United States EPA-600/7-82-038b
Environmental Protection '
Agency May 1982
v°/EPA Research and
Development
ENVIRONMENTAL ASSESSMENT OF
A LOW-EMESDN OIL-FIRED
RESIDENTIAL HOT WATER
CONDENSING HEATING SYSTEM
Volume IL Data Supplement
Prepared for
Office of Air Quality Planning and Standards
Prepared by
Industrial Environmental Research
Laboratory
Research Triangle Park NC 27711
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the INTERAGENCY ENERGY-ENVIRONMENT
RESEARCH AND DEVELOPMENT series. Reports in this series result from the
effort funded under the 17-agency Federal Energy/Environment Research and
Development Program. These studies relate to EPA's mission to protect the public
health and welfare from adverse effects of pollutants associated with energy sys-
tems. The goal of the Program is to assure the rapid development of domestic
energy supplies in an environmentally-compatible manner by providing the nec-
essary environmental data and control technology. Investigations include analy-
ses of the transport of energy-related pollutants and their health and ecological
effects; assessments of, and development of, control technologies for energy
systems; and integrated assessments of a wide range of energy-related environ-
mental issues.
EPA REVIEW NOTICE
This report has been reviewed by the participating Federal Agencies, and approved
for publication. Approval does not signify that the contents necessarily reflect
the views and policies of the Government, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
ACUREX TECHNICAL REPORT TR-81-78/EE
ENVIRONMENTAL ASSESSMENT OF A LOW EMISSION OIL-FIRED RESIDENTIAL
HOT WATER CONDENSING HEATING SYSTEM
VOLUME II: DATA SUPPLEMENT
April 1982
Acurex Project 7600
Contract 68-02-3188
For
EPA Project Officer - R. E. Hall
Combustion Research Branch
Energy Assessment and Control Division
Industrial Environmenta Research Laboratory
Research Triangle Park, North Carolina 27711
By
C. Castaldini
Acurex Corporation
Energy & Environmental Division
485 Clyde Avenue
Mountain View, California 94042
-------
CONTENTS
Section Page
1 Introduction 1
References
2 Preliminary Equipment Calibration 2
3 Complete Furnace Operating Data 7
4 Sampling Data Sheets 39
5 Analytical Laboratory Results 56
5.1 Fuel Analysis Report
5.2 Particulate Gravimetric Analyses and
Mass Emission Calculations 59
5.3 Sulfur Analysis Report and Emission
Calculation 64
5.4 Trace Element Analysis Report 66
5.5 Organic Analysis Report 88
5.6 Bioassay Analyses Report 112
6 SAM IA Work Sheets 180
iii
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SECTION 1
INTRODUCTION
The purpose of this Data Supplement is to document data in greater
detail than was practical in the final report (reference 1-1). It is
intended to provide sufficient detail for researchers to perform their own
analysis of the data. Readers are referred to the final report for
objectives, description of the emission results, interpretations, and
conclusions.
The data supplement contains the following information:
• Preliminary equipment calibration — calibration data for the
dry gas meters used to sample flue gas for both EPA Method 5
and Source Assessment Sampling System (SASS) trains
t Complete furnace operating data — furnace water and flue gas
temperatures throughout each burner-on/burner-off cycle;
cooling water flowrate, and thermostat settings
0 Sampling data sheets — emission data obtained with continuous
monitoring instrumentation: operating data tables for EPA
Method 5 (for particulate mass emissions), EPA Method 8 (for
S02 and S03 sampling), and SASS (for particulate, trace
element, and organic sampling)
a Analytical laboratory results -- ultimate analysis of
distillate fuel oil used in the test program: laboratory
-------
analysis reports on participate levels by gravimetric
analysis; sulfur by turbimetric analysis; trace elements
by Spark Source Mass Spectronetry (SSMS) analysis and
Atomic Absorption (AA) analysis; anions by ion chromatography;
Total Chromatographable Organic (TCO) emissions by onsite Gas
Chromatography (GC); speciation of organic emissions by Gas
Chromatography/Mass Spectrometry (GC/MS); biological assays of
the extract and water discharge samples
t SAM IA work sheets — Source Assessment Model IA evaluation of
emission and effluent data to determine the potential
hazard posed by both flue gas and water discharge samples
REFERENCE FOR SECTION 1
1-1. Castaldini, C., "Environmental Assessment of a Low-NO
Residential Hot Water Condensing Heating System Burnifig Distillate
Oil, Volume 1: Technical Results."
-------
SECTION 2
PRELIMINARY EQUIPMENT CALIBRATION
-------
--7-8 C
Dm
BeroKtrlc rVetiure 3o-l0
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2.4
2.6
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rrtairy Calibration Utter
Control nodule*
Operiton
Met lulb Teaperature
HETER CALIBRATION DATA
Orifice
Manometer
(1n.*9.)
Orifice
Magnehellc
Prlwry
Meter
Dry Test
Meter
Cts Volim
PrlHry Meter
fiei Voluw
Dry Teit Meter
(ft.1)
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t
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70.0
fit
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Average
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Drt.
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-------
SECTION 3
COMPLETE FURNACE OPERATING DATA
-------
FURNACE OPERATING DATA
Cycle
No.
±
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Flowrate
(gpm)
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Cycle
No.
5.
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Flowrate
(gpm)
/7
tf
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53
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Cycle
No.
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(gpm)
t-7
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Setting
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63,
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Cycle
No.
3
Cooling Water
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(gpm)
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17
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(gpm)
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6'3.5-
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Cycle
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10
Cycle
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(gpm)
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(°C)
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(gpm)
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Cycle
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(9Pm)
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Cycle
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(gpn)
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FURNACE OPERATING DATA
17
Cycle
No.
II
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(gpm)
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Exhaust Duct
Temperature
7^
73
•7^k
7a
ya
73
^3
-------
FURNACE OPERATING DATA
Cycle
No.
Cooling Hater
Flowrate
(gpm)
/ 7
\J
Thermostat
Setting
t°C)
5
-ft,
•>'<>
£<*
A' 4
£~t,
J<~
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9 +
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&0
7V
7?
77
77
7.<
7^
7^
7V
73
7A
73
7/
71
71
Tank Hater
Temperature
/"**'
/v-/
/3«
/a-r
y.w
/39
/J7
/o
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f?7
9/
fite
s^
*&
•79
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»^a
9G
tf6.
»7
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8--S"
S?/A
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. #3
ro
ui
-------
FURNACE OPERATING DATA
Cycle
No.
/£
Cooling Water
Flowrate
(91*)
/.7
J
Thermostat
Setting
rcj
•Z5
\)
Inlet Water
Tempgrature
£>' 7
&<~
*t,
<7
s 7
A' 7
A'6-
S-7
a-r
SI
,<-7
£7
*7
47
~f7
S7
*'(.
.6-7
A~&
X-7
£7
A' -7
.•T7
Outlet Water
Tempgrature
7t
<3o
«3
»7
*9
ft
»/
&o
79
?*f
77
Tank Hater
Tempgrature
iol
107
//a
J/7
/a.3.
/A7
/3i
/3H.
/3V
tn.3.
/4*o"
S+3
/•f-0
/37
t*3
S3&
/as?
I2H-
/aa
/\ao
us
///„
//*!.
Exhaust Duct
Tempgrature
6>7
71
7*4
77
78
79
79
fito
&o
8/
K2.
SI
79
St*
79
79
ffo
»/
7»
77
&>
Sfc>
S'o
Ambient
Tempgrature
80
?2
»a
9V-
g*a
0v
?&
ff6
i?7
SV-
*/
9a-
. «?/
ro
en
-------
FURNACE OPERATING DATA
Cycle
No.
/3
Cooling Mater
Flowrate
(gpm)
t,7
\
[
/.r
\
/
Thermostat
Setting
rcj
55-
V
1
fS
I
(
Inlet Mater
Tempgrature
6" 7
.<6>
f •*+
•5" 7
&'7
A'-T
.V7
«T7
ft"7
-T7
o-7
i>~ 7
*'t.
A' 7
X7
£7
T7
A'6
o'7
A' 7
A* 7
A* 7
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Outlet Mater
Temperature
7
7A"
7**
7-y.
•ra
/a
73
7f
7/
70
7o
Vt
i><*
i»<*
7f-
7%
«
1,7
-re,
73
7to
77
77
77
7^
Ambient
Tempgrature
^0
97
99
fiL.
Sfc
87
«5
8-r
ffi-
«a
»/*
H3
6>4
fi-3
83
?0
ff3
*2
.'SO
e-o
so
9^
. «/ '
ro
-------
FURNACE OPERATING DATA
Cycle
No.
Cooling Water
Flowrate
(gpnO
i,1
\
/
Thermostat
Setting
m
££~
J
Inlet Mater
Tempgrature
A'C»
£-(c,
-5" 7
&
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toy.
^7
9J
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*v
»A'
ft-^
«3
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80
TV
77
77
7t-
7-5"
7.T
-7.3
73
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Tempgrature
/3k
Ht-O
/HM.
/«7
M?
/*-«
tt+U-
f*-c>
/*7
,3 +
/3i
/as
/3S'
/J*
yjy
y//
«/
«?0
7^
7-7
7V
7,f
7*
7S"
77
Ambient
Tempgrature
«/
«3
«3
»7
/oo
ion.
ma.
99
tOCf
/oo
9S
S-/
»8
i?9
9J
S7
S>£,
fft
84
»7
83"
fff-
. 94C '
rsa
-------
FURNACE OPERATING DATA
Cycle
No.
M-
Coollng Hater
Flowrate
(gpm)
1.7
/
t.7
i
Thermostat
Setting
t°C)
SS
i
J
££
v/
Inlet Hater
Temperature
A-&
4' 7
A" 7
S~(*
i>-t>
&<*>
*,'<*
6~<~
A' -7
*~t>
6' 7
5" 7
-5'&
*>' 7
6" 6
5-<^
6" 6
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s-t
ft 7
4' 7
a- 7
A- 7
Outlet Hater
Tempgrature
•ra
7/
71
•70
•70
&ff
6t
&.K
-//
7^
go
fc-3
?
So
Sfc>
»o
6tf
t~~7
"70
73
76,
77
79
7
-------
FURNACE OPERATING DATA
Cycle
No.
»*
Cooling Hater
Flowrate
(gem)
'•7
I
//
Thermostat
Setting
l°0
*55*
i
y
s*
Inlet Mater
Tempgrature
-6'7
.•>- 7
*<°
A'C,
A'<~
g~>
g (a
A' 7
^6,
g <~
£7
S7
s-r
A 7
*7
S-J
S7
t>'(*
S -7
s<*
f7
£7
4-6
Outlet Water
Temperature
?/
»8
<47
3£
S'S
S3
&/
So
-71
7S
77
~7l*
7:>
73-
73
73
72
73
7/
y/
70
iff
71
Tank Water
Temperature
/^/
/.^-7
yj^.
131
/as
ta.L,
/33
/a/
//s-
//4»
//y.
HA
/to
/o
-------
FURNACE OPERATING DATA
Cycle
No.
Cooling Hater
Flowrate
(9pm)
1,7
\
f
Thermostat
Settinq
t°C)
*Z$
\
V
Inlet Water-
Temperature
•f L.
A" 7
•y 7
ft*
j-c.
t? (-
A~ 7
t>'7
A" 7
£•<-
*,-<*,
*:<-
A"<~
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A' 7
S7
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A-4
A- 7
S7
6't>
Outlet Mater
Temperature
Tfc
£o
8 <+
&7
e$
ft
V3
9 if
1 7
¥1
/oo
/CO
<*<.
93
9&
&8
&7
8*
«3
S3.
&i
Vo
7H
Tank Matnr
Tempgrature
totj.
/0a
ga.
sa
«-a
ffS
»a
s-/
ffCi
va
79
7«?
/y
ffo
79
So
Ambient
Temperature
SV
ff/
ffa
/?y
«a
»a
ga
SI
S>^3
»a
»a
5^
98
9^
4>3
9a
«?a
<« j
90
9/
*/
S>7
,54 '
-------
FURNACE OPERATING DATA
Cycle
No.
Cooling Water
Flowrate
(9P«i)
/'7
>
//
v
Thermostat
Sgttlns
£g
\
55
\j
,
Inlet Water
Temperature
-•T6.
^-£
57
i>~7
s(j>
£•<*
A't,
fT
A- 7
X7
fT7
SI
£7
.•S-7
&t>
A'C,
&(*
*£,
£<*
£7
S7
£7
«*
Outlet Water
Tempgrature
79
77
7fe
7 .V
73
7*4-
73
73
7*
71
7I
73
7«
83
a-.-v
sa
Vo
vz
(i*
1C,
C1$
/CO
103.
Tank Water
Temperature
IHa
//v-
//a
HO
;o&
/o 7
yoa"
/CiU-
/Ol
JOO
/o2.
/07
I/SL
117
/ja
/as
Xvl/
/..-?.-r
/3«?
/¥-"
(A)
ro
-------
FURNACE OPERATING DATA
Cycle
No.
Cooling Hater
Flowrate
(9Pm)
1.7
\
Thermostat
Setting
l°C)
6V
ti
Inlet Water
Temperature
4-4.
S7
&1
4L,
£-?
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6'(*
t>'7
3-C*
£3-7
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3-7
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d~6
5-6
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59
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83
6'«2
Sto
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^S1
77
77
76,
7-5"
7^
7.3
73
>*
71
7/
Tank Hater
Temperature
/SI
/*-%
/V-G-
/*-/
/^tf
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A27
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/^2/
/V9
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//s~
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///
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/o3
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Exhaust Duct
Temperature
S13
6*3
»/
»/
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77
Sfc»
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So
^0
£7
ffa
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fi'^Z.
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Ambient
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9^r
9
%3
S>7
ff-7
ff7
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fi»?
S>7
ffd,
6>»
6*7
g-7
ff6.
9$
89
97
97
?£>
#7
,96
CO
CO
-------
FURNACE OPERATING DATA
Cycle
No.
17
Cooling Mater
Flowrate
(9P-0
/7
'.7
\f
Thermostat
Settinq
rO
fs
fsf
-<7
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So
3
• S3
S3.
S*
83
-------
FURNACE OPERATING DATA
Cycle
No.
/*
Cooling Hater
Flowrate
(gpm)
1,7
V
//
V
Thermostat
Setting
l°C)
£*
\
56
/
v/
Inlet Mater
Temperature
57
_<6
A 7
S<~
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Outlet Water
Temperature
6?/
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79
77
77
76
74*
73
73
rJ
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7/
73
78
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V^
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/aa
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117
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H3
til
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/c-3
/oi
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IdS
1 13
US
I33>
/ax
IZI
/3k>
/3&
/^3>
/V-
-------
FURNACE OPERATING DATA
3ft
Cycle
No.
Cooling Mater
Flowrate
<9P»)
1.1
\
J
Thermostat
Setting
l°C)
S-5-
V
Inlet Hater
Temperature
°F
£<*.
£(»
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Outlet Mater
Tempgrature
10 /
/CM.
ioS
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/03
W
<*<*
V3
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87
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S3
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no
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TV
77
7t>
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7f-
Tank Hater
Temperature
»&
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IS*
is -7
y
//4
1'+
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//o
Exhaust Duct
Tempgrature
S/
82
6>3
J?3
S>3
»3
»/
ffo
78
77
77
77
77
77
77
77
77
77
77
77
77
77
79
Ambient
Tempgrature
sa
ff/
So
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<5to
9/
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90
S9
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9/
85
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ffA-
ffi.
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tfa
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g-A
79
Sb
S>^
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CO
(71
-------
FURNACE OPERATING DATA
Cycle
No.
ft
Cooling Mater
Flowrate
(gpm)
/.7
,
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1.7
V
Thermostat
Setting
l°C)
3-V
5S
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Tempgrature
£C
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Outlet Mater
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7*
73
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76.
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Exhaust Duct
Tempgrature
78
r^'
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S
?*-
fft
S>6
-------
FURNACE OPERATING DATA
Cycle
No.
Cooling Water
Flowrate
(go*)
/, 7
i
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Thermostat
Settinq
l°C)
*V
1
V
Inlet Water
Tempgrature
6'C"
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«"7
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i."^
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Outlet Water
Tempgrature
77
7t-
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7 a
73
7 a
73
71
70
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93
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Exhaust Duct
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7-iT
7*
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76.
69
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63
Ambient
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g-3
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83
79
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7*
77
77
77
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75^
76.
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70
,
CO
CO
-------
SECTION 4
SAMPLING DATA SHEETS
39
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^^Corporat
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£\ ACUREX
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^^Corporation
PARTICULATE TEST FIELD DATA SHEET
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-------
SECTION 5
ANALYTICAL LABORATORY RESULTS
56
-------
5.1 FUEL ANALYSIS REPORT
57
-------
LABORATORY CERTIFICATE
_
CURTIS & TOM PKINS, LTD.
orrci*k
ANO'Oo tAMPiiat re* MANY
COMMODITY AND T»API e»8ANUATlQNl
ANALYTICAL. CH EM I SXS-CONSULTING
•AMPLER* —INSPECTOR*
t»O DIVISION »THICT
•AN rHANCIBCO. CALIF. B41O3
U.S.A.
•«•!.»•£•
— IMVMTIftATIOMS
VITAMIN *•»*¥• - •lOCMIMIfTIlT
•VCCWUSTS IN BULK COMMOOITKI
' Laboratory No. SOklO
Preliminary No. 4132
For
ACUREX CORPORATION
Reported 10/6/80
Sampled
Received 9/8/80
Report on 5 8an,pies of Oil
\ ' ,
Sample # Sample Type
Mark (5) 80-1562, Fuel Oil
(All) Project No.: 7601.22, Customer No.: RB 59186A, rel. 01,
Prime Contract: 68-02, 3188, Acurex Project: 7601, Date:
September 5, 1980, Subcontract No.: RB59186A.
(5)
Carbon
Hydrogen (H), T. —
Oxygen (0), by
difference,?.
