-------
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.
-------
EPA-600/7-87-Ol2b
March 1987
ENVIRONMENTAL ASSESSMENT OF A WOOD-WASTE-FIRED
INDUSTRIAL WATERTUBE BOILER
Volume II: Data Supplement
by
C. Castaldini and L. R. Water!and
Acurex Corporation
Environmental Systems Division
485 Clyde Avenue
Mountain View, California 94039
EPA Contract No. 68-02-3188
Project Officer: R. E. Hall
Air and Energy Engineering Research Laboratory
Research Triangle Park, North Carolina 27711
Prepared for:
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
-------
ACKNOWLEDGMENTS
The authors wish to extend their gratitude to P. B. Vlalnwright of the
North Carolina Department of Natural Resources and Community Development and
to R. Weeks of the Ethan Allen Corporation. Their Interest and cooperation
in working with Acurex are gratefully acknowledged. The cooperation of D. B.
Harris and J. Montgomery of EPA/AEERL and R. Encke of GCA was also
Instrumental to the success of the test program. Special recognition 1s also
extended to the Acurex field test team under the supervision of B. C. DaRos,
assisted by M. Chips, R. Best, and 0. Holm.
11
-------
CONTENTS
Section Page
1 INTRODUCTION 1-1
2 PRELIMINARY TESTS 2-1
3 BOILER OPERATING DATA 3-1
4 SAMPLING DATA SHEETS 4-1
4.1 CONTINUOUS MONITORING EMISSION DATA
(BY GCA AND EPA) 4-3
4.2 FIELD DATA SHEETS FOR EPA METHOD 5, SASS, AND
CONTROLLED CONDENSATION 4-7
5 ANALYTICAL LABORATORY RESULTS .. . 5-1
5.1 FUEL ANALYSIS 5-3
5.2 PARTICULATE EMISSIONS FROM SASS SAMPLES 5-7
5.3 PARTICULATE EMISSIONS FROM EPA METHOD 5 SAMPLES . . 5-15
5.4 SULFUR OXIDE EMISSIONS FROM CONTROLLED
CONDENSATION SAMPLES 5-25
5.5 TRACE ELEMENT AND LEACHABLE ANION ANALYSES 5-29
5.6 GASEOUS (q to C6) HYDROCARBONS 5-49
5.7 TOTAL CHROMATOGRAPHABLE (TCO) AND GRAVIMETRIC
ORGANICS, INFRARED SPECTRA (IR), AND GAS
CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS) OF
TOTAL SAMPLE EXTRACTS 5-77
5.8 LIQUID CHROMATOGRAPHY (LC) SEPARATION AND INFRARED
SPECTRA OF LC FRACTIONS 5-99
5.9 LOW RESOLUTION MASS SPECTROMETRY (LRMS) OF SELECTED
TOTAL SAMPLE EXTRACTS AND LC FRACTIONS 5-127
5.10 RADIOMETRIC ANALYSIS RESULTS 5-145
5.11 BIOLOGICAL ASSAY RESULTS 5-149
iii
-------
SECTION 1
INTRODUCTION
The purpose of this data supplement is to provide sufficient detail for
researchers to perform their own analysis of the data obtained. Readers are
referred to Volume I (Technical Results) for objectives, description of the
source tested, results, interpretations, and conclusions.
This data supplement contains the following information:
Section 2: Preliminary Tests — Stack velocity traverse and gas
composition tests.
Section 3: Boiler Operating Data ~ Field data sheets of boiler
operating conditions from available test meters; boiler
efficiency calculation using ASME abbreviated test forms.
Section 4: Sampling Data Sheets — Emission data obtained with
continuous monitoring instrumentation operated by EPA and
GCA. Operating data tables for EPA Method 5 (for
particulate mass emissions), Source Assessment Sampling
Systems (SASS) (for particulate mass and size
fractionation, trace elements, and organic emissions), and
controlled condensation (for S02 and $03 sampling).
1-1
-------
Section 5: Analytical Laboratory Results ~ Fuel analyses; laboratory
analysis reports on particulate emissions by gravimetric
analysis; sulfur emissions by turbidimetric analysis; trace
element emissions by spark source mass spectrometry (SSMS)
and atomic absorption spectroscopy (AAS), and Teachable
anion analyses by specific ion electrode; C^ to Cg
hydrocarbons by gas chromatography; total chromatographable
organic (TCO) and gravimetric (6RAV) results; determination
of organic compounds by gas chromatography/mass
spectrometry (GC/MS) in total sample extracts; liquid
chromatography (LC) separation; low resolution mass
spectrometry (LRMS) of selected total extracts and LC
fractions; radiological assay reports for flue gas
particulate and flyash samples; biological assay reports
for flue gas and solid flyash samples for both test 1 (dry
wood) and test 2 (green wood).
1-2
-------
SECTION 2
PRELIMINARY TESTS
2-1
-------
TRAVERSE POINT LOCATION FOR CIRCULAR DUCTS
.0 Q
PLANT .
DATE . V--it-
SAMPLING LOCATION
INSIDE OF FAR WALL TO ^ ~~
OUTSIDE OF NIPPLE, (DISTANCE A) _
INSIDE OF NEAR WALL TO
OUTSIDE OF NIPPLE, (DISTANCE B) _
STACK I.D., (DISTANCE A - DISTANCE B).
NEAREST UPSTREAM DISTURBANCE
NEAREST DOWNSTREAM DISTURBANCE _
CALCULATOR £^05 p. f ? y-
SCHEMATIC OF SAMPLING LOCATION
TRAVERSE
POINT
NUMBER
FRACTION
OF STACK 1.0.
STACK 1.0.
PRODUCT OF
COLUMNS 2 AND 3
go NEAREST 1/8 INCH)
DISTANCE B
TRAVERSE POINT LOCATION
FROM OUTSIDE OF NIPPLE
(SUM OF COLUMNS 4 & 5)
50
\
\
10.
12.
\
3t-
\
37-
/. 13
U-M . o
*•¥-.
7
**•
-------
PRELIMINARY VELOCITY TRAVERSE
PLANT plTH*r*0 PtQ t? r£T . *
DATE m--fT-&;
LOCATION £TVVC.I<_ - eat)£
STACK 1.0. 4 .
^ /
UT" /C.i4-l^< C IWr-PrtCl
TRAVERSE
POINT
NUMBER
/
(
)
\
/
V
)
/
\
j
/
\
)
I
i
\
I
\
1
\
)
*
(f
tL fJ
AVERAGE
VELOCITY
HEAD
(Aps),in.H20
££~
• rr
,(,,0
.90
•bo
•u^
«*-^
•rr*
»yr*
•H"
.*K>
»3^
•3"
•'S^
,/o
• |0
,05"
,05"^
.»s"
• OS
.03
.03
• O 7,
x£7
STACK
TEMPERATURE
(Ts), *F
S30
22£
3Z3
323
3Z3
323
?2.3
J2J
f2?
J23
J2^
JZ2.
32Z
520
5;^
j>2:
3'£
5/7
^/5-
5/S'
5/V-
?0*r
fj/&
o/s^
EPA (Our) 233
4/72
SCHEMATIC OF TRAVERSE POINT LAYOUT
TRAVERSE
POINT
NUMBER
/
]
/
)
/
\
;
(
\
)
i
\
}
i
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\
/
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*
Y*
AVERAGE
VELOCITY
HEAD
^ps), in.H20
,^D
^0
-30
,30
>3to
,30
.20
,2.0
»20
• 20
-2o
•2o
.2o
«2o
.20
>2d
*Zo
.10
.20
• /3T
.10
STACK
TEMPERATURE
(Ts), «F
30
31*7
320
5z/
ZZZ
$22.
523
3z$
22.**-
12+
3-2*
sa^
se-5
32T
J-IJ
^32
52^
52;
^Zt>
3/7
3/k
^2.
V/A.
/°/£.
~~~
2-4
-------
Plant pT.
Date
ISOKINECTIC SAMPLING WORKSHEET
j Reformed by /Z>£c>T~.
Sample Location
Test No./Type A?-
K * 782.687 (Cp)2 (1-BWO)2 Ps Md
M
where: K = Contant of fixed and assumed parameters (dimension!ess)
Pitot coefficient (dimension less)
Water vapor in the gas stream
(proportion by volume)
Absolute stack gas pressure (in. Hg)
Molecular weight, stack gas dry
(Ib/lb-mole)
Orifice coefficient (dimensionless)
Molecular weight, stack gas wet
(Ib/lb-mole) Md(l-BWQ) + 18(8^)
Abolute meter pressure (in. Hg)
782.687 ( )2 (1- )2 ( ) ( )
( )Z ( ) ( )
/w«, //
CP
B«o
• S" hjO
Md
Ko
MS
Pm
K
.-.-,
.10
afc.io
™
.n,i<.
^
Wi
^
2-5
-------
Plant
Date
ISOKINECTIC NOZZLE CALCULATION
AND
SAMPLING RATE CALCULATION
c Performed by_gi£_
Sample Location
Test No. /Type
/v> - S'
k.25
where: N^ ( +460)( )/
AH
Ts
Tm
AP
Nd
,Cp
AH
(AP)
where: AH = Pressure differential across the orifice meter (in
Nozzel diameter
Temperature of
Temperature of
, actual (inches)
gas meter (°F)
stack gas (°F)
Stack gas velocity pressure (in H20)
(._» L
Magic number
x4 ( + 460) / A
; ( + 460) { ' J
i )4
N(j
'm
Ts
AP
AH
K(Nd)4
.Jcsr u
I.Zt*
2-6
-------
ISOKINECTIC SAMPLING WORKSHEET
Ai.t
.t.«r.o
Date
Sample Location
Test No. /Type Z
K = 782.687 (Cp)2 (1-BWO)2 Ps Md
M
where: K = Contant of fixed and assumed parameters (d intension less)
Pitot coefficient (dimensionless)
Water vapor in the gas stream
(proportion by volume)
Tfe= 5O.UO
Absolute stack gas pressure (in. Hg)
Molecular weight, stack gas dry
(Ib/lb-mole)
Orifice coefficient (dimensionless)
Molecular weight, stack gas wet
(Ib/lb-mole) Md(l-BWQ) + 18(BWQ)
Abolute meter pressure (in. Hg)
782.687 ( )2 (1- )2 ( ) ( )
( )Z ( ) ( )
CP
BWO
-^0
Md
KO
MS
Pm
K
.ri
A Z
2<7.r?
^C.u
.01
^"l.d^
in .10
Sriv-.^;
f "t.VAc,*'-'^-^ \
r^L.. Jb
2-7
-------
ISOKINECTIC NOZZLE CALCULATION
AND
SAMPLING RATE CALCULATION
PI ant g^
Date
&r(ir
Performed
Sample Location_
Test No./Type
Nd •
/ AH Ts Y
\KTmAP/
where: N^ - Nozzel diameter (inches)
Average pressure differential across the
orifice meter (in. ^0}
Temperature stack gas, average (°F)
Temperature of gas meter, average (°F)
Stack gas velocity pressure (in ^0)
/ ( ) ( +460) V25
VfrmV ( + 460) ( )/
AH
TS
Tm
AP
Nd
!*
-------
SECTION 3
BOILER OPERATING DATA
3-1
-------
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load. (»o* \\./*,}
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o
tto
/So
$00
S/S
3-3
-------
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7e*r
/o: so
\oadL (icMuAw)
/7
- /i
- /s
0.6
J.o
J.O
1.3
r«lU_UKJ»
3.5
3.S
- OJS
- Atf
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-------
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3-5
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3-6
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3-9
-------
SUMMARY SHEET
ASME TEST FORM m
FOR ABBREVIATED EFFICIENCY TEST
PTC 4.1-o (1964)
TEST MO. If-ir, u.. BOILER NO. 4- DATE - -•*."' C- • OBJECTIVE OF TEST f^.JA. ^ j /?e.,t DURATION g 4fl
BOILER. MAKE * TYPE ^t,*C f*,y** I*:/*, H>*\ j-kfc^ tf^JflcJi k O...1. -£•>.. R»T" CAPACITY **,.*— !«./«.,
STOKER. TYPE i SIZE ^"/^x^ ^-.^r/r t^rU Si^J>£* ft.af a C. f* KO.TJ
PULVERIZER. TYPE ft SIZE j MINE COUNTY STATE SIZE AS FIRED
PRESSURES 1> TEMPERATURES
1
,
1
S
*
7
§
,
IB
11
ta
13
14
IS
'•
17
IB
I*
IB
at
a
n
24
as
M
27
2B
a*
IB
11
aa
33
14
IS
*
STEAM PRESSURE W BOILER DRUM
STEAM PRESSURE AT S. H. OUTLET
STEAM PRESSURE AT R. H. INLET
STEAM PRESSURE AT R. H. OUTLET
STEAM TEMPERATURE AT S. H. OUTLET
STEAM TEMPERATURE AT R.H. INLET
STEAM TEMPERATURE AT R.H. OUTLET
STEAM QUALITY* MOISTURE OR P P M
AIR TEMP. AROUND BOILER (AMBIENT)
TEMP. AIR FOR COMBUSTION
fThiB) IB) Kt)
TOTAL M*AT IUM1T [l*f-, 21 « I»-_41J
1000
HEAT OUTPUT IN BLOW-DOWN WATER
Sit*1 III— J4.U— 20MIKM 27-lta. 21).. VI
OUTPUT 1000
M«.
••••
•*!•
•li.
F
F
F
F
F
F
F
F
F
liw/lk
Bki/lk
Blu^k
km/Ik
M**
IWIk
IWIk
IWIk
iM/lk
Ik/Ik
Ik/Ik
Ik/Vr
IkAr
IkAr
kB/w
kB/W
kBA,
tSA '
/£&
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4fi?3
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60
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—
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/^C^
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PLUI GAS ANAL. (BOILSRHECON) (AIR HTR) OUTLET
CO,
0,
CO
M, (BY DIFFERENCE)
EXCESS AIR
• VOL
• VOL
• VOL
• VOL
T? s*y
4-«/
0_i^
"^9^>
_»_ !.-..?'-<••
CoACOAirXS FIRED
PROX. ANALYSIS
J7
IB
^
40
41
n
MOISTURE
VOL MATTER
FIXED CARBON
ASM
TOTAL
Itu »f Ik AS FIRED
ASH SOFT TEMP.*
ASTM METHOD
FUEL
««t
//,4>i
O.17
in
77/1 '
C8/.L OR Q4L AS FIRED fuo*a i
ULTIMATE ANALYSIS '
43
44
4S
44
1T
40
)7
CARBON
HYDROGEN
OXYGEN
NITROGEN
SULPHUR
ASH
MOISTURE
TOTAL
fe.iT
S.Ji
3>7>73
Ci.li
cicJt
a 5=
//.£><-
COAL PULVIRIZATION
40
4*
SO
44
4S
44
47
4i
4f
70
71
n
GRIMDABILITY
INDEX*
FINENESS STMRU
JOM»
FINENESS • THRU
aeoM*
DATA
51
52
,,
M
41
OIL
FLASH POINT F*
S*. Gravity DM. API*
BURNER SSF
TOTAL HYDROGEN
» «
Bo •» Ik
CAS
S4
ss
54
57
M
]*
40
41
42
43.
41
CO
CH, METHANE
C.H, ACETYLENE
C,H. ETHYLENE
C.M, ETHANE
N,S
CO,
H, HVOROGEM
TOTAL
TOTAL HYDROGEN
• M
»VOL
DENSITY 4B F
ATM. PRESS.
B* PER CU FT
BM PER LB
INPUT.OUTPUT ITEM 31 . 100 _ //V— •»
EFFICIENCY OF UNIT • ITEM 2» ~ ^^ • '*>
HEAT LOSS EFFICIENCY A. F. FUEL
HEAT LOSS DUE TO DRY GAS j?i- 6 V
HEAT LOSS DUE TO MOISTURE IN FUEL / } •?
MEAT LOSS DUE TO H,0 FROM COMB. OF H, <^V g
HEAT LOSS DUE TO COMBUST. IH REFUSE
HEAT LOSS DUE TO RADIATION
UNMEASURED LOSSES
TOTAL
EFFICIENCY • (100 . IM. 71)
*N« R«4»ln4 Iff EHiciWMV T««n««
• •4 A. F
PUEL
""3 J-
1.7V
7 7"f
o
t."3
/ .,-
*/*/. 7
^v3
3-10
-------
ASME TEST FORM
CALCULATION SHEET FOR ABBREVIATED EFFICIENCY
OWNER Of PLANT *r/yy/fln/ fli t gj TEST NO. J.
M
It
u
»
u
14
a
M
t*
»
n
n
HlAT OUTPUT IN BOILER BLOW-OOtm VATER «L§ Or WATE* tLOw-OOm PER HR
tf impractical to wtigA rt/w*t, rfus
ilwn eon it «tMiW«rf e< fo//o«x
».. nfrul, r,n , § Of ,, ,..,,„ rut, . * *SM IN AS FIRED COAL
IWT REFUSE PER LB oc AS HMO run. . 1M ^ s C0iii |N tf ru$f. MMW>f ^
ITEM 41 ITTEK 22 . ITEM 23 "I
CARBON tu*Nco 4-5.27 IS-lxo"5 . £$"*> 1 A«/C ,
rueL "• L u-sco J
D*T CAS rt» k> nco, « 10, • TIN, « coi
•UKNED *COi * C01 / \
ITEHJ2 ITEM 31 [ITEM IS ITEM M 1
. " » .^.r 4 •• ./^.y. • »\7*4. • ft-.X ./
(IT EM 12 ITEM 14 \
.3,*.* .°'."h)
TEST R...W S«p*m»*T. 1965
•OILER NO. OATEV'/Vft
' ITEM 11 ITEM 17
1000
kBAt
NOTEl IP PLUE DUST t ASH
PIT REPUSroTTFER MATERIALLY
IN COMBUSTIBLE CONTENT, THEY
SHOULD BE ESTIMATED
SEPARATELY. SEE SECTION 7.
COMPUTATIONS.
S PIRED PUEI
[ITEM 24
t •"
EXCESS 0, . " ,TE«» - J!!"Ji
.2«2N, - (0, . CO , '" '
^ 3 .2M2 (ITEM IS) . OTEM 31 .
ITEM 14 ,
2
HEAT LOSS EmCIENCY
MEAT LOSS DUE LB OUT CAS ITEM 21 flTEMHI.IITEMIII
T00.t OA, . PERU, AL '^^-^'^^0^:t"^-
-Sr,TTUt.T,ND'ilETL0 • AS* W.?^ • t ««^.« 0, VAPOR AT , P«A » T GA, t»0,
.(ENTNAkPT Of LIOUIOAT T AIR)] • II*M_lL >(( ENTHALPY Of VAPOR
u.a»./,o3( u-so-ia; >••
AT 1 PWA « T1TEM 11) .IEMTHALPT OP LIQUID AT T ITEM ID] •
HEAT LOSS DUE TO H,0 FROM COMB. Of N, • «N, * (
-------
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load.
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load. (io*U./h,)
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ILKnCt^iVt dn* ^ t*>. TTi
0.7
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rr«jx O<-(c^<. «oll. (i
-------
SUMMARY SHEET
ASME TEST FORM *
FOR ABBREVIATED EFFICIENCY TEST
PTC 4. La (1964)
TEST NO. 2 fact Ai^^j BOILER NO. 1 DATE V- /e -£*V
OWNER OF PLANT £r/v.4*J /QJi£*S LOCATION Qt-O i*c.t f , .*L A xt. RATED CAPACITY — #»>-- /&a/i-^-
STOKER. TYPE * SIZE ^"..ei /k^,-^* rs<, A-Jes^.*,** JL Jf/) r^a.-J
PULVERIZER. TYPE » SIZE *&«£T BURNER. TYPE
FUEL USED MINE COUNTY STATE
» uzitfixsi&e*
SIZE AS FIRED
PRESSURES 4 TEMPERATURES PUEL DATA
1
t
1
4
5
4
7
f
10
II
II
1]
14
STEAM PRESSURE IN BOILER DRUM
STEAM PRESSURE AT S. H. OUTLET
STEAM PRESSURE AT R. H. IHLET
STEAM PRESSURE AT R. H. OUTLET
STEAM TEMPERATURE AT R.H. OUTLET
WATER TEMP. ENTERING (ECON.) (BOILER)
STEAMOUALITY* MOISTURE OR P. P.M.
AIR TEMP. AROUND BOILER (AMBIENT)
TEMP. AIR FOR COMBUSTION
TEMPERATURE OP FUEL
GAS TEMP. LEAVING (B».l«r) (Et**.) (Alt Mir.)
GAS TEMP. EMTERIHG AH (H M»«Nm M to
P.I.
MM
M..
f
F
F
F
F
F
F
F
Hft
11$
VIA
&*
SSc
~^A~
/fl
C
fio
AH ft
u $ 7
&
UNIT QUANTITIES
IS
1*
17
1*
1*
10
21
22
21
14
:s
24
27
M
21
M
II
n
n
14
is
14
ENTHALPY OP SAT. LIQUID (TOTAL HEAT)
ENTHALPY OP (SATURATED) (SUPERHEATED)
STM.
ENTHALPY OP SAT. PEED TO (BOILER)
(ECON.)
ENTHALPY OP REHEATED STEAM R.H. INLET
ENTHALPY OP REHEATED STEAM R. H.
OUTLET
HEAT ABS/LBOF STEAM (ITEM 14. ITEM 17)
HEAT ABS/LB R.H. STEAMQTEM If .ITEM 1M
DRY REFUSE (ASH PIT « PLY ASH) PER LB
AS FIRED FUEL
(IK PER LB IN REFUSE (WEIGHTED AVERAGE)
CARBON BURNED PER LB AS FIRED FUEL
DRY GAS PER LB AS FIRED FUEL BURNED
HOURLY QUANTITIES
ACTUAL WATER EVAPORATED
REHEAT STEAM FLOW
RATE OF FUEL FIRING (AS FIRED -rt)
TOT it, UCAT IMfVr BJTSJHJ'J.V" *>')
tooa
HEAT OUTPUT IN BLOW-DOWN WATER
Z°TlTk(lM.*4.lM.»awM27-.M-211tlnnm
OUTPUT 1000
Im/lk
BWIk
BWIk
tWIk
IWIk
IWIk
ktwlk
Ik/Ik
IWIk
Ik/Ik
Ik/Ik
Ik/kt
Ik A.
Ik/lit
kB/W
kB/k.
kBA,
ix1/
U0
—
-
il77
-
^j.i.'/
eftx**
iH\
ffjjtfP1
&XI.
~~ A/A
IC**1
PLUE GAS ANAL. (BOILERI(ECON) (AIR NTR) OUTLET ,..
CO,
0,
CO
H, (BY DIFFERENCE)
EXCESS AIR
* VOL
» VOL
*VOL
c.V
/?.9
« -'{r-is
A'«i)Cfl.£>
n
ULTIMATE ANALYSIS
41
44
45
44
47
40
J7
CARBON
HYDROGEN
OXYGEN
NITROGEN
SULPHUR
ASH
MOISTURE
TOTAL
3».»7
5. fed
tt.ott
n.te)
0.01
/>l'-t
33 &*
COAL PULVERIZATION
40
40
SO
44
GRIND-ABILITY
INOEX*
FINENESS XTHRU
SOM*
FINENESS » THRU
200 M*
INPUT.OUTPUT
EFFICIENCY OP UNIT *
si
SI
5?
44
OIL
PLASH
POINT F*
S».GM«IT D*^-API*_
VISCOSITY AT SSU*
BURNER SSF
TOTAL HYDROGEN
% wt
6AS
^
55
54
57
SO
Sf
40
41
CO
CH, METHANE
C.H, ACETYLENE
C.H, ETHYLEHE
C,Ho ETHANE
H,S
CO,
M, HYDROGEN
TOTAL
42
„
41
TOTAL HYDROGEN
%VOL
DENSITY 40 P
ATM. PRESS.
Bh. PER CU PT
Bw PER LB
ITEM 11 •
ITEM ?
HEAT LOSS EFFICIENCY
M
44
47
,t
4«
70
71
71
HEAT LOSS DUE TO DRY GAS
HEAT LOSS DUE TO MOISTURE IN FUEL
HEAT LOSS DUE TO H,O FROM COMB. OFH,
HE AT LOSS DUE TO COMBUST. IN REFUSE
HEAT LOSS DUE TO RADIATION
UNMEASURED LOSSES
TOTAL
JOO ^ .,-
1 ~ ' *
BtWIk
A. P. PUEL
//"/ *
<4o7
581
ff
•
EFFICIENCY • (100 - IM 71)
(fi fofi y 1 »f •"^t*"' •< Pw. 7.2.1 .1-PYC 4.l-If44
3-16
-------
ASME TEST FORM
:ALCULATION SHEET FOR ABBREVIATED EFFICIENCY
OWNER OF PLANT £r»,)J /»V*f?A/ TEST NO. 2
M
M
a
M
M
M
AT
M
M
TO
71
77
MEAT OUTPUT m tout* ILOW.OOVN WATER «L» OF WATER ILOW.DOWM PER MR
U inprKfico/ to w,igh nlum. rfwi
iMMi eon fct •*• imofvrf o* fe/b»s
».. Bf.w Pf(( L§ or „ n--B -„, . « ASM IN AS FIRED COAL
™ 100 - » COM*. IN REFUSE SAMPLE
ITEM 41 HTBM a ITEM »~]
CARBON IURNEO SJ.Ob" mccbi. . 6ooa \ £3>"
PfR 1 • >t rifrn . '.',.' !?•»••• » 1 77 ,
FUEL »• I '000 J
DRY CAS PER LI nco, « oo, • 7m, « co>
•URNED *eo> * CO) . - - * .
ITEMM ITEM 11 [ITEM 11 ITEM 14]
. " " .67X4 • « . .«M * » V.7?. ? • . I?/. ./
/ITEM» ITEM 14 \
1 » i . .-£"'•?» • . f?*/. . J
EXCESS °» - 4r~ ITEMS - "'***
.2M7N, - to _ CO .
^ j .M01 (ITEM IS) . (ITEM M .
ri«.4.i-» live*'
TEST ftwiW Scprmnfcor. 1 965
•OILER NO. DATE ^- Id-Si
' ITEM IS ITEM 17
1000
NOTEi IP FLUE DUST t ASH
PIT REFUSFBIFFER MATERIALLY
m COMIUSTIILE CONTENT. THEY
SHOULD IE ESTIMATED
SEPARATELY. SEE SECTION 7.
COMPUTATIONS.
S PIREO FUEL • * S)
[ITEM 14 ITEM 47 j
....?. * fft&fs iJ^rt^J
\
ITEM 14 ,
1
NEAT LOIS EFFICIENCY
NEAT LOSS OUE L» DRY CAS ITEMM (ITIMIII-IITCy III
TO DRY CAS * PER LI AS «C, » <«l.| - > • l^T ?aOJ< \ft ,7 ™ /Ji '
. (ENTHALPY OP LIQUID »T T AIR)] • .!I£!L*L « [(ENTHALPY OP VAPOR
ij.gf/too (life- *&) «•
HEAT LOSS OUE TO H.O FROM COM!. OF N, • IN, • ((ENTHALPY OP VAPOR AT 1 PSIA 4 T CAS
LVG) - (ENTHALPY OP LIOUIO AT T AIR)]
• t I ITtM ** 1 [(ENTHALPY OF VAPOR AT 1 PSIA t T ITEM 11) . (ENTHALPY OF LIOUIO AT
100 T ITEM 11)]*
-------
SECTION 4
SAMPLING DATA SHEETS
4.1 CONTINUOUS MONITORING EMISSION DATA (BY GCA AND EPA)
4.2 FIELD DATA SHEETS FOR EPA METHOD 5, SASS, AND CONTROLLED CONDENSATION
4-1
-------
4.1 CONTINUOUS MONITORING EMISSION DATA (BY GCA AND EPA)
Emission results were compiled by GCA into summary tables.
4-3
-------
• VST J. (*
FIFTEEN-MINUTE AVERAGE DATA FOR APRIL 15, 1981
Elapsed
T1__ time
Tlme (min)
1304
1319
1334
1349
1401
1413
1425
1440
1455
1510
1525
1540
1555
1610
1625
1640
1655
1710
1725
1740
1755
1816
1825
1840
1855
1910
1925
1940
1955
274
299
' 314
329
341
353
365
380
395
410
425
440
455
470
485
500
515
530
545
560
575
590
605
620
635
650-
665
680
695
02 (MV)
6.267
6.243
6.258
6.273
6.589
6.281
6.442
6.440
6.613
6.481
6.400
6.519
6.334
6.444
6.232
6.488
6.682
6.397
6.688
6.406
6.382
6.555
6.564
6.476
6.563
6.464
6.315
6.682
6.861
NOX (MV)
2.592
2.665
2.443
2.207
1.899
2.211
2.196
2.237
1.595
1.926
2.043
1.925
2.042
1.777
2.237
2.463
1.548
1.962
1.433
2.072
2.101
1.734
1.720
1.888
1.777
2.196
2.048
1.103
0.846
CO (MV)
3.340
2.444
3.431
4.520
5.707
4.073
4.681
4.785
6.828
6.036
5.188
5.712
5.387
6.407
4.773
5.103
7.593
6.581
7.195
7.085
6.336
7.288
7.778
7.053
7.684
6.764
6.759
7.580
8.375
02 (%)
15.93
15.87
15.91
15.95
16.75
15.97
16.38
16.37
16.81
16.48
16.27
16.57
16.10
16.38
15.85
16.50
16.99
16.26
17.00
16.29
16.23
16.70
16.72
16.50
16.72
16.31
16.09
17.02
17.48
NOX (ppm) CO (ppm)
59.8
61.5
56.3
50.7
43.5
50.8
50.5
51.4
36.3
44.1
46.9
44.1
46.8
40.6
51.4
56.7
35.2
45.0
32.5
47.5
48.2
39.6
39.3
43.2
40.5
50.5
47.0
24.7
18.7
1366
996
1403
1852
2342
1668
1919
1962
2804
2478
2128
2344
2210
2631
1957
2093
3120
2703
2956
2910
2602
2994
3196
2897
3158
2778
2776
3115
3443
4-5
-------
FIFTEEN-MINUTE AVERAGE DATA FOR APRIL 16, 1981
Time
1112
1127
1142
1157
1212
1227
1242
1257
1312
1327
1342
1357
1412
1427
1442
1457
1612
1627
1642
1657
1712
1727
1742
1757
Elapsed
time
(rain)
1502
1517
1532
1547
1562
1577
1592
1607
1622
1637
1652
1667
1682
1697
1712
1727
1802
1817
1832
1847
1862
1877
1892
1907
02 (MV)
5.392
4.956
4.774
4.671
5.111
4.742
5.231
5.141
6.286
6.313
5.365
5.141
5.244
5.794
5.794
5.565
5.549
5.749
6.220
5.762
5.970
6.050
6.011
5.744
NOX (MV)
3.693
3.891
5.596
4.245
3.884
3.983
3.759
3.584
2.385
2.420
3.230
3.747
3.677
2.998
2.814
CO (MV)
1.806
1.116
0.811
0.716
1.108
0.991
2.909
1.254
5.438
5.512
2.657
1.065
1.201
2.913
2.441
02 (%!
13.53
12.43
12.00
11.71
12.82
11.89
13.13
13.00
15.77
15.84
13.46
12.90
13.16
14.54
14.54
3.327 1.312 13.96
tions performed 1500-1600
3.534 1.555 13.92
2.906
1.630
3.073
3.275
2.724
2.861
2.828
2.120
4.255
2.991
2.597
4.183
4.479
2.866
14.42
15.61
14.46
14.98
15.18
15.08
14.41
) NOX (ppm)
85.8
90.4
130.6
98.8
90.3
92.6
87.2
83.2
54.9
55.7
78.4
87.0
85.4
69.4
65.0
77.1
82.0
67.2
37.1
71.1
75.9
- 62.9
66.1
«5.4
CO (ppm)
731
447
322
283
444
396
1185
504
2227
2257
1082
426
482
1187
993
528
628
861
1740
1219
1057
1710
1832
1168
4-6
-------
4.2 FIELD DATA SHEETS FOR EPA METHOD 5, SASS, AND CONTROLLED
CONDENSATION
4-7
-------
r\ ACUREX
£CT^
-------
i
o
SAMPLE
POINT
2l
(o
_f-
/O
/i-
ii
L/l
4
TIME
Sfc.fl
£L5.
57,^
£0.0
U.f
67.S
75*0
;»*-
VELOCITY
HCAO
AP m. wg
ivfl
^c.
.56
5^6
.SO
S
ORIFICE
METER
&H in wg
^
-76
A93
GAS
METER
VOLUME FT*
111. 9
-335*. 6
33.9
35^, S
STACK
306
3-06.
III
30*7
30
PROBE
J25S.
351
27^
^70
35O
TEMPERATURES *F
IMPINGER
ORGANIC
MODULE
OVEN
9V- 1
2,71
*LL
273
J.JO
GAS METER
IN OUT
22.
7JL
7?
7,?
t
^~
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7L
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7/
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62.
2S-
7/T
*5*
7
3
2L
1L
PUMP
VACUUM
f
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-------
ACUREX
PARTICULATE TEST FIELD DATA SHEET
T*« toe Alton .. ^J*£>
/\yt
Run NunilNti ffW
Slack Owmvloi inclMt .
Duel Otjnun&MJnft in « H
3%.'.?
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POINT
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CLOCK
TIME
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55? #
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FILTER DATA
NUMBER
TARE
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IMPINGER
VOLUMES
SILICA
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TEMPERATURES *F
STACK
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813714
813713
813712
PAHTICULATE TEST FIELD DATA SHEET
Tm LocilNHt -
Hun Numb*
Slack OuuiwMr ffichn JjLs..
Duel Dinwntiont m. * m. I^ZZ
Sun Tim* .
Haioowlnc Prauui*.
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NUMBER
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TEMPERATURES 'F
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TEMPERATURES *F
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3/6
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-------
ACUREX
I
4*
PARTICULATE TEST FIELD DATA SHEET
Pmuum
9-lb-?)
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Nonto Sin « NumbM _/*£?_.
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I
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ACUREX.
Corporation
PARTICULATE TEST FIELD DATA SHEET
T.ii LocalMHi Smc* -Our^ff"
Run Number A - S,
. SWIG •««.....« •~O'3"
. SUct Prnur*
Nouto Sin » Number tft*t
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Off
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NUMBER
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TIME
tf;n'fl
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17-38
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ORIFICE
METER
4H in. wg
£7 So
•
co>
Ol
CO
TEMPERATURES *F
STACK
308
a/a
3/3
pfloae
M-OO
H£2_
Hoo
MOO
MOO
Moo
U-oo
Uoo
too
IMPINGER
ORGANIC
MODULE
(.0
(,2.
ta.
Ao
JkSL.
60
ft
OVEN
boo
Hoo
too
AGO
4-00
**9Q .
itloo
uao
Mol
GAS METER
BA
S*
69
JB*L.
OUT
ai
81
fff
St.
8s
8U
16-
ffo
79
» Hg
In.
in-
/ff
ZZ.
78
•JSf
A/rr>t« ntffus
A A*/*
0
/a Vo »*•»>
/i.OS fa**fo»
/3-v» fyiuuoi. > \
'33
r>.«n rrn
-------
poration
PARTICULATE TEST FIELD DATA SHEET
I
*vl
Plant
OaM
Slalte Pf«^u« ,, ...
Tail location - fiUeb »IMI.,I»
Run NumbM Pinh. w..™*..
Slack Diaim
1.1 inclm _. p.lnl riMlliriMil
Ouci DimaniMMt in. • HI
Slid THM
UUU B_. U._l».
OPMMOI __ Ofilic* CMlhciMt ,
SAMPLE
POINT
3170
AUU/lOIAl
CLOCK
TIME
It. S3
VELOCITY
HEAD
AP HI. wg
o-/r
ORIFICE
METER
AH in. wg
ts-s
GAS
METER
VOLUME FT'
/7/P-38S
I0»9-»*f
He
Me
BV
xito Sin • »
lumlMf
UKUlM Waigni
in
FILTER DATA
NUMBER
*
TARE
FINAL WT.
IUPIN
VOL
SIL
G
TEMPERATURES *F
STACK
3lO
3oS
PROBE
H-oo
IMPINQER
ORGANIC
MODULE
*V
OVEN
Hoi
GAS METER
IN
/?U
•
ft
OUT
7?
