0.259)
4.090
(
0.237)
4.121
(
0.368)
2.5
0.092
151.2
36.0
0.994
high
Yes
Test
tube
T4
opt
2.855
(
0.293)
3.409
(
0.332)
3.822
(
0.303)
4.330
(
0.471)
2.5
0.044
26.2
35.5
0.281
low
*
Values
in
parentheses
are
standard
errors
i.
If
the
coded
value
for
shaker
speed
was
between
­
1
and
­
0.5
then
the
shaker
speed
was
defined
as
"
none".
If
the
coded
value
for
shaker
speed
was
between
­
0.5
and
0.5
then
the
shaker
speed
was
defined
as
"
low".
If
the
coded
value
for
shaker
speed
was
between
0.5
and
1
then
the
shaker
speed
was
defined
as
"
high".
9­

25
Section
9
Phase
II
Table
49.
Predicted
Values
for
Specified
Optimum
Factor
Combinations
for
Models
with
Baseline
Concentration
Removed
hCG
Stimulation
Vessel
Type
Time
Predicted
Log(
Testosterone
Concentration)

Media
Volume
hCG
Conc
Fragment
Size
Temp
Shaker
Speed
Code
Shaker
Speed
T1
T2
T3
T4
No
Scintillation
vial
Optimal
3.243
(
0.228)
3.257
(
0.258)
3.687
(
0.268)
3.758
(
0.280)
5.0
175.0
36.0
0.0
low
No
Scintillation
vial
Optimal
3.137
(
0.288)
3.012
(
0.326)
3.082
(
0.339)
3.150
(
0.354)
5.0
175.0
36.0
1.0
high
Yes
Scintillation
vial
Optimal
4.586
(
0.261)
4.888
(
0.316)
5.609
(
0.390)
5.914
(
0.397)
2.5
0.100
175.0
36.0
0.0
low
Yes
Scintillation
vial
Optimal
5.178
(
0.308)
5.700
(
0.374)
6.283
(
0.461)
6.468
(
0.469)
2.5
0.100
175.0
36.0
1.0
high
*
Values
in
parentheses
are
standard
errors
i.
If
the
coded
value
for
shaker
speed
was
between
­
1
and
­
0.5
then
the
shaker
speed
was
defined
as
"
none".
If
the
coded
value
for
shaker
speed
was
between
­
0.5
and
0.5
then
the
shaker
speed
was
defined
as
"
low".
If
the
coded
value
for
shaker
speed
was
between
0.5
and
1
then
the
shaker
speed
was
defined
as
"
high".
Section
10.0
Testes
Preparation
Experiment
10­
1
10.0
TESTES
PREPARATION
EXPERIMENT
10.1
Statistical
Analysis
of
Phase
II
Testis
Preparation
Optimization
Experiment
10.1.1
Objectives
The
objectives
of
the
testis
preparation
optimization
experiment
were
to
determine
the
effect
of
various
testis
preparation
methods
and
aliquot
volumes
on
testosterone
production
using
the
sliced
testis
assay
and
to
identify
the
factor
settings
that
maximize
the
amount
of
testosterone
production.
These
experiments
tested
the
effect(
s)
of
four
factors:
time
delay,
organ
preparation
technique,
sample
aliquot
volume
and
hCG
concentration.
Table
50
presents
the
testis
preparation
factor
settings
used
in
this
experiment.
Each
trial
in
the
experiment
was
run
with
and
without
hCG
stimulation
and
repeated
measurements
were
taken
at
baseline
(
time
0)
and
at
1,
2,
3,
and
4
hours
after
baseline.

10.1.2
Data
A
SAS
data
set
was
constructed
from
the
raw
Excel
data
files
and
two
fundamental
types
of
dependent
variables
were
used
in
the
analyses:
the
original
testosterone
concentrations
and
the
(
natural)
logarithm
of
the
testosterone
concentrations.
Each
observation
includes
dependent
variable
values
for
4
time
points
and
a
corresponding
baseline
level.
Each
observation
also
includes
data
identifying
the
levels
of
the
pertinent
factors.
The
data
used
in
the
analysis
are
displayed
in
Tables
51
and
52.

10.1.2
Statistical
Analysis
Methods
In
order
to
assess
the
effects
of
each
testis
preparation
factor
on
the
amount
of
testosterone
production,
analysis
of
variance
(
ANOVA)
models
were
fit
to
the
data
separately
for
the
trials
with
and
without
hCG
stimulation.
For
each
hCG
stimulation
type,
the
ANOVA
models
were
fit
to
each
individual
time
point.
All
main
effects
and
two­
factor
interactions
(
2fi)
of
the
four
factors
were
initially
included
in
the
models.
Tests
for
interactions
were
conducted
and
where
they
were
not
detected
as
statistically
significant
(
p=
0.10),
a
reduced
model
was
employed
that
retained
the
main
effects,
the
baseline
covariate
and
only
those
2fi's
deemed
to
have
significant
effects.
Once
the
final
ANOVA
model
was
determined,
the
data
were
fit
using
a
response
surface
regression
analysis
to
determine
the
maximum
predicted
value
of
testosterone
and
the
incubation
factor
levels
associated
with
the
maximum.
Additional
details
are
provided
in
the
Results
section.

10.1.4
Results
To
assess
the
effects
of
each
testis
preparation
factor
on
testosterone
production,
an
ANOVA
model
was
fit
separately
to
the
set
of
data
for
each
hCG
stimulation
type
and
each
time
Section
10.0
Testes
Preparation
Experiment
10­
2
point
beyond
the
baseline.
The
ANOVA
models
containing
all
main
effects
and
2fi's
were
fit
to
the
original
testosterone
concentrations
and
the
logarithms
of
testosterone
concentrations
using
PROC
GLM.
The
logarithm
of
the
baseline
testosterone
concentration
(
T0)
was
also
added
to
the
model
as
a
dependent
variable.
Upon
examining
the
residuals
from
fitting
the
full
ANOVA
models,
the
residuals
for
the
original
testosterone
concentrations
showed
signs
of
heteroskedasticity.
The
residuals
for
the
logarithms
of
testosterone
concentrations
appear
homoskedastic
and
randomly
distributed
around
0,
thereby
satisfying
the
assumptions
necessary
for
valid
ANOVA
modeling
results.
The
logarithms
of
the
testosterone
concentrations
were
therefore
chosen
as
the
more
appropriate
dependent
variables
for
the
analysis
of
the
incubation
experiment
data.

For
the
full
ANOVA
models
using
the
logarithm
of
testosterone
concentration
as
the
dependent
variable,
we
examined
the
t­
tests
for
the
significance
of
each
factor
in
the
model.
In
the
first
round
of
model
evaluation,
we
examined
the
t­
test
results
for
the
2fi's
to
see
if
any
of
those
terms
could
be
removed
from
the
models.
Any
2fi's
with
p­
values
greater
than
0.1
were
removed
from
the
models.
The
reduced
models,
one
model
for
the
data
without
hCG
stimulation
and
one
model
for
the
data
with
hCG
stimulation,
were
fit
to
the
logarithms
of
testosterone
concentration
again
and
the
t­
tests
of
the
model
terms
were
re­
evaluated
for
significance.
After
2
rounds
of
removing
insignificant
2fi's,
we
arrived
at
the
final
reduced
models
containing
only
significant
2fi's
and
first
order
factors.

To
obtain
the
optimal
combination
of
factors
for
each
time
point,
the
data
were
fit
using
response
surface
regression
models
and
the
SAS
procedure
RSREG.
(
The
PROC
GLM
modeling
results
were
used
to
determine
which
factors
and
2fi's
needed
to
remain
in
the
models.
Any
factors
which
did
not
have
any
associated
2fi's
in
the
final
models
were
treated
as
covariates
in
the
response
surface
regression
models.)
The
ANOVA
results
for
the
response
surface
regressions
are
summarized
in
Table
53.
The
parameter
estimates
from
the
response
surface
regression
models
for
the
data
without
hCG
stimulation
are
presented
in
Table
54
and
the
parameter
estimates
for
the
data
with
hCG
stimulation
are
presented
in
Table
55.
Because
the
organ
preparation
technique
is
a
categorical
variable,
we
needed
to
run
separate
models
for
each
organ
preparation
technique
in
PROC
RSREG.
Differentiation
fo
the
prediction
equation
is
used
to
determine
the
location
of
the
stationary
point;
in
most
cases
the
stationary
point
was
not
a
point
of
maximal
response.
Under
these
circumstances
the
RIDGE
technique
in
SAS
is
used
to
find
a
point
of
maximal
response
within
the
experimental
region.
Starting
at
the
center
of
the
experimental
region,
a
hypersphere
having
a
given
radius
is
considered
and
the
point
on
the
hypersphere
with
the
maximal
response
is
determined.
The
radius
is
then
incremented
and
the
maximum
on
that
hypersphere
is
determined.
This
process,
when
repeated,
traces
out
a
"
ridge
of
maximal
response."
The
iterations
are
terminated
when
one
of
the
factors
reaches
the
extremity
of
its
range,
as
defined
by
the
levels
used
in
the
experiment.
We
determined
the
factor
combination
that
produced
the
maximum
predicted
value
of
logarithm
of
testosterone
concentration
for
each
time
point
at
each
combination
of
with/
without
hCG
stimulation
and
for
Section
10.0
Testes
Preparation
Experiment
3
This
suggests
that
the
baseline
levels
are
affected
by
(
some
of)
the
factors
and
that
their
resultant
effect
on
later
times
is
diminished
if
adjustment
for
baseline
level
are
made.

4
This
indicates
that
baseline
adjustments
for
the
latter
cases
were
unnecessary,
as
was
also
apparent
from
Table
4
(
since
baseline
effects
was
not
significant).

10­
3
each
organ
preparation
technique.
The
optimal
value
factor
combinations
were
plugged
back
into
the
response
surface
regression
models
to
obtain
predicted
values
for
the
optimal
factor
combinations
at
all
time
points.
For
example,
we
used
the
RIDGE
regression
results
to
determine
the
optimal
factor
combinations
for
the
first
time
point
(
T1)
for
the
scintillation
vial
without
hCG
stimulation.
We
then
used
the
optimal
T1
combination
to
obtain
the
predicted
values
for
logarithm
of
testosterone
concentration
and
their
standard
errors
for
the
scintillation
vial
without
hCG
stimulation
for
that
time
point
and
for
all
the
other
time
points
(
T2,
T3
and
T4).
The
optimal
factor
results,
presented
in
Table
55,
show
the
effects
of
the
optimal
factors
for
one
time
point
on
the
other
time
points
in
evaluating
the
best
combination
of
factors
to
use
for
further
experimentation.

After
examining
the
possible
optimal
factors
for
each
time
point,
organ
preparation
technique
and
hCG
stimulation,
the
chosen
optimal
values
and
their
predicted
values
and
standard
errors
are
presented
in
Table
56.

In
order
to
assess
the
effects
of
the
baseline
testosterone
concentrations
on
the
effects
of
factors,
the
same
analysis
steps
that
were
performed
above
were
also
repeated
on
the
logarithms
of
testosterone
concentration
but
with
the
baseline
concentration
removed
from
the
models.
The
ANOVA
results
for
the
final
reduced
model
from
PROC
RSREG
are
shown
in
Table
57.

Comparing
the
results
in
Table
57
with
those
in
Table
53
(
results
for
models
with
baseline
concentration
in
the
models)
indicates
that
there
are
many
more
significant
terms
for
the
models
without
the
baseline
concentrations.
3
The
root
mean
square
errors
(
RMSEs)
in
Table
57
are
higher
than
Table
53
for
the
cases
with
no
hCG
stimulation,
especially
for
the
cases
of
warm
buffered
saline,
while
the
RMSEs
for
the
cases
with
hCG
stimulation
vary
as
to
whether
the
higher
values
are
in
Table
53
or
Table
57.4
The
response
surface
regression
results
from
PROC
RSREG
are
presented
in
Tables
58
and
59.
The
optimal
values
for
each
time
point
are
shown
in
Table
60.
The
factor
combinations
that
produced
the
optimal
values
for
each
time
point
in
Table
60
are
more
stable
within
the
organ
preparation
techniques
than
those
in
Table
55.
The
standard
errors
of
the
predicted
values
for
Table
60
are
similar
to
those
in
Table
55,
except
for
the
cases
involving
warm
buffered
saline.
For
the
warm
buffered
saline
cases,
the
data
without
hCG
stimulation
has
higher
standard
errors
in
Table
57
whereas
the
data
with
hCG
stimulation
has
much
higher
standard
errors
in
Table
55.
The
predicted
values
for
the
chosen
optimal
combination
of
factors
are
presented
in
Table
61.
Section
10.0
Testes
Preparation
Experiment
10­
4
Section
10.0
Testes
Preparation
Experiment
10­
5
Table
50.
Testis
Preparation
Factor
Settings
and
Coded
Values
Factor
Identification
Units
Variable
Name
Factor
Experimental
Levels
Coded
Experimental
Levels
1
2
3
1
2
3
hCG
Concentration
IU/
ml
hCG_
Conc
X5
0.01
0.1*
1
­
1
0
+
1
Time
Delay
hr
Time_
Delay
X7
0.5
1*
2
­
1
0
+
1
Organ
Preparation
Technique
NAa
Organ_
Prep_
Tec
h
X8
Cold
buffered
saline
Warm
buffered
saline
Cold
media*
­
1
0
+
1
Sample
Aliquot
Volume
ml
Sample_
vol
X9
0.125
0.25
0.5*
­
1
0
+
1
*
Prototypical
value.
a
NA
­
not
applicable.
Section
10.0
Testes
Preparation
Experiment
10­
6
Table
51.
Data
Listing
for
Incubation
Experiment
SET
Organ
Prep
Technique
hCG
Conc
Time
Delay
Sample
Vol
Weight
Test
0
Test
1
Test
2
Test
3
Test
4
P
Warm
Buffered
Saline
0.01
0.5
0.125
0.2270
3.35
15.02
14.10
21.23
31.63
PC
Warm
Buffered
Saline
0.01
0.5
0.125
0.2292
4.01
16.58
36.13
62.83
83.64
Q
Warm
Buffered
Saline
0.01
0.5
0.500
0.2311
7.53
18.69
18.26
26.66
30.98
QC
Warm
Buffered
Saline
0.01
0.5
0.500
0.2308
4.81
34.45
121.92
161.61
31.63
AA
Warm
Buffered
Saline
0.10
0.5
0.250
0.2275
3.69
16.75
22.68
25.36
32.66
AAC
Warm
Buffered
Saline
0.10
0.5
0.250
0.2255
5.72
20.80
84.12
133.04
166.03
T
Warm
Buffered
Saline
1.00
0.5
0.125
0.2285
3.28
16.76
24.07
21.23
25.73
TC
Warm
Buffered
Saline
1.00
0.5
0.125
0.2262
3.05
19.01
29.09
48.10
72.06
U
Warm
Buffered
Saline
1.00
0.5
0.500
0.2375
1.89
15.20
21.09
24.04
30.57
UC
Warm
Buffered
Saline
1.00
0.5
0.500
0.2315
4.41
33.05
84.02
123.97
31.63
X
Warm
Buffered
Saline
0.10
1.0
0.250
0.2311
14.54
55.56
72.70
80.10
89.05
XC
Warm
Buffered
Saline
0.10
1.0
0.250
0.2294
18.00
92.94
X*
Warm
Buffered
Saline
0.10
1.0
0.250
0.2269
20.45
54.96
65.93
73.29
76.51
XC*
Warm
Buffered
Saline
0.10
1.0
0.250
0.2489
14.42
72.16
141.90
21.23
31.63
CC
Warm
Buffered
Saline
0.10
1.0
0.125
0.2484
17.31
49.32
67.95
74.40
86.84
CCC
Warm
Buffered
Saline
0.10
1.0
0.125
0.2262
15.78
81.74
130.11
169.14
190.63
DD
Warm
Buffered
Saline
0.10
1.0
0.500
0.2302
21.33
63.99
89.88
93.53
100.65
DDC
Warm
Buffered
Saline
0.10
1.0
0.500
0.2387
21.37
68.50
103.64
124.01
137.91
DD*
Warm
Buffered
Saline
0.10
1.0
0.500
0.2588
16.38
59.12
74.46
86.32
96.10
DDC*
Warm
Buffered
Saline
0.10
1.0
0.500
0.2313
19.71
82.45
130.31
172.55
212.02
Z
Warm
Buffered
Saline
1.00
1.0
0.250
0.2519
2.18
11.47
16.12
28.62
19.02
ZC
Warm
Buffered
Saline
1.00
1.0
0.250
0.2389
4.48
15.32
33.74
53.24
62.58
Z*
Warm
Buffered
Saline
1.00
1.0
0.250
0.2385
6.46
21.72
25.49
19.96
36.77
ZC*
Warm
Buffered
Saline
1.00
1.0
0.250
0.2602
3.54
13.72
35.59
46.85
54.88
R
Warm
Buffered
Saline
0.01
2.0
0.125
0.2254
12.64
14.15
16.64
12.20
RC
Warm
Buffered
Saline
0.01
2.0
0.125
0.2262
7.82
7.91
11.72
7.74
S
Warm
Buffered
Saline
0.01
2.0
0.500
0.2263
22.14
26.78
30.67
7.07
SC
Warm
Buffered
Saline
0.01
2.0
0.500
0.2265
21.24
38.54
52.32
42.83
BB
Warm
Buffered
Saline
0.10
2.0
0.250
0.2258
2.21
11.56
18.95
18.87
33.22
BBC
Warm
Buffered
Saline
0.10
2.0
0.250
0.2269
2.56
45.84
21.82
77.39
95.28
BB*
Warm
Buffered
Saline
0.10
2.0
0.250
0.2257
2.57
17.63
21.75
54.76
57.60
BBC*
Warm
Buffered
Saline
0.10
2.0
0.250
0.2251
2.71
24.34
44.96
71.04
68.01
V
Warm
Buffered
Saline
1.00
2.0
0.125
0.2262
3.01
10.52
15.78
15.92
18.52
VC
Warm
Buffered
Saline
1.00
2.0
0.125
0.2262
2.08
8.97
15.12
25.95
22.41
W
Warm
Buffered
Saline
1.00
2.0
0.500
0.2269
3.75
16.22
15.03
18.38
19.00
WC
Warm
Buffered
Saline
1.00
2.0
0.500
0.2251
2.13
17.95
33.14
49.31
60.46
Section
10.0
Testes
Preparation
Experiment
10­
7
Table
51.
Data
Listing
for
Incubation
Experiment
(
Continued)

SET
Organ
Prep
Technique
hCG
Conc
Time
Delay
Sample
Vol
Weight
Test
0
Test
1
Test
2
Test
3
Test
4
A
Cold
Buffered
Saline
0.01
0.5
0.125
0.2284
2.41
40.67
59.81
62.04
68.96
AC
Cold
Buffered
Saline
0.01
0.5
0.125
0.2272
2.07
50.57
102.55
135.52
173.20
B
Cold
Buffered
Saline
0.01
0.5
0.500
0.2283
51.82
78.32
90.93
98.82
BC
Cold
Buffered
Saline
0.01
0.5
0.500
0.2263
15.73
70.35
161.33
246.00
307.07
L
Cold
Buffered
Saline
0.10
0.5
0.250
0.2280
11.58
75.92
126.84
163.90
189.87
LC
Cold
Buffered
Saline
0.10
0.5
0.250
0.2369
4.69
53.19
124.61
183.50
190.92
L*
Cold
Buffered
Saline
0.10
0.5
0.250
0.2335
7.02
59.87
67.45
78.07
89.46
LC*
Cold
Buffered
Saline
0.10
0.5
0.250
0.2310
3.38
43.07
102.99
142.42
134.94
E
Cold
Buffered
Saline
1.00
0.5
0.125
0.2268
4.41
42.86
62.70
76.85
91.36
EC
Cold
Buffered
Saline
1.00
0.5
0.125
0.2268
3.26
63.05
153.13
F
Cold
Buffered
Saline
1.00
0.5
0.500
0.2268
4.54
32.10
30.64
36.68
45.06
FC
Cold
Buffered
Saline
1.00
0.5
0.500
0.2293
5.49
92.63
180.20
302.97
365.94
J
Cold
Buffered
Saline
0.01
1.0
0.250
0.2553
2.23
14.10
19.94
22.13
24.09
JC
Cold
Buffered
Saline
0.01
1.0
0.250
0.2400
1.25
13.04
27.50
33.38
39.83
J*
Cold
Buffered
Saline
0.01
1.0
0.250
0.2605
1.31
19.27
27.91
32.48
34.36
JC*
Cold
Buffered
Saline
0.01
1.0
0.250
0.2305
1.43
24.60
78.92
125.12
183.51
N
Cold
Buffered
Saline
0.10
1.0
0.125
0.2277
1.41
14.45
19.85
22.97
23.50
NC
Cold
Buffered
Saline
0.10
1.0
0.125
0.2357
1.78
15.91
37.89
47.77
N*
Cold
Buffered
Saline
0.10
1.0
0.125
0.2348
1.92
14.99
16.87
21.76
26.06
NC*
Cold
Buffered
Saline
0.10
1.0
0.125
0.2605
1.88
19.00
39.50
54.74
65.57
O
Cold
Buffered
Saline
0.10
1.0
0.500
0.2457
5.90
26.41
30.20
40.37
40.09
OC
Cold
Buffered
Saline
0.10
1.0
0.500
0.2461
4.51
34.54
76.55
100.16
128.81
K
Cold
Buffered
Saline
1.00
1.0
0.250
0.2432
4.61
24.59
32.36
32.48
34.91
KC
Cold
Buffered
Saline
1.00
1.0
0.250
0.2551
6.23
31.75
61.11
95.61
117.72
C
Cold
Buffered
Saline
0.01
2.0
0.125
0.2334
12.04
59.68
76.22
70.52
92.46
CC
Cold
Buffered
Saline
0.01
2.0
0.125
0.2303
11.59
59.75
81.37
119.97
149.41
D
Cold
Buffered
Saline
0.01
2.0
0.500
0.2391
17.69
57.88
69.30
78.59
91.47
DC
Cold
Buffered
Saline
0.01
2.0
0.500
0.2417
10.76
58.50
88.99
119.57
107.65
I
Cold
Buffered
Saline
0.10
1.0
0.250
0.2685
1.27
13.52
17.58
20.41
22.16
IC
Cold
Buffered
Saline
0.10
1.0
0.250
0.2313
1.95
25.25
66.36
96.89
104.37
I*
Cold
Buffered
Saline
0.10
1.0
0.250
0.2354
3.14
13.85
11.55
20.65
25.45
IC*
Cold
Buffered
Saline
0.10
1.0
0.250
0.2265
1.72
14.17
33.77
47.90
71.04
M
Cold
Buffered
Saline
0.10
2.0
0.250
0.2507
13.48
78.30
94.58
96.41
99.88
MC
Cold
Buffered
Saline
0.10
2.0
0.250
0.2573
8.05
61.52
126.00
129.11
119.20
G
Cold
Buffered
Saline
1.00
2.0
0.125
0.2555
7.20
60.31
54.68
69.59
48.77
GC
Cold
Buffered
Saline
1.00
2.0
0.125
0.2594
8.02
67.27
112.80
140.52
151.50
H
Cold
Buffered
Saline
1.00
2.0
0.500
0.2472
8.58
74.23
94.58
110.32
112.10
HC
Cold
Buffered
Saline
1.00
2.0
0.500
0.2585
12.42
69.05
100.97
136.25
149.67
EE
Cold
Media
0.01
0.5
0.125
0.2247
3.56
22.47
32.80
41.57
43.04
EEC
Cold
Media
0.01
0.5
0.125
0.2353
2.72
44.16
73.18
145.13
176.80
FF
Cold
Media
0.01
0.5
0.500
0.2311
4.89
58.24
53.05
46.04
59.89
FFC
Cold
Media
0.01
0.5
0.500
0.2258
6.91
31.31
19.93
17.98
67.01
PP
Cold
Media
0.10
0.5
0.250
0.2264
3.67
29.99
41.21
48.32
52.87
PPC
Cold
Media
0.10
0.5
0.250
0.2406
2.91
45.39
105.94
24.02
180.22
PP*
Cold
Media
0.10
0.5
0.250
0.2254
4.84
30.43
31.77
43.26
51.77
PPC*
Cold
Media
0.10
0.5
0.250
0.2327
6.10
56.51
105.50
110.10
232.14
II
Cold
Media
1.00
0.5
0.125
0.2289
5.37
27.13
118.92
48.27
54.74
IIC
Cold
Media
1.00
0.5
0.125
0.2321
3.58
40.07
79.15
111.68
35.55
JJ
Cold
Media
1.00
0.5
0.500
0.2301
4.48
37.29
49.76
54.89
67.88
JJC
Cold
Media
1.00
0.5
0.500
0.2315
3.67
43.97
83.71
126.00
166.95
NN
Cold
Media
0.01
1.0
0.250
0.2428
4.32
32.62
41.80
49.30
52.47
Section
10.0
Testes
Preparation
Experiment
Table
51.
Data
Listing
for
Incubation
Experiment
(
Continued)