Nitrogen (N) ,7. —
Sulfur (S),%
Heating Value:
BTU/Pound —
Gravity, •
°API @ 60°F
*Less Than
1st 2nd
Test Test
86.94
12.90
19,190
33.75
3rd
Test
0.01*
0.03 0.04 0.05
0.18 0.20 0.20
58
-------
5.2 PARTICULATE GRAVIMETRIC ANALYSES AND MASS EMISSION CALCULATIONS
59
-------
/•S ACUREX
T >
Corporation PARTICULATE CALCULATIONS
Too. \\lfr* VW*ME? ^T*CK SAMPLE ' hpTTHoO 5
1. Volume of dry gas sampled at standard conditions, 68° F, 29.92 inch Hg (set)
Vm / Pb * 13.6
r 1764-irlT ^460"
llm,avg
i + v«i "\ /Ts,avg + 460\pn_\ (acf at stack conditions)
or Vmstack' \~5~ vwstd/\T ~" " '
2. Stack gas moisture condensed at standard conditions (scf)
Vwstd = 0.04707 Vlc
3. Stack gas proportion of water vapor, by volume
o
B
wo - Vwstd * Vmstd
4. Stack gas dry molecular weight (Ib/lb-mole)
MWd = 0.44 (%CO2) + 0.32 (%O2) + 0.28 (%N2 + %CO)
5. Stack gas molecular weight (Ib/lb-mole)
MWS = MWd (1 - Bwo) + 18 (Bwo)
6. Pressure stack, in. Hg
s - rb T 13.6
60
-------
7. Stack gas velocity at stack conditions (ft/sec)
tft
V -B549(C 46°
s- •
/Ts,avB * 46
^ psMW5
8. Stack gas volume at standard conditions (scfm)
Q8 . 60 (1 - Bwo) Vsavg A8 (
or Qa = 60VsavoAs(acfm)=
8 avg"s»
9. Test percent isokinetic
-
s ' Vmstd " 1S6 .4
MD
or Cfl = 15.432 -n— -^ — (gr/acf) =
vmstack
11. Emission rate of participate matter. Ib/hr
ER = 0.00857 (Qs) Cs
12. Percent excess air at sampling point
100 [%02 - 0.5
= 0.264 %N2 - (%02 - 0.5 x %CO)
13. Emission rate of paniculate matter, lb/106 Btu
E = 2.679 x 10*
PL.W
VVmstd/ \
VVmstd/ \20-9 - %02
17-33 (T5[8vg ^ 460) [0.04707 (W|c) * Vmstdl
• vs ps Dn2
10. Participate matter concentration, gr/scf
15 432
'
61
-------
PART1CULATE CALCULATIONS
1. Volume of dry gas sampled at standard conditions, 68° F, 29.92 inch Hg (scf)
Vmstd
m.avg
or Vmstack
2. Stack gas moisture condensed at standard conditions (scf)
Vwstd = 0.04707 Vlc
= ^l.^ll
3. Stack gas proportion of water vapor, by volume
.2C.Vf 3\,3M
4. Stack gas dry molecular weight (Ib/lb-mole)
MWd = 0.44 (%C02) + 0.32 (%O2) + 0.28 (%N2 + %CO)
5. Stack gas molecular weight (Ib/lb-mole)
MWS = MWd (1 - Bwo) + 18 (Bwo)
= JO-iH (l-3.o)-»-l8(l..o)
6. Pressure stack, in. Hg
62
-------
7. Stack gas velocity at stack conditions (ft/sec) ., ., .
VtUa^j uss. 4. j-^jit -k i*
Vs = 85.49 (CD) T * °
P
8. Stack gas volume at standard conditions (scfm)
D
or C_ = 15.432 T,—-E— (gr/acf)
vmstack
11. Emission rate of paniculate matter, Ib/hr
ER = 0.00857 (Qs) Cs
r o.
12. Percent excess air at sampling point
-- l-
100 [%O2 - 0.5 (%CO)]
% ^ = 0.264 %N2 - (%O2 - 0.5 x %CO)
13. Emission rate of paniculate matter, lb/10" Btu
E = 2.679x10*'—K ' = ' ?P9
VrnStd/ \20.9-%02
avg PSMWS ^Siow^t,, . s^acfc -YI ^fcloe.'-fj
or Qa = 60 Vsavg AS (acfm) = 60 (i-0.oi)^ £* f*<3 £i£? - I fe. S '
9. Test percent isokinetic
,,. . 17.33 (Ts.avg ^ 460) [0.04707 (Wlc) + Vn^J
fl~V—P—D~»~
s s n
= \v? %
10. Paniculate matter concentration, gr/scf
Cc = 15.432 -n-*- = It
63
-------
5.3 SULFUR ANALYSIS REPORT AND EMISSION CALCULATION
64
-------
Mist and SO2
Clean-Up and Analysis
Plant
Oate
5".
Project No.
760$ '
Recorded by
H23C>4 Mist and SC>2 Train Description
Impinger Sequence 1 2 3
Impinger Number
G-S Standard ( )
G-S Modified ( )
Contents *?/) &0i- MA
Concentration ("'•} _£p ^ ^
Initial Volume vO /ft? /&/ 2 Anal
Sample Number Aft£ /// &0t/
Ttt. Vol. of Soln. (ml) 2$o £5o
Volume of Sample .
Aliquot Titrated (ml) *7*? *>o
Performed by (V'''4^»«. vOu&ru*
and Recovery Data
456
- ^^ ^-
e jlso.oji '•
3 jl?6.iK>
J
** Mt
•* [
6*. 07 \
>rSis
1
65
-------
5.4 TRACE ELEMENT ANALYSIS REPORT
66
-------
COMMERCIAL TESTING & ENGINEERING CO.
MNltAL OFFieCt- IM HOtTH It, SAUt STRUT, CHICMO. IU.IDOI* W*0i • MI* COPt (It TM-M14
••ply 10 MS1IUMINTM AMMTMS MVWON. MM WBT MIN AVtNUf. OOUKN. COUMAOO «MO). MONt; MMM4S11
T* Mr. Brent Higgln
Acurex Corporati
485 Clyde Ave.
Mountain View, C
f. O. NO.:
Simpto No.: 80-1535
ELEMENT CONC.
Uranium
Thorium
Bismuth <0.1
Lead 0.6
Thallium
Mercury NR
Gold
Platinum
IHdium
Osmium
Rhenium
Tungsten
Tantalum <0.6
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Holnlum
Dysprosium
tat - Not ((ported
Alt MWIMRtl llOf OVtt0BO^
ibotham ^\
on ^S
A 94042
•WWK SOURCE MASS IPB
CONCENTRATION
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymium
Praseodymium 0.2
Cerium 0.9
Lanthanum 2
Barium 1
Cesium
Iodine <0.1
Tellurium
Antimony 0.2
Tin 0.1
Indium STD
Cadmium
Silver
Palladium
Rhodium
* Heterogeneous
c 0.1 ppm
k
IN PPM WEIGHT
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Geranium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
Appro*): 'I/
m
CONC.
0.4
1
<0.1
0.2
<0.1
0.4
0.2
<0.1
0.2
0.1
6
13
MC
3
120
39
29
M -
Me/
°»* September 23, 1980
Analyst: T. Bouts
IAD No.: 97-E697-116-28
ELEMENT
•Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
N1 trogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
%«£-*n*-
CONC
0.3
2
<0.1
170
24
1
9
26
*MC
13
17
98
=3
NR
NR
NR
<0.1
0.1
NR
£
MC - Mtjer Compenwtt
•UT
mi ^
67
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
eiNEIAL OmClt IM MOUTH LA IALLC «T*IET, CHICAGO. ILLI«OI» IMOI • A»f A COOC «1S T»»-»4»4
IMTIUMNTAl ANAmil tXVIVOH, IOU WIST MTH AVfNUE. 6OIMM. CtXOtADO IMOI. fMONi: 103-1714991
T»= Mr. Brent H1gg1nbotham
Acurex Corporation
485 Clyde Avenue
Mountain View, California 94042
September 26, 1980
T. Bouts
P. O. No.:
Sempte No.: M75 5pARK SOURCE MASS SPEaROGRAPHIC ANALYSIS IAD No- 97-E697-116-28
l^.TW.'Ur.i "&».ft*ite CONCENTRATION IN vg/ml
ELEMENT
Uranium
Thorium
Bismuth
Lead
Thallium
Mercury
Gold
Platinum
Iridlum
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Holmium
Dysprosium
CONC. ELEMENT CONC.
Terbium
Gadolinium
Europium
<0.02 Samarium
Neodymi urn
NR Praseodymium
Cerium
Lanthanum
Barium * 1
Cesium
Iodine 0.01
Tellurium
Antimony
Tin 0.2
Indium STD
Cadmium
Silver 0.5
Palladium
Rhodium
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC. ELEMENT
Vanadium
0.05 Titanium
Scandium
0.01 Calcium
Potassium
0.006 Chlorine
Sulfur
0.02 Phosphorus
Silicon
Aluminum
Magnesium
Sodium
0.07 Fluorine
0.07 oxygen
0-03 Nitrogen
0.004 carbon
0.09 Boron
0.006 Beryllium
0.006 uthlum
Hydrogen
CONC
0.002
0.01
0.001
6
2
0.3
0.2
3
0.4
0.3
0.08
* 6
=0.1
NR
NR
NR
0.001
0.002
NR
™" ^ ww Reported
AH elements not detected^
•wt ••• Me|or Component ',
INT — InlerfeieiiLe
0.004
g/ml
i
Approved:
63
-------
••ply*
COMMERCIAL TESTING & ENGINEERING CO.
MNMAL OFFICO: tit NOKTM LA IAU.C »T»MT. CHICAGO. ILLINOIS MUi • AMA CODC HI TM-MM
MSTMMINTAl ANAiniS MVWON, 1AM WOT MIM AVINUf. GOIHN. COtOIAOO «M01. MtONt; >QH>MSi1
To: Mr. Brent Higginbotham ^Bk
Acurex Corporation ^*5flS^^
485 Clyde Ave.
Mountain View. CA 94042
P. 0. NO.:
$wnpi« NO.: A-45 Blank SPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
D*** September 25. 1980
Ai»ly«f: T. BOUtS
IAD NO.: 97-E697-116-28
^n.Tc 10 vg/cm2
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
Approved; /
>
CONC.
0.005
0.01
0.05
0.003
0.04
<0.001
0.03
0.005
0.001
0.008
0.01
0.01
0.01
< 0.001
0.2
0.003
0.02
st // /
VI 9
i
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
'#/
'JUg^&fl
CONC
0.02
0.9
0.002
MC
0.5
0.4
0.8
0.07
MC
MC
1
>2
•0.005
NR
NR
NR
0.9
<0.001
NR
tf
-~fa
69
-------
COMMERCIAL TESTING & ENGINEERING CO.
•cmMI orrict»: in HORTH i* *ALLI trncT, CNICABO, lumoi* M«OI • *«• eoei nt ?t»-««M
••ply tO MKIUWN1M MttlVSU WVMION. 1AM WIST *OH AVtNUI. OOUXN. COtOMOO tO»l. MOM: JBM7MSJI
To: Mr. Brent Hlgglnbotham
Acurex Corporation
485 Clyde Ave.
Mountain View. CA 94042
V*llL.
mmrnmm.
P. O. No.:
StmpteNo.: 80-1312
TAP DATcf, i
SPARK SOURCE MASS SPEaROGRAPHIC ANALYSIS
CONCENTRATION IN jig/ml
September 24, 1980
An«iy*t:L. Jacobs
IAD No.: 97-E697-116-28
ELEMENT
Uranium
Thorium
Bismuth
Lead
Thallium
Mercury
Gold
Platinum
Iridium
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetian
Ytterbium
Thulium
Erbium
Holmlum
Dysprosium
STD - Mm
NK-Noth
INT «« hitarfi
CONC. ELEMENT CONC.
Terbium
Gadolinium
Europium
0.01 Samarium
Neodymium
Praseodymium
Cerium
Lanthanum
Barium 0.02
Cesium
Iodine
Tellurium
Antimony
Tin
Indium STD
Cadmium
Silver
Palladium
Rhodium
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
21 nc
Copper
Nickel
CONC.
0.1
0.005
0.02
0.02
10.002
0.1
0.004
0.009
0.04
0.002
0.003
Cobalt *<0.002
Iron
Manganese
Chromium
0.2
0.006
0.07
«l Snnctant * Meter°9en«>US
^^>io°d/SW Appw-: ''1M ^
tranc*
r
l( (/A
ELEMENT
Vanadium
Titanium
Scandi urn
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
//
ft^e-i
CONC.
<0.001
0.1
10
0.05
MC
0.4
0.9
0.2
0.004
MC
0.2
"0.004
NR
NR
NR
<0.001
INT
NR
&
70
-------
••ply to
COMMERCIAL TESTING & ENGINEERING CO.
•MCtAL OmCM: ttt W*TN IACAUI »TIKT, CHICAGO. IU.IMOII IMOi • AM* CODE ttt »•••«•«
•muMMTAi ANMTSn MVWON. MM WOT 4«IH AVWUf. SOIMN. COlOtAOO M«l. tHOHt
To: Mr. Brent Hlgglnbotham
Acurex Corporation
485 Clyde Ave.
Mountain View. CA 94042
9. O. No.:
SwnphNo.: 80-1313
WftTcft T
SPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
CONCENTRATION IN
: September 24. 1980
A««iytt:T. gouts
IAD No.: 97-E697-116-28
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead 0.03
Thallium
Mercury NR
Gold
Platinum
IHdium
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetlum
Ytterbium
Thulium
Erbium
Holmlum
Oysproslum
STD - hitaffwl $».nd»rd
* * NR ^ Not Rcpofftd
All •!• • •nil m*ut j^t^*^^
All •••menu nor avi^cwn
MC- M»jor Compoowrt
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymium
Praseodymium
Cerium
Lanthanum
Barium 0.2
Cesium
Iodine °-01
Tellurium
Antimony
Tin
Indium STD
Cadmium
Silver
Palladium
Rhodium
* Heterogeneous
i<0.003 v9/ml
> 10 vg/ml
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Z1nc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
Approved: /
CONC.
*0.1
0.02
0.03
0.006
0.3
0.002
<0.007
*2
4
0.04
0.005
0.3
0.01
0.008
WL
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
/A~
CONC
0.003
0.1
<0.001
10
2
8
2
0.4
2
0.2
0.5
2
•0.1
NR
NR
NR
0.002
0.004
NR
-A
•UT
mi ^
71
-------
COMMERCIAL TESTING & ENGINEERING CO.
•iNt»i ernctt: tt* HO»TM u «»u.« §T«HT, CHICACO. IU.IMOI* MMI • »•§• coot >tt >*«.M*«
R>ply 10 MHMMNTM. AHAtTSII WVIilON. 14BS WOT 44TH AVINUI. 6OIMN. CMOMOO Ml. »MOtW M-XTMBI
T<* Mr. Brent H1gg1nbotham
Acurex Corporation
485 Clyde Ave.
Mountain View, CA 94042
f. O. NO.:
Swnpte No.: 80-1368
SPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
o.t«: September 24, 1980
An«ly»t T. BOUtS
IAD No,: 97-E697-116-28
-—. — _,_ bunwENiKAimn IN Ma/1"1
ELEMENT
Uranium
Thorium
Bismuth
Lead
Thallium
Mercury
Gold
Platinum
Irldlum
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetian
Ytterbium
Thulium
Erbium
Holmium
Dysprosium
STD- Men
m«j— •*.
•"* NO? mt
All^tonwnta
MT-kMrft
CONC. ELEMENT CONC.
Terbium
Gadolinium
Europium
0.1 Samarium
Neodymium
NR Praseodymium
Cerium 0.008
Lanthanum 0.01
Barium 0.07
Cesium
Iodine
0.02 Tellurium 0.02
Antimony
Tin
Indium STD
Cadmium
Silver 0.03
Palladium
Rhodium
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Z1nc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC.
*0.4
0.05
0.05
0.05
<0.001
0.4
0.1
INT
0.002
0.004
MC
MC
1
0.02
MC
0.2
0.7
* Heterogeneous
wl Standard
•W **cMd< 0.002 ng/ml
JJJ2"""1* > 10 ug/ml
Approved: -'y
?n
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine *
Oxygen
Nitrogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
/ /
-------
COMMERCIAL TESTING & ENGINEERING CO.
•ENMAL OFFICE*: »• NORTH LA «AklE (TVEET, CHICAGO, ILLINOIS «M01 • AKA CODE Itl Tl«-«4»4
Reply to msnuMiNTAi ANAITSU DIVISIOM. MBS WEST MTH AVINUE. GOUKN. COIMAOO «M». FHONE: aa-i7**si
To: Mr. Brent H1gg1nbotham
Acurex Corporation
485 Clyde Avenue
Mountain View, California 94042
o*te: September 26. 1980
An«h/*T: T. BOUtS
9. O. NO.:
Ssmpte No.: 1471
HArj >t«.r
~^f \\-~f_tL
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead 0.2
Thallium
Mercury NR
Gold
Platinum
Indium
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Hoi ml urn
SPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
CONCENTRATION IN v9/cm2
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymium 0.005
Praseodymium 0.003
Cerium 0.03
Lanthanum 0.04
Barium * 0.9
Cesium
Iodine
Tellurium
Antimony
Tin 0-02
Indium STD
Cadmium
Silver °-002
Palladium
Rhodium
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC.
0.05
0.005
0.006
0.008
0.08
<0.001
0.04
<0.001
<0.006
0.009
0.6
0.3
0.04
0.02
0.7
0.02
0.1
IAD No.: 97-E697-116-28
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Ni trogen
Carbon
Boron
Beryllium
Lithium
U \fft *tAM API
CONC
0.2
1
<0.001
MC
0.5
0.3
8
0.5
MC
MC
4
MC
=1
NR
NR
NR
MC
0.02
NR
(TO - Intonwl Standard
NR - Not Iteportod 2
All •tenwnti not dMMMd< 0.001 UO/Cm
NIC - Mijor Component >10ug/Cffl
IUT t
mi ^
* Heterogeneous
73
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
•tNCML orriet t: it* NORTH t» «*ut •TMIT, CMIC»«O, ILLINOIS Mtoi • »«» CODE tti ?f t-»4i«
msnuMiNTM MUITSIS DIVISION. i«tt WIST MTM AVINUE. GOiofN. COIMADO «woi. mowf, JOJ-ITMSJI
To: Mr. Brent HIgglnbotham
Acurex Corporation
485 Clyde Avenue
Mountain View, California 94042
Septenter 26, 1980
Arwlyit:
f. O. No.:
Simple No.:
ELEMENT
Uranium
Thorium
Bismuth
Lead
Thallium
Mercury
Gold
Platinum
Irldlum
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutefium
Ytterbium
Thulium
Erbium
Holmlum
Dysprosium
sm _ *— *
1476 SPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
¥ OKC, CONCENTRATION IN vg/ml
CONC. ELEMENT CONC.
Terbium
Gadolinium
Europium
0.04 Samarium
Neodymlum
NR Praseodymium
Cerium <0.001
Lanthanum
Barium 0.05
Cesium <0.001
Iodine
Tellurium 0.01
<0.002 Antimony
Tin 0.05
Indium STD
Cadmium
Silver 0.03
Palladium
Rhodium
t e*«^___i
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC.
0.008
0.002
0.002
<0.001
0.004
0.006
0.002
0.2
0.2
0.2
0.004
0.4
0.04
0.08
IAD NO.: 97-E697-116-28
ELEMENT
Vanadium
Titanium
Scandi urn
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
CONC.
0.001
0.3
<0.001
1
3
0.04
MC
3
1
>0.7
0.6
MC
=0.3
NR
NR
NR
0.003
0.005
NR
NR - No* *(porl*d
AII •iwMnh not d>i»cf*d< 0.001 vg/ml
MC - Mijer Component >1Q
INT — bntrftrcna
Approved:
74
-------
COMMERCIAL TESTING & ENGINEERING CO.
•EMIIAL OPFICM: t»i NOVTH Uk IAU.C tTKET, CMICAOO, ILLINOIS «0«OI • »«» CODC 111 T»l-*4»4
••ply tO INtnuMNTM. AMAiniS DIVISION. I«UJ «WST «TH AVfNlrt. GOIDCN. COlOtADO KM01. PHONE: 10.17*4531
To: Mr. Brent Higginbotham
Acurex Corporation
485 Clyde Avenue
Mountain View, California 94042
P. O. NO.:
September 26, 1980
*nily*t:
Simple No.^ i35g spARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
TA *~-'-' T* ' - A— -- - CONCENTRATION IN vg/ml
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead 0.09
Thallium
Mercury NR
Gold
Platinum
Indium
Osmium
Rhenium
Tungsten
Tantalum <0.04
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Hqlmium
Dysprosium
$TO - IM*m«l Standard
NR - Not Reported
All wwwnti not otTtctvo ^
MC - Mijor Component •
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samari um
Neodymi um
Praseodymium
Cerium
Lanthanum
Barium 0.02
Cesium
Iodine 0.004
Tellurium 0.006
Antimony 0.02
Tin <0.006
Indium STD
Cadmium 0.006
Silver
Palladium
Rhodium
0.002 pg/ml
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC.