•6
GEH
MES
:A
/ACUL
m. HI
TIME
•
»
Ntl
<9d
COi Oj CO
«,
.»**
SSs
>J ftfMf
t**»«i rftr
»r if so
A-X-H^
"Z*^<£?,9
Is/* o-r
^ttdT-^a
i «/«l J>
x££?
^//tilf«»»
/-7-.J3
/..»»- A«^
^jH&ILf ^*"*
/<» '3^ yAjs
I/
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
FIELD DATA SHEET
Plant
Date
Sample Location
Run No. /
Ambient Temperature
Barometric Pressure
Meter Box Number
Operator
Meter Orifice Coefficient .-?oSx
Meter a Factor /.or>') /
Clock Time
(24-hr)
clock
c
Sam-
pling
Time,
min
Gas
Meter
Reading
Init.
Temperature (°F)
Stack
Probe
Filter
Skin
Out
Recirc
Water
Exit
Coil
Dry Gas
Meter
In
Out
. (TO
5V)
5*32
V
-77.7
6-0
5V/
5-5Z?
to
Average
4-18
-------
ISOKINETIC PERFORMANCE WORKSHEET & PARTICULATE CALCULATIONS
Plant p-r^y^o AY ;r.o Performed by_
Date v--,c-.^..
Sample Location
Test No./Type \ I cc.<;
Barometric Pressure (in
Meter volume (std),
17 fid I .. a 1 1 .
i / . u1* i -r rv iir
• Hg)
«/i\Ab + ^X
«.,"* 'i'V
13.6 I
_%±) + /
Volume of liquid collected (grams)
Volume of liquid at standard condition (scf)
Vlc x 0.04707
Stack gas proportion of
vw std
water vapor
Vw std + Vm std ( J * ( }
Molecular weight, stack
(Ib/lb-mole)
(% C02x 0.44) + (% 02x
(^.o_ x 0.44) + ( k.o x
Molecular weight, stack
(Ib/lb-mole)
Md(l-Buin) + 18(Bwn), (
x wo wo -
Absolute stack pressure
Pstack (in' H
rb 13.6
gas dry
0.32) + (% N2+ % CO x 0.28)
0.32) + {#0,0+ — x 0.28)
gas wet
(In. Hg)
2°) ^ (-'*)
» '., .....' + 1^ fi
>*
Vm std
vie
vw std
Bwo
Md
Ms
%
^oc
17,26
,-
-
Fr:/~
«.
»,v
7602/5/81/Rev 1
4-19
-------
Temperature stack gas, average (°F)
Stark velocity ffns) • .. .
/T.avg + 460
n^ 4Q (r \ {J~A$ \ 1 --
05.40 (C ) (/4Ps ) / p
^55
/( ) + 460
or /tn i \ ij~ \ /_,... , , . ,
05.49 ( W ) J( )( }
Total sample time (minutes)
Nozzle diameter, actual (inches)
Percent isokinetic (%)
17.33 (Ts + 460)(VW std + Vm std)
e vs PS Nd2
17.33 ( + 460) (( ) + ( ))
( )( )( )( 2 )
Area of stack (ft2) »= 3.1416
»r2H-144-f »(_ }2-H44
Stack gas volume at standard conditions (dscfm)
60 <] * Bwo)Vsavg A- / 528 \ / Ps\
wo avg . / T^ avg + 4gQ i i 2g 921
60 (1 - )( )( )/ 528 \ (( )\
I + 460 / \( 29.92)7
\ ~ ' \ i
Particulate matter concentration, dry (gr/dscf)
15.432 V^ams) lg 432 ( )
Vmstd ( >
Emission rate of particulate matter (Ib/hr)
0.00857 (QJ Cc , 0.00857 ( )( )
J(stdJ
TS
Vs(avg)
e
Nd
%I
AS
QS
r
^c
S(std)
Ep
3iO
i\^
* n* '••"'• -•;
^ a'
^dhiit* 3 4
it wit l*j ;
^^
H\
c-V
Ayy\^,. b\ u
4-20
7602/5/81/Rev 1
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
FIELD CHECKPOINT SHEET
Checkpoint
Supervisor
Initials
QA
Inspector
Remarks
LABORATORY PREPARATION
• Inspect and clean CCC. Both filter holder and CCC
are cleaned with hot chromic acid solution and
O.I. HaO.
• Rinse with acetone and air dry CCC.
• Place Tlssuequartz filter In filter housing.
• Check seal between end of joint and filter.
• Do not use grease on joints.
• Inspect and clean all glass joints.
SITE SETUP
• Rinse the Inside of probe prior to run.
• Rinse probe with acetone until rinse solution 1s
clear.
t Perform leak test.
• Leak rate must be less than 80 ml/mln (0.003 cfm).
• Thermocouple leads attched to probe and filter.
• CCC water bath held at 60°C (140«F) +1<>C.
• Leak test train.
• Probe temperature maintained at 316°C (600°F)
• Gas temperature out of filter holder held at
228°C (550°F).
• Fresh solutions placed 1n Impingers.
• Fresn absorbent replaced 1n final implnger.
• Adjust flowrate in system to 3 1pm.
4-21
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
CHECKPOINT SHEET — Continued
Checkpoint
SAMPLING RUN
t Turn vacuum pump on just before Inserting probe
1n stack.
• Check seal between probe and port to prevent any
outside air from entering stack.
• Run test for 1 hour or until colls are frosted to
1/2 or 2/3 their length.
• After run, cap both ends of probe and lay In
horizontal position.
• Rinse the CCC coils Into the modified Erlenmeyer
flask with a maximum of 40 ml D.I. HgO.
• Was any of the solution lost (Sm\ estimated)?
• After probe has cooled, 1t Is rinsed with a maximum of
40 ml D.I. H20 Into a 25-ml Erlenmeyer flask.
- Was any solution lost (/"ml estimated)?
- Clean support equipment priot to next run.
- Save filter for tltratlon.
Initials
Supervisor
QA
Inspector
—
^
*^
t^~
^"
~^'
-^
*-""
^
Remarks
^— '
Comments:
4-22
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
FIELD DATA SHEET
Plant
Date
Sample Location vmac
Run No. g.
Operator
Ambient Temperature ^
Barometric Pressure
Meter Box Number
Meter Orifice Coefficient
Meter a Factor ;.t>o"?
Clock Time
(24-hr)
clock
Sam-
pi ing
Time,
min
vCZ.
Average
Gas
Meter
Reading
(Vm)>
Init.
(Of, 50
. 10
Temperature (°F)
Stack
Probe
Filter
Skin
Viz
Out
Recirc
Water
to
Exit
Coil
Dry Gas
Meter
In
XI
Out
8-u
-------
ISOKINETIC PERFORMANCE WORKSHEET & PARTICIPATE CALCULATIONS
Plant gVfr*^j A^CAJ Performed by
Date «+ -H fri
Sample Location
Test No./Type o /<•-<-.^
Barometric Pressure (in. Hg)
Meter volume (std),
"•"/iV'b*^
\°Y\Tm + 460/
M «&/(»,*<* + <-ii£>\
17 61 [ if 13'6
1 • yAflfl2.)/y-22J + 46° /
Volume of liquid collected (grams)
Volume of liquid at standard condition (scf)
Vlc x 0.04707
Stack gas proportion of water vapor
Vw std . ( )
v + \i i i + f I
vw std + vm std ( ' ( >
Molecular weight, stack gas dry
(Ib/lb-mole)
(% C02x 0.44) + (% 02x 0.32) + (% N2+ % CO x 0.28)
(.££> 0.44) + (/^x 0.32) + ($A^+ x 0.28)
Molecular weight, stack gas wet
(Ib/lb-mole)
Md(l-BWQ) + 18(BWQ), ( )(1- ) + I8(.oy )
Absolute stack pressure (in. Hg)
Pstack (in' H20) (- ^)
p StaCK + 13.6
Pb
Vm std
Vlc
vw std
Bwo
wu
Md
Ms
Ps
b
2*. go
n.*2(*
-
—
.0 Y
C <^
Cu^^— ~~Y ^
u«-*-i*~- ^ {>
r^ -
-------
Temperature stack gas, average (°F)
Stack vplnritv (frc;^ ...
/T.avg + 460
R^ AQ (T \ tJl\D } / .
85.49 (Cp) (yMP$ avg) / p M
v s s
/( ) + 460
05.10 ( )( ).J( )( }
Total sample time (minutes)
Nozzle diameter, actual (inches)
Percent isokinetic (%)
17.33 (T. + 460)^ std + Vm std)
5 * w rn
0 Vs Ps Nd2
17.33 ( + 460) (( ) + ( ))
( )( )( )( z )
Area of stack (ft2) »= 3.1416
,rr2 -S- 1 44 , it (_ ^2H-1 44
Stack gas volume at standard conditions (dscfm)
60 (1 - Bw)Vs A. / 528 \ / Ps \
wo avg o j ay + 4gQ i (29.92)
Vs / \ /
60 (1 - )( )( )/ 528 \ ((—]\
\ + 460 / V(29.92)/
\-- - / \ /
Particulate matter concentration, dry (gr/dscf)
15 132 MD^rams) 15 /)32 ( )
Vrn-td < }
Emission rate of particulate matter (Ib/hr)
0.00857 (QJ Cc , 0.00857 ( )( )
ista)
Ts
Vs(avg)
0
Nd
XI
AS
^
r
i-c
S(std)
E.
P
2°«
10
4-25
7602/5/81/Rev 1
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
FIELD CHECKPOINT SHEET
Checkpoint
Supervisor
Initials
QA
Inspector
Remarks
LABORATORY PREPARATION
• Inspect and clean CCC. Both filter holder and CCC
are cleaned with hot chromic acid solution and
D.I. H20.
• Rinse with acetone and air dry CCC.
• Place Tissuequartz filter In filter housing.
• Check seal between end of joint and filter.
• Do not use grease on Joints.
* Inspect and clean all glass joints.
SITE SETUP
• Rinse the Inside of probe prior to run.
• Rinse probe with acetone until rinse solution 1s
clear.
• Perform leak test.
a Leak rate must be less than 80 ml/min (0.003 cfm).
• Thermocouple leads attched to probe and filter.
• CCC water bath held at 60°C (140<>F) +1°C.
• Leak test train.
• Probe temperature maintained at 316°C (600°F)
• Gas temperature out of filter holder held at
228°C (550°F).
• Fresh solutions placed In 1mp1ngers.
• Fresh absorbent replaced in final impinger.
• Adjust flowrate in system to 3 1pm.
4-26
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
FIELD CHECKPOINT SHEET - Continued
Checkpoint
SAMPLING RUN
• Turn vacuum punp on just before Inserting probe
In stack.
• Check seal between probe and port to prevent any
outside air from entering stack.
• Run test for 1 hour or until colls are frosted to
1/2 or 2/3 their length.
• After run, cap both ends of probe and lay 1n
horizontal position.
• Rinse the CCC colls Into the modified Erlenmeyer
flask with a maximum of 40 ml O.I. HgO.
• Was any of the solution lost (tf ml estimated)?
• After probe has cooled, 1t 1s rinsed with a maximum of
40 ml D.I. HeO Into a 25-ral Erlenmeyer flask.
- Mas any solution lost ( V'ml estimated)?
- Clean support equipment pHot to next run.
- Save filter for tltratlon.
Initials
Supervisor
QA
Inspector
—
— "
—
^
\~^
^
^
U
U-"
Remarks
--^
Cooments:
4-27
-------
SECTION 5
ANALYTICAL LABORATORY RESULTS
5.1 FUEL ANALYSIS
5.2 PARTICULATE EMISSIONS FROM SASS SAMPLES
5.3 PARTICULATE EMISSIONS FROM EPA METHOD 5 SAMPLES
5.4 SULFUR OXIDE EMISSIONS FROM CONTROLLED CONDENSATION SAMPLES
5.5 TRACE ELEMENT AND LEACHABLE ANION ANALYSES
5.6 GASEOUS (Cj to C6) HYDROCARBONS
5.7 TOTAL CHROMATOGRAPHABLE (TCO) AND GRAVIMETRIC ORGANICS, INFRARED
SPECTRA (IR), AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS)
OF TOTAL SAMPLE EXTRACTS
5.8 LIQUID CHROMATOGRAPHY (LC) SEPARATION AND INFRARED SPECTRA
OF LC FRACTIONS
5.9 LOW RESOLUTION MASS SPECTROMETRY (LRMS) OF SELECTED TOTAL SAMPLE
EXTRACTS AND LC FRACTIONS
5.10 RADIOMETRIC ANALYSIS RESULTS
5.11 BIOLOGICAL ASSAY RESULTS
5-1
-------
5.1 FUEL ANALYSIS
5-3
-------
LABORATORY CKRTinCATK
CURTIS & TOMPKIIVS.LxD.
ANALYTICAL. CH EM I STS -CONSULTING
•AMPLJCIIS — IMCPCCTOMJi
t»o omstoM vrmcT
«AM riuNeiico. CALIF. »«io»
U.S.A.
• M1MMMM3
SlblOO Reported 8/13/81
6437 Sampled
Received 7/07/8!
For ACUREX CORPORATION
Report om 5 samples of Fuel Product
Project So. 7734.12, 7/06/81, Blanket Subcontract RB59186A,
Release Ho. 2.
DRY BASIS EXCZPT AS NOTED
813661 813743
lit 2nd 3ro~~ 1st 2nd 3rd
Test Teat Teat Test Test Test
Carbon (C), < 50.88 53.02
Hydrogen (H), % 6.U 5.44
Oxygen (0), (by
difference), % 42.46 39.40
Witrogen (H), % 0.14 0.08 O.l6 O.l6 0.09 0.20
Sulfur (S),«J 0.04 0.04 0.03 0.03 0.02 0.03
Heating Value:
BTO/Pound : 8,675 8,675
density Ibs/cu ft.
(u ree'd) 14.52 11.95
Ash, 1 0.37 1.95
SAMPLES DISCARDED 3COAVS AFTER RECEIFT UNLESS OTHERWISE REQUESTED
5-5
-------
5.2 PARTICIPATE EMISSIONS FROM SASS SAMPLES
5-7
-------
^Corporation
ANALYSIS LABORATORIES
DATA REPORTING FORM
CUSTOMER OCA
DATE Ally 13. 1981
CUSTOMER CONTRACT NO. 307736.12
RESULTS REPORT TO L. Hater-land
ADDRESS
ACUREX CONTRACT NO. A81-05-030
TELEPHONE
JEthan Allen - 1
SAMPLE KXCiJSTOMEN)
Probe
1u
3u
10U
Filter
XAD
SAMPLE |P (LAB)
648
€44
645
646
660
650
PARAMETER
Weight
0.2308 0.4975 0.8016 0.7865 1.1061 130
gram
ANALYST
ton* tfO-OH «/•>
"^VIEWER.
J.'labash
G. Nlcoll
-------
1SOKIKETIC PERFORMANCE WORKSHEET fc PARTICULATE CALCULATIONS
Plant £-rm* /ki&JPerformed by
Date
Sample Location_
Test No./Type / -
Barometric Pressure (in. Hg)
Keter volume (std),
17.64 /U/Pb4^\
\tt/\V460/
/(^y^/D^o) 4 t^J\
1? 61 ( If 13'6 )
* W.*»7)/U*'g. ) * 460 /
Volume of liquid collected (grams)
Volume of liquid at standard condition (scf)
Vlc x 0.04707
Stack gas proportion of water vapor
Vw$td ^ <3/£y
Vw std 4 Vm std *&& * <2£*fcl
Molecular weight, stack gas dry
(Ib/lb-mole)
(X C02x 0.44) 4 (X 02x 0.32) 4 (X N£+ X CO x 0.28)
//-/«•**" v n ^d^ A f/> T, • rt ^9A A r^\*^A -^-^" -•• n ^B<>
1 7-s * U.^*»J * yjj;^ * U.JIJ + t/7-» * ^Sr X U.£OJ
Molecular weight, stack gas wet
(Ib/lb-mole)
Md(l-8wo) 4 18(8^). Q2^.)(l-^v/) 4 18(^0^)
Absolute stack pressure (1n. Hg)
PstackXln. H.O) (-.1)
f + S18CK.. a * _ fV'1>'^t -^
rb 13.5 • vdL*il * 13.6"
^b
vm std
V1C
Vwstd
Bwo
ww
"d
"s
p.
s
Pf5o
"7^s. $~^;L_
&*>.*?
3/- s-i
o. 07
6v VC
-
7602/5/81/Rev 1
5-10
-------
Temperature stack gas, average (°F)
Stark wploritv ffnO _-
/.avg 4 460
» .,
s $ *
/(?//-£) 4 460
85 49 f /OS 1 ( "n «i/ "1 / ~ • •-
Total sample time (minutes)
Nozzle diameter, actual (inches)
Percent isokinetic (X)
17.33 (T$ 4 460) (Vw std 4 vm std)
9 V$ P$ Nd2
17.33 (3//.£ 4 460)((l£O) 4 Pag))
( ,3."^ )( -TS.t,'* )( -26-^C, )( 2,>^_)
Area of sti>ck (ft2) *« 3.1416
irr2 -T- 144 , ir (J i2-ri-144
Stack gas volume at standard conditions (dscfm)
60 (1 - Bwo)Vs A / 528 \ / Ps \
wo avg > [ 7 jvg 4 -455 1 1 29 92 )
\$ / \ /
60 (1 -.^JCtf^CP.-f?)/ 528 \ f$¥Q\
\J?/-^_ 4 460/ \C 29. 92)7
Particulate matter concentration, dry (gr/dscf )
15.432 V9rara$). 15.432 «
*n:<
0.0 '
/?/s°
C?. O(» ? ^
-C. •
' ^ 't) ^
7602/5/81/Rev 1
5-11
-------
^* Corporation
ANALYSIS LABORATORIES
DATA REPORTING FORM
CUSTOMER CHEA
July 13. 1981
CUSTOMER CONTRACT NO. 307736.12..
RESULTS REPORT TQ L. Water land
ADDRESS
DATE
ACUREX CONTRACT NO. Ml-05-030
TELEPHONE
Ethan Allen - 2
en
ro
Weight
Q.3QB9
0.7075
UflT
oram
foim tCO-061 4/M
ANALYST
J. Labash
G. Nlcoll
REVIEWER .
-------
ISOKINET1C PERFORMANCE WORKSHEET & PARTICIPATE CALCULATIONS
Plant prtfaj #L Performed by
Date ..
Sanple Location
Test No./Type_p .„
Barometric Pressure (in. Hg)
Keter volume (std).
/{/O2*i\ fo$^
vw std * vro std (ti£3 * (2SS3"?7
Molecular weight, stack gas dry
(Ib/lb-mole)
(X C02x 0.44) 4 (X 02x 0.32) + (X N£+ X CO x 0.28)
. (£<_* 0.44) * (/'T'x 0.32) + (7X> —>rD72Bi;
Molecular weight, stack gas wet
( Ib/lb-mole)
Absolute stack pressure (in. Hg)
Pstack 41n- H20) Q^ <^1^
rb u.e . CLJT I3<6
^b
Vm std
Vlc
Vwstd
Bwo
ww
%
"»
P,
s
^.«s"
?sV.->->?
w
«.^
,5.0V/
*1V-«.
7602/5/81/Rev 1
5-13
-------
Temperature stack gas, average (°F)
Stack vploritv ffnO _- —
/T.avg 4 460
ft1; AQ If \ tJ~AP \ IJz* » . •
Oj.49 (Cp) (/4P$ aygj / P$ M$
/( 36-0 4 460
Of. AQ />'')"1C»W O t/~C&( I ., .,_
O^O^KC.^ ji^igg
Total sample time (minutes)
Nozzle diameter, actual (inches)
Percent isokinetic (X)
17.33 (T$ * 460) (Vw std * Vm std)
0 Vs P$ Nd2
17.33 (3&t 4 460)((|22) 4 &&-y$)
( ^o )( ^5..<( )Ctf3x-7( 2x7V/)
Area of stack (ft2) »« 3.1416
irr? -*- 144 , w (__ l2->f-144
Stack gas volume at standard conditions (dscfm)
60 (1 - Bw0)Vsa A / 528 \ / Ps \
wo avg s r T^ ayg + 4&0 i I 25 g?J
60 (1 -0»ittto4fert)/ 528 \ /^^\
^ 7av> 4 460/ \l[29.§2)/
Particulate matter concentration, dry (gr/dscf )
15.432 V9ram$). 15.432 &™<$
vmstd (?«.•> >^1
Emission rate of particulate matter (Ib/hr)
0.00857 (QJ C« , , 0.00857 (/?.^/ )(.$./ $-,?
A ^ .j ^
P*-*Y
p-*>^
o-t **l
m
/ >VW? /
/CK-I
/7.S-7
/£ ?yy
o./Co^
•
X"?.?^/
7602/5/8VRev
5-14
-------
5.3 PARTICULATE EMISSIONS FROM EPA METHOD 5 SAMPLES
5-15
-------
ACUREX
ANALYTICAL REPORT
Sample of:
Sample Pt*
/?
t.99
-------
ACUREX
ANALYTICAL REPORT
Sample of:
Sample
Requested By:
I.D. Number:
Analytical Method:
Date of Analysis
/9f/
Lab I.D. Number
Component
Analytical Result
Unit
NOT
-.or
6
TV of
TW-IT
Analysis
Date
082 i/31
-------
ACUREX
ANALYTICAL REPORT
Sample of:
Sample Pat*
Requested By:
1.0. Number
Analytical Method:
Date Of Analy^ie
Lab I.D. Number
Component
Analytical Result
Unit
. 03
5-ig Analysis By
O
-------
tn
ro
O
: ETItfir* AU.CIO
i >
i >
F-
r7
C.«*JT «
-------
1SOKIKET1C PERFORMANCE WORKSHEET & PARTICIPATE CALCULATIONS
P 1 ant £T**A/ &€*^ ____ Performed by
Date — ^r^
Sample location
Test No./Type /-
Barometric Pressure (1n. Hg)
Keter volume (std),
™(±\h*&\
\a/\V460/
/KtttfV/t*^* (^\
17 6fl( I! 13*6 )
1 ' • e Uxn^J / vbav) * *w y
Volume of liquid collected (grams)
Volume of liquid at standard condition (scf)
Vlc x 0.04707
Stack gas proportion of "water vapor
*..td . <^3
v« std * v» std <£S2> * «*&
Holecular weight, stack gas dry
(Ib/lb-mole)
(X C02x 0.44) 4 (X 02x 0.32) 4 (% N£+ X CO x 0.28)
/.., * t\ AA\ + t ss «n79)^ f«V. A *• A 9A-)
1 JJ.Q x u.^1*/ * i//e * \t*Atj * \yp * •— ~ x V*£QI
Molecular weight, stack gas wet
(Ib/lb-mole)
Hd(l-Bwo) * 18(8^), (^3vKl-o^^ 4 18(o
-------
Temperature stack gas, average (°F)
Stack veloritv ffosl , - -
/T avg * 460
fie; AQ If \ /J"/iD \ /_5 . —
OJ.49 (Cp) &MPS ayg) J ^ M$
/(3oV) * 4SO
Rl AQ f^S V j-i jj-t.i'l /~- ,_ ______
8,.49 (/)X)( 0.^3 JigzjH^
Total sample time (minutes)
Nozzle diameter, actual (inches)
Percent isokinetic (X)
17.33 (T$ * 460)(VW std * Vm std)
6 V$ P$ Nd2
17.33 (?<*> * 460)((|o41) 4 (^c^)
(/3o JC^-JT)^^.^ )( z,^r-6)
Area of Stfck (ft?) »« ,3.1416
*r' -T- 144 , ir (J iz-ri-144
Stack gas volume at standard conditions (dscfm)
60 (1 - Bwo)Vs A / 528 \ / Ps \
wo avg s f ^ ayg 4 4W i ( 2g 92 1
60 (1 -xtfvflfc^t;^/ 528 \ / fe^ \
\_2Sil4 4*0/ \(29.d2)/
Particulate matter concentration, dry (gr/dscf )
15.432 V9ram$). 15.432 <-^i>
Vmstd (^r^iJ
Emission rate of particulate matter (Ib/hr)
0.00857 (QJ C« , , 0.00857 (/- v^ }(.H )
*»a> .^
T$
Vs(avg)
6
»d
XI
AS
's
C.
$(std)
ED
P
Ictf
^•>S"
/*o
o.3o*t
»
?6-l**-
/-).O
/xv^
O'CDl^f S*/n
o/fu
•
%\ ^Y/O *
0-&S~^ ^
?r.v5^? TS
.c/^.to^^
7602/5/BVRev 1
5-22
-------
ISOKIKETIC PERFORMANCE WORKSHEET & PARTICIPATE CALCULATIONS
Plant^^^t/ /&+£*/ . Performed fay_/Wx
Sample Location
Test No./Type ^L -
Barometric Pressure (in. Hg)
Keter volume (std),
17.64 AA/Vi^A
W\V460/
/fcc»:i\ /(*«» * tetf> \
17 61 ( if 13'6 )
17-6 y.^vyy?-?) 4 460 /
Volume of liquid collected (grams)
Volume of liquid at standard condition (scf)
Vlc x 0.04707
Stack gas proportion of water vapor
Wd % <£<£)
vw std 4 v« std l^> 4 «<£2^
Molecular weight, stack gas dry
(Ib/lb-mole)
(X C02x 0.44) 4 (X 02x 0.32) 4 (X Hf % CO x 0.28)
( cO 0.44) * (^Tc> x 0.32) + (7?.-T* — K 0.28)
Molecular weight, stack gas wet
(Ib/lb-mole)
Md(l-Bwo) * 18(8^), L?V>in- ^ ) 4 1B( .oVJ
Absolute stack pressure (in. Hg)
•Wk*1"* H2°) (' 2)
p stacx ^ r-iT
^>-W
x^c.
^•^^
7602/5/81/Rev 1
5-23
-------
Temperature stack gas, average (°F)
Stack velocity (fps)
(cp)
fi
.avg + 460
1.49
O-y?o)
+ 460
Total sample time (minutes)
Nozzle diameter, actual (inches)
Percent isokinetic (*)
17.33 (T$ * 460) (Vw std * Vm std)
$ $
17.33 (>?V_ + 460)((«^C) +
Area of Stack (ft2) »%3.1416
*r < -r- 1 44 , ir (__ _ f+1 44
Stack gas volume at standard conditions (dscfm)
60 (1 - Bwp)Vsavg A. / 528 ' *
^ (VarFU) (*&)
60
/ \ / tZY-^ \
/——-^J ^(?975I)/
s(avg)
/;.
Particulate matter concentration, dry (gr/dscf )
15.432 Vgr*ms)a 15.432
O. /»// /
¥m
std
'$($td)
Emission rate of particulate matter (Ib/hr)
0.00857 (Q ) C. , , 0.00857 L/2* Js. )( .%;/ )
(std) ^ ^
./s
O.i/"J «o «^/<-»'SSW/
5 72-
7602/5/81/Rev 1
5-24
-------
5.4 SULFUR OXIDE EMISSIONS FROM CONTROLLED CONDENSATION SAMPLES
5-25
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
LABORATORY DATA SHEET
Date U.— IV ~4f\
Sample Location
Run No. /-2 64 ( , ¥„)( , PM)
( ,0=)
ppm S02 - j6
5-27
-------
Plant
CONTROLLED CONDENSATION SYSTEM (CCS)
LABORATORY DATA SHEET
r M.i.Ana1yst R.C. .
u - (fc,-?!
Date Lab Analysis Completed
Sample Location
Run No. 2
Method
Titrant
Titration Data
Normality
Indicator
Sample
Description
Sample No.
Vol. of Sample
Vol. of Aliquot
Vol. of Titrant
Used
Average Vol. of
Titrant Used
Probe,
Nozzle
and
Filter
Rinse
A *?>
*KO
/o.o
'**".*?
•osr
G/R
Coil
Rinse
**»
70.0
lo.O
«'« -
.05-
Impinger
Contents
and
Rinse
t»v*
,*.*
10.0
.of
—
.of
Blank
S I ^ ~Q
r
,0.0
'^ .V
' 0^
3%
HoO?
Blank
« JttT
\
/o.o
.05-
t
Calculations
Vol
Meter
. of Gas Sampled (VM) /?.ggp ft3, Avg. Meter Temp (TM) &1 °F
er Pressure (P^) ^?».^ "Hg, Meter a Factor /.g07 dimension
ionless
48.15 ( , MgSOJ(
PPM *
50- 96 1. , VM)(
_. TM+460)
b \ ( tOt)
1 M'
ppm 504 = d
48.15 ( , MgSO,)(
PPM '
500 64 1.. , VM)(
_, TM+460)
P^ \ »'*/
, - MJ
ppm S02 = 0
5-28
-------
5.5 TRACE ELEMENTS AND LEACHABLE ANION ANALYSES
5-29
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
•tNtMl OMICtl: »• NOtTM LA IAIIC (TICIT, CHICAGO. ILLINOH «0«0t • A«CA COOt 111 t».(414
INStMMMNtAt ANAITJIJ DIVISION. 141U WIST MTH AVINUI. COIOIN. CO1OUOO 10401. »HON(. XJ.J7»-«JI
Te! Mr. Roy A. Belletto >*
Acurex Corporation ^5
485 Clyde Avenue
Mountain View, CA 94042
Release No. 5
P.O. No.: Subcontract SW 591 59 A
k
•i •#. •
Date August 21, 1981
Analyst: J. Old ham
Sample No.: A81-05-030-642spARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
EA Filter Blank ,
CONCENTRATION IN yg/cm2
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead *o.04
Thallium
Mercury NR
Gold
Platinum
IHdlum
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetlum
Ytterbium
Thulium
Erbium
Holmlum
Dysprosium
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymlum <0.001
Praseodymium <0.001
Cerium 0.007
Lanthanum 0.008
Barium 0.1
Cesium
Iodine 0.001
Tellurium
Antimony NR
Tin <0.001
Indium STO
Cadmium
Silver
Palladium
Rhodium
•Heterogeneous
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Z1nc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC.
<0.001
0.002
0.02
0.003
0.02
<0.001
0.07
NR
0.003
0.08
0.009
0.005
0.002
0.3
0.007
0.009
IAO No.:97-G852-1 16-25
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium *
Fluorine
Oxygen
N1 trogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
CONC.
0.006
0.8
0.002
MC
0.06
0.07
0.03
0.1
MC
>Q.3
*MC
>0.8
•1
NR
NR
NR
2
0.002
NR
JTO - Intanwl Standard
Nt — Not »»ooft«d
All ctwrwnn net d«t*cnd<
MC - Mtoier Compemnt >
INT ^
0 .
001 ug/cm2
u/cm2
Approved:
5-31
-------
B«ply to
COMMERCIAL TESTING & ENGINEERING CO.
(HlUi QrnClt: 11* NO*TM I* lAUf ITtCCT. CM>C*aO. IILINOK «0«OI • «*IA COOI 111 M».»O4
IMTtVMfNtM AMMTSli OIVIKON. 14139 WflT til* AVINUI. 6O10IN. COIOIAOO 10401. fMONI; J01.27l.fJ2l
>v
Te: Mr. Roy A. Belletto ^^EJJIv
Acurex Corporation ^"*~r..T.
485 Clyde Avenue
Mountain View.CA 94942
Release No. 5
P. O. No.: Subcontract
SW59159A
Sampl* No. A81 -05-030- 651 jPARK SOURCE AAASS SPKTROGRAPHIC ANALYSIS
EA XAO Blank
CONCENTRATION IN PPM WEIGHT
ELEMENT CONC.
Uranium ^0.3
Thorium
Bismuth
Lead 0.4
Thallium
Mercury NR
Gold
Platinum *4
Iridium
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetlum
Ytterbium
Thulium
Erbium
Holmlum
Dysprosium
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymium
Praseodymium
Cerium
Lanthanum
Barium 0.5
Cesium <0.1
Iodine <0.1
Tellurium
Antimony NR
Tin
Indium STO
Cadmium
Silver <0.1
Palladium
Rhodium
•Heterogeneous
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC.
0.4
0.2
<0.1
<0.1
0.3
NR
0.1
3
3
8
<0.1
12
0.6
*2
Date. August 20, 1981
Analyst: J.
Oldham
IAD No.=97-G852-1 16-25
ELEMENT.
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Ni trogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
CONC.
<0.1
6
37
24
7
6
4
17
1
2
4
•0.4
NR
NR
NR
<0.1
<0.1
NR
NR - Net Reported
All «l«m«ot» ngt d«t*a«d<
MC ^ wMjor Cofnoonvnt
INT — Inrarf w*nc*
0.1 ppm
Approved:
5-32
-------
COMMERCIAL TESTING & ENGINEERING CO.
Roplyto
MMMAt OrriCIl: IIS NO«TM LA (All! IT*f IT. CHIC AS 0. H.UNOH (0*01 • »»CA COOC 111
AMAITUS DIVISION. \au WIST «TH AVINUI. OOIOIN. COIOIAOO 10401. maN* »j.j7«.fjji
To: Mr. Roy A. Bellett
Acurex Corporation
485 Clyde Avenue
Mountain View, CA
A
O J^
94942
»
Release No 5
P. 0. No.:Subcontract SM59159A
Swnpte No.: A81-05-030-654SPARK SOURCE AAASS SPECTROGRAPHIC
EA Imp 1 Blank
CONCENTRATION IN ug/ml
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead 0.003
Thallium
Mercury NR
Gold
Platinum
IHdlum
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetlum
Ytterbium
Thulium
Erbium
Holmlum
Dysprosium
STO - Inttrrul Stindvd
NB — Net *»oort«d
All •IwiMMi net d«t«eT«d<
MC — Major Cemeefwnt *
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymlum
Praseodymium
Cerium
Lanthanum
Barium 0.008
Cesium
Iodine 0.002
Tellurium
Antimony NR
Tin 0.02
Indium STO
Cadmium
Silver
Palladium
Rhodium
•Heterogeneous
W/mr1
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
Approved: />(
ANALYSIS
CONC.
0.008
0.001
<0.001
*0.05
NR
0.02
0.005
0.05
0.003
0.01
<0.001
0.004
•I" JCU
_ ./
One August
20, 1981
An»iy»t:j. Old ham
IAD No.: 97-G852-1 16-25
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
CONC.
0.003
0.04
<0.001
0.5
0.1
0.04
0.1
0.1
0.6
0.04
0.03
0.6
-0.7
NR
NR
NR
<0.001
0.01
NR
INT - lnt«rf«rwK*
5-33
-------
COMMERCIAL TESTING & ENGINEERING CO.
• (NIMt. OrriCIt: J»« »0»TM LA SAILC 1TMIT. CMlCAOO. IIUMOIS .0.01 • »tCA COOC 111 »»«••«».
Rtply to wurauMNfM AHMTSIS DIVISION. i«u wist *a» AVINUI. COIOIN. COXMAOO KHOI. >MONI. MTMMI
To: Mr. Roy A. Belletto /^iFa.
Acurex Corporation JkceBk!^
485 Clyde Avenue
Mountain View, CA 94942
Release No. 5
f. 0. No.: Subcontract SW59159A
Swnpl. No.:A81-05-030-661 SPARK SOURCE MASS SPEaROGRAPHIC ANALYSIS
EA-1 fuel CONCENTRATION IN PPM WEIGHT
ELEMENT CONC
Uranium ^.03
Thorium *0.04
Bismuth
Lead 0.4
Thallium 0.03
Mercury NR
Gold
Platinum
Irldlum
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetlum
Ytterbium
ThulHi£CElVES
Erbium 3EP08RECT
Holmlum
Dysprosium
STD - Intwiwl Sf.txi.rd
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium _<0.02
Neodymlum «0.01
Praseodymium 0.02
Cerium 0.1
Lanthanum 0.2
BaHum 21
Cesium 0.06
Iodine 0.09
Tellurium 0.03
Antimony NR
Tin <0.01
Indium STD
Cadmium 0.03
*\
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Z1nc
Copper
Nickel
Cobalt
HaZnese
Rhodium Chromium
•Heterogeneous
Note: Sample low temperature oxygen
ashed prior to analysis.
CONC.
^0.01
<0.01
0.07
0.04
7
0.4
0.4
0.01
NR
0.01
29
6
0.08
0.1
11
>45
0.1
plasma
0«ie August 25, 1981
Analyst: J. Old ham
IAD NO.: 97-6852-116-25
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
* 4
CONC.