SET
Organ
Prep
Technique
hCG
Conc
Time
Delay
Sample
Vol
Weight
Test
0
Test
1
Test
2
Test
3
Test
4
10­
8
NNC
Cold
Media
0.01
1.0
0.250
0.2415
7.54
54.99
88.65
117.52
223.81
NN*
Cold
Media
0.01
1.0
0.250
0.2272
3.79
47.80
53.13
58.10
62.76
NNC*
Cold
Media
0.01
1.0
0.250
0.2554
7.20
46.44
107.44
120.83
155.01
MM
Cold
Media
0.10
1.0
0.250
0.2396
9.22
44.45
52.13
61.56
60.60
MMC
Cold
Media
0.10
1.0
0.250
0.2466
2.96
58.48
115.98
154.06
238.12
MM*
Cold
Media
0.10
1.0
0.250
0.2271
4.89
39.10
54.65
50.07
57.64
MMC*
Cold
Media
0.10
1.0
0.250
0.2506
6.58
64.37
107.42
248.92
280.29
RR
Cold
Media
0.10
1.0
0.125
0.2599
8.31
43.13
87.69
40.44
48.71
RRC
Cold
Media
0.10
1.0
0.125
0.2382
6.76
57.98
RR*
Cold
Media
0.10
1.0
0.125
0.2397
4.84
22.40
40.13
39.88
RRC*
Cold
Media
0.10
1.0
0.125
0.2247
3.47
50.96
SS
Cold
Media
0.10
1.0
0.500
0.2347
6.43
34.04
40.90
43.42
49.34
SSC
Cold
Media
0.10
1.0
0.500
0.2404
5.57
50.46
105.91
223.00
244.55
OO
Cold
Media
1.00
1.0
0.250
0.2470
4.29
48.58
54.94
62.47
63.36
OOC
Cold
Media
1.00
1.0
0.250
0.2550
12.63
53.96
123.92
210.00
226.71
KK
Cold
Media
1.00
2.0
0.125
0.2386
2.05
11.61
15.17
17.64
20.41
KKC
Cold
Media
1.00
2.0
0.125
0.2493
16.69
34.86
43.00
31.33
LL
Cold
Media
1.00
2.0
0.500
0.2311
2.64
14.63
17.27
21.12
21.29
LLC
Cold
Media
1.00
2.0
0.500
0.2459
3.09
20.46
41.32
96.38
101.38
GG
Cold
Media
0.01
2.0
0.125
0.2488
3.26
16.60
23.71
27.17
20.86
GGC
Cold
Media
0.01
2.0
0.125
0.2298
2.31
19.41
28.37
43.43
49.56
HH
Cold
Media
0.01
2.0
0.500
0.2294
3.62
15.87
19.62
26.07
30.25
HHC
Cold
Media
0.01
2.0
0.500
0.2527
2.06
24.93
67.12
135.46
195.84
QQ
Cold
Media
0.10
2.0
0.250
0.2252
2.71
16.96
19.94
23.22
32.06
QQC
Cold
Media
0.10
2.0
0.250
0.2247
1.78
27.99
80.02
102.67
22.16
Section
10.0
Testes
Preparation
Experiment
10­
9
Table
52.
Data
Listing
of
Coded
Factor
Values
and
Logarithm
of
Testosterone
Concentrations
SET
Organ
Prep
Technique
hCG
Stimulation
hCG
Conc
Code
Time
Delay
Code
Sample
Vol
Code
L
Test
0
L
Test
1
L
Test
2
L
Test
3
L
Test
4
P
Warm
Buffered
Saline
0
­
1
­
1
­
1
1.21
2.71
2.65
3.06
3.45
PC
Warm
Buffered
Saline
1
­
1
­
1
­
1
1.39
2.81
3.59
4.14
4.43
Q
Warm
Buffered
Saline
0
­
1
­
1
1
2.02
2.93
2.90
3.28
3.43
QC
Warm
Buffered
Saline
1
­
1
­
1
1
1.57
3.54
4.80
5.09
3.45
AA
Warm
Buffered
Saline
0
0
­
1
0
1.31
2.82
3.12
3.23
3.49
AAC
Warm
Buffered
Saline
1
0
­
1
0
1.74
3.03
4.43
4.89
5.11
T
Warm
Buffered
Saline
0
1
­
1
­
1
1.19
2.82
3.18
3.06
3.25
TC
Warm
Buffered
Saline
1
1
­
1
­
1
1.12
2.94
3.37
3.87
4.28
U
Warm
Buffered
Saline
0
1
­
1
1
0.64
2.72
3.05
3.18
3.42
UC
Warm
Buffered
Saline
1
1
­
1
1
1.48
3.50
4.43
4.82
3.45
X
Warm
Buffered
Saline
0
0
0
0
2.68
4.02
4.29
4.38
4.49
XC
Warm
Buffered
Saline
1
0
0
0
2.89
4.53
X*
Warm
Buffered
Saline
0
0
0
0
3.02
4.01
4.19
4.29
4.34
XC*
Warm
Buffered
Saline
1
0
0
0
2.67
4.28
4.96
3.06
3.45
CC
Warm
Buffered
Saline
0
0
0
­
1
2.85
3.90
4.22
4.31
4.46
CCC
Warm
Buffered
Saline
1
0
0
­
1
2.76
4.40
4.87
5.13
5.25
DD
Warm
Buffered
Saline
0
0
0
1
3.06
4.16
4.50
4.54
4.61
DDC
Warm
Buffered
Saline
1
0
0
1
3.06
4.23
4.64
4.82
4.93
DD*
Warm
Buffered
Saline
0
0
0
1
2.80
4.08
4.31
4.46
4.57
DDC*
Warm
Buffered
Saline
1
0
0
1
2.98
4.41
4.87
5.15
5.36
Z
Warm
Buffered
Saline
0
1
0
0
0.78
2.44
2.78
3.35
2.95
ZC
Warm
Buffered
Saline
1
1
0
0
1.50
2.73
3.52
3.97
4.14
Z*
Warm
Buffered
Saline
0
1
0
0
1.87
3.08
3.24
2.99
3.60
ZC*
Warm
Buffered
Saline
1
1
0
0
1.26
2.62
3.57
3.85
4.01
R
Warm
Buffered
Saline
0
­
1
1
­
1
2.54
2.65
2.81
2.50
Section
10.0
Testes
Preparation
Experiment
10­
10
Table
52.
Data
Listing
of
Coded
Factor
Values
and
Logarithm
of
Testosterone
Concentrations
(
Continued)

SET
Organ
Prep
Technique
hCG
Stimulation
hCG
Conc
Code
Time
Delay
Code
Sample
Vol
Code
L
Test
0
L
Test
1
L
Test
2
L
Test
3
L
Test
4
RC
Warm
Buffered
Saline
1
­
1
1
­
1
2.06
2.07
2.46
2.05
S
Warm
Buffered
Saline
0
­
1
1
1
3.10
3.29
3.42
1.96
SC
Warm
Buffered
Saline
1
­
1
1
1
3.06
3.65
3.96
3.76
BB
Warm
Buffered
Saline
0
0
1
0
0.79
2.45
2.94
2.94
3.50
BBC
Warm
Buffered
Saline
1
0
1
0
0.94
3.83
3.08
4.35
4.56
BB*
Warm
Buffered
Saline
0
0
1
0
0.94
2.87
3.08
4.00
4.05
BBC*
Warm
Buffered
Saline
1
0
1
0
1.00
3.19
3.81
4.26
4.22
V
Warm
Buffered
Saline
0
1
1
­
1
1.10
2.35
2.76
2.77
2.92
VC
Warm
Buffered
Saline
1
1
1
­
1
0.73
2.19
2.72
3.26
3.11
W
Warm
Buffered
Saline
0
1
1
1
1.32
2.79
2.71
2.91
2.94
WC
Warm
Buffered
Saline
1
1
1
1
0.76
2.89
3.50
3.90
4.10
A
Cold
Buffered
Saline
0
­
1
­
1
­
1
0.88
3.71
4.09
4.13
4.23
AC
Cold
Buffered
Saline
1
­
1
­
1
­
1
0.73
3.92
4.63
4.91
5.15
B
Cold
Buffered
Saline
0
­
1
­
1
1
3.95
4.36
4.51
4.59
BC
Cold
Buffered
Saline
1
­
1
­
1
1
2.76
4.25
5.08
5.51
5.73
L
Cold
Buffered
Saline
0
0
­
1
0
2.45
4.33
4.84
5.10
5.25
LC
Cold
Buffered
Saline
1
0
­
1
0
1.54
3.97
4.83
5.21
5.25
L*
Cold
Buffered
Saline
0
0
­
1
0
1.95
4.09
4.21
4.36
4.49
LC*
Cold
Buffered
Saline
1
0
­
1
0
1.22
3.76
4.63
4.96
4.90
E
Cold
Buffered
Saline
0
1
­
1
­
1
1.48
3.76
4.14
4.34
4.51
EC
Cold
Buffered
Saline
1
1
­
1
­
1
1.18
4.14
5.03
F
Cold
Buffered
Saline
0
1
­
1
1
1.51
3.47
3.42
3.60
3.81
FC
Cold
Buffered
Saline
1
1
­
1
1
1.70
4.53
5.19
5.71
5.90
J
Cold
Buffered
Saline
0
­
1
0
0
0.80
2.65
2.99
3.10
3.18
JC
Cold
Buffered
Saline
1
­
1
0
0
0.22
2.57
3.31
3.51
3.68
J*
Cold
Buffered
Saline
0
­
1
0
0
0.27
2.96
3.33
3.48
3.54
JC*
Cold
Buffered
Saline
1
­
1
0
0
0.36
3.20
4.37
4.83
5.21
N
Cold
Buffered
Saline
0
0
0
­
1
0.34
2.67
2.99
3.13
3.16
NC
Cold
Buffered
Saline
1
0
0
­
1
0.58
2.77
3.63
3.87
N*
Cold
Buffered
Saline
0
0
0
­
1
0.65
2.71
2.83
3.08
3.26
NC*
Cold
Buffered
Saline
1
0
0
­
1
0.63
2.94
3.68
4.00
4.18
O
Cold
Buffered
Saline
0
0
0
1
1.78
3.27
3.41
3.70
3.69
OC
Cold
Buffered
Saline
1
0
0
1
1.51
3.54
4.34
4.61
4.86
K
Cold
Buffered
Saline
0
1
0
0
1.53
3.20
3.48
3.48
3.55
KC
Cold
Buffered
Saline
1
1
0
0
1.83
3.46
4.11
4.56
4.77
C
Cold
Buffered
Saline
0
­
1
1
­
1
2.49
4.09
4.33
4.26
4.53
CC
Cold
Buffered
Saline
1
­
1
1
­
1
2.45
4.09
4.40
4.79
5.01
D
Cold
Buffered
Saline
0
­
1
1
1
2.87
4.06
4.24
4.36
4.52
DC
Cold
Buffered
Saline
1
­
1
1
1
2.38
4.07
4.49
4.78
4.68
I
Cold
Buffered
Saline
0
0
0
0
0.24
2.60
2.87
3.02
3.10
IC
Cold
Buffered
Saline
1
0
0
0
0.67
3.23
4.20
4.57
4.65
I*
Cold
Buffered
Saline
0
0
0
0
1.15
2.63
2.45
3.03
3.24
IC*
Cold
Buffered
Saline
1
0
0
0
0.54
2.65
3.52
3.87
4.26
Section
10.0
Testes
Preparation
Experiment
Table
52.
Data
Listing
of
Coded
Factor
Values
and
Logarithm
of
Testosterone
Concentrations
(
Continued)

SET
Organ
Prep
Technique
hCG
Stimulation
hCG
Conc
Code
Time
Delay
Code
Sample
Vol
Code
L
Test
0
L
Test
1
L
Test
2
L
Test
3
L
Test
4
10­
11
M
Cold
Buffered
Saline
0
0
1
0
2.60
4.36
4.55
4.57
4.60
MC
Cold
Buffered
Saline
1
0
1
0
2.09
4.12
4.84
4.86
4.78
G
Cold
Buffered
Saline
0
1
1
­
1
1.97
4.10
4.00
4.24
3.89
GC
Cold
Buffered
Saline
1
1
1
­
1
2.08
4.21
4.73
4.95
5.02
H
Cold
Buffered
Saline
0
1
1
1
2.15
4.31
4.55
4.70
4.72
HC
Cold
Buffered
Saline
1
1
1
1
2.52
4.23
4.61
4.91
5.01
EE
Cold
Media
0
­
1
­
1
­
1
1.27
3.11
3.49
3.73
3.76
EEC
Cold
Media
1
­
1
­
1
­
1
1.00
3.79
4.29
4.98
5.17
FF
Cold
Media
0
­
1
­
1
1
1.59
4.06
3.97
3.83
4.09
FFC
Cold
Media
1
­
1
­
1
1
1.93
3.44
2.99
2.89
4.20
PP
Cold
Media
0
0
­
1
0
1.30
3.40
3.72
3.88
3.97
PPC
Cold
Media
1
0
­
1
0
1.07
3.82
4.66
3.18
5.19
PP*
Cold
Media
0
0
­
1
0
1.58
3.42
3.46
3.77
3.95
PPC*
Cold
Media
1
0
­
1
0
1.81
4.03
4.66
4.70
5.45
II
Cold
Media
0
1
­
1
­
1
1.68
3.30
4.78
3.88
4.00
IIC
Cold
Media
1
1
­
1
­
1
1.27
3.69
4.37
4.72
3.57
JJ
Cold
Media
0
1
­
1
1
1.50
3.62
3.91
4.01
4.22
JJC
Cold
Media
1
1
­
1
1
1.30
3.78
4.43
4.84
5.12
NN
Cold
Media
0
­
1
0
0
1.46
3.48
3.73
3.90
3.96
NNC
Cold
Media
1
­
1
0
0
2.02
4.01
4.48
4.77
5.41
NN*
Cold
Media
0
­
1
0
0
1.33
3.87
3.97
4.06
4.14
NNC*
Cold
Media
1
­
1
0
0
1.97
3.84
4.68
4.79
5.04
MM
Cold
Media
0
0
0
0
2.22
3.79
3.95
4.12
4.10
MMC
Cold
Media
1
0
0
0
1.09
4.07
4.75
5.04
5.47
MM*
Cold
Media
0
0
0
0
1.59
3.67
4.00
3.91
4.05
MMC*
Cold
Media
1
0
0
0
1.88
4.16
4.68
5.52
5.64
RR
Cold
Media
0
0
0
­
1
2.12
3.76
4.47
3.70
3.89
RRC
Cold
Media
1
0
0
­
1
1.91
4.06
RR*
Cold
Media
0
0
0
­
1
1.58
3.11
3.69
3.69
RRC*
Cold
Media
1
0
0
­
1
1.24
3.93
SS
Cold
Media
0
0
0
1
1.86
3.53
3.71
3.77
3.90
SSC
Cold
Media
1
0
0
1
1.72
3.92
4.66
5.41
5.50
OO
Cold
Media
0
1
0
0
1.46
3.88
4.01
4.13
4.15
OOC
Cold
Media
1
1
0
0
2.54
3.99
4.82
5.35
5.42
KK
Cold
Media
0
1
1
­
1
0.72
2.45
2.72
2.87
3.02
KKC
Cold
Media
1
1
1
­
1
2.81
3.55
3.76
3.44
LL
Cold
Media
0
1
1
1
0.97
2.68
2.85
3.05
3.06
LLC
Cold
Media
1
1
1
1
1.13
3.02
3.72
4.57
4.62
GG
Cold
Media
0
­
1
1
­
1
1.18
2.81
3.17
3.30
3.04
GGC
Cold
Media
1
­
1
1
­
1
0.84
2.97
3.35
3.77
3.90
HH
Cold
Media
0
­
1
1
1
1.29
2.76
2.98
3.26
3.41
HHC
Cold
Media
1
­
1
1
1
0.72
3.22
4.21
4.91
5.28
QQ
Cold
Media
0
0
1
0
1.00
2.83
2.99
3.15
3.47
Section
10.0
Testes
Preparation
Experiment
Table
52.
Data
Listing
of
Coded
Factor
Values
and
Logarithm
of
Testosterone
Concentrations
(
Continued)

SET
Organ
Prep
Technique
hCG
Stimulation
hCG
Conc
Code
Time
Delay
Code
Sample
Vol
Code
L
Test
0
L
Test
1
L
Test
2
L
Test
3
L
Test
4
10­
12
QQC
Cold
Media
1
0
1
0
0.58
3.33
4.38
4.63
3.10
Section
10.0
Testes
Preparation
Experiment
10­
13
Table
53.
ANOVA
results
for
PROC
RSREG
hCG
Stimulation
Organ
Prep.
Error
df
Time
R2
hCG
Conc.
Time
Delay
Aliquot
Volume
Baseline
RMSE
no
Cold
Buffered
Saline
11
T1
93
NA
***
**
0.220
T2
85
NA
**
0.342
T3
87
NA
**
**
0.293
T4
90
NA
***
**
0.262
Cold
Media
11
T1
89
NA
***
*
0.197
T2
79
NA
*
0.318
T3
89
NA
***
0.156
T4
92
NA
***
*
0.140
Warm
Buffered
Saline
9
T1
92
NA
***
0.238
T2
83
NA
***
0.355
T3
69
NA
*
0.468
T4
71
NA
**
0.423
yes
Cold
Buffered
Saline
6
T1
91
*
0.285
T2
77
0.404
T3
76
0.443
T4
71
0.473
Cold
Media
5
T1
97
***
0.119
T2
94
*
**
**
0.227
T3
74
0.649
T4
81
0.589
Warm
Buffered
Saline
4
T1
91
0.412
T2
87
0.499
T3
89
*
**
0.407
T4
90
*
*
**
0.414
*
Statistically
significant
F
test
at
0.10
level
of
significance.
**
Statistically
significant
F
test
at
0.05
level
of
significance.
***
Statistically
significant
F
test
at
0.01
level
of
significance.
Note:
R2
is
the
percentage
of
variation
accounted
for
by
the
model.
RMSE
=
square
root
of
the
residual
(
error)
mean
square
NA
=
not
applicable
Section
10.0
Testes
Preparation
Experiment
10­
14
Table
54.
Response
Surface
Regression
Results
for
No
hCG
Stimulation
Organ
Prep
Tech
Model
Terms
L
Test_
1
L
Test
2
L
Test
3
L
Test
4
Beta
P
Value
Beta
P
Value
Beta
P
Value
Beta
P
Value
Cold
Buffered
Saline
Time_
Delay_
Code
0.05
0.536
0.02
0.890
­
0.04
0.740
­
0.12
0.252
Sample_
vol_
code
­
0.04
0.625
­
0.11
0.422
­
0.08
0.471
­
0.08
0.428
Time_
Delay_
Code*
Time_
Delay_
Code
0.83
0.001
0.84
0.016
0.70
0.018
0.63
0.018
Sample_
vol_
code*
Time_
Delay_
Code
0.07
0.434
0.21
0.152
0.20
0.105
0.23
0.047
Sample_
vol_
code*
Sample_
vol_
code
­
0.12
0.343
­
0.19
0.317
­
0.16
0.334
­
0.16
0.278
LTest_
0
0.33
0.022
0.34
0.107
0.38
0.043
0.43
0.014
Cold
Media
Time_
Delay_
Code
­
0.33
0.002
­
0.37
0.013
­
0.32
0.000
­
0.36
<.
0001
Sample_
vol_
code
0.12
0.090
­
0.13
0.215
0.04
0.423
0.09
0.058
Time_
Delay_
Code*
Time_
Delay_
Code
­
0.46
0.012
­
0.39
0.142
­
0.34
0.018
­
0.30
0.019
Sample_
vol_
code*
Time_
Delay_
Code
­
0.14
0.065
0.03
0.811
­
0.01
0.791
­
0.02
0.694
Sample_
vol_
code*
Sample_
vol_
code
­
0.08
0.504
0.13
0.501
­
0.13
0.164
­
0.15
0.079
LTest_
0
0.26
0.237
0.49
0.167
0.12
0.462
0.13
0.392
Warm
Buffered
Saline
Time_
Delay_
Code
­
0.02
0.784
0.01
0.966
0.04
0.828
0.01
0.943
Sample_
vol_
code
0.08
0.347
0.01
0.918
0.08
0.660
0.02
0.874
Time_
Delay_
Code*
Time_
Delay_
Code
­
0.12
0.547
­
0.26
0.413
­
0.21
0.608
0.06
0.866
Sample_
vol_
code*
Time_
Delay_
Code
0.08
0.427
­
0.02
0.893
­
0.01
0.976
­
0.01
0.962
Sample_
vol_
code*
Sample_
vol_
code
­
0.03
0.817
­
0.05
0.814
­
0.13
0.657
­
0.22
0.398
LTest_
0
0.64
0.000
0.58
0.008
0.50
0.052
0.60
0.015
Note:
Ltest_
J
=
log
testosterone
concentration
at
time
J
Beta
=
regression
coefficient,
as
applied
to
coded
factors
Pvalue
=
significance
level
for
t­
test
of
hypothesis
that
the
true
Beta
=
0
Section
10.0
Testes
Preparation
Experiment
10­
15
Table
55.
Response
Surface
Regression
Results
for
hCG
Stimulation
Organ
Prep
Tech
Model
Terms
L
Test
1
L
Test
2
L
Test
3
L
Test
4
Beta
P
Value
Beta
P
Value
Beta
P
Value
Beta
P
Value
Cold
Buffered
Saline
Time_
Delay_
Code
­
0.07
0.602
­
0.14
0.473
­
0.25
0.249
­
0.28
0.234
Sample_
vol_
code
0.05
0.695
0.14
0.485
0.12
0.574
0.13
0.571
hCG_
conc_
code
0.11
0.343
0.06
0.702
0.09
0.624
0.04
0.828
Time_
Delay_
Code*
Time_
Delay_
Code
0.69
0.028
0.67
0.098
0.54
0.201
0.32
0.456
Sample_
vol_
code*
Time_
Delay_
Code
­
0.04
0.797
­
0.12
0.569
­
0.12
0.596
­
0.20
0.417
Sample_
vol_
code*
Sample_
vol_
code
0.12
0.499
0.00
0.994
0.01
0.979
0.06
0.853
hCG_
conc_
code*
Time_
Delay_
Code
­
0.03
0.840
0.05
0.774
­
0.01
0.959
0.05
0.811
hCG_
conc_
code*
Sample_
vol_
code
0.05
0.706
0.02
0.926
0.03
0.880
0.12
0.585
hCG_
conc_
code*
hCG_
conc_
code
0.06
0.722
­
0.02
0.940
0.08
0.778
0.14
0.622
LTest_
0
0.23
0.289
0.10
0.720
0.17
0.590
0.17
0.607
Cold
Media
Time_
Delay_
Code
­
0.33
0.001
­
0.30
0.029
0.04
0.890
­
0.47
0.124
Sample_
vol_
code
0.04
0.467
0.17
0.157
0.09
0.770
0.66
0.055
hCG_
conc_
code
­
0.03
0.569
0.01
0.898
0.05
0.863
­
0.48
0.102
Time_
Delay_
Code*
Time_
Delay_
Code
­
0.42
0.013
­
0.45
0.085
­
0.76
0.256
­
0.48
0.415
Sample_
vol_
code*
Time_
Delay_
Code
0.13
0.088
0.46
0.011
0.69
0.087
0.70
0.064
Sample_
vol_
code*
Sample_
vol_
code
­
0.16
0.133
­
0.50
0.034
0.25
0.632
­
0.18
0.710
hCG_
conc_
code*
Time_
Delay_
Code
­
0.11
0.119
­
0.40
0.018
­
0.46
0.219
­
0.51
0.147
hCG_
conc_
code*
Sample_
vol_
code
0.09
0.205
0.26
0.070
0.43
0.237
0.73
0.054
hCG_
conc_
code*
hCG_
conc_
code
­
0.17
0.134
­
0.22
0.280
­
0.36
0.512
­
0.38
0.453
LTest_
0
0.07
0.601
­
0.03
0.884
0.39
0.580
0.59
0.364
Warm
Buffered
Saline
Time_
Delay_
Code
0.49
0.396
­
0.24
0.721
­
1.71
0.029
­
1.64
0.034
Sample_
vol_
code
0.13
0.505
0.21
0.387
0.52
0.043
0.27
0.212
hCG_
conc_
code
­
0.57
0.157
­
0.06
0.880
­
0.15
0.665
0.26
0.469
Time_
Delay_
Code*
Time_
Delay_
Code
0.21
0.861
0.22
0.882
­
3.64
0.032
­
3.53
0.037
Sample_
vol_
code*
Time_
Delay_
Code
0.00
0.999
­
0.14
0.626
­
0.42
0.113
0.21
0.373
Sample_
vol_
code*
Sample_
vol_
code
0.15
0.835
­
0.11
0.899
2.46
0.019
2.12
0.032
hCG_
conc_
code*
Time_
Delay_
Code
­
0.64
0.147
0.04
0.935
0.30
0.441
0.58
0.178
hCG_
conc_
code*
Sample_
vol_
code
0.02
0.936
0.05
0.833
0.20
0.357
0.15
0.501
hCG_
conc_
code*
hCG_
conc_
code
0.18
0.884
­
0.11
0.940
­
4.10
0.024
­
4.40
0.020
LTest_
0
0.69
0.568
0.70
0.631
­
3.70
0.028
­
3.36
0.040
Note:
Ltest_
J
=
log
testosterone
concentration
at
time
J
Beta
=
regression
coefficient,
as
applied
to
coded
factors
Pvalue
=
significance
level
for
t­
test
of
hypothesis
that
the
true
Beta
=
0
10­