0.2
0.03
<0.2
0.009
0.1
0.1
<0.009
0.002
MC
MC
* 0.7
* 0.07
7
0.1
0.5
IAD No.: gy.
ELEMENT
Vanadium
Titanium
Scandi um
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodi um
Fluorine
Oxygen
Ni trogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
•E697-116-28
CONC
0.002
0.2
0.01
MC
3
* MC
MC
0.8
6
0.7
2
2
«0.2
NR
NR
NR
* 0.03
0.003
NR
f Heterogeneous /?*.
t
w, c
f if -^«-
^ "
75
-------
••ply le
COMMERCIAL TESTING & ENGINEERING CO.
•fNt*AL OrriCtt: tte MOtTH LA MU.C tTKCT, CHICAGO. lUIHOIt eoeot • AMA COOi MI fl»-»4»4
, ANAinu WVIIION. uns urtji *m» ATOM. oou*H coiotAoo *MOI. moNi: jowrwai
T«* Mr. Brent H1ggi
Acurex Corporat
485 Clyde Ave.
Mountain View.
P. O. No.:
Simple NO.: 80-1473
yA£> ~ MrtrJ TEST
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead 0.3
Thallium
Mercury NR
Gold
Platinum
Iridlum
Osmium
Rhenium
Tungsten
Tantalum 0.7
Hafnium
Lutetian
Ytterbium
Thulium
Erbium
Holmlum
Dysprosium
fTO - Internal Standard
N« - Not Reported
All •tonwnti not delected <
MC - Meter Comment
nbotham J
1on ^"
CA 94042
••ARK SOURCE MASS W
ft
•ECTBOOHAPrtC ANALYSIS
D.I. September 23, 1980
*n«iy»t: T. Bouts
IAD No, 97-E697-116-28
CONCENTRATION IN PPM WEIGHT
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymium
Praseodymium
Cerium 0.3
Lanthanum 0.4
Barium 0.9
Cesium
Iodine
Tellurium
Antimony
Tin
Indium STO
Cadmium
Silver
Palladium
Rhodium
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC.
1
<0.1
0.2
0.8
<0.1
<0.1
*<0.1
6
6
180
0.7
26
1
11
* Heterogeneous
* t
0.1 ppm
/}/
ri|f|iiuv*Mj. u A
I/I ( /
'/, 1 / A
ELEMENT
Vanadium
Titanium
Scandi urn
Calcium
Potassium _
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryllium
Lithium
Uyd/bgen
y/
r* ^
L£~^**-*
CONC
<0.1
*2
*<0.1
62
22
1
14
15
*500
5
8
22
**0.3
NR
NR
NR
<0.1
<0.1
NR
s?
&i
7A.
BAIT —
•«* ^
76
-------
toply to
COMMERCIAL TESTING & ENGINEERING CO.
•ENttAL OFFICES: SSS NO«TM LASALLE STKET, CHICAGO. ILLINOIS SMOI • AIEA CODE S1I TM-MI4
WSnUMCNTM ANAirSH DIVISION. MM WIST «TM AVEMUt. COiBCN. COIOIADO SMOt. PHONE* M*-«S«S9I
To: Mr. Brent H1gg1
Acurex Corporal
485 Clyde Ave.
Mountain View,
f. O. No.:
tempi* No.: 80-1562
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead 0.2
Thallium
Mercury
Gold
Platinum
Irldium
Osmium
Rhenium
Tungsten
Tan tali urn
Hafnium
Lutetian
Ytterbium
Thulium
Erbium
Hplmlum
Dysprosium
iTD - M*rn*l StwidMd
NR *•> Not teportvd
All •!••!• Ill* BkM* JaW^M^M^
mi vwvnvnT* n*7* OTI^O^V
MC - Major Component
nbotham ^i
:1on "•.
CA 94042
__ SPARK SOURCE MASS SPEC1
|T '»' CONCENTRATION 1
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymium
Praseodymium
Cerium
Lanthanum
Barium 0.3
Cesium
Iodine
Tellurium 0.2
Antimony
Tin 0.03
Indium STD
Cadmium 0-02
Silver
Palladium
Rhodium
* Heterogeneous
i< 0.01 ppm
> 100 ppm
k
1C*
TROGAAPMtC AMALYI
IN PPM WEIGHT
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
Approved: / >
/
77
MS
CONC
0.1
0.04
0.02
<0.01
0.3
0.02
0.02
0.7
0.5
1
0.04
10
0.2
0.4
VY //
°*e: September 22, 1980
Analyst: L. Jacobs
IAD No.: 97-E697-116-28
ELEMENT
Vanadium
Titanium
Scandium
Cal ci urn
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
CONC
0.02
5
<0.01
MC
7
*70
0.2
1
25
MC
2
4
=1
NR
NR
NR
0.04
0.02
NR
-------
00
T^ Corporation
ANALYSIS LABORATORIES
DATA REPORTING FORM
CUSTOMER
CMEA
nATF July 10. 1980
CUS
RES
AC
TOMER COI
ULTS REPOI
lORPSS ....
MTRACT NO
UTTO
307605.22
ACUREX C(
TELEPHONE
)NTRACT N
n
Mountain View May 21, 1980 MfliO TU.«J/»tC \TWrEM
SAMPLE ID (CUSTOMER)
SAMPLE ID (LAB)
PARAMETER
Blank
Sample Aliquot
Total Sample
Total Sample
Total Sample
Fuel Hg
-!W--.-.-.mrr::--:r:.--i-----!:--?--
FMF_ — -i— ~
Less 1
Less 1
Less 0.1
XAD Hg
Less 1
Less 1
Less 0.1
Less 10
1 Imp Hg
Less 1
Less 1
Less 1
Less 1
2&3 Hg
Less 1
Less 1
Less 1
Less 1
2&3 As
' i " . L • " _ •
;:;T:;-V— .«:: • —_-.._ —
Less 10
Less 10
Less 10
Less 10
2&3 Sb
..'- -_^_^'_ • '• '
!-r'-g-L-;v"E" '
...^-M-.— .... - -.
Less 3
Less 3
Less 3
Less 3
*A11 water tank samples were below 1 ug/L
form EED-06T 4/60
Water Hg
=5==ss=EE±=E==
Less 1*
Less 1
Less 1
^=
3
f^^^^^^mmummfmtmnflff
UNITS
ug/L
ug/L
ug/L
ug/g
ug
ANAIVST (r- fa* 4 (/
RFMIFWPR ^£*U& ~>t*^€f
-------
COMMERCIAL TESTING & ENGINEERING CO.
OtMtlUl OfHCtS m MOXTM U JAUt ITMtT, CM.CAaO. ILUHOU M«ei . ME* COOlT " ^
Reply to
Instrume
490 Orch
Golden. CO 80401
Imtrumentil An»Jytis Division m^. ao3.27B „-„
490 Orchard Street ^)ne 303-278-9521
November 18. 1980
Mr. Brent Higginbotham
Acurex Aerotherm Corp.
485 Clyde Avenue
Mountain View, CA 94042
RE: IAD #97-E697-116-28
Analytical Report
We were asked as per phone conversation with Carlo Castaldini, on
October 20, 1980, to check by atomic absorption, several elemental
values on samples that were analyzed by spark source mass spectrometry
(SSMS) and reported September 24, 1980.
Nickel was checked on the XAD Resins Acurex ID Nos. 80-1473 & 1533-37.
Nickel contamination was due to combustion in the Parr combustion
apparatus as they were prepared for SSMS. The samples were reprepped
by ashing at approximately 600° C, dissolving the ash in aqua regia,
and diluting with deionized water. Nickel was determined from this
solution as well by flame atomic absorption.
Copper was found to be a major component on samples 80-1368 and 80-1369
by SSMS. This was quantitated by flame atomic absorption on a diluted
portion of each of the samples.
The results of these analyses are presented in Table No. 1 and are re-
ported in the appropriate units on the samples "as received".
I. OH . Olimo. CB > OOLMN. CO • Mm«ON. IT . JU>». >t •
. MUVN.ll.«» 'MUM. UT •». NOIUU*. IL.TOtteO.O". MHOMim. l£. CM.
79
-------
Table No. 1
Nickel
Sample » lug/9)
80-1473 fW« **> 2.4
80-1368 To~*fc»*»* Prior 4* — 505
80-1369 Tcut-oA^af^-U^- — 480
If there are any questions concerning these results, please call
Bruce A. Hale M. L. Jacaps. Ph.D., l»Ingr.
Section Supervisor Instrumental Analysis Div.
as
COMMERCIAL TMTINO ft •NaiNEIKINO CO.
Onginil Copy Wittrmtrlod
for You' Prottclion
Hit
80
-------
1C ANALYSIS SHEET
Acurex F.Cl.Br.NOg.NQ-,
Contractor _^
SimphSH. Mountain View Simple Acquisition Oite ** 21* 198°
Typi of Source
TtstNumbtr - , — _ _ Simple ID Number _
. , „ ... Water Tank
Simple Description . - , _ _ _
. , „ ... E. Hagmann „ _ July 21, 1980 8pm - 2am
Anilyst Responsible - T - Dite Analyzed _ Time _
G. Nicoll July 23, 1980
Calculations and Report Reviewed By . Report Dite
Micromeritics - Wescan Conductivity
Instrument
PI....* 0.0025M Sodium Benzoate @ pH 6.0
1.50 ml/mi n _.„ 1.08 KPSIp.M^g... *>* cm/min
Simple Size IUW "' Attenuator Setting
X
Original Simple Volume or Mass Multiple Stindird Addition: Yes No
Observations
30 min run, good separation
81
-------
1C ANALYSIS SHEET
so3,so4
Contactor Acurex
Sample s.a Mountain View sample Ambition Oat. ^ 21, 1980
Typt of Source -
T«$t Number Sample ID Number
Water Tank
Sample Description
Analyst Responsible E' H«gnann D.t.An.ly»d J"^ "' 198° Tin,.
Micromeritics - Wescan Conductivity
C.,cu«,tion,.ndR,portR8view«.By G' N1C011 Report D.t.
Instrument.
0.0047M KHP @ pH 4.5
E luent 1
2.00 ml/min 0.79 KPSI 1 cm/min
Column Flow Rate Pressure Recorder Speed
10 Ul 4
Sample Size __ __ Attenuator Setting
Original Sample Volume or Mass „ Multiple Standard Addition: Yes No
Observations Spike and recovery: average recovery at 88%
82
-------
1C ANALYSIS SHEET
PO,
Contactor Acurex
* ,.«.. Mountain View . lm ... „ May 21, 1980
SainpltSitt _ Sample Acquisition Date J
Type «f Source
Test Number Simple ID Number
. Water Tank
Sample Description , -
E. Hagmann July 22, 1980 8am - 3pm
Analyst Responsible _ Dite Analyzed Time
Calculations and Report Reviewed By G. NJCOll _ Report Date July 23, 1980
instrument Micromeri tics - Wescan Conductivity _
x 10"3M Phthalic Acid
2.00ml/min 1-43 KPSI _ ,^,^ 0.5 cm/mi n
Sample Size u' _ Attenuator Setting
Original Sample Volume or Mass , Multiple Standard Addition: Yes No
Spike and recovery: 106% recovery
Observations r ±
-------
Water Tank Blank- Tap Water
Ion
F ~
d~
Br~
NOJ
NOJ
so3=
504
POf
Uncorrected
Simpli Value
ND
2
ND
ND
ND
ND
ND
ND
Blink
Value
ND
ND
ND
ND
ND
ND
ND
ND
Corrected
Sample Value
2
High/Low Calibration
Standards or Con-
ctntration Addad
5, 50
5, 50
5, 50
5, 50
5, 50
50
50, 500, 1000
10, 50
Dilution
Factor
Astignad
Concantration*
ND
2
\ ND
ND
ND
ND
ND
ND
Detection
Limit*
1
1
5
2
5
10
10
3
'Results: Hlg/L values (in original sample or I - Interference; MC - major constituent, not quantified; NC - not computed; NG - sample value below
blank; ND - not detectable (<2 a blank or baseline).
-------
Water Tank- Sample Blank
oo
Ol
ton
F~
a"
Br~
M<£
N0§"
so3=
soj
»•
tin ••«••• ait
URCvrracmi
Simplt Value
ND
2
ND
ND
ND
ND
ND
ND
Blank
Vilut
ND
ND
ND
ND
ND
ND
ND
ND
Corrected
Simplt Vilui
2
High/Low Calibration
Standards or Con-
cantration Added
5, 50
5, 50
5, 50
5, 50
5, 50
50
50, 500, 1000
10, 50
Dilution
Factor
Alined
Concentration*
ND
2
ND
ND
ND
ND
ND
ND
Detection
Limit"
1
1
5
2
5
10
10
3
"Results: mj/L values (in original sample or I - Interference; MC - major constituent, not quantified; NC - not computed; N6 - sample value below
blank; NO - not detectable (<2 a blank or baseline).
-------
Water Tank - Prior to Test
CO
en
Ion
F ~
d~
Br~
NOf
NOT,
sof
$04
POf
Itacomcted
Sample Value
ND
3
ND
ND
7
ND
990
ND
Blank
Value
ND
ND
ND
ND
ND
ND
ND
ND
Corrected
Sample Vilut
3
7
990
High/Low Calibration
Standard! or Con-
contrition Addad
5, 50
5, 50
5, 50
5, 50 .
5, 50
50
50, 500, 1000
10, 50
Dilution
Factor
Astignad
Concentration*
ND
3
'. ND
ND
7
ND
990
ND
Detection
Limit"
3
2
10
10
5
10
10
20
•Rtjulti: mj/L values (in original sample or I - Interference; MC - major constituent, not quantified; NC - not computed; N6 - sample value below
blank; ND - not detectable (<2 a blank or baseline).
-------
Water Tank - Sample
00
Ion
F~
a~
Br~
NOJ
N0£
so3=
804
POf
Uacornctid
Simph Value
ND
3
ND
ND
7
ND
1000
ND
Blank
Valin
ND
ND
ND
ND
ND
ND
ND
ND
Comcttd
Simple Vilui
3
7
\ooo ^
High/Low Calibrttion
Standards or Con-
centration Added
5, 50
5, 50
5, 50
5, 50
5, 50
50
50, 500, 1000
10, 50
Dilution
Factor
aaai-aaii
fuufMB
Concentration*
ND
ND
ND
ND
7
ND
1000
ND
Detection
Limit*
3
2
10
10
5
10
10
20
"Results: rtB/L values (in original sample or I - Interference; MC - major constituent, not quantified; NC - not computed; NG - sample value below
blank; NO - not detectable (<2 a blank or baseline).
-------
5.5 ORGANIC ANALYSIS REPORT
88
-------
ORGANIC COMPOUNDS (bp <10(r C)
rl^tAVtf v
Ito rfOu>»Tt«i'« l/iViJ
fy.wi J
M«W*B Hri RtHK Ktrim^ IT
&
Tim
Workup
1 MSohtaV)
1.0
I EhanMOrlMlAtaVl
7.
&
It
U.
K
1 bikmiMDm
1
Ai
11.
mi irin (a wipnl atnph) »t I -
•D - MI <«tKHMi «le M«k ir
iuafamm; HC - MI c*m»«4: NG - ami* rtut Mm Mmk;
Ttaw
•( Nik
IK)
CMC.
K)0
0.7
0.087
Uo
OM
wo
11 fc.,-
MO
Ll_^_
K!D
U
•00
VJC
89
-------
ORGANIC COMPOUNDS (bp Mnnil.M(aiUi.M
It O
& EbcMaMBtalA/aV)
7. kKphttntaU
4. CahMPmajnbnil-
L MtnliaaDaM —
FkuMFInRanitail^aW: Hj-
1 OHM TtMHiiUn fO
Air
11
11
11.
>•» t2.
/ /*,O9D -^
mi nte (» w<|iMl MiipM if I - tturttima: MC
•0 - Ml JmcttMi « J 0 Wnk ir Mint).
- MI unf nN: KG - Miplt urin Mm Mnik;
ThM
no
M|l/Laitealikntin
AID
0.7 HM
0.067
UO
Ml)
LO
an
2-l.pp,
U *,.;.
K)J)
0.17
H.X
m«
.V/J)
C.28
l.i
WD
Aid
o.n
b
MO
III
U6
^-
90
-------
ORGANIC COMPOUNDS (bp <100* C)
TypirflMfw
tatlkmtw
AV/g
o
-------
ACUREX.
Corporation
ANALYSIS LABORATORIES
CUSTOMER
CNEA
DATA REPORTING FORM
DATE July 15. 1980
CUSTOMER CONTRACT NO. 307605.22
RESULTS REPORT TO
ADDRESS
ACUREX CONTRACT NO.
TELEPHONE
Mountain View May 21. 1980
SAMPLE 10 (CUSTOMER)
SAMPLE ID (LAB)
TCO Blank
TCO Sample
TCO, Corrected
GRAV Blank
GRAV Sample
GRAV, Corrected
TCO & GRAV
TCO & GRAV
Volume Analyzed
Filter
Less 2
3
3
3
0.1
XAD
0.07
74
74
4
22
18
92
3.2
OMC
Less 0.02
Less 0.02
Less 0.02
Less 2
5
5
5
0.2
Tank
Blank
Less 0.02
Less 0.02
Less 0.02
Less 2
6
6
6
0.2
3665
Tap
Water
Less 0.02
Less 0.02
Less 0.02
Less 2
Less 2
Less 2
Less 2
Less 0.1
3720
Prior to
Test
— r~ r — • -a— rr:
Less 0.02
Less 0.02
Less 0.02
Less 2
Less 2
Less 2
Less 2
Less 0.1
1300
Tank
Sample
Less 0.02
0.50
0.50
Less 2
Less 2
Less 2
Less 2
Less 0.1
10,000
. — ...i-i. . TTTT •-;;
mg
mg
mg
mg
mg
mg
mg
mg/dscm
ml
vo
FormEEO-097 t to
ANALYST .
REVIEWER
M. Gardner, G. Nlcoll
-------
Ill REPORT
Filter Mountain View 5/21/80
Ho Peaks
93
-------
Ill ftC'OUT
Filter Blank
We* R«>tat
hmwiiy
No Peaks
94
-------
IM MPOftT
SAMPLE:
XU> Mountain View 5/21/80
!W
3575-3070
2905
2845
2750-2450
1725
1445.
1265
1095
805
tafMitr
w
s
s
M
M
w
w
N
W
Anipment ComnMBti
O-H
C-B Alkane
C-H Alkane
O-H
OO
C-H Alkane
C-0 .
C-0
Not Assigned
•
•• •
95
-------
IAMPLI: XAD BlanX
totraity
No Peaks
96
-------
in REPORT
OMC Mountain View 5/21/80
tamwity
Mo Peaks
97
-------
1AMELI: Tap Water Mountain Vi«* 5/21/80
No Peaks
CemmMi*
98
-------
Ill REPORT
Tank Blank Mountain View 4/21/80
«-•'»
brevity
Mo Peaks
99
-------
Tank Sample Mountain Vi«* 5/2X/80
!~j*r
2905
2845
1190
.
•_*,
S
S
S
•
M&*^tfMMt BWWWfli
C-B Alkane
C-B Alkane
Sulfonamide Found in GC/MS PHA
.
•• •
•
•
•
.