0.08
0.05
MC
>54
10
>27
19
MC
>4
MC
>11
»0.6
NR
NR
NR
*0.04
0.03
NR
N« - MM RcperMd
All •IwtMnn net d«Mer«d< 0. Olppm
MC - Mijor Cofflperwnl >100ppm
Approved:
5-34
-------
COMMERCIAL TESTING & ENGINEERING CO.
atxtiAi orncti: »• »O«TM u »*nl »TMM. CHICAAO. IU.IMOI* «o«oi - «CA coot 111 ?i«.(O4
R«0ly tO IMiNUMINtM UtM.ni DIVISION. I4MJ WIST 4JTH AVlNUI. OOIOIN. COlOtAOO KUOI. W.OHI: MMTMfll
To: Mr. Roy A. Belletto y^l-K.
Acurex Corporation AdzuhAX
485 Clyde Avenue
Mountain View, CA 94942
Release No. 5
P.O. No, Subcontract No. SW59159A
StmpJt No: A81-05-030-646SpAR|{ so^g MASS SPECTROGRAPHIC ANALYSIS
EA-1 lOtf + 3u
CONCENTRATION IN PPM WEIGHT
ELEMENT CONC.
Uranium i
Thorium 4
Bismuth
Lead 41
Thallium
Mercury NR
Gold
Platinum
IHdlum
.Osmium
Rhenium
Tungsten 5
Tantalum
Hafnium
Lute t1 urn 0.1
Ytterbium 0.9
Thulium 0.1
Erbium 0.4
Holmlum 0.5
Dysprosium 2
ELEMENT
Terbium
Gadolinium
Europium
Samarium
Neodymium
Praseodymium
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmium
Silver
Palladium
Rhodium
CONC.
1
1
0.5
5
4
2
13
42
MC
0.6
1
0.3
NR
0.4
STD
0.7
4
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Z1nc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC.
10
5
6
7
MC
79
8
0.5
NR
0.5
7
MC
98
17
2
MC
MC
26
•Heterogeneous
o«'e August 19, 1981
Analyst: j. 01 dham
IAD No, 97-G852-1 16-25
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Ni trogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
CONC.
17
MC
0.5
MC
MC
680
MC
MC
MC
MC
MC
MC
*MC
NR
NR
NR
190
<0.1
3
NR
JTO — InMrn*' Sr«nd«rd
NR - Net ««eert«d
All •i«nwnti net d*net«d<
MC - M*(er Component
INT -
O.lpprn
Approved,
5-35
-------
COMMERCIAL TESTING & ENGINEERING CO.
•INUAL OrriCCI: !»• "OITH I* 1*1.1.1 tT»»T. CMICAOO. IILINOI* «0«0t • *•(* COOC 111 TM-1414
Reply to iNitmiMiNUi ANMTSII WVUIOM. ims wist *
ELEMENT
Terbium
Gadolinium
Europium
Samarium
Neodymlum
Praseodymium
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmium
Silver
Palladium
Rhodium
0.001ug/cm2
10ug/cm
-------
COMMERCIAL TESTING & ENGINEERING CO.
R«ply ID
ornett: HI NO»TM i« «ALLI »T»CIT. CHICAGO. IU.INOI* «o«oi • *«i» coot in
INITIUUINTAI ANMVSIl OIVI1IOM. U3M Will 44TH AVlNUI. COIDIN. COIOIAOO MMI. MONI: Xa.J7i.WJ1
To: Mr. Roy A. Belletto /
Acurex Corporation *"
485 Clyde Avenue
Mountain View, CA 94942
Release No. 5
f. O. No.: Subcontract
No. SW59159A
4f-
j=Lk.*s&.
5.mpl« No.A-81 -05-030-6505PARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
EA-1 XAD
CONCENTRATION IN PPM WEIGHT
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead a. 5
Thallium
Mercury NR
Gold
Platinum *2
IHdlum
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetian
Ytterbium
Thulium
Erbium
Holmlum
Dysprosium
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymlum
Praseodymium
Cerium
Lanthanum £.2
Barium 0.4
Cesium 0.3
Iodine *0.2
Tellurium
Antimony NR
Tin
Indium STD
Cadmium
Silver 130
Palladium
Rhodium
•Heterogeneous
ELEMENT CONC.
Ruthenium
Molybdenum 0.4
Niobium
Zirconium 2
Yttrium
Strontium <0.1
Rubidium
Bromine 2
Selenium
Arsenic NR
Germanium
Gallium <0.1
Z1nc 5
Copper 1
Nickel 27
Cobalt 1
Iron 18
Manganese 0.4
Chromium 0.9
0*te August 20, 1981
Analyst: j. Old ham
IAD No.:97_6852-n 6-25
ELEMENT CONC.
Vanadium <0.1
Titanium 2
Scandium <0.1
Calcium 25
Potassium 46
Chlorine 34
Sulfur 160
Phosphorus 3
Silicon 11
Aluminum 2
Magnesium *8
Sodium 64
Fluorine *0.3
Oxygen NR
N1 trogen NR
Carbon NR
Boron <0.1
Beryllium
Lithium 0.1
Hydrogen "R
JTO — lnt*m«l Standard
Nt - Not *«oort«d
All •tafiwnn net dctKMd < O.I ppttl
M€ ••* ^^41 of Cowoofl^rtt
INT — Interference
Approved:
5-37
-------
COMMERCIAL TESTING & ENGINEERING CO.
•CNl**l OFHCC1: «• »O»tN U1AU.C IT«[CT. CMlCAOO. lltlHOIt «0«OI • • «• COOt »« ?3«-t4(«
R*ply «O INSTIUWIN1M ANMTSIS DIVISION. 14319 WIST *it« »VI»»U«. OOIOIN. COtOtADO U49I. MONI, »1 J7HUI
To: Mr. Roy A. Belletto ^^Ft^
Acurex Corporation ^"SrsT"*1
485 Clyde Avenue
Mountain View. CA 94042
Release No. 5
P. O. No.: Subcontract
SW 591 59A
Sampl* No.A81 -05-030-652 SPARK SOURCE MASS SPECTROGRAPHIC
EA-1 Imp 1
CONCENTRATION IN PPM WEIGHT
ELEMENT CONC
Uranium
Thorium
Bismuth
Lead
Thallium
Mercury NR
Gold
Platinum
IHdlum
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetlum
Ytterbium
•Thulium
Erbium
Holmlum ^C^l
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymlum
Praseodymium
Cerium
Lanthanum
Barium 0.05
Cesium
Iodine
Tellurium
Antimony NR
Tin
Indium STD
Cadmium
Silver 0.2
Palladium
wWllrUffl
Dysprosium 3t? Q o flEW""™"^ ^
STO — Internal SKndtuj. itjgjt
All tltnwnt* not d»met«d < 0 . 002 ug/ml
MC - M.,or Component >1 Ouq/ml
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Z1nc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
1981
Approved: X^/
Date September 2, 1981
Analyst: J,
Oldham
ANALYSIS 'AO No.: 97-6852-116-25
CONC. ELEMENT
Vanadium
0.2 Titanium
0.005 Scandium
0.03 Calcium
Potassium
0.004 Chlorine
0.08 Sulfur
0.08 Phosphorus
Silicon .
NR Aluminum
Magnesium
<0.001 Sodium
*1 Fluorine
0.01 Oxygen
0.08 Nitrogen
Carbon
0.1 Boron
0.005 Beryllium
0.2 Lithium
Hydrogen
pc/^
CONC.
0.007
0.1
£0.002
0.4
*0.6
0.4
MC
0.09
MC
0.08
0.7
MC
*3
NR
NR
NR
0.01
<0.001
NR
^
INT — lm*rf*r*n«
5-38
-------
COMMERCIAL TESTING & ENGINEERING CO.
Reply to
U: 13* NO«TH LA 1AILC ITIKT, CMlCABO. llUNOIt «««OI • A«€A COOC 111 Tl|.*41«
iNinuoiNTM ANALYSIS DIVISION, t
-------
COMMERCIAL TESTING & ENGINEERING CO.
•CNI»*i ornctt: it* »O«T« t* SAU.I IT»ctT. CHICAGO, utmoil «040i • A*CA coot lit »»«-«4»4
Reply to iNintiWNfAt ANAITSU DIVISION. i«iu wist urn AVINUI. GOIOIN. coiotAoo KMOI. mONi, roi7i.f»i
TO! Mr. Roy A. Belletto ^r[^
Acurex Corporation ^^r-rv.
485 Clyde Avenue
Mountain Vlew.CA 94042
Release No.
P. o. NO.: Subcontract
6 Exhibit A
No. SM59159A
Sample No.;A81-07-033-1 spARK soURCE MASS SPECTROGRAPHIC
£/) riya^i, JfOefaft, CONCENTRATION IN wg/mL
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead 0.08
Thallium
Mercury NR
Gold
Platinum £0.005
Iridium
Osmium
Rhenium
Tungsten 0.09
Tantalum 0.009
Hafnium
Lutetlum
Ytterbium
Thulium
Erbium
Holmlum
Dysprosium
STO - tnteriul Sf«nd«rd
N* - Net Rmrtcd
All •l«ffi«nt» net d«f«t«d<
MC — Maier Comoonmt >
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymium
Praseodymium
Cerium 0.002
Lanthanum 0.003
Barium MC
Cesium
Iodine 0.05
Tellurium £0.008
Antimony NR
Tin <.0.009
Indium STD
Cadmium 0.002
Silver
Palladium
Rhodium
0.001 wg/mL
1 0 ug/mL
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt .
Iron
Manganese
Chromium
Approved: S
ANALYSIS
CONC.
0.003
0.002
<0.001
MC
MC
0.4
NR
<0.001
0.003
0.02
0.03
0.02
£0.002
6
0.005
0.1
?jf:
D,te October 12, 1981
Analyst: J.
Oldham
IADNo.S7-H437-ll 6-1 3
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
N1 trogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
CONC.
0.08
2
<0.001
MC
MC
MC
MC
0.2
MC
0.1
0.05
MC
«4
NR
NR
NR
0.01
0.01
NR
^&
5-40
-------
cn
r > Corporation
ANALYSIS LABORATORI
CUS
cus
RES
AC
Etl
DATA REPORTING FORM
ES '
TOUCH CMEA nATP
TOMER COI
JLTS REPO
IHRFRS
MTRACTNO
mtff
307736.12
L. Waterland
ACUREX O
TELEPHON
ONTRACT K
f
n A81 -07-033
ian Allen fly ash
SAMPLE 10 (CUSTOMER)
SAMPIE >D (LABI
PARAMETER
r
CL-
Br~
N03-
N02-
so3s
so4-
P04" as P
Nil 4* as N
Units
662
033-1
0.2
140
10
25
59
<2
200
0.04
1.2
mg/l
Blank
<5
<1
<0.1
<0r1
<2
<5
<0.02
<0.5
mg/l
662
033-1
0.8
560
40
100
240
-------
COMMERCIAL TESTING & ENGINEERING CO.
• INOAl OmCCI: III NOITH LA (AlLf ITIItT. CHICAGO. IlLIHOIf «0»OI • AHA COOt 111 ?!••«•»«
R«ply ID INJTtOMINIM ANALYSIS DIVISION. 14115 WIST *t1H AVINUI. OOIOIN. COtOfAOO H*H. fttOHl- XJ.TO-M1I
To: Mr. Roy A. Belletto >^1^
Acurex Corporation ^SSi?^
485 Clyde Avenue
Mountain View, CA 94942
Release No. 5
P. 0. No.: Subcontract SW59159A
S.mpl. No.: A81-05-031-743SPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
EA-2 fuel CONCENTRATION IN PPM WEIGHT
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead 0.3
Thallium 0.04
Mercury NR
Gold
Platinum
IHd1uni
Osmium
Rhenium
Tungsten
Tantalum
Hafnium 0.08
Lutetlum
Ytterbium
Thulium
ELEMENT
Terbium
Gadolinium
Europium
Samarium
Neodymium
Praseodymium
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmium
Silver
Erbiuni* k£ C E 1 V EBal ladium
Holmium i£p 0 8 REC'D Rhodium
Oysprosiui^CUK*^ Note: Sample
STD - lKMrn«l Stindwd plasma
ftjA *j_> a.»__.^...j
CONC.
0.02
0.04
0.2
0.2
36
0.03
0.04
0.05
NR
0.03
STO
0.1
0.08
ELEMENT CONC.
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
low temperature oxygen
ashed prior to analysis
0.07
0.1
0.5
0.06
12
3
0.07
<0.02
NR
<0.01
0.1
22
3
0.3
0.5
MC
>76
0.04
0«i«. August 25, 1981
Analyst: J. Oldham
IAD NO.: 97-G852- 116-25
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
/
CONC.
0.6
9
0.01
MC
>92
4
>47
MC
MC
>7
MC
>19
-7
NR '
NR
NR
0.03
<0.01
0.05
NR
All «l«ifwnt« net d»t«cr«d< O.OlPpm
MC - M«ior Coffloeiwnf >100ppm
INT - Inurforcnc*
Approved:
5-42
-------
feply to
COMMERCIAL TESTING & ENGINEERING CO.
tt: Ml HO*TM IA tALLt ITHIIT. CHICAQO. ItLINOIt «0«OI • A«CA COOC II* ?1*.*«14
ANAlTJIt DIVISION. 14)13 WflT «TH AVINUt. OOIOIN. COtOIAOO MMOI. MONI XJ.J«.»SJI
>.
Te: Mr. Roy A. Belletto ,/tDsL
Acurex Corporation ^SW^
485 Clyde Avenue .*ik_m
Mountain View, CA 94942 RECB.lVc.l-'
Release No. 5 ALJG &8 RECD
P. O. .No.: Subcontract SW591 59A ACUKHX
Swnpta No.A-81-05-030-672SpAR|c SOURCE MASS SPECTROGRAPHIC ANALYSIS
EA-2 10u+ 3u
ELEMENT
Uranium
Thorium
Bismuth
Lead
Thallium
Mercury
Gold
Platinum
Irldlum
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetlum
Ytterbium
Thulium
Erbium
Holmlum
Dysprosium
CONC.
2
3
0.3
63
NR
•2
0.2
2
0.2
0.6
0.8
3
0««e August 21, 1981
A*ulyi*j. Oldham
IAON°-:97-G852-l 16-25
CONCENTRATION IN PPM WEIGHT
ELEMENT
Terbium
Gadolinium
Europium
Samarium
Neodynrium
Praseodymium
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmium
Silver
Palladium
Rhodium
CONC.
0.7
2
0.7
3
4
8
41
72
MC
1
1
NR
1
STO
3
<1
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Z1nc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC.
4
6
*22
13
MC
300
14
2
NR
0.4
5
460
84
6
3
MC
MC
8
•Heterogeneous
ELEMENT
Vanadium
Titanium
Scandi urn
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
N1 trogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
CONC.
29
MC
0.9
MC
MC
880
MC
KC
MH
MC '
MC
MC
-55
NR
NR
NR
150
0.3
22
NR
STD - InMriul Standard
Nl - Not R«oorWd
All •Imwnti
MC - Maior
INT - lnf«rf«
net d»*end<
Camoorwnt
iranc*
O.lppm
Approved: M.-
L -
-------
COMMERCIAL TESTING & ENGINEERING CO.
•CNtMl OFFlCt. 1» I.O«M lAlALLC *T««T, CH.CAOO. ILLINOIS «040. • A.CA CO DC 111 **S-S414
fttply 10 .NStltUMNfM ANAtTSIS DIVISION. 1401 WIST -TM AV|Ng|. OOIOIN. COlOtAOO KMfll. WOM, M9.i7S.tSll
>.
Te: Mr. Roy A. Belletto ^^liLSk.
Acurex Corporation -^ «,. .JS1^
485 Clyde Avenue
Mountain View, CA 94942
Release No. 5
P.O. No.: Subcontract SW 591 59A
Ssmpl. No.-. A81-05-030-674SpARK SOURCE MASS SPECTROGRAPHIC ANALYSIS'
EA-2 lu+ filter
CONCENTRATION IN Vg/cm2
ELEMENT
Uranium
Thorium
Bismuth
Lead
Thallium
Mercury
Gold
Platinum
Irldlum
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetlum
Ytterbium
Thulium
Erbium
Holmium
Dysprosium
CONC
0.002
0.002
0.004
0.2
<0.001
NR
.
0.005
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
ELEMENT
Terbium
Gadolinium
Europium
Samarium
Neodymium
Praseodymium
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmium
Silver
Palladium
Rhodium
CONC.
<0.001
<0.001
<0.001
0.004
0.005
0.002
0.01
0.02
MC
<0.001
0.002
NR
0.004
STD
0.002
0.02
ELEMENT
: Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC.
0.005
3.001
0.03
0.004
0.8
0.3
0.02
0.01
NR
0.001
0.01
6
0.2
0.04
0.002
MC
>0.5
0.02
Dste August 20, 1981
Ansiystrj. oidham
/
IAD No.: 97-6852-116-25
ELEMENT
Vanadium
Titanium
Scandi urn
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Ni trogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
CONC.
0.009
0.6
<0.001
MC
>0.6
0.3
>0.3
>2
>3
>0.05
>4
>0.1
*0.2
NR
NR
NR
>0.9
<0.001
0.007
NR
NR - Net R«eerMd
All tlciTwnti nor d«Mci«d<
MC - Mjiof Cemeerant >
INT — lnr«rf«r*fxa
0.001ug/cm2
Approved:
,-L.
iY 4-V V/
5-44
-------
COMMERCIAL TESTING & ENGINEERING CO.
• INf**k OrriCIS: 121 HOITH I* »ALlf ITCItT. CMlCAOO. IIUNOI* *0«OI • «•«» COOC lit »J«-«O4
Rtply tO INlTIUMtNIM ANAlTSll OIVI1ION. I4MJ Wljf *«» AVINUi. OOIDIN. COtOIAOO KHOI »HONI M3.17t.f91l
T<» Mr. Roy A. Belletto ./>£*
Acurex Corporation ^SfR??^
485 Clyde Avenue
Mountain View. CA 94942
Release No. 5
0«ie August 20. 1981
An«ly$t:J. Old ham
P.O. No.: subcontract SW59159A
S.mpl« No.: A81-05-030-676SPARK soyRcg /y^ASS SPEaRCXSRAPHlC ANALYSIS
CONCENTRATION IN PPM WEIGHT
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead 0.4
Thallium
Mercury NR
Gold
Platinum 2
Ir1d1um
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetlum
Ytterbium
Thulium
Erbium
Holmlum
Dysprosium
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymlum
Praseodymium
Cerium
Lanthanum
Barium 0.8
Cesium
Iodine 0.1
Tellurium
Antimony NR
Tin
Indium STD
Cadmium
Silver *2
Palladium
Rhodium
•Heterogeneous
ELEMENT CONC.
Ruthenium
Molybdenum 1
Niobium
Zirconium 0.5
Yttrium
Strontium 0.2
Rubidium
Bromine 2
Selenium
Arsenic NR
Germanium
Gallium
Zinc 25
Copper 10
Nickel 67
Cobalt
-------
COMMERCIAL TESTING & ENGINEERING CO.
•INtlAl OrriCIl: »• MO«TN LAtAlLI «T»HT. CHICAGO. ILLINOI* «fl«l • AMA COOl «1J Tl*-*4>4
Reply to uariuMNTAi AHAIOIS DIVISION. mii wist *a* AVINUI. GOIOIN. COIOIAOO IMOI. WON* 303 wwji
To: Mr. Roy A. Belletto >
Acurex Corporation A^
485 Clyde Avenue
Mountain Vlew.CA 94942
Release No. 5
P. 0. No.: Subcontract SW591 59A
4p.
=LL=k
MM* **r*
0(te August 21. 1981
Analyst: J. Oldham
S*mpl* No.: A81-05-030-678spARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
CONCENTRATION IN Jig/ml
ELEMENT CONC.
Uranium
Thorium
Bismuth
Lead 0.01
Thallium
Mercury NR
Gold
Platinum
Irldlum
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetlum
Ytterbium
Thulium
Erbium
Holmlum
Dysprosium
STO — Internal Standard
All •Icnwnti not d»f«ct»d<
MC - M«jor Comporwnt >
ELEMENT
Terbium
Gadolinium
Europium
Samarium
Neodymlum
CONC.
0.009
Praseodymium 0.002
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmium
Silver
Palladium
Rhodium
O.OOIpg/ml
1 Ovg/mT
0.005
0.006
0.2
5
0.1
MC
>0.8
0.9
>2
-3
NR
NR
NR
0.002
0.001
NR
S2^^
INT — lnl«rf«r*nc*
Y4~?v *i
5-46
-------
R«ply to
COMMERCIAL TESTING & ENGINEERING CO.
NttAl OrriCti )» «0«TM k* lAlK ITICCT. CMICAOO. IlLINOK iO«01 • A«A COOt 119 ?J«-««1«
INttniMINIM ANAiniS DIVISION. IOU WH» 44«« AVfNUl. OOlDtW. COIOIAOO KMOI. »
-------
5.6 GASEOUS (Cj to C6) HYDROCARBONS
5-49
-------
Onsite Gas Chromatography Results3
Dry Wood Fuel, 4-15-81
Time
18:53
18:56
Run No.
16
17
Cl
39.5
16.9
C2
43.2
18.0
C3
2.8
2.9
C4
5.7
1.3
C5
4.9
0.8
C6
1.3
1.9
Met Wood Fuel, 4-16-81
Time
12:45
13:01
16:35
16:40d
Run No.
1
2
3
4
Cl
5.2
4.6
1.3
6.1
C2
5.9
2.0
4.0
3.0
C3
1.8
2.6
—
2.0
C4
7.1
__c
0.4
0.4
C5
<3.8b
0.1
—
0.6
ce
<8.5b
<4.6b
<1.5b
<7.7b
aAll ppm values ±10 percent
bVa1ue is higher than actual due to excessive noise
CNot detected
^Burnout in furnace
5-51
-------
GAS CHROMATOv .APH OPERATING CONDITIONS AND FIELD LOG
Client
_, Location
Job No. <3o">bO7..''M
Injection Date w.-.;T-.?i . Time ij^
Recorder/Printout Reference No. ^
Purpose of Run OuJ.^/z v->Xo
. Instrument ID
, Recorder ID
C. -£., /-r-
Sample Description
,* -r TV
GC CONDITIONS
Amount Injected 2.Ois*>_ . Inj. Port or Sample Loop Used 2.Qf*i Loof
Detector Used: FID x « ECD _ t FPD _ , TCD _ (Current _ J
Detector Attenuation \ _ , Amplifier or Range to"* _
Column: Liquid Phase _ , Solid Phase
Length /„' O.D. V* " 1.0.
Temperature: Injector \ *^o ° C, Oven
Temperature Program
, Material
, Detector
SAMPLE RUN
Sampling Method
RT
Area
Peak Height
Amount
Component
,5*4,37-
Name of Operator AT K.
, Date
- 5ir
5-53
-------
Column No. .................. length. ..... t.f. ...... Ota....^.". ........
Cottlng. ............. ...«....« ........... .Conen*... .......................
Support...E»tAWr.K..O ...... ......... .....Mwh.V.o/iQ...
TEMP: Cot lirit ............... f.30t...*G Fkwl....l.to ..... °C
Ritt ........... "0/11*1. DeLV^O....^ In). ..... AJa....°C
CARRIER OAS..H*« ............... R«
PratMirM! Inlrt, —
DETECTOR E.C. -------------- .T.C.
Scivcnf 6f . ..**
Svnt......
F.I.O..X. ...............
*i»ifnL/nrin.
, ..... mv.
Solvmt
.Conen ................. '. ................
tn
l
in
R
a
! J
iiiniii -i
I is p-f^P* Is
-------
GAS CHROMATOv >APH OPERATING CONDITIONS AND FIELD LOG
Client
_, Location j
Job No.
Injection Date t-.^x ! , Time ,?:'+2: /v . Instrument ID
Recorder/Printout Reference No. /5" , Recorder ID
Purpose of Run C^L.K £i«vr,/-'vo
-c_
Sample Description
'TTV
f'VJ) 'v-T '. « f
IN't , y — 1* '
GC CONDITIONS
Amount Injected 2.O/SAL . Inj. Port or Sample Loop Used ^.o^>. Loof
Detector Used: FID x . ECD _ , FPD _ , TCD _ (Current _ )
Detector Attenuation \ _ , Amplifier or Range IQ-* _
Column: Liquid Phase _ , Solid Phase
Length {„• O.D.
Temperature: Injector
Temperature Program
I.D.
c, Oven
, Material
c , Detector
SAMPLE RUN
Sampling Method p
RT
Area
Peak Height
Amount
Component
0s?
v.r;
Name of Operator M. K. r.**/
, Date
^ fr
5-55
-------
. ........ Dat«..Y-!£-.Sr.{
Column No ........... . ....... Length ...... tx' ...... Dla....
Coating ................................... .Concn..
TEMP: Col: Intt ......... . ...... /.3CL..°C Flnal..f.3o
Rat* ............ °C/mln. Det/3JL..°C \n\...i3ff.
CARRIER GAS.. N*- .............. Rate...Cf.Of6,|.
Pressures: ln!ei...» .................. Outlet
DETECTOR E.C ............... .T.C ................
Scavenger .................... .s..Rate .......... ................mL/mln.
Sens. ........ v ................. Rec.Range ............................ mv.
SAMPLE..OAM&JR*».T.IflAi ........ .
Solvent .............................. Conen
ID
to
I
I
I
3"
f
If
I
to
la
-------
GAS CHROMATG. .APH OPERATING CONDITIONS AND FIELD LOG
Client
_, Location
Job No.
, »o,«-.
Injection Date H -.£"-?! . Time iT-SJ.'H6? , Instrument ID
Recorder/Printout Reference No. ;u . Recorder ID
Purpose of Run . -c A
.? « -
Sample Description
GC CONDITIONS
Amount Injected 2,Ot*+L. . Inj. Port or Sample Loop Used 2.o«**i loof
Detector Used: FID x . ECD _ , FPD _ , TCO _ (Current _ )
Detector Attenuation \ _ , Amplifier or Range IQ-* _
Column: Liquid Phase _ , Solid Phase Py>e«p»>v<^ n •
Length („• O.D. */<» " I.D. _ , Material
Temperature: Injector \-*>Q c. Oven
Temperature Program
c . Detector
SAMPLE RUN
Sampling Method
RT
Area
Peak Height
Amount
Component
/.l-D
Name of Operator /vi.K. r
.. Date
19
5-57
-------
RT
4,. It
i.rr
3-1.70
Area
tip*2C
V.^'Z^
ktTr.~
Peak Height
\
;
Amount (p^4
*•
-------
Column No.. Length kf. Dta..../Jr.?.
Coating.... , .Goncri*
Support..R*fefc«*rt«».6l. .....Me
TEMP: Col: Inlt j.3o..°C final
Rate °C/mln. Det/la....°C InJ. 13.9. °C
CARRIER GAS..H£~. Rate..4».tl>ti
Pressures: Inlet;..... .Outlet........
Hydrogen.H.Q.|ttr/....mli/mln. Alr.fiK).^.SJ....
DETECTOR E.C .T.C F.I.O..X
Scavenger ....Rate «..............ml./mln.
Sens Rec.Range mv.
SAMPLE ijQ£S......?t?.r.&«lU& Slze^JC?jft1A«...
Solvent Concn '.
in
ir>
-
B
|
i
j—
-------
GAS CHROMATG. -APH OPERATING CONDITIONS AND FIELD LOG
Client
_, Location
Job No.
Injection Date 4--.-.r-.y/ . Time g.t-.
c, Oven 1*30 °c , Detector C%o °c
SAMPLE RUN
Sampling Method r_ £>*•<*
RT
Area
Peak Height
Amount
Component
or
h li^
i (y
t 3.
Name of Operator yvi. K. ^.^,'
» Date
5-60
-------
RT
Area
Peak Height
Amount
Component
1
5-61
-------
Operator. n,X>....C.rttPS ........ Date.Mr:1$>fcl ..................
Column No. .................. Length....**.'. ........ Dla..^>" ...........
Coating ................................. ...Concn ............................
Support.. .fcttfcfrHM^. .Q.... ................. Mesh.Vs!>/**--
TEMP: Col: Inlt ................... I.'5.Q.OC Flnai...lSO ....... °C
Rate ............ °C/mln. Det...».3p...°C ln| ..... I3.Q ...... °C
CARRIER GAS..|fc~. .............. Rate..<*9.JtS'l. ....... r
Pressures: Inlet ........................ Outlet ...........................
DETECTOR E.C ............... .T.C ......... .. ..... F.I.D. .*. ................
Scavenger ........................ Rate .......................... ml./mln.
Sens. ........................... Rec.Range ............................ mv.
SAMPLE...II>JT.6» ....... .«*:. .........................
Solvent ....... ...... .......... . ...... Concn...
i
in
w «1 •
T ? •
38 S5 ?
H H «
f
I
JM«V r»
£c
n
is B
-------
GAS CHROMATOv-APH OPERATING CONDITIONS AND FIELD LOG
Client
_, Location
Job No.
Injection Date *t-»r-!
Recorder/Printout Reference No.
Purpose of Run C.*,~.K a AT'»>+> C - c
* Time //; c -/-."»<=. , Instrument ID
21
» Recorder ID 3 3*10
Sample Description
>-. j.--r'.r.n*-.*»g*j.g.o
Amount Injected
GC CONDITIONS
. Inj. Port or Sample Loop Used i.o^i t.ot»P
Detector Used: FID x . ECD _ , FPD _ , TCD _ (Current _ )
Detector Attenuation _J _ , Amplifier or Range IQ-' _
, Solid Phase
Column: Liquid Phase
Length f,' O.D.
Temperature: Injec
Temperature Program
"
1.0.
D
, Material 5*5..
Temperature: Injector 1*^0 °C, Oven |-?o °c , Detector (*%o °c
SAMPLE RUN
Sampling Method
RT
Area
Peak Height
Amount
Component
c ~
Name of Operator /NO. K. r.^/
.. Date
5-63
-------
... ...... Dite..».r.lVrJt.l. ................
Column No. .................. Length.....*).? ......... «•.../«.* ..........
CoaHng ................................. ...Concn.
Support. .F.fcKA.f.MK.fiL ......................
TEMP: Cote Intt ........... ......AlD..eC Fhnl
Kite ........... .*<:/"*«. D»U3a....0C InJ. ......
CARRIER GAS ..... fc*— • ........ Ril*..Jftr...flSi ....... mh/iflln.
Pft"Msims! Intel........ ...... ..........Outtrt...... ...»». .............
Hy*o»«n..H:Of.*.V..B*/n*i. Mr...(.O.M) ..... mfc/rrtn.
DETECTOR C.C. ............... T.C. ........ . ------ FJ.O...X. ...............
Sc*vin|*r. ....................... Rita ........... ... ............ ml/mln.
Sotv»n»
Concn
I
in
s
-------
GAS CHROMATOv .APH OPERATING CONDITIONS AND FIELD LOG
Client
_, Location
Job No.
Injection Date M-/fc-
Recorder/Printout Reference No. {
Purpose of Run C — c ,-tv p>^~'C..w.rr'0
. Time <5";cq'.V7 , Instrument ID
, Recorder ID 33*10*
Sample Description , .iy^J V
GC CONDITIONS
. Inj. Port or Sample Loop Used 2.o«~c Lo&?
Detector Used: FID x , ECD _ , FPD _ , TCD __ (Current _ )
Amount Injected
Detector Attenuation \
, Amplifier or Range to"1
, Solid Phase
» Material
Column: Liquid Phase
Len9th L' O.t
Temperature: Injector 1*^0 °c, Oven >-?o °c . Detector
Temperature Program
r>
SAMPLE RUN
Sampling Method
RT
Area
Peak Height
Amount
Component
2.
-------
RT
Area
Peak Height
Amount (pp( \
Component
2.0!
53.
^ T T '«
r. -)i
5-66
-------
.. ........ D«t« ...
Column No. ................. Un«th....fc'. ......... U^.'/ff.
Cortng..... ......... .. ................. ...Concn. ............ .......
TEMP: Cot Intt .............. J.3». _____ °C Fkwl....l3Gl.....0C
Rit* ........... ."C/mhi. Det.|3.0....°C ln|......«aft ..... °C
CARRIER GAS ..... R.KU. .......... Rito..lff5\ ............. mh/mhi.
PfMMIFM!
OCTECTmC.C.
Scjv«nt»r.
T.C. ............... F.I.O..X .................
Rita .......................... mL/mfei.
in
I 5 SB R
j g
r
! iff
. ,.
•"•-"--«i«i"»-22E2
22E22«»lBRRX!9|5| S
-------
GAS CHROMATO. .APH OPERATING CONDITIONS AND FIELD LOG
Client
_, Location
Job No.
Injection Date V-/*-& . Time &r ^|T' --
Sample Description
Amount Injected
GC CONDITIONS
, Inj. Port or Sample Loop Used 2.o^\. Loef
Detector Used: FID x « ECD , FPD , TCD (Current )
Detector Attenuation I , Amplifier or Range to"*
Column: Liquid Phase , Solid Phase_
Length {„' O.D. '/»" 1.0.
Temperature: Injector
Temperature Program
C, Oven
, Material
Jc, Detector
Sampling Method
SAMPLE RUN
RT
Area
Peak Height
Amount
Component
a.c
Name of Operator
., Date
5-68
-------
RT
2/.3>-
3**--t3
3-). To
Area
2V t^o
Peak Height
Amount (f^r-X
Cy.fc.
Component
\
\
""fe
/
5-69
-------
Oper«tor...tA,j>.....C,Hl>5- Oa
Column No.. Length. .
CoiUng.... .CM
Support.f.»R*MlK,.q ....M.th.*«Kro....
TEMP: Cofe Intt tSR.'C FlniUaO. °C
Rite 'C/mhi. Dct.t?.P....aC N...J.3.P °C
CARRIER OAS.....*l.1^. Rit«..llTf S'l mfc/mtn.
Prmwm! Wet,— > .OuHrt.
Hydrogen.>.P>.v:i...mWn*i. Mr.trflfii nrt/Mbi.
DCTtCTORE.C. .T.C. F.I.D.X
Sc*v«n(«r. Rite ................inL/inln.
S«ni. Rec.R»ng« mv.
I
in
n P =
N • *
B
E
rtSi
1 s
-------
GAS CHROMATOv .APH OPERATING CONDITIONS AND FIELD LOG
Client
_, Location
Injection Date y— te-»/ Time y^ty-fr: Sf • Instrument ID
Recorder/Printout Reference No. g _ , Recorder ID
Purpose of Run c -C.. t-Kv
/v*o>v<.-s'.
Sample Description , u A
-------
Column No. .................. lwif«t....fef ........ Oto...Vfr..'.; ........
CMttni ................................. ...Conen. ......... ... ...............
Support..fhfc.&l>*tX;..& ................. MMH.M/KQ....
TEMP: C* ln« ................. ».IJo.°C HiMl ....... «ft..°C
Rite ........... .°C/mln. 0»I..J.TO..°C N~ ....... .«?ft..eC
CARRIER OAS ...... HC- ......... R«U..I*TfJft ......... .nrnk/mhi.
Praitunc h*t ....................... OirtW ...........................
HydroKn^JD.fV/. ..... nH^mki. Nr.*o.|k&)l ..... iqMMbi.
DETECTOR E.C. ............... T.C ................ F.I.O.X ..................
Solwnt
sm.a.neM_..
*....* ...... Concji,..,. ..... „ .......................
4 a 4 *
iii JH
11 5 k
CM
i
LT>
I?
.
g 5 §
-------
GAS CHROMATO. -APH OPERATING CONDITIONS AND FIELD LOG
Client
_, Location
frccfA Job No.
Injection Date 'l->*-'•'*»
/• . -i
"/ * •* *
Area
3»5
-------
RT
fS.ci
i<~T£
Ji-li
3 3. -77
xY. C1
3 (.or
Area
IJC<«
>fe-t-C/
S^SZL
3±-n«
>t^i5^
iS2-JlC
Peak Height
\
\
\
1
/
Amount (pp/v)
< n.i
Component
\
\
1 C.c.
1
/
f
5-74
-------
Operator.. ./M
Column No ................... Length ..... .fa.'. ....... Dla ..... ./$.* ........
Coating ....................... . ........... .Concn.( ..........................
Support.fc*.fcKfc**»..Q. ................... ..Mesh ................
TEMP: Col: Inlt ........ . ......... l3o..°C Flnal...A.3.O ..... °C
Rate ............ °C/mln. Det.l.ljO....°C ln| ..... ,.\7.P.....°C
CARRIER
Pressures: Inlet..... ................. .Outlet
DETECTOR E.C ..... . .......... T.C. --------- .......F.I.D...X. ................
Scavenger.............. •..•(. .*..Rate.......*.«>««*«»««,««,,,,.mL/iitlf)«
Sens ............................ Rec.Range ................... . ........ mv.