16
Section
10
Testes
Preparation
Experiment
Table
56.
Maximum
Values
for
Each
Time
Point
hCG
Stimulation
Organ
Prep
Technique
Predicted
Log(
Testosterone
Concentration)*
Sample
Volume
hCG
Conc
Time
Delay
log(
T0)
T1
T2
T3
T4
No
Cold
Buffered
T1
opt
3.977
(
0.187)
4.181
(
0.291)
4.212
(
0.249)
4.186
(
0.223)
0.253
2.0
1.5058
No
Cold
Buffered
T2
opt
3.976
(
0.187)
4.182
(
0.291)
4.214
(
0.249)
4.190
(
0.223)
0.258
2.0
1.5058
No
Cold
Buffered
T3
opt
3.879
(
0.122)
4.183
(
0.189)
4.324
(
0.162)
4.469
(
0.145)
0.224
0.5
1.5058
No
Cold
Buffered
T4
opt
3.876
(
0.120)
4.182
(
0.188)
4.324
(
0.161)
4.470
(
0.144)
0.221
0.5
1.5058
No
Cold
Media
T1
opt
3.823
(
0.142)
3.876
(
0.230)
3.942
(
0.113)
4.092
(
0.101)
0.499
0.7
1.4503
No
Cold
Media
T2
opt
3.484
(
0.145)
4.156
(
0.234)
3.842
(
0.115)
3.872
(
0.103)
0.125
0.8
1.4503
No
Cold
Media
T3
opt
3.746
(
0.086)
3.876
(
0.139)
4.028
(
0.068)
4.153
(
0.061)
0.283
0.7
1.4503
No
Cold
Media
T4
opt
3.753
(
0.083)
3.853
(
0.134)
4.020
(
0.066)
4.162
(
0.059)
0.318
0.7
1.4503
No
Warm
Buffered
T1
opt
3.273
(
0.177)
3.490
(
0.264)
3.680
(
0.348)
3.615
(
0.315)
0.499
1.2
1.7231
No
Warm
Buffered
T2
opt
3.226
(
0.120)
3.542
(
0.178)
3.740
(
0.235)
3.804
(
0.212)
0.274
1.0
1.7231
No
Warm
Buffered
T3
opt
3.237
(
0.119)
3.539
(
0.177)
3.745
(
0.233)
3.794
(
0.211)
0.308
1.1
1.7231
No
Warm
Buffered
T4
opt
3.072
(
0.171)
3.291
(
0.254)
3.562
(
0.335)
3.877
(
0.303)
0.255
2.0
1.7231
Yes
Cold
Buffered
T1
opt
3.963
(
0.174)
4.829
(
0.246)
5.176
(
0.270)
5.178
(
0.289)
0.264
0.127
0.5
1.4834
Yes
Cold
Buffered
T2
opt
3.966
(
0.168)
4.858
(
0.238)
5.193
(
0.261)
5.212
(
0.279)
0.282
0.101
0.5
1.4834
Yes
Cold
Buffered
T3
opt
3.986
(
0.170)
4.868
(
0.241)
5.210
(
0.264)
5.223
(
0.282)
0.284
0.122
0.5
1.4834
Yes
Cold
Buffered
T4
opt
4.010
(
0.168)
4.916
(
0.238)
5.253
(
0.261)
5.293
(
0.279)
0.326
0.112
0.5
1.4834
Yes
Cold
Media
T1
opt
4.133
(
0.062)
4.914
(
0.118)
4.975
(
0.336)
5.273
(
0.305)
0.247
0.111
0.8
1.4291
Yes
Cold
Media
T2
opt
4.087
(
0.068)
4.951
(
0.130)
4.902
(
0.372)
5.104
(
0.338)
0.262
0.375
0.7
1.4291
Yes
Cold
Media
T3
opt
3.865
(
0.098)
4.572
(
0.187)
5.640
(
0.535)
5.699
(
0.486)
0.497
0.166
1.2
1.4291
Yes
Cold
Media
T4
opt
3.927
(
0.101)
4.577
(
0.193)
5.565
(
0.550)
5.706
(
0.500)
0.498
0.142
1.1
1.4291
Yes
Warm
Buffered
T1
opt
5.429
(
2.298)
3.878
(
2.781)
­
2.568
(
2.272)
­
3.233
(
2.312)
0.266
0.010
2.0
1.6640
10­

17
Section
10
Testes
Preparation
Experiment
Table
56.
Maximum
Values
for
Each
Time
Point,
(
Continued)

hCG
Stimulation
Organ
Prep
Technique
Predicted
Log(
Testosterone
Concentration)*
Sample
Volume
hCG
Conc
Time
Delay
log(
T0)
T1
T2
T3
T4
Yes
Warm
Buffered
3.107
(
0.504)
4.555
(
0.610)
5.826
(
0.498)
5.623
(
0.507)
0.321
0.082
0.5
1.6640T
Yes
Warm
Buffered
T3
opt
3.521
(
1.594)
4.159
(
1.929)
10.264
(
1.575)
9.685
(
1.603)
0.499
0.100
0.9
1.6640
Yes
Warm
Buffered
T4
opt
3.526
(
1.580)
4.140
(
1.912)
10.193
(
1.561)
9.655
(
1.589)
0.497
0.106
0.9
1.6640
*
Values
in
parentheses
are
standard
errors
Table
57.
Predicted
values
for
Specified
Optimum
Factor
Combinations
hCG
Stimulation
Organ
Prep
Technique
Predicted
Log(
Testosterone
Concentration)*
Sample
Volume
hCG
Concent
Time
Delay
log(
T0)
T1
T2
T3
T4
No
Cold
Buffered
Optimal
2.935
(
0.151)
3.027
(
0.235)
3.312
(
0.201)
3.436
(
0.180)
0.500
1.0
1.5058
No
Cold
Buffered
Optimal
3.643
(
0.199)
3.641
(
0.310)
3.846
(
0.265)
3.959
(
0.237)
0.500
0.5
1.5058
Yes
Cold
Buffered
Optimal
3.366
(
0.213)
4.155
(
0.302)
4.499
(
0.331)
4.748
(
0.354)
0.500
0.100
1.0
1.4834
Yes
Cold
Buffered
Optimal
4.168
(
0.272)
5.076
(
0.385)
5.406
(
0.422)
5.545
(
0.451)
0.500
0.100
0.5
1.4834
*
Values
in
parentheses
are
standard
errors
Section
10.0
Testes
Preparation
Experiment
10­
18
Table
58.
ANOVA
results
for
PROC
RSREG
with
the
Baseline
Concentration
Removed
from
Model
hCG
Stimulation
Organ
Prep.
Error
df
Time
R2
hCG
Conc.
Time
Delay
Aliquot
Volume
RMSE
no
Cold
Buffered
Saline
13
T1
89
NA
***
0.261
T2
80
NA
***
0.374
T3
80
NA
***
0.342
T4
81
NA
***
0.334
Cold
Media
12
T1
87
NA
***
0.201
T2
75
NA
***
0.333
T3
89
NA
***
0.153
T4
92
NA
***
*
0.139
Warm
Buffered
Saline
12
T1
66
NA
***
0.438
T2
63
NA
***
0.471
T3
55
NA
**
0.510
T4
48
NA
*
0.646
yes
Cold
Buffered
Saline
7
T1
89
***
0.292
T2
77
**
0.378
T3
74
*
0.421
T4
69
0.449
Cold
Media
7
T1
97
***
0.109
T2
86
**
0.304
T3
73
*
0.590
T4
69
0.698
Warm
Buffered
Saline
7
T1
84
**
0.450
T2
87
**
0.455
T3
67
0.664
T4
80
*
*
0.578
*
Statistically
significant
F
test
at
0.10
level
of
significance.
**
Statistically
significant
F
test
at
0.05
level
of
significance.
***
Statistically
significant
F
test
at
0.01
level
of
significance.
Note:
R2
is
the
percentage
of
variation
accounted
for
by
the
model.
RMSE
=
square
root
of
the
residual
(
error)
mean
square
NA
=
not
applicable
Section
10.0
Testes
Preparation
Experiment
10­
19
Table
59.
Response
Surface
Regression
Results
for
No
hCG
Stimulation
with
Baseline
Concentration
Removed
Organ
Prep
Tech
Model
Terms
Ltest_
1
Ltest_
2
Ltest_
3
Ltest_
4
Beta
P
value
Beta
P
value
Beta
P
value
Beta
P
value
Cold
Buffered
Saline
Time_
Delay_
Code
0.16
0.065
0.09
0.437
0.06
0.604
0.00
0.981
Sample_
vol_
code
0.07
0.394
0.05
0.682
0.08
0.474
0.09
0.398
Time_
Delay_
Code*
Time_
Delay_
Code
1.25
<.
0001
1.28
<.
0001
1.19
<.
0001
1.19
<.
0001
Sample_
vol_
code*
Time_
Delay_
Code
0.03
0.766
0.11
0.411
0.12
0.356
0.15
0.238
Sample_
vol_
code*
Sample_
vol_
code
­
0.16
0.240
­
0.20
0.314
­
0.18
0.318
­
0.20
0.263
Cold
Media
Time_
Delay_
Code
­
0.39
<.
0001
­
0.49
0.000
­
0.35
<.
0001
­
0.39
<.
0001
Sample_
vol_
code
0.12
0.079
­
0.12
0.272
0.04
0.381
0.10
0.047
Time_
Delay_
Code*
Time_
Delay_
Code
­
0.60
0.000
­
0.66
0.003
­
0.41
0.000
­
0.37
0.000
Sample_
vol_
code*
Time_
Delay_
Code
­
0.14
0.081
0.04
0.733
­
0.01
0.834
­
0.02
0.743
Sample_
vol_
code*
Sample_
vol_
code
­
0.03
0.793
0.22
0.243
­
0.11
0.208
­
0.13
0.106
Warm
Buffered
Saline
Time_
Delay_
Code
­
0.04
0.769
­
0.02
0.871
0.00
0.982
­
0.22
0.280
Sample_
vol_
code
0.15
0.284
0.12
0.437
0.16
0.335
0.02
0.901
Time_
Delay_
Code*
Time_
Delay_
Code
­
0.99
0.001
­
1.04
0.001
­
0.90
0.005
­
0.91
0.018
Sample_
vol_
code*
Time_
Delay_
Code
0.11
0.495
0.06
0.735
0.05
0.784
­
0.08
0.719
Sample_
vol_
code*
Sample_
vol_
code
0.28
0.237
0.21
0.398
0.09
0.736
­
0.13
0.710
Note:
Ltest_
J
=
log
testosterone
concentration
at
time
J
Beta
=
regression
coefficient,
as
applied
to
coded
factors
Pvalue
=
significance
level
for
t­
test
of
hypothesis
that
the
true
Beta
=
0
Section
10.0
Testes
Preparation
Experiment
10­
20
Table
60.
Response
Surface
Regression
Results
for
hCG
Stimulation
with
Baseline
Concentration
Removed
Organ
Prep
Tech
Model
Terms
Ltest_
1
Ltest_
2
Ltest_
3
Ltest_
4
Beta
Pvalu
e
Beta
Pvalu
e
Beta
Pvalue
Beta
Pvalue
Cold
Buffered
Saline
Time_
Delay_
Code
0.02
0.888
­
0.10
0.496
­
0.19
0.262
­
0.21
0.233
Sample_
vol_
code
0.14
0.244
0.18
0.254
0.19
0.279
0.20
0.283
hCG_
conc_
code
0.13
0.258
0.07
0.625
0.10
0.529
0.06
0.737
Time_
Delay_
Code*
Time_
Delay_
Cod
e
0.91
0.001
0.76
0.008
0.70
0.019
0.48
0.091
Sample_
vol_
code*
Time_
Delay_
Code
­
0.10
0.458
­
0.15
0.409
­
0.17
0.395
­
0.25
0.250
Sample_
vol_
code*
Sample_
vol_
code
0.18
0.325
0.02
0.917
0.05
0.846
0.10
0.717
hCG_
conc_
code*
Time_
Delay_
Code
­
0.01
0.922
0.06
0.730
0.00
0.999
0.06
0.760
hCG_
conc_
code*
Sample_
vol_
code
0.03
0.848
0.01
0.971
0.01
0.949
0.10
0.619
hCG_
conc_
code*
hCG_
conc_
code
0.07
0.689
­
0.01
0.950
0.08
0.745
0.15
0.583
Cold
Media
Time_
Delay_
Code
­
0.33
<.
0001
­
0.16
0.133
0.05
0.790
­
0.35
0.145
Sample_
vol_
code
0.02
0.584
­
0.01
0.949
­
0.01
0.962
0.37
0.173
hCG_
conc_
code
0.00
0.980
0.14
0.174
0.20
0.309
­
0.14
0.534
Time_
Delay_
Code*
Time_
Delay_
Cod
e
­
0.46
0.000
­
0.41
0.066
­
1.01
0.028
­
0.85
0.090
Sample_
vol_
code*
Time_
Delay_
Code
0.09
0.056
0.28
0.033
0.49
0.052
0.25
0.351
Sample_
vol_
code*
Sample_
vol_
code
­
0.16
0.073
­
0.36
0.129
0.26
0.552
­
0.06
0.899
hCG_
conc_
code*
Time_
Delay_
Code
­
0.07
0.096
­
0.22
0.076
­
0.25
0.262
­
0.05
0.835
hCG_
conc_
code*
Sample_
vol_
code
0.05
0.246
0.08
0.464
0.23
0.297
0.29
0.279
hCG_
conc_
code*
hCG_
conc_
code
­
0.13
0.089
­
0.22
0.285
­
0.17
0.665
­
0.07
0.879
Section
10.0
Testes
Preparation
Experiment
Table
60.
Response
Surface
Regression
Results
for
hCG
Stimulation
with
Baseline
Concentration
Removed
(
Continued)

Organ
Prep
Tech
Model
Terms
Ltest_
1
Ltest_
2
Ltest_
3
Ltest_
4
Beta
Pvalu
e
Beta
Pvalu
e
Beta
Pvalue
Beta
Pvalue
Warm
Buffered
Saline
Time_
Delay_
Code
­
0.19
0.209
­
0.53
0.007
­
0.46
0.058
­
0.31
0.118
Sample_
vol_
code
0.26
0.099
0.41
0.021
0.38
0.104
0.09
0.624
hCG_
conc_
code
­
0.02
0.916
­
0.04
0.805
0.10
0.668
0.23
0.285
Time_
Delay_
Code*
Time_
Delay_
Cod
e
­
0.39
0.173
­
0.46
0.124
0.13
0.736
­
0.14
0.677
Sample_
vol_
code*
Time_
Delay_
Code
0.05
0.758
0.01
0.946
0.03
0.899
0.56
0.029
Sample_
vol_
code*
Sample_
vol_
code
0.45
0.126
0.24
0.383
0.39
0.346
0.27
0.448
hCG_
conc_
code*
Time_
Delay_
Code
­
0.02
0.924
0.14
0.427
0.16
0.521
0.19
0.373
hCG_
conc_
code*
Sample_
vol_
code
­
0.06
0.715
­
0.12
0.483
­
0.11
0.664
­
0.07
0.738
hCG_
conc_
code*
hCG_
conc_
code
­
1.08
0.005
­
0.79
0.024
­
0.82
0.079
­
1.14
0.013
Note:
Ltest_
J
=
log
testosterone
concentration
at
time
J
Beta
=
regression
coefficient,
as
applied
to
coded
factors
Pvalue
=
significance
level
for
t­
test
of
hypothesis
that
the
true
Beta
=
0
10­

22
Section
10
Testes
Preparation
Experiment
Table
61.
Maximum
Values
for
Models
with
Baseline
Concentration
Removed
hCG
Stimulation
Organ
Prep
Technique
Predicted
Log(
Testosterone
Concentration)*
Sample
Volume
hCG
Conc.
Time
Delay
T1
T2
T3
T4
No
Cold
Buffered
Saline
T1
opt
4.313
(
0.156)
4.495
(
0.224)
4.574
(
0.205)
4.613
(
0.200)
0.256
2.0
No
Cold
Buffered
Saline
T2
opt
4.313
(
0.156)
4.496
(
0.224)
4.576
(
0.205)
4.616
(
0.200)
0.259
2.0
No
Cold
Buffered
Saline
T3
opt
4.312
(
0.156)
4.495
(
0.223)
4.576
(
0.204)
4.617
(
0.199)
0.262
2.0
No
Cold
Buffered
Saline
T4
opt
4.309
(
0.155)
4.494
(
0.223)
4.576
(
0.204)
4.617
(
0.199)
0.265
2.0
No
Cold
Media
T1
opt
3.926
(
0.120)
4.084
(
0.200)
3.991
(
0.092)
4.137
(
0.083)
0.498
0.8
No
Cold
Media
T2
opt
3.595
(
0.122)
4.346
(
0.202)
3.888
(
0.093)
3.917
(
0.084)
0.126
0.8
No
Cold
Media
T3
opt
3.916
(
0.117)
4.052
(
0.194)
3.987
(
0.089)
4.146
(
0.081)
0.497
0.7
No
Cold
Media
T4
opt
3.888
(
0.117)
4.007
(
0.194)
3.969
(
0.089)
4.138
(
0.081)
0.499
0.6
No
Warm
Buffered
Saline
T1
opt
3.957
(
0.233)
4.151
(
0.251)
4.233
(
0.271)
4.086
(
0.344)
0.500
1.0
No
Warm
Buffered
Saline
T2
opt
3.957
(
0.233)
4.151
(
0.251)
4.233
(
0.271)
4.091
(
0.344)
0.500
1.0
No
Warm
Buffered
Saline
T3
opt
3.957
(
0.233)
4.151
(
0.251)
4.233
(
0.271)
4.088
(
0.344)
0.500
1.0
No
Warm
Buffered
Saline
T4
opt
3.902
(
0.228)
4.103
(
0.245)
4.189
(
0.265)
4.122
(
0.336)
0.497
0.9
Yes
Cold
Buffered
Saline
T1
opt
3.950
(
0.209)
4.611
(
0.270)
4.758
(
0.301)
4.708
(
0.321)
0.255
0.118
2.0
Yes
Cold
Buffered
Saline
T2
opt
3.971
(
0.172)
4.870
(
0.223)
5.202
(
0.248)
5.224
(
0.265)
0.288
0.102
0.5
Yes
Cold
Buffered
Saline
T3
opt
3.974
(
0.171)
4.866
(
0.221)
5.206
(
0.246)
5.229
(
0.262)
0.294
0.118
0.5
Yes
Cold
Buffered
Saline
T4
opt
4.045
(
0.171)
4.937
(
0.221)
5.282
(
0.246)
5.329
(
0.262)
0.334
0.109
0.5
Yes
Cold
Media
T1
opt
4.034
(
0.075)
4.848
(
0.208)
5.070
(
0.404)
5.251
(
0.478)
0.292
0.978
0.7
Yes
Cold
Media
T2
opt
4.006
(
0.076)
4.876
(
0.214)
4.967
(
0.414)
4.952
(
0.490)
0.217
0.999
0.6
Yes
Cold
Media
T3
opt
3.895
(
0.088)
4.558
(
0.246)
5.649
(
0.478)
5.683
(
0.565)
0.498
0.245
1.1
Yes
Cold
Media
T4
opt
3.967
(
0.090)
4.554
(
0.252)
5.556
(
0.489)
5.724
(
0.578)
0.498
0.214
1.0
Yes
Warm
Buffered
Saline
T1
opt
4.595
(
0.268)
5.182
(
0.271)
5.106
(
0.395)
5.011
(
0.344)
0.499
0.095
1.0
Yes
Warm
Buffered
Saline
T2
opt
4.591
(
0.266)
5.265
(
0.269)
5.207
(
0.393)
4.933
(
0.342)
0.499
0.083
0.8
Yes
Warm
Buffered
Saline
T3
opt
4.554
(
0.270)
5.290
(
0.273)
5.317
(
0.398)
4.870
(
0.347)
0.497
0.094
0.7
Yes
Warm
Buffered
Saline
T4
opt
4.020
(
0.326)
4.463
(
0.329)
4.758
(
0.480)
5.423
(
0.418)
0.126
0.112
0.6
*
Values
in
parentheses
are
standard
errors
10­

23
Section
10
Testes
Preparation
Experiment
Table
62.
Predicted
values
for
Specified
Optimum
Factor
Combinations
for
Models
with
Baseline
Concentration
Removed
hCG
Stimulation
Organ
Prep
Technique
Predicted
Log
(
Testosterone
Concentration)*
Sample
Volume
hCG
Concent
Time
Delay
T1
T2
T3
T4
No
Cold
Buffered
Optimal
2.815
(
0.157)
2.973
(
0.225)
3.227
(
0.205)
3.317
(
0.200)
0.5
1.0
No
Cold
Buffered
Optimal
3.872
(
0.169)
4.048
(
0.243)
4.242
(
0.222)
4.359
(
0.217)
0.5
0.5
Yes
Cold
Buffered
Optimal
3.344
(
0.218)
4.145
(
0.282)
4.482
(
0.314)
4.731
(
0.334)
0.5
0.100
1.0
Yes
Cold
Buffered
Optimal
4.337
(
0.236)
5.154
(
0.305)
5.535
(
0.339)
5.676
(
0.361)
0.5
0.100
0.5
*
Values
in
parentheses
are
standard
errors
Section
11.0
Equilibration
11­
1
11.0
EQUIIBRATION
The
optimal
values
for
factors
were
selected
based
upon
the
results
of
the
statistical
analyses
of
the
incubation
and
testes
preparation
experiments.
Further
considerations
were
the
practical
aspects
of
the
assay
and
how
it
will
be
used
as
a
screen
for
endocrine
disruptors.
The
temperature
chosen
was
36
?
C
which
is
about
3
degrees
lower
than
the
literature
value
for
the
rat
body
temperature.
This
correlates
with
the
human
situation
where
the
testicular
temperature
is
34
?
C
which
is
3
degrees
below
the
average
body
temperature.
The
media
chosen
is
M­
199
without
phenol
red
gassed
with
95%
O
2
:
5%
CO
2.
Five
mL
of
this
media
is
placed
in
a
scintillation
vial
and
a
0175
mg
slice
of
testicular
tissue
is
added
and
incubated
for
one
hour.
At
the
end
of
the
hour,
the
media
is
removed
and
replaced
with
fresh
media,
samples
are
removed
for
the
determination
of
LDH
and
testosterone
baselines
and
the
same
amount
of
media
is
replaced
with
either
plain
media
or
media
with
hCG.
The
vials
are
then
incubated
for
a
total
of
4
hours
with
aliquots
removed
at
hourly
intervals.