100
-------
COMPOUNDS SOUGHT
Detection Limit in nanograms
8
1
2
8
40
1
1
8
4
40
3
3
1
2
2
2
5
7
8
8
5
1
1
40
2
1
40
* 4-bromophenyl phenyl ether
* bis (2-chloroisopropyl) ether
* bis (2-chloroethoxy) methane
* hexachlorobutadiene
* hexachlorocyclopentadiene
* isophorone
* naphthalene
* nitrobenzene
* N-nitrosodiphenylamine
* N-nitrosodi-n-propylaroine
* bis (2-ethylhexyl) phthalate
* butyl benzyl phthalate
* di-n-bujyl phthalate
* di-n-octyl phthalate
* diethyl phthalate
* dimethyl phthalate
* benzo(a)anthracene
* benzo(a)pyrene
* 3,4-benzofluoranthene
* benzo(k)fluoranthene
* chrysene
* acenaphthylene
* anthracene
* benzo(ghi)perylene
* fluorene
* phenanthrene
* dibenzo(a,h)anthracene
40
2
20
2
100
8
8
8
3
2
4
8
4
20
10
10
1
2
4
40
40
9
@
40
40
40
40
* 1ndeno(1,2,3-cd)pyrene
* pyrene
2,3,7,8-tetrachlorodibenzo-p-dioxin
* acenaphthene
* benzidine
* 1,2,4-trichlorobenzene
* hexachlorobenzene
* hexachloroethane
* bis(2-chloroethyl)ether
* 2-chlpronaphthalene
* 1,2-dichlorobenzene
* 1,3-dichlorobenzene
* 1,4-dichlorobenzene
* 3,3-dichlorobenzindine
* 2,4-dinitrotoluene
* 2,6-dinitrotoluene
* 1,2-diphenylhydrazine (as azobenzene)
* fluoranthene
* 4-chlorophenyl phenyl ether
anthanthrene
benzo(e)pyrene
dibenzo(a.H)pyrene
dibenzo(a,i)pyrene
dibenzo(c,g}carbozole
7,12 dimethyl benz(a)anthracene
3-methyl cholanthrene
perylene
*Authenic standard ran
Wtolecular weight too high for direct
40 Benzo(c)phenanthrene
analysis by Base/Neutral run
101
-------
^Corporation
ANALYSIS LABORATORIES
CMEA
CUSTOMER
CUSTOMER CONTRACT NO. 307605.22
RESULTS REPORT TO
DATA REPORTING FORM
July 21. 1980
DATE
ACUREX CONTRACT NO.
TELEPHONE
ADDRESS
Mountain View 5/21/80
28.5 dscm
1 SAMPLE ID (CUSTOMER)
SAMPLE 10 (LAB)
PARAMETER
Amount Infected
Naphthalene*
Phenanthrene/
Anthracene*
Naphthalene
Phenanthrene/
Anthracene
Volume analyzed
Filter Bk
— — -7T- •
"'"'L,.1™1.' ."'._•::.::
^^
2
Less 0.5
Less 0.5
Filter
2
Less 0.5
Less 0.5
Less 0.02
Less 0.02
XAD Blank
••sr7sr:B-,;^T-!BT-.-;.'.-r-r:
2
Less 0.5
Less 0.5
Xad
-'-"'-':: '--:'•• •"•-":"'' ' WS""-jr ;
1
94
4
36
2
OMC
2
Less 0.5
Less 0.5
Less 0.0;
Less 0.0;
9
Mater Blk
- -'--„-- v- i 'vi j -a .""— " •
3
Less 0.3
Less 0.3
Tank watei
.1
9
2
0.4
0.08
10,000
• '"• '^i~" i .. ' . ....
L _g
UNITS
II •l-IIM
uL
mg/L
mg/L
ug/dscm
ug/dscm
mL
o
ro
*Sample Aliquot 0A11 water tank blanks are clean
FwmEED-057 4/M
ANALYST _
REVIEWER .
-------
SAMPLE: Man XAD 11473
o
CO
Mta*'
T*MtorLC*
i
TCO
•1
74
36
26
BMV
"•1
18
9
14
TCO+GIIAV
Tcttlnf
92
45
40
mg/dscm
3.2
1.6
1.4
wVHRMw
1 .
i
i
4
I
•
f
SMI
TtfthMj
FfKtMft
25
0.09
0.41
0.06
<0.01
<0.01
<0.01
26
MM*
<0.02
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.08
tor-
fMAM
25
0.09
0.41
0.06
cO.Ol
eO. 01
cO.01
26
W
52
0.2
' 0.85
0.1
<0.02
<0.02
-------
II
I
1 REPORT
AMPLE: '
WvM MMMF
'«-•')
2920
2840
'
.
.
.-
. *
IAN XAD 1473 F1
knmftr
*
s
s
.
, •
t
... . _
•
"
.•
• Mf^_^_u^ ^^MMIkMlM
jWB^ViiiMii* w^^nnwniB
CH A3 iphatic hydrocarbons
CH Aliphatic hydrocarbons
•
-
r
•
.-
•• •
-"
.. ._ * - ._
.
•
•
104
-------
•ft REPORT
MM XAD 1473 F2
No Peaks
105
-------
Ill KPQftT
Mo Peak*
106
-------
. HAN XAD 1473 F4
No Peaks
107
-------
MftEPOKT
•AMPU;
HAN XAD 1473 F5
tto Peaks
108
-------
IftKEPONT
. MAN XAD 1473 F6
o Peaks
109
-------
tmttro*T
SAMPLE:
WN XAD 1473 F7
No Peaks
no
-------
LftMS REPORT
SAMPLE:
1473 Fl Mountain View XAD
tenmifv
1
e»iw»
Aliphatic hydrocarbons
.
.
MWR.V
Sub-Ciwgofiw. Specific Campoundt
Ifitsfwty
Ctngery
m/i ComDotition
Oth«r
111
-------
5.6 BIOASSAY ANALYSES REPORT
112
-------
GENETICS ASSAY NO. 5397
WiMMMMBHB
LBI SAFETY NO. 6391
CYTOTOXIC EVALUATION OF
SAMPLE #1473
(CMEA MOUNTAIN VIEW)
IN THE
RODENT CELL (CHO)
CLONAL TOXICITY ASSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20795
LBI PROJECT NO. 20933
REPORT DATE: DECEMBER, 1980
113
-------
PREFACE
This report contains a summary of the data compiled during the evaluation
of the test compound. The report is organized to present the results in
a concise and easily interpretable manner. The first part contains items
I - IX. Items I - IV provide sponsor and compound identification infor-
mation, type of assay, and the assay design reference number. All assay
design references indicate a standard procedure described in the Litton
Bionetics, Inc. "Screening Program for the Identification of Potential
Mutagens and Carcinogens." Item V provides the initiation and completion
dates for the study, and Item VI provides identification of supervisory
personnel. Item VII identifies the tables and figures containing the data
used by the study director in interpreting the test results. The interpre-
tation itself is in Item VIII. Item IX provides the conclusion and evalua-
tion.
The second part of the report, entitled Assay Design, describes the
materials and procedures employed in conducting the assay. This part
of the report also contains any appendices, as well as evaluation
criteria used by the study director. The evaluation criteria are
included to acquaint the sponsor with the methods used to develop and
analyze the test results.
All test and control results presented in this report are supported by
fully documented raw data which are permanently maintained in the files
of the Department of Genetics and Cell Biology or in the archives of
Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington, Maryland, 20795.
Copies of raw data will be supplied to the sponsor upon request.
114
-------
I. SPONSOR: Acurex Corporation
II. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5397
A. Identification: Sample #1473 (CMEA Mountain View)
B. Date Received: October 9, 1980
C. Physical Description: Brown, opaque liquid
III. TYPE OF ASSAY: Rodent Cell (CHO) Clonal Toxicity Assay
IV. ASSAY DESIGN NUMBER: 442
V. STUDY DATES:
A. Initiation: October 20, 1980
B. Completion: November 24, 1980
VI. SUPERVISORY PERSONNEL:
A. Study Director: Brian C. Myhr, Ph.D.
B. Laboratory Supervisor: Robert Young, M.S.
VII. RESULTS:
The data are presented in Table 1 on page 4 and in Figure 1
on page 5.
VIII. INTERPRETATION OF RESULTS:
The test material, Sample No. 1473, was supplied as a SASS
train organic extract in 10 ml of methylene chloride. The
total organic content was given as 26 mg. A portion of the
sample (8.20 ml) was solvent exchanged into an equal volume
of dimethylsulfoxide (DMSO). The concentration of organics
was therefore unchanged, and the value of 2.6 mg/ml (or
2.6 yg/vl) was used to convert the doses obtained on a
volume basis into the equivalent amount of organics per
milliliter of culture medium.
The test material remained completely soluble after exchange
into DMSO. Diluted stocks were prepared with DMSO just prior
to testing, and the treatments were initiated with culture
media containing 1:100 dilutions of the stocks. The highest
test concentration was achieved with a 1:50 dilution of the
solvent-exchanged sample, which introduced 2% DMSO into the
medium. Vetiicle control cultures were therefore exposed to
115
-------
VIII. INTERPRETATION OF RESULTS: (continued)
culture media containing 1% and 2% DMSO in order to provide the
reference points for determining the survival of the cells to
treatments with the test material. No precipitation of test
material in the culture medium was observed at any of the tested
concentrations.
Seven test concentrations from 0.05 ul/ml to 20 pi/ml were
evaluated for their effects on colony survival. As shown in
Table 1 and Figure 1, none of the assayed concentrations caused
significant changes in the numbers of colonies. This lack of
observable toxicity was not conclusive, however, because the
highest concentration corresponded to only 52 ^g of organics/ml,
which was essentially the midpoint of the concentration range
where an EC50 would yield a moderate toxicity classification (see
Evaluation Criteria). A sharp increase in toxicity could occur
with a small increase in concentration, but it is reasonable to
assume that 50% survival would not have been observed had the high
dose been doubled. This concentration would have fallen just
within the low toxicity region. Thus, the test material was
evaluated as possibly having low toxicity or no detectable toxicity
to CHO cells. The position of the EC50 could be further investi-
gated by concentrating the extracted organic material.
116
-------
IX. CONCLUSIONS:
The test material, Sample No. 1473, was evaluated as having low or
nondetectable toxicity in the CHO Clonal Toxicity Assay. No
toxicity was observed for concentrations up to the highest testable
level of 52 yg of organics/ml.
Submitted by:
Study Director
Brian C. Myhr,'Ptf.D7
Section Chief
Mammalian Genetics
Department of Genetics
and Cell Biology
Reviewed by:
U/i/Vo
Date
David J. Brusick, Ph.D.
Di rector
Department of Genetics
and Cell Biology
117
-------
TABLE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
Sample Ide
MOUNTAIN V
ntity: Samole
'IEW
Description of Test Sam
yellow solution In DMSO
LBI Assay
No.: 5397
f!473. CMEA
iple: Clear, Dale
i.
Date Received: October 9, 1980
Test Date:
Vehicle:
Cell Type:
November 17, 1980
DMSO
CHO-K1
Cells Seeded per Dish:
200
EC50
Value: >2(
Toxicity
Classification
3 yl/ml (>5I
2 yg/ml)
: Probablv Low or Non-
detectable
pH Alterations
Comments on Tr<
: NONE
jatment:
'
CLONAL TOXICITY DATA
Sampl e
NC
VC, 1%
VC, 2%
Test
Test
Test
Test
Test
Test
Test
Applied
Concentration
pi /ml
10.0
20.0
0.05
0.2
0.5
2.0
5.0
10.0
20.0
Dish
#1
207
180
173
183
C
185
161
161
166
.170
Dish
#2
185
177
162
192
192
189
187
190
178
171
Dish
#3
192
172
166
189
183
179
183
164
180
154
Average
Count
194.7
176.3
167.0
188.0
187.5
184.3
177.0
171.7
174.7
165.0
Relative
Survival*
%
100.0
100.0
106.6
106.4
104.5
100.4
97.4
99.1
98.8
Cloning
Efficiency
-------
FIGURE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
EC50 DETERMINATION
SAMPLE #1473, CMEA MOUNTAIN VIEW
140
130
120
110
100
90
»« 80
•
§ 70
be
i
60
50
40
30
20
10
0.1
10
100
CONCENTRATION, pi/ml
119
-------
ASSAY DESIGN NO. 442
RODENT CELL CLONAL (CHO) TOXICITY ASSAY
This assay conforms to one of the bioassay tests in the US Environmental Protec-
tion Agency (EPA) IERL - RTP Procedures Manual: Level 1 Environmental Assessment
Biological Tests (September, 1980).Level 1 bioassays obtain preliminary infor-
mation on the harmful biological effects of chemicals found in industrial feed
and waste streams by using mammalian cells in culture to measure metabolic
impairment and cellular death.
1. OBJECTIVE
The objective of this assay is to determine the concentration of a test
article that will reduce the colony forming ability of Chinese hamster
cells by 50% after a 24-hour exposure. This concentration is referred to
as the EC50 value.
2. MATERIALS
A. Indicator Cells
The indicator cells used in this study are Chinese hamster CHO-K1
cells (American Type Culture Collection No. CCL 6l). This cell type
was derived from ovarian tissue and has spontaneously transformed to
a stable, hypodiploid line of rounded, fibroblastic cells with un-
limited growth potential. Monolayer cultures have a fast doubling
time of 11 to 14 hours, and untreated cells can normally be cloned
with an efficiency of 80% or greater. Permanent stocks are main-
tained in liquid nitrogen and laboratory cultures are maintained by
serial subculturing. Laboratory cultures are periodically checked
by culturing methods for the absence of mycoplasma contamination.
This test system is specified by the IERL- RTP Procedures Manual.
B. Medium
The CHO-K1 cell line has an absolute requirement for proline and
therefore must.be maintained in a culture medium containing suf-
ficient amounts of this amino acid. Ham's F12 medium, which contains
3 x 10-«M L-proline, is used, supplemented with 10% fetal bovine
serum, 100 units per milliliter of penicillin, 100 ug streptomycin
per milliliter, and 0.5 ug amphotericin B (Fungizone) per milliliter.
C. Controls
Untreated cells are cloned to establish the control cloning effi-
ciency. If the test article is dissolved in an organic solvent
(usually dimethylsulfoxide), cells exposed to solvent in the medium
120
-------
2. MATERIALS (Continued)
are cloned to provide the reference cloning efficiency for the effect
of the test article. The final concentration of solvent in the growth
nedium is generally IX or less. All controls are performed in tripli-
C A v6 •
D. Sample Forms
Solid samples are tested as a solution or are ground to fine particles
(less than 5 urn) and tested as a suspension in growth medium. Dry
particulate articles, aqueous liquids, suspensions and slurries are
added directly to the growth medium and tested as a suspension or
solution. Liquids containing less than 0.2% organic solvent are
generally tested directly; samples dissolved in organic solvents are
solvent exchanged in dimethylsulfoxide (DMSO) before testing. Original
sample volumes may be reduced a maximum of 10-fold during solvent
exchange, and the concentration factor is used to convert assayed
volumes into equivalent original sample volumes. All sample manipu-
lations are performed as described in the IERL-RTP Level 1 Procedures
Manual.
3. 'EXPERIMENTAL DESIGN
A. Dose Selection
Unless the approximate toxicity is already known or the sample size
is limiting, the following dose ranges are tested for different sample
forms. Dry particulate articles are dissolved or suspended in growth
medium and tested at five dose levels from 1000 ug/ml to 10 pg/ml.
Aqueous samples, suspensions or slurries are tested from 600 Hliters/ml
to 6 |jliters/ml in five dose steps. Samples that are solvent exchanged
into DMSO are tested from 20 uliters/ml to 0.2 uliters/ml, also in five
dose steps. Solvent concentrations are IX for all dose levels except
the maximum applicable dose (MAD) of 20 ul/ml, which contains 2% solvent.
A second dose study is performed with an appropriate dose range if the
EC50 has not been properly located in the initial test; EC50 values
greater than 1,000 ug/ml, 600 |jliters of aqueous sample/ml or 20 uliters
nonaqueous sample/ml are not determined.
B. Clonal Toxicity Assay
Cells from monolayer stock cultures in logarithmic growth phase are
trypsinized, counted by hemacytometer and reseeded into a series of
60- or 100-mm culture dishes at 200 cells per dish. The cultures
are incubated for 6 to 16 hours at 37°C to allow attachment of the
cells and recovery of growth rate.
Test article is then applied (three dishes per dose), and the cultures
returned to the incubator. If the test article causes a color change,
an additional dish is treated with the high dose and the pH of the
121
-------
3. EXPERIMENTAL DESIGN (Continued)
medium 1s determined. The pH is also recorded for the highest dose
which results in a slight color change. After a 24-hour exposure
period the medium is aspirated and the cells washed with Dulbecco s
phosphate buffered saline (PBS; prewarmed to 37°C). The pH of the
discarded medium for which initial pH measurements were made is again
recorded. Fresh medium is placed on each culture and Incubation con-
tinued for an additional 6 days to allow colony development. Medium
is drained from the cultures and the surviving colonies are washed
with PBS, fixed in methanol, and stained with Giemsa. Colony counting
is performed by eye; colonies smaller than 50 to 100 cells are not
counted.
4. ASSAY ACCEPTANCE CRITERIA
The assay is considered acceptable for evaluation of the test results if
the following criteria are met:
The average cloning efficiency of the CHO-K1 cells in the nega-
tive controls is 70% or greater, but not exceeding 115%.
The distribution of colonies in the treated cultures is generally
uniform over the surface of the culture dish.
The data points for each test concentration critical to the loca-
tion of the ECSO are .the averages of at least two treated cultures.
A sufficient number of test concentrations are available to
clearly locate the EC50 within a toxicity region as defined
under Assay Evaluation Criteria.
If the EC50 value is greater than 1000 ug/ml, 600 uliters Of
aqueous sample/ml, or 20 uliters of nonaqueous sample/ml, the
plotted curve does not exceed 110% of the negative control.
5. ASSAY EVALUATION CRITERIA
The screened doses, pH values (if appropriate), colony counts, percent
survivals (colony counts relative to control colony counts) and ECSO
values are provided. The percent survival is plotted as a function of
applied concentration and the EC50 value determined graphically by fitting
a curve by eye through the data points. The EC50 1s used to rank the test
material using the standard evaluation criteria defined in the table below.
Sorbent extracts of known organic content are evaluated as nonaqueous
liquids and In terms of their solid contents (ug organics/ml), and are
ranked using the more sensitive parameter. Where data on the original
liquid or gas sample volumes is provided by the Sponsor, the EC50 value
is calculated in terms of these equivalent volumes per mill inter of
culture medium; however, no evaluations are made as yet on this basis.
122
-------
5. ASSAY EVALUATION CRITERIA (Continued)
Toxicity*
Solids
(EC50 in ug/ml)
Aqueous Liquids
(EC50 in
Nonaqueous Liquids
(EC50 in ul/ml)
High
Moderate
Low
Not Detectable
10 to 100
100 to 1000
>1000
<6
6 to 60
60 to 600
>600
.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests.
Criteria for nonaqueous liquids are tentative and under evaluation.
RECORDS TO BE MAINTAINED
All raw data, protocols, protocol modifications, test article weight and
dispensation records and correspondence between LBI and the Sponsor are
being maintained in a central file within the Department of Genetics and
Cell Biology. These records will be filed under departmental assay number
and held up to 2 years following submission of the final report to the
Sponsor. After 2 years they will be transferred to the LBI archives for
permanent storage.
123
-------
Q.A. Inspection Statement
(reference 21 CFR 58.35(b)(7))
PROJECT J0993 '" LBI Assay No.