SAMPLE...|.V.H.O....«S ..............................
Solvent .............................. Concn
in
i
10
is ni —
ft*'*
8 IB 96 8
S3t2Cr"*S'rv* — «
(nvf^^>«o^A«rj.'i
sSSsasts^R
-------
5.7 TOTAL CHROMAT06RAPHABLE (TCO) AND GRAVIMETRIC ORGANICS, INFRARED
SPECTRA (IR), AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS) OF TOTAL
SAMPLE EXTRACTS
5-77
-------
ACUREX
Corporation
Energy & Environmental Division
Acurex August 3, 1981
M.S. 2-2260 Acurex ID #A81-05-030
A81-05-031
Client P.O. #307736.12
Attention: L. Waterland
Sample: 2 SASS Train, received 5/11/81
1 SASS Train, received 5/29/81
The above referenced samples were analyzed per Level 1 protocol. Arsenic
and antimony were determined by furnace AAS.
Polynuclears were determined by a modified EPA method 625. 1 ul of sample
was injected onto a SE-54, J and W 30 meter capillary column using Grob
injection. The column was held at 30°C. No polynuclears were detected in
the GC/MS blanks above 1 ng.
The XAD-2 resin samples and XAO-2 resin blank contained about 120 mg TCO
of material that appears to be a product of acetone reacting with XAD-2
resin. Through the use of the TCO and GC/MS chromatograms, the blank TCO
runs were subtracted to give the data in the report.
Benzo (c) phenanthrene, dibenzo (c,g) carbozole, 7, 12-dimethylbenz (a) anthra-
cene, 3-methyl chloranthene, and perylene were not detected (<40 ng/ul
injection) in any sample analyzed by GC/MS.
Prepared by:
Authorized
Greg Ni/oll
Progpatn Director
-linda K. Bohannon
Sample Control Manager
5-79
485 CLYDE AVENUE. MOUNTAIN VIEW. CA 94042 PHONE (415) 964-3200 TELEX: 34-6391 TWX: 910-3796593
-------
V^ Corporation
ANALYSIS LABORATORIES
DATA REPORTING FORM
Acurex E/S (CHEA)
A81-05-030
Page 2 of 5
CHEA
October 25. 1982
en
00
/
cus
RES
AC
TOMER COI
LJLTS REPOI
prtRf-SS .
MTRACT NO
»TTO L
307736.12
. Water land
ACUREX C<
TELEPHONI
DNTRACT N
n A81 -05-030
Ethan A11en-1 21.62 dscm
SAMPLE 10 (CUSTOMER)
SAMPLE 10 (LABI
PARAMETER
GRAV Aliquot
GRAV (Blank)
GRAV
TCO Al Iquot
TCO (Blank)
TCO
Mercury Aliquot
Mercury (Blank)
Mercury
Ant intony Al Iquot
Ant Imony
Arsenic Al Iquot
Arsenic
10»i * 3u
646
<4
<4
<0.2
_.
__
.-
<)
^cl
<0. 00007
—
—
--
..
ly+Fllter
644 .
<4
<4
<0.3
..
__
—
<1
<1
<0. 00007
—
«
--
—
XAD
650
;^£5S=r^-Tf-7
181
<4
9.1
(150)130*
( 120)0 64 <
6.0*
2
<1
0.0006
—
--•
—
—
OHC
652
8
<4
0.4
4.9
<0.1
0.23
--
--
—
--
—
—
—
tap I
653
..
..
.-
..
«
7
<1
0.0005
—
—
—
—
Imp 2+3
655
-.
..
..
—
--
<1
<1
<0. 00006
<10
<0.0006
<10
<0.0006
Fly Ash
662
14
<4
700ng/kg
0.4
<0.1
20mg/kg
<1
<1
<0.05mo/k«
—
—
—
—
Fuel
661
—
-.
--
~
—
<1
<1
<0.05mg/ki
—
—
—
—
UNITS
mg
mg
mg/dscm
mg
mg
mg/dscm
ug/L
M9/L
mg/dscm
M9/L
mg/dscm
wg/L
mg/dscm
Corrected for resin contamination -- unconnected number in parentheses
ANAI v$T
RFVIFWFR
-------
in
642 ^ T Filter
I ' -
Imtmity
ComnMna
No Peaks
5-82
-------
SAMPLE: 651 EA I XAO Blank
ilRtrwty
ComnMntt
No Peaks
5-83
-------
IK REPORT
646 EA I IQu S 3u
Inanity
Anigpnwnt
Commwm
No Peaks
5-84
-------
IN REPORT
tAMPLE:
EA I lu & Filter
lnt»n»Jty
Alignment
Cemmanti
No Peaks
5-85
-------
Ift REPORT
tAMPtJE:
650 EA I XAD
W**1U»»fcar
- (-'»
3600-3000
2900
2820
1790
1600
1440
1180
Imraity
S
S
S
S
w
M
M
•
Anignrrwnt Commma
0-H COOH
OH Alkane
C-H Alkane
OO COOH
C-H Alkane
C-H Alkane
Not assigned
.
• i
• • •
•
5-86
-------
Ift
fAMfLE: 652 EA I OMC
Vmft
- I-'1)
Immity
Anignirwnt
Comnuntl
No Peaks
5-87
-------
Ift
SAMPLE:
662
Wp*
'1)
Untnvty
Cemmwitt
2900
C-H Alkane
2820
C-H Alkane
5-5
-------
^Corporation
ANALYSIS LABORATORIES
CMEA
DATA REPORTING FORM
nATB July 31. 1981
(
in
OO
cus
RES
AC
TOMER CO
ULTS REPO
>DRESS
NTRACT NO
AT TO L.
an773fi.i2
Waterfand
ACUREX C<
TELEPHON
ONTRACT N
P
nA31 -05-030
Ethan Allen - 1 21.62 .
| SAMPLE 10 (CUSTOMER)
SAMPLE ID (IAB|
PARAMETER
Phenol Aliquot
Naphthalene Aliquot
Ace naphthalene All quo
Phenanthrene Aliquot
Pyrene Aliquot
Fluorene Aliquot
Benzo/J+K/Fluoranther
Aliquot
10u+3u
646
mm
< i
< i
t < i
< i
< i
< i
es < 1
lu+Flltei
644
zm
< i
< i
< i
< i
< i
< i
< i
XAD
650
•
94
90
10
140
5
13
2
OHC
652
< 1
< 1
< 1
< 1
< 1
< 1
< 1
Fly Ash
662
•
< 1
< 1
< 1
< 1
< 1
< 1
< 1
•
«
s==g
•
UNITS
ng
ng
ng
ng
ng
ng
ng
1KIA| VST
fu.ni tEO <*7 460 HFVIFWFR
-------
/N ACUREX
^T > Corporation
ANALYSIS LABORATORIES
CHEA
DATA REPORTING FORM
July 31, 1981
01
1
VO
0
cus
RES
A[
iTOMERCO
ULTS HERO
vuiESfi
NTRACT NO
HTTr»
307736.12 •
L. Waterland
ACUREX C<
TELEPHONI
3NTRACT N
F
n A81 -05-030
Ethan Allen - 1 21.62 dscm
1 SAMPLE 10 (CUSTOMER)
SAMPIEID(LAB)
PARAMETER
Phenol
Naphthalene
Acenapthalene
Phenanthrene
Pyrene
Fluorene
Benxo/ J*K/F1 uoranthene
Others with a detect io
Umli of 1 ng
10u+3u
646
< 90
< 90
< 90
< 90
< 90
< 90
5 < 90
n< 90
lu+FUtei
644
^S
< 80
< 80
< 80
< 80
< 80
< 80
< 80
< 80
XAD
650
H
4700
4500
500
7000
300
650
100
< 50
OHC
652
•
< 50
< 50
< 50
< 50
< 50
< 50
< 50
< 50
Fly Ash
662
11 i,7 SSSS5SSS
_•".'...! '____^1=_
<0.05mg/k
<0.05mg/k
<0.05mg/l
<0.05mg/l
<0.05mg/l
<0.05mg/i
<0.05mg/l
<0.05mg/l
••
9
9
9
9
9
9
9
9
«
•
•
UNITS
ng/dscm
ng/dscm
ng/dscm
ng/dscm
ng/dscm
ng/dscm
ng/dscm
ng/dscm
ANAIVST
FaimEEO-DAI 4M
REVIEWER
-------
k
A
en
1
10
^VACUREX
r> Corporation
NALVSIS LABORATORI
COS
cus
RES
AI
DATA REPORTING FORM A81-05-030 (<*EA)
ES Page 3 of 5
TOMF* CMEA OATF October 25. 1982
TOMER Cd
ULTS HEPO
1DRESS
NTRACTNO
BT TO L.
307736.12
Waterland
ACUREX Cl
TELEPHONI
3NTRACT N
f
n A81 -05-030
Ethan Allen-2 27.06 dscm
SAMPLE 10 (CUSTOMER)
SAMPLE 10 (LAB)
PARAMETER
GRAV Aliquot
GRAV (Blank)
GRAV
TCO Aliquot
TCO (Blank)
TCO
Hercury Aliquot
Hercury (Blank)
Hercury
Antimony Aliquot
Ant Iroony
Arsenic Aliquot
Arsenic
^'Tjffff"^"
10U+3U
672
<4
<4
<0.3
._
».
..
<1
<1
<0.0003
—
—
—
—
lu* Ftltei
674
<4
<4
<0.3
..
._
--
<1
<1
<0. 00006
—
—
—
—
XAD
35
<4
1.4
(140) 20*
(120)0.64-
0.72*
<1
<1
<0.0002
«
—
—
--
OHC
677
<4
<4
<0.1
0.2
<0.1
0.007
—
—
.-
~
--
—
—
Imp 1
678
__
._
..
..
..
cl
<1
<0. 00007
«
--
—
—
Imp 2+3
679
.v
..
..
..
..
<1
<1
<0. 00005
<10
<0.0005
<10
<0.0005
Fly Ash
744
13
<4
BSOmg/kg
0.3
(0.1
20mg/kg
<1
o,_tca«r tto
-------
Ift REPORT
672 ** IT 10u S 3U
I.'1)
Imtnuty
Anignmcnt
ContriMna
No Peaks
5-92
-------
Ill REPORT
SAMPLE:
EA II lu s Filter
Vvtftmfar
- I-'1)
Anignnwnt
Comments
2900
C-H Alkane
5-93
-------
Ift REPORT
SAMPLE:
676
tnttraity
Comnnnti
2900
C-H Alkane
2820
1710
M
C-H Alkane
OO
5-94
-------
IM KEPOKT
SAMPLE: 677 OMC EA II
I ' •
bntmity
Cemmtna
3680-3200
0-H
.nnn-?7nn
w
C-H Alkane
5-95
-------
IR REPORT
744 EA II Flyash
hromity
Alignment
Commana
2900
C-H Alkane
2820
C-H Alkane
5-96
-------
^T^ Corporation
ANALYSIS LABORATORIES
DATA REPORTING FORM
CMEA
July 31. 1981
Ol
4
iO
CDS
RES
AI
•TOMER CO
ULTS REPO
inRPSs ..
MTRACT NO
BT rn L.
307736.12
Waterland
ACUREX C<
TELEPHON
ONTRACT N
P
in A81 -05-030
Ethan Allen - 2 27.06 dscm
1 SAMPLE ID (CUSTOMER)
SAMPLE ID (LAB)
PARAMETER
Acenaphthylene Allquo
Acenaphthene Aliquot
Phenanthrene Aliquot
Anthracene Aliquot
Fluoranthene Aliquot
Pyrene Aliquot
Chrysene Aliquot
Phenol Aliquot
10u*3u
672
H
: < 1
< 1
< 1
< 1
< 1
< 1
< 1
< 1
lu+Fllter
674
7~J^linmiir!^^^'g=g=
- __ --ggagggg^j^
< 1
< 1
< 1
< 1
< 1
< 1
< 1
< 1
XAD
676
•
130
3
49
5
7
5
1
< 1
OMC
677
< 1
< 1
< 1
< 1
< 1
< 1
< 1
26
Fly Ash
744
< 1
< 1
< 1
< 1
< 1
< 1
< 1
< 1
•
•
B
•
UNITS
ng
ng
ng
ng
ng
ng
ng
ng
ANAIVST
REVIEWER
-------
tn
i
CO
ACUREX
Corporation
ANALYSIS LABORATORIES
CUSTOMER
DATA REPORTING FORM
CMEA
July 31. 1981
cus
RES
Al
•TOMER CO
ULTS REPO
3DRESS
NTRACT NO
BTTft L.
307736.12
HAterland
ACUREX Cl
TELEPHONI
DNTRACT N
f
n A81 -05-030
Ethan Allen - 2 27.06 dscn
SAMPLE ID {CUSTOMER)
SAMPLE 10 (LAB)
PARAMETER
Acenaphthylene
Acenaphthene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Chrysene
Phenol
Others with a detectl
limit uf 1 ny
10u+3u
672
B
< SO
< SO
< 50
< SO
< SO
< 50
< 50
< 50
n < SO
lu+F11ter
674
< 50
< 50
< 50
< 50
< 50
< 50
< 50
< 50
< 50
XAD
676
H
5200
100
2000
200
300
200
40
<40
<40
OMC
677
•
< 40
< 40
< 40
< 40
<.40
< 40
< 40
960
< 40
Fly Ash
744
< O.OSmg/
< O.OSmg/
< O.OSmg/
< O.OSmg/
< O.OSmg/
< O.OSmg/
< O.OSmg/
< O.OSmg/
< O.OSmg/
eg
eg
eg
eg
eg
eg
eg
eg
eg
- -__ -—Ssssssss
•
.
*
UNITS
g/dscm
g/dscm
ig/dscm
ig/dscm
ig/dscm
ig/dscm
ig/dscm
ig/dscm
ig/dscm
AMAIVRT
(o.mtEDtti? tin REVIEWER
-------
5.8 LIQUID CHROMATOGRAPHY (LC) SEPARATION AND INFRARED SPECTRA
OF LC FRACTIONS
5-99
-------
ACUREX
Corporation
Energy & Environmental Division
CMEA/Acurex October 5, 1981
Lab ID Number: A81-08-023
Customer P.O. Number: 307736.12
ATTENTION: L. Waterland
Samples: Ethan Allen XAD extracts (3)
The above referenced samples from earlier work were analyzed by Level 1
procedures. The TCO, GRAV and IR results from the LC fractions are
included.
Prepared by: ' * A* ^ T?u
-------
SAMPLE: EAI XAD #650
Acurex E/S (CMEA)
A81-05-030
Page 4 of 5
TCO
MAY
mg/dscm
130
196
326
15
47
71
118
5.5
16
50
66
3.1
en
i
o
CO
J,
4.
FnriM
1
t
1
4
1
I
f
few
TCOb«f
FMrftn
» — . —
rfMtiw
0.45
2.1
2.1
1.3
4.2
1.3
4.6
16
MM*
cO. 05
cO. 02
cO.02
<0.02
<0.02
<0.02
0.1
Q.I
e»
IMIMl
0.45
2.1
2.1
1,3
4.2
1.3
4.5
16
T.««
1.2
5.8
5.8
3.6
12
3.6
12
44
•HAVhiN
fM*hi
1.2
<0.8
1.8
l.R
4.n
1.4
41.?
51
toMk
-------
IR REPORT
SAMPLE; 651 Blank XAD F1
WIM Number
Innraity
Assignment
No Peaks
Commwits
5-104
-------
IR REPORT
SAMPLE:
651 Blank XAD F2
Wmltambw
Intensity
Assignment
Commtnts
No Peaks
5-105
-------
IR REPORT
SAMPLE; 651 Blank XAD F3
te»
Inttraity
Assignment
No Peaks
Commtnti
5-106
-------
|R REPORT
SAMPLE: 651 Blank XAD F4
WmNwnbw
Intimity
Assignment
No Peaks
Comments
5-107
-------
IR REPORT
SAMPLE:
651 Blank XAD F5
ffm Noinbw
- fa-'1)
Intensity
Assignment
No Peaks
Commtnts
5-108
-------
IR REPORT
SAMPLE:
651 Blank XAD F6
nttfflMf
limmity
Assignment
No Peaks
Commmti
5-109
-------
REPORT
651 Blank XAD F7
I VtoTii
Intimity
Commwm
3600-3300
OH
5-rio
-------
IN REPORT
SAMPLE: 650 EAI XAD Fl
int»mity
Conimwm
No Peaks
5-111
-------
IR REPORT
SAMPLE:
650 EAI XAD F2
WIM Numbw
- fa.'1)
Inttmity
Auignmtnt
Commtnts
No Peaks
5-112
-------
IR REPORT
SAMPLE: 650 EAI XAD F3
Wn« ffamto
Inttmity
Assignment
Commtnts
3450
OH
Aliphatic
5-113
-------
IR REPORT
SAMPLE: 65° EAI XAD F4
Wm
te»1)
Inttnsity
Auignmtnt
Commtnti
No Peaks
5-114
-------
IR REPORT
650 EAI XAD F5
Win Number
Inttraity
Assignment
Commtnts
No Peaks
5-115
-------
IR REPORT
SAMPLE:
650 EAI XAD F6
WIM Numb*
, to.'1)
Intimity
Assignment
Comments
No Peaks
5-116
-------
IK REPORT
tAMPLC:
650 EAI XAD F7
| mi2*r
3230
2880
2810
1650
1190
.
tnttmity
M
M
M
S
S
•
Attigprnwrt Comnwrm
OH Carboxylic acid
CH Carboxylic acid
CH Carboxylic acid
C=O Carboxylic acid
CO Carboxylic acid
•
.
«• •
--..
5-117
-------
SAMPLE: EAII XAD #676
Acurex E/S (CMEA)
A81-05-030
Page 5 of 5
CO
TCO
20
n
1.4
•HAV
38
22
12
Ufal M|
58
33
13
mg/dscm
2.1
1.2
0.48
t.
4. T«Mlw|
*Corrected for resin contamination
1C «•!•<•«,
-------
IAMPLI: 676 EAII XADF1
tmtiwity
Commcna
No Peaks
5-119
-------
IR REPORT
SAMPLE: 676 EAII XAD F2
WIM Mwnbv
Inttraity
Assignment
No Peaks
Cemmtnts
5-120
-------
IR REPORT
SAMPLE: 676 EAII XAD F3
Wiw Numb*
< W')
3450
InttreitY
OH
Assignment
Aliphatic
Commtnts
5-121
-------
IR REPORT
SAMPLE: 676 EAII XAD F4
N«fflb«
Inttraity
Assignment
No Peaks
Comments
5-122
-------
IR REPORT
SAMPLE; 676 EAII XAD F5
Intensity
AsiignnMnt
Commtnts
No Peaks
5-123
-------
IR REPORT
SAMPLE: 675EAII XAD F6
WIM Nambw
Intensity
Assignment
No Peaks
Commtnts
5-124
-------
|R REPORT
SAMPLE:
676 EAII XAD F7
WiM NWHMf
< te»1)
Intaraity
Assignment
No Peaks
Cotnmtnts
5-125
-------
5.9 LOW RESOLUTION MASS SPECTROMETRY (LRMS) OF SELECTED TOTAL
SAMPLE EXTRACTS AND LC FRACTIONS
5-127
-------
ACUREX
Corporation
CMEA/ACUREX
ATTENTION: L. Water-land
Energy & Environmental Division
December 4, 1981
Acurex ID*: A81-10-011, A81-10-022
Client P.0.#: 307605
Samples: 9 extracts from Tosco and Ethan Allen
The above referenced samples were analyzed by direct probe mass spectrometry.
Searches have been made for classes of compounds most likely to be found in
the various LC fractions, according to procedures described in the "IERL-RTP
Procedures Manual: Level 1 Environmental Assessment". The following frag-
ment ions used for search are given below:
Compound Class
Polycyclic aromatic hydrocarbons
Aliphatic hydrocarbons
Halogenated aliphatics
Aromatic hydrocarbons
Ethers
Alcohols
Phenols
Nitriles
Phthalate esters
Amines
Ketones
N-heterocyclics
Mercaptans, sulfides
Benzothiophenes
Carboxylic acids
Amides
Fragment ions (m/e-)
178,202,216,228,252,276
57,71
79,81,93,95,107,109;49,63
50,51,77,78,79,91
45*59,73
45,59,61,73,75
51,77,94
54,68,82
61,59,71,87
44,58
51,71
117,167;129,179
47,61,75
57,58,59,69,70,85,97,111,125
60,73,149
58,72,86,100
To test the analysis procedure, a standard mixture containing ethers, amines,
polycyclic aromatic hydrocarbons, nitrosamines, phenols, etc., was analyzed
under identical conditions as the samples. Losses of the very volatile
compounds such as naphthalene, bis(2-chloroethy!)ether, low molecular weight
nitrosamines were observed, however the higher molecular weight compounds in
a particular class were recovered.
Prepared by:
Greg UTcoll
Program Director
Approved by:
Vionca Lopez-
Technical Director
GN/VLA:es
5-129
485 CLYDE AVENUE, MOUNTAIN VIEW. CA 34042 PHONE (415) 964-3200 TELEX: 34-6391 TWX: 910-3796593
-------
LRMS REPORT
SAMPLE; Ethan Allen IXAD 650 F2& F3
Major Cmgoriti
Intimity
10 ;
1
Cittoory
Carfyoxvlic aciH
PAH
t
MWR»no»
.<216
Sub>Cat>s0rif4. Sptdfie Compound*
Inttnaty
Ctttfory
m/t
Composition
Other
5-131
-------
L*MS REPORT
Ethan Allen I XAD 650 F4 & F5
Major C*t*goriM
Intimity
Oratory MW Ranoi
None detected
*
.
Su^Citvgoriflt. Specific Compound* -
Intimity
•
Cragory
•
.
m/«
Campoaition
_. •
: '
Other
•
5-132
-------
L*MS REPORT
Major Cmgorin
Intimity
f
Ccttgory MW Rang*
None detected
•
.
.
Sub-Cattgoritt . Specific Compound* ,
Intimity
.
•
Ctttgory
.
m/t
«
Composition
.. •
Other
•
5-133
-------
REPORT
Ethan Allen I OMC 652
Mijor Cmgorit*
Inunsity
100 .
TOO '
100
100
100
10
Crtr^ory
Ethers
Nitriles
Ami nes
Heterocyclic sulfur compounds
Carboxylic acids
Haloaenated aliohatics
MW bngB
Sub-dt»50f>«. Sp*df»e Compounds
Inttrtsty
m/t
Campositien
Other
5-134
-------
LRMS REPORT
IAMPH; Ethan Allen I OMC 652 (cont)
Major CittgoriM
Intensity
10
10
10
10
1
CrttffOfy
Aromatic hydrocarbons
Phenols
Ketones
Heterocyclic nitrogen compounds
PAH
MV» Rjno*
•
<216
Inttnaty
Compounds
Cattgory
m/t
Composition
Oth«r
5-135
-------
LRMS REPORT
SAMPLE:
Ethan Allen I Flv Ash 662
Ctrtgerits
Inunsity
1
C«t70fy
PAH
.
'
MWRanp
<216
it». Sp*eifie Cempoundt
Inttnsty
dttgory
m/t
Compeiitien
Oth«r
5-136
-------
REPORT
I AMPLE:Ethan Allen II XAD 676
Major Cmgoritt
Intensity
.
Gaftfcoory iwnfT Rain^B
None detected
^ *
i
t
Sub-Cat»sor!»«. Specific Compounds .
Intensity
Canary
.
m/t
Composition
• -
i •
Oth«r
-~*
•
5-137
-------
REPORT
SAMPLE: Ethan Allen II Fly Ash 744
Major Caitgorin
Intamity
100 .
10
Crt*flory
Carboxylic acids
PAH
»
MWRangi
- <216
it*. Sp«esfie Compound*
Inttraity
m/t
Composition
Other
5-138
-------
Acorn Park
Cambridge, Massachusetts 02140
617 864-5770 Telex 921436
/111 Arthur D. Little, Inc.
July 8, 1982
Dr. Larry Waterland
M2S-2260
Accurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
Dear Larry: 1-7641
We have completed the batch inlet LRMS analysis of your ten Level
1 samples. The data obtained in the analysis of these samples is
reported on the enclosed, standard EPA Level 1 LRMS report forms.•
The intensity levels are reported for this (batch) analysis only,
as though it were the complete LRMS analysis. Presumably you will
integrate the data from these analyses with your own direct probe
LRMS analysis of the same samples.
We have reported the "sample" content of the samples as though
the solvent(s) were not present; a component reported as intensity
100 is a major component of the non-solvent portion of the spectrum;
one as intensity 10 when it is present and identifiable in the non-
solvent portion of the spectrum, and so on. Intensity level 1
components appeared to be present in some of the samples, but were
not specifically identifiable. When they occur they are included
in the unclassified category.
All samples were analyzed by direct injection of 4 yL of sample
into the three liter glass inlet of the mass spectrometer. The
mass spectrometer was operated in the electron impact ionization
mode, at 70 eV. Low energy ionization was not used due to the
low level of sample material as compared to the solvent content of
the sample. Instrument blanks were obtained by direct injection of
4 yL of spectra grade methylene chloride. One sample (AC009) was
5-139
Brussels Paris Tokyo
Houston Rio de Janeiro Toronto
London San Francisco Washington
Madrid Sao Paulo Wiesbaden
-------
/k Arthur D. Little, Inc.
July 8, 1932
-2-
Dr. Larry Waterland
Accurex Corporation
concentrated 3X and given an additional direct injection analysis.
This was done only to clarify some spectral ambiguities. The
reported data is from the unconcentrated initial analysis.
If you have any questions about any of this work, please feel free
to call me.
Yours Truly,
/laf
enclosures
James L. Stauffer
5-140
-------
LRMS REPORT
SAMPLE: g
±
LC-«3 * t-C-3
Major Categories
Inttniity
Category
MWRanoe
IOO
» ^ ' *^ ^^ ^ *"1^
Sub-Categories, Specific Compounds
Intensity
Category
m/e
Composition
IOO
Other
5-141
-------
LRMS REPORT
SAMPLE:
VA 0 ^ SO
Major Categories
Intensity
Category
MW Range
100
10
10
IO
10
Sub-Categories, Specific Compounds
Intensity
Category
m/e
Composition
\DO
tf
\o
&
OLl
fa#
Other
I
bf_rr*,r L
Oi
i «
5-142
-------
LRMS REPORT
SAMPLE; fj^rx AI Ux 1 XAD Lurvd<
— Wb
10
Sub-Categories, Specific Compounds
Intensity
Category
m/e
Composition
100
Prm-nol
Dimg>Ku |
/ Puj-dUeKydt.
Other
10
5-143
-------
LRMS REPORT
SAMPLE: £j-
yyy.
1
OHC.
Major Categories
Intensity
IOO
10
K)
/O
Category
PKtnolrs
H-*. *r, no CAJ «i IA^_ Oy u *l *-**%. ^*^ i~-»|(9 c u^w.
-------
5.10 RADIOMETRIC ANALYSIS RESULTS
5-145
-------
SAFETY SPECIALISTS, Inc.
3284 F Edward Avenue. Santa Clara. California 95050 • Telephone (408) 988-1111
ASSAY REPORT
Acurex Corporation
Attn: Mr. Larry Water!and
485 Clyde Avenue
Mountain View, California 94042
SSI No.
812280
E
F
G
Client Description
A81-05-030-646 ^
A81-05-030-662(j^t i
A81-05-030-674(Ter» z
A81-05-030-7^4(r^tz
Date: August. 13, 1981
•Date Samples Received: 6/29/81
Customer Order No.: RB59185A, Rel. 15
Activity*
Gross Alpha
pCi/g
20.2 ± 12.1
17.6 ± 4.2
22.2 ± 9.6
15.6 ± 3.9
iross Beta
pCT/g
218.8 ± 18.5
119.0 ± 38.0
164.3 ± 30.5
93.3 ± 35.0
Analyst: Pamela S. Shreve
Approved: T. C. Noble, Director
Safety and Health Services Division
*The ± values are the two sigma Poisson standard deviation of the counting
error.
The <. values are equal to or less than three sigma of the counting error.
5-147
-------
SAFETY SPECIALISTS, Inc.
3284F Edward Avenue. Santa Clara. California 95050 - Telephone (408) 988-1111
ASSAY REPORT
Acurex Corporation
Attn: Mr. Larry Waterland
485 Clyde Avenue <
Mountain View, California 94042
Date: August 13, 1981
Date Samples Received: 6/29/81
Customer Order No.: RB59185A, Rel. 15
SSI No.
81228
Client Description
Activity*
Gross Gamma
pCi/L
Gross Gamma
pCi/g
D
E
F
G
A81-05-030-646
A81-05-030-662
A81-05-030-674
A81-05-030-774
-415 ± 734
4 ± 419
161 ± 679
163 t 476
Analyst: Pamela S. Shreve
Approved: T. C. Noble, Director
Safety and Health Services Division
*The ± values are the two sigma Poisson standard deviation of the counting
error.
The £ values are equal to or less than three sigma of the counting error.
5-148
-------
5.11 BIOLOGICAL ASSAY RESULTS
5-149
-------
GENETICS ASSAY NO.: 5882
LBI SAFETY NO.: 7155
MUTAGENICITY EVALUATION OF
A81-05-030-646
(EA-1 10+3+1+FILTER)
IN THE
EPA~LEWL 1
AMES SAP35NETIA7MT.CROSOME
PLATE TEST
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
Litton
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
5-151
BIONETICS
-------
ffl
Litton
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Ames
Saltnonella/microsome mutagenesis assay as described in "IERL-RTP Proce-
dures Manual: Level 1 Environmental Assessment Biological Tests l. The
data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting"2.
The Ames Salmonella/microsome mutagenesis assay has been shown to be a
sensitive method for detecting mutagenic activity for a variety of chemi-
cals representing various chemical classes3. This assay is one of several
recommended by EPA to identify, categorize and rank the pollutant potential
of influent and effluent streams from industrial and energy-producing pro-
cesses. This assay has been well validated with a wide range of positive
and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good Labora-
tory Practices Regulations of 1979. Deviations from standard procedure
shall be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives, of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon .request.
5-152
BIONETICS
-------
Litton
TABLE OF CONTENTS
Page No.
PREFACE i
I. ASSAY SUMMARY 1
II. OBJECTIVE 2
III. TEST MATERIAL 3
A. Description 3
B. Handling and Preparation 3
IV. MATERIALS . 4
A. Indicator Microorganisms 4
B. Media 4
C. Activation System 5
1. S9 Homogenate 5
2. S9 Mix 5
V. EXPERIMENTAL DESIGN 6
A. Dose Selection 6
B. Mutagenicity Test 6
1. Nonactivation Assay 6
2. Activation Assay 6
C. Control Compounds 7
D. Recording and Presenting Data 7
VI. RESULTS ..... 9
A. Interpretation 9
B. Tables 9
VII. EVALUATION CRITERIA 11
A. Surviving Populations 11
B. Dose-Response Phenomena 11
C. Control Tests 11
D. Evaluation Criteria for Ames Assay 12
1. Strains TA-1535 and TA-1537 12
2. Strains TA-98 and TA-100 12
3. Pattern 12
4. Reproducibility 12
E. Relation Between Mutagenicity and
Card nogeni city 13
F. Criteria for Ranking Samples in the Ames Assay . . 13
VIII. REFERENCES 14
5-153
BIONETICS i i
-------
I. ASSAY SUMMARY
A. Sponsor: Acurex Corporation
B. Material (Test Compound): Genetics Assay Number: 5882
1. Identification: A81-05-030-646 (EA-1 10+3+1+Filter)
2. Date Received: August 26, 1981
3. Physical Description: Fine, brown powder and fiberglass
filter with embedded particles.
C. Type of Assay: EPA Level 1 Ames Salmonella/Microsome Plate Test
D. Assay Design Number: 401 (EPA Level 1)
E. Study Dates:
1. Initiation: October 1, 1981
2. Completion: October 26, 1981
F. Supervisory Personnel:
A. Study Director: D.R. Jagannath, Ph.D.
G. Evaluation:
The test material, A81-05-030-646 (EA-1 10+3+1+Filter), was
tested for activity in the Ames Salmonella mutagenicity assay
over a concentration range of 0.05 mg/plate to 5.0 mg/plate.
The test was performed in duplicate under nonactivation and
activation test conditions with strains TA-1535, TA-1537, TA-98,
and TA-100.
The sample was not mutagenic under the test conditions employed
and was ranked as having nondetectable (ND) mutagenic activity
as defined by the IERL-EPA Level 1 criteria for the Ames bio-
assay1.
Submitted by:
Study Director
O.R. Jagannath, Ph.U;-
Section Chief,
Submammalian Genetics,
Department of Molecular
Toxicology
Litton
BIONET1CS
Date
5-154
Reviewed by:
.
David J. Brusick, pfr.D.
Director,
Department of Molecular
Toxicology
Date
-------
II. OBJECTIVE
The objective of this study was to determine the genetic activity of
A81-05-030-646 (EA-1 10+3+1+filter) in the Salmonella/ microsome assay
with and without the addition of mammalian metabolic activation prepara-
tions. The genetic activity of a sample is measured in these assays by
its ability to revert the Salmonella indicator strains from histidine
dependence to histidine independence. The degree of genetic activity of
a sample is reflected in the number of revertants that are observed on
the histidine-free medium.
Litton
5-155
BIONET1CS
-------
m
Litton
III. TEST MATERIAL
A. Description
The test material, as received, was comprised of two separate components.
The first component, a fine, brown powder, was the 1 urn, 3 pm and 10 urn
SASS train participate catch. The second component was a fiberglass
filter with embedded particulate material. This brown participate material
represented participates less than 1 urn collected in the SASS train sample.
Both components were supplied together in a Nalgene screw-top bottle.
B. Handling and Preparation
The test material was received at LBI on August 26, 1981. The sample
was assigned LBI safety number 7166 and LBI assay number 5882. The
sample was stored at +4°C in the dark.
The filter portion of the sample required removal of the embedded parti -
culates before testing could begin. The uncut filter was sonicated in
cyclohexane as recommended by current IERL-EPA pretest sample preparation
procedures1. The decanted particulate suspension from three successive
sonication treatments were combined and evaporated to dryness. The parti-
culate material was weighed and combined with the 1 urn particulate catch
portion of the sample. A total of 215.14 mg of combined test material
available for testing was comprised of 37.78 mg (17.6%) of <1 urn particu-
lates removed from the filter and 177.36 mg (82.4%) of 1 urn, 3 urn and
10 urn particulates.
Appproximately 181 mg of test material were used for the trial in the
Ames Salmonella Assay. The test material was suspended at 100 mg/ml in
dimethylsuIfoxide (DMSO) and incubated overnight at 37°C on a rotary
shaker. This stock suspension was used to make dilutions in DMSO to be
used for dosing in the EPA Level 1 Ames Salmonella Assay.
5-156
BIONETICS
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IV.
A.
MATERIALS
Indicator Microorganisms
The Salmonella typhimurium strains used In this assay were obtained from
Dr. Bruce Ames, University of California at Berkeley.4-8 The following
four strains were used.
Strain
Designation
Gene
Additional Mutations
Affected Repai r LPS R Factor
Mutation Type
Detected
TA-1535
TA-1537
TA-98
TA-100
his G A uvr B rfa
his C A uvr B rfa
his D A uyi B rfa pKMlOl
his G A uvr B rfa pKMlOl
Base-pair
substitution
Frameshift
Frameshi ft
Base-pair
substitution
All the above strains have, in addition to the mutation in the histidine
operon, mutation (rfa-) that leads to defective lipopolysaccharide coat,
a deletion that covers genes involved in the synthesis of vitamin biotin
(bio-) and in the repair of ultraviolet (uv) - induced 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 physi-
cally~3amaged 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 mutagens.8
In addition, plasmid pKMlOl confers resistance to the antibiotic ampi-
cillin, 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 supplemented
with a trace of biotin and an excess of histidine. In addition, the
plates with plasmid-carrying strains contain ampicillin (25 ug/ml) to
ensure stable maintenance of plasmid pKMlOl. New stock culture plates
are made as often as necessary from the frozen master cultures or from
single colony reisolates that were checked for their genotypic character-
istics (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) and used.
B.
Media
The bacterial strains were cultured in Oxoid Media #2 (Nutrient Broth).
The selective medium was Vogen Bonner Medium E with 2% glucose.10 The
Litton
5-157
BIONETICS
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overlay agar consisted of 0.6% purified agar with 0.05 mM histidine,
0.05 mM biotin and 0.1M NaCl according to the methods of Ames et a_L9
C. Activation System
1. S9 Homogenate
A 9,000 x c[ supernatant prepared from Sprague-Dawley adult male rat liver
induced by Aroclor 1254 (Ames et aj.9) was purchased commercially and
used in these assays.