11.1
Statistical
Analysis
of
Phase
II
Equilibration
Period
Determination
11.1.1
Objectives
The
objective
of
the
equilibration
experiment
was
to
determine
whether
LDH
can
be
used
as
a
marker
to
determine
the
period
of
time
needed
for
parenchymal
equilibration
(
baseline).
The
assay
was
performed
with
and
without
hCG
stimulation
at
various
equilibration
times
before
baseline
sampling:
15
minutes,
30
minutes,
45
minutes,
60
minutes,
90
minutes
and
120
minutes.
Concentrations
of
LDH
and
testosterone
for
each
sample
were
measured
at
baseline
and
at
1,
2,
3
and
4
hours
after
baseline.

11.1.2
Data
A
SAS
data
set
was
constructed
from
the
raw
Excel
data
file.
Each
observation
includes
concentration
measurements
for
LDH
and
testosterone
and
the
corresponding
sampling
time
and
repeated
measurement
times.
The
data
used
in
the
analysis
are
displayed
in
Tables
63
and
64.

11.1.3
Statistical
Analysis
Methods
and
Results
In
order
to
examine
the
effects
of
the
sampling
times
on
the
concentrations
of
LDH
and
testosterone,
summary
statistics
were
calculated
for
the
combinations
of
sampling
times
and
repeated
measurements.
Results
of
the
means,
standard
deviations,
sample
sizes
and
coefficients
of
variation
for
LDH
concentrations
are
shown
in
Tables
65
through
72
and
the
results
for
testosterone
concentrations
are
shown
in
Tables
73
through
80.
Graphs
of
the
means
and
coefficients
of
variation
for
LDH
and
testosterone
concentrations
are
presented
at
the
end
of
the
section
report.
The
summary
of
these
data
is
shown
in
Tables
81
through
84.
Section
11.0
Equilibration
11­
2
Table
63.
Equilibration
Data
for
No
hCG
Stimulation
Sample
ID
Timepoint
Equilibration
time
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)
A
Baseline
15
min.
233.2
0.0029
A
T­
1
(
1
hr)
15
min.
1355.7
0.0300
A
T­
2
(
2
hr)
15
min.
1420.8
0.0350
A
T­
3
(
3
hr)
15
min.
1480.5
0.0322
A
T­
4
(
4
hr)
15
min.
1556.4
0.0306
B
Baseline
15
min.
331.8
0.0030
B
T­
1
(
1
hr)
15
min
1117.6
0.0314
B
T­
2
(
2
hr)
15
min.
1146.7
0.0132
B
T­
3
(
3
hr)
15
min.
1152.5
0.0291
B
T­
4
(
4
hr)
15
min.
1146.7
0.0295
C
Baseline
15
min.
394.1
0.0038
C
T­
1
(
1
hr)
15
min.
1092.3
0.0303
C
T­
2
(
2
hr)
15
min.
1064.2
0.0304
C
T­
3
(
3
hr)
15
min.
1013.5
0.0276
C
T­
4
(
4
hr)
15
min.
929.1
0.0271
D
Baseline
30
min
218.1
0.0041
D
T­
1
(
1
hr)
30
min
839.7
0.0261
D
T­
2
(
2
hr)
30
min
932.4
0.0274
D
T­
3
(
3
hr)
30
min
970.6
0.0275
D
T­
4
(
4
hr)
30
min
970.6
0.0260
E
Baseline
30
min
135.6
0.0043
E
T­
1
(
1
hr)
30
min
672.3
0.0289
E
T­
2
(
2
hr)
30
min
785.3
0.0302
E
T­
3
(
3
hr)
30
min
853.1
0.0299
E
T­
4
(
4
hr)
30
min
870.1
0.0295
F
Baseline
30
min
110.6
0.0023
F
T­
1
(
1
hr)
30
min
504.0
0.0293
F
T­
2
(
2
hr)
30
min
522.4
0.0314
F
T­
3
(
3
hr)
30
min
565.5
0.0301
F
T­
4
(
4
hr)
30
min
596.2
0.0315
G
Baseline
45
min
262.6
0.0031
G
T­
1
(
1
hr)
45
min
1061.3
0.0176
G
T­
2
(
2
hr)
45
min
1198.0
0.0211
Section
11.0
Equilibration
11­
3
Table
63.
Equilibration
Data
for
No
hCG
Stimulation
(
Continued)

Sample
ID
Timepoint
Equilibration
time
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)
G
T­
3
(
3
hr)
45
min
1247.3
0.0187
G
T­
4
(
4
hr)
45
min
1187.1
0.0183
H
Baseline
45
min
300.6
0.0039
H
T­
1
(
1
hr)
45
min
1043.7
0.0252
H
T­
2
(
2
hr)
45
min
1111.7
0.0288
H
T­
3
(
3
hr)
45
min
1179.8
0.0251
H
T­
4
(
4
hr)
45
min
1174.1
0.0252
I
Baseline
45
min
1055.4
0.0070
I
T­
1
(
1
hr)
45
min
1032.1
0.0351
I
T­
2
(
2
hr)
45
min
1061.2
0.0334
I
T­
3
(
3
hr)
45
min
1008.7
0.0308
I
T­
4
(
4
hr)
45
min
262.4
0.0287
J
Baseline
60
min
159.4
0.0080
J
T­
1
(
1
hr)
60
min
615.7
0.0521
J
T­
2
(
2
hr)
60
min
698.2
0.0476
J
T­
3
(
3
hr)
60
min
830.1
0.0437
J
T­
4
(
4
hr)
60
min
874.1
0.0398
K
Baseline
60
min
300.9
0.0096
K
T­
1
(
1
hr)
60
min
886.2
0.0463
K
T­
2
(
2
hr)
60
min
809.6
0.0408
K
T­
3
(
3
hr)
60
min
820.6
0.0359
K
T­
4
(
4
hr)
60
min
820.6
0.0327
L
Baseline
60
min
124.9
0.0057
L
T­
1
(
1
hr)
60
min
772.9
0.0326
L
T­
2
(
2
hr)
60
min
796.7
0.0368
L
T­
3
(
3
hr)
60
min
909.6
0.0346
L
T­
4
(
4
hr)
60
min
1076.1
0.0348
M
Baseline
90
min
179.8
0.0104
M
T­
1
(
1
hr)
90
min
859.6
0.0408
M
T­
2
(
2
hr)
90
min
955.1
0.0402
M
T­
3
(
3
hr)
90
min
1005.6
0.0349
M
T­
4
(
4
hr)
90
min
988.8
0.0392
N
Baseline
90
min
140.1
0.0111
N
T­
1
(
1
hr)
90
min
786.6
0.0530
N
T­
2
(
2
hr)
90
min
856.7
0.0466
N
T­
3
(
3
hr)
90
min
878.2
0.0418
N
T­
4
(
4
hr)
90
min
862.1
0.0330
Section
11.0
Equilibration
Table
63.
Equilibration
Data
for
No
hCG
Stimulation
(
Continued)

Sample
ID
Timepoint
Equilibration
time
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)

11­
4
O
Baseline
90
min
139.6
0.0054
O
T­
1
(
1
hr)
90
min
722.5
0.0198
O
T­
2
(
2
hr)
90
min
777.2
0.0251
O
T­
3
(
3
hr)
90
min
710.4
0.0208
O
T­
4
(
4
hr)
90
min
1135.4
0.0214
P
Baseline
120
min
192.8
0.0089
P
T­
1
(
1
hr)
120
min
1000.0
0.0396
P
T­
2
(
2
hr)
120
min
1174.7
0.0403
P
T­
3
(
3
hr)
120
min
626.5
0.0211
P
T­
4
(
4
hr)
120
min
1741.0
0.0404
Q
Baseline
120
min
943.1
0.0135
Q
T­
1
(
1
hr)
120
min
1406.3
0.0454
Q
T­
2
(
2
hr)
120
min
1484.4
0.0493
Q
T­
3
(
3
hr)
120
min
1523.4
0.0411
Q
T­
4
(
4
hr)
120
min
948.7
0.0332
R
Baseline
120
min
318.6
0.0073
R
T­
1
(
1
hr)
120
min
950.6
0.0314
R
T­
2
(
2
hr)
120
min
918.7
0.0297
R
T­
3
(
3
hr)
120
min
892.2
0.0297
R
T­
4
(
4
hr)
120
min
897.5
0.0258
Section
11.0
Equilibration
11­
5
Table
64.
Equilibration
Data
for
hCG
Stimulation
Sample
ID
Timepoint
Equilibrationtime
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)

AC
Baseline
15
min.
195.4
0.0026
AC
T­
1
(
1
hr)
15
min.
770.9
0.0545
AC
T­
2
(
2
hr)
15
min.
830.6
0.1024
AC
T­
3
(
3
hr)
15
min.
879.5
0.1979
AC
T­
4
(
4
hr)
15
min.
928.3
0.2350
BC
Baseline
15
min.
535.8
0.0044
BC
T­
1
(
1
hr)
15
min.
1147.4
0.0674
BC
T­
2
(
2
hr)
15
min.
1170.6
0.2153
BC
T­
3
(
3
hr)
15
min.
1176.5
0.1449
BC
T­
4
(
4
hr)
15
min.
1071.6
0.2566
CC
Baseline
15
min.
332.0
0.0041
CC
T­
1
(
1
hr)
15
min.
797.9
0.0620
CC
T­
2
(
2
hr)
15
min.
862.0
0.1501
CC
T­
3
(
3
hr)
15
min.
885.3
0.2394
CC
T­
4
(
4
hr)
15
min.
850.3
0.2543
DC
Baseline
30
min
178.9
0.0030
DC
T­
1
(
1
hr)
30
min
773.3
0.0428
DC
T­
2
(
2
hr)
30
min
804.8
0.0854
DC
T­
3
(
3
hr)
30
min
804.8
0.1385
DC
T­
4
(
4
hr)
30
min
799.6
0.2101
EC
Baseline
30
min
353.7
0.0034
EC
T­
1
(
1
hr)
30
min
1024.4
0.0486
EC
T­
2
(
2
hr)
30
min
1085.4
0.1080
EC
T­
3
(
3
hr)
30
min
1274.4
0.1755
EC
T­
4
(
4
hr)
30
min
1146.3
0.2261
FC
Baseline
30
min
133.8
0.0018
FC
T­
1
(
1
hr)
30
min
711.6
0.0512
FC
T­
2
(
2
hr)
30
min
775.8
0.1221
FC
T­
3
(
3
hr)
30
min
802.6
0.1801
FC
T­
4
(
4
hr)
30
min
936.3
0.2385
Section
11.0
Equilibration
11­
6
Table
64.
Equilibration
Data
for
hCG
Stimulation
(
Continued)

Sample
ID
Timepoint
Equilibrationtime
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)

GC
Baseline
45
min
341.0
0.0043
GC
T­
1
(
1
hr)
45
min
1221.1
0.0448
GC
T­
2
(
2
hr)
45
min
1281.6
0.0811
GC
T­
3
(
3
hr)
45
min
1199.1
0.1266
GC
T­
4
(
4
hr)
45
min
1276.1
0.1431
HC
Baseline
45
min
383.4
0.0053
HC
T­
1
(
1
hr)
45
min
1053.0
0.0620
HC
T­
2
(
2
hr)
45
min
1156.4
0.1326
HC
T­
3
(
3
hr)
45
min
1241.6
0.2413
HC
T­
4
(
4
hr)
45
min
1284.2
0.2879
IC
Baseline
45
min
1056.3
0.0085
IC
T­
1
(
1
hr)
45
min
1098.7
0.0630
IC
T­
2
(
2
hr)
45
min
1098.7
0.1382
IC
T­
3
(
3
hr)
45
min
1045.6
0.1700
IC
T­
4
(
4
hr)
45
min
376.9
0.2271
JC
Baseline
60
min
204.3
0.0085
JC
T­
1
(
1
hr)
60
min
859.1
0.1175
JC
T­
2
(
2
hr)
60
min
1000.5
0.1923
JC
T­
3
(
3
hr)
60
min
1052.9
0.2481
JC
T­
4
(
4
hr)
60
min
1094.8
0.2962
KC
Baseline
60
min
274.4
0.0096
KC
T­
1
(
1
hr)
60
min
533.0
0.0952
KC
T­
2
(
2
hr)
60
min
723.0
0.1664
KC
T­
3
(
3
hr)
60
min
781.0
0.2142
KC
T­
4
(
4
hr)
60
min
854.9
0.2057
LC
Baseline
60
min
148.4
0.0072
LC
T­
1
(
1
hr)
60
min
679.2
0.1074
LC
T­
2
(
2
hr)
60
min
724.9
0.2139
LC
T­
3
(
3
hr)
60
min
724.9
0.2438
LC
T­
4
(
4
hr)
60
min
707.8
0.3026
MC
Baseline
90
min
201.7
0.0093
MC
T­
1
(
1
hr)
90
min
690.0
0.0731
MC
T­
2
(
2
hr)
90
min
843.9
0.1478
Section
11.0
Equilibration
Table
64.
Equilibration
Data
for
hCG
Stimulation
(
Continued)

Sample
ID
Timepoint
Equilibrationtime
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)

11­
7
MC
T­
3
(
3
hr)
90
min
966.0
0.1636
MC
T­
4
(
4
hr)
90
min
1008.5
0.1847
NC
Baseline
90
min
229.2
0.0106
NC
T­
1
(
1
hr)
90
min
648.4
0.1025
NC
T­
2
(
2
hr)
90
min
760.2
0.1874
NC
T­
3
(
3
hr)
90
min
877.6
0.2360
NC
T­
4
(
4
hr)
90
min
944.7
0.2666
OC
Baseline
90
min
208.0
0.0053
OC
T­
1
(
1
hr)
90
min
582.3
0.2442
OC
T­
2
(
2
hr)
90
min
635.8
0.4070
OC
T­
3
(
3
hr)
90
min
760.5
0.4769
OC
T­
4
(
4
hr)
90
min
837.8
0.4797
PC
Baseline
120
min
1188.5
0.0089
PC
T­
1
(
1
hr)
120
min
1842.7
0.0711
PC
T­
2
(
2
hr)
120
min
2046.8
0.1131
PC
T­
3
(
3
hr)
120
min
2028.8
0.1545
PC
T­
4
(
4
hr)
120
min
1200.5
0.1628
QC
Baseline
120
min
789.9
0.0125
QC
T­
1
(
1
hr)
120
min
1190.9
0.2552
QC
T­
2
(
2
hr)
120
min
1268.7
0.4781
QC
T­
3
(
3
hr)
120
min
1238.8
0.4377
QC
T­
4
(
4
hr)
120
min
784.0
0.4604
RC
Baseline
120
min
403.1
0.0082
RC
T­
1
(
1
hr)
120
min
1019.0
0.1031
RC
T­
2
(
2
hr)
120
min
985.4
0.1619
RC
T­
3
(
3
hr)
120
min
946.2
0.1889
RC
T­
4
(
4
hr)
120
min
1192.6
0.2415
Section
11.0
Equilibration
11­
8
Table
65.
Means
of
LDH
Concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
hCG
Stimulation
Time
point
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
319.7
154.8
539.5
195.1
153.2
484.8
T­
1
(
1
hr)
1,188.6
672.0
1,045.7
758.3
789.6
1,119.0
T­
2
(
2
hr)
1,210.6
746.7
1,123.7
768.2
863.0
1,192.6
T­
3
(
3
hr)
1,215.5
796.4
1,145.3
853.4
864.7
1,014.0
T­
4
(
4
hr)
1,210.7
812.3
874.5
923.6
995.4
1,195.7
Table
66.
Standard
Deviations
of
LDH
concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes).
No
hCG
Stimulation
Time
point
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
81.2
56.2
447.2
93.3
23.0
401.8
T­
1
(
1
hr)
145.3
167.9
14.7
135.8
68.6
250.0
T­
2
(
2
hr)
186.7
207.7
69.2
60.9
89.1
283.2
T­
3
(
3
hr)
239.8
208.4
123.0
48.9
148.1
460.7
T­
4
(
4
hr)
318.5
193.8
530.2
134.8
136.8
472.9
Table
67.
Sample
sizes
of
LDH
concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
No
hCG
Stimulation
Time
point
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
3
3
3
3
3
3
T­
1
(
1
hr)
3
3
3
3
3
3
T­
2
(
2
hr)
3
3
3
3
3
3
T­
3
(
3
hr)
3
3
3
3
3
3
T­
4
(
4
hr)
3
3
3
3
3
3
Section
11.0
Equilibration
11­
9
Table
68.
Coeff
of
Variation
of
LDH
concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
No
hCG
Stimulation
Time
point
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
25.4
36.3
82.9
47.8
15.0
82.9
T­
1
(
1
hr)
12.2
25.0
1.4
17.9
8.7
22.3
T­
2
(
2
hr)
15.4
27.8
6.2
7.9
10.3
23.8
T­
3
(
3
hr)
19.7
26.2
10.7
5.7
17.1
45.4
T­
4
(
4
hr)
26.3
23.9
60.6
14.6
13.7
39.5
Table
69.
Means
of
LDH
concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
Time
point
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
354.4
222.1
593.6
209.0
213.0
793.9
T­
1
(
1
hr)
905.4
836.4
1,124.3
690.4
640.2
1,350.9
T­
2
(
2
hr)
954.4
888.7
1,178.9
816.1
746.6
1,433.7
T­
3
(
3
hr)
980.4
960.6
1,162.1
852.9
868.1
1,404.6
T­
4
(
4
hr)
950.1
960.8
979.1
885.8
930.3
1,059.0
Table
70.
Standard
Deviations
of
LDH
concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
Time
point
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
171.3
116.2
401.3
63.1
14.4
392.7
T­
1
(
1
hr)
210.0
165.7
86.9
163.3
54.3
434.5
T­
2
(
2
hr)
187.9
171.0
93.5
159.7
104.8
549.6
T­
3
(
3
hr)
169.8
271.8
103.1
175.4
103.1
560.0
T­
4
(
4
hr)
112.3
174.7
521.6
195.4
86.3
238.2
Section
11.0
Equilibration
11­
10
Table
71.
Sample
sizes
of
LDH
concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
Time
point
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
3
3
3
3
3
3
T­
1
(
1
hr)
3
3
3
3
3
3
T­
2
(
2
hr)
3
3
3
3
3
3
T­
3
(
3
hr)
3
3
3
3
3
3
T­
4
(
4
hr)
3
3
3
3
3
3
Table
72.
Coeff
of
Variation
of
LDH
concentration
(
mU/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
Timepoint
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
48.3
52.3
67.6
30.2
6.8
49.5
T­
1
(
1
hr)
23.2
19.8
7.7
23.7
8.5
32.2
T­
2
(
2
hr)
19.7
19.2
7.9
19.6
14.0
38.3
T­
3
(
3
hr)
17.3
28.3
8.9
20.6
11.9
39.9
T­
4
(
4
hr)
11.8
18.2
53.3
22.1
9.3
22.5
Table
73.
Means
of
Testosterone
concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
No
hCG
Stimulation
Timepoint
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
0.0032
0.0036
0.0047
0.0078
0.0090
0.0099
T­
1
(
1
hr)
0.0306
0.0281
0.0259
0.0437
0.0379
0.0388
T­
2
(
2
hr)
0.0262
0.0297
0.0277
0.0417
0.0373
0.0398
T­
3
(
3
hr)
0.0297
0.0292
0.0249
0.0381
0.0325
0.0307
T­
4
(
4
hr)
0.0291
0.0290
0.0241
0.0358
0.0312
0.0331
Section
11.0
Equilibration
11­
11
Table
74.
Standard
Deviations
of
Testosterone
concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
No
hCG
Stimulation
Time
point
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
0.0005
0.0011
0.0021
0.0019
0.0031
0.0032
T­
1
(
1
hr)
0.0007
0.0017
0.0088
0.0100
0.0168
0.0070
T­
2
(
2
hr)
0.0115
0.0021
0.0062
0.0054
0.0110
0.0098
T­
3
(
3
hr)
0.0023
0.0014
0.0061
0.0049
0.0107
0.0100
T­
4
(
4
hr)
0.0018
0.0028
0.0053
0.0036
0.0091
0.0073
Table
75.
Sample
sizes
of
Testosterone
concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
No
hCG
Stimulation
Timepoint
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
3
3
3
3
3
3
T­
1
(
1
hr)
3
3
3
3
3
3
T­
2
(
2
hr)
3
3
3
3
3
3
T­
3
(
3
hr)
3
3
3
3
3
3
T­
4
(
4
hr)
3
3
3
3
3
3
Table
76.
Coeff
of
Variation
of
Testosterone
concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
No
hCG
Stimulation
Timepoint
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
15.7
31.3
44.4
25.0
34.7
32.8
T­
1
(
1
hr)
2.4
6.2
33.9
22.8
44.4
18.1
T­
2
(
2
hr)
43.8
7.0
22.3
13.0
29.5
24.6
T­
3
(
3
hr)
7.9
4.9
24.4
12.8
33.0
32.7
T­
4
(
4
hr)
6.1
9.5
22.0
10.2
29.0
22.1
Section
11.0
Equilibration
11­
12
Table
77.
Means
of
Testosterone
concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
Timepoint
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
0.0037
0.0027
0.0061
0.0084
0.0084
0.0099
T­
1
(
1
hr)
0.0613
0.0475
0.0566
0.1067
0.1400
0.0871
T­
2
(
2
hr)
0.1559
0.1052
0.1173
0.1909
0.1676
0.1375
T­
3
(
3
hr)
0.1941
0.1647
0.1793
0.2354
0.1998
0.2604
T­
4
(
4
hr)
0.2486
0.2385
0.2271
0.2994
0.2256
0.3510
Table
78.
Standard
Deviations
of
Testosterone
concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
Timepoint
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
0.0010
0.0008
0.0022
0.0012
0.0028
0.0023
T­
1
(
1
hr)
0.0065
0.0043
0.0102
0.0112
0.0915
0.0226
T­
2
(
2
hr)
0.0566
0.0185
0.0315
0.0238
0.0280
0.0345
T­
3
(
3
hr)
0.0474
0.0228
0.0579
0.0184
0.0512
0.1545
T­
4
(
4
hr)
0.0118
.
.
0.0045
0.0579
0.1548
Table
79.
Sample
sizes
of
Testosterone
concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
Time
point
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
3
3
3
3
3
3
T­
1
(
1
hr)
3
3
3
3
3
2
T­
2
(
2
hr)
3
3
3
3
2
2
T­
3
(
3
hr)
3
3
3
3
2
3
T­
4
(
4
hr)
3
1
1
2
2
2
Section
11.0
Equilibration
11­
13
Table
80.
Coeff
of
Variation
of
Testosterone
concentration
(
ng/
mg)
by
Equilibration
Time
(
minutes)
with
hCG
Stimulation
Timepoint
15
minutes
30
minutes
45
minutes
60
minutes
90
minutes
120
minutes
Baseline
25.9
29.5
36.3
14.3
33.0
23.3
T­
1
(
1
hr)
10.6
9.0
18.0
10.5
65.4
26.0
T­
2
(
2
hr)
36.3
17.6
26.8
12.5
16.7
25.1
T­
3
(
3
hr)
24.4
13.9
32.3
7.8
25.6
59.4
T­
4
(
4
hr)
4.8
.
.
1.5
25.7
44.1
11­