TYPE of STUDY fydi^lJr Csjfi fat)) C£jnu>JL.
This final study report was reviewed by the LBI Quality
Assurance Unit on /UatnukeA^ I iQfo. A report of findings was
;
submitted to the Study Director and to Management on
The short-term nature' of this study precluded inspection while
it was in process. The Quality Assurance Unit inspects an in-process
study of this type approximately once per month to assure that no
significant problems exist that are likely to affect the integrity of
this type of study.
iuubu,*ju
Auditor, Quality Assurance Unit
124
-------
GENETICS ASSAY NO. 5397
LBI SAFETY NO. 6391
MUTA6ENICITY EVALUATION OF
SAMPLE #1473
IN THE
AMES SALMORELOTHICROSOME
"~PlATE"TEST
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20795
LBI PROJECT NO. 20988
REPORT DATE: DECEMBER, 1980
125
-------
PREFACE
This report contains a summary of the data compiled during the
evaluation of the test compound. The report is organized to
present the results in a concise and easily interpretable manner.
The first part contains items I-IX. Items I-IV provide sponsor
and compound identification information, type of assay, and the
protocol reference number. All protocol references indicate a
standard procedure described in the Litton Bionetics, Inc.
"Screening Program for the Identification of Potential Mutagens
and Carcinogens." Item V provides the initiation and completion
dates for the study, and Item VI provides identification of super-
visory personnel. Item VII identifies the tables and/or figures
containing the data used by the study director in interpreting
the test results. The interpretation itself is in Item VIII.
Item IX provides the conclusion and evaluation.
The second part of the report describes the materials and procedures
employed in conducting the assay. This part of the report also
contains evaluation criteria used by the study director, and any
appendices. The evaluation criteria are included to acquaint the
sponsor with the methods used to develop and analyze the test
results.
All test and control results presented in this report are supported
by fully documented raw data which are permanently maintained in
the files of the Department of Genetics and Cell Biology or in the
archives of Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington,
Maryland 20795.
126
-------
I. SPONSOR: Acurex Corporation
II. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5397
A. Identification: Sample #1473
B. Date Received: October 9, 1980
C. Physical Description: Pale yellow liquid
III. TYPE OF ASSAY: Ames Salmonella/microsome Mutagenesis Assay
IV. PROTOCOL NUMBER: 401 (EPA-Level I)
V. STUDY DATES:
A. Initiation: November 7, 1980
B. Completion: November 13, 1980
VI. STUDY DIRECTOR: D.R. Jagannath, Ph.D.
VII. RESULTS:
The results of this assay are presented in Table 1.
VIII. INTERPRETATION OF RESULTS:
The test material, Sample No. 1473, was supplied as a SASS train
organic extract in the 10 ml of methylene chloride. The total
organic content was given as 26.0 mg. A portion of the sample
(8.20 ml) was solvent exchanged into dimethylsulfoxide (DMSO) to
a final volume of 8.20 ml. This solvent exchanged sample (test
material) was examined for mutagenic activity in the Ames/Salmonella
assay in the presence and absence of liver microsomal enzyme pre-
parations from Aroclor-induced rats.
A negative control consisting of the solvent DMSO and-specific
positive compounds were also assayed concurrently with the test
material.
DOSE RANGE:
The dose range employed for the evaluation of this test material
was from 10.0 yl to 200.0 yl per plate. Based on the organic
content of the sample the doses employed would equal to 26.0 vg to
520.0 yg per plate. The tests were conducted using two plates per
dose level.
127
-------
VIII. INTERPRETATION OF RESULTS: (continued)
TOXICITY
The test material did not exhibit toxicity with any of the
indicator strains used in this assay.
The results of the tests conducted on the test material in the
absence of an activation system was positive with the strain TA-98.
The lowest dose at which the response was observed was at 100 yl
or 260.0 yg organics per plate.
The results of the test conducted on the test material in the
presence of rat liver activation system were negative.
IX. CONCLUSIONS:
(1) The SASS train organic extract sample #1473 exhibited genetic
activity with the strain TA-98 in the nonactivation assays conducted
in this evaluation and is considered as mutagenic under these test
conditions. (2) Based on the EPA's 'Definition of Toxicity
Categories for Health Effects Tests' the mutagenic activity of the
sample is classified as 'Moderate1 (see Table A). The specific Acti-
vity at the Minimum Effective Concentration (which is the number of >e-
vertants minus the background revertants divided by yg organics x 1000) of
the sample (260.0 yg) based on the results from TA-98 (nonactivation)
was 92.31 revertants per mg of organics.
The classification of the mutagenic activity (item 2 above) and the
Specific Activity of the sample (item 3 above) are given here to
compare the potency of various samples belonging to the same class.
Submitted by:
Study Director
D.R. Jagannath, Ph.D. Date
Section Chief
Submammalian Genetics
Department of Genetics
and Cell Biology
Reviewed by:
f^f f - - - i i^
_ Ph.D. Tate
Director
Department of Genetics
and Cell Biology
128
-------
TABLE A
DEFINITION OF TOXICITY CATEGORIES FOR HEALTH EFFECTS ASSAYS*
PO
Assay"
Ames
RAN
CHO
WAT
"standard
Ames:
Activity Measured6
NEC
(mutagenesls)
(lethality)
EC50
(lethality)
LD80
(lethality and
toxic signs)
test abbreviations are as
Ames Salmonella/microsome
Sample
Typec
S
AL.NAL
S
AL
NAL
S
AL
NAL
S
AL.NAL
follows:
mutagenesls
MADd
5
200
1
600
20
1
600
20
5
5
assay
Range of Concentration or
Units
mg/plate
Ml/plate
Ml/ml
Ml/ml
mg/ml
Ml /ml
Ml /ml
gm/kg
ml/kg
High
<0.05
<2
<0.01
<6
<0.2
<0.01
<6
<0.2
<0.05
<0.05
Node rate
0.05-0.5
2-20
0.01-0.1
6-60
0.2-2
0.01-0.1
6-60
0.2-2
0.05-0.5
0.5-0.5
Low
0.5-5
20-200
0.1-1
60-600
2-20
0.1-1
60-600
2-20
0.5-5
0.5-5
Dosage
Not Detectable
(ND)
ND at >5
ND at >200
ND at >1
ND at >600
ND at >20
ND at >1
ND at <600
NO at >20
ND at <5
ND at <5
RAN: Rabbit alveolar macrophage cytotoxicity assay
CHO: Rodent cell clonal toxlcity assay
WAT: Acute |n vivo test In rodents (whole animal test)
Standard abbreviations for measured endpoints are as follows:
NEC: Minimum effective concentration
EC90: Calculated concentration expected to produce effect 1n 50 percent of population
LDSO: Calculated dose expected to kill 50 percent of population
CS = Solid, AL - Aqueous liquid, NAL = Nonaqueous liquid
-------
RESULTS
TABLE I
NAME OR CODE DESIONATION OP THE TEST COMPOUND: SIMPLE f!4W
SOLVENT: OHSO
TEST INITIATION DATES: tl/OT/HO
TEST COUPLET ION DATE: 11/13/RO
s-9 LOTR: 00009
NOTE: CONCENTRATIONS *NE OIVEN IN NICROLITERS PER PLATE
TEST
NONACTIVAT10N
SPECIES TISSUE
TA-1935
1 2
TA-153T
1 2
PLATE
T»-90
1 2
TA-100
I 2
SOLVENT CONTROL
PCSITIVE CONTROL**
UL
VL
12 11
«90 to;*
6 9
191 225
CO
O
TEST C
10.000000
23.000000
SO.OOOOOO UL
100.000000 UL
200.000000 UL
ACTIVATION
SOLVENT CONTROL RAT
POSITIVE CONTROL**• RAT
TEST CO
10.000000 UL RAT
29.000000 Ul RAT
SO.000000 UL RAT
100.000000 UL RAT
200.000000 UL RAT
22
34
IT
21
24
21
24
21
23
20
10
9
10
10
14
10
«
3
R
9
LIVER 9 9
LIVER 110 1*0
9 9
114 161
22 10
861 T2T
28 21
30 71
30 44
98 30
63 33
14 15
900 1066
ICO 193
1303 12T4
195 141
I«9 IS?
173 152
IT* 140
153 14*
190 190
1942 1T46
LIVEN
LIVER
LIVER
LIVER
LIVEN
11
6
19
10
10
10
II
14
1C
6
8
3
1
15
9
5
3
•i
32
3T
33
23
24
21
29
1*
29
24
191
152
145
193
223
110
135
152
101
22 R
TA-S9S9 SODIUM AZIOE
TA-193T 9-ANINOACRIOIKE
TA-90 2-NITROFLUORCNE
TA-100 SODIUM AZIOE
SOLVENT 200 UL/PLATE
10 US/PLATE
50 Uf/PUTE
10 UGSPLATE
10 US/PLATE
TA-1535
T A-1531
TI-90
TA-100
2-ANTHRANINC
2-ANTHRAHINC
2-ANTHRANINE
2-ANTHRANINC
2.5 UO/PLATFI
2.5 U6/PLATE
2.5 UC/PLATE
?.5 HO/PLATE
-------
AMES SALMONELLA/MICROSOME PLATE ASSAY
1. OBJECTIVE
The objective of this study was to evaluate a test material for mutagenic
activity in a bacterial assay with and without a mammalian S9 activation
system.
2. RATIONALE
The Salmonella typhimurium strains used at LBI are all histidine
auxotrophs by virtue of mutations in the histidine operon. When
these histidine-dependent cells are grown in a minimal media petri
plate containing a trace of histidine, only those cells that revert
to histidine independence (his*) are able to form colonies. The
trace amount of histidine allows all the plated bacteria to undergo
a few divisions; this growth is essential for mutagenesis to occur.
The hi£+ revertants are easily scored as colonies against the slight
background growth. The spontaneous mutation frequency of each strain
is relatively constant, but when a mutagen is added to the agar the
mutation frequency is increased 2- to 100-fold. Cells which grow to
form colonies on the minimal media petri plates are therefore assumed
to have reverted, either spontaneously or by the action of a test
substance to his+ genotype.
3. MATERIALS
A. Indicator Microorganisms
The Salmonella typhimurium strains.used in this assay were obtained
from Dr. Bruce Ames, University of California at Berkeley.1'5 The
following 4 strains were used:
Strain
Designation
TA-1535
Gene
Affected
his S
Additional Mutations
Repair
A uvr B
LPS R Factor
rfa
Mutation Type
Detected
Base-pair
substitution
TA-1537 his C A uvr B rfa - Frameshift
TA-98 his D AuvrB rfa pKMIOl Frameshift
TA inn his G A uvr B rfa pKMTOl. Base pair
IA IUU substitution
131
-------
3. MATERIALS (Continued)
The aforementioned strains have, in addition to the mutation in the
histidine operon, a mutation (rfa-) that leads to defective 1'POpoly-
saccharide coat, a deletion that covers genes involved 1n the synthesis
of vitamin biotln (bio-) and in the repair of ultraviolet (uv) - in-
duced DNA damage (uvrB-). The rfa- mutation makes the strains more
permeable to many large molecules. The uvrB- mutation decreases repair
of some types of chemically or physically damaged DNA and thereby
enhances the strain's sensitivity to some mutagenic agents. The
resistant transfer factor plasmid (R factor) pKMlOl in TA-98 and
TA-100 is believed to cause an increase in error-prone DNA repair
that leads to many more mutations for a given dose of most mutagens5.
In addition, plasmid pKMlOl confers resistance to the antibiotic
ampicillin, which is a convenient marker to detect the presence of
plasmid in the cells.
All indicator strains are kept at 4°C on minimal medium plates supple-
mented with a trace of biotin and an excess of histidine. The plates
with plasmid-carrying strains contain in addition ampicillin (25 pg/ml)
to ensure stable maintenance of plasmid pKMlOl. New stock culture
plates are made as often as necessary from frozen master cultures
or from single colony reisolates that were checked for their genotypic
characteristics (his, rfa. uvrB. bio) and for the presence of plasmid.
For each experiment, an inoculum from the stock culture plates is
grown overnight at 37°C in nutrient broth (Oxoid CM67).
B. Media
The bacterial strains were cultured in Oxoid Media #2 (nutrient Broth).
The selective medium was Vogel Bonner Medium E with 2% glucose7. The
overlay agar consisted of 0.6% purified agar with 0.5 mM histidine,
0.05 mM biotin and 0.1 M NaCl according to the methods of Ames et. al..6
C. Activation System
(1) 59 Homogenate
A 9,000 x s supernatant prepared from Sprague-Dawley adult
male rat liver induced by Aroclor 1254 (described by Ames
et. al..6) was purchased from Bionetics Laboratory Products,
Litton Bionetics, Inc. and used in this assay.
(2) 59 Mix
Concentration per Mi Hi liter
Components S9 Mix
NADP (sodium salt) 4 umoles
D-glucose-6-phosphate 5 umoles
MgCl2 8 umoles
KC1 33 umoles
Sodium phosphate buffer
pH 7.4 100 umoles
Organ homogenate from rat
liver (S9 fraction) 100 uliters
132
-------
EXPERIMENTAL DESIGN
A. Dosage Selection
All tests are run at a minimum of four concentrations. In the
Standard EPA Level I Ames assays, five dose levels of the test
material, dissolved in a suitable solvent, are added to the test
system. The standard test doses for the extracted material are
10, 25, 50, 100 and 200 yliters per plate. The solids are tested
up to 5 mg per plate and at lower concentrations of 2.5, 1, 0.5,
0.1 and 0.05 mg per plate. The samples are retested over a
narrower range of concentrations with strains showing positive
results if there is enough sample.
B. Mutagenicitv Testing
The procedure used is based on the paper published by Ames et. al.6
and is performed as follows:
(1) Nonactivation Assay
To a sterile 13 x 100 mm test tube placed in a 43°C water bath
the following is added in order:
(a) 2.00 ml of 0.6% agar containing 0.05 mM histidine
and 0.05 mM biotin.
(b) 0.05 ml of a solution of the test chemical to give
the appropriate dose.
(c) 0.1 ml - 0.2 ml.of indicator organism(s).
(d) 0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture is swirled gently and then poured onto minimal
agar plates (see 3B, Media). After the top agar has set, the
plates are incubated at 37°C for approximately 2 days. The
number of his+ revertant colonies growing on the plates is
counted and recorded.
(2) Activation Assay
The activation assay is run concurrently with the nonactiva-
tion assay. The only difference is the addition of 0.5 ml of
S9 mix (see 3C:2, Activation System) to the tubes in place of
0.5 ml of phosphate buffer which is added in nonactivation
assays. All other details are similar to the procedure for
nonactivation assays.
A detailed flow diagram for the plate incorporation assay is
provided in Figure 1.
133
-------
FIGURE 1
REVERSE MUTATION ASSAY
[Agar Incorporation Method]
Aliquot of 0.5 ml
buffer
I
-S9
Molten [43 to 45°C] overlay agar
appropriately supplemented
0.05 ml
<
0.1 ml to 0.2 ml
]
T
Test article, positive
control or solvent control
Aliquot of an overnight
culture of bacteria
0.5 ml S9 mix [hepatic
•*— homogenate from
PCS pretreated rat
plus necessary
cof actors]
Overlay poured on selective
bottom agar medium
Plates incubated at 37*C for approximately 2 days
Number of revertants per plate counted
I
Data entered onto preprinted forms
Interpretation/conclusion
134
-------
4. EXPERIMENTAL DESIGN (Continued)
C. Control Compounds
A negative control consisting of the solvent used for the test
material 1s performed 1n all cases. For negative controls, step
'b1 of Nonactlvatlon Assays Is replaced by 0.05 ml of the solvent.
The negative controls are employed for each Indicator strain and
are performed In the absence and presence of S9 mix. The solvent
used to prepare the stock solution of the test material 1s given
1n the Results section of this report. All dilutions of the test
material are made using this solvent. The amount of solvent used
1s equal to the maximum volume used to give the appropriate test
dose.
Specific positive control compounds known to revert each strain are
also used 1n the assays. The concentrations and specificities of
these compounds to specific strains are given In the following table:
Concentration
per Plate Salmonella
Assay Chemical Solvent (yg) strains
Nonactlvatlon Sodium azlde Water 1 TA-1535, TA-100
2-Nitrofluorene Dimethyl- 10 TA-98
(NF) sulfoxide
9-aminoacridine Ethanol 50 TA-1537
(9AA)
Activation 2-anthramine Dimethyl- 2.5 For all strains
(ANTH) sulfoxlde
135
-------
5. EVALUATION CRITERIA
Statistical methods are not currently used and evaluation is based
on the criteria included in this protocol.
Plate test data consist of direct revertant colony counts obtained
from a set of selective agar plates seeded with populations of mutant
cells suspended in a semisol id overlay. Because the test material
and the cells are incubated in the overlay for approximately 2 days
and a few cell divisions occur during the incubation period, the
test is semiquantitative in nature. Although these features reduce
the quantitation of result, they provide certain advantages not con-
tained in a quantitative suspension test:
The small number of cell divisions permits potential mutagens
to act on replicating DNA, which is often more sensitive
than nonreplieating DNA.
The combined incubation of the test article and the cells
in the overlay permits constant exposure of the indicator
cells for approximately 2 days.
A. Surviving Populations
Plate test procedures do not permit exact quantitation of the number
of cells surviving chemical treatment. At low concentrations of
the test material, the surviving population on the treatment plates
is essentially the same as that on the negative control plate. At
high concentrations, the surviving population is usually reduced by
some fraction. Our protocol will normally employ several doses
ranging over two or three log concentrations. This does not apply
to spot tests and tests performed on fabrics and like materials which
are tested at a single concentration.
B. Dose-Response Phenomena i
The demonstration of dose-related increases in mutant counts is an
important criterion in establishing mutagenicity. A factor that
might modify dose-response results for a mutagen would be the selec-
tion of doses that are too low (usually mutagenicity and toxicity
are related). If the highest dose is far lower than a toxic concen-
tration, no increases may be observed over the dose range selected.
Conversely, if the lowest dose employed is highly cytotoxic, the
test material may kill any mutants that are induced, and the test
material will not appear to be mutagenic.
136
-------
5. EVALUATION CRITERIA (Continued)
C. Control Tests
Positive and negative control assays are conducted with each experi-
ment and consist of direct-acting mutagens for nonactivation assays
and mutagens requiring metabolic biotransformation in activation
assays. Negative controls constst of the test material solvent in
the overlay agar together with the other essential components. The
negative control plate for each strain gives a reference point to
• which the test data is compared. The positive control assay is con-
ducted to demonstrate that the test systems are functional with known
mutagens.
D. Evaluation Criteria for Ames Assay
Because the procedures used to evaluate the mutagenicity of the test
material are semiquantitative, the criteria used to determine positive
effects are inherently subjective and are based primarily on a his-
torical data base. Most data sets are evaluated using the following
criteria:
(1) Strains TA-1535, TA-1537
If the solvent control value is within the normal range, a test
material producing a positive response equal to three times
the solvent control value is considered mutagenic.
(2) Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test
material producing a positive response equal to twice the solvent
control value for TA-98 and TA-100 is considered mutagenic.
(3) Pattern
Because TA-1535 and TA-100 are both derived from the same parental
strain (6-46) and because TA-1538 and TA-98 are both derived
from the same parental strain (D3052), to some extent there is
a built-in redundancy in the microbial assay. In general, the
two strains of a set respond to the same mutagen and such a
pattern is sought. Generally, if a strain responds to a mutagen
in nonactivation tests, it will do so in activation tests.