2. S9 Mix
S9 mix used in these assays consisted of the following components:
Components
Concentration per Milliliter
S9 Mix
NADP (sodium salt)
D-glucose-6-phosphate
MgCl2
KC1
Sodium phosphate buffer
pH 7.4
Organ homogenate from rat
liver (S9 fraction)
4 umoles
5 umoles
8 umoles
33 umoles
100 umoles
100 uliters
5-158
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3IONETICS
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Litton
V. EXPERIMENTAL DESIGN
A. Dosage Selection
Test strategy and dose selection depend upon sample type and sample avail-
ability. The Level 1 manual1 recommends solids to be initially tested
at the maximum applicable dose (MAD) of 5 mg per plate and at lower con-
centrations of 2.5, 1, 0.5, 0.1 and 0.05 mg per plate. Liquids are tested
initially at the MAD of 200 pi per plate, and at lower concentrations of
100, 50 and 10 ul per plate. Samples are retested over a narrower range
of concentrations with strains showing positive results initially. Alter-
nate dose are employed if sample size is limiting or at the direction of
the sponsor.
Doses selected to test this sample covered the recommended dose range
for solids. The highest dose was at the MAD level of 5 mg per plate and
included five lower dose levels of 2.5, 1, 0.5, 0.1 and 0.05 mg per plate.
B. Mutagem'city Testing
The procedure used was based on the paper published by Ames et. aj_.9 and
was performed as follows:
1. Nonactivation Assay
To a sterile 13 x 100 mm test tube placed in a 43°C water bath the fol-
lowing was added in order:
2.00 ml of 0.6% agar containing 0.05 mM histidine and
0.05 mM biotin.
0.05 ml of a suspension of the test chemical to give the
appropriate dose.
0.1 ml to 0.2 ml of indicator organism(s).
0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured onto minimal agar plates
(see IV B, Media). After the top agar had set, the plates were incubated
at 37°C for approximately 2 days. The number of his+ revertant colonies
growing on the plates were counted with an automatic colony counter and
recorded.
2. Activation Assay
The activation assay was run concurrently with the nonactivation assay.
The only difference was the addition of 0.5 ml of S9 mix (see IV C, Acti-
vation System) to the tubes in place of 0.5 ml of phosphate buffer which
was added in nonactivation assays. All other details were similar to
the procedure for nonactivation assays.
5-159
BIONETICS
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A detailed flow diagram for the plate incorporation assay is provided in
Figure 1.
C.
Control Compounds
A negative control consisting of the solvent used for the test material
was also assayed concurrently with the test material. For negative con-
trols, step 'b' of Nonactivation Assays was replaced by 0.05 ml of the
solvent. The negative controls were employed for each indicator strain
and were 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 were
made using this solvent. The amount of solvent used was equal to the
maximum volume used to give the appropriate test dose.
Specific positive control compounds known to revert each strain were also
used and assayed concurrently with the test material. The concentrations
and specificities of these compounds to specific strains are given in
the following table:
Concentration
per plate Salmonella
Assay
Nonactivation
Chemical
Sodium azide
2-Nitrofluorene
(NF)
9-aminoacridine
(9AA)
Solvent (ug)
Water
Dimethyl-
sulf oxide
Ethanol
10. 0
10.0
50.0
Strains
TA-1535,
TA-98
TA-1537
TA-100
Activation
2-anthramine
(ANTH)
Dimethyl-
sulfoxide
2.5
For all strains
D.
Recording and Presenting Data
The number of colonies on each plate were counted and recorded on printed
forms. These raw data were analyzed in a computer program and reported
on a printout. The results are presented as revertants per plate for
each indicator strain employed in the assay. The positive and solvent
controls are provided as reference points.
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AMES ASSAY (PLATE INCORPORATION METHOD]
Aliquot of
saline
0.5 ml
-S-9
Molten [45*C] overlay agar
appropriately supplemented
0.1 ml
Test, positive or solvent
control chemical
Aliquot of an overnight culture
of bacterial 10$ cells/ml]
0.5 ml S-9 mix [hepatic
S-9—— homogenate from PCB
pretreated rat plus
necessary cofactors
Overlay poured on selective
bottom agar medium
Plated incubated at 37*C for 48 hours
The numbers of revertants/plate counted
Data analyzed
Interpretation / C onclusion
Figure 1 AMES .SALMONELLA/MICROSOME MUTAGENESIS ASSAY
5-161
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LJtton
VI. RESULTS
A. Interpretations
The test material, A81-05-030-646 (EA-1 10+3+1+fliter), was dissolved in
DMSO at a stock concentration of 100 mg/ml and leached overnight on a
shaker at 37°C. Additional dilutions were prepared in DMSO for testing.
The maximum test level was 5.0 mg/plate except for the activation portion
of the assay with strain TA-1535 which used a maximum dose of 1 mg/plate
because of limited test material. There was no evidence of toxicity at
this level.
Reverse mutation was measured in strains TA-1535, TA-1537, TA-98 and
TA-100. The test was conducted in duplicate both with and without rat
liver S9 mix for metabolic activation.
There was no mutagenic activity associated with the test material treat-
ment and the sample was considered nonmutagenic and non toxic. The sample
was ranked as having nondetectable (NO) mutagenic activity using the
IERL-EPA Level 1 evaluation criteria for the Ames Assay1.
Solvent control and positive control values were within acceptable ranges.
These results achieved assay acceptance criteria and provided confidence
in the assumptions that the recorded data represented typical responses
to the test material.
B. Tables
This report is based on the data provided in Table 1.
5-162
BIONETICS
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RE SUMS
TABLE 1
en
i
co
NAME OR CODE DESIGNATION OF THE TEST COHPOUNC: A-81-05-020-646 (EA-1 10+3+HFILTER)
SOLVENT: OHSO
TEST INITIATION DATES: 10/22/81
TEST COMPLETION DATE: 10/2C/81
S-9 LOT«: REF050
CONCENTRATIONS ARE 6IVEN IN MILLIGRAMS PER PLATE
A.
B.
C.
D.
t.
NOTE:
TEST
NONACTIVATION
REVERTANTS FER FLATE
SPECIES TISSUE TA-1535 TA-1537 TA-9B TA-10B
123 123 123 12
SOLVENT CONTROL
POSITIVE CONTROL**
TEST COMPOUND
0.050 HG
0.100 HG
0.500 MG
1.000 MG
2.500 HG
5.COO HG
ACTIVATION
8 10
1021 1034
IS
18
19
19
20
29
14
19
1!
18
23
20
7 8
117 190
7
4
10
a
9
7
26 20
552 845
21
13
28
30
32
22
35
25
30
32
25
25
119 104
1543 1515
124 132
111 141
112 112
121 128
116 121
102 126
o
SOLVENT
POSITIVE
CONTROL RAT
CONTROL*** RAT
LIVER 11 10
LIVER 140 145
13
118
16
181
37
810
24
950
94
1586
101
1833
TEST COMPOUND
0.
0.
0.
1.
2.
5.
• *
050
100
500
000
500
000
TA-1535
TA-IS
TA-98
37
HG
HG
HG
HG
HG
HG
SODIUM
RAT
RAT
RAT
RAT
RAT
RAT
AZIOE
LIVER 6 10
LIVER 12 9
LIVER 17 7
LIVER C 14
LIVER
LIVER
10
6
11
14
17
10
13
5
12
4
1
10
!5
43
37
27
28
29
**•
10 UG/PLATE
9-APINOACRIDINE
2-NITROFLUORENE
IA-JOO
SOLVENT
SOOIUH
AZIOE
£0 UG/PLATE
10 UG/PLATE
10 UG/PLATE
35
49
38
34
28
37
TA-1535
TA-1537
I A -98
TA-100
95
100
126
100
107
93
129
116
121
90
122
103
2-ANTHRAHINE
2-ANTHRAHINE
2-ANTHRAMINC
2-ANTHRAMINE
2.5 UG/PLATE
2.5 UG/PLATE
2.5 UG/PLATE
2.5 UG/PLATE
50 UL/PLATE
- INDICATES TEST UAS NOT DONE
C INDICATES CONTAMINATION
-------
VII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA
Statistical methods are not currently used, and evaluation is based on
the criteria included in this protocol.
Plate test data consists of direct revertant colony counts obtained from
a set of selective agar plates seeded with populations of mutant cells
suspended in a semi sol 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 semiquanti-
tative in nature. Although these features of the assay reduce the quanti-
tation of results, they provide certain advantages not contained in a
quantitative suspension test:
The small number of cell divisions permits potential
mutagens to act on replication 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 quantisation 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 concen-
trations, the highest of these doses being selected to show slight toxicity
as determined by subjective criteria.
B. Dose-Response Phenomena
The demonstration of dose-related increased in mutant counts is an impor-
tant criterion in establishing metagenicity. A factor that might modify
dose-response results for a mutagen would be the selection of doses that
are too low (usually mutagenicity and toxicity are related). If the
highest dose is far lower than a toxic concentration, 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.
C. Control Tests
Positive and negative control assays were conducted with each experiment
and consisted of direct-acting inutagens for nonactivation assays and
mutagens that require metabolic biotransformation in activation assays.
m
Litton
5-164
BIONETICS 11
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Negative controls consisted of the test material solvent in the overlay
agar together with the other essential components. The negative control
plate for each strain gave a reference point to which the test data was
compared. The positive control assay was conducted to demonstrate that
the test systems were functional with known mutagens.
The following normal range of revertants for solvent controls are generally
considered acceptable.
TA-1535: 8-30
TA-1537: 4-30
TA-98: 20-75
TA-100: 80-250
D. Evaluation Criteria for Ames Assay
Because the procedures to be used to evaluate the mutagenicity of the
test material are semi quantitative, the criteria to be used to determine
positive effects are inherently subjective and are based primarily on a
historical data base. Most data sets will be evaluated using the following
criteria.
1. Strains TA-1535 and TA-1537
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to three times the solvent control value will
be considered to be mutagenic.
2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to twice the solvent control value for TA-98
and TA-100 will be considered to be mutagenic.
3. Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain
(G-46), 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 that.cannot be
reproduced in additional runs, the initial positive test data lose signi-
ficance.
The preceding criteria are not absolute, and other extenuating factors
may enter into a final evaluation decision. However, these criteria
will be applied to the majority of situations and are presented to aid
those individuals not familar with this procedure. As the data base is
increased, the criteria for evaluation can be more firmly established.
5-165
BIONETICS 12
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E. Relation Between Mutaqenlcity and Carcinoqenicity
It must be emphasized that the Ames Salmonella/Microsome Plate Assay is
not a definitive test for chemical carcinogens. It is recognized, however,
that correlative and functional relations have been demonstrated between
these two endpoints. The results of comparative tests on 300 chemicals
by McCann et a_L4 show an extremely good correlation between results of
microbial mutagenesis tests and HI vivo rodent earcinogenesis 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 muta-
genic activity.
F. Criteria for Ranking Samples in the Ames Assay
The goal of EPA Level 1 Ames testing is to rank source streams by relative
degree of genetic toxicity (mutagenicity). Samples are first identified
as mutagenic or nonmutagenic by the criteria in Section D above and
then ranked using the mutagenicity categories presented in the table
below. The lowest concentration giving a positive response in any strain,
with or without metabolic activation, is identified as the minimum effec-
tive concentration (MEC) for that sample. The mutagenicity of the sample
is evaluated as high (H), moderate (M), low (L), or nondetectable (ND)
according to the evaluation criteria developed in the Level 1 manual1
and summarized below. Samples with no detectable activity at the maximum
applicable dose (MAD) are ranked nondetectable (ND).
Ames Assay Mutagenicity Ranking Criteria1
Mutagenic
Activity
High (H)
Moderate (M)
Low (L)
Not Detectable (ND)
Solids
(MEC in ug/plate)
<50
50-500
500-5000
>5000
(MEC
<2
2-20
Liquids3
in ul/plate)
20-200
. >200
•
Concentration of organic extracts is based upon organic content (ug
organics per plate) and not volume (ul extract per plate) of sample
tested.
Utton
5-166
BIONETICS 13
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VIII. REFERENCES
1. Brusick, D.J., et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment Biological Tests.EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetlcs, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD, August
1981, 52 pp.
4. 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.
5. Ames, B.N., Gurney, E.G., Miller, J.A. and Bartsch, H.: Carcinogens
as frameshift mutagens: Metabolites and derivatives of 2-acetylamino-
fluorene and other aromatic amine carcinogens. Proc. Nat. Acad.
Sci., USA 69:3128-3132, 1972.
6. 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.
7. 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.
8. 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.
9. Ames, B.N., McCann, J. and Yamasaki, E.: Methods for detecting
carcinogens and mutagens with the Salmone11 a/mammalian-microsome
mutagenicity test. Mutation Res., 3_l:347-364, 1975.
10. Vogel, H.J. and Bonner, D.M.: Acetylornithinase of E. coli partial
purification and some properties. J. Biol. Chem., 218:91^106. 1966.
Litton
5-167
BIONETICS 14
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GENETICS ASSAY NO.:
LBI SAFETY NO.:
MUTAGENICITY EVALUATION OF
A81-05-030-650
(EA-1 XAD EXTRACT)
1F"THF
EPA~LEVlL 1
AMES SALR5NETLA7MTCROSOME
PLATE TEST
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
ffl
Litton
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
L§1 PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
5-168
BIONETICS
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PREFACE
This assay conforms to the standard EPA Level I procedure for the Ames
Salmonella/microsome mutagenesis assay as described in "IERL-RTP Proce-
dures Manual: Level 1 Environmental Assessment Biological Tests"1. The
data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting"2.
The Ames Salmonel1 a/mi crosome mutagenesis assay has been shown to be a
sensitive method for detecting mutagenic activity for a variety of chemi-
cals representing various chemical classes3. This assay is one of several
recommended by EPA to identify, categorize and rank the pollutant potential
of influent and effluent streams from industrial and energy-producing pro-
cesses. This assay has been well validated with a wide range of positive
and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good Labora-
tory Practices Regulations of 1979. Deviations from standard procedure
shall be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives, of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
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5-169
BIONETICS
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LLJ BIONETICS
Utton
TABLE OF CONTENTS
Page No.
PREFACE • 1
I. ASSAY SUMMARY 1
II. OBJECTIVE 2
III. TEST MATERIAL 3
A. Description 3
B. Handling and Preparation 3
IV. MATERIALS 4
A. Indicator Microorganisms 4
B. Media 4
C. Activation System 5
1. S9 Homogenate 5
2. S9 Mix 5
V. EXPERIMENTAL DESIGN 6
A. Dose Selection 6
B. Mutagenicity Test • 6
1. Nonactivation Assay : 6
2. Activation Assay 7
C. Control Compounds 7
D. Recording and Presenting Data 7
VI. RESULTS 9
A. Interpretation 9
B. Tables 9
VII. EVALUATION CRITERIA 12
A. Surviving Populations 12
B. Dose-Response Phenomena 12
C. Control Tests 12
D. Evaluation Criteria for Ames Assay 13
1. Strains TA-1535 and TA-1537 13
2. Strains TA-98 and TA-100 13
3. Pattern 13
4. Reproducibility 13
E. Relation Between Mutagenicity and
Carcinogenicity 14
F. Criteria for Ranking Samples in the Ames Assay ! . 14
VIII. REFERENCES 15
. 5-170
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I. ASSAY SUMMARY
A. Sponsor: Acurex Corporation
B. Material (Test Compound): Genetics Assay Number: 5879
1. Identification: A81-05-030-650 (EA-1 XAD Extract)
2. Date Received: August 26, 1981
3. Physical Description: Clear, amber/brown liquid.
C. Type of Assay: EPA Level 1 Ames Salmonella/Microsome Plate Test
D. Assay Design Number: 401 (EPA Level 1)
E. Study Dates:
1. Initiation: September 23, 1981
2. Completion: October 16, 1981
F. Supervisory Personnel:
A. Study Director: D.R. Jagannath, Ph.D.
G, Evaluation:
The test material, A81-05-030-650 (EA-1 XAD extract), contained
18.3 mg organics per ml after solvent exchange into dimethyl sul-
foxide (DMSO). The solvent exchanged sample was evaluated for
its genetic activity in the EPA Level 1 Ames assay, directly,
and in the presence of S9 metabolic activation mix. The test
sample was mutagenic to TA-1537, TA-98 and TA-100 in the activa-
tion and nonactivation assays. The tests indicate that the
test material contains both base-pair and frameshift type muta-
gens. The dose-related mutagenic response was observed at a
minimum concentration of 2.5 ul (or 45.75 ug organics) per
plate with TA-1537 and TA-98 in the activation assays. The
MEC of 45.75 ug/plate, while in the high mutagenicity category,
closely approached the high/moderate boundary. The test
material, therefore, was ranked as having high/moderate (H/M)
borderline mutagenicity using the IERL-EPA Level I evaluation
criteria for the Ames Assay1.
Submitted by: Reviewed by:
StudtJi rector
m
Litton
D.R. Jajjannath, Ph.D. Date DavTd J. Brusick, Ph.D. (Date
Section Chief, Director,
Submammalian Genetics, Department of Molecular
Department of Molecular Toxicology
5-171
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II. OBJECTIVE
The objective of this study was to determine the genetic activity of
A81-05-030-650 (EA-1 XAD Extract) in the Salmonella/ microsome assay
with and without the addition of mammalian metabolic activation prepara-
tions. The genetic activity of a sample is measured in these assays by
its ability to revert the Salmonella indicator strains from histidine
dependence to histidine independence. The degree of genetic activity of
a sample is reflected in the number of revertants that are observed on
the histidine-free medium.
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BIONETICS
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III. TEST MATERIAL
A. Description
The test material was received as a clear, amber-brown solution in
methylene chloride. The sample contained 75.0 milligrams of organic
material in 0.7 ml of methylene chloride. No information on the sampling
parameters (such as the equivalent volume of stack gas represented by
the sample) was provided.
B. Handling and Preparation
The test material was received at LBI on August 26, 1981. The sample
was assigned LBI safety number 7163 and LBI assay number 5879. The sample
was stored at +4°C in the dark.
Pretest sample preparation consisted of solvent exchanging the sample
into dimethylsulfoxide (DMSO). The sample was transferred with methylene
chloride rinses into a graduated conical tube. The methylene cholride
was gradually evaporated (50°C under a stream of nitrogen) and DMSO was
sequentially added. The sample was brought to volume in 4.1 ml of DMSO,
giving a sample concentration of 18.3 mg organics per ml DMSO. The sample
was transferred to a glass vial and sealed with a teflon-coated rubber
septum.
Approximately 3.0 ml of test material was used for testing in two trials.
Varying aliquots of the test material were added directly to the test
mixtures to give the desired concentration. The amount of sample used
in Trial 1 was 2.9 ml and 75 ul was used in Trial 2.
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5-173
BIONETICS
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IV.
A.
MATERIALS
Indicator Microorganisms
The Salmonella typhimurium strains used in this assay were obtained from
Dr. Bruce Ames, University of California at Berkeley.4-8 The following
four strains were used.
Strain
Designation
TA-1535
Gene
Affected
his G
Additional Mutations
Repair
A uvr B
IPS R Factor
rfa
Mutation Type
Detected
Base-pair
substitution
TA-1537
TA-98
TA-100
his C A uvr B rfa
his D A uvr B rfa pKMlOl
his G A uvr B rfa pKMlOl
Frameshift
Frameshi ft
Base-pair
substitution
All the above strains have, in addition to the mutation in the histidine
operon, mutation (rfa-) that leads to defective lipopolysaccharide coat,
a deletion that covers genes involved in the synthesis of vitamin biotin
(bio-) and in the repair of ultraviolet (uv) - induced 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 physi-
cally 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 mutagens.8
In addition, plasmid pKMlOl confers resistance to the antibiotic ampi-
cillin, 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 supplemented
with a trace of biotin and an excess of histidine. In addition, the
plates with plasmid-carrying strains contain ampicillin (25 ug/ml) to
ensure stable maintenance of plasmid pKMlOl. New stock culture plates
are made as often as necessary from the frozen master cultures or from
single colony reisolates that were checked for their genotypic character-
istics (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) and used.
B.
Media
The bacterial strains were cultured in Oxoid Media #2 (Nutrient Broth).
The selective medium was Vogen Bonner Medium E with 2% glucose.10 The
m
IJtton
5-174
BIONETICS
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overlay agar consisted of 0.6% purified agar with 0.05 mM histidine,
0.05 mM biotin and 0.1M NaCl according to the methods of Ames et a_[.9
C.
1.
Activation System
S9 Homogenate
A 9,000 x £ supernatant prepared from Sprague-Dawley adult male rat liver
induced by Aroclor 1254 (Ames et aj.9) was purchased commercially and
used in these assays.
2. S9 Mix
59 mix used in these assays consisted of the following components:
Components
Concentration per Mi Hi liter
S9 Mix
NADP (sodium salt)
D-glucose-6-phosphate
MgCl2
KC1
Sodium phosphate buffer
pH 7.4
Organ homogenate from rat
liver (S9 fraction) •
4 umoles
5 umoles
8 umoles
33 umoles
100 umoles
100
5-175
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BIONETICS
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V. EXPERIMENTAL DESIGN
A. Dosage Selection
Test strategy and dose selection depend upon sample type and sample avail-
ability. The Level 1 manual1 recommends solids to be initially tested
at the maximum applicable dose (MAD) of 5 mg per plate and at lower con-
centrations of 2.5, 1, 0.5, 0.1 and 0.05 mg per plate. Liquids are tested
initially at the MAD of 200 ul per plate, and at lower concentrations of
100, 50 and 10 ul per plate. Samples are retested over a narrower range
of concentrations with strains showing positive results initially. Alter-
nate dose are employed if sample size is limiting or at the direction of
the sponsor.
Doses selected for the initial test of sample covered the recommended
dose range for liquid samples. The highest dose was at the MAD level of
200 ul/ml per plate and included three lower dose levels of 100, 50 and
10 pi per plate. These dose levels corresponded to 3660, 1830, 915, and
183 ug organics per plate. The second trial, using a lower range of
doses, was conducted using dose levels of 5, 2.5 and 1.0 ul per plate.
These doses corresponded to 91.5, 45.75 and 18.3 ug organics per plate.
B. Mutagenicity Testing
The procedure used was based on the paper published by Ames et. aJL9 and
was performed as follows:
1. Nonactivation Assay
To a sterile 13 x 100 mm test tube placed in a 43°C water bath the fol-
lowing was added in order:
2.00 ml of 0.6% agar containing 0.05 mM histidine and
0.05 mM biotin.
0.01 ml to 0.2 ml of a solution of the test chemical to
give the appropriate dose.
0.1 ml to 0.2 ml of indicator organism(s).
0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured onto minimal agar plates
(see IV B, Media). After the top agar had set, the plates were incubated
at 37°C for approximately 2 days. The number of his+ revertant colonies
growing on the plates were counted with an automatic colony counter and
recorded.
ffi
Litton
5-176
BIONETICS
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2.
Activation Assay
The activation assay was run concurrently with the nonactivation assay.
The only difference was the addition of 0.5 ml of S9 mix (see IV C, Acti-
vation System) to the tubes in place of 0.5 ml of phosphate buffer which
was added in nonactivation assays. All other details were similar to
the procedure for nonactivation assays.
A detailed flow diagram for the plate incorporation assay is provided in
Figure 1.
C.
Control Compounds
A negative control consisting of the solvent used for the test material
was also assayed concurrently with the test material. For negative con-
trols, step 'b1 of. Nonactivation Assays was replaced by 0.05 ml of the
solvent. The negative controls were employed for each indicator strain
and were 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 were
made using this solvent. The amount of solvent used was equal to the
maximum volume used to give the appropriate test dose.
Specific positive control compounds known to revert each strain were also
used and assayed concurrently with the test material.. The concentrations
and specificities of these compounds to specific strains are given in
the following table:
Concentration
per plate SalmoneT
Assay
Nonactivation
Chemical
Sodium azide
2-Nitrofluorene
(NF)
9~ ami noacri dine
(9AA)
Solvent (ug)
Water
Dimethyl-
sulfoxide
Ethanol
10.0
10.0
50.0
Strains
TA-1535,
TA-98
TA-1537
la
TA-100
Activation
2-anthramine
(ANTH)
Dimethyl-
sulfoxide
2.5
For all strains
D.
Recording and Presenting Data
The number of colonies on each plate were counted and recorded on printed
forms. These raw data were analyzed in a computer program and reported
on a printout. The results are presented as revertants per plate for
each indicator strain employed in the assay. The positive and solvent
controls are provided as reference points.
5-177
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BIONETICS
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AMES ASSAY [PLATE INCORPORATION METHOD]
Aliquot of
saline
0.5 ml
-S-9
Molten [45'C] overlay agar
appropriately supplemented
0.1 ml
Test, positive or solvent
control chemical
Aliquot of an overnight culture
of bacterial 109 cells/ml]
0.5 ml S-9 mix [hepatic
— homogenate from PCB
pretreated rat plus
necessary cofactors
Overlay poured on selective
bottom agar medium
Plated incubated at 37'C for 48 hours
The numbers of revertants/plate counted
Data analyzed
Interpretation/Conclusion
Figure 1 AMES SALMONELLA/MICROSOME MUTAOENESIS ASSAY
5-178
-------
VI. RESULTS
A. Interpretations
The test material, A81-05-030-650 (EA-1 XAD extract), in methylene chloride
was solvent exchanged to DMSO and this solvent exchanged material was
tested for its genetic activity in the EPA Level 1 Ames assays. The
organic content, after solvent exchange, was 18.3 mg per ml. Initially,
the test was performed only with TA-98 and TA-100 at four dose levels
from 10.0 ul per plate to 200.0 (jl per plate doses due to the limited
quantity of the test sample.
The initial results with TA-98 and TA-100 exhibited positive response at
the lowest dose of 10.0 ul per plate with both strains. The test sample
was toxic to both strains at doses of 50.0 pi and above in the nonactiva-
tion assays. As such, repeat tests were conducted using all the four
Salmonella strains at 1, 2.5 and 5.0 ul/plate in the activation and
nonactivation assays.
The repeat tests conducted on the test sample were positive with TA-1537
and TA-98 in the activation and nonactivation assays and with TA-100 in
the activation assays. The minimum effective concentration that exhibited
the mutagenic response was at 2.5 ul per plate (45.75 ug organics/plate)
in the activation assays with TA-1537 and TA-98. This response, while
in the high mutagenicity category, closely approached, the high/moderate
borderline. The test material, therefore, was ranked as having high/
moderate (H/M) mutagenicity using the IERL-EPA Level 1 evaluation criteria
for the Ames Assay1. These tests indicate that the XAD extract of the
test material, A81-05-030-650 (EA-1 XAD extract), contains both base-pair
and frameshift type mutagens.
Solvent control and positive control values were within acceptable ranges.
These results achieved assay acceptance criteria and provided confidence
in the assumptions that the recorded data represented typical responses
to the test material.
B. Tables
This report is based on the data provided in Tables 1 and 2.
Litton
5-179
BIONETICS
-------
RESULTS
TABLE 1
NAME OR COOt DESIGNATION OF THE TEST COHPOUNC:
SOLVENT: NONE
TEST INITIATION DATES: 10/01/81
TEST COMPLETION OATC: 10/Q«/81
S-9 LOT*: REF030
CONCENTRATIONS ARE GIVEN IN NICROLITERS FER
A.
B.
C.
0.
£.
NOTE:
TEST
NONACTIVATION
A81-05-030-650 (EA-1 XAD EXTRACT)
SPECIES TISSUE
PLATE
REVERTANTS FER
TA-98 TA-100
123 123
F L A T E
cn
i
oo
o
SOLVENT CONTROL
POSITIVE CONTROL**
TEST COMPOUND
10.00 UL
£0.00 UL
100.00 UL
200.00 UL
ACTIVATION
30
760
93
0
0
0
30
119
0
0
0
124 128
1192 1362
303
0
0
0
249
0
0
0
SOLVENT CONTROL RAT
POSITIVE CONTROL*** RAT
TEST COMPOUND
10.00 UL
SO. 00 UL
100.00 UL
200.00 UL
* •
•
TA-98
TA-100
RAT
RAT
RAT
RAT
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
38
2036
466
315
185
0
45
301
218
110
0
2-NITROFLUORENE
SODIUM AZIOE
127
2074
397
251
0
0
132
214S
245
248
0
G
10 UG/PLATE
10 UG/PLATE
»*»
TA-98 2-ANTHRAHINE 2.5 UG/PLATE
TA-100 2-ANTHR*r>INE 2.5 UG/PLATE
SOLVENT SO UL/PLATE
- INDICATES TEST HAS NOT DONE
-------
RESULTS
TABLE 2
A. NAME OR CODE DESIGNATION OF THE TEST COMPOUND: A81-05-03C-650 IEA-IXAD EMTRACTJ
B. SCLVENT: DMSO
C. TEST INITIATION DATES: 10/13/81
D. TEST COMPLETION DATE: 10/16/81
E. S-9 LOTH: REF050
NCTE: CONCENTRATIONS ARE GIVEN IN NICROLITERS PER PLATE
en
l
00
TEST SPECIES TISSUE
NONACTIVATION
SOLVENT CONTROL
SOLVENT CONTROL
POSITIVE CONTROL**
POSITIVE CONTROL**
TEST COMPOUND
1.00 UL
2.50 UL
5.00 UL
ACTIVATION
SOLVENT CONTROL RAT
SOLVENT CONTROL RAT
POSITIVE CONTROL*** RAT
POSITIVE CONTROL*** RAT
TEST COMPOUND
1.00 UL RAT
2.50 UL RAT
5.00 UL RAT
• •
TA-1S35 SODIUM AZIOE
TA-1S3T 9-AHINOACRIOINE
TA-98 2-NITROFLUORENE
TA-100 SODIUM AZIOE
SOLVENT 50 UL/PLATE
— -
__-
---
--.
.__
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
TA-1535
1 2
12
16
158
1007
14
19
a
a
11
391
351
10
11
13
TA-1537
TA-9B
3 123 12
9
9
256
220
7
12
28
6
7
431
416
15
27
46
10 UG/PLATE
SO UG/PLATE
10 UG/PLATE
10 UG/PLATE
27
22
958
933
38
35
50
38
37
1680
1758
62
111
170
**«
TA-1535
TA-1537
TA-98
TA-100
TA-100
312
123
109
1404
1370
112
139
191
97
105
2113
1997
134
173
249
2-ANTHR4MINC
2-ANTHRAHINE
2-ANTHRAMINE
2-ANTHRAMINE
3
2.5 U6/PLATE
2.5 UG/PLATE
2.5 UG/PLATE
2.5 UG/PLATE
- INDICATES TEST UAS NOT DONE
-------
VII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA
Statistical methods are not currently used, and evaluation is based on
the criteria included in this protocol.
Plate test data consists of direct revertant colony counts obtained from
a set of selective agar plates seeded with populations of mutant cells
suspended in a semi sol 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 semi quanti-
tative in nature. Although these features of the assay reduce the quanti-
tation of results, they provide certain advantages not contained in a
quantitative suspension test:
The small number of cell divisions permits potential
mutagens to act on replication DNA, which is often more
sensitive than nonreplicating 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 quantitatiom 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 concen-
trations, the highest of these doses being selected to show slight toxicity
as determined by subjective criteria.
B. Dose-Response Phenomena
The demonstration of dose-related increased in mutant counts is an impor-
tant criterion in establishing metagenicity. A factor that might modify
dose-response results for a mutagen would be the selection of doses that
are too low (usually mutagenicity and toxicity are related). If the
highest dose is far lower than a toxic concentration, 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.
C. Control Tests
Positive and negative control assays were conducted with each experiment
and consisted of direct-acting mutagens for nonactivation assays and
mutagens that require metabolic biotransformation in activation assays.
ffl
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5-182
BIONET1CS
12
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Negative controls consisted of the test material solvent in the overlay
agar together with the other essential components. The negative control
plate for each strain gave a reference point to which the test data was
compared. The positive control assay was conducted to demonstrate that
the test systems were functional with known mutagens.
The following normal range of revertants for solvent controls are generally
considered acceptable.
TA-1535: 8-30
TA-1537: 4-30
TA-98: 20-75
TA-100: 80-250
D. Evaluation Criteria for Ames Assay
Because the procedures to be used to evaluate the mutagenicity of the
test material are semiquantitative, the criteria to be used to determine
positive effects are inherently subjective and are based primarily on a
historical data base. Most data sets will be evaluated using the following
criteria.
1. Strains TA-1535 and TA-1537
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to three times the solvent control value will
be considered to be mutagenic.
2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to twice the solvent control value for TA-98
and TA-100 will be considered to be mutagenic.
3. Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain
(G-46), 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 that cannot be
reproduced in additional runs, the initial positive test data lose signi-
ficance.
The preceding criteria are not absolute, and other extenuating factors
may enter into a final evaluation decision. However, these criteria
will be applied to the majority of situations and are presented to aid
those individuals not familar with this procedure. As the data base is
increased, the criteria for evaluation can be more firmly established.
5-183
__ BIONETICS 13
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-------
E. Relation Between Mutaqenicity and Carcinogenicity
It must be emphasized that the Ames Salmonella/Microsome Plate Assay is
not a definitive test for chemical carcinogens. It is recognized, however,
that correlative and functional relations have been demonstrated between
these two endpoints. The results of comparative tests on 300 chemicals
by McCann et al_.4 show an extremely good correlation between results of
microbial mutagenesis tests and |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 muta-
genic activity.
F. Criteria for Ranking Samples in the Ames Assay
The goal of EPA Level 1 Ames testing is to rank source streams by relative
degree of genetic toxicity (mutagenicity). Samples are first identified
as mutagenic or nonmutagenic by the criteria in Section D above and
then ranked using the mutagenicity categories presented in the table
below. The lowest concentration giving a positive response in any strain,
with or without metabolic activation, is identified as the minimum effec-
tive concentration (MEC) for that sample. The mutagenicity of the sample
is evaluated as high (H), moderate (M), low (L), or nondetectable (ND)
according to the evaluation criteria developed in the Level 1 manual1
and summarized below. Samples with no detectable activity at the maximum
applicable dose (MAD) are ranked nondetectable (ND).
Ames Assay Mutagenicity Ranking Criteria1
Mutagenic
Activity
High (H)
Moderate (M)
Low (L)
Not Detectable (ND)
Solids
(MEC in ug/plate)
<50
50-500
500-5000
>5000
(MEC
<2
2-20
Liquids3
in ul/plate)
20-200
>200
a
'Concentration of organic extracts is based upon organic content (ug
organics per plate) and not volume (ul extract per plate) of sample
tested.
Litton
5-184
BIONET1CS
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VIII. REFERENCES
1. Brusick, D.J., et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment Biological Tests.EPA Contract No. 68-02-2681,
technical Directive No. 501, Litton Bionetics, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD, August
1981, 52 pp.
4. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N.: Detection of
carcinogens as mutagens in the Salmonena/microsome test: Assay of
300 chemicals. Proc. Nat. Acad. Sci., USA 72:5135-5139, 1975.
5. Ames, B.N., Gurney, E.G., Miller, J.A. and Bartsch, H.: Carcinogens
as frameshift mutagens: Metabolites and derivatives of 2-acetylamino-
fluorene and other aromatic amine carcinogens. Proc. Nat. Acad.
Sci., USA 69:3128-3132, 1972.
6. 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.
7. 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.
8. 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.
9. 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.
10. Vogel, H.J. and Bonner, D.M.: Acetylornithinase of E. coli partial
purification and some properties. J. Biol. Chem., 218:9"7r106, 1966.
Litton
5-185
BIONETICS 15
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m
Litton
GENETICS ASSAY NO.: 5880
LBI SAFETY NO.: 7164
CYTOTOXIC EVALUATION OF
(EA-1 XAD EXTRACT)
TORE
RODENT"CHT (CHO)
CLQNAL ToncTTY~Ss3AY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO. 22064
REPORT DATE: NOVEMBER 1981
5-186
BIONETICS
-------
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Chinese
hamster ovary cell (CHO) clonal toxicity assay as described in "IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests" (1).
The data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting" (2).