14
Section
11
Equilibration
11­

15
Section
11
Equilibration
11­

16
Section
11
Equilibration
11­

17
Section
11
Equilibration
11­

18
Section
11
Equilibration
11­

19
Section
11
Equilibration
11­

20
Section
11
Equilibration
11­

21
Section
11
Equilibration
11­

22
Section
11
Equilibration
11­

23
Section
11
Equilibration
11­

24
Section
11
Equilibration
Table
81.
Summary
of
Equilibration
Experiment:
Testosterone
Concentration*(
ng/
mg)
by
Time
Point
With
No
hCG
Stimulation
Equilibration
Time
(
minutes)
Baseline
One
Hour
Two
Hours
Three
Hours
Four
Hours
15
0.0032
+
0.0005
0.0306
+
0.0007
0.0262
+
0.0115
0.0297
+
0.0023
0.0291
+
0.0018
30
0.0036
+
0.0011
0.0281
+
0.0017
0.0297
+
0.0021
0.0292
+
0.0014
0.0290
+
0.0028
45
0.0047
+
0.0021
0.0259
+
0.0088
0.0277
+
0.0062
0.0249
+
0.0061
0.0241
+
0.0053
60
0.0078
+
0.0019
0.0437
+
0.0100
0.0417
+
0.0054
0.0381
+
0.0049
0.0358
+
0.0036
90
0.0090
+
0.0031
0.0379
+
0.0168
0.0373
+
0.0110
0.0325
+
0.0107
0.0312
+
0.0091
120
0.0099
+
0.0032
0.0388
+
0.0070
0.0398
+
0.0098
0.0307
+
0.0100
0.0331
+
0.0073
*
Mean
+
Standard
Deviation
Table
82.
Summary
of
Equilibration
Experiment:
Testosterone
Concentration*(
ng/
mg)
by
Time
Point
With
hCG
Stimulation
Equilibration
Time
(
minutes)
Baseline
One
Hour
Two
Hours
Three
Hours
Four
Hours
15
0.0037
+
0.0010
0.0613
+
0.0065
0.1559
+
0.0566
0.1941
+
0.0474
0.2486
+
0.0118
30
0.0027
+
0.0008
0.0475
+
0.0043
0.1052
+
0.0185
0.1647
+
0.0228
0.2385
45
0.0061
+
0.0022
0.0566
+
0.0102
0.1173
+
0.0315
0.1793
+
0.0579
0.2271
60
0.0084
+
0.0012
0.1067
+
0.0112
0.1909
+
0.0238
0.2354
+
0.0184
0.2994
+
0.0045
90
0.0084
+
0.0028
0.1400
+
0.0915
0.1676
+
0.0280
0.1998
+
0.0512
0.2256
+
0.0579
120
0.0099
+
0.0023
0.0871
+
0.0226
0.1375
+
0.0345
0.2604
+
0.1545
0.3510
+
0.1548
*
Mean
+
Standard
Deviation
11­

25
Section
11
Equilibration
Table
83.
Summary
of
Equilibration
Experiment:
LDH
Concentration*(
mU/
mg)
by
Time
Point
With
hCG
Stimulation
Equilibration
Time
(
minutes)
Baseline
One
Hour
Two
Hours
Three
Hours
Four
Hours
15
354.41
+
171.29
905.38
+
209.98
954.41
+
187.92
980.40
+
169.82
950.10
+
112.25
30
222.09
+
116.15
836.43
+
165.68
888.67
+
170.96
960.60
+
271.75
960.75
+
174.67
45
593.58
+
401.26
1124.30
+
86.95
1178.90
+
93.51
1162.10
+
103.09
979.07
+
521.55
60
209.03
+
63.14
690.43
+
163.34
816.12
+
159.70
852.93
+
175.44
885.82
+
195.37
90
212.95
+
14.40
640.24
+
54.33
746.64
+
104.75
868.05
+
103.07
930.31
+
86.25
120
793.85
+
392.68
1350.90
+
434.53
1433.7
+
549.58
1404.60
+
560.01
1059.00
+
238.24
*
Mean
+
Standard
Deviation
Table
84.
Summary
of
Equilibration
Experiment::
LDH
Concentration*(
mU/
mg)
by
Time
Point
Without
hCG
Stimulation
Equilibration
Time
(
minutes)
Baseline
One
Hour
Two
Hours
Three
Hours
Four
Hours
15
319.70
+
81.15
1188.60
+
145.34
1210.60
+
186.70
1215.50
+
239.77
1210.70
+
318.54
30
154.78
+
56.24
672.00
+
167.85
746.71
+
207.69
796.37
+
208.42
812.27
+
193.76
45
539.53
+
447.15
1045.70
+
14.70
1123.7
+
69.18
1145.30
+
122.95
874.54
+
530.18
60
195.05
+
93.26
758.28
+
135.84
768.16
+
60.95
853.44
+
48.90
923.59
+
134.76
90
153.17
+
23.04
789.57
+
68.56
862.97
+
89.11
864.74
+
148.08
995.41
+
136.79
120
484.83
+
401.82
1119.00
+
250.03
1192.60
+
283.24
1014.00
+
460.71
1195.70
+
472.9
*
Mean
+
Standard
Deviation
Section
12.0
Phase
II
Sampling
Time
Factors
Determination
12­
1
12.0
SAMPLING
TIMES
12.1
Statistical
Analysis
of
Phase
II
Sampling
Time
Factors
Determination
12.1.1
Objectives
The
objective
of
the
sampling
time
factors
experiment
were
to
determine
the
effect
of
sampling
times
on
testosterone
production
using
the
sliced
testis
assay.
Concentrations
of
LDH
and
testosterone
were
measured
at
the
following
time
points:
baseline
and
0.5,
1,
2,
3,
4,
8,
12
and
24
hours.
The
sampling
points
of
interest
in
this
experiment
are
those
at
which
the
average
concentration
remains
linear
and
at
which
the
coefficient
of
variation
settles
down
to
an
approximately
constant
value.

12.1.2
Data
A
SAS
data
set
was
constructed
from
the
raw
Excel
data
file.
Each
observation
includes
concentration
measurements
for
LDH
and
testosterone
and
the
corresponding
repeated
measurement
times.
The
data
used
in
the
analysis
are
displayed
in
Tables
85
and
86.

12.1.3
Statistical
Analysis
Methods
and
Results
In
order
to
examine
the
effects
of
the
sampling
times
on
the
concentrations
of
LDH
and
testosterone,
summary
statistics
were
calculated
for
each
sampling
time.
Results
of
the
means,
standard
deviations,
sample
sizes
and
coefficients
of
variation
for
LDH
concentrations
are
shown
in
Tables
87
through
90
and
the
results
for
testosterone
concentrations
are
shown
in
Tables
91
through
94.
Graphs
of
the
means
and
coefficients
of
variation
for
LDH
and
testosterone
concentrations
are
presented
at
the
end
of
the
report.
Section
12.0
Phase
II
Sampling
Time
Factors
Determination
12­
2
Table
85.
Sampling
Time
Factors
Data
for
No
hCG
Stimulation
Sample_
ID
Timepoint
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)
A
Baseline
147.9
0.0033
A
T­
1
(
0.5
hr)
834.3
0.0318
A
T­
2
(
1
hr)
822.5
0.0338
A
T­
3
(
2
hr)
964.5
0.0420
A
T­
4
(
3
hr)
1142.0
0.0404
A
T­
5
(
4
hr)
1355.0
0.0402
A
T­
6
(
8
hr)
2763.3
0.0376
A
T­
7
(
12
hr)
3171.6
0.0373
A
T­
8
(
24
hr)
4053.3
0.0270
B
Baseline
84.9
0.0027
B
T­
1
(
0.5
hr)
928.7
0.0270
B
T­
2
(
1
hr)
860.7
0.0300
B
T­
3
(
2
hr)
1002.3
0.0370
B
T­
4
(
3
hr)
1126.8
0.0366
B
T­
5
(
4
hr)
1228.8
0.0307
B
T­
6
(
8
hr)
2463.2
0.0341
B
T­
7
(
12
hr)
2769.0
0.0260
B
T­
8
(
24
hr)
3023.8
0.0191
C
Baseline
135.3
0.0041
C
T­
1
(
0.5
hr)
641.2
0.0361
C
T­
2
(
1
hr)
770.6
0.0348
C
T­
3
(
2
hr)
852.9
0.0462
C
T­
4
(
3
hr)
1023.5
0.0474
C
T­
5
(
4
hr)
1370.6
0.0448
C
T­
6
(
8
hr)
3441.2
0.0448
C
T­
7
(
12
hr)
3811.8
0.0358
C
T­
8
(
24
hr)
3747.1
0.0314
D
Baseline
198.0
0.0043
D
T­
1
(
0.5
hr)
741.0
0.0295
D
T­
2
(
1
hr)
763.6
0.0314
D
T­
3
(
2
hr)
893.7
0.0592
D
T­
4
(
3
hr)
1295.2
0.0413
D
T­
5
(
4
hr)
1583.7
0.0390
Section
12.0
Phase
II
Sampling
Time
Factors
Determination
12­
3
Table
85.
Sampling
Time
Factors
Data
for
No
hCG
Stimulation,
(
Continued)

Sample_
ID
Timepoint
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)
D
T­
6
(
8
hr)
2856.3
0.0371
D
T­
7
(
12
hr)
3535.1
0.0314
D
T­
8
(
24
hr)
2867.6
0.0243
E
Baseline
140.5
0.0041
E
T­
1
(
0.5
hr)
696.9
0.0305
E
T­
2
(
1
hr)
659.1
0.0317
E
T­
3
(
2
hr)
756.3
0.0347
E
T­
4
(
3
hr)
956.2
0.0348
E
T­
5
(
4
hr)
1053.5
0.0332
E
T­
6
(
8
hr)
2582.4
0.0365
E
T­
7
(
12
hr)
3446.8
0.0280
E
T­
8
(
24
hr)
3463.0
0.0216
Section
12.0
Phase
II
Sampling
Time
Factors
Determination
12­
4
Table
86.
Sampling
Time
Factors
Data
for
hCG
Stimulation
Sample
ID
Timepoint
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)
AC
Baseline
104.0
0.0029
AC
T­
1
(
0.5
hr)
630.4
0.0278
AC
T­
2
(
1
hr)
636.5
0.0348
AC
T­
3
(
2
hr)
795.6
0.0636
AC
T­
4
(
3
hr)
1003.7
0.0867
AC
T­
5
(
4
hr)
1211.8
0.1035
AC
T­
6
(
8
hr)
2894.7
0.1228
AC
T­
7
(
12
hr)
3237.5
0.1045
AC
T­
8
(
24
hr)
3445.5
0.0797
BC
Baseline
160.6
0.0047
BC
T­
1
(
0.5
hr)
725.4
0.0352
BC
T­
2
(
1
hr)
664.5
0.0352
BC
T­
3
(
2
hr)
858.3
0.0815
BC
T­
4
(
3
hr)
952.4
0.0406
BC
T­
5
(
4
hr)
1096.3
0.1039
BC
T­
6
(
8
hr)
2530.5
0.1297
BC
T­
7
(
12
hr)
3028.8
0.0782
BC
T­
8
(
24
hr)
3693.2
0.0683
CC
Baseline
234.2
0.0047
CC
T­
1
(
0.5
hr)
626.4
0.0294
CC
T­
2
(
1
hr)
697.2
0.0401
CC
T­
3
(
2
hr)
866.0
0.0566
CC
T­
4
(
3
hr)
1029.4
0.1016
CC
T­
5
(
4
hr)
1345.3
0.1039
CC
T­
6
(
8
hr)
2963.0
0.1166
CC
T­
7
(
12
hr)
3507.6
0.0731
CC
T­
8
(
24
hr)
3104.6
0.0650
DC
Baseline
187.4
0.0050
DC
T­
1
(
0.5
hr)
871.0
0.0382
DC
T­
2
(
1
hr)
815.9
0.0476
DC
T­
3
(
2
hr)
970.2
0.0828
DC
T­
4
(
3
hr)
1102.5
0.1125
DC
T­
5
(
4
hr)
1185.2
0.1355
Section
12.0
Phase
II
Sampling
Time
Factors
Determination
12­
5
Table
86.
Sampling
Time
Factors
Data
for
hCG
Stimulation,
(
Continued)

Sample
ID
Timepoint
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)
DC
T­
6
(
8
hr)
2817.0
0.2103
DC
T­
7
(
12
hr)
3847.9
0.1407
DC
T­
8
(
24
hr)
2932.7
0.1113
EC
Baseline
112.9
0.0041
EC
T­
1
(
0.5
hr)
807.0
0.0270
EC
T­
2
(
1
hr)
807.0
0.0318
EC
T­
3
(
2
hr)
953.7
0.0501
EC
T­
4
(
3
hr)
1038.4
0.0562
EC
T­
5
(
4
hr)
1179.5
0.0884
EC
T­
6
(
8
hr)
2590.3
0.0980
EC
T­
7
(
12
hr)
3820.5
0.0870
EC
T­
8
(
24
hr)
2844.2
0.0678
Section
12.0
Phase
II
Sampling
Time
Factors
Determination
12­
6
Table
87.
Means
of
LDH
Concentration
(
mU/
mg)
by
hCG
Stimulation
Time
point
No
hCG
Stimulation
With
hCG
Stimulation
Baseline
141.3
159.8
T­
1
(
0.5
hr)
768.4
732.0
T­
2
(
1
hr)
775.3
724.2
T­
3
(
2
hr)
893.9
888.8
T­
4
(
3
hr)
1,108.8
1,025.3
T­
5
(
4
hr)
1,318.3
1,203.6
T­
6
(
8
hr)
2,821.3
2,759.1
T­
7
(
12
hr)
3,346.8
3,488.5
T­
8
(
24
hr)
3,430.9
3,204.1
Table
88.
Standard
Deviations
of
LDH
Concentration
(
mU/
mg)
by
hCG
Stimulation
Time
point
No
hCG
Stimulation
With
hCG
Stimulation
Baseline
40.2
53.9
T­
1
(
0.5
hr)
114.1
107.8
T­
2
(
1
hr)
76.1
82.5
T­
3
(
2
hr)
96.6
72.4
T­
4
(
3
hr)
129.2
54.6
T­
5
(
4
hr)
195.4
90.2
T­
6
(
8
hr)
378.9
189.8
T­
7
(
12
hr)
395.7
358.5
T­
8
(
24
hr)
492.8
357.3
Section
12.0
Phase
II
Sampling
Time
Factors
Determination
12­
7
Table
89.
Sample
sizes
of
LDH
concentration
(
mU/
mg)
by
hCG
stimulation
Time
point
No
hCG
Stimulation
With
hCG
Stimulation
Baseline
5
5
T­
1
(
0.5
hr)
5
5
T­
2
(
1
hr)
5
5
T­
3
(
2
hr)
5
5
T­
4
(
3
hr)
5
5
T­
5
(
4
hr)
5
5
T­
6
(
8
hr)
5
5
T­
7
(
12
hr)
5
5
Section
12.0
Phase
II
Sampling
Time
Factors
Determination
Time
point
No
hCG
Stimulation
With
hCG
Stimulation
12­
8
T­
8
(
24
hr)
5
5
Table
90.
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
hCG
Stimulation
Time
point
No
hCG
Stimulation
With
hCG
Stimulation
Baseline
28.5
33.7
T­
1
(
0.5
hr)
14.8
14.7
T­
2
(
1
hr)
9.8
11.4
T­
3
(
2
hr)
10.8
8.2
T­
4
(
3
hr)
11.7
5.3
T­
5
(
4
hr)
14.8
7.5
T­
6
(
8
hr)
13.4
6.9
T­
7
(
12
hr)
11.8
10.3
T­
8
(
24
hr)
14.4
11.2
Section
12.0
Phase
II
Sampling
Time
Factors
Determination
12­
9
Table
91.
Means
of
Testosterone
concentration
(
ng/
mg)
by
hCG
stimulation
Time
point
No
hCG
Stimulation
With
hCG
Stimulation
Baseline
0.0037
0.0043
T­
1
(
0.5
hr)
0.0310
0.0315
T­
2
(
1
hr)
0.0323
0.0379
T­
3
(
2
hr)
0.0438
0.0669
T­
4
(
3
hr)
0.0401
0.0795
T­
5
(
4
hr)
0.0376
0.1071
T­
6
(
8
hr)
0.0380
0.1369
T­
7
(
12
hr)
0.0317
0.1013
T­
8
(
24
hr)
0.0247
0.0784
Table
92.
Standard
Deviations
of
Testosterone
concentration
(
ng/
mg)
by
hCG
Stimulation
Time
point
No
hCG
Stimulation
With
hCG
Stimulation
Baseline
0.0007
0.0008
T­
1
(
0.5
hr)
0.0034
0.0049
T­
2
(
1
hr)
0.0020
0.0062
T­
3
(
2
hr)
0.0097
0.0147
T­
4
(
3
hr)
0.0049
0.0303
T­
5
(
4
hr)
0.0056
0.0172
T­
6
(
8
hr)
0.0040
0.0501
T­
7
(
12
hr)
0.0049
0.0292
T­
8
(
24
hr)
0.0048
0.0192
Section
12.0
Phase
II
Sampling
Time
Factors
Determination
12­
10
Table
93.
Sample
sizes
of
Testosterone
concentration
(
ng/
mg)
by
hCG
Stimulation
Timepoint
No
hCG
Stimulation
With
hCG
Stimulation
Baseline
5
5
T­
1
(
0.5
hr)
5
5
T­
2
(
1
hr)
5
5
T­
3
(
2
hr)
5
5
T­
4
(
3
hr)
5
5
T­
5
(
4
hr)
5
5
T­
6
(
8
hr)
5
4
T­
7
(
12
hr)
5
4
T­
8
(
24
hr)
5
5
Table
94.
Coeff
of
Variation
of
Testosterone
concentration
(
ng/
mg)
by
hCG
stimulation
Timepoint
No
hCG
Stimulation
With
hCG
Stimulation
Baseline
18.6
19.6
T­
1
(
0.5
hr)
10.8
15.6
T­
2
(
1
hr)
6.1
16.3
T­
3
(
2
hr)
22.1
22.0
T­
4
(
3
hr)
12.1
38.1
T­
5
(
4
hr)
14.9
16.1
T­
6
(
8
hr)
10.5
36.6
T­
7
(
12
hr)
15.3
28.8
T­
8
(
24
hr)
19.4
24.5
12­

11
Section
12
Statistical
Analysis
of
Phase
II
Sampling
Time
Factors
Determination
12­

12
Section
12
Statistical
Analysis
of
Phase
II
Sampling
Time
Factors
Determination
12­

13
Section
12
Statistical
Analysis
of
Phase
II
Sampling
Time
Factors
Determination
12­

14
Section
12
Statistical
Analysis
of
Phase
II
Sampling
Time
Factors
Determination
12­

15
Section
12
Statistical
Analysis
of
Phase
II
Sampling
Time
Factors
Determination
Table
95.
Summary
of
Sampling
Times
Experiment:
Testosterone
Concentration*
(
ng/
mg)
by
Sampling
Time
Points
over
24
Hours
hCG
Stimulation
Baseline
0.5
hr
1
hr
2
hr
3
hr
4
hr
8
hr
12
hr
24
hr
No
0.0037
+
0.0007
0.0310
+
0.0034
0.0323
+
0.0020
0.0438
+
0.0097
0.0401
+
0.0049
0.0376
+
0.0056
0.0380
+
0.0040
0.0317
+
0.0049
0.0247
+
0.0048
Yes
0.0043
+
0.0008
0.0315
+
0.0049
0.0379
+
0.0062
0.0669
+
0.0147
0.0795
+
0.0303
0.1071
+
0.0172
0.1369
+
0.0501
0.1013
+
0.0292
0.0784
+
0.0192
*
Means
+
Standard
Deviation
Table
96
.
Summary
of
Sampling
Times
Experiment:
LDH
Concentration*
(
mU/
mg)
by
Sampling
Time
Points
over
24
Hours
hCG
Stimulation
Baseline
0.5
hr
1
hr
2
hr
3
hr
4
hr
8
hr
12
hr
24
hr
No
141.32
+
40.21
768.40
+
114.10
775.29
+
76.13
893.94
+
96.60
1108.8
+
129.17
1318.3
+
195.36
2821.3
+
378.87
3346.8
+
395.67
3430.9
+
492.78
Yes
159.82
+
53.88
732.02
+
107.8
724.19
+
82.55
888.76
+
72.43
1025.3
+
54.63
1203.6
+
90.24
2759.1
+
189.79
3488.5
+
358.51
3204.1
+
357.28
*
Means
+
Standard
Deviation
Section
13.0
Induced
Parenchymal
Fragment
Injury
13­
1
13.0
INDUCED
PARENCHYMAL
FRAGMENT
INJURY
13.1.
Statistical
Analysis
of
Phase
II
LDH
Determination
 
Induced
Parenchymal
Fragment
Injury
13.1.1
Objectives
The
objective
of
the
induced
parenchymal
fragment
injury
experiment
is
to
determine
the
effect
of
various
types
of
injuries
on
LDH
concentration.
The
assay
was
performed
with
and
without
hCG
stimulation
under
three
different
inflicted
injury
types:
chemical,
heat
and
trauma.
Concentrations
of
LDH
and
testosterone
for
each
sample
were
measured
at
baseline
and
at
1,
2,
3
and
4
hours
after
baseline.