(4) Reproducibility
If a test material produces a response in a single test which
cannot be reproduced in additional runs, the Initial positive
test data lose significance.
137
-------
5. EVALUATION CRITERIA (Continued)
E.. Evaluation Criteria for Toxlclty
(1) Complete toxicity
When there are no revertants observed on the plate(s) treated
with the test compound, the test compound 1s defined as toxic
to all or any of the Indicator strains at that (those) particular
dose(s).
(2) Slight toxicity
When there are fifty or less percent revertants on the plate(s)
treated with the test compound as compared to the solvent control
plate(s), the test compound is defined as slightly toxic to
all or any of the indicator strains at that (those) particular
dose(s).
F. Relation Between Mutagenicity and Cardnogenicity
It must be emphasized that the Ames Salmonella/Microsome Plate Assay
is not a definitive test for chemical carcinogens. It is recognized,
howerver, that correlative and functional relations have been demon-
strated between these two endpoints. The results of comparative
tests on 300 chemicals by McCann et. aJL x show an extremely good
correlation between results of microbial mutagenesis tests and jrj
vivo rodent carcinogenesis assays.
All evaluations and interpretation of the data to be presented in
the final report will be based only on the demonstration, or lack,
of mutagenic activity.
138
-------
REFERENCES
1. McCann,J., Choi, E., Yamasaki, E. and Ames, B.N.: Detection of
carcinogens as mutagens in the Salmonella/microsome test: Assay
of 300 chemicals. Proc. Nat. Acad. Sci. USA, 72:5135-5139, 1975.
2. Ames, B.N., Gurney, E.G., Miller, J.A. and Bartsch, H.: Carcinogens
as frameshift mutagens: Metabolites and derivatives of 2-acetyl-
aminof1uorene and other aromatic amine carcinogens. Proc. Nat. Acad.
Sci. USA, 69:3128-3132, 1972.
3. Ames, B.N, Lee, F.D. and Durston, W.E.: An improved bacterial
test system for the detection and classification of*.mutagens and
carcinogens. Proc. Nat. Acad. Sci. USA, 70:782-786, 1973.
4. Ames, B.N, Durston, W.E., Yamasaki, E. and Lee, F.D.: Carcinogens
are mutagens: A simple test system combining liver homogenates
for activation and bacteria for detection. Proc. Nat. Acad. Sci.
USA, 70:2281-2285, 1973.
5. McCann, J., Springarn, N.E., Kobori, J. and Ames, B.N.: Detection of
carcinogens as mutagens: Bacterial tester strains with R factor
plasmids. Proc. Nat. Acad. Sci. USA, 72:979-983, 1975.
6. Ames, B.N., McCann, J. and Yamasaki, E.: Methods for detecting
carcinogens and mutagens with the Salmonella/mammalian-microsome
mutagenicity test. Mutation Res., 31:347-364, 1975.
7. Vogel, H.J. and Bonner, D.M.: Acetylornithinase off. col i; Partial
purification and some properties. J. Biol. Chem., 218:97-106, 1956.
139
-------
Q.A. Inspection Statement
(reference 21 CFR 58.35(b)(7))
PROJECT
LBX Assay No. S3 9?
TYPE of STUDY ftwrs ~PI*
-------
GENETICS ASSAY NO. 5394
•^•••••OT
LBI SAFETY NO. 6378
CYTQTOXIC EVALUATION OF
SAMPLE f 1452
(WATER TANK DRAIN, END OF TEST)
IN THE
RODENTCELL (CHO)
CLONAL TOXICITY ASSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20795
LBI PROJECT NO. 20933
REPORT DATE: DECEMBER 1980
141
-------
PREFACE
This report contains a summary of the data compiled during the evaluation
of the test compound. The report is organized to present the results in
a concise and easily Interpretable manner. The first part contains Items
I - IX. Items I - IV provide sponsor and compound identification infor-
mation, type of assay, and the assay design reference number. All assay
design references indicate a standard procedure described in the Litton
Bionetics, Inc. "Screening Program for the Identification of Potential
Mutagens and Carcinogens." Item V provides the initiation and completion
dates for the study, and Item VI provides identification of supervisory
personnel. Item VII identifies the tables and figures containing the data
used by the study director in interpreting the test results. The interpre-
tation itself is in Item VIII. Item IX provides the conclusion and evalua-
tion.
The second part of the report, entitled Assay Design, describes the
materials and procedures employed in conducting the assay. This part
of the report also contains any appendices, as well as evaluation
criteria used by the study director. The evaluation criteria are
included to acquaint the sponsor with the methods used to develop and
analyze the test results.
All test and control results presented in this report are supported by
fully documented raw data which are permanently maintained in the files
of the Department of Genetics and Cell Biology or in the archives of
Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington, Maryland, 20795.
Copies of raw data will be supplied to the sponsor upon request.
142
-------
I. SPONSOR: Acurex Corporation
II. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5394
A. Identification: Sample #1452 (Man Test 5/21/80, #5 Gal, .
Water Tank Drain, End of Test)
B. Date Received: October 6, 1980
C. Physical Description: Clear, colorless liquid
III. TYPE OF ASSAY: Rodent Cell (CHO) Clonal Toxicity Assay
IV. ASSAY DESIGN NUMBER: 442
V. STUDY DATES:
A. Initiation: November 19, 1980
B. Completion: December 1, 1980
VI. SUPERVISORY PERSONNEL:
A. Study Director: Brian C. Myhr, Ph.D.
B. Laboratory Supervisor: Robert Young, M.S.
VII. RESULTS:
The data are presented in Table 1 on page 3 and in Figure 1
on page 4.
VIII. INTERPRETATION OF RESULTS:
The test material, Sample No. 1452, was supplied as a colorless,
aqueous solution that did not contain any obvious suspended
material. A portion of the sample (18 ml) was combined with
10X F10 medium and supplements to yield 30 ml of culture
medium containing the test material at a concentration of
600 yl/ml. This was the highest concentration applied to the
CHO cells. Lower concentrations were achieved by serial dilu-
tion with F10 culture medium. The treatments were initiated
by replacing the F12 medium in the cell cultures with F10
SdlS containing the test material. After the 24 hour expo-
suVe period, the cultures were returned to Fl2 medium and
incubated for colony development. The test material did not
cause any visible precipitation of culture medium components,
but I thift to a slightly acidic PH of 6.26 was observed for
the 600 yl/ml concentration. The pH remained normal (above
7.0) for the other test concentrations.
143
-------
VIII. INTERPRETATION OF RESULTS: (continued)
Two trials of the assay were performed. The first trial was
considered insufficient for evaluation because the average
cloning efficiency of the negative controls was 67.4%, which
was slightly below the 70% criterion used for an acceptable
assay. The results of the second trial are presented in Table
1 and Figure 1 .
Seven test concentrations from 6 yl/ml to 600 yl/ml were
evaluated for their effects on colony survival. As shown in
Table 1, complete lethality was obtained for treatments in the 60
to 600 yl/ml concentration range. In contrast, the treatments
with 6 to 30 vl/ml were essentially nontoxic to the cells. Thus,
an extremely sharp survival curve was obtained, as shown in Figure
1, with an EC50 apparently located at 45 yl/ml. [In the first trial
(results not shown), an EC50 value of 52 yl/ml was obtained for a
somewhat broader survival curve]. The EC50 was therefore located
at the upper end of the 6-60 yl/ml concentration range, which is
the range defined for moderate toxicity for aqueous liquids (see
Evaluation Criteria). Since complete lethality was obtained at the
boundary between moderate and low toxicity, an evaluation of moderate
toxicity to CHO cells appeared appropriate.
IX. CONCLUSIONS:
The test material, Sample No. 1452, was evaluated as having moderate
toxicity in the CHO Clonal Toxicity Assay. The EC50 value was
45 yl/ml.
Submitted by:
Study Director:
C. ftk 13,/3/fr?
Brian C. Myhr, PKjD. Date
Section Chief
Mammalian Genetics
Department of Genetics
and Cell Biology
Reviewed by:
David J. /Brusick, Ph.D. Date
Director
Department of Genetics
and Cell Biology
144
-------
TABLE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
Sample Identity: Sample 11452r
Tank Drain. End of Test)
Description of Sample: Clear, colorless
aqueous liquid
LBI Assay No.: 5394
EC50 Value: 45 ,,i/mi
Toxicity
Classification: Moderate
Date Received: October 6, 1980
Test Date: November 25. 1980 (Trial 2)
Vehicle: None
Cell Type: CHO-K1
Cells Seeded per Dish: 200
pH Alterations: pH 6.26 at 600 n
Comments on Treatment: Normal DH
for treatments with 6 to 300 ul/ml
CLONAL TOXICITY DATA
Sample
NC
Test
Test
Test
Test
Test
Test
Test
Applied
Concentration
yl/ml
—
6.0
10.0
30.0
60.0
100.0
300.0
600.0
Dish
#1
187
190
214
167
0
0
0
0
Dish
#2
193
219
210
190
0
0
0
0
Dish
#3
187
183
181
178
0
0
0
0
Average
Count
189.0
197.3
201.7
178.3
0
0
0
0
Relative
Survival
%
100.0
104.4
106.7
94.3
0
0
0
0
Cloning
Efficiency
%
94.5
NC = Negative Control, F12 medium.
145
-------
FIGURE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
EC50 DETERMINATION
SAMPLE #1452 (WATER TANK DRAIN, END OF TEST)
10
1000
CONCENTRATION, pi/ml
146
-------
ASSAY DESIGN NO. 442
RODENT CELL CLONAL (CHO) TOXICITY ASSAY
This assay conforms to one of the bioassay tests in the US Environmental Protec-
tion Agency (EPA) IERL - RTP Procedures Manual: Level 1 Environmental Assessment
Biological Tests (September, 1980). Level 1 bioassays obtain preliminary infor-
•ation on the harmful biological effects of chemicals found in industrial feed
and waste streams by using mammalian cells in culture to measure metabolic
impairment and cellular death.
1. OBJECTIVE
The objective of this assay is to determine the concentration of a test
article that will reduce the colony forming ability of Chinese hamster
cells by 50% after a 24-hour exposure. This concentration is referred to
as the EC50 value.
2. MATERIALS
A. Indicator Cells
The indicator cells used in this study are Chinese hamster CHO-K1
cells (American Type Culture Collection No. CCL 61). This cell type
was derived from ovarian tissue and has spontaneously transformed to
a stable, hypodiploid line of rounded, fibroblastic cells with un-
limited growth potential. Monolayer cultures have a fast doubling
time of 11 to 14 hours, and untreated cells can normally be cloned
with an efficiency of 80% or greater. Permanent stocks are main-
tained in liquid nitrogen and laboratory cultures are maintained by
serial subculturing. Laboratory cultures are periodically checked
by culturing methods for the absence of mycoplasma contamination.
This test system is specified by the IERL- RTP Procedures Manual.
B. Medium
The CHO-K1 cell line has an absolute requirement for proline and
therefore must be maintained in a culture medium containing suf-
ficient amounts of this amino acid. Ham's F12 medium, which contains
3 x 10-4M L-proline, is used, supplemented with 10% fetal bovine
serum, 100 units per milliliter of penicillin, 100 ug streptomycin
per milliliter, and 0.5 pg amphotericin B (Fungizone) per milliliter.
C. Controls
Untreated cells are cloned to establish the control cloning effi-
ciency. If the test article is dissolved in an organic solvent
(usually dimethylsulfoxide), cells exposed to solvent fn the medium
147
-------
2. MATERIALS (Continued)
are cloned to provide the reference cloning efficiency for the effect
of the test article. The final concentration of solvent in the growth
medium is generally IX or less. All controls are performed in tripli-
cate.
D. Sample Forms
Solid samples are tested as a solution or are ground to fine particles
(less than 5 urn) and tested as a suspension in growth medium. Dry
particulate articles, aqueous liquids, suspensions and slurries are
added directly to the growth medium and tested as a suspension or
solution. Liquids containing less than 0.2% organic solvent are
generally tested directly; samples dissolved in organic solvents are
solvent exchanged in dimethylsulfoxide (DMSO) before testing. Original
sample volumes may be reduced a maximum of 10-fold during solvent
exchange, and the concentration factor is used to convert assayed
volumes into equivalent original sample volumes. All sample manipu-
lations are performed as described in the IERL-RTP Level 1 Procedures
Manual.
3. EXPERIMENTAL DESIGN
A. Dose Selection
Unless the approximate toxicity is already known or the sample size
is limiting, the following dose ranges are tested for different sample
forms. Dry particulate articles are dissolved or suspended in growth
medium and tested at five dose levels from 1000 ug/ml to 10 ug/ml.
Aqueous samples, suspensions or slurries are tested from 600 uliters/ml
to 6 uliters/ml in five dose steps. Samples that are solvent exchanged
into DMSO are tested from 20 uliters/ml to 0.2 uliters/ml, also in five
dose steps. Solvent concentrations are IX for all dose levels except
the maximum applicable dose (MAD) of 20 ul/ml, which contains 2% solvent.
A second dose study is performed with an appropriate dose range if the
EC50 has not been properly located in the initial test; EC50 values
greater than 1,000 ug/ml, 600 uliters of aqueous sample/ml or 20 uliters
nonaqueous sample/ml are not determined.
B. Clonal Toxicity Assay
Cells from monolayer stock cultures in logarithmic growth phase are
trypsinized, counted by hemacytometer and reseeded into a series of
60- or 100-mm culture dishes at 200 cells per dish. The cultures
are incubated for 6 to 16 hours at 37°C to allow attachment of the
cells and recovery of growth rate.
Test article is then applied (three dishes per dose), and the cultures
returned to the incubator. If the test article causes a color change,
an additional dish is treated with the high dose and the pH of the
148
-------
3. EXPERIMENTAL DESIGN (Continued)
medium Is determined. The pH is also recorded for the highest dose
which results in a slight color change. After a 24-hour exposure
period the medium is aspirated and the cells washed with Dulbecco's
phosphate buffered saline (PBS; prewarmed to 37°C). The pH of the
discarded medium for which initial pH measurements were made is again
recorded. Fresh medium is placed on each culture and incubation con-
tinued for an additional 6 days to allow colony development. Medium
is drained from the cultures and the surviving colonies are washed
with PBS, fixed in methanol, and stained with Giemsa. Colony counting
is performed by eye; colonies smaller than 50 to 100 cells are not
counted.
4. ASSAY ACCEPTANCE CRITERIA
The assay is considered acceptable for evaluation of the test results if
the following criteria are met:
The average cloning efficiency of the CHO-K1 cells in the nega-
tive controls is 70% or greater, but not exceeding 115%.
The distribution of colonies in the treated cultures is generally
uniform over the surface of the culture dish.
The data points for each test concentration critical to the loca-
tion of the EC50 are .the averages of at least two treated cultures.
A sufficient number of test concentrations are available to
clearly locate the EC50 within a toxicity region as defined
under Assay Evaluation Criteria.
If the EC50 value is greater than 1000 M9/">1, 600 jjliters of
aqueous sample/ml, or 20 uliters of nonaqueous sample/ml, the
plotted curve does not exceed 110% of the negative control.
5. ASSAY EVALUATION CRITERIA
The screened doses, pH values (if appropriate), colony counts, percent
survivals (colony counts relative to control colony counts) and EC50
values are provided. The percent survival is plotted as a function of
applied concentration and the EC50 value determined graphically by fitting
a curve by eye through the data points. The EC50 is used to rank the test
•aterial using the standard evaluation criteria defined in the table below.
Sorbent extracts of known organic content are evaluated as nonaqueous
liquids and in terms of their solid contents (pg orgamcs/ml), and are
ranked using the more sensitive parameter. Where data on the original
liquid or gas sample volumes is provided by the Sponsor the-EC50 value
is calculated in terms of these equivalent volumes per mill11 ter of
culture medium; however, no evaluations are made as yet on this basis.
149
-------
ASSAY EVALUATION CRITERIA (Continued)
Toxicity3
Solids
(EC50 in ug/ml)
Aqueous Liquids
(EC50 in Ml/ml)
Nonaqueous Liquids
(EC50 in Ml/ml)
High
Moderate
Low
Not Detectable
10 to 100
100 to 1000
>1000
<6
6 to 60
60 to 600
>600
.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests.
Criteria for nonaqueous liquids are tentative and under evaluation.
RECORDS TO BE MAINTAINED
All raw data, protocols, protocol modifications, test article weight and
dispensation records and correspondence between LBI and the Sponsor are
being maintained in a central file within the Department of Genetics and
Cell Biology. These records will be filed under departmental assay number
and held up to 2 years following submission of the final report to the
Sponsor. After 2 years they will be transferred to the LBI archives for
permanent storage.
150
-------
Q.A. Inspection Statement
(reference 21 CFR 58.35(b)(7))
LSI Assay No.
TYPE Of STUDY £&.&!+> OtJiQ. (
J i^LOuLJ
This final study report was reviewed by the LBI Quality
Assurance Unit on /Qi. u^3
The short-term nature' of this study precluded inspection while
it was in process. The Quality Assurance Unit inspects an in-process
study of this type approximately once per month to assure that no
significant problems exist that are likely to affect the integrity of
this type of study.
Auditor, Quality Assurance Unit
151
-------
GENETICS ASSAY NO. 5394
LBI SAFETY NO. 6378
MUTAGENICITY EVALUATION OF
SAMPLE #1452
IN THE
AMES SALMONEIEATMICROSOME
PLATE TEST
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20795
LBI PROJECT NO. 20988
REPORT DATE; DECEMBER, 1980
152
-------
PREFACE
This report contains a summary of the data compiled during the
evaluation of the test compound. The report is organized to
present the results in a concise and easily interpretable manner.
The first part contains items I-IX. Items I-IV provide sponsor
and compound identification information, type of assay, and the
protocol reference number. All protocol references indicate a
standard procedure described in the Litton Bionetics, Inc.
"Screening Program for the Identification of Potential Mutagens
and Carcinogens." Item V provides the initiation and completion
dates for the study, and Item VI provides identification of super-
visory personnel. Item VII identifies the tables and/or figures
containing the data used by the study director in interpreting
the test results. The interpretation itself is in Item VIII.
Item IX provides the conclusion and evaluation.
The second part of the report describes the materials and procedures
employed in conducting the assay. This part of the report also
contains evaluation criteria used by the study director, and any
appendices. The evaluation criteria are included to acquaint the
sponsor with the methods used to develop and analyze the test
results.
All test and control results presented in this report are supported
by fully documented raw data which are permanently maintained in
the files of the Department of Genetics and Cell Biology or in the
archives of Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington,
Maryland 20795.
153
-------
I. SPONSOR: Acurex Corporation
II. MATERIAL (TEST COMPOUND): GENETIC ASSAY NUMBER: 5394
A. Identification: Sample Number 1452 and its Extract
B. Date Received : October 6, 1980
C. Physical Description: Neat Sample: Colorless Liquid
Extract : Green Liquid
III. TYPE OF ASSAY : Ames Salmonella/microsome Mutagenesis Assay
IV. PROTOCOL NUMBER: 401 (EPA-LEVEL I)
V. STUDY DATES:
A. Initiation: November 21, 1980
B. Completion: November 25, 1980
VI. SUPERVISORY PERSONNEL:
A. Study Director: D.R. Jagannath, Ph.D.
VII. RESULTS:
The results of this are presented in Tables 1 and 2.