The CHO clonal toxicity assay has been shown to be a sensitive method for
detecting cytotoxic activity for a variety of chemicals representing
various chemical classes (3). This assay is one of several recommended
by EPA to identify, categorize and rank the pollutant potential of
influent and effluent streams from industrial and energy-producing
processes. This assay has been well validated with a wide range of posi-
tive and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration,, testing and evaluation of the test material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good
Laboratory Practices Regulations of 1979. Deviations from standard
procedure shall be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
Litton
5-187
BIONETICS
-------
TABLE OF CONTENTS
Page No.
PREFACE
I. ASSAY SUMMARY
II. OBJECTIVE
III. TEST MATERIAL
A. Description
B. Handling and Preparation
IV. MATERIALS
A. Indicator Cells ....
B. Media
C. Controls
V. EXPERIMENTAL DESIGN
A. Dose Selection
B. Clonal Toxicity Assay .
VI. ASSAY ACCEPTANCE CRITERIA . .
VII. RESULTS
A. Interpretation
B. Tables and Figures . . .
VIII. ASSAY EVALUATION CRITERIA . .
IX. REFERENCES
i
1
2
3
3
3
4
4
4
5
5
7
8
8
8
12
13
m
Litton
BIONETICS
5-188
-------
I. ASSAY SUMMARY
A. SPONSOR: Acurex Corporation
B. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5879
1. Identification: A81-05-030-650 (EA-1 XAD Extract)
2. Date Received: August 26, 1981
3. Physical Description: Clear, amber-brown liquid
C. TYPE OF ASSAY: Rodent Cell (CHO) Clonal Toxicity Assay
D. ASSAY DESIGN NUMBER: 442
E. STUDY DATES:
1. Initiation: September 23, 1981
2. Completion: November 24, 1981
F. SUPERVISORY PERSONNEL:
1. Study Director: Brian C. Myhr, Ph.D.
2. Laboratory Supervisor: Robert Young, M.S.
G. EVALUATION:
The test material was assayed, as a DMSO extract, over the concen-
tration range of 0.01 ul/ml to 20 ul/ml. A very sharp increase in
toxicity occurred in the vicinity of 0.1 ul/ml in the course of two
trials. The EC50 was estimated to be 0.1 ul/ml, which was equivalent
to 1.8 ug of organics/ml. Although the exact position of the EC50
appeared to vary between the two trials, the values remained in the
high (H) toxicity category defined by the evaluation criteria for
the IERL-EPA Level I CHO Clonal Toxicity Assay1.
Submitted by:
Study Director
Brian Myhr, P&/.D.
Associate Director,
Department of Molecular
Toxicology
Reviewed by:
David J. Brusick, Ph.D.
Director,
Department of Molecular
Toxicology
5-189
Litton
BIONETICS
-------
II. OBJECTIVE
The objective of this study was to determine and rank the cytotoxicity
of A81-05-030-650 (EA-1 XAD extract) to cultured Chinese hamster cells
(CHO-K1 cell line). The measure of cytotoxicity was the reduction in
colony-forming ability after a 24-hour exposure to the test material.
After a period of recovery and growth, the number of colonies that
developed in the treated cultures was compared to the colony number in
unexposed vehicle control cultures. The concentration of test material
that reduced the colony number by 50% was estimated graphically and
referred to as the EC50 value. Standard EPA Level 1 toxicity evaluation
criteria for the CHO clonal toxicity assay were used to rank the toxicity
potential of the test material.
m
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5-190
BIONETICS
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III. TEST MATERIAL
A. Description
The test material was received as a clear, amber-brown solution in
methylene chloride. The sample contained 75.0 milligrams of organic
material in 0.7 ml of methylene chloride. No information on the sampling
parameters (such as the equivalent volume of stack gas represented by
the sample) was provided.
B. Handling and Preparation
The test material was received at LBI on August 26, 1981. The sample
was assigned LBI safety number 7163 and LBI assay number 5879. The sample
was stored at +4°C in the dark.
Pretest sample preparation consisted of solvent-exchanging the sample
into dimethylsulfoxide (DMSO). The sample was transferred with methylene
chloride rinses into a graduated conical tube. The methylene chloride
was gradually evaporated (50°C under a stream of nitrogen) and DMSO was
sequentially added. The sample was brought to volume in 4.1 ml of DMSO,
giving a sample concentration of 18.3 mg organics per ml DMSO. The sample
was then transferred to a glass vial and sealed with a teflon-coated rubber
septum.
A total volume of 0.45 ml of test sample was used in the CHO assay. The
maximum concentration of 20 pi/ml was obtained by adding 0.12 ml of sample
to 5.88 ml of F12 medium; this resulted in 2% (v/v) DMSO in the medium
and effectively limited the concentration of test material that could be
assayed. Only two plates were exposed to the high dose in order to con-
serve sample. Another 0.12 ml aliquot of sample was used to prepare the
10 ul/ml test concentration. An additional 0.21 ml of test sample was
used to prepare a series of dilutions in DMSO from which 1:100 dilutions
into growth medium were performed to obtain the lower assayed concentra-
tions. Thus, except for the 20 ul/ml test concentration, the final DMSO
concentration was constant at 1% (v/v).
Litton
5-191
BIONETICS
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IV. MATERIALS
A. Indicator Cells
The indicator cells for this study were Chinese hamster CHO-K1 cells
(ATCC No. CCL 61) obtained from Flow Laboratories, Inc., Rockville, MD.
This cell type was derived from ovarian tissue and has spontaneously
transformed to a stable, hypodiploid line of rounded, fibroblastic cells
with unlimited 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. Laboratory stock are maintained by
routine serial subpassage. Cells are cultivated in Ham's F-12 nutrient
medium at 37°C in 5 percent C02 with saturated humidity. Stocks are
continually observed macroscopically and microscopically for possible
microbial contamination. Laboratory cultures are periodically checked
by culturing and staining methods for the absence of mycoplasma contami-
nation. Laboratory cultures are discarded every three months and new
cultures started from mycoplasma-free, long-term frozen cultures.
B. Media
The CHO-K1 cell line has an absolute requirement for proline and therefore
must be maintained in culture medium containing sufficient amounts of
this amino acid. Ham's F12 medium, which contains 3 x 10-4 M L-proline
was used, supplemented with 10% fetal bovine serum, 2mM L-glutamine,
100 units/ml of penicillin, 100 ug/ml of streptomycin, and 0.9 ul/ml of
amphotericin B. A 10X formulation of Ham's F10 is available commercially
and was used for the testing of aqueous test samples in order to avoid
the dilution of medium components. This medium contains 1 x 10-4 L-proline
and was supplemented in the same manner as F12, except that kanamycin at
40 (jg/ml is included for additional protection against bacterial contami-
nation. Both media formulations support the growth and cloning of CHO
cells equally well.
C. Controls
The negative control consisted of three untreated cultures carried through
the same experimental time period as the treated cells. Since the test
material was tested as a solution in an organic vehicle (DMSO) and was
diluted into the medium to provide each test concentration, two sets of
vehicle control cultures containing the organic solvent at 1% and 2% by
volume were prepared in triplicate.
The average number of colonies in the negative control established the
cloning efficiency of the CHO cells used in the assay, and the appropriate
vehicle controls provided the reference points for determining the effects
of different concentrations of the test material on cell survival.
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BIONET1CS
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Litton
V. EXPERIMENTAL DESIGN
A. Dose Selection
Unless the approximate toxicity is already known or the sample size is
limiting, the following dose ranges are usually tested for different
sample forms. Aqueous samples, suspensions, or slurries are tested from
600 ul/ml to 3 pi/ml, usually in six dose steps. Eight doses are often
used when the amount of test sample is limited to provide a more precise
description of toxicity in the event of sharp dose-response curves. Dry,
particulate material is dissolved or suspended in DMSO, diluted into
growth medium, and tested at six dose levels from 1000 MO/1""! to 3 ug/ml.
Samples that are solvent-exchanged into DMSO are tested from 20 ul/ml
(2% DMSO in growth medium) to 0.2 Ml/ml, also in six dose steps. A
second dose study is performed with an adjusted dose range if the EC50
was not located properly in the initial test. However, EC50 values
greater than 1000 ug/ml for particulate material, 600 ul/ml for aqueous
samples, or 20 pi/ml for organic solutions will not be determined.
This sample, A81-05-030-650 (EA-1 XAD extract), was tested at eight dose
levels. The concentrations started with the maximum applicable dose (MAD)
of 20 ul/ml and included 10, 6, 3, 1, 0.6, 0.3, and 0.1 pi/ml. The
corresponding concentration of organics at the MAD level was 366 ug/ml;
the lower doses were equivalent to 183, 109.8, 54.9, 18.3, 11.0, 5.5, and
1.8 ug of organics/ml.
B. Clonal Toxicity Assay
Cells from monolayer stock cultures in logarithmic growth phase were tryp-
sinized with 0.1% trypsin plus 0.01% versene for 4 minutes and the density
of the resulting cell suspension determined by hemocytometer. A number
of 60-mm culture dishes were then seeded with 200 cells and 4 ml of culture
medium per dish. The cultures were incubated for approximately 6 hours
at 37°C in a humidified atmosphere containing 5% C02 to allow attachment
of the cells. The 6-hour attachment period was used in order to avoid
cell division and the subsequent formation of two-cell colonies prior to
treatment.
The medium was aspirated from the cultures and 4 ml medium containing
the test material applied. Three cultures were exposed to each test con-
centration. After an exposure time of 24 hours at 37°C, the medium was
removed by aspiration and each culture washed three times with approxi-
mately 4 ml aliquots of Dulbecco's phosphate buffered saline (pre-warmed
to 37°C). Fresh culture medium (5 ml) was placed in each dish and incuba-
tion at 37°C is continued for an additional 6 days to allow colony develop-
ment.
The test material caused a color change in the culture medium, the pH of
the medium containing the high dose would be determined at the time of
treatment. The pH at the lowest dose that results in a slight color change
would also recorded. At the end of the treatment period, the pH values
of the discarded media from the two described treatments would be recorded
again. No sample related pH effects were noted.
5-193
BIONETICS 5
-------
After the incubation period, the medium was drained from the cultures
and the surviving colonies fixed with 100% ethanol and stained with
Giemsa. Colonies were counted by eye; tiny colonies of approximately
50 cells or less were arbitrarily excluded from the counts.
EH
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5-194
BIONETICS
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VI. 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
negative 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 location 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 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.
Litton
5-195
BIONETICS
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ffl
VII. RESULTS
A. Interpretation
The test material, A81-05-030-650 (EA-1 XAD extract), was highly toxic
to the CHO cells in the first trial. As shown in Table 1, only the low
dose of 0.1 pi/ml resulted in any surviving colonies (15.6% survival).
These results indicated that the EC50 was less than 0.1 ul/ml or 1.8 ug
of organics/ml. Since EC50 values below 10 ug/ml are in the high toxicity
region defined for the IERL-EPA CHO clonal toxicity bioassay1, the test
material was clearly categorized as having high (H) toxicity to CHO cells.
A very small amount of the test material was available for a second trial,
so an attempt was made to locate the EC50. Concentrations from 0.01 ul/ml
to 0.3 ul/ml were tested, and the results are presented in Table 2.
Also, the relative survivals were plotted along with the results from
the first trial in Figure 1. A comparison of the two trials indicated
that the EC50 had shifted to a value greater than 0.1 ul/ml in the second
trial. The survival curve was very sharp. It is not unusual for sharp
curves to shift between trials, so the results were analyzed by considering
a curve that appeared to be intermediate between the two tirals (dashed
line in Figure 1). Thus, a sharp break in survival was estimated to be
centered, on the average, at an EC50 of 0.1 ul/ml (1.8 ug organics/ml).
Individual trials might yield values ranging from 0.06 to 0.16 pi/ml
(1.1 to 2.9 ug orgaincs/ml).
The cells used for the assay were in logarithmic growth phase and were
greater than 98 percent viable for both trials. About 73 percent of the
seeded cells in trial 1 and 77 percent of the seeded cells in trial 2
were able to form colonies as shown by the negative control results.
Colony growth was normal and well distributed on the culture dishes.
The combined results were considered to achieve assay acceptance criteria
and provided confidence in the assumption that the recorded data repre-
sented typical responses to the test material.
B. Tables and Figures
This report is based on the data provided in Tables 1 and 2 and Figure 1.
5-196
BIONETICS
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TABLE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
Sample Identity: A81-05-030-650
(EA-1 XAD Extract")
Description .of Sample: Clear,
amber-brown liquid
EC50 Value: <1.8 pg/ml
Toxicity
.Classification:
High
pH Alterations: None
LBI Assay No.:
Date Received:
5879
August 26. 1981
Test Date: September 28. 1981 (Trial 1)
Vehicle: DMSO
Cell Type: CHQ-K1
Cells Seeded per Dish: 200
Comments on
Treatment: Sample prepared in DMSO
at a concentration of 18.3 ug
organics/ul
COLONY COUNTS
Sample
NCb r
VC, 1%C
VC, 2%
TEST
TEST
TEST
TEST
TEST
TEST
TEST
TEST
Applied
Concentration
Ml /ml
• **
10
20
0.1
0.3
0.6
1.0
3.0
6.0
10.0
20.0
Dish
146
143
112
21
0
0
0
0
0
0
0
Dish
#2
152
125
110
26
0
0
0
0
0
0
0
Dish
#3
140
155
121
19
0
0
0
0
0
°d
sd
Average
Count
146.0
141.0
114.3
22.0
0
0
0
0
0
0
0
Relative
Survival
•»__
100.0
100.0
15.6
0
0
0
0
0
0
0
Cloning
Efficiency
73.0
70.5
57.2
^Relative to 2% VC for 20 ul/ml treatment and to 1% VC for other treatments.
NC = Negative Control, F12 medium.
jVC = Vehicle Control, percent DMSO given.
Only two plates dosed to conserve limited test material.
5-197
Litton
BIONETICS
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TABLE 2
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
Sample Identity: A81-05-030-650
(EA-1 XAD Extract)
Description of Sample: Clear, amber
brown liquid
LBI Assay No.: 5879
Estimated
EC50 Value: 0.1 ul/ml (1.8
organics /ml)
Toxicity
Classification:
pH Alterations:
High
None
Date Received: August 26. 1981
Test Date: November 17, 1981
(Trial 2)
Vehicle: F12 Medium
Cell Type: CHO-K1
Comments on
Treatment: Sample prepared in DMSO
in DMSO at a concentration of
18.3 ug organics/ul
Cells Seeded per Dish:
200
CLONAL TOXICITY DATA
Sample
NCb
TEST
TEST
TEST
TEST
TEST
TEST
TEST
Applied
Concentration
Ml /ml
—
0.01
0.02
0.04
0.06
0.08
0.1
0.3
Dish
#1
141
154
130
140
137
141
134
10
Dish
#2
155
155
137
135
136
130
125
6
Dish
#3
167
144
138
139
133
131
127
8
Average
Count
154.3
151.0
135.0
138.0
135.3
134.0
128.7
8.0
Relative
Survival
%
100.0
97.9
87.5
89.4
87.7
86.8
83.4
5.2
Cloning
Efficiency
%
77.2
^Relative to F12 negative control for all treatments.
NC = Negative Control, F12 medium.
m
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5-198
BIONETICS
10
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FIGURE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
EC50 DETERMINATION
A81-05-030-650
(EA-1 XAD EXTRACT)
140
11
-------
VIII. ASSAY EVALUATION CRITERIA
The EC50 value represents the concentrations of test material that reduces
the colony-forming ability of CHO cells to 50% of the vehicle or negative
control value. EC50 values are determined graphically by fitting a curve
by eye through relative survival data plotted as a function of the loga-
rithm of the applied concentration. Each data point normally represents
the average of three culture dishes. In order to indicate the variability
of the data, the high and low colony counts for each concentration are
used to calculate the relative survivals, and the range is shown by a
bar at the position of the plotted average. If no bar is shown, the
variability was within the size of the symbol. Statistical analysis is
unnecessary in most cases for evaluation.
The toxicity of the test material is evaluated as high, moderate, low,
or nondetectable according to the range of EC50 values defined in the
following table.
SolidsAqueous LiquidsNonaqueous Liquids
Toxicity3 (EC50 in ug/ml) (EC50 in Ml/ml) (EC50 in ul/ml)
High
Moderate
Low
Not Detectable
<10
10 to 100
100 to 1000
>1000
<6
6 to 60
60 to 600
>600
<0.2
0.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level 1 Environmental Assessment BiologicalTests?
Criteria for nonaqueous liquids are tentative and under evaluation.
If the organic or solids content is known, the sample is evaluated under
the solids criteria.
Another evaluation scheme is proposed for extracts obtained from SASS
train gas volumes. The proportion of the total gas volume corresponding
to the volume of extract used in the bioassay is calculated and expressed
as L/ml of culture medium (or DSCF/ml of culture medium). A criterion
of 1000 L/ml is set as the limit for nondetectable toxicity. This gas
volume corresponds to the average volume breathed by humans over a 2-hour
period. The subsequent toxicity ranges are defined by 10-fold dilution
steps to conform to standard procedure. The toxicity ranges are defined
in the following table for liter and dry standard cubic feet units:
Toxicity
High
Moderate
Low
Nondetectable
ECSO In
Liters/ml (L/ml)
<10
10-100
100-1000
>1000
EC50 In
Dry Standard Cubic Feet/ml (DSCF/ml)
<0.35 DSCF
0.35-3.5
3.5-35
>35
Litton
5-200
BIONETICS 12
-------
IX. REFERENCES
1. Brusick, D.J., et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment Biological Tests.EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD,
August 1981, pp 52.
Litton
5-201
BIONETICS 13
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GENETICS ASSAY NO.: 5886
LBI SAFETY NO.: TITO
MUTAGENICITY EVALUATION OF
A81-05-030-662
TEA-1 FLYASH)
~~IN THE
EPA~LEVlL 1
AMES SAlFiO'NELWMT.CROSOME
PLATE TEST
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
E
Utton
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
5-202
BIONETICS
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PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Ames
Salmonella/mlcrosome mutagenesis assay as described in "IERL-RTP Proce-
dures Manual: Level 1 Environmental Assessment Biological Tests"1. The
data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting"2.
The Ames Salmonel1 a/mi crosome mutagenesis assay has been shown to be a
sensitive method for detecting mutagenic activity for a variety of chemi-
cals representing various chemical classes3. This assay is one of several
recommended by EPA to identify, categorize and rank the pollutant potential
of influent and effluent streams from industrial and energy-producing pro-
cesses. This assay has been well validated with a wide range of positive
and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good Labora-
tory Practices Regulations of 1979. Deviations from standard procedure
shall be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives, of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
Litton
5-203
BIONETICS
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TABLE OF CONTENTS
Page No.
PREFACE 1
I. ASSAY SUMMARY l
11. OBJECTIVE 2
III. TEST MATERIAL 3
A. Description 3
B. Handling and Preparation 3
IV. MATERIALS 4
A. Indicator Microorganisms 4
B. Media ..... 4
C. Activation System 5
1. S9 Homogenate ; 5
2. S9 Mix 5
V. EXPERIMENTAL DESIGN 6
A. Dose Selection 6
B. Mutagenicity Test 6
1. Nonactivation Assay 6
2. Activation Assay 6
C. Control Compounds 7
D. Recording and Presenting Data 7
VI. RESULTS 9
A. Interpretation 9
B. Tables 9
VII. EVALUATION CRITERIA 11
A. Surviving Populations 11
B. Dose-Response Phenomena 11
C. Control Tests 11
D. Evaluation Criteria for Ames Assay 12
1. Strains TA-1535 and TA-1537 12
2. Strains TA-98 and TA-100 12
3. Pattern 12
4. Reproducibility 12
E. Relation Between Mutagenicity and
Carcinogenicity 13
F. Criteria for Ranking Samples in the Ames Assay . . 13
VIII. REFERENCES 14
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BIONETICS ,;
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I. ASSAY SUMMARY
A. Sponsor: Acurex Corporation
B. Material (Test Compound): Genetics Assay Number: 5886
1. Identification: A81-05-030-662 (EA-1 Flyash)
2. Date Received: August 26, 1981
3. Physical Description: Black and gray particles
C. Type of Assay: EPA Level 1 Ames Sal monell a/Mi crosome Plate Test
D. Assay Design Number: 401 (EPA Level 1)
E. Study Dates:
1. Initiation: September 23, 1981
2. Completion: September 28, 1981
F. Supervisory Personnel:
A. Study Director: D.R. Jagannath, Ph.D.
G. Evaluation:
The test material , 'A81-05-030-662 (EA-1 flyash), was evaluated
for its genetic activity in the EPA Level 1 Ames Salmonella
assay directly and in the presence of a metabolic activation
system. The test material was preincubated in dimethyl sulfoxide
at 37°C overnight in a rotary shaker before testing. Testing
was conducted over a concentration range of 0.05 mg/plate to
5.0 mg/plate. The test was performed in duplicate under non-
activation and activation test conditions with strains TA-1535,
TA-1537, TA-98, and TA-100.
The results of the nonactivation and activation assays were
negative. Based on the mutagenicity results, the mutagenic
activity of the test material was ranked as nondetectable (ND)
according to the EPA Level 1 evaluation criteria for the Ames
Assay1 .
Submitted by: Reviewed by:
StudyQj rector ^—
D.R. Jagannath, Ph.D. Date David J. Brusick, Ph.D. Date
Section Chief, Director,
Submammalian Genetics, Department of Molecular
Department of Molecular Toxicology
m Toxicology
BIONET1CS 5-205
Litton
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II. OBJECTIVE
The objective of this study was to determine the genetic activity of
A81-05-030-662 (EA-1 flyash) in the Salmonella/ microsome assay with and
without the addition of mammalian metabolic activation preparations.
The genetic activity of a sample is measured in these assays by its ability
to revert the Salmonella indicator strains from histidine dependence to
histidine independence. The degree of genetic activity of a sample is
reflected in the number of revertants that are observed on the histidine-
free medium.
ffl
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BIONETICS
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III. TEST MATERIAL
A. Description
The test material was received as black and gray particles (15 gm) and
was used without further preparation. No information on actual particle
size distribution or on sampling parameters was received.
B. Handling and Preparation
The test material was received at LBI on August 26, 1981. The sample
was assigned LBI safety number 7170 and LBI assay number 5886. The
sample was stored at +4°C in the dark.
A total of 313.08 mg of test material was weighed and suspended in 3.13 ml
of dimethylsulfoxide. The sample formed an opaque suspension that settled
upon standing. The suspension was incubated at 37°C on a shaker overnight
to help leach material out of the particulates. Serial dilutions were
made in DMSO such that 50 ul aliquots of each dilution give the desired
concentration. The suspension was well mixed when aliquots were removed
for dosing.
BIONET1CS
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5-207
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IV.
A.
MATERIALS
Indicator Microorganisms
The Salmonella typhimurium strains used in this assay were obtained from
Dr. Bruce Ames, University of California at Berkeley.4-8 The following
four strains were used.
Strain
Designation
Gene Additional Mutations
Affected Repair ITSR Factor
Mutation Type
Detected
TA-1535 his G A uvr B rfa
TA-1537 his C A uvr B rfa
TA-98 his D A uvr B rfa pKMlOl
TA-100 his G A uvr B rfa pKMlOl
Base-pair
substitution
Frameshift
Frameshift
Base-pair
substitution
m
Litton
All the above strains have, in addition to the mutation in the histidine
operon, mutation (rfa-) that leads to defective lipopolysaccharide coat,
a deletion that covers genes involved in the synthesis of vitamin biotin
(bio-) and in the repair of ultraviolet (uv) - induced DNA damage (uvrB-).
TfiFVfa- mutation makes the strains more permeable to many large molecules.
The uvrB- mutation decreases repair of some types of chemically or physi-
cally 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 mutagens.6
In addition, plasmid pKMlOl confers resistance to the antibiotic ampi-
cillin, 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 supplemented
with a trace of biotin and an excess of histidine. In addition, the
plates with plasmid-carrying strains contain ampicillin (25 ug/ml) to
ensure stable maintenance of plasmid pKMlOl. New stock culture plates
are made as often as necessary from the frozen master cultures or from
single colony reisolates that were checked for their genotypic character-
istics (tm, rfa uvrB, bio) and for the presence of plasmid. For each
experiment, an inoculum from the stock culture plates is qrown overniqht
at 37°C in nutrient broth (Oxoid CM67) and used
B.
Media
The bacterial strains were cultured in Oxoid Media #2 (Nutrient Broth).
The selective medium was Vogen Bonner Medium E with 2% glucose.10 The
5-208
BIONETICS
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overlay agar consisted of 0.6% purified agar with 0.05 mM histidine,
0.05 mM biotin and 0.1M NaCl according to the methods of Ames et aj.9
C.
1.
Activation System
59 Homogenate
A 9,000 x 2 supernatant prepared from Sprague-Dawley adult male rat liver
induced by Aroclor 1254 (Ames et aj.9) was purchased commercially and
used in these assays.
2. S9 Mix
S9 mix used in these assays consisted of the following components:
Components
Concentration per Milliliter
S9 Mix
NADP (sodium salt)
D-glucose-6-phosphate
MgCl2
KC1
Sodium phosphate buffer
pH 7.4
Organ homogenate from rat
liver (S9 fraction)
4 umoles
5 umoles
8 umoles
33 umoles
100 umoles
100 (jliters
5-209
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BIONETICS
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V.
A.
EXPERIMENTAL DESIGN
Dosage Selection
Test strategy and dose selection depend upon sample type and sample avail-
ability. The Level 1 manual1 recommends solids to be initially tested
at the maximum applicable dose (MAD) of 5 mg per plate and at lower con-
centrations of 2.5, 1, 0.5, 0.1 and 0.05 mg per plate. Liquids are tested
initially at the MAD of 200 pi per plate, and at lower concentrations of
100, 50 and 10 ul per plate. Samples are retested over a narrower range
of concentrations with strains showing positive results initially. Alter-
nate dose are employed if sample size is limiting or at the direction of
the sponsor.
Doses selected to test this sample covered the recommended dose range
for solids. The highest dose was at the MAD level of 5.0 mg per plate
and included five lower dose levels of 2.5, 1, 0.5, 0.1 and 0.05 mg per
plate.
B.
Mutagenicity Testing
The procedure used was based on the paper published by Ames et. a_L9 and
was performed as follows:
1.
Nonactivation Assay
To a sterile 13 x 100 mm test tube placed in a 43°C water bath the fol-
lowing was added in order:
2.00 ml of 0.6% agar containing 0.05 mM histidine and
0.05 mM biotin.
0.05 ml of a suspension of the test chemical to give the
appropriate dose.
0.1 ml to 0.2 ml of indicator organism(s).
0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured onto minimal agar plates
(see IV B, Media). After the top agar had set, the plates were incubated
at 37 C for approximately 2 days. The number of his+ revertant colonies
growing on the plates were counted with an automatic colony counter and
recorded.
2.
Activation Assay
The activation assay was run concurrently with the nonactivation assay
The only difference was the addition of 0.5 ml of S9 mix (see IV C Acti-
vation System) to the tubes in place of 0.5 ml of phosphate buffer'which
was added in nonactivation assays. All other details were similar to
the procedure for nonactivation assays.
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5-210
BIONET1CS
-------
A detailed flow diagram for the plate incorporation assay is provided in
Figure 1.
C.
Control Compounds
A negative control consisting of the solvent used for the test material
was also assayed concurrently with the test material. For negative con-
trols, step 'b' of Nonactivation Assays was replaced by 0.05 ml of the
solvent. The negative controls were employed for each indicator strain
and were 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 were
made using this solvent. The amount of solvent used was equal to the
maximum volume used to give the appropriate test dose.
Specific positive control compounds known to revert each strain were also
used and assayed concurrently with the test material. The concentrations
and specificities of these compounds to specific strains are given in
the following table:
Assay
Nonactivation
Chemical
Sodium azide
2-Nitrofluorene
(NF)
9-aminoacridine
(9AA)
Concentratio
per plate
Solvent (ug)
Water
Dimethyl-
sulf oxide
Ethanol
10. 0
10.0
50.0
n
Salmonella
Strains
TA-1535,
TA-98
TA-1537
TA-100
Activation
2-anthramine
(ANTH)
Dimethyl-
sulfoxide
2.5
For all strains
D.
Recording and Presenting Data
The number of colonies on each plate were counted and recorded on printed
forms. These raw data were analyzed in a computer program and reported
on a printout. The results are presented as revertants per plate for
each indicator strain employed in the assay. The positive and solvent
controls are provided as reference points.
5-211
Litton
BIONETICS
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AMES ASSAY [PLATE INCORPORATION METHOD]
Molten [45*C] overlay igar
appropriately supplemented
Te$t, positive or solvent
control chemical
0.1 ml
Aliquot of an overnight culture
of bacterial 10» cells/ml]
Aliquot of
saline
0.5 ml
-S-9
0.5 ml S-9 mix [hepatic
4-S-9—— homogenate from PCB
pretreated rat plus
necessary cofactors
Overlay poured on selective
bottom agar medium
Plated incubated at 37'C for 48 hours
The numbers of revertants/plate counted
Data analyzed
Interpretation/Conclusion
Figure 1 AMES SALMONELLA/MICROSOME MUTAGENESIS ASSAY
5-212
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VI. RESULTS
A. Interpretations
The test material, A81-05-030-662 (EA-1 flyash), was dissolved in DMSO
at a stock concentration of 100 mg/ml and leached overnight on a shaker
at 37°C. Additional dilutions were prepared in DMSO for testing. The
maximum test level was 5.0 mg/plate.
Reverse mutation was measured in strains TA-1535, TA-1537, TA-98 and
TA-100. The test was conducted in duplicate both with and without rat
liver S9 mix for metabolic activation.
There was no mutagenic activity associated with the test material treat-
ment and the sample was considered nonmutagenic and non toxic. The sample
was ranked as having nondetectable (NO) mutagenic activity using the
IERL-EPA Level 1 evaluation criteria for the Ames Assay1.
Solvent control and positive control values were within acceptable ranges.
These results achieved assay acceptance criteria and provided confidence
in the assumptions that the recorded data represented typical responses
to the test material.
B. Tables
This report is based on the data provided in Table 1..
Litton
BIONETICS 5'213
-------
RESULTS
TABLE 1
tn
ro
A.
a.
c.
D.
C.
NOTE:
TEST
NAME OR cooc DESIGNATION OF THE TEST COMPOUND: Aai-os-030-662 (EA-l FLYASH)
SOLVENT: OMSO
TEST INITIATION DATES: 09/24/81
TEST COMPLETION DATE: 09/28/81
s-9 LOTH: s-9-ii
CONCENTRATIONS ARE GIVEN IN MILLIGRAMS PER PLATE
REVERTANTS PER PLATE
SPFCIES TISSUE TA-1535 TA-1531 TA-98 TA-100
123 123 123 12
NONACTIVATION
SOLVENT CONTROL
POSITIVE CONTROL**
TEST COMPOUND
0.050 M6
0.100 MG
0.500 MG
1.000 M6
2 .500 MG
5.000 MG
ACTIVATION
12
1076
IB
13
14
15
17
961
12
14
IS
IS
12
15
9 12
621 628
7
9
R
R
12
14
12
11
11
12
14
6
46 38
745 811
47
32
44
46
47
41
27
46
33
33
29
53
132 106
1308 1359
145 130
126 137
156 166
133 165
149 168
143 157
SOLVENT CONTROL RAT
POSITIVE CONTROL*** RAT
LIVER
LIVER
17
308
11
254
13
339
a
372
45
1562
34
1600
101 123
2065 1832
TEST COMPOUND
0.050
0.100
0.500
1.000
2.500
5.000
TA-1535
TA-1537
TA-98
TA-100
SOLVENT
MG
MG
MG
MG
NG
MG
SODIUM
RAT
RAT
RAT
RAT
RAT
RAT
AZIDE
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
7
7
13
10
14
10
11
3
9
7
7
6
11
12
13
10
13
15
a
13
14
12
11
20
51
44
38
43
48
53
10 UG/PLATE
9-AMINOACRIOINE
2-NITROFLUORENE
SODIUM
AlIDE
50 UG/PLATE
10 UG/PLATE
10 UG/PLATE
49
57
47
48
41
55
TA-1S35
TA-1537
TA-98
TA-100
138 115
118 127
109 126
121 128
128 125
143 129
2-ANTHRAHINE
2-ANTHRAMINE
2-ANTHRAMINE
2-ANTHRAMINE
2.5 UG/PLATE
2.5 UG/PLATE
2.5 UG/PLATL
2.5 UG/PLATE
50 UL/PLATE
-------
VII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA
Statistical methods are not currently used, and evaluation is based on
the criteria included in this protocol.
Plate test data consists of direct revertant colony counts obtained from
a set of selective agar plates seeded with populations of mutant cells
suspended in a semi sol 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 semiquanti-
tative in nature. Although these features of the assay reduce the quanti-
tation of results, they provide certain advantages not contained in a
quantitative suspension test:
The small number of cell divisions permits potential
mutagens to act on replication DNA, which is often more
sensitive than nonreplicating 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 quantisation 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 concen-
trations, the highest of these doses being selected to show slight toxicity
as determined by subjective criteria.
B. Dose-Response Phenomena
The demonstration of dose-related increased in mutant counts is an impor-
tant criterion in establishing metagenicity. A factor that might modify
dose-response results for a mutagen would be the selection of doses that
are too low (usually mutagenicity and toxicity are related). If the
highest dose is far lower than a toxic concentration, 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.
C. Control Tests
Positive and negative control assays were conducted with each experiment
and consisted of direct-acting mutagens for nonactivation assays and
mutagens that require metabolic biotransformation in activation assays.
Litton
5-215
BIONETICS 11
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Negative controls consisted of the test material solvent in the overlay
agar together with the other essential components. The negative control
plate for each strain gave a reference point to which the test data was
compared. The positive control assay was conducted to demonstrate that
the test systems were functional with known mutagens.
The following normal range of revertants for solvent controls are generally
considered acceptable.
TA-1535: 8-30
TA-1537: 4-30
TA-98: 20-75
TA-100: 80-250
D. Evaluation Criteria for Ames Assay
Because the procedures to be used to evaluate the mutagenicity of the
test material are semiquantitative, the criteria to be used to determine
positive effects are inherently subjective and are based primarily on a
historical data base. Most data sets will be evaluated using the following
criteria.
1. Strains TA-1535 and TA-1537
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to three times the solvent control value will
be considered to be mutagenic.
2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to twice the solvent control value for TA-98
and TA-100 will be considered to be mutagenic.
3. Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain
(G-46), 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 that cannot be
reproduced in additional runs, the initial positive test data lose signi-
ficance.
The preceding criteria are not absolute, and other extenuating factors
may enter into a final evaluation decision. However, these criteria
will be applied to the majority of situations and are presented to aid
those individuals not familar with this procedure. As the data base is
increased, the criteria for evaluation can be more firmly established.
BIONETICS 12
Litton
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E. Relation Between Mutagenicity and Carclnogenicity
It must be emphasized that the Ames Salmonella/Microsome Plate Assay is
not a definitive test for chemical carcinogens. It is recognized, however,
that correlative and functional relations have been demonstrated between
these two endpoints. The results of comparative tests on 300 chemicals
by McCann et a_L4 show an extremely good correlation between results of
microbial mutagenesis tests and in 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 muta-
genic activity.
F. Criteria for Ranking Samples in the Ames Assay
The goal of EPA Level 1 Ames testing is to rank source streams by relative
degree of genetic toxicity (mutagenicity). Samples are first identified
as mutagenic or nonmutagenic by the criteria in Section D above and
then ranked using the mutagenicity categories presented in the table
below. The lowest concentration giving a positive response in any strain,
with or without metabolic activation, is identified as the minimum effec-
tive concentration (MEC) for that sample. The mutagenicity of the sample
is evaluated as high (H), moderate (M), low (L), or nondetectable (ND)
according to the evaluation criteria developed in the Level 1 manual1
and summarized below. Samples with no detectable activity at the maximum
applicable dose (MAD) are ranked nondetectable (ND).
Ames Assay Mutagenicity Ranking Criteria1
Mutagenic
Activity
High (H)
Moderate (M)
Low (L)
Not Detectable (ND)
Solids
(MEC in ug/plate)
<50
50-500
500-5000
>5000
(MEC
<2
2-20
Liquids3
in ul/plate)
20-200
>200
Concentration of organic extracts is based upon organic content (ug
organics per plate) and not volume (ul extract per plate) of sample
tested.
Litton
5-217
BIONETICS 13
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VIII. REFERENCES
1. Brusick, D.J. , et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment Biological Tests. EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics Inc., Kensington, MD, April 1980 ,
100 pp.
3. Brusick, D.J. and Young, R.R. : Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD, August
1981, 52 pp.