13.1.2
Data
A
SAS
data
set
was
constructed
from
the
raw
Excel
data
file.
Each
observation
includes
concentration
measurements
for
LDH
and
testosterone,
the
type
of
injury
inflicted
on
the
sample
and
the
corresponding
repeated
measurement
times.
The
data
used
in
the
analysis
are
displayed
in
Tables
97
and
98.

13.1.3
Statistical
Analysis
Methods
and
Results
In
order
to
examine
the
effects
of
the
injury
types
on
the
concentrations
of
LDH
and
testosterone,
summary
statistics
were
calculated
for
each
injury
type.
Results
of
the
means,
standard
deviations,
sample
sizes
and
coefficients
of
variation
for
LDH
concentrations
are
shown
in
Tables
99
through
106
and
the
results
for
testosterone
concentrations
are
shown
in
Tables
107
through
114.
Graphs
of
the
means
and
coefficients
of
variation
for
LDH
and
testosterone
concentrations
are
presented
at
the
end
of
the
section
report.
Section
13.0
Induced
Parenchymal
Fragment
Injury
13­
2
Table
97.
Induced
Parenchymal
Fragment
Injury
Data
for
No
hCG
Stimulation
Sample
ID
Timepoint
Injury
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)

A
Baseline
Heat
390.9
0.0012
A
T­
1
(
1
hr)
Heat
1455.3
0.0081
A
T­
2
(
2
hr)
Heat
1742.2
0.0095
A
T­
3
(
3
hr)
Heat
1950.1
0.0087
A
T­
4
(
4
hr)
Heat
2241.2
0.0083
B
Baseline
Heat
132.2
0.0011
B
T­
1
(
1
hr)
Heat
941.5
0.0078
B
T­
2
(
2
hr)
Heat
1121.8
0.0078
B
T­
3
(
3
hr)
Heat
1370.2
0.0081
B
T­
4
(
4
hr)
Heat
1718.8
0.0075
C
Baseline
Heat
310.9
0.0012
C
T­
1
(
1
hr)
Heat
779.3
0.0053
C
T­
2
(
2
hr)
Heat
1028.8
0.0061
C
T­
3
(
3
hr)
Heat
1220.7
0.0056
C
T­
4
(
4
hr)
Heat
1493.3
0.0054
D
Baseline
Heat
788.2
0.0012
D
T­
1
(
1
hr)
Heat
1216.4
0.0060
D
T­
2
(
2
hr)
Heat
1356.6
0.0069
D
T­
3
(
3
hr)
Heat
1583.9
0.0059
D
T­
4
(
4
hr)
Heat
1879.5
0.0058
E
Baseline
Heat
541.4
0.0017
E
T­
1
(
1
hr)
Heat
1094.2
0.0068
E
T­
2
(
2
hr)
Heat
1361.0
0.0070
E
T­
3
(
3
hr)
Heat
1608.8
0.0072
E
T­
4
(
4
hr)
Heat
1849.0
0.0061
F
Baseline
Heat
255.9
0.0015
F
T­
1
(
1
hr)
Heat
1012.4
0.0066
F
T­
2
(
2
hr)
Heat
1309.7
0.0070
F
T­
3
(
3
hr)
Heat
1614.6
0.0070
F
T­
4
(
4
hr)
Heat
1825.4
0.0063
Section
13.0
Induced
Parenchymal
Fragment
Injury
13­
3
Table
97.
Induced
Parenchymal
Fragment
Injury
Data
for
No
hCG
Stimulation,
(
Continued)

Sample
ID
Timepoint
Injury
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)

G
Baseline
Trauma
243.9
0.0213
G
T­
1
(
1
hr)
Trauma
815.8
0.0196
G
T­
2
(
2
hr)
Trauma
811.6
0.0339
G
T­
3
(
3
hr)
Trauma
832.6
0.0327
G
T­
4
(
4
hr)
Trauma
878.9
0.0325
H
Baseline
Trauma
493.2
0.0030
H
T­
1
(
1
hr)
Trauma
978.0
0.0309
H
T­
2
(
2
hr)
Trauma
949.0
0.0330
H
T­
3
(
3
hr)
Trauma
944.9
0.0322
H
T­
4
(
4
hr)
Trauma
1036.1
0.0274
I
Baseline
Trauma
432.5
0.0040
I
T­
1
(
1
hr)
Trauma
774.2
0.0381
I
T­
2
(
2
hr)
Trauma
778.5
0.0456
I
T­
3
(
3
hr)
Trauma
787.2
0.0407
I
T­
4
(
4
hr)
Trauma
847.8
0.0378
J
Baseline
Trauma
242.8
0.0016
J
T­
1
(
1
hr)
Trauma
595.1
0.0410
J
T­
2
(
2
hr)
Trauma
665.6
0.0484
J
T­
3
(
3
hr)
Trauma
743.9
0.0491
J
T­
4
(
4
hr)
Trauma
947.5
0.0498
K
Baseline
Trauma
514.2
0.0034
K
T­
1
(
1
hr)
Trauma
913.8
0.0325
K
T­
2
(
2
hr)
Trauma
933.5
0.0330
K
T­
3
(
3
hr)
Trauma
925.6
0.0344
K
T­
4
(
4
hr)
Trauma
969.1
0.0299
L
Baseline
Trauma
460.7
0.0019
L
T­
1
(
1
hr)
Trauma
790.3
0.0364
L
T­
2
(
2
hr)
Trauma
802.2
0.0419
L
T­
3
(
3
hr)
Trauma
849.9
0.0415
L
T­
4
(
4
hr)
Trauma
961.1
0.0386
M
Baseline
Chemical
919.7
High
M
T­
1
(
1
hr)
Chemical
1554.2
0.1108
M
T­
2
(
2
hr)
Chemical
1421.7
0.1279
M
T­
3
(
3
hr)
Chemical
1160.6
0.0972
Section
13.0
Induced
Parenchymal
Fragment
Injury
Table
97.
Induced
Parenchymal
Fragment
Injury
Data
for
No
hCG
Stimulation,
(
Continued)

Sample
ID
Timepoint
Injury
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)

13­
4
M
T­
4
(
4
hr)
Chemical
947.8
0.1133
N
Baseline
Chemical
1256.3
0.0854
N
T­
1
(
1
hr)
Chemical
2103.7
0.1341
N
T­
2
(
2
hr)
Chemical
1943.5
0.1162
N
T­
3
(
3
hr)
Chemical
1648.4
0.1165
N
T­
4
(
4
hr)
Chemical
1340.6
0.1006
O
Baseline
Chemical
884.0
0.1111
O
T­
1
(
1
hr)
Chemical
1629.7
0.1258
O
T­
2
(
2
hr)
Chemical
1223.3
0.1149
O
T­
3
(
3
hr)
Chemical
1206.5
0.1142
O
T­
4
(
4
hr)
Chemical
988.7
0.1055
P
Baseline
Chemical
863.9
High
P
T­
1
(
1
hr)
Chemical
1500.2
0.1366
P
T­
2
(
2
hr)
Chemical
1234.7
0.1540
P
T­
3
(
3
hr)
Chemical
1015.6
0.1290
P
T­
4
(
4
hr)
Chemical
813.3
0.1075
Q
Baseline
Chemical
929.9
0.1242
Q
T­
1
(
1
hr)
Chemical
1483.0
0.1092
Q
T­
2
(
2
hr)
Chemical
1210.4
0.1108
Q
T­
3
(
3
hr)
Chemical
973.9
0.1016
Q
T­
4
(
4
hr)
Chemical
793.6
0.0966
R
Baseline
Chemical
855.8
0.0997
R
T­
1
(
1
hr)
Chemical
1539.7
0.1058
R
T­
2
(
2
hr)
Chemical
1363.8
0.1232
R
T­
3
(
3
hr)
Chemical
1133.3
0.1277
R
T­
4
(
4
hr)
Chemical
937.9
0.0825
Section
13.0
Induced
Parenchymal
Fragment
Injury
13­
5
Table
98.
Induced
Parenchymal
Fragment
Injury
Data
for
hCG
Stimulation
Sample_
ID
Timepoint
Injury
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)
AC
Baseline
Heat
195.5
0.0014
AC
T­
1
(
1
hr)
Heat
1280.2
0.0079
AC
T­
2
(
2
hr)
Heat
1767.0
0.0084
AC
T­
3
(
3
hr)
Heat
2027.6
0.0084
AC
T­
4
(
4
hr)
Heat
2453.0
0.0079
BC
Baseline
Heat
121.0
0.0010
BC
T­
1
(
1
hr)
Heat
1147.7
0.0063
BC
T­
2
(
2
hr)
Heat
1644.4
0.0068
BC
T­
3
(
3
hr)
Heat
1944.9
0.0074
BC
T­
4
(
4
hr)
Heat
2220.4
0.0065
CC
Baseline
Heat
312.1
0.0016
CC
T­
1
(
1
hr)
Heat
1526.3
0.0081
CC
T­
2
(
2
hr)
Heat
1898.2
0.0094
CC
T­
3
(
3
hr)
Heat
2227.4
0.0087
CC
T­
4
(
4
hr)
Heat
2548.1
0.0086
DC
Baseline
Heat
272.3
0.0013
DC
T­
1
(
1
hr)
Heat
1214.1
0.0074
DC
T­
2
(
2
hr)
Heat
1633.9
0.0077
DC
T­
3
(
3
hr)
Heat
2015.9
0.0070
DC
T­
4
(
4
hr)
Heat
2341.1
0.0078
EC
Baseline
Heat
231.6
0.0013
EC
T­
1
(
1
hr)
Heat
1330.5
0.0070
EC
T­
2
(
2
hr)
Heat
1650.5
0.0082
EC
T­
3
(
3
hr)
Heat
1898.9
0.0083
EC
T­
4
(
4
hr)
Heat
2143.2
0.0099
FC
Baseline
Heat
281.5
0.0014
FC
T­
1
(
1
hr)
Heat
1441.2
0.0087
FC
T­
2
(
2
hr)
Heat
1705.9
0.0078
FC
T­
3
(
3
hr)
Heat
2016.8
0.0085
FC
T­
4
(
4
hr)
Heat
2357.1
0.0108
GC
Baseline
Trauma
845.5
0.0037
GC
T­
1
(
1
hr)
Trauma
778.8
0.0341
GC
T­
2
(
2
hr)
Trauma
853.8
0.0537
GC
T­
3
(
3
hr)
Trauma
903.8
0.0733
GC
T­
4
(
4
hr)
Trauma
1082.9
0.0947
Section
13.0
Induced
Parenchymal
Fragment
Injury
13­
6
Table
98.
Induced
Parenchymal
Fragment
Injury
Data
for
hCG
Stimulation
(
Continued)

Sample_
ID
Timepoint
Injury
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)
HC
Baseline
Trauma
646.6
0.0025
HC
T­
1
(
1
hr)
Trauma
814.7
0.0589
HC
T­
2
(
2
hr)
Trauma
866.4
0.1228
HC
T­
3
(
3
hr)
Trauma
931.0
0.1460
HC
T­
4
(
4
hr)
Trauma
1060.3
0.2081
IC
Baseline
Trauma
342.0
0.0027
IC
T­
1
(
1
hr)
Trauma
731.1
0.0438
IC
T­
2
(
2
hr)
Trauma
786.7
0.0703
IC
T­
3
(
3
hr)
Trauma
795.2
0.0956
IC
T­
4
(
4
hr)
Trauma
961.9
0.1105
JC
Baseline
Trauma
388.7
0.0022
JC
T­
1
(
1
hr)
Trauma
837.2
0.0358
JC
T­
2
(
2
hr)
Trauma
965.4
0.0679
JC
T­
3
(
3
hr)
Trauma
1008.1
0.0891
JC
T­
4
(
4
hr)
Trauma
1213.2
0.1214
KC
Baseline
Trauma
454.7
0.0031
KC
T­
1
(
1
hr)
Trauma
770.7
0.0296
KC
T­
2
(
2
hr)
Trauma
820.8
0.0492
KC
T­
3
(
3
hr)
Trauma
828.5
0.0625
KC
T­
4
(
4
hr)
Trauma
1009.6
0.0874
LC
Baseline
Trauma
349.8
0.0023
LC
T­
1
(
1
hr)
Trauma
718.3
0.0262
LC
T­
2
(
2
hr)
Trauma
815.0
0.0447
LC
T­
3
(
3
hr)
Trauma
841.1
0.0576
LC
T­
4
(
4
hr)
Trauma
908.1
0.0771
MC
Baseline
Chemical
968.4
0.1205
MC
T­
1
(
1
hr)
Chemical
1616.6
0.1083
MC
T­
2
(
2
hr)
Chemical
1347.1
High
MC
T­
3
(
3
hr)
Chemical
1089.4
0.2762
MC
T­
4
(
4
hr)
Chemical
870.8
0.2551
NC
Baseline
Chemical
927.3
0.0891
NC
T­
1
(
1
hr)
Chemical
2142.9
0.1298
NC
T­
2
(
2
hr)
Chemical
1783.6
0.1312
NC
T­
3
(
3
hr)
Chemical
1453.6
0.1772
NC
T­
4
(
4
hr)
Chemical
1173.8
0.2370
OC
Baseline
Chemical
1142.4
0.1318
OC
T­
1
(
1
hr)
Chemical
2003.1
0.1368
OC
T­
2
(
2
hr)
Chemical
1811.4
0.1433
OC
T­
3
(
3
hr)
Chemical
1561.0
High
Section
13.0
Induced
Parenchymal
Fragment
Injury
Table
98.
Induced
Parenchymal
Fragment
Injury
Data
for
hCG
Stimulation
(
Continued)

Sample_
ID
Timepoint
Injury
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)

13­
7
OC
T­
4
(
4
hr)
Chemical
1291.1
0.2996
PC
Baseline
Chemical
1038.3
0.1282
PC
T­
1
(
1
hr)
Chemical
1223.8
0.0971
PC
T­
2
(
2
hr)
Chemical
1046.2
0.1090
PC
T­
3
(
3
hr)
Chemical
884.3
0.2131
PC
T­
4
(
4
hr)
Chemical
730.4
0.1683
QC
Baseline
Chemical
1005.4
0.1091
QC
T­
1
(
1
hr)
Chemical
1416.7
0.1160
QC
T­
2
(
2
hr)
Chemical
1117.6
0.1032
QC
T­
3
(
3
hr)
Chemical
951.4
0.2807
QC
T­
4
(
4
hr)
Chemical
756.1
0.3867
RC
Baseline
Chemical
885.7
0.1446
RC
T­
1
(
1
hr)
Chemical
2396.5
0.1189
RC
T­
2
(
2
hr)
Chemical
2223.9
0.1296
RC
T­
3
(
3
hr)
Chemical
1894.2
High
RC
T­
4
(
4
hr)
Chemical
1549.1
0.1813
Section
13.0
Induced
Parenchymal
Fragment
Injury
13­
8
Table
99.
Means
of
LDH
concentration
(
mU/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
951.6
403.3
397.9
T­
1
(
1
hr)
1,635.1
1,083.2
811.2
T­
2
(
2
hr)
1,399.6
1,320.0
823.4
T­
3
(
3
hr)
1,189.7
1,558.1
847.4
T­
4
(
4
hr)
970.3
1,834.5
940.1
Table
100.
Standard
Deviations
of
LDH
concentration
(
mU/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
152.2
232.9
122.9
T­
1
(
1
hr)
235.2
234.0
131.8
T­
2
(
2
hr)
279.9
247.2
105.3
T­
3
(
3
hr)
241.5
248.7
77.7
T­
4
(
4
hr)
197.4
243.9
67.6
Table
101.
Sample
sizes
of
LDH
concentration
(
mU/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
6
6
6
T­
1
(
1
hr)
6
6
6
T­
2
(
2
hr)
6
6
6
T­
3
(
3
hr)
6
6
6
T­
4
(
4
hr)
6
6
6
Section
13.0
Induced
Parenchymal
Fragment
Injury
13­
9
Table
102.
Coeff
of
Variation
of
LDH
concentration
(
mU/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
16.0
57.7
30.9
T­
1
(
1
hr)
14.4
21.6
16.2
T­
2
(
2
hr)
20.0
18.7
12.8
T­
3
(
3
hr)
20.3
16.0
9.2
T­
4
(
4
hr)
20.3
13.3
7.2
Table
103.
Means
of
LDH
concentration
(
mU/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
994.6
235.7
504.6
T­
1
(
1
hr)
1,799.9
1,323.3
775.1
T­
2
(
2
hr)
1,555.0
1,716.7
851.4
T­
3
(
3
hr)
1,305.7
2,021.9
884.6
T­
4
(
4
hr)
1,061.9
2,343.8
1,039.3
Table
104.
Standard
Deviations
of
LDH
concentration
(
mU/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
90.5
69.4
201.3
T­
1
(
1
hr)
453.2
141.3
46.1
T­
2
(
2
hr)
460.2
102.0
62.7
T­
3
(
3
hr)
395.8
112.6
78.5
T­
4
(
4
hr)
329.3
147.9
106.4
Section
13.0
Induced
Parenchymal
Fragment
Injury
13­
10
Table
105.
Sample
sizes
of
LDH
concentration
(
mU/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
6
6
6
T­
1
(
1
hr)
6
6
6
T­
2
(
2
hr)
6
6
6
T­
3
(
3
hr)
6
6
6
T­
4
(
4
hr)
6
6
6
Table
106.
Coeff
of
Variation
of
LDH
concentration
(
mU/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
9.1
29.4
39.9
T­
1
(
1
hr)
25.2
10.7
5.9
T­
2
(
2
hr)
29.6
5.9
7.4
T­
3
(
3
hr)
30.3
5.6
8.9
T­
4
(
4
hr)
31.0
6.3
10.2
Table
107.
Means
of
Testosterone
concentration
(
ng/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
0.1051
0.0013
0.0059
T­
1
(
1
hr)
0.1204
0.0068
0.0331
T­
2
(
2
hr)
0.1245
0.0074
0.0393
T­
3
(
3
hr)
0.1144
0.0071
0.0384
T­
4
(
4
hr)
0.1010
0.0066
0.0360
Section
13.0
Induced
Parenchymal
Fragment
Injury
13­
11
Table
108.
Standard
Deviations
of
Testosterone
concentration
(
ng/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
0.0165
0.0002
0.0076
T­
1
(
1
hr)
0.0135
0.0010
0.0076
T­
2
(
2
hr)
0.0157
0.0012
0.0069
T­
3
(
3
hr)
0.0130
0.0012
0.0066
T­
4
(
4
hr)
0.0107
0.0011
0.0081
Table
109.
Sample
sizes
of
Testosterone
concentration
(
ng/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
4
6
6
T­
1
(
1
hr)
6
6
6
T­
2
(
2
hr)
6
6
6
T­
3
(
3
hr)
6
6
6
T­
4
(
4
hr)
6
6
6
Table
110.
Coeff
of
Variation
of
Testosterone
concentration
(
ng/
mg)
by
Type
of
Injury
Induced
No
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
15.7
16.4
129.6
T­
1
(
1
hr)
11.2
15.5
22.9
T­
2
(
2
hr)
12.6
15.8
17.6
T­
3
(
3
hr)
11.4
17.1
17.1
T­
4
(
4
hr)
10.6
16.7
22.4
Section
13.0
Induced
Parenchymal
Fragment
Injury
Table
111.
Means
of
Testosterone
concentration
(
ng/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
0.1206
0.0013
0.0027
T­
1
(
1
hr)
0.1178
0.0076
0.0381
T­
2
(
2
hr)
0.1233
0.0081
0.0681
T­
3
(
3
hr)
0.2368
0.0081
0.0873
T­
4
(
4
hr)
0.2547
0.0086
0.1165
Table
112.
Standard
Deviations
of
Testosterone
concentration
(
ng/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
0.0194
0.0002
0.0006
T­
1
(
1
hr)
0.0143
0.0008
0.0119
T­
2
(
2
hr)
0.0167
0.0009
0.0287
T­
3
(
3
hr)
0.0503
0.0007
0.0323
T­
4
(
4
hr)
0.0808
0.0016
0.0476
Table
113.
Sample
sizes
of
Testosterone
concentration
(
ng/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
6
6
6
T­
1
(
1
hr)
6
6
6
T­
2
(
2
hr)
5
6
6
T­
3
(
3
hr)
4
6
6
T­
4
(
4
hr)
6
6
6
Table
114.
Coeff
of
Variation
of
Testosterone
concentration
(
ng/
mg)
by
Type
of
Injury
Induced
with
hCG
Stimulation
Timepoint
Chemical
Heat
Trauma
Baseline
16.1
15.5
20.2
T­
1
(
1
hr)
12.2
10.9
31.1
T­
2
(
2
hr)
13.5
10.8
42.1
T­
3
(
3
hr)
21.2
8.5
37.0
T­
4
(
4
hr)
31.7
18.3
40.8
13­

14
13­

16
13­

18
13­

20
13­

22
13­

24
13­

26
13­

28
Table
115.
Summary
of
Injury
Experiment:
Testosterone
Concentrations*
(
ng/
mg)
by
Sampling
Timepoints
Injury
Type**
Baseline
1
Hr
2
Hr
3
Hr
4
Hr
Chemical
(
Triton­
X
100)
0.1051
+
0.0165
0.1204
+
0.0135
0.1245
+
0.0157
0.1144
+
0.0130
0.1010
+
0.0107
Heat
(
42
?
C)
0.0013
+
0.0002
0.0068
+
0.0010
0.0074
+
0.0012
0.0071
+
0.0012
0.0066
+
0.0011
Trauma
(
Mincing)
0.0059
+
0.0076
0.0331
+
0.0076
0.0393
+
0.0069
0.0384
+
0.0066
0.0360
+
0.0081
*
Mean
+
standard
deviation
**
Injuries
occurred
during
the
1
hour
incubation
period
before
the
baseline
sample
was
taken.
***
No
hCG
stimulation
occurred
Table
116.
Summary
of
Injury
Experiment
:
Testosterone
Concentrations*
(
ng/
mg)
by
Sampling
Timepoints***

Injury
Type**
Baseline
1
hr
2
hr
3
hr
4
hr
Chemical
(
Triton­
X
100)
0.1206
+
0.0194
0.1178
+
0.0143
0.1233
+
0.0167
0.2368
+
0.0503
0.2547
+
0.0808
Heat
(
42
?
C)
0.0013
+
0.0002
0.0076
+
0.0008
0.0081
+
0.0009
0.0081
+
0.0007
0.0086
+
0.0016
Trauma
(
Mincing)
0.0027
+
0.0006
0.0381
+
0.0119
0.0681
+
0.0287
0.0873
+
0.0323
0.1165
+
0.0476
*
Mean
+
standard
deviation
**
Injuries
occurred
during
the
1
hour
incubation
period
before
the
baseline
sample
was
taken.
***
hCG
stimulation
occurred
Table
117.
Summary
of
Injury
Experiment
:
LDH
Concentrations*
(
mU/
mg)
by
Sampling
Timepoints***

Injury
Type**
Baseline
1
Hr
2
Hr
3
Hr
4
Hr
Chemical
(
Triton­
X
100)
951.58
+
152.18
1635.10
+
235.21
1399.6
+
279.88
1189.70
+
241.49
970.32
+
197.44
Heat
(
42
?
C)
403.25
+
232.85
1083.20
+
234.00
1320.00
+
247.17
1558.10
+
248.69
1834.50
+
243.94
Trauma
(
Mincing)
397.88
+
122.91
811.21
+
131.80
823.43
+
105.27
847.36
+
77.69
940.08
+
67.56
*
Mean
+
standard
deviation
**
Injuries
occurred
during
the
1
hour
incubation
period
before
the
baseline
sample
was
taken.
***
No
hCG
stimulation
occurred
Table
118.
Injury
Experiment
:
LDH
Concentrations*
(
mU/
mg)
by
Sampling
Timepoints***

Injury
Type**
Baseline
1
Hr
2
Hr
3
Hr
4
Hr
Chemical
(
Triton­
X
100)
994.59
+
90.49
1799.90
+
453.24
1555.00
+
460.20
1305.70
+
395.79
1061.90
+
329.34
Heat
(
42
?
C)
235.67
+
69.37
1323.30
+
141.25
1716.70
+
102.04
2021.90
+
112.60
2343.80
+
147.89
Trauma
(
Mincing)
504.56
+
201.32
775.14
+
46.10
851.35
+
62.73
884.63
+
78.50
1039.30
+
106.44
Mean
+
standard
deviation
**
Injuries
occurred
during
the
1
hour
incubation
period
before
the
baseline
sample
was
taken.
***
hCG
stimulation
occurred
30
14.0
VEHICLE
EFFECT
EXPERIMENT
14.1
Statistical
Analysis
of
Phase
II
Vehicle
and
Vehicle
Concentration
Determination
14.1.1
Objective
The
objective
of
the
vehicle
and
vehicle
concentration
experiment
is
to
determine
the
effect
of
various
types
of
vehicles
and
vehicle
concentrations
on
assay
performance.
The
assay
was
performed
with
and
without
hCG
stimulation
under
three
different
vehicle
types:
DMSO,
ethanol
and
Tween
20.
Each
vehicle
was
evaluated
at
each
of
five
different
concentrations:
0
(
control),
0.5,
1,
5
and
10
percent
(
v/
v).
Concentrations
of
LDH
and
testosterone
for
each
sample
were
measured
at
baseline
and
at
1,
2,
3
and
4
hours
after
baseline.