VIII. INTERPRETATION OF RESULTS:
The test material, Sample No. 1452 was received as a clear colorless
liquid (1 gallon) with no visible suspended particulates. An aliquot
of the neat sample (3250 ml ) was concentrated 500 fold by passing
through the XAD-2 resin column at a flow rate of 3 ml per minute. The
resin was then extracted with methylene chloride in a Soxhlet apparatus
for 24 h. The extract (250 ml ) was concentrated to approximately 5 ml
using a Kuderna Concentrator. The concentrated extract was then solvent
exchanged by sequential addition of Dimethylsulfoxide (DMSO) and evapo-
ration of residual methylene chloride in a warm water bath under a
stream of nitrogen. Final sample size in DMSO was 6.5 ml which repre-
sents a 500 fold concentration factor. The concentrate was a clear
solution with a pale blue/green color.
The test materials, neat sample #1452 and its extract were examined for
mutagenic activity in the Ames Salmonella assay in the presence and
absence of liver microsomal enzymes from Aroclor induced rats.
Solvent controls, distilled water for the neat sample and DMSO for the
extract of the sample, and specific positive controls were also assayed
concurrently with the test materials.
154
-------
MNGE: The dose range employed for the neat sample was from 10.0 ul
to 200.0 ul per plate and for the extract it was from 5.0 ul to 100 0 ul
per plate (due to the limited amount of the extract). The organic content
of the sample was not supplied by the sponsor.
TOXICITY; The test materials, neat sample and its extract were not toxic to
any of the strains used in this assays.
RESULTS:
The results of the tests conducted on the test material, neat sample, in the
absence of an activation system was negative. However, the extract of the
sample exhibited genetic activity with the strain TA-98 in the absence of an
activation system. The response was slightly above two times the background
level and was observed at the highest concentration of 100 ul per plate.
The results of the tests conducted on the test materials neat sample and its
extract were all negative in the presence of rat liver activation system.
Dose- related increases in the number of revertants were also observed with the
base-pair substitution strain TA-100 in the non-activation and activation
assays performed on the extract. However these increases did not meet the
criteria for a positive response.
IX. CONCLUSION:
The test material, neat sample #1452 and its extract were tested in the
Ames/Salmonella assay and were compared for their mutagenic activities. The
neat sample did not exhibit genetic activity in any of the assays conducted
in this evaluation and was considered not mutagenic under these conditions.
However, the extract of the sample #1452 exhibited genetic activity with
the strain TA-98 in the nonactivation assays conducted in this evaluation
and is considered as mutagenic. Based on the EPA's "Definition of Toxicity
Categories for Health Effects Tests" the mutagenic activity of the neat
sample #1452 is classified as 'ND1 and the mutagenic activity of the extract
of the sample #1452 is classified as 'low'. Since organic content of the
sample was not available the specific activity has not been calculated.
Reviewed by:
-
JLf
"
Date
Director
Department of Genetics
and Cell Biology
Submitted by:
Study Director
D.R. Jagannath, Ph.D.
Section Chief
Submammalian Genetics
Department of Genetics
and Cell Biology
Date
155
-------
RESULTS
TAHIE I
O1
». MANE OR CODE O'SICNATION OF THC TEST COMPOUND: SAHPLF §14«2 (EXTRACT)
n. SOLVENT: ON2C
c. TCSI INITMTIOW DATES: u/2i/*o
D. TESI COMPLETION OATr: H/It/HO
£• s-9 LOTI: 0*009
NCTC: CONCENTRATIONS *RE 6t«CN IN NICROLITER! PER PLATE
TEST SPECIES TISSUC
NOMCTIVATION
SOLVENT CONTROL .
POSITIVE CONTROL** -~
TEST COMPOUND
10.000000 UL
25.000000 UL —
50.000000 UL —
100.000000 UL
200.000000 UL
ACTIVATION
SOLVENT CONTROL HAT LIVER
POSITIVE CONTROL*** RAT LIVER
TESI COMPOUND
10.000000 Ul RAT LIVER
25.000000 UL RAT LIVER
•0.000000 UL RAT LIvrR
100.000000 UL RAT LIVER
200.000000 UL "AT LIVEN
*•
U-I53S SODIUM A/IDE
TA-153T 9-AHINOACRI01NE
IA-9R 2-NITHI)FLUORTN?
TA-IOO snoiu" tr'.ot
SOLVtNT 200UL/PLAU
TA-1531:
21 39
6?0 662
11 2«
31 :T
32 •!
29 Ifl
22 19
2T 31
163 164
2T 11
?T 19
4? 1*
2H IK
'1 14
»A-1«3» TA-98
13
TIT
R
1
16
R
Q
10
262
10
*
f.
*
T
It
84 R
11
9
^
«
12
16
210
T
1
1.1
t
*
2«
1013
20
3«
22
79
33
46
130?
16
79
?f
2«
16
* * *
21
*90
??
20
??
31
70
•in
1263
2M
3'
tT
•>*,
74
TA-IOA
3*
I
126
T01
142
146
115
12R
139
145
1505
ion
TT
RT
•iy
106
10 UI5/PHT* T«-tS3« 2-ANTMl
sp \f.
tO IJG
/PI 'T'
/PLA^f
10 l|f/PL»Tr
T»-11ST
T A-"R
TA-109
2t(
5
1T6
654
119
142
11?
150
12«
115
1«21
TR
91;
RT
It.
9-»
•AMI NT 2.5 UC/PLATr
7-tKTH'AMMC J.5 U«/PlATr
?*I*JTHI
1AMINC ?.5 UH/PLAf
7-ANTHPAMINr 2.« UG/PLATf
-------
NAPE OP CODE OFSICNATION OP THE TEST COMPOUND: SAMPLE
SOLVENT: ONSO
TEST INITIATION DATES: ll'2!'4n
TEST COMPLETION DATE: ll/2*/HO
S-9 LOTt: 01009
CONCENTRATIONS ARE SIVEN IN MICRCLITER* PER PLATE
A.
B. I
C.
0.
E.
NOTE:
TEST
NCNACTIVATION
(EXTRACT)
SPECIES TISSUE
1A-153S
1 ?
TI-153T
t ?
L A T f
TA-98
1 f
TA-10J
1 2
SCLVEkT CONTROL
POSITIVE CONTROL**
TEST CORPOUN1
5.000000 UL
10.000000 UL
25.000000 UL
50.000000 UL
100.000000 UL
ACTIVATICk
32 35
620 6*2
T T
797 84ft
II
IT
20
23
19
22
!2
23
10
II
1?
16
1"
4
13
14
31
1013
3D
3ft
44
*9
33
31
12
46
144 141
Tfll 654
164 162
1T4 1T3
lift 164
213 199
233 220
Ul
-si
SOLVENT CONTROL RAT
POSITIVE CONTROL*** RAT
LIVER 34 57
LIVER 163 1M
1R 9
262 210
39
1302
3T
153 143
15(« l«23
TEST COMPOUND
5.000000 UL
10.0COOOO UL
25.000006 UL
•0.000000 UL
100.000000 UL
RAT
RAT
RAT
RAT
RAT
LIVER
LIVER
LIVER
LIVER
LIVfR
42
53
26
9*i
32
42
40
3*
4«
34
7
1
13
!
-------
TABLE A
DEFINITION OF TOXICITY CATEGORIES FOR HEALTH EFFECTS ASSAYS*
en
00
Assay9
Ames
RAM
CHO
WAT
'Standard
Ames:
RAM:
CHO:
WAT:
Activity Measured6 Type5 MADd
MEC S 5
(Mutagenesls) AL.NAL 200
ECjo s !
(lethality) AL 600
NAL 20
ECSO S 1
(lethality) AL 600
NAL 20
LDSO S 5
(lethality and AL.NAL 5
toxic signs)
test abbreviations are as follows:
Ames SalMonella/nicrosone Mutagenesls assay
Rabbit alveolar macrophage cytotoxicity assay
Rodent cell clonal toxicity assay
Acute in vivo test in rodents (whole animal test)
Ranae of Concentration or Dosage
Units
ing/plate
pi/plate
mg/ml
Ml /ml
pi /ml
mg/ml
Ml/ml
Ml/ml
gm/kg
ml /kg
High
<0.05
<2
<0.01
<6
<0.2
<0.01
<6
<0.2
<0.05
<0.05
Moderate
0.05-0.5
2-20
0.01-0.1
6-60
0.2-2
0.01-0.1
6-60
0.2-2
0.05-0.5
0.5-0.5
Low Not Detectable
(ND)
0.5-5
20-200
0.1-1
60-600
2-20
0.1-1
60-600
2-20
0.5-5
0.5-5
ND at >5
ND at >200
ND at >1
ND at >600
ND at >20
ND at >1
ND at <600
ND at >20
ND at <5
ND at <5
Standard abbreviations for measured endpoints are as follows:
MEC: Minimum effective concentration
ECSO: Calculated concentration expected to produce effect in 50 percent of population
LD50: Calculated dose expected to kill 50 percent of population
CS = Solid, AL = Aqueous liquid, NAL = Nonaqueous liquid
dMAD = Maximum applicable dose
"Evaluation criteria formulated by Litton Bionetics, Inc. for 1ERL-RTP
Procedures Manual: Level 1 Environmental Assessment BiologicaT Tests.
-------
AMES SALMONELLA/MTCRQSOME PLATE A«;AV
1. OBJECTIVE
assay with and without a mammalian S9 activation"
2. RATIONALE
luxof?^?^ at Ul are all histidine
n
DUte cotMln PJ"dent 5*1 S *re 9rown 1n a m1nimal ^1. petri
to MitldlSi J2.I I?" 1?1s'1d1ne. only those cells that revert
t?ace Liolnt I! !?%"•;•" CMi*) *re able to fon" "lonies. The
h'?Vdlne a11?« all the plated bacteria to undergo
; ?hls-9fowtl? 1s essential for mutagenesis to occu?.
"I6 "Slly $C°red " C0lon1es a9a1nst the sll'9ht
* J6 5pontaneous -utitlon frequency of each strain
const?nt! but when a "«"tagen is added to the agar the
™™?" *- to 100-fold. Cells which grow to
* "inimal media petri P1ates are therefore assumed
to have reverted, either spontaneously or by the action of a test
substance to hj_s+ genotype.
3. MATERIALS
A. Indicator Microorganisms
The Salmonella typhimurium strains.used in this assay were obtained
from Dr. Bruce Ames, University of California at Berkeley.3
following 4 strains were used:
Strain
Designation
TA-1535
TA-1537
TA-98
TA-100
Gene
Affected
his G
his C
his D
his G
Additional Mutations
Repair
A uvr B
A uvr B
A uvr B
A uvr B
IPS R Factor
rfa
rfa.
rfa pKMIOl
rfa pKMlOT
l'5 The
Mutation Type
Detected
Base-pair
substitution
Frameshift
Frameshift
Base pair
substitution
159
-------
3. MATERIALS (Continued)
The aforementioned strains have, in addition to the mutation in the
histidine operon, a mutation (rfa-) that leads to defective lipopoly-
saccharide coat, a deletion that covers genes involved in the synthesis
of vitamin biotin (bio-) and in the repair of ultraviolet (uv) - in-
duced DNA damage (uvrB-). The rfa- mutation makes the strains more
permeable to many large molecules. The uvrB- mutation decreases repair
of some types of chemically or physically damaged DNA and thereby
enhances the strain's sensitivity to some mutagenic agents. The
resistant transfer factor plasmid (R factor) pKMlOl in TA-98 and
TA-100 is believed to cause an increase in error-prone DNA repair
that leads to many more mutations for a given dose of most mutagens5.
In addition, plasmid pKMlOl confers resistance to the antibiotic
ampicillin, which is a convenient marker to detect the presence of
plasmid in the cells.
All indicator strains are kept at 4°C on minimal medium plates supple-
mented with a trace of biotin and an excess of histidine. The plates
with plasmid-carrying strains contain in addition ampicillin (25 ug/ml)
to ensure stable maintenance of plasmid pKMlOl. New stock culture
plates are made as often as necessary from frozen master cultures
or from single colony reisolates that were checked for their genotypic
characteristics (his, rfa. uvrB. bio) and for the presence of plasmid.
For each experiment, an inoculum from the stock culture plates is
grown overnight at 37°C in nutrient broth (Oxoid CM67).
B. Media
The bacterial strains were cultured in Oxoid Media #2 (nutrient Broth).
The selective medium was Vogel Bonner Medium E with 2% glucose7. The
overlay agar consisted of 0.6% purified agar with 0.5 mM histidine,
0.05 mM biotin and 0.1 M Nad according to the methods of Ames et. al_.6
C. Activation
(1) S9 Homogenate
A 9,000 x £ supernatant prepared from Sprague-Dawley adult
male rat liver induced by Aroclor 1254 (described by Ames
et. al_.6) was purchased from Bionetics Laboratory Products,
Litton Bionetics, Inc. and used in this assay.
(2) S9 Mix
Concentration per Milliliter
Components 59 Mix
NADP (sodium salt) 4 umoles
D-glucose-6-phosphate 5 umoles
MgCl2 8 umoles
KC1 . 33 umoles
Sodium phosphate buffer
pH 7.4 100 umoles
Organ homogenate from rat
liver (S9 fraction) 100 uliters
160
-------
4. EXPERIMENTAL DESIGN
A. Dosage Selection
All tests are run at a minimum of four concentrations. In the
Standard EPA Level I Ames assays, five dose levels of the test
material, dissolved in a suitable solvent, are added to the test
system. The standard test doses for the extracted material are
10, 25, 50, 100 and 200 ^liters per plate. The solids are tested
up to 5 mg per plate and at lower concentrations of 2.5, 1, 0.5,
0.1 and 0.05 mg per plate. The samples are retested over a
narrower range of concentrations with strains showing positive
results if there is enough sample.
B. Mutaqenicitv Testing
The procedure used is based on the paper published by Ames et. aj.6
and is performed as follows:
(1) Nonactivation Assay
To a sterile 13 x 100 m test tube placed in a 43°C water bath
the following is added in order:
(a) 2.00 ml of 0.6% agar containing 0.05 mM histidine
and 0.05 mM biotin.
(b) 0.05 nl of a solution of the test chemical to give
the appropriate dose.
(c) 0.1 ml • 0.2 ml of indicator organism(s).
(d) 0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture is swirled gently and then poured onto minimal
agar plates (see 3B, Media). After the top agar has set, the
plates are incubated at 37°C for approximately 2 days. The
number of his-*- revertant colonies growing on the plates is
counted and recorded.
(2) Activation Assay
The activation assay is run concurrently with the nonactiva"
tion assay. The only difference is the addition of 0.5 ml of
S9 mix (see 3C:2, Activation System) to the tubes in place of
0.5 ml of phosphate buffer which is added in nonactivation
assays. All other details are similar to the procedure for
nonactivation assays.
A detailed flow diagram for the plate incorporation assay is
provided in Figure 1.
161
-------
FIGURE 1
REVERSE MUTATION ASSAY
[Agar Incorporation Method]
Molten [43 to 45* C] overlay agar
appropriately supplemented
0.05 ml
0.1 ml to 0.2 ml
Aliquot of 0.5 ml
buffer —
-S9
Test article i positive
control or solvent control
Aliquot of an overnight
culture of bacteria
0.5 mi S9 mix [hepatic
4- SB
** •*— homogenate from
PCS pretreated rat
plus necessary
co factors]
Overlay poured on selective
bottom agar medium
Plates incubated at 37* C for approximately 2 days
Number of revertants per plate counted
\
Data entered onto preprinted forms
Interpretation /conclusion
162
-------
4. EXPERIMENTAL DESIGN (Continued)
C. Control Compounds
C°nt!r01 Con!1st1n9 * the solvent used for the test
« perfofmedA1n a11 "ses. For negative controls, step
«! N°nactlvat10n Assays is replaced by 0.05 ml of the solvent.
* "fSJtive controls are employed for each indicator strain and
are performed In the absence and presence of S9 mix. The solvent
used to prepare the stock solution of the test material is given
in the Results section of this report. All dilutions of the test
material are made using this solvent. The amount of solvent used
is equal to the maximum volume used to give the appropriate test
Specific positive control compounds known to revert each strain are
also used in the assays. The concentrations and specificities of
these compounds to specific strains are given in the following table:
Concentration
A ^ Per Plate Salmonella
Assay Chemical Solvent (ug) strains
Nonactivation Sodium azide Water 1 TA-1535, TA-100
2-Nitrofluorene Dimethyl- 10 TA-98
(NF) sulfbxlde
9-aminoacridine Ethanol 50 TA-1537
(9AA)
Activation 2-anthramine Dimethyl- 2.5 For all strains
(ANTH) sulfoxide
163
-------
5. EVALUATION CRITERIA
Statistical methods are not currently used and evaluation is based
on the criteria included in this protocol.
Plate test data consist of direct revertant colony counts obtained
from a set of selective agar plates seeded with populations of mutant
cells suspended in a semisol id overlay. Because the test material
and the cells are incubated in the overlay for approximately 2 days
and a few cell divisions occur during the incubation period, the
test is semiquantitative in nature. Although these features reduce
the quantitation of result, they provide certain advantages not con-
tained in a quantitative suspension test:
The small number of cell divisions permits potential mutagens
to act on replicating DNA, which is often more sensitive
than nonreplieating DNA.
The combined incubation of the test article and the cells
in the overlay permits constant exposure of the indicator
cells for approximately 2 days.
A. Surviving Populations
Plate test procedures do not permit exact quantitation of the number
of cells surviving chemical treatment. At low concentrations of
the test material, the surviving population on the treatment plates
is essentially the same as that on the negative control plate. At
high concentrations, the surviving population is usually reduced by
some fraction. Our protocol will normally employ several doses
ranging over two or three log concentrations. This does not apply
to spot tests and tests performed on fabrics and like materials which
are tested at a single concentration.
B. Dose-Response Phenomena
The demonstration of dose-related increases in mutant counts is an
important criterion in establishing mutagenicity. A factor that
might modify dose-response results for a mutagen would be the selec-
tion of doses that are too low (usually mutagenicity and toxicity
are related). If the highest dose is far lower than a toxic concen-
tration, no increases may be observed over the dose range selected.
Conversely, if the lowest dose employed is highly cytotoxic, the
test material may kill any mutants that are induced, and the test
material will not appear to be mutagenic.
164
-------
5. EVALUATION CRITERIA (Continued)
C. Control Tests
Positive and negative control assays are conducted with each experi-
ment and consist of direct-acting mutagens for nonactivation assays
and mutagens requiring metabolic biotransformation in activation
assays. Negative controls consist of the test material solvent in
the overlay agar together with the other essential components. The
negative control plate for each strain gives a reference point to
• which the test data is compared. The positive control assay is con-
ducted to demonstrate that the test systems are functional with known
mutagens.
D. Evaluation Criteria for Ames Assay
Because the procedures used to evaluate the mutagenicity of the test
material are semi quantitative, the criteria used to determine positive
effects are inherently subjective and are based primarily on a his-
torical data base. Most data sets are evaluated using the following
criteria:
(1) Strains TA-1535, TA-1537
If the solvent control value is within the normal range, a test
material producing a positive response equal to three times
the solvent control value is considered mutagenic.
(2) Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test
material producing a positive response equal to twice the solvent
control value for TA-98 and TA-100 is considered mutagenic.
(3) Pattern
Because TA-1535 and TA-100 are both derived from the same parental
strain (G-46) and because TA-1538 and TA-98 are both derived
from the same parental strain (D3052), to some extent there is
a built-in redundancy in the microbial assay. In general, the
two strains of a set respond to the same mutagen and such a
pattern is sought. Generally, if a strain responds to a mutagen
in nonactivation tests, it will do so in activation tests.