4. McCann, J. , Choi, E. , Yamasaki , E. and Ames, B.N.: Detection of
carcinogens as mutagens in the Salmonel 1 a/mi crosotne test: Assay of
300 chemicals. Proc. Nat. Acad. Sci., USA 72:5135-5139, 1975.
5. Ames, B.N. , Gurney, E.G., Miller, J.A. and Bartsch, H. : Carcinogens
as frameshift mutagens: Metabolites and derivatives of 2-acetylamino-
fluorene and other aromatic amine carcinogens. Proc. Nat. Acad.
Sci., USA 69:3128-3132, 1972.
6. 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.
7. 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.
8. 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.
9. Ames, B.N., McCann, J. and Yamasaki, E.: Methods for detecting
carcinogens and mutagens with the Salmonella/mammal ian-microsome
mutagemcity test. Mutation Res., 31:347-364, 1975.
ffl
10' nSJlkV' and*onner' D-M': Acetylornithinase of E. coli partial
and some properties. J. Biol. Chem. , 218:9T:106, 1966.
5-218
BIONETICS
Litton 14
-------
GENETICS ASSAY NO.: 5886
LBI SAFETY NO.: 7T70"
CYTOTOXIC EVALUATION OF
A8TrO'5-Q30-662
"TEA7! FLYASH)
IFTTRE RABBIT
ALVEOLAl MlgROPHAGE (RAM)
EYTDTOXICITY ASSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LSI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
5-219
BiONETICS
LJtton
-------
ffl
Litton
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the rabbit
alveolar macrophage (RAM) cytotoxicity assay as described in "IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests" (1).
The data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting" (2).
The RAM cytotoxicity assay has been shown to be a sensitive method for
detecting cytotoxic activity for a variety of chemicals representing
various chemical classes (3). This assay is one of several recommended
by EPA to identify, categorize and rank the pollutant potential of influent
and effluent streams from industrial and energy-producing processes.
This assay has been well validated with a wide range of positive and
negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepara-
tion, testing and evaluation of the test material shall conform to Litton
Bionetics, Inc. standard operating procedures and the Good Laboratory
Practices Regulations of 1979. Deviations from standard procedure shall
be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
5-220
BIONETICS
-------
TABLE OF CONTENTS
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
PREFACE
ASSAY SUMMARY
OBJECTIVE
TEST MATERIAL
A. Description
B. Handling and Preparation
MATERIALS
A. Indicator Cells
B. Media
C. Negative Controls
EXPERIMENTAL DESIGN
A. Procurement of Cells
B. Sample Forms
C. Dose Selection
D. Treatment
E. Cell Viability Assay
F. ATP Assay
ASSAY ACCEPTANCE CRITERIA
RESULTS
A. Interpretation
B. Tables and Figures
ASSAY EVALUATION CRITERIA
REFERENCES
Page No.
i
1
2
3
3
3
4
4
4
4
5
5
5
6
6
6
7
8
9
9
9
16
17
Litton
5-221
BIONETICS i i
-------
I.
A.
B.
C.
D.
E.
F.
G.
ASSAY SUMMARY
SPONSOR: Acurex Corporation
MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5886
1. Identification: A81-05-030-662 (EA-1 Flyash)
2. Date Received: August 26, 1981
3. Physical Description: Gray, black powder with small chunks
TYPE OF ASSAY: Rabbit Alveolar Macrophage (RAM) Cytotoxicity
Assay
ASSAY DESIGN NUMBER: 443
STUDY DATES:
1. Initiation: September 23, 1981
2. Completion: October 23, 1981
SUPERVISORY PERSONNEL:
1. Study Director: Brian Myhr, Ph.D.
2. Laboratory Supervisor: Robert Young, M.S.
EVALUATION:
The test material, after being ground to a fine powder, caused
a dose-related increase in toxicity for applied concentrations
greater than 300 ug/ml. The viability index and ATP content
were the most sensitive assay parameters and both indicated an
EC50 near 1000 ug/ml. This result was evaluated as showing
low/nondetectable (L/ND) borderline toxicity for the test
material in the RAM Cytotoxicity Assay, according to the IERL-EPA
Level 1 toxicity categories.
ffl
Litton
BIONETICS
Submitted by:
Study Director
I!
Brian Myhr, Wi.D. Date"
Associate Director,
Department of Molecular
Toxicology
5-222
David J. Brusick, Ph.D.
Director,
Department of Molecular
Toxicology
ate
-------
II. OBJECTIVE
The objective of this study was to determine and rank the cytotoxicity
of A81-05-030-662 (EA-1 Flyash) to cultured rabbit alveolar macrophage
(RAM) cells. The measure of cytotoxicity was the reduction in cell
viability and adenosine triphosphate (ATP) content of the cultures after
a 20 hour exposure to the test material. At the conclusion of the exposure
period, the number of viable cells and total ATP content in the treated
cultures were compared to the corresponding values in unexposed control
cultures. The concentration of test material that reduced each experi-
mental parameter by 50% was estimated graphically and referred to as the
EC50 value. Standard EPA Level 1 toxicity evaluation criteria for the
RAM cytotoxicity assay were used to rank the toxicity potential of the
test material based upon the most sensitive parameter.
Litton
5-223
BIONETICS
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m
Litton
III. TEST MATERIAL
A. Description
The test material was received as a gray powder containing small, black
particles. The total amount of sample supplied was 15 grams. No informa-
tion on the sampling parameters was provided.
B- Handling and Preparation
The test material was received on August 26, 1981, and was assigned LBI
assay number 5886 and LBI safety number 7170. The sample was stored at
+4°C in the dark.
Approximately 28 mg of the test material was tested as supplied. Then,
on October 1, 1981, the remaining sample was ground in a mortar and pestle
to fine gray powder. Approximately 3.0 grams of the ground sample was
further pulverized on October 8, 1981, to a very fine, gray powder of
which 36 mg was used in the second trial of the assay. For both trials,
the test material was suspended in serum-free EMEM culture medium at a
concentration of 2000 ug/ml and incubated at 37°C on a roller drum for
8 hours. The original material settled quickly on standing, but the
suspension formed from the pulverized powder remained well-dispersed for
dilutions. No pH changes were observed. The suspensions were serially
diluted with EMEM (serum-free) and applied to the cultures at a maximum
concentration of 1000 ug/ml in the presence of 10% serum.
5-224
BIONETICS
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IV. MATERIALS
A. Indicator Cells
Both assay trials employed short-term primary cultures of alveolar macro-
phage cells obtained by lung lavage of male New Zealand white rabbits
(2.0-2.5 kg). The rabbits were maintained on Purina Lab Rabbit Chow 5321
and water ad libitum and were examined for the absence of respiratory
illnesses prior to use.
B. Media
The cells were maintained and treated in Eagle's Minimum Essential Medium
(EMEM) with Earle's salts and supplemented with 10% fetal bovine serum
(heat-inactivated), 100 units/ml penicillin, 100 ug/ml streptomycin,
17.6 ug/ml kanamycin, and 0.4 ug/ml amphotericin B.
C. Negative Controls
The negative control for the first trial consisted of six untreated
cultures carried through the same experimental time period as the treated
cells. Six cultures were used because a large number of cells was obtained
by pooling the yield from two rabbits in order to run two concurrent
assays. Only one animal was used for the second trial, and the usual
three untreated cultures were prepared. The average .viability and ATP
content of the negative controls provided the reference points for deter-
mining the effects of different concentrations of the test material on
the assay parameters.
Litton
5-225
BIONET1CS
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Litton
V. EXPERIMENTAL DESIGN
A. Procurement of Cells
(8)
The rabbits were sacrificed by injection of Nembutal (60 mg/ml) into the
marginal ear vein, and sterile operating techniques were used to perform
a tracheostomy. Prewarmed normal saline (30 ml) was then introduced
into the lungs via a catheter and allowed to stand for 15 minutes. This
lavage fluid was removed and placed into a 50-ml sterile centrifuge tube
on ice. Nine additional lavages were similarly performed and collected,
except the saline was removed shortly after its introduction into the
lungs. Any lavage fluid containing blood or mucous was discarded. The
lavages were centrifuged at 365 x g for 15 minutes and the cells resus-
pended in cold 0.85% saline. After two washes in saline by centrifugation,
the cell pellets were resuspended in cold EMEM containing 20% serum and
then combined. A cell count was obtained by hemocytometer and the suspen-
sion diluted to between 5 x 105 and 106 cells/ml. Viability was determined
by trypan blue staining and the cells were not used if less than 95%
viable. Also, a differential cell count from Wright-stained smears was
performed to verify that the macrophage content was above 90%.
B. Sample Forms
The usual sample form for application to the cells is a suspension of
particulate material. Solid samples are ground to fine particles and a
weighed portion is suspended in a known volume of EMEM (0% FBS) for about
eight hours to help leach any water-soluble material. Finely-divided
test material may'be suspended directly in culture medium without further
grinding. Aqueous liquids, suspensions, or slurries containing less
than 0.5% organic solvent are added by volume to culture medium.
Samples supplied as solutions in organic solvents are usually solvent-
exchanged into DMSO before testing. Original sample volumes may first
be reduced a maximum of 10-fold in a Kuderna-Danish concentrator, and
the concentrative factor is used to convert assayed volumes into equi-
valent original sample volumes in the absence of information about solute
concentration. An aliquot of the reduced volume is exchanged into DMSO
by repeated, partial evaporation under a stream of nitrogen in a warm
water bath (50°C); the evaporated volumes are replaced with equal volumes
of DMSO.
Samples adsorbed on XAD-2 resin are extracted with methylene chloride
or acetone in a Soxhlet apparatus for. 24 hours. The extract is then
concentrated and solvent-exchanged into DMSO. Alternatively, acetone
extracts can be assayed directly at concentrations up to 2% by volume in
the culture medium.
Samples impregnated on fiber glass or teflon filters are repeatedly soni-
cated in cyclohexane to remove particulates. The resulting cyclohexane
particulate suspension is then evaporated to dryness and the particulates
resuspended in EMEM culture medium at the desired concentration.
5-226
BIONETICS
-------
Utton
Sponsor-specified handling of sample materials will be followed if the
above procedures are not applicable or a specific procedure is desired.
C. Dose Selection
Unless the approximate toxicity is already known or the sample size is
limiting, the following usual dose ranges are tested for different sample
forms. Dry, particulate material is tested at six dose levels from
1000 ug/ml to 3 ug/ml. Aqueous samples, suspensions, or slurries are
tested from 600 ul to 3 ul/ml in six dose steps. Samples that are solvent-
exchanged into DMSO are tested from 20 ul/ml (2% DMSO in growth medium)
to 0.2 Ml/mli also in six dose steps. A second dose study is performed
with an adjusted dose range if the EC50 was not located properly in the
initial test. However, EC50 values greater than 1000 ul/ml for particulate
material, 600 ul/ml for aqueous samples, or 20 ul/ml for organic solutions
will not be determined.
This test material, A81-05-030-662 (EA-1 flyash), was tested as supplied
at 6 dose levels, starting at the maximum applicable dose (MAO) of
1000 ug/ml and including 600, 300, 100, 60 and 30 jjg/ml. The second
trial was performed with only three doses of the finely ground test
material: 1000, 600 and 300 ug/ml.
D. Treatment
A series of 25 cm2 culture flasks were prepared, each containing 2.0 ml
of serum-free medium at 37°C and the test material at twice the desired
final concentration. Three flasks were prepared for each test concen-
tration. Aliquots of cell suspension (2 ml) were then added; each flask,
therefore, contained from 1 to 2 x 106 viable cells in a 4-ml volume of
media containing 10% serum. The flasks were placed on a rocker platform
in a 37°C incubator with a humidified atmosphere containing 5% C02.
After sitting for about 30 minutes, the flasks were slowly rocked for
the remainder of a 20-hour exposure period.
If the test substance causes a color change in the growth medium, the pH
is determined in additional treated flasks. After the exposure period,
the pH of the medium in the experimental flasks is again recorded.
E. Cell Viability Assay
At the end of the treatment period, the medium containing unattached
cells was decanted into a centrifuge tube on ice. The attached cells
were rinsed with 1 ml of 0.1% trypsin/0.01% versene and then incubated
with 2 ml of the trypsin/versene solution for about 5 minutes at 37°C.
The trypsinates and decanted media were combined for each culture to
yield a 7-ml cell suspension for subsequent analysis.
A 0.5 ml aliquot of the cell suspension was removed for cell count and
viability determination. The aliquot was combined with 1.0 ml of 0.4%
trypan blue and counted by hemocytometer about 5 to 15 minutes later.
5-227
BIONETICS
-------
The total number of cells counted per culture was the sum of the numbers
found in five squares for each chamber of the hemocytometer (1 ul total
volume). The numbers of live (colorless) and dead (blue) cells were
recorded.
F. ATP Assay
ATP was immediately analyzed by extraction of a 0.1-ml sample of cell
suspension with 0.9 ml of 90% DMSO. After 2 minutes at room temperature
5.0 ml cold MOPS buffer (0.01 M morpholinopropane sulfonic acid) at pH 7.4
was added and the extract mixed well and placed on ice. Aliquots of
10 ul were injected into a cuvette containing a luciferin-luciferase
reaction mixture in a DuPont Model 760 Luminescence Biometer. The Biometer
was calibrated daily with standard ATP solutions to provide a direct
read-out of the ATP content. Each test sample was assayed at least twice
to obtain repeatable readings.
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Litton
5-228
BIONETICS
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VI. ASSAY ACCEPTANCE CRITERIA
The assay will be considered acceptable for evaluation of the test results
if the following criteria are met:
1. The macrophage population is 90% or greater of the total nucleated
cells collected by lung lavage.
2. The percent viability of the macrophages used to initiate the assay
is 95% or greater.
3. The survival of viable macrophages in the negative control cultures
over the 20 hour treatment priod is 70% or greater.
4. A sufficient number of data points (for five test concentrations or
less) are available to clearly locate the EC50 of the most sensitive
test parameter within a toxicity region as defined under Assay Eval-
uation Criteria.
5. The data points critical to the location of the EC50 for the most
sensitive parameter are the averages of at least two treated cultures.
6. If all the test parameters yield EC50 values greater than 1000 ug/ml,
600 ul/ml for aqueous solutions, or 20 ul/ml for organic solutions,
the plotted curves for ATP content and viability index parameters
do not exceed 120% of the negative control.
Litton
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BIONETICS
-------
VII. RESULTS
A. Interpretation
The original test material, which consisted of many coarse particles,
did not interact appreciably with the macrophages. As shown in Table 1
and Figures 1 and 2, the assay parameters remained near the negative
control values for all tested doses up to 1000 ug/ml. However, when the
test material was pulverized to a fine powder, a toxic response was obser-
ved at concentrations above 300 pg/ml. The results for the fine powder
are presented in Table 2 and Figures 3 and 4. Absolute and relative
assay parameters are provided in the tables, whereas the relative values
are plotted in the Figures to determine EC50 positions.
The viability index (which measures cell survival) and the culture ATP
content usually tend to parallel each other, and an inspection of the
curves in Figures 3 and 4 show this to be the case for the current assay.
Both parameters were essentially equally sensitive and indicated an EC50
near 1000 ug/ml. This value is on the borderline between the low (L)
and nondetectable (ND) toxicity categories defined for the IERL-EPA Level 1
RAM assay1. Since the EC50 position will shift slightly in either direc-
tion with repeated trials, the toxicity was evaluated as low/nondetectable
(L/ND) borderline. The results from the second trial were considered
more relevant than those from the first trial because the small particle
size allowed ingestion by the macrophages.
The percent viability assay parameter was unaffected in both trials.
This behavior indicated the cells disrupted soon after ingesting the
particles. The decline in cell count was shown by the decreased viability
index and was probably responsible for the lack of response for the
ATP/106 cells assay parameter.
The macrophages collected for the assays had normal morphology and appeared
to be in a healthy state. The initial viability was approximately 99%
and the survival of viable cells in the negative controls was also about
99% in both trials. The average cellular ATP content (ATP/106 total
-cells) for the negative controls was within the historical range for
acceptable cultures. These results achieved the assay acceptance criteria
and provided confidence in the assumption that the collected data repre-
sented typical responses to the test material.
B. Tables and Figures
This report is based on the data provided in Tables 1 and 2 and Figures 1
to 4.
Litton
5-230
BIONETICS
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TABLE 1
RABBIT ALVEOLAR MACROPHAGE (RAN) CYTOTOXICITY ASSAY DATA
ro
IBI Assay No.: 5886 (Trial I, Unground sample)
Test Material Identity: A81-05-030-662 (EA-1 Flyash)
Test Date: September 23, 1981
Vehicle: EMEM
Sample
NCC
TEST
TEST
TEST
TEST
TEST
TEST
apH change
Concentration9
Mg/ml
...
30
60
100
300
600
1000
in culture medium:
Average Values
Viable Cells
10* Units
2.14
2.06
2.21
2.16
1.82
2.02
1.95
None observed
?er Culture
otal Cells
106 Units
2.16
2.08
2.25
2.20
1.84
2.03
2.02
Flask
ATP ,,
108fgD
66.4
65.1
67.2
66.8
64.3
62.6
60.5
Initial Cell Viability: 98.8%
Viable Macrophage Seeded/Flask: 2.0 x 10* cells/flask
Macrophage Population Percentage: >90.0%
Survival of Negative Control
Macrophage Over Treatment Time: 99. 1%
ATP Per
106 Cells
10s fg
30.
31.
29.
30.
34.
30.
30.
dEC50
7
3
9
4
9
8
0
VALUES:
Viability
%
99.1
99.0
98.2
98.2
98.9
99.5
96.5
Expressed
Viability
100.0
99.9
99.1
99.1
99.8
100.4
97.4
as Percent
Viability
Index
100.0
96.3
103.3
100.9
85.0
94.4
91.1
of Negative Control
ATP
100.0
98.0
101.2
100.6
96.8
94.3
91.1
ATP Per
10° Cells
100.0
102.0
97.4
99.0
113.7
100.3
97.7
bfg = Femtogram (10-IS gram).
CNC = Negative Control, EMEM culture medium.
>1000
>1000 >1000
>1000
Determined from data plots in Figures 1 and 2.
Toxicity
Classification: Nondetectable
-------
TABLE 2
RABBIT ALVEOLAR MACROPHAGE (RAH) CYTOTOXICITY ASSAY DATA
LBI Assay No.: 5886 (Trial II, Ground sample)
Test Material Identity: A81-05-030-662 (EA-1 Flyash)
Test Date: October 22, 1981
Vehicle:
01
i ,
ro Sample
CJ
ro
NCC
TEST
TEST
TEST
EMEM
Concentration9
Mg/«l
—
300
600
1000
Initial Cell Viability: 94.4%
Viable Hacrophage Seeded/Flask: 1.03 x 106 cells/flask
Hacrophage Population Percentage: >90%
Survival of Negative Control
Hacrophage Over Treatment Time: 98.9%
Average Values
Viable Cells
106 Units
0.89
0.83
0.72
0.42
fer Culture
otal Cells
10* Units
0.90
0.86
0.74
0.44
Flask
ATP K
10«fgb
26.1
23.1
19.9
14.2
ATP Per
106 Cells Viability
10" fg %
29.0 98.9
26.9 - 96.5
26.9 97.3
32.3 95.5
Expressed
Viability
100.0
97.6
98.4
96.6
as Percent of
Negative Control
Viability ATP Per
Index ATP 106 Cells
100.0 100
93.3 88
80.9 76
47.2 54
.0 100.0
.5 92.8
.2 92.8
.4 111.4
pH change in culture medium: None observed
fg = Femtogram (10-IS gram).
CNC = Negative Control, EHEH culture medium.
Determined from data plots in Figures 1 and 2.
EC50 VALUES:
>1000
-1000 -1000
>1000
Toxiclty
Classification: Low/Nondetectable Borderline
-------
FIGURE 1
EC50 DETERMINATION FOR
PERCENT VIABILITY (0) AND VIABILITY INDEX (•)
A81-05-030-662
(EA-1 FLYASH)
TRIAL 1
1000
CONCENTRATION, JJG/ML
5-233
12
-------
FIGURE 2
EC50 DETERMINATION FOR
ATP/FLASK (0) AND ATP/106 CELLS (•)
A81-05-030-662
(EA-1 FLYASH)
TRIAL 1
120
10 TOO
CONCENTRATION, JJG/ML
1000
5-234
13
-------
FIGURE 3
EC50 DETERMINATION FOR
PERCENT VIABILITY (0) AND VIABILITY INDEX {•)
A81-05-030-662
(EA-1 FLYASH)
TRIAL 2
1000
CONCENTRATION, J)G/ML
5-235
14
-------
5
LU
FIGURE 4
EC50 DETERMINATION FOR
ATP/FLASK (0) AND ATP/106 CELLS (I)
A81-05-030-662
(EA-1 FLYASH)
TRIAL 2
10 100
CONCENTRATION, JJG/ML
1000
5-236
15
-------
ASSAY EVALUATION CRITERIA
The EC50 value represents the concentration of test material that reduces
the most sensitive parameter of the RAM assay to 50% of the vehicle or
negative control value. EC50 values are determined graphically by fitting
a curve by eye through relative toxicity data plotted as a function of
the logarithm of the applied concentration. Each data point normally
represents the average of three culture dishes. Statistical analysis is
unnecessary in most cases for evaluation.
The toxicity of the test material is evaluated as high, moderate, low,
or nondetectable according to the range of EC50 values defined in the
following table.
Solids
Toxicity (EC50 in ug/ml)
High <10
Moderate 10 to 100
Low 100 to 1000
Not Detectable >1000
Aqueous Liquids
(EC50 in Ml/ml)
<6
6 to 60
60 to 600
>600
Nonaqueous Liquids"
(EC50 in pi/ml)
<0.2
0.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests1.
Criteria for nonaqueous liquids are tentative and under evaluation. If
the organic or solid content is known, the solid evaluation criteria
are applied.
Another evaluation scheme is proposed for extracts obtained from SASS
train gas volumes. The proportion of the total gas volume corresponding
to the volume of extract used in the bioassay is calculated and expressed
as L/ml of culture medium (or DSCF/ml of culture medium). A criterion
of 1000 L/ml is set as the limit for nondetectable toxicity. This gas
volume corresponds to the average volume breathed by humans over a 2-hour
period. The subsequent toxicity ranges are defined by 10-fold dilution
steps to conform to standard procedure. The toxicity ranges are defined
in the following table for liter and dry standard cubic feet units:
EC50 InEC50 In
Toxicity Liters/ml (L/ml) Dry Standard Cubic Feet/ml (DSCF/ml)
HTgR<10<0.35 DSCF'
Moderate 10-100 0.35-3.5
Low 100-1000 3.5-35
Nondetectable >1000 >35
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5-237
BIONETICS 16
-------
IX. REFERENCES
1. Brusick, D.J., et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment Biological Tests.EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington,
MD, September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD, August
1981, pp. 52.
Litton
5-238
BIONETICS
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GENETICS ASSAY NO.: 5886
LBI SAFETY NO.: 7170
TOXIC EVALUATION OF
A81-05-030-662
l FLYASH)
""IN THE
EPA LEVEL 1 ACUTE IN VIVO
RODENT TOXICITY ASSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
Utton
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MD 20795
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
5-239
BIONETICS
-------
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the acute
in vivo toxicity test in rodents as described in "IERL-RTP Procedures
Manual? Level 1 Environmental Assessment Biological Tests ». Jhe data
were evaluated and formatted as recommended in "Level 1 Biological Testing
Assessment and Data Formatting"2. The organisms used in this assay were
male and female weanling mice as recommended by the Level 1 Manual.
The advantages of in vivo toxicity assays are embodied mainly in the fact
that the toxicologTcaFassessment is performed in whole animals. There
is a significant background of test data on a wide range of toxicants
for the rodent systems, thus supplying needed information for the reliable
interpretation of results with complex effluents3. The main disadvantage
of an acute rodent toxicity study is a possibly unsatisfactory prediction
of toxicity induced by long-term/ low-level exposures. An additional
consideration is the need for multi-gram quantities of test material
which may prohibit testing where small amounts of sample are available,
such as from source streams containing gaseous and particulate material.
Since the major objective of the Level 1 biological testing procedures
is to identify toxicological problems at minimal cost, a two-step approach
was developed for the initial acute jji vivo toxicological evaluation of
unknown compounds. The first step is based on the quantal (all-or-none)
response of dosing animals only at the maximum applicable dose. If no
animals die in the quantal test, further jm vivo testing is not initiated
and the sample toxicity is categorized as not detectable. If any animals
die in the quantal screening, a multiple dose quantitative test is initiated
to determine the dose that kills 50 percent of the animals (LD50). The
toxicity potential of the test material is then ranked using standard
EPA Level 1 toxicity evaluation criteria for the acute jjn vivo rodent
toxicity assay1.
All procedures and documents pertaining to the receipt, storage, prepara-
tion, testing and evaluation of the test material shall conform to Litton
Bionetics, Inc. standard operating procedures and the Good Laboratory
Practices Regulations of 1979. Deviations from standard procedure shall
be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology 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.
ffl
Litton
5-240
BIONETICS
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TABLE OF CONTENTS
Page No.
PREFACE 1
LIST OF TABLES iii
I. ASSAY SUMMARY ' 1
II. OBJECTIVES 2
III. TEST MATERIAL 3
A. Description 3
B. Handling and Preparation 3
IV. MATERIALS 4
A. Test Organisms 4
V. EXPERIMENTAL DESIGN 5
A. Quanta! Test 5
B. Quantitative Test 5
VI. RESULTS
A. Interpretation 7
B. Tables 7
VII. EVALUATION CRITERIA 10
VIII. REFERENCES 11
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BIONETICS
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LIST OF TABLES
Table Title Page No.
1 Definition of Pharmacological Toxic Signs .... 6
2 Quanta1 Toxicity Data with Weanling Mice .... 8
3 Acute In Vivo Rodent Toxicity Assay
Evaluation Criteria 10
ffl
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BIONETICS
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I. ASSAY SUMMARY
A. SPONSOR: Acurex Corporation
B. MATERIAL (TEST COMPOUND): GENETICS ASSAY NO.: 5886
1. Identification: A81-05-030-662 (EA-1 Flyash)
2. Date Received: August 26, 1981
3. Physical .Description: Gray and white powder with small
black particles.
C. TYPE OF ASSAY: EPA Level 1 Rodent Quanta! Toxicity Assay
D. STUDY DATES:
A. Initiation: October 5, 1981
B. Completion: October 23, 1981
E. SUPERVISORY PERSONNEL:
A. Study Director: David J. Brusick, Ph.D.
B. Senior Technician: Joan McGowan
F. EVALUATION:
The test substance, A81-05-030-662 (EA-1 Flyash), was not lethal
or toxic to weanling mice following an oral gavage dose of
5 gm/kg body weight. There were no unusual findings upon
necropsy that would suggest test substance related toxicity.
The test sample response was evaluated as being in the nondetect-
able (ND) range as defined for the IERL-EPA Level 1 Rodent
Quantal Toxicity Assay1.
Submitted by:
Litton
_
David J. Brusick, Ph.D. Date
Director
Department of Molecular
Toxicology
5-243
BIONETICS
-------
II. OBJECTIVES
The objective of this assay was to evaluate the acute toxicity of
A81-05-030-662 (EA-1 flyash) when administered by oral gavage to male
and female weanling mice. Test strategy involved initial testing of the
sample at the maximum applicable dose in the quanta1 assay. If lethality
was observed in the quantal assay, additional testing would be initiated
at lower doses to identify the LDSO-
The assay consisted of recording any lethality and toxic signs that occur-
red initially and over a 14-day period following a single treatment.
Additional information was collected from necropsy observations on animals
that died during the course of the experiment or were killed at the end
of the 14-day observation period.
Litton
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BIONETICS
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III. TEST MATERIAL
A. Description
The test material, A81-05-030-662 (EA-1 flyash), was received as a gray
and white powder containing small, black particles. The amount of sample
supplied was 15.0063 grams. No information on the sampling parameters
was provided.
B. Handling and Preparation
The test material was received at LBI on August 26, 1981. The sample
was assigned LBI safety number 7170 and LBI assay number 5886. The sample
was stored at +4°C in the dark. :
On October 1, 1981, the test material was ground in a mortar and pestle
to a fine, gray powder. The primary dosing suspension was prepared
24 hours in advance to permit water soluble materials in the flyash to
leach into the water at room temperature. A total of 1716.83 rag of test
material was suspended in 17.07 ml of sterile distilled water giving a
stock concentration of 101 mg/ml. This suspension would not pass freely
through a 24G gavage needle so it was discarded. On October 8, 1981,
approximately 3.0 gm of the previously ground sample was pulverized a
second time in a mortar and pestle. The suspension prepared 24 hours in
advance of dosing, passed through the gavage needle without difficulty.
A total of 1815.5 mg of test material was suspended in 12.0 ml of sterile
water giving a stock concentration of 151 mg/ml.
Litton
5-245
BIONETICS
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IV. MATERIALS
A. Test Organisms
The test organisms for this study were weanling Charles River CD-I mice.
Weanlings were used because they are likely to be more sensitive to toxic
effects of test materials than adult mice. In addition, significantly
less test material is required for dosing.
Eight nursing female Charles River CD-I mice with ten pups each (5 male
and 5 female) were obtained from Charles River Breeding Laboratories,
Inc., Wilmington, MA on September 30, 1981. The birth date of the pups
was September 13, 1981. The animals were quarantined for 5 days upon
receipt. The litters were individually housed on Ab-sorb-dri bedding in
polycarbonate cages and were cared for according to Litton Bionetics,
Inc., Department of Molecular Toxicology and LAMS Standard Operating
Procedures. Purina certified laboratory chow and water (pH 2.5) were
provided ad libitum. The pups were maintained with mothers until weaned.
The animals were identified by eartags and cage cards and were released
for study on October 9, 1981.
Litton
5-246
BIONETICS
-------
V. EXPERIMENTAL DESIGN
A. Quanta! Test
Ten male and ten female weanling CD-I mice were used in the initial quanta!
screening test. The pups appeared to be in good health with no physical
or behavioral problems noted. Pups that were selected were of similar
size. The pups were 26 days old at the time of dosing.
Prior to dosing, each animal was individually weighed and the mean weight
calculated for each sex. The volume of test material to be administered
was based on the mean weight if all animals were within plus or minus
15 percent of the average for the sex. If any animals were outside that
range, they were then excluded from the average, a new mean calculated
for the remaining animals and individual dosing volumes calculated for
each outlying animals.
The test material was administered by gavage to the pups at the rate of
5 gm/kg. The average weight of the males was 15.1 gm and that of the
females was 13.3 gm. All animals were within ±15 percent of the average
for the sex. The test material, suspended at the concentration of 151 mg
per ml, was applied to the animals in two equal doses that totaled 0.50 ml
for the males and 0.44 ml for the females.
Immediately following administration of the test substance and during
the first day, observations of the frequency and severity of all toxic
signs or pharmacological effects (Table 1) were recorded. Particular
attention was paid to time of onset and disappearance of signs. Observa-
tions were made and recorded on all animals through a 14-day period. At
termination of the observation period, all surviving animals were weighed,
killed, and then gross necropsies performed. Necropsies were also per-
formed on all animals that died during the course of this study.
B. Quantitative Test
Since no animals died during the preliminary quantal screening test, the
quantitative test to determine the LD50 was unneccessary.
Litton
5-247
BIONETICS
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TABLE 1. DEFINITION OF PHARMACOLOGICAL TOXIC SIGNS
Organ System
Observation and
Examination
Common Signs of Toxicity
CNS and
somatomotor
Autonomic
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
Peri anal region
Color, turgor,
integrity
Conjunctiva, mouth
Eyeball
Transparency
Rectal or paw skin
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, flaccid skinfold,
eruptions, piloerection
Discharge, congestion,
hemorrhage cyanosis, jaundice
Exophthalmus, nystagmus
Opacities
Subnormal, increased temperature
Abnormal posture, emaciation
m
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BIONETICS
5-248
-------
VI. RESULTS
A. Interpretation
The test material, A81-05-030-662 (EA-1 flyash), was tested and evaluated
in the EPA Level 1 Acute In Vivo Rodent Toxicity Assay. The first phase
of testing was the quanta'TtoxTcity test in which 10 male and 10 female
weanling CD-I mice were exposed to an oral dose of the test material.
This dose was at the maximum applicable dose (MAD) of 5 gm/kg as recom-
mended by the EPA Level 1 procedures manual1.
All twenty animals survived the exposure with no evidence of any compound-
related behavioral or toxic signs (see Table 1 for definitions). The
only visable sign related to test material dosing was staining of the
muzzle noted in some animals immediately after dosing. Both male and
female mice showed good weight gains during the 14-day observation period.
At the end of the observation period, the mice were sacrificed and necro-
psied. Gross necropsy showed no evidence of compound-related lesions.
The results of the study are summarized in Table 2.
The test material was evaluated as having nondetectable (NO) toxicity at
the MAD of 5 gm/kg in the acute w vivo rodent toxicity assay. No deaths,
toxic signs or gross lesions at necropsy were noted. Because no toxic
effects were observed at the MAD, the quantitative study (LD50 determina-
tion) was unnecessary.
B. Tables
This report is based on the data provided in Table 2.
Litton
5-249
BIONETICS
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TABLE 2
QUANTAL TOXICITY DATA WTH WEANLING MICE
Quantal Toxicity: Weanling CD-I mice
Sponsor: Acurex Corporation
Test Article: A81-05-030-662 (EA-1 flyash)
Description: Gray and white powder with black particles. The test
material was ground to a fine, gray powder that was
used to prepare the dosing suspension.
Vehicle: Sterile, deionized water
Study Dates: October 8, 1981 to October 23, 1981
Animals: Charles River CD-I mice, P.O. 106949
Dose: 5 gm/kg administered by oral gavage
Animal No.
Males
9022
9023
9024
9025
9026
9027
9028
Initial
Weight
gm
14.8
16.0
15.6
14.7
14.0
13.9
15.0
Final
Weight
gm
23.4
23.9
24.4
24.5
20.4
24.6
27.1
Visible
Toxic,
Signs3
NTS5
NTS
NTS
NTS
Scruffy
after dosing
NTS
NTS
Gross
NSLC
Necropsy Findings
Large intestine filled
with yellow fluid
NSL
NSL
White mucous plug in
urinary bladder
NSL
White
mucous plug in
9029
9030
9031
Mean Body
15.4
15.7
15.4
23.1
25.1
25.4
NTS
NTS
NTS
urinary bladder
Hard lymph node in
mammary tissue in lower
right intestinal area
NSL
NSL
Weight: Initial = 15.1 ± 0.7 gm (Standard Deviation)
Final = 24.2 ± 1.7 gm (Standard Deviation)
uAnimals observed over 14 days.
°NTS = No Toxic Signs.
NSL = No Significant Lesions
5-250
Litton
BIONETICS
Note: Staining of the muzzle from
the test material was noted
in some animals immediately
after dosing.
8
-------
TABLE 2 (Continued)
QUANTAL TOXICITY DATA WTH WEANLING MICE
Animal No.
Females
9032
9033
9034
9035
9036
9037
9038
9039
9040
9041
Mean Body
Initial
Weight
gm
14.7
13.8
14.2
13.7
13.4
12.1
12.7
12.0
12.6
13.3
Weight:
Initial
Final
Final
Weight
gm
19.3
20.4
23.3
19.8
19.6
18.5
19.5
17.4
18.2
21.4
= 13.3 ± 0.9
= 19.7 ± 1.7
Visible
Toxic.
Signs3
NTS5
NTS
NTS
NTS
NTS
NTS
NTS
NTS
NTS
NTS
gm (Standard
gm (Standard
Gross Necropsy Findings
NSLC
NSL
NSL
NSL
NSL
NSL
One mesenteric
node slightly
NSL
NSL
lymph
enlarged
Mesenteric lymph nodes
slightly enlarged.
Deviation)
Deviation)
^Animals observed over 14 days.
°NTS = No Toxic Signs.
CNSL = No Significant Lesions
Note: Staining of the muzzle from
the test material was noted
in some animals immediately
after dosing.