14.1.2
Data
A
SAS
data
set
was
constructed
from
the
raw
Excel
data
file.
Each
observation
includes
concentration
measurements
for
LDH
and
testosterone,
the
type
of
vehicle
used,
the
concentration
of
the
vehicle
and
the
corresponding
repeated
measurement
times.
The
data
used
in
the
analysis
are
displayed
in
Tables
153
and
154.

14.1.3
Statistical
Analysis
Methods
and
Results
In
order
to
examine
the
effects
of
the
vehicles
and
their
concentrations
on
the
concentrations
of
LDH
and
testosterone,
summary
statistics
were
calculated
for
each
vehicle
type
and
each
concentration
level.
Results
of
the
means,
standard
deviations,
sample
sizes
and
coefficients
of
variation
for
LDH
concentrations
are
shown
in
Tables
155
through
170
and
the
results
for
testosterone
concentrations
are
shown
in
Tables
171
through
186.
Graphs
of
the
means
and
coefficients
of
variation
for
LDH
and
testosterone
concentrations
are
presented
at
the
end
of
the
section
report.
31
Table
119.
Vehicle
and
Vehicle
Concentrations
Data
for
No
hCG
Stimulation
Sample_
ID
Timepoint
Vehicle
Vehicle
Type
Vehicle
Concentration
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)
A
Baseline
0.5%
ETOH
ETOH
0.5%
207.7
0.0021
A
T­
1
(
1
hr)
0.5%
ETOH
ETOH
0.5%
708.6
A
T­
2
(
2
hr)
0.5%
ETOH
ETOH
0.5%
1001.8
0.0166
A
T­
3
(
3
hr)
0.5%
ETOH
ETOH
0.5%
1166.8
0.0199
A
T­
4
(
4
hr)
0.5%
ETOH
ETOH
0.5%
1258.4
0.0205
B
Baseline
1.0%
ETOH
ETOH
1.0%
231.7
0.0029
B
T­
1
(
1
hr)
1.0%
ETOH
ETOH
1.0%
842.5
0.0167
B
T­
2
(
2
hr)
1.0%
ETOH
ETOH
1.0%
1369.1
0.0238
B
T­
3
(
3
hr)
1.0%
ETOH
ETOH
1.0%
1448.1
0.0243
B
T­
4
(
4
hr)
1.0%
ETOH
ETOH
1.0%
1542.9
0.0224
C
Baseline
5.0%
ETOH
ETOH
5.0%
303.9
0.0026
C
T­
1
(
1
hr)
5.0%
ETOH
ETOH
5.0%
1157.2
0.0122
C
T­
2
(
2
hr)
5.0%
ETOH
ETOH
5.0%
1408.5
0.0137
C
T­
3
(
3
hr)
5.0%
ETOH
ETOH
5.0%
1525.4
0.0150
C
T­
4
(
4
hr)
5.0%
ETOH
ETOH
5.0%
1654.0
0.0125
D
Baseline
10.0%
ETOH
ETOH
10.0%
361.2
0.0029
D
T­
1
(
1
hr)
10.0%
ETOH
ETOH
10.0%
679.2
0.0148
D
T­
2
(
2
hr)
10.0%
ETOH
ETOH
10.0%
927.2
0.0156
D
T­
3
(
3
hr)
10.0%
ETOH
ETOH
10.0%
1148.2
0.0160
D
T­
4
(
4
hr)
10.0%
ETOH
ETOH
10.0%
1390.8
0.0150
E
Baseline
0.5%
DMSO
DMSO
0.5%
317.1
0.0033
E
T­
1
(
1
hr)
0.5%
DMSO
DMSO
0.5%
764.4
0.0203
E
T­
2
(
2
hr)
0.5%
DMSO
DMSO
0.5%
1030.6
0.0249
E
T­
3
(
3
hr)
0.5%
DMSO
DMSO
0.5%
1313.7
0.0242
E
T­
4
(
4
hr)
0.5%
DMSO
DMSO
0.5%
1528.9
0.0232
F
Baseline
1.0%
DMSO
DMSO
1.0%
244.2
0.0022
F
T­
1
(
1
hr)
1.0%
DMSO
DMSO
1.0%
573.2
0.0189
F
T­
2
(
2
hr)
1.0%
DMSO
DMSO
1.0%
759.0
0.0279
F
T­
3
(
3
hr)
1.0%
DMSO
DMSO
1.0%
1003.2
0.0253
F
T­
4
(
4
hr)
1.0%
DMSO
DMSO
1.0%
1380.0
0.0207
G
Baseline
5.0%
DMSO
DMSO
5.0%
152.4
0.0016
G
T­
1
(
1
hr)
5.0%
DMSO
DMSO
5.0%
1306.5
0.0239
G
T­
2
(
2
hr)
5.0%
DMSO
DMSO
5.0%
1589.5
0.0279
G
T­
3
(
3
hr)
5.0%
DMSO
DMSO
5.0%
1937.9
0.0264
G
T­
4
(
4
hr)
5.0%
DMSO
DMSO
5.0%
2319.0
0.0286
H
Baseline
10.0%
DMSO
DMSO
10.0%
236.7
0.0026
H
T­
1
(
1
hr)
10.0%
DMSO
DMSO
10.0%
1479.3
0.0219
H
T­
2
(
2
hr)
10.0%
DMSO
DMSO
10.0%
2242.6
0.0217
H
T­
3
(
3
hr)
10.0%
DMSO
DMSO
10.0%
3047.3
0.0189
H
T­
4
(
4
hr)
10.0%
DMSO
DMSO
10.0%
3461.5
0.0167
Sample_
ID
Timepoint
Vehicle
Vehicle
Type
Vehicle
Concentration
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)

32
I
Baseline
0.5%
Tween
Tween
0.5%
321.0
0.0023
I
T­
1
(
1
hr)
0.5%
Tween
Tween
0.5%
1444.7
0.0230
I
T­
2
(
2
hr)
0.5%
Tween
Tween
0.5%
1938.2
0.0179
I
T­
3
(
3
hr)
0.5%
Tween
Tween
0.5%
1973.8
0.0174
I
T­
4
(
4
hr)
0.5%
Tween
Tween
0.5%
2098.7
0.0140
J
Baseline
1.0%
Tween
Tween
1.0%
304.3
0.0023
J
T­
1
(
1
hr)
1.0%
Tween
Tween
1.0%
1206.6
0.0234
J
T­
2
(
2
hr)
1.0%
Tween
Tween
1.0%
1478.9
0.0232
J
T­
3
(
3
hr)
1.0%
Tween
Tween
1.0%
1804.6
0.0215
J
T­
4
(
4
hr)
1.0%
Tween
Tween
1.0%
2151.6
0.0143
K
Baseline
5.0%
Tween
Tween
5.0%
128.5
0.0018
K
T­
1
(
1
hr)
5.0%
Tween
Tween
5.0%
2631.3
0.0344
K
T­
2
(
2
hr)
5.0%
Tween
Tween
5.0%
3240.2
0.0246
K
T­
3
(
3
hr)
5.0%
Tween
Tween
5.0%
3921.8
0.0245
K
T­
4
(
4
hr)
5.0%
Tween
Tween
5.0%
3217.9
0.0245
L
Baseline
10.0%
Tween
Tween
10.0%
95.4
0.0023
L
T­
1
(
1
hr)
10.0%
Tween
Tween
10.0%
3784.3
0.0630
L
T­
2
(
2
hr)
10.0%
Tween
Tween
10.0%
4445.8
0.0570
L
T­
3
(
3
hr)
10.0%
Tween
Tween
10.0%
4368.3
0.0580
L
T­
4
(
4
hr)
10.0%
Tween
Tween
10.0%
4004.8
0.0267
M
Baseline
ETOH
Control
ETOH
0.0%
215.4
0.0021
M
T­
1
(
1
hr)
ETOH
Control
ETOH
0.0%
491.4
0.0231
M
T­
2
(
2
hr)
ETOH
Control
ETOH
0.0%
668.1
0.0395
M
T­
3
(
3
hr)
ETOH
Control
ETOH
0.0%
938.7
0.0560
M
T­
4
(
4
hr)
ETOH
Control
ETOH
0.0%
1325.2
0.0670
N
Baseline
DMSO
Control
DMSO
0.0%
234.9
0.0030
N
T­
1
(
1
hr)
DMSO
Control
DMSO
0.0%
1089.2
0.0247
N
T­
2
(
2
hr)
DMSO
Control
DMSO
0.0%
1377.5
0.0262
N
T­
3
(
3
hr)
DMSO
Control
DMSO
0.0%
1580.4
0.0265
N
T­
4
(
4
hr)
DMSO
Control
DMSO
0.0%
1713.8
0.0254
O
Baseline
Tween
Control
Tween
0.0%
174.9
0.0026
O
T­
1
(
1
hr)
Tween
Control
Tween
0.0%
683.1
0.0295
O
T­
2
(
2
hr)
Tween
Control
Tween
0.0%
956.3
0.0346
O
T­
3
(
3
hr)
Tween
Control
Tween
0.0%
1327.9
0.0348
O
T­
4
(
4
hr)
Tween
Control
Tween
0.0%
1590.2
0.0335
33
Table
120.
Vehicle
and
Vehicle
Concentrations
Data
for
hCG
Stimulation
Sample
ID
Timepoint
Vehicle
Vehicle
Type
Vehicle
Concentration
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)
AC
Baseline
0.5%
ETOH
ETOH
0.5%
217.8
0.0021
AC
T­
1
(
1
hr)
0.5%
ETOH
ETOH
0.5%
876.6
0.0200
AC
T­
2
(
2
hr)
0.5%
ETOH
ETOH
0.5%
1172.5
0.0412
AC
T­
3
(
3
hr)
0.5%
ETOH
ETOH
0.5%
1328.9
0.0629
AC
T­
4
(
4
hr)
0.5%
ETOH
ETOH
0.5%
1591.3
0.0835
BC
Baseline
1.0%
ETOH
ETOH
1.0%
332.6
0.0034
BC
T­
1
(
1
hr)
1.0%
ETOH
ETOH
1.0%
548.8
0.0198
BC
T­
2
(
2
hr)
1.0%
ETOH
ETOH
1.0%
765.0
0.0330
BC
T­
3
(
3
hr)
1.0%
ETOH
ETOH
1.0%
936.8
0.0467
BC
T­
4
(
4
hr)
1.0%
ETOH
ETOH
1.0%
1064.3
0.0595
CC
Baseline
5.0%
ETOH
ETOH
5.0%
183.1
0.0023
CC
T­
1
(
1
hr)
5.0%
ETOH
ETOH
5.0%
754.7
0.0196
CC
T­
2
(
2
hr)
5.0%
ETOH
ETOH
5.0%
937.8
0.0355
CC
T­
3
(
3
hr)
5.0%
ETOH
ETOH
5.0%
1132.1
0.0613
CC
T­
4
(
4
hr)
5.0%
ETOH
ETOH
5.0%
1320.8
0.1082
DC
Baseline
10.0%
ETOH
ETOH
10.0%
229.3
0.0026
DC
T­
1
(
1
hr)
10.0%
ETOH
ETOH
10.0%
715.1
0.0206
DC
T­
2
(
2
hr)
10.0%
ETOH
ETOH
10.0%
1031.7
0.0210
DC
T­
3
(
3
hr)
10.0%
ETOH
ETOH
10.0%
1250.0
0.0265
DC
T­
4
(
4
hr)
10.0%
ETOH
ETOH
10.0%
1452.0
0.0309
EC
Baseline
0.5%
DMSO
DMSO
0.5%
168.8
0.0019
EC
T­
1
(
1
hr)
0.5%
DMSO
DMSO
0.5%
906.0
0.0169
EC
T­
2
(
2
hr)
0.5%
DMSO
DMSO
0.5%
1249.3
0.0268
EC
T­
3
(
3
hr)
0.5%
DMSO
DMSO
0.5%
1525.0
0.0409
EC
T­
4
(
4
hr)
0.5%
DMSO
DMSO
0.5%
1778.3
0.0542
FC
Baseline
1.0%
DMSO
DMSO
1.0%
285.7
0.0020
FC
T­
1
(
1
hr)
1.0%
DMSO
DMSO
1.0%
1074.3
0.0361
FC
T­
2
(
2
hr)
1.0%
DMSO
DMSO
1.0%
1537.1
0.0916
FC
T­
3
(
3
hr)
1.0%
DMSO
DMSO
1.0%
1662.9
0.1271
FC
T­
4
(
4
hr)
1.0%
DMSO
DMSO
1.0%
1874.3
0.2407
GC
Baseline
5.0%
DMSO
DMSO
5.0%
157.3
0.0019
GC
T­
1
(
1
hr)
5.0%
DMSO
DMSO
5.0%
971.9
0.0172
GC
T­
2
(
2
hr)
5.0%
DMSO
DMSO
5.0%
1219.1
0.0270
GC
T­
3
(
3
hr)
5.0%
DMSO
DMSO
5.0%
1544.9
0.0418
GC
T­
4
(
4
hr)
5.0%
DMSO
DMSO
5.0%
1977.5
0.0470
HC
Baseline
10.0%
DMSO
DMSO
10.0%
135.2
0.0022
HC
T­
1
(
1
hr)
10.0%
DMSO
DMSO
10.0%
723.1
0.0362
HC
T­
2
(
2
hr)
10.0%
DMSO
DMSO
10.0%
1469.7
0.0388
HC
T­
3
(
3
hr)
10.0%
DMSO
DMSO
10.0%
2369.2
0.0467
HC
T­
4
(
4
hr)
10.0%
DMSO
DMSO
10.0%
3004.1
0.0521
Sample
ID
Timepoint
Vehicle
Vehicle
Type
Vehicle
Concentration
LDH
(
mU/
mg)
Testosterone
(
ng/
mg)

34
IC
Baseline
0.5%
Tween
Tween
0.5%
148.8
0.0017
IC
T­
1
(
1
hr)
0.5%
Tween
Tween
0.5%
1002.8
0.0180
IC
T­
2
(
2
hr)
0.5%
Tween
Tween
0.5%
1603.3
0.0226
IC
T­
3
(
3
hr)
0.5%
Tween
Tween
0.5%
1840.2
0.0217
IC
T­
4
(
4
hr)
0.5%
Tween
Tween
0.5%
2099.2
0.0243
JC
Baseline
1.0%
Tween
Tween
1.0%
242.1
0.0024
JC
T­
1
(
1
hr)
1.0%
Tween
Tween
1.0%
962.5
0.0237
JC
T­
2
(
2
hr)
1.0%
Tween
Tween
1.0%
1302.6
0.0225
JC
T­
3
(
3
hr)
1.0%
Tween
Tween
1.0%
1608.1
0.0232
JC
T­
4
(
4
hr)
1.0%
Tween
Tween
1.0%
1907.8
0.0184
KC
Baseline
5.0%
Tween
Tween
5.0%
93.5
0.0013
KC
T­
1
(
1
hr)
5.0%
Tween
Tween
5.0%
2523.4
0.0238
KC
T­
2
(
2
hr)
5.0%
Tween
Tween
5.0%
5461.4
0.0367
KC
T­
3
(
3
hr)
5.0%
Tween
Tween
5.0%
5654.2
0.0273
KC
T­
4
(
4
hr)
5.0%
Tween
Tween
5.0%
5566.6
0.0372
LC
Baseline
10.0%
Tween
Tween
10.0%
106.1
0.0026
LC
T­
1
(
1
hr)
10.0%
Tween
Tween
10.0%
2882.7
0.0868
LC
T­
2
(
2
hr)
10.0%
Tween
Tween
10.0%
4352.0
0.0408
LC
T­
3
(
3
hr)
10.0%
Tween
Tween
10.0%
4374.3
0.0447
LC
T­
4
(
4
hr)
10.0%
Tween
Tween
10.0%
4379.9
0.0417
MC
Baseline
ETOH
Control
ETOH
0.0%
222.3
0.0021
MC
T­
1
(
1
hr)
ETOH
Control
ETOH
0.0%
803.9
0.0146
MC
T­
2
(
2
hr)
ETOH
Control
ETOH
0.0%
935.0
0.0213
MC
T­
3
(
3
hr)
ETOH
Control
ETOH
0.0%
1214.4
0.0290
MC
T­
4
(
4
hr)
ETOH
Control
ETOH
0.0%
1413.9
0.0344
NC
Baseline
DMSO
Control
DMSO
0.0%
488.2
0.0082
NC
T­
1
(
1
hr)
DMSO
Control
DMSO
0.0%
1418.7
0.0285
NC
T­
2
(
2
hr)
DMSO
Control
DMSO
0.0%
1596.8
0.0291
NC
T­
3
(
3
hr)
DMSO
Control
DMSO
0.0%
1614.0
0.0341
NC
T­
4
(
4
hr)
DMSO
Control
DMSO
0.0%
1660.0
0.0310
OC
Baseline
Tween
Control
Tween
0.0%
254.5
0.0035
OC
T­
1
(
1
hr)
Tween
Control
Tween
0.0%
735.3
0.0639
OC
T­
2
(
2
hr)
Tween
Control
Tween
0.0%
1402.7
0.1699
OC
T­
3
(
3
hr)
Tween
Control
Tween
0.0%
2013.6
0.1944
OC
T­
4
(
4
hr)
Tween
Control
Tween
0.0%
2177.6
0.2324
35
Table
121.
Means
of
LDH
concentration
(
mU/
mg)
by
Vehicle
Type
No
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
237.1
264.0
204.8
T­
1
(
1
hr)
1,042.5
775.8
1,950.0
T­
2
(
2
hr)
1,399.8
1,075.0
2,411.9
T­
3
(
3
hr)
1,776.5
1,245.5
2,679.3
T­
4
(
4
hr)
2,080.7
1,434.3
2,612.6
Table
122.
Standard
Deviations
of
LDH
concentration
(
mU/
mg)
by
Vehicle
Type
No
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
58.4
66.4
102.6
T­
1
(
1
hr)
374.2
247.3
1,249.2
T­
2
(
2
hr)
568.7
312.5
1,417.4
T­
3
(
3
hr)
789.3
239.4
1,368.0
T­
4
(
4
hr)
850.5
161.9
978.1
Table
123.
Sample
sizes
of
LDH
concentration
(
mU/
mg)
by
Vehicle
Type
No
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
5
5
5
T­
1
(
1
hr)
5
5
5
T­
2
(
2
hr)
5
5
5
T­
3
(
3
hr)
5
5
5
T­
4
(
4
hr)
5
5
5
36
Table
124.
Coeff
of
Variation
of
LDH
concentration
(
mU/
mg)
by
Vehicle
Type
No
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
24.6
25.1
50.1
T­
1
(
1
hr)
35.9
31.9
64.1
T­
2
(
2
hr)
40.6
29.1
58.8
T­
3
(
3
hr)
44.4
19.2
51.1
T­
4
(
4
hr)
40.9
11.3
37.4
Table
125.
Means
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
No
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
208.4
282.0
260.1
194.9
231.1
T­
1
(
1
hr)
754.6
972.6
874.1
1,698.3
1,980.9
T­
2
(
2
hr)
1,000.6
1,323.5
1,202.4
2,079.4
2,538.5
T­
3
(
3
hr)
1,282.3
1,484.8
1,418.6
2,461.7
2,854.6
T­
4
(
4
hr)
1,543.1
1,628.7
1,691.5
2,397.0
2,952.4
Table
126.
Standard
Deviations
of
LDH
concentration
(
mU/
mg)
by
Vehicle
Concentration
No
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
30.6
64.3
38.8
95.1
133.0
T­
1
(
1
hr)
305.2
409.8
317.9
811.4
1,612.1
T­
2
(
2
hr)
356.7
532.5
387.8
1,009.3
1,777.8
T­
3
(
3
hr)
323.2
429.9
401.5
1,281.2
1,618.7
T­
4
(
4
hr)
198.5
428.9
406.7
784.9
1,379.3
37
Table
127.
Sample
sizes
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
No
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
3
3
3
3
3
T­
1
(
1
hr)
3
3
3
3
3
T­
2
(
2
hr)
3
3
3
3
3
T­
3
(
3
hr)
3
3
3
3
3
T­
4
(
4
hr)
3
3
3
3
3
Table
128.
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
No
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
14.7
22.8
14.9
48.8
57.6
T­
1
(
1
hr)
40.4
42.1
36.4
47.8
81.4
T­
2
(
2
hr)
35.7
40.2
32.3
48.5
70.0
T­
3
(
3
hr)
25.2
29.0
28.3
52.0
56.7
T­
4
(
4
hr)
12.9
26.3
24.0
32.7
46.7
38
Table
129.
Means
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Type
with
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
247.1
237.0
169.0
T­
1
(
1
hr)
1,018.8
739.8
1,621.3
T­
2
(
2
hr)
1,414.4
968.4
2,824.4
T­
3
(
3
hr)
1,743.2
1,172.4
3,098.1
T­
4
(
4
hr)
2,058.8
1,368.4
3,226.2
Table
130.
Standard
Deviations
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Type
with
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
146.9
56.3
75.4
T­
1
(
1
hr)
257.6
122.6
1,000.8
T­
2
(
2
hr)
170.9
149.3
1,943.9
T­
3
(
3
hr)
354.2
149.5
1,812.5
T­
4
(
4
hr)
541.3
195.9
1,652.0
Section
14.0
VEHICLE
EFFECT
EXPERIMENT
39
Table
131.
Sample
sizes
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Type
with
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
5
5
5
T­
1
(
1
hr)
5
5
5
T­
2
(
2
hr)
5
5
5
T­
3
(
3
hr)
5
5
5
T­
4
(
4
hr)
5
5
5
Table
132.
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Type
with
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
59.5
23.8
44.6
T­
1
(
1
hr)
25.3
16.6
61.7
T­
2
(
2
hr)
12.1
15.4
68.8
T­
3
(
3
hr)
20.3
12.8
58.5
T­
4
(
4
hr)
26.3
14.3
51.2
40
Table
133.
Means
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
321.7
178.5
286.8
144.6
156.9
T­
1
(
1
hr)
986.0
928.5
861.9
1,416.7
1,440.3
T­
2
(
2
hr)
1,311.5
1,341.7
1,201.6
2,539.5
2,284.4
T­
3
(
3
hr)
1,614.0
1,564.7
1,402.6
2,777.1
2,664.5
T­
4
(
4
hr)
1,750.5
1,822.9
1,615.5
2,955.0
2,945.3
Table
134.
Standard
Deviations
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
145.1
35.5
45.3
46.2
64.4
T­
1
(
1
hr)
376.4
66.0
276.8
964.6
1,249.2
T­
2
(
2
hr)
340.2
229.8
395.9
2,534.4
1,803.9
T­
3
(
3
hr)
399.6
258.0
404.3
2,500.2
1,582.9
T­
4
(
4
hr)
389.8
256.9
477.6
2,285.5
1,464.8
Section
14.0
VEHICLE
EFFECT
EXPERIMENT
41
Table
135.
Sample
sizes
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
3
3
3
3
3
T­
1
(
1
hr)
3
3
3
3
3
T­
2
(
2
hr)
3
3
3
3
3
T­
3
(
3
hr)
3
3
3
3
3
T­
4
(
4
hr)
3
3
3
3
3
Table
136.
Coeff
of
Variation
of
LDH
Concentration
(
mU/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
45.1
19.9
15.8
31.9
41.0
T­
1
(
1
hr)
38.2
7.1
32.1
68.1
86.7
T­
2
(
2
hr)
25.9
17.1
32.9
99.8
79.0
T­
3
(
3
hr)
24.8
16.5
28.8
90.0
59.4
T­
4
(
4
hr)
22.3
14.1
29.6
77.3
49.7
42
Table
137.
Means
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
No
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
0.0025
0.0025
0.0023
T­
1
(
1
hr)
0.0219
0.0162
0.0346
T­
2
(
2
hr)
0.0257
0.0218
0.0315
T­
3
(
3
hr)
0.0243
0.0262
0.0312
T­
4
(
4
hr)
0.0229
0.0275
0.0226
Table
138.
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
No
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
0.0006
0.0004
0.0003
T­
1
(
1
hr)
0.0024
0.0042
0.0165
T­
2
(
2
hr)
0.0026
0.0106
0.0155
T­
3
(
3
hr)
0.0032
0.0171
0.0163
T­
4
(
4
hr)
0.0045
0.0225
0.0084
Section
14.0
VEHICLE
EFFECT
EXPERIMENT
43
Table
139.
Sample
sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
No
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
5
5
5
T­
1
(
1
hr)
5
5
5
T­
2
(
2
hr)
5
5
5
T­
3
(
3
hr)
5
5
5
T­
4
(
4
hr)
5
5
5
Table
140.
Coeff
of
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
No
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
25.4
15.9
12.3
T­
1
(
1
hr)
11.0
26.1
47.7
T­
2
(
2
hr)
10.1
48.4
49.3
T­
3
(
3
hr)
13.0
65.0
52.1
T­
4
(
4
hr)
19.7
81.7
37.2
44
Table
141.
Means
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
No
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
0.0026
0.0025
0.0025
0.0020
0.0026
T­
1
(
1
hr)
0.0258
0.0191
0.0197
0.0235
0.0332
T­
2
(
2
hr)
0.0334
0.0198
0.0250
0.0221
0.0314
T­
3
(
3
hr)
0.0391
0.0205
0.0237
0.0220
0.0309
T­
4
(
4
hr)
0.0420
0.0192
0.0191
0.0219
0.0195
Table
142.
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
No
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
0.0004
0.0007
0.0004
0.0005
0.0003
T­
1
(
1
hr)
0.0033
0.0046
0.0034
0.0111
0.0260
T­
2
(
2
hr)
0.0067
0.0044
0.0025
0.0074
0.0224
T­
3
(
3
hr)
0.0152
0.0034
0.0020
0.0061
0.0235
T­
4
(
4
hr)
0.0221
0.0048
0.0043
0.0084
0.0063
Section
14.0
VEHICLE
EFFECT
EXPERIMENT
45
Table
143.
Sample
sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
No
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
3
3
3
3
3
T­
1
(
1
hr)
3
3
3
3
3
T­
2
(
2
hr)
3
3
3
3
3
T­
3
(
3
hr)
3
3
3
3
3
T­
4
(
4
hr)
3
3
3
3
3
Table
144.
Coeff
of
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
No
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
17.4
25.6
14.3
24.4
11.5
T­
1
(
1
hr)
12.8
24.0
17.4
47.3
78.4
T­
2
(
2
hr)
20.1
22.4
10.1
33.6
71.2
T­
3
(
3
hr)
38.9
16.7
8.2
27.7
75.9
T­
4
(
4
hr)
52.6
24.7
22.3
38.3
32.3
46
Table
145.
Means
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
with
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
0.0032
0.0025
0.0023
T­
1
(
1
hr)
0.0270
0.0189
0.0432
T­
2
(
2
hr)
0.0427
0.0304
0.0585
T­
3
(
3
hr)
0.0581
0.0452
0.0623
T­
4
(
4
hr)
0.0850
0.0633
0.0708
Table
146.
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
with
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
0.0028
0.0005
0.0009
T­
1
(
1
hr)
0.0096
0.0024
0.0305
T­
2
(
2
hr)
0.0278
0.0089
0.0628
T­
3
(
3
hr)
0.0388
0.0172
0.0744
T­
4
(
4
hr)
0.0875
0.0328
0.0908
Section
14.0
VEHICLE
EFFECT
EXPERIMENT
47
Table
147.
Sample
sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
with
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
5
5
5
T­
1
(
1
hr)
5
5
5
T­
2
(
2
hr)
5
5
5
T­
3
(
3
hr)
5
5
5
T­
4
(
4
hr)
5
5
5
Table
148.
Coeff
of
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Type
with
hCG
Stimulation
Timepoint
DMSO
ETOH
Tween
Baseline
85.8
21.6
37.2
T­
1
(
1
hr)
35.4
12.9
70.5
T­
2
(
2
hr)
65.1
29.4
107.3
T­
3
(
3
hr)
66.8
38.1
119.6
T­
4
(
4
hr)
103.0
51.9
128.3
48
Table
149.
Means
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
0.0046
0.0019
0.0026
0.0018
0.0025
T­
1
(
1
hr)
0.0356
0.0183
0.0265
0.0202
0.0478
T­
2
(
2
hr)
0.0734
0.0302
0.0490
0.0331
0.0336
T­
3
(
3
hr)
0.0858
0.0418
0.0657
0.0434
0.0393
T­
4
(
4
hr)
0.0992
0.0540
0.1062
0.0641
0.0416
Table
150.
Standard
Deviations
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
0.0032
0.0002
0.0007
0.0005
0.0002
T­
1
(
1
hr)
0.0254
0.0015
0.0085
0.0034
0.0346
T­
2
(
2
hr)
0.0836
0.0098
0.0372
0.0053
0.0109
T­
3
(
3
hr)
0.0941
0.0206
0.0545
0.0171
0.0112
T­
4
(
4
hr)
0.1153
0.0296
0.1183
0.0384
0.0106
Section
14.0
VEHICLE
EFFECT
EXPERIMENT
49
Table
151.
Sample
sizes
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
3
3
3
3
3
T­
1
(
1
hr)
3
3
3
3
3
T­
2
(
2
hr)
3
3
3
3
3
T­
3
(
3
hr)
3
3
3
3
3
T­
4
(
4
hr)
3
3
3
3
3
Table
152.
Coeff
of
Variation
of
Testosterone
Concentration
(
ng/
mg)
by
Vehicle
Concentration
with
hCG
Stimulation
Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
70.1
10.8
27.2
28.7
7.8
T­
1
(
1
hr)
71.3
8.4
32.0
16.6
72.4
T­
2
(
2
hr)
113.8
32.3
76.0
16.0
32.5
T­
3
(
3
hr)
109.6
49.3
83.0
39.3
28.4
T­
4
(
4
hr)
116.2
54.8
111.4
59.9
25.4
Section
14.0
VEHICLE
EFFECT
EXPERIMENT
50
80
Table
153.
Summary
of
Vehicle
Experiment
:
LDH
Concentrations*
(
mU/
mg)
by
Vehicle
Type**