(4) Reproducibility
If a test material produces a response in a single test which
cannot be reproduced in additional runs, the Initial positive
test data lose significance.
165
-------
5. EVALUATION CRITERIA (Continued)
E: Evaluation Criteria for Toxldty
(1) Complete toxicity
When there are no revertants observed on the plate(s) treated
with the test compound, the test compound is defined as toxic
to all or any of the indicator strains at that (those) particular
dose(s).
(2) Slight toxicity
When there are fifty or less percent revertants on the plate(s)
treated with the test compound as compared to the solvent control
plate(s), the test compound is defined as slightly toxic to
all or any of the indicator strains at that (those) particular
dose(s).
F. Relation Between Mutagenicity and Carcinogenicity
It must be emphasized that the Ames Salmonella/Microsome Plate Assay
is not a definitive test for chemical carcinogens. It is recognized,
howerver, that correlative and functional relations have been demon-
strated between these two endpoints. The results of comparative
tests on 300 chemicals by McCann et. aJL1 show an extremely good
correlation between results of microbial mutagenesis tests and j_n
vivo rodent carcinogenesis assays.
All evaluations and interpretation of the data to be presented in
the final report will be based only on the demonstration, or lack,
of mutagenic activity.
166
-------
REFERENCES
1. McCann,J., Choi, E., Yamasaki, E. and Ames, B.N.: Detection of
carcinogens as mutagens in the Salmonella/microsome test: Assay
of 300 chemicals. Proc. Nat. Acad. Sci. USA, 72:5135-5139, 1975.
2. Ames, B.N., Gurney, E.G., Miller, J.A. and Bartsch, H.: Carcinogens
as frameshift mutagens: Metabolites and derivatives of 2-acetyl-
aminofluorene and other aromatic amine carcinogens. Proc. Nat. Acad.
Sci. USA, 69:3128-3132, 1972.
3. Ames, B.N, Lee, F.D. and Durston, W.E.: An improved bacterial
test system for the detection and classification of mutagens and
carcinogens. Proc. Nat. Acad. Sci. USA, 70:782-786, 1973.
4. Ames, B.N, Durston, W.E., Yamasaki, E. and Lee, F.D.: Carcinogens
are mutagens: A simple test system combining liver homogenates
for activation and bacteria for detection. Proc. Nat. Acad. Sci.
USA, 70:2281-2285, 1973.
5. McCann, J., Springarn, N.E., Kobori, J. and Ames, B.N.: Detection of
carcinogens as mutagens: Bacterial tester strains with R factor
plasmids. Proc. Nat. Acad. Sci. USA, 72:979-983, 1975.
6. Ames, B.N., McCann, J. and Yamasaki, E.: Methods for detecting
carcinogens and mutagens with the Salmonel1a/mammalian-microsome
mutagenicity test. Mutation Res., 31:347-364, 1975.
7. Vogel, H.J. and Bonner, D.M.: Acetylornithinase of E. coli; Partial
purification and some properties. J. Biol. Chem., 218:97-106, 1956.
167
-------
Q.A. Inspection Statement
(reference 21 CFR 58.35(b)(7))
PROJECT 3-C*!?? LBI Assay No.
TYPE of STUDY ft Mrs 7/^-rr Tfsr~
This final study report was reviewed by the LBI Quality
Assurance Unit on /£ / ^ / fo A report of findings was
submitted to the Study Director and to Management on /3~/ s
The short-term nature of this study precluded inspection while
it was in process. The Quality Assurance Unit inspects -an in-process
study of this type approximately once per month to assure that no
significant problems exist that are likely to affect the integrity of
this type of study.
Auditor, Quality Assurance Unit
168
-------
GENETICS ASSAY NO. 5394
LBI SAFETY NO. 6378
TOXIC EVALUATION OF
SAMPLE #1452
(WATER TANK DRAIN, END OF TEST)
IN THE
RODENT QUANTAL TOXICITY
ASSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20795
LBI PROJECT NO. 22064
REPORT DATE: DECEMBER, 1980
169
-------
PREFACE
This report contains a summary of the data compiled during the
evaluation of the test compound. The report is organized to
present the results in a concise and easily interpretable manner.
The first part contains items I - VIII. Items I - III provide
sponsor and compound identification information and identify the
type of assay. The assay was conducted and evaluated according to
procedures recommended in IERL-RTP Procedures Manual: Level 1
Environmental Assessment Biological Tests (Litton Bionetics, Inc.,
Kensington, MD, September, 1980, in press). Item IV provides the
initiation and completion dates for the study, and Item V provides
identification of supervisory personnel. Item VI identifies the
tables and figures containing the data used by the study director
in interpreting the test results. The interpretation itself is in
Item VII. Item VIII provides the conclusion and evaluation.
The second part of the report, entitled Assay Design, describes the
materials and procedures employed in conducting the assay. This
part of the report also contains evaluation criteria used by the
study director, and any appendices. The evaluation criteria are
included to acquaint the sponsor with the methods used to develop
and analyze the test results.
All test and control results presented in this report are supported
by fully documented raw data which are permanently maintained in
the files of the Department of Genetics and Cell Biology or in the
archives of Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington
Maryland, 20795.
Copies of raw data will be supplied to the sponsor upon request.
170
-------
I. SPONSOR: Acurex Corporation
II. MATERIAL (TEST COMPOUND): GENETICS ASSAY NO.: 5394
A. Identification: Sample #1452 (Man Test 5/21/80, #5 Gal, water tank
D n + B • j « drain, end of test)
B. Date Received: October 6, 1980
C. Physical Description: Clear, colorless liquid
III. TYPE OF ASSAY: Rodent Quantal Toxicity Assay
IV. STUDY DATES:
A. Initiation: October 22, 1980
B. Completion: November 24, 1980
V. SUPERVISORY PERSONNEL:
A. Study Director: David J. Brusick, Ph.D.
B. Laboratory Supervisor: Joan McGowan
VI. RESULTS:
The data are presented in Table 1 on pages 3 and 4.
VII. INTERPRETATION OF RESULTS:
The test material, Sample No. 1452, was concentrated 10-fold (as
described under Assay Design) prior to administration by oral gavage
to 10 male and 10 female weanling CD-I mice. A single average dose
of 5 ml/kg was given, which corresponded to 0.08 ml for the males
and 0.07 ml for the females. The animals were observed for 14 days
for toxic signs or lethality. Survivors were weighed and necropsied
on Day 14 after dosing.
All twenty animals survived the exposure with no evidence of any
compound-related toxic signs. Both the male and female mice showed
good weight gains, and no observations were obtained at necropsy
that indicated compound-related lesions (Table 1). The test material
was therefore considered to have no detectable toxicity in this assay,
and according to the Evaluation Criteria, an LD50 determination was
unnecessary.
171
-------
VIII. CONCLUSIONS:
The test material, Sample No. 1452, concentrated 10-fold, had no
detectable toxicity to weanling CD-I mice in the Rodent Quantal
Toxicity Assay. The applied dose was 5 ml/kg.
Submitted by:
Study Director
, £ ^) U/3/lo
JLf '. David J. Brusick, Wi.D. Date
Di rector
Department of Genetics
and Cell Biology
172
-------
TABLE 1
QUANTAL TOXICITY DATA WITH WEANLING MICE
Quanta! Toxicity: Weanling mice
Sponsor: Acurex Corporation
Test Article: Sample No. 1452
Vehicle: Not Applicable
Study Dates: 11/10/80 to 11/24/80
Animal Room No.: 4S
Animals: Charles River CD-I mice, P.O. 100760
Dose: 5 ml/kg administered P.O.
Initial
Height
Animal No. gm
Final Visible
Weight Toxic
gm Signs3
Gross Necropsy Findings
Males
1101
1102
1103
1104
1105
1106
1107
1108
1109
1110
24.2 NTSb Small mucoid lump in bladder
25.5 NTS Both eyes opaque
22.1 NTS Small mucoid lump in bladder
21.7 NTS NSLC
23.2 NTS NSL
22.2 NTS Both eyes opaque
23.7 NTS NSL
23.7 NTS NSL
23.8 NTS -Both eyes opaque
•Mesenteric lymph nodes
slightly enlarged
•Left kidney slightly pale
16.6 25.2 NTS Both eyes opaque
16.1
16.7
14.2
14.4
15.6
13.
16.
13.0
14.8
.5
.2
Mean Body Weight:
Initial 15.1 ± 1.3 gm (Standard Deviation)
Final 23.5 ± 1.3 gm (Standard Deviation)
a Animals observed for 14 days.
b NTS = No Toxic Signs.
c NSL = No Significant Lesions.
173
-------
TABLE 1 (continued)
QUANTAL TOXICITY DATA WITH WEANLING MICE
Animal No.
Females
1111
1112
1113
1114
1115
1116
1117
1118
1119
1120
Initial
Weight
gm
14.1
14.4
14.3
14.5
12.5
14.0
13.3
12.1
13.5
11.9
Final
Weight
gm
19.8
20.0
20.4
20.2
19.0
21.2
18.7
19.9
19.7
16.7
Visible
Toxi c
Signs3
NTSb
NTS
Possible nasal
irritation
NTS
NTS
NTS
NTS
NTS
NTS
NTS
Gross Necropsy
NSLC
NSL
Uterine horns
fluid
NSL
NSL
Uterine horns
fluid
NSL .
NSL
Left kidney si
smaller than
NSL
Findings
enlarged/
enlarged/
ightly
right
Mean Body Weight:
Initial
Final
13.5 ± 1.0 gm (Standard Deviation)
19.6 ± 1.2 gm (Standard Deviation)
aAnimals observed for 14 days.
bNTS - No Toxic Signs.
CNSL = No Significant Lesions.
174
-------
ASSAY DESIGN
PJJANTAL IOXICITY ASSAY
SllLa+-ay S0nf0rm?rJ^°?fn?f the b1oassay tests in the US Environmental
Protection Agency (EPA) IERL-RTP Procedures Manual : Level 1 Environ-
mental Assessment Biological Tests (September. TQan) »o»0i i Mna
obtain preliminary information on the harmful biological effects of
chemicals found in industrial feed and waste streams.
1. OBJECTIVE
The objective of this assay was to evaluate the acute toxicity of a
test material when administered by oral gavage to male and female
weanling mice.
The assay consisted of recording any lethality and toxic signs that
occurred over a 14-day period following a single treatment and then
collecting necropsy information on animals that died or were killed
at the end of the observation period.
2. TEST MATERIAL
A test material described as Sample #1452 (Man Test 5/21/80, #5 Gal,
Water Tank Drain, End of Test) was received as a clear, aqueous
liquid containing no obvious particulate material. The sample was
stored at 4°C in its original one-gallon amber glass container.
An aliquot of 600 ml of test material was concentrated 10-fold to
60 ml by lyophilization prior to testing. A total volume of 1.5 ml
of the concentrate was then utilized for oral dosing of a group of
20 weanling mice. The concentrate was stored at 4°C in an amber
glass bottle.
3. EXPERIMENTAL DESIGN
Nine nursing female Charles River CD-I mice with six pups each (three
male and three female) were obtained from Charles River Breeding
Laboratories, Inc., Wilmington, Massachusetts on November 5, 1980.
The birth date of the pups was October 19, 1980. The animals were
quarantined for 5 days upon receipt. The litters were individually
housed on Absorb-Dri bedding in polycarbonate cages and were cared
for according to Litton Bionetics, Inc., Department of Genetics and
LAMS Standard Operating Procedures. Purina certified laboratory
chow and water (pH 2.5) were provided ad libitum. The pups were
175
-------
3. EXPERIMENTAL DESIGN (continued)
maintained with mothers until weaned. The animals were identified by
eartags and cage cards and were released for study on November 10,
1980.
Prior to initiation of dosing, ten male and ten female weanling mice
were individually weighed; the volume of the test material to be
administered was based on the mean weight values for each sex. The
test material (10-fold concentrate of Acurex Sample #1452) was ad-
ministered once at 5 ml/kg on November 10, 1980. The weanling mice
were 23 days old. The animals were observed at the time of dosing,
2 hours later, and daily thereafter. On day 14, the surviving animals
were fasted overnight. They were subjected to gross necropsy the
next day.
4. ASSAY EVALUATION CRITERIA
Immediately following administration of the test material and at
frequent intervals during the first day, observations of the frequency
and severity of all toxic signs or pharmacological effects (as listed
in Table A) will be recorded. Particular attention will be paid to
the time of onset and disappearance of the signs. Daily observations
will be made and recorded on all animals through a 14-day period. At
termination of the observation period, all surviving animals will be
weighed, killed, and then gross necropsies performed. Necropsies will
also be performed on all animals that die during the course of this
study.
If no mortality occurs in the quantal study, no further studies will
be performed with the test substance and the LDso should be reported
as greater than 5 ml/kg or 5 g/kg. The test material is ranked as
having nondetectable toxicity (ND) at the maximum applicable dose (MAD),
Effluent samples which produce harmful effects in vivo and do not
result in deaths will be noted in the results summary. Such observa-
tions are difficult to quantitate but provide insight into the sub-
lethal effects of a sample on rodents. Further investigations may be
recommended from observations of nonlethal toxic effects.
If a single animal in the quantal study dies in the 14-day observation
period, a quantitative study will be performed. An LDso will be
calculated by the method of Litchfield and Wilcoxin. If the data are
not suitable for calculation of a precise LDso, i.e., total mortality
occurs for the lowest dose, an estimate of the LDso could be made or
the LDso could be expressed as 0.05 ml/kg or 0.05 g/kg or less. Oc-
casionally, it may be necessary to use a different series of dosages
in a repeat study to accurately locate the LDso. The calculated LDso
value is used to rank the toxicity of the test material according to
the dose ranges presented in Table B.
176
-------
TABLE A. DEFINITION OF PHARMACOLOGICAL TOXIC SIGNS
Organ System
Observation and
Examination
Common Signs of Toxicity
CNS and
somatomotor
Autonomi c
nervous system
Respiratory
Cardiovascular
Gastrointestinal
Skin and fur
Mucous membranes
Eye
Others
Behavior
Movements
Reactivity to various
stimuli
Cerebral and spinal
reflexes
Muscle tone
Pupil size
Secretion
Nostrils
Character and rate
of breathing
Palpation of cardiac
region
Events
abdominal shape
feces consistency
and color ,
Vulva, mammary
glands
Penis
Perianal region
Color, turgor,
integrity
Conjunctiva, mouth
Eyeball
Transparency
Rectal or paw skin
Injection site
General Condition
Change in attitude to observer,
unusual vocalization, restless-
ness, sedation
Twitch, tremor, ataxia, cata-
tonia, paralysis, convulsion,
forced movements
Irritability, passivity,
anaesthesis, hyperaesthesis
Sluggishness, absence
Rigidity, flaccidity
Myosis, mydriasis
Salivation, lacrimation
Discharge
Bradypnoea, dyspnoea, Cheyne-
Stokes breathing, Kussmaul
breathing
Thrill, bradycardia, arrhy-
thmia, stronger or weaker
beat
Diarrhea, constipation,
Flatulence, contraction
Unformed, black or clay colored
Swelling
Prolapse
Soiled
Reddening
eruptions
flaccid skinfold,
pi 1oerection
Discharge, congestion,
hemorrhage cyanosis, jaundice
Exophthalmus, nystagmus
Opacities
Subnormal, increased temperature
Swelling
Abnormal posture, emaciation
177
-------
4. ASSAY EVALUATION CRITERIA (continued)
Observations are also made and recorded daily on all animals through
the 14-day period. As in the quantal phase, no attempt is made to
quantitate or rank the observations. The average animal body weight
of each group is determined initially and at the termination of the
experiment. The average weights and the weights as fractions of the
control are reported for each dose level. Necropsy observations are
recorded and reported.
TABLE B. ACUTE IN VIVO RODENT ASSAY EVALUATION CRITERIA
Solids Liquids
Toxicity3 (LDso in g/kg) (LDso in ml/kg)
High
Moderate
Low
Not Detectable
<0.05
0.05 to 0.5
0.5 to 5
>5
<0.05
0.05 to 0.
0.5 to 5
>5
5
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-
RTP Procedures Manual: Level 1 Environmental Assessment Biological
Tests.
5. RECORDS TO BE MAINTAINED
All raw data, protocols, protocol modifications, test article weight
and dispensation records and correspondence between LBI and the Sponsor
are being maintained in a central file within the Department of
Genetics and Cell Biology. These records will be filed under depart-
mental assay number and held up to 2 years following submission of the
final report to the Sponsor. After 2 years they will be transferred
to the LBI archives for permanent storage.
178
-------
Q.A. Inspection Statement
(reference 21 CFR 58.35(b)(7))
PROJECT 1G LBi Assay No.
TYPE of STUDY ?0e/eW Quwr*t.TaK,c;
This final study report was reviewed by the LBI Quality
Assurance Unit on )£ //'/So _ . A report of findings was
submitted to the Study Director and to Management on t
The short-term nature' of this study precluded inspection while
it was in process. The Quality Assurance Unit inspects .an in-process
study of this type approximately once per month to assure that no
significant problems exist that are likely to affect the integrity of
this type of study.
Auditor, Quality Assurance Unit
179
-------
SECTION 6
SAM IA WORK SHEETS
180
-------
I
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tgVR. t
LEVEL 2
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CODE NO.
NAME
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-------
TECHNICAL REPORT DATA
(Pleau nod Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/7-82-038b
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Environmental Assessment of a Low-emission oil-
fired Residential Hot Water Condensing Heating
System; Volume II Data Supplement
5. REPORT DATE
May 1982
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
C. Castaldini
B. PERFORMING ORGANIZATION REPORT NO
10. PROGRAM ELEMENT NO.
9. PERFORMING OROANIZATION NAME AND ADDRESS
Acurex Corporation
485 Clyde Avenue
Mountain View, California 94042
11. CONTRACT/GRANT NO.
68-02-3188
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Office of Research and Development
Industrial Environmental Research Laboratory
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final: 7/80-2/81
14. SPONSORING AGENCY CODE
EPA/600/13
is SUPPLEMENTARY NOTES !ERL.RTp project officer is Robert E. Hall, Mail Drop 65, 919/
541-2477. Volume I. gives technical results.
i6. ABSTRACT The report gives results of a test program measuring air and water emis-
sions from a high-efficiency hot-water residential heating system of European
design, utilizing a condensing flue gas system and a low emission burner. Criteria
and noncriteria emissions, including trace elements and organic species in both flue
gas and condensate waste water streams, were measured. NO (as NO2), CO, total
UHC (as propane), and total particulate emissions measured about 37, 12, 1. 5, and
2.7 ng/J heat input, respectively. Absorption of sulfates and nitrates in the waste
water resulted in a constant pH of 3.0. Total organic emissions in the flue gas mea-
sured 3.5 mg/dscm; they were below the detectable limit in the waste water. Several
inorganic trace elements, including chromium, copper, iron, and nickel, in the
waste water were attributed to leaching of heat transfer metal surfaces by the warm
acidic water. Bioassays were also performed to evaluate the potential health hazard
of the streams. Results indicate nondetectable to moderate toxicity and mutagenicity.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Pollution Waste Water
Assessments Condensates
Hot Water Heating Measurement
Residential Buildings
Fuel Oil
Flue Gases
Pollution Control
Stationary Sources
Environmental Assess-
ment
13B
14B
13A
13M
21D
21B
07D
13. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
193
20. SECURITY CLASS (Thitotge)
Unclassified
22. PRICE
EPA Form 2220-1 (t-7J)
190
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