Timepoint
DMSO
ETOH
Tween
20
Baseline
237.1
+
58.4
264.0
+
66.4
204.8
+
102.6
1
hr
1,042.5
+
374.2
775.8
+
247.3
1,950.0
+
1,249.2
2
hr
1,399.8
+
568.7
1,075.0
+
312.5
2,411.9
+
1,417.4
3
hr
1,776.5
+
789.3
1,245.5
+
239.4
2,679.3
+
1,368.0
4
hr
2,080.7
+
850.5
1,434.3
+
161.9
2,612.6
+
978.1
*
Mean
+
standard
deviation
**
No
hCG
stimulation
occurred
81
Table
154.
Summary
of
Vehicle
Experiment
:
LDH
Concentrations*
(
mU/
mg)
by
Vehicle
Type**

Timepoint
DMSO
ETOH
Tween
20
Baseline
247.1
+
146.9
237.0
+
56.3
169.0
+
75.4
1
hr
1,018.8
+
257.6
739.8
+
122.6
1,621.3
+
1,000.8
2
hr
1,414.4
+
170.9
968.4
+
149.3
2,824.4
+
1,943.9
3
hr
1,743.2
+
354.2
1,172.4
+
149.5
3,098.1
+
1,812.5
4
hr
2,058.8
+
541.3
1,368.4
+
195.9
3,226.2
+
1,652.0
*
Mean
+
standard
deviation
**
hCG
stimulation
occurred
82
Table
155.
Summary
of
Vehicle
Experiment
:
LDH
Concentrations*
(
mU/
mg)
by
Vehicle
Concentration**

Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
208.4
+
30.6
282.0
+
64.3
260.1
+
38.8
194.9
+
95.1
231.1
+
133.0
1
hr
754.5
+
305.2
972.6
+
409.8
874.1
+
317.9
1,698.3
+
811.4
1,980.9
+
1,612.1
2
hr
1,000.6
+
356.7
1,323.5
+
532.5
1,202.4
+
387.8
2,079.4
+
1,009.3
2,538.5
+
1,777.8
3
hr
1,282.3
+
323.2
1,484.8
+
429.9
1,418.6
+
401.5
2,461.7
+
1,281.2
2,854.6
+
1,618.7
4
hr
1,543.1
+
198.5
1,628.7
+
428.9
1,691.5
+
406.7
2,397.0
+
784.9
2,952.4
+
1,379.3
*
Mean
+
standard
deviation
**
No
hCG
stimulation
occurred
83
Table
156.
Summary
of
Vehicle
Experiment
:
LDH
Concentrations*
(
mU/
mg)
by
Vehicle
Concentration**

Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
321.7
+
145.1
178.5
+
35.5
286.8
+
45.3
144.6
+
46.2
156.9
+
64.4
1
hr
986.0
+
376.4
928.5
+
66.0
861.9
+
276.8
1,416.7
+
964.6
1,440.3
+
1,249.2
2
hr
1,311.5
+
340.2
1,341.7
+
229.8
1,201.6
+
395.9
2,539.5
+
2,534.4
2,284.4
+
1,803.9
3
hr
1,614.0
+
399.6
1,564.7
+
258.0
1,402.6
+
404.3
2,777.1
+
2,500.2
2,664.5
+
1,582.9
4
hr
1,750.5
+
389.8
1,822.9
+
256.9
1,615.5
+
477.6
2,955.0
+
2,285.5
2,945.3
+
1,464.8
*
Mean
+
standard
deviation
**
hCG
stimulation
occurred
84
Table
157.
Summary
of
Vehicle
Experiment
:
Testosterone
Concentrations*
(
ng/
mg)
by
Vehicle
Type**

Timepoint
DMSO
ETOH
Tween
20
Baseline
0.0025
+
0.0006
0.0025
+
0.0004
0.0023
+
0.0003
1
hr
0.0219
+
0.0024
0.0162
+
0.0042
0.0346
+
0.0165
2
hr
0.0257
+
0.0026
0.0218
+
0.0106
0.0315
+
0.0155
3
hr
0.0243
+
0.0032
0.0262
+
0.0171
0.0312
+
0.0163
4
hr
0.0229
+
0.0045
0.0275
+
0.0225
0.0226
+
0.0084
*
Mean
+
standard
deviation
**
No
hCG
stimulation
occurred
85
Table
158.
Summary
of
Vehicle
Experiment
:
Testosterone
Concentrations*
(
ng/
mg)
by
Vehicle
Type**

Timepoint
DMSO
ETOH
Tween
20
Baseline
0.0032
+
0.0028
0.0025
+
0.0005
0.0023
+
0.0009
1
hr
0.0270
+
0.0096
0.0189
+
0.0024
0.0432
+
0.0305
2
hr
0.0427
+
0.0278
0.0304
+
0.0089
0.0585
+
0.0628
3
hr
0.0581
+
0.0388
0.0452
+
0.0172
0.0623
+
0.0744
4
hr
0.0850
+
0.0875
0.0633
+
0.0328
0.0708
+
0.0908
*
Mean
+
standard
deviation
**
hCG
stimulation
occurred
86
Table
159.
Summary
of
Vehicle
Experiment
:
Testosterone
Concentrations*
(
ng/
mg)
by
Vehicle
Concentration**

Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
0.0026
+
0.0004
0.0025
+
0.0007
0.0025
+
0.0004
0.0020
+
0.0005
0.0026
+
0.0003
1
hr
0.0258
+
0.0033
0.0191
+
0.0046
0.0197
+
0.0034
0.0235
+
0.0111
0.0332
+
0.0260
2
hr
0.0334
+
0.0067
0.0198
+
0.0044
0.0250
+
0.0025
0.0221
+
0.0074
0.0314
+
0.0224
3
hr
0.0391
+
0.0152
0.0205
+
0.0034
0.0237
+
0.0020
0.0220
+
0.0061
0.0309
+
0.0235
4
hr
0.0420
+
0.0221
0.0192
+
0.0048
0.0191
+
0.0043
0.0219
+
0.0084
0.0195
+
0.0063
*
Mean
+
standard
deviation
**
No
hCG
stimulation
occurred
87
Table
160.
Summary
of
Vehicle
Experiment
:
Testosterone
Concentrations*
(
ng/
mg)
by
Vehicle
Concentration**

Timepoint
0.0%
0.5%
1.0%
5.0%
10.0%

Baseline
0.0046
+
0.0032
0.0019
+
0.0002
0.0026
+
0.0007
0.0018
+
0.0005
0.0025
+
0.0002
1
hr
0.0356
+
0.0254
0.0189
+
0.0015
0.0265
+
0.0085
0.0202
+
0.0034
0.0478
+
0.0346
2
hr
0.0734
+
0.0836
0.0302
+
0.0098
0.0490
+
0.0372
0.0331
+
0.0053
0.0336
+
0.0109
3
hr
0.0858
+
0.0941
0.0418
+
0.0206
0.0657
+
0.0545
0.0434
+
0.0171
0.0393
+
0.0112
4
hr
0.0992
+
0.1153
0.0540
+
0.0296
0.1062
+
0.1183
0.0641
+
0.0384
0.0416
+
0.0106
*
Mean
+
standard
deviation
**
hCG
stimulation
occurred
88
15.0
DISCUSSION
15.1.
Incubation
Factors
Experiments
The
test
tube
results
had
smaller
root
mean
squared
errors
(
RMSEs)
than
vial
results
both
with
and
without
hCG.
This
may
be
the
result
of
the
tissue
not
breaking
up
as
much
since
there
is
less
space
for
tissue:
media
interaction
than
in
the
vial.
For
the
cases
with
no
hCG,
the
shaker
speed
was
the
dominant
factor.
However,
for
situations
with
hCG,
incubation
volume
and
fragment
size
were
important
for
the
incubations
in
vials
but
shaker
speed
and
incubation
temperature
also
became
important
for
test
tubes.

Vials
consistently
resulted
in
higher
testosterone
levels
than
test
tubes.
The
optimum
values
occurred
at
the
extremes
of
the
experimental
regions
in
all
cases.
The
optimum
fragment
size
generally
fell
in
the
middle
portion
of
its
range.
For
vials,
the
optimal
fragment
size
with
no
hCG
was
around
80
mg,
while
with
hCG
the
optimal
size
was
approximately
200
mg.
Therefore
it
is
felt
that
100­
175
mg
of
testicular
tissue
is
a
range
that
would
be
appropriate
to
use
in
the
slice
testis
assay
when
vials
are
used.
The
fragment
size
for
test
tubes
appeared
to
be
optimal
at
130­
170
mg.

The
optimal
hCG
concentration
fell
in
the
middle
of
the
range
of
tested
concentrations.
Although
that
range
was
0.05­
0.08
for
some
cases,
it
was
felt
that
the
0.1
concentration
would
be
the
most
easily
adapted
for
a
screening
assay.
The
concentration
for
hCG
concentration
in
the
assay
has
to
be
calculated
for
the
final
concentration
in
the
volume
of
media
to
be
used
in
each
vial
and
the
0.1
concentration
is
more
easily
calculated
and
weighed
for
a
general
assay.

The
optimal
temperature
fell
between
34
and
37
°
C
and
was
approximately
36
°
C
.
When
shaker
speed
was
treated
as
a
continuous
factor,
the
optimal
speed
was
low
(
135
rpm)
with
vials
with
no
hCG,
but
high
(
200
rpm)
with
the
other
cases.
It
is
felt
that
the
overall
optimal
speed
is
around
175
rpm
based
on
our
studies
and
those
of
others.
We
found
that
200
rpm
is
difficult
to
maintain
in
the
incubator,
especially
over
the
24
hour
period
without
movement
of
the
shaker
itself
and
results
in
dissociation
of
the
testicular
fragments.

The
optimal
volume
of
the
media
for
test
tubes
was
at
the
lower
extreme
of
2.5
ml
but
in
all
other
cases
it
was
approximately
4
ml.
Therefore
we
kept
the
incubation
volume
at
5.0
ml
for
ease
of
the
other
technical
operations
that
had
to
be
performed
in
later
assays.
This
included
larger
aliquots
for
LDH
determinations
than
had
originally
been
planned.

15.2
Organ
Preparation
Factors
Experiments
In
the
organ
preparation
series
of
experiments,
the
use
of
warm
and
cold
Delbecco's
Phosphate
Buffered
Saline
(
DPBS)
as
well
as
cold
media
was
evaluated
as
well
as
the
time
the
tissue
spent
in
these
solutions
,
the
aliquot
volume
and
the
hCG
concentration.
In
the
cases
with
89
hCG,
there
were
very
small
degrees
of
freedom
for
error,
resulting
in
difficulty
in
testing
for
the
significance
of
factors.
In
the
cases
with
no
hCG,
there
were
somewhat
smaller
RMSEs
for
cold
media
than
for
cold
or
warm
buffered
saline.
For
the
cold
solutions,
time
delay
(
the
time
from
placing
the
tissue
in
the
holding
solution
until
placing
it
in
the
media
to
start
the
incubations)
was
the
dominant
factor
as
would
be
expected.
Baseline
was
important
for
the
buffered
saline
cases.

Cold
buffered
saline
cases
yielded
slightly
higher
testosterone
values
than
cold
media
and
the
cold
media
values
were
slightly
higher
than
the
warm
buffered
saline.
The
time
delays
were
inconsistent
when
considered
across
time
points.
Aliquot
volumes
were
also
inconsistent
and
often
at
the
extremes
of
the
regions.
The
LDH
experiments
performed
with
the
optimized
factors
appeared
to
confirm
that
the
time
delay
should
not
be
more
than
one
hour
before
the
starting
assay
incubation.
These
data
combined
with
the
results
from
the
previous
experiments
to
determine
the
hCG
concentration
to
be
used
in
the
optimized
assay.
The
cold
DPBS
appeared
to
be
the
best
solution
to
collect
the
testes
in
prior
to
the
initial
assay
incubation.
90
16.0
CONCLUSIONS
Based
upon
the
incubation
experiments,
the
optimized
assay
factors
that
yielded
the
maximum
testosterone
concentrations
were
the
use
of
scintillation
vials
with
testicular
fragments
of
100
to
175
mg.
This
fragment
size
should
allow
for
at
least
8­
10
fragments
per
testis
for
use
in
the
pre­
validation
of
the
sliced
testis
assay.
The
volume
of
incubation
media
should
be
4­
5
ml.

It
was
difficult
to
determine
the
optimal
factors
from
the
testis
preparation
experiments.
There
should
have
been
more
determinations
using
hCG.
The
small
number
used
made
the
degrees
of
freedom
for
error
very
small.
The
cases
without
hCG
however
showed
that
cold
buffered
saline
with
no
more
than
one
hour
of
delay
before
the
first
assay
incubation
in
media
199
was
optimal.
The
aliquot
volume
can
be
less
than
0.5
ml
(
the
optimal
appeared
to
be
around
0.3ml)
but
the
larger
volume
is
necessary
if
more
than
one
determination
is
to
be
made
from
the
aliquot.
When
smaller
aliquots
were
used,
there
was
not
enough
sample
if
repeat
determinations
for
testosterone
were
necessary.

Therefore,
the
optimal
assay
was
run
using
Delbecco's
PBS
for
collection
of
testes
for
a
period
less
than
one
hour.
The
testis
fragment
was
then
placed
in
a
scintillation
vial
with
5
ml
of
media
199
and
incubated
at
36
°
C
for
one
hour.
The
media
was
then
replaced
with
fresh
media
and
the
baseline
samples
were
taken.
Then
media
removed
was
replaced
with
the
same
volume
of
media
or
media
plus
hCG.
The
sample
was
then
re­
incubated
and
hourly
samples
were
taken.
The
optimal
assay
was
run
as
part
of
the
24
hour
sampling
assay
also
and
the
sampling
after
4
hours
was
not
optimal
in
terms
of
the
rise
in
both
testosterone
and
LDH
concentration.
This
probably
is
an
indication
of
cell
death
and
release
of
testosterone.

The
injury
experiment
showed
the
differences
in
LDH
and
testosterone
concentration
with
the
different
types
of
injury.
Chemical
injury
with
Triton
X­
100
caused
the
cell
to
lyse
resulting
in
high
LDH
values
and
the
cellular
testosterone
was
released
at
the
one
hour
time
point.
Heat
(
42
°
C)
caused
the
LDH
to
continually
rise
but
the
testosterone
remained
low
indicating
that
the
cellular
testosterone
production
was
minimal
(
probably
due
to
denaturation
of
enzymatic
proteins)
during
incubation.
The
trauma
of
mincing
the
tissue
resulted
in
both
the
testosterone
and
LDH
concentrations
maximal
at
the
1
hour
time
point.
Only
the
heated
sample
showed
no
additional
testosterone
release
after
hCG
stimulation.

The
vehicle
experiment
should
help
us
to
determine
the
influence
of
vehicle
concentration
influence
on
the
assay
system.
The
testosterone
assays
for
these
experiments
need
to
also
include
vehicle
blanks
to
determine
if
the
vehicle
itself
is
affecting
the
assay.

It
should
be
noted
that
all
of
these
optimization
experiments
were
performed
on
control
testicular
fragments
(
albeit
+
hCG)
.
There
is
no
guarantee
that
the
same
optimized
conditions
established
in
this
work
would
necessarily
hold
for
testicular
tissue
incubated
with
LH,
FSH
or
an
anti­
androgen
with
the
mechanism
of
action
of
inhibition
of
testosterone
biosynthesis.
91
APPENDIX
1
LDH
Validation
and
Verification
with
Media
199
without
Phenol
Red
(
See
Draft
Letter
Report
for
Phase
I)