5-251
Litton
BIONETICS
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VII. EVALUATION CRITERIA
If no mortality occurs in the quanta! study, no further studies will be
performed with the test substance and the LD50 should be reported as
greater than 5 ml/kg or 5 g/kg. The test material is then ranked as
having nondectable toxicity (ND) at the maximum applicable dose (MADJ-
Effluent samples which produce harmful effects In vivo and do not result
in deaths will be noted in the results summary. Such observations are
difficult to quantitate but provide insight into the sublethal 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 LD50 will be calculated
by the method of Litchfield and Wilcoxin4. If the data are not suitable
for calculation of a precise LD50, i.e., total mortality occurs for the
lowest dose, an estimate of the LD50 could be made or the LD5o could be
expressed as 0.05 ml/kg or 0.05 g/kg or less. Occasionally, it may be
necessary to use a different series of dosages in a repeat study to
accurately locate the LD50. The calculated LD50 value is used to rank
the toxicity of the test material according to the dose ranges presented
in Table 3.
Frequent observations are also made and recorded on all animals through
the 14-day period. As in the quantal phase, no attempt is made to quanti-
tate or rank 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 3
ACUTE IN VIVO RODENT TOXICITY ASSAY EVALUATION CRITERIA
Toxicity3
High
Moderate
Low
Not Detectable
Solids
(LDSO in g/kg)
<0.05
0.05 to 0.5
0.5 to 5
>5
Liquids
(LD50 in ml /kg)
<0.05
0.05 to 0.5
0.5 to 5
>5
Litton
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests.1
5-252
BIONETICS 10
-------
VIII. REFERENCES
1. Brusick, D.J., et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment Biological Tests.EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington,
MD, September 1980, 177 pp., in press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc., Kensington, MD, April
1980, 100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA 600/7-81-135 Litton Bionetics, Inc., Kensington, MD, August
1981, 52 pp.
4. Litchfield, J.T. and Wilcoxin, F.: "A Simplified Method of Evaluation
Dose-Effect Experiments." J. Pharmac. Exp. Ther., Vol. 96, 1949,
pp. 99-113.
Litton
5-253
BIONETICS 11
-------
GENETICS ASSAY NO.:
LBI SAFETY NO.:
MUTAGENICITY EVALUATION OF
A81-05-030-672
~-2 10+3 )
__~
EPFLEVEL 1
AMES SAWNEIIA7MICROSOME
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 20895
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
IJiton
5-254
BIONETICS
-------
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Ames
Salmonella/mlcrosome mutagenesis assay as described in "IERL-RTP Proce-
dures Manual: Level 1 Environmental Assessment Biological Tests"1. The
data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting"2.
The Ames Salmonella/microsome mutagenesis assay has been shown to be a
sensitive method for detecting mutagenic activity for a variety of chemi-
cals representing various chemical classes3. This assay is one of several
recommended by EPA to identify, categorize and rank the pollutant potential
of influent and effluent streams from industrial and energy-producing pro-
cesses. This assay has been well validated with a wide range of positive
and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good Labora-
tory Practices Regulations of 1979. Deviations from standard procedure
shall be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives, of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
Litton
5-255
BIONETICS
-------
m
LU BIONETICS
Litton
TABLE OF CONTENTS
Page No.
PREFACE ........................ i
I. ASSAY SUMMARY ..................... 1
II. OBJECTIVE ................. ....... 2
III. TEST MATERIAL ..................... 3
A. Description ................... 3
.B. Handling and Preparation ............. 3
IV. MATERIALS ....................... 4
A. Indicator Microorganisms ............. 4
B. Media ...................... 4
C. Activation System ................ 5
1. 59 Homogenate ................ 5
2. S9 Mix ................... 5
V. EXPERIMENTAL DESIGN .................. 6
A. Dose Selection .................. 6
B. Mutagenicity Test ................ 6
1. Nonactiyation Assay ............. 6
2. Activation Assay .............. 6
C. Control Compounds ................ 7
D. Recording and Presenting Data .......... 7
VI. RESULTS ........................ 9
A. Interpretation .................. 9
B. Tables ...................... 9
VII. EVALUATION CRITERIA .................... 12
A. Surviving Populations .............. 12
B. Dose-Response Phenomena ............. 12
C. Control Tests .................. 12
D. Evaluation Criteria for Ames Assay ........ 13
1. Strains TA-1535 and TA-1537 ......... 13
2. Strains TA-98 and TA-100 .......... 13
3. -Pattern ................... 13
4. Reproducibility ............... 13
E. Relation Between Mutagenicity and
Carcinogenic! ty ................ 14
F. Criteria for Ranking Samples in the Ames Assay ! ! 14
VIII. REFERENCES .................... 15
5"255
-------
I. ASSAY SUMMARY
A. Sponsor: Acurex Corporation
B. Material (Test Compound): Genetics Assay Number: 5883
1. Identification: A81-05-030-672 (EA-2 10+3)
2. Date Received: August 26, 1981
3. Physical Description: Fine, gray powder..
C. Type of Assay: EPA Level 1 Ames Salmonella/Microsome Plate Test
D. Assay Design Number: 401 (EPA Level 1)
E. Study Dates:
1. Initiation: October 26, 1981
2. Completion: November 9, 1981
F. Supervisory Personnel:
A. Study Director: D.R. Jagannath, Ph.D.,
G. Evaluation:
The test material, A81-05-030-672 (EA-2 10+3), was tested for
activity in the Ames Salmonella mutagenicity assay over a concen-
tration range of 0.05 mg/plate to 5.0 mg/plate. The test was
performed in duplicate under nonactivation and activation test
conditions with strains TA-1535, TA-1537, TA-98, and TA-100.
The sample was not mutagenic under the test conditions employed
and was ranked as having nondetectable (ND) mutagenic activity
as defined by the IERL-EPA Level 1 criteria for the Ames bio-
assay1.
Submitted by:
Study Director
D.R. Jagannath, Ph.Q.
Section Chief,
Submammalian Genetics,
Department of Molecular
Toxicology
Date
Reviewed by:
avid J. Brusick, Ph.D.
Director,
Department of Molecular
Toxicology
te
Utton
BIONETICS
5-257
-------
II. OBJECTIVE
The objective of this study was to determine the genetic activity of
A81-05-030-672 (EA-2 10+3) in the Salmonella/ microsome assay with and
without the addition of mammalian metabolic activation preparations.
The genetic activity of a sample is measured in these assays by its
ability to revert the Salmonella indicator strains from histidine depen-
dence to histidine independence. The degree of genetic activity of a
sample is reflected in the number of revertants that are observed on the
histidine-free medium.
EH
Litton
5-258
BIONETICS
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III. TEST MATERIAL
A. Description
The test material was received as a fine gray powder (1.5 gin) and was
used without further preparation. The sample consisted of the 3 (Jin and
10 urn SASS train particulate catch.
B. Handling and Preparation
The test material was received at LSI on August 26, 1981. The sample
was assigned LBI safety number 7167 and LBI assay number 5883. The sample
was stored at +4°C in the dark.
A total of 476.58 mg of test material were used for two trials of the
Ames Salmonella Assay. The test material was suspended at 100 mg/ml in
dimethylsulfoxide (DMSO). The sample formed an opaque suspendion that
settled upon standing. The suspension was incubated at 37°C on a shaker
overnight to help leach material out of the particulates. Serial dilutions
were made in OMSO such that 50 ul aliquots of each dilution give the
desired concentration. The suspension was well mixed when aliquots were
removed for dosing.
Utton
5-259
BIONETICS
-------
IV.
A.
MATERIALS
Indicator Microorganisms
The Salmonella typhimurium strains used in this assay were obtained from
Dr. Bruce Ames, University of California at Berkeley.4-8 The following
four strains were used.
Strain
Designation
Gene Additional Mutations
Affected RepairLP§R Factor
Mutation Type
Detected
TA-1535
TA-1537
TA-98
TA-100
his G
his C
Ml D
his G
A uvr B rfa
A uvr B
A uvr B
A uvr B
rfa
rfa
rfa
-
pKMlOl
pKMlOl
Base-pair
substitution
Frameshift
Frameshift
Base-pair
substitution
All the above strains have, in addition to the mutati.on in the histidine
operon, mutation (rfa-) that leads to defective lipopolysaccharide coat,
a deletion that covers genes involved in the synthesis of vitamin biotin
(bio-) and in the repair of ultraviolet (uv) - induced 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 physi-
callyaamaged 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 mutagens.8
In addition, plasmid pKMlOl confers resistance to the antibiotic ampi-
cillin, 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 supplemented
with a trace of biotin and an excess of histidine. In addition, the
plates with plasmid-carrying strains contain ampicillin (25 ug/ml) to
ensure stable maintenance of plasmid pKMlOl. New stock culture plates
are made as often as necessary from the frozen master cultures or from
singl-e colony reisolates that were checked for their genotypic character-
istics (his, rfa uvrB, bio) and for the presence of plasmid. For each
experiment, an inoculum from the stock' culture plates is grown overniqht
at 37°C in nutrient broth (Oxoid CM67) and used.
8.
Media
The bacterial strains were cultured in Oxoid Media #2 (Nutrient Broth)
The selective medium was Vogen Bonner Medium E with 2% glucose.10 The
ffl
Lrtton
5-260
BIONET1CS
-------
overlay agar consisted of 0.6% purified agar with 0.05 mM histidine,
0.05 mM biotin and 0.1M NaCl according to the methods of Ames et a_L9
C.
1.
Activation System
S9 Homogenate
A 9,000 x c[ supernatant prepared from Sprague-Dawley adult male rat liver
induced by Aroclor 1254 (Ames et aj.9) was purchased commercially and
used in these assays.
2. S9 Mix
S9 mix used in these assays consisted of the following components:
Components
Concentration per Milliliter
S9 Mix
NAOP (sodium salt)
D-glucose-6-phosphate
MgCl2
KC1
Sodium phosphate buffer
pH 7.4
Organ homogenate from rat
liver (S9 fraction)
4 pinoles
5 umoles
8 umoles
33 umoles
100 umoles
100 uliters
5-261
Litton
BIONETICS
-------
V. EXPERIMENTAL DESIGN
A. Dosage Selection
Test strategy and dose selection depend upon sample type and sample avail-
ability. The Level 1 manual1 recommends solids to be initially tested
at the maximum applicable dose (MAO) of 5 mg per plate and at lower con-
centrations of 2.5, 1, 0.5, 0.1 and 0.05 mg per plate. Liquids are tested
initially at the MAD of 200 ul per plate, and at lower concentrations of
100, 50 and 10 ul per plate. Samples are retested over a narrower range
of concentrations with strains showing positive results initially. Alter-
nate dose are employed if sample size is limiting or at the direction of
the sponsor.
Doses selected to test this sample covered the recommended dose range
for solids. The highest dose was at the MAD level of 5 mg per plate and
included five lower dose levels of 2.5, 1, 0.5, 0.1, and 0.05 mg per
plate. A repeat trial with strain TA-98 without activation used the
same dose levels.
B. Mutagenicity Testing
The procedure used was based on the paper published by Ames et. aj.9 and
was performed as follows:
1. Nonactivation Assay
To a sterile 13 x 100 mm test tube placed in a 43°C water bath the fol-
lowing was added in order:
2.00 ml of 0.6% agar containing 0.05 mM histidine and
0.05 mM biotin.
0.05 ml of a suspension of the test chemical to give the
appropriate dose.
0.1 ml to 0.2 ml of indicator trganism(s).
0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured onto minimal agar plates
(see IV B, Media). After the top agar had set, the plates were incubated
at 37 C for approximately 2 days. The number of his+ revertant colonies
growing on the plates were counted with an automatic colony counter and
recorded.
2. Activation Assay
The activation assay was run concurrently with the nonactivation assay
The only difference was the addition of 0.5 ml of S9 mix (see IV C Acti-
vation System) to the tubes in place of 0.5 ml of phosphate buffer'which
was added in nonactivation assays. All other details were similar to
the procedure for nonactivation assays.
BIONET1CS s-zez
Utton
-------
A detailed flow diagram for the plate incorporation assay is provided in
Figure 1.
C.
Control Compounds
A negative control consisting of the solvent used for the test material
was also assayed concurrently with the test material. For negative con-
trols, step 'b' of Nonactivation Assays was replaced by 0.05 ml of the
solvent. The negative controls were employed for each indicator strain
and were 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 were
made using this solvent. The amount of solvent used was equal to the
maximum volume used to give the appropriate test dose.
Specific positive control compounds known to revert each strain were also
used and assayed concurrently with the test material. The concentrations
and specificities of these compounds to specific strains are given in
the following table:
Assay
Nonactivation
Concentration
per plate Salmonella
Chemical
Sodium azide
2-Nitrofluorene
(NF)
9-aminoacridine
(9AA)
Solvent (ug)
Water
Dimethyl-
sulf oxide
Ethanol
10. 0
10.0
50.0
Strains
TA-1535,
TA-98
TA-1537
TA-100
Activation
2-anthramine
(ANTH)
Dimethyl-
sulfoxide
2.5
For all strains
D.
Recording and Presenting Data
The number of colonies on each plate were counted and recorded on printed
forms. These raw data were analyzed in a computer program and reported
on a printout. The results are presented as revertants per plate for
each indicator strain employed in the assay. The positive and solvent
controls are provided as reference points.
5-263
Litton
BIONETICS
-------
AMES ASSAY (PLATE INCORPORATION METHOD]
Molten [45CC] overlay agar
appropriately supplemented
Te$t, positive or solvent
control chemical
0.1 ml
Aliquot of an overnight culture
of bacterial 1Q9 cells/ml]
Aliquot of
saline
0.5 ml
-S-9
0.5 ml S-9 mix [hepatic
-S-9—— homogenate from PCS
pretreated rat plus
necessary cofactors
Overlay poured on selective
bottom agar medium
Plated incubated at 37'C for 48 hours
The numbers of revertants/plate counted
Data analyzed
Interpretation/Conclusion
Figure 1 AMES SALMONELLA/MICROSOME MUTAGENESIS ASSAY
5-264
-------
VI. RESULTS
A. Interpretations
The test material, A81-05-030-672 (EA-2 10+3), was dissolved in DMSO at
a stock concentration of 100 mg/ml and leached overnight on a shaker at
37°C. Additional dilutions were prepared in DMSO for testing. The maximum
test level was 5.0 mg/plate. There was no evidence of toxicity at this
level.
Reverse mutation was measured in strains TA-1535, TA-1537, TA-98 and
TA-100. The test was conducted in duplicate both with and without rat
liver S9 mix for metabolic activation. The trial with strain TA-98 without
activation was repeated using the same test conditions, because in the
first trial, one of the positive control plates was lost due to contamina-
tion.
There was no mutagenic activity associated with the test material treatment
and the sample was considered nonmutagenic and non toxic. The sample
was ranked as having nondetectable (ND) mutagenic activity using the
IERL-EPA Level 1 evaluation criteria for the Ames Assay1.
Solvent control and positive control values were within acceptable ranges.
These results achieved assay acceptance criteria and provided confidence
in the assumptions that the recorded data represented typical responses
to the test material.
B. Tables
This report is based on the data provided in Tables 1 and 2.
B1ONETICS s-265
Litton
-------
RESULTS
I ABLE 1
A. NAHL OH COOt DESIGNATION OF THE TEST COMPOUND: A81-05-U30-672 EA-2 10*3
B. SOLVENT: ONSO
c. itsi INITIATION DATES: io/26/ai
0. TEST COMPLETION DATE: 10/29/81
E. S-9 LOT»: REF050
NOTE: CONCENTRATIONS ARE GIVEN IN MILLIGRAMS PER PLATE
in
i
ro
TEST
SPECIES TISSUE
NONACTIVATION
SOLVENT CONTROL
POSITIVE CONTROL**
TEST
COMPOUND
0.050 MG
0.100 MG
0.500 HG
1.000 MG
2.500 HG
5.000 HG
...
TA-1535
1
16
1239
10
12
15
10
13
11
2
19
1032
16
14
20
12
R
14
TA-1537
3 1
7
733
9
12
14
6
1C
12
2 3
4
650
8
13
5
12
6
14
TA-98
1
24
060
24
27
26
34
28
22
TA-100
231
'c
30
21
33
23
2a
30
116
1196
144
126
131
117
105
78
2
11H
1080
104
129
87
92
72
86
3
ACTIVAT ION
SOLVENT CONTROL RAT
POSITIVE CONTROL*** RAT
TEST
* *
TA
T«
TA
TA
COMPOUND
0.050 HG RAT
0.100 MG RAT
0.500 HG RAT
1.000 HG RAT
2.500 HG RAT
5.000 MG RAT
-1535 iOOIUH AZIOE
-153/ 9-AHINOACRIDINE
-•*8 2-MIROFLUORENE
-10- SODIUM AZIOE
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
15
479
16
8
14
H
12
12
11
509
12
9
13
10
14
9
6
459
11
R
13
a
7
9
7
445
11
12
9
A
a
13
41
645
34
27
29
33
41
30
* * ft
10 UG/PLATE
50 U&/PLATE
M
1991
34
ib
41
43
T3
33
TA-1535
TA-1537
10 UG/PLATF TA-*8
10 UG/PLATE
TA-10C
92
2371
120
103
100
101
95
93
101
1861
100
87
98
112
98
103
2-ANTHRAMINE
2-ANTHNAHINE
2-ANTHRAMINE
2-ANTHRAMINE:
2.5 UG/PLATt
2.5 UG/PLATE
2.5 UG/PLATf
2.5 UG/PLATrZ
SOLVENT tO UL/PLATE
C IND1CAILS CONTAMINATION
-------
RC&ULTS
TABLE 2
en
IM
NAME OK CODE DESIGNATION OF THE TEST COMPOUND: A81-05-330-672 EA-2 10*3
SOLVENT: DNSO
TEST INITIATION DATES: 11/S3/BI
TEST COMPLETION DATE: 11/09/81
S-9 LOIN: REF050
CONCENTRATIONS ARE GIVEN IN MILLIGRAMS PER PLATE
A.
e.
c.
o.
E.
NOTE;
TEST
NONACTIWAI ION
SPECIES TISSUE
SOLVENT CONTROL
POSITIVE CONTROL**
TEST COMPOUND
R?VERTANTS
IA-98
123
22 21
1128 1M3
PER
PLATE
' o.Q^o
0.100
O.'iOO
1 .000
2.500
5.000
• *
TA-98
SOLVENf
Ma
MG
MG
MG
MG
MG
-_- _-_
... ...
...
— _
...
25
23
19
11
23
ia
2-Nf TROFLUORENE
33
20
22
20
2*
21
10 UG/PLATE
50 UL/PLATE
-------
VII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA
Statistical methods are not currently used, and evaluation is based on
the criteria included in this protocol.
Plate test data consists of direct revertant colony counts obtained from
a set of selective agar plates seeded with populations of mutant cells
suspended in a semi sol 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 semiquanti-
tative in nature. Although these features of the assay reduce the quanti-
tation of results, they provide certain advantages not contained in a
quantitative suspension test:
The small number of cell divisions permits potential
mutagens to act on replication 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 quantisation 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 concen-
trations, the highest of these doses being selected to show slight toxicity
as determined by subjective criteria.
B. Dose-Response Phenomena
The demonstration of dose-related increased in mutant counts is an impor-
tant criterion in establishing metagenicity. A factor that might modify
dose-response results for a mutagen would be the selection of doses that
are too low (usually mutagenicity and toxicity are related). If the
highest dose is far lower than a toxic concentration, 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.
C. Control Tests
Positive_and negative control assays were conducted with each experiment
and consisted of direct-acting mutagens for nonactivation assays and
mutagens that require metabolic biotransformation in activation assays.
Litton
5-268
BIONETICS
-------
Negative controls consisted of the test material solvent In the overlay
agar together with the other essential components. The negative control
plate for each strain gave a reference point to which the test data was
compared. The positive control assay was conducted to demonstrate that
the test systems were functional with known mutagens.
The following normal range of revertants for solvent controls are generally
considered acceptable.
TA-1535: 8-30
TA-1537: 4-30
TA-98: 20-75
TA-100: 80-250
D. Evaluation Criteria for Ames Assay
Because the procedures to be used to evaluate the mutagenicity of the
test material are semiquantitative, the criteria to be used to determine
positive effects are inherently subjective and are based primarily on a
historical data base. Most data sets will be evaluated using the following
criteria.
1. Strains TA-1535 and TA-1537
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to three times the solvent control value will
be considered to be mutagenic.
2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to twice the solvent control value for TA-98
and TA-100 will be considered to be mutagenic.
3. Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain
(G-46), 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 that cannot be
reproduced in additional runs, the initial positive test data lose signi-
ficance.
The preceding criteria are not absolute, and other extenuating factors
may enter into a final evaluation decision. However, these criteria
will be applied to the majority of situations and are presented to aid
those individuals not familar with this procedure. As the data base is
increased, the criteria for .evaluation can be more firmly established.
5-269
BIONETICS 13
Litton
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EB
Litton
E. Relation Between Mutaqenicity and Carcinogenlcity
It must be emphasized that the Ames Salmonella/Microsome Plate Assay is
not a definitive test for chemical carcinogens. It is recognized, however,
that correlative and functional relations have been demonstrated between
these two endpoints. The results of comparative tests on 300 chemicals
by McCann et al.4 show an extremely good correlation between results of
microbial mutagenesis tests and in 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 muta-
genic activity.
F. Criteria for Ranking Samples in the Ames Assay
The goal of EPA Level 1 Ames testing is to rank source streams by relative
degree of genetic toxicity (mutagenicity). Samples are first identified
as mutagenic or nonmutagenic by the criteria in Section D above and
then ranked using the mutagenicity categories presented in the table
below. The lowest concentration giving a positive response in any strain,
with or without metabolic activation, is identified as the minimum effec-
tive concentration (MEC) for that sample. The mutagenicity of the sample
is evaluated as high (H), moderate (M), Tow (L), or nondetectable (ND)
according to the evaluation criteria developed in the Level 1 manual1
and summarized below. Samples with no detectable activity at the maximum
applicable dose (MAD) are ranked nondetectable (ND).
Ames Assay Mutagenicity Ranking Criteria1
Mutagenic Solids Liquids3
Activity (MEC in ug/plate) (MEC in pi/plate)
High (H)
Moderate (M)
Low (L)
Not Detectable (NO)
<50
50-500
500-5000
>5000
<2
2-20
20-200
>200
Concentration of organic extracts is based upon organic content (|jg
organics per plate) and not volume (pi extract per plate) of sample
tested.
5-270
BIONETICS 14
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VIII. REFERENCES
1. Brusick, D.J., et a"L: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment ITological Tests.EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD, August
1981, 52 pp.
4. 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.
5. Ames, B.N., Gurney, E.G., Miller, J.A. and Bartsch, H.: Carcinogens
as frameshift mutagens: Metabolites and derivatives of 2-acetylamino-
fluorene and other aromatic amine carcinogens. Proc. Nat. Acad.
Sci., USA 69:3128-3132, 1972.
6. 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.
7. 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.
8. 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.
9. 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.
10. Vogel, H.J. and Bonner, D.M.: Acetylornithinase of E. coli partial
purification and some properties. J. Biol. Chem., 218:97IT06, 1966.
Litton
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GENETICS ASSAY NO.:
LBI SAFETY NO.:
CYTOTOXIC EVALUATION OF
A81-05-030-672
(EA-2 10+3)~
INTHE RABBIT
ALVEOLA MAEROPHAGE (RAM)
CYTOTOXICITY ASSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
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PREFACE
This assay conforms to the standard EPA Level 1 procedure for the rabbit
alveolar macrophage (RAM) cytotoxicity assay as described in "IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests" (1).
The data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting" (2).
The RAM cytotoxicity assay has been shown to be a sensitive method for
detecting cytotoxic activity for a variety of chemicals representing
various chemical classes (3). This assay is one of several recommended
by EPA to identify, categorize and rank the pollutant potential of influent
and effluent streams from industrial and energy-producing processes.
This assay has been well validated with a wide range of positive and
negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepara-
tion, testing and evaluation of the test material shall conform to Litton
Bionetics, Inc. standard operating procedures and the Good Laboratory
Practices Regulations of 1979. Deviations from standard procedure shall
be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
Litton
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BIONETICS
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TABLE OF CONTENTS
Page No.
PREFACE ...... . ............... i
I. ASSAY SUMMARY ................... 1
II. OBJECTIVE .................... 2
III. TEST MATERIAL ...... . ............ 3
A. Description ................. 3
B. Handling and Preparation ........... 3
IV. MATERIALS
A. Indicator Cells ............... 4
B. Media .................... 4
C. Negative Controls ............. - . 4
V. EXPERIMENTAL DESIGN
A. Procurement of Cells ............. 5
B. Sample Forms ................. 5
C. Dose Selection ................. 6
D. Treatment .................. 6
E. Cell Viability Assay ............. 6
F. ATP Assay .................. 7
VI. ASSAY ACCEPTANCE CRITERIA ............. 8
VII. RESULTS ...................... 9
A. Interpretation ................ 9
B. Tables and Figures .............. 9
VIII. ASSAY EVALUATION CRITERIA ............. 13
IX. REFERENCES .................... 14
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I. ASSAY SUMMARY
A. SPONSOR: Acurex Corporation
B. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5883
1. Identification: A81-05-030-672 (EA-2 10+3)
2. Date Received: August 26, 1981
3. Physical Description: Fine, gray powder
C. TYPE OF ASSAY: Rabbit Alveolar Macrophage (RAM) Cytotoxicity Assay
D. ASSAY DESIGN NUMBER: 443
E. STUDY DATES:
1. Initiation: October 22, 1981
2. Completion: October 23, 1981
F. SUPERVISORY PERSONNEL:
1. Study Director: Brian Myhr, Ph.D.
2. Laboratory Supervisor: Robert Young, M.S.
G. EVALUATION:
The test material, which was tested as supplied, caused a dose-related
increase in toxicity for concentrations above approximately 200 ug/ml.
The viability index and ATP content assay parameters were about
equally sensitive and indicated an EC50 would be achieved at approxi-
mately the maximum applicable dose (MAD) of 1000 |jg/ml. Since
toxicity was clearly observed in the low toxicity region of 100-
1000 ug/ml, as defined by the IERL-EPA evaluation criteria, and the
projected EC50 values were essentially on the borderline between
the low and nondetectable toxicity regions, the test material was
best evaluated as having low/nondetectable (L/ND) toxicity to cultured
RAM cells.
Submitted by:
Study Director
u
_ _ _
Jrian Myhr, Phyu: Date ' David J. Brusick, Ph.D.
Associate Director, Director,
Department of Molecular Department of Molecular
Toxicology Toxicology
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II. OBJECTIVE
The objective of this study was to determine and rank the cytotoxicity
of A81-05-030-672 (EA-2 10+3) to cultured rabbit alveolar macrophage
(RAM) cells. The measure of cytotoxicity was the reduction in cell
viability and adenosine triphosphate (ATP) content of the cultures after
a 20 hour exposure to the test material. At the conclusion of the exposure
period, the number of viable cells and total ATP content in the treated
cultures were compared to the corresponding values in unexposed control
cultures. The concentration of test material that reduced each experi-
mental parameter by 50% was estimated graphically and referred to as the
EC50 value. Standard EPA Level 1 toxicity evaluation criteria for the
RAM cytotoxicity assay were used to rank the toxicity potential of the
test material based upon the most sensitive parameter.
m
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III. TEST MATERIAL
A. Description
The test material was received as a fine, gray powder (1.5 gin). No infor-
mation on particle size distribution or sampling parameters was provided.
B. Handling and Preparation
The test material was received on August 26, 1981, and was assigned LBI
assay number 5883 and LBI safety number 7167. The sample was stored at
+4°C in the dark.
Approximately 33 mg of test material was used as supplied, without
grinding, for the assay. The test material was suspended in serum-free
EMEM culture medium at a concentration of 2000 ug/ml and incubated at
37°C on a roller drum for 8 hours. A fine, gray suspension was formed
that settled upon standing. No pH changes were noted. The suspension
was serially diluted with EMEM (serum-free) and applied to the cultures
at a maximum concentration of 1000 ug/ml in the presence of 10% serum.
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IV. MATERIALS
A. Indicator Cells
The assay employed short-term primary cultures of alveolar macrophage
cells obtained by lung lavage of a male New Zealand white rabbit (2.4 kg).
The rabbit was maintained on Purina Lab Rabbit Chow 5321 and water ad
libitum and was examined for the absence of respiratory illnesses prior
to use.
B. Media
The cells were maintained and treated in Eagle's Minimum Essential Medium
(EMEM) with Earle's salts and supplemented with 10% fetal bovine serum
(heat-inactivated), 100 units/ml penicillin, 100 ug/ml streptomycin,
17.6 ug/ml kanamycin, and 0.4 ug/ml amphotericin B.
C. Negative Controls
The negative control consisted of three untreated cultures carried through
the same experimental time period as the treated cells. The average
viability and ATP content of the negative control provided the reference
points for determining the effects of different concentrations of the
test material on the assay parameters.
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V. EXPERIMENTAL DESIGN
A. Procurement of Cells
A rabbit was sacrificed by injection of Nembutal (60 mg/ml) into the
marginal ear vein, and sterile operating techniques were used to perform
a tracheostomy. Prewarmed normal saline (30 ml) was then introduced
into the lungs via a catheter and allowed to stand for 15 minutes. This
lavage fluid was removed and placed into a 50-ml sterile centrifuge tube
on ice. Nine additional lavages were similarly performed and collected,
except the saline was removed shortly after its introduction into the
lungs. Any lavage fluid containing blood or mucous was discarded. The
lavages were centrifuged at 365 x g for 15 minutes and the cells resus-
pended in cold 0.85% saline. After two washes in saline by centrifugation,
the cell pellets were resuspended in cold EMEM containing 20% serum and
then combined. A cell count was obtained by hemocytometer and the suspen-
sion diluted to between 5.13 x 105 and 106 cells/ml. Viability was deter-
mined by trypan blue staining and the cells were not used if less than
95% viable. Also, a differential cell count from Wright-stained smears
was performed to verify that the macrophage content was above 90%.
B. Sample Forms
The usual sample form for application to the cells is a suspension of
particulate material. Solid samples are ground to fine particles and a
weighed portion is suspended in a known volume of EMEM (0% FBS) for about
eight hours to help leach any water-soluble material. Finely-divided
test material may be suspended directly in culture medium without further
grinding. Aqueous liquids, suspensions, or slurries containing less
than 0.5% organic solvent are added by volume to culture medium.
Samples supplied as solutions in organic solvents are usually solvent-
exchanged into DMSO before testing. Original sample volumes may first
be reduced a maximum of 10-fold in a Kuderna-Danish concentrator, and
the concentrative factor is used to convert assayed volumes into equi-
valent original sample volumes in the absence of information about solute
concentration. An aliquot of the reduced volume is exchanged into DMSO
by repeated, partial evaporation under a stream of nitrogen in a warm
water bath (50°C); the evaporated volumes are replaced with equal volumes
of DMSO.
Samples adsorbed on XAD-2 resin are extracted with methylene chloride
or acetone in a Soxhlet apparatus for 24 hours. The extract is then
concentrated and solvent-exchanged into DMSO. Alternatively, acetone
extracts can be assayed directly at concentrations up to 2% by volume in
the culture medium.
Samples impregnated on fiber glass or teflon filters are repeatedly soni-
cated in cyclonexane to remove particulates. The resulting cyclohexane
particulate suspension is then evaporated to dryness and the particulates
resuspended in EMEM culture medium at the desired concentration.
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Utton
Sponsor-specified handling of sample materials will be followed if the
above procedures are not applicable or a specific procedure is desired.
C. Dose Selection
Unless the approximate toxicity is already known or the sample size is
limiting, the following usual dose ranges are tested for different sample
forms. Dry, particulate material is tested at six dose levels from
1000 ug/ml to 3 \jg/m1. Aqueous samples, suspensions, or slurries are
tested from 600 ul to 3 ul/ml in six dose steps. Samples that are solvent-
exchanged into DMSO are tested from 20 Ml/ml (2% DMSO in growth medium)
to 0.2 pi/ml, also in six dose steps. A second dose study is performed
with an adjusted dose range if the EC50 was not located properly in the
initial test. However, EC50 values greater than 1000 ul/ml for particulate
material, 600 ul/ml for aqueous samples, or 20 ul/ml for organic solutions
will not be determined.
This test material, A81-05-030-672 (EA-2 10+3), was tested at 6 dose
levels, starting at the maximum applicable dose (MAD) of 1000 ug/ml and
including 600, 300, 100, 60 and 30 ug/ml.
D. Treatment
A series of 25 cm2 culture flasks were prepared, each containing 2.0 ml
of serum-free medium at 37°C and the test material at twice the desired
final concentration. Three flasks were prepared for each test concen-
tration. Aliquots of cell suspension (2 ml) were then added; each flask,
therefore, contained from 1.03 to 2 x 106 viable cells in a 4-ml volume
of media containing 10% serum. The flasks were placed on a rocker plat-
form in a 37°C incubator with a humidified atmosphere containing 5% C02.
After sitting for about 30 minutes, the flasks were slowly rocked for
the remainder of a 20-hour exposure period.
If the test substance causes a color change in the growth medium, the pH
is determined in additional treated flasks. After the exposure period,
the pH of the medium in the experimental flasks is again recorded.
E. Cell Viability Assay
At the end of the treatment period, the medium containing unattached
cells was decanted into a centrifuge tube on ice. The attached cells
were rinsed with 1 ml of 0.1% trypsin/0.01% versene and then incubated
with 2 ml of the trypsin/versene solution for about 5 minutes at 37°C.
The trypsinates and decanted media were combined for each culture to
yield a 7-ml cell suspension for subsequent analysis.
A 1.0 ml aliquot of the cell suspension was removed for cell count and
• viability determination. The aliquot was combined with 1.0 ml of 0.4%
trypan blue and counted by hemocytometer about 5 to 15 minutes later.
The total number of cells counted per culture was the sum of the numbers
found in five squares for each chamber of the hemocytometer (1 ul total
volume). The numbers of live (colorless) and dead (blue) cells were
recorded.
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BIONETICS fi
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f- ATP Assay
ATP was immediately analyzed by extraction of a 0.1-ml sample of cell
suspension with 0.9 ml of 90% DMSO. After 2 minutes at room temperature
5.0 ml cold MOPS buffer (0.01 M morpholinopropane sulfonic acid) at pH 7.4
was added and the extract mixed well and placed on ice. Aliquots of
10 ul were injected into a cuvette containing a luciferin-luciferase
reaction mixture in a DuPont Model 760 Luminescence Biometer. The Biometer
was calibrated daily with standard ATP solutions to provide a direct
read-out of the ATP content. Each test sample was assayed at least twice
to obtain repeatable readings.
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VI. ASSAY ACCEPTANCE CRITERIA
The assay will be considered acceptable for evaluation of the test results
if the following criteria are met:
1. The macrophage population is 90% or greater of the total nucleated
cells collected by lung lavage.
2. The percent viability of the macrophages used to initiate the assay
is 95% or greater.
3. The survival of viable macrophages in the negative control cultures
over the 20 hour treatment priod is 70% or greater.
4. A sufficient number of data points (for five test concentrations or
less) are available to clearly locate the EC50 of the most sensitive
test parameter within a toxicity region as defined under Assay Eval-
uation Criteria.
5. The data points critical to the location of the EC50 for the most
sensitive parameter are the averages of at least two treated cultures.
6. If all the test parameters yield EC50 values greater than 1000 ug/ml,
600 ul/ml for aqueous solutions, or 20 ul/ml for organic solutions,
the plotted curves for ATP content and viability index parameters
do not exceed 120% of the negative control.
E
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VII. RESULTS
A. Interpretation
The test material was ingested by the macrophages and caused a general
decline in their viability when the applied concentration was increased
above approximately 200 vg/ml. Absolute values for the assay parameters
are given in Table 1, as well as the parameters relative to the negative
control average value, and the relative values are plotted in Figures 1
and 2.
The viability index (which measures cell survival) and the culture ATP
content usually tend to parallel each other, and an inspection of the
results in Figures 1 and 2 show this to be the case for the current assay.
Both parameters were about equally sensitive and showed declines in ATP
and the numbers of viable cells in the 100-1000 ug/ml concentration range.
Both parameters also indicated the EC50 values would be achieved for
concentrations at or just above the MAD level of 1000 ug/ml. Therefore,
strict application of the IERL-EPA evaluation criteria would result in a
nondetectable toxicity classification. However, toxicity was clearly
evident in the low toxicity region (100-1000 ug/ml), and repeat assays
could be expected to result in variations in the ECSO positions such
that borderline responses could fall within either the low or nondetect-
able categories. The percent viability and ATP/106 cells parameters
were essentially nonresponsive and did not contribute to an evaluation
of the test material. On the basis of the responsive parameters, the
test material was evaluated as having low/nondetectable (L/ND) borderline
toxicity to the RAM cells.
The macrophages collected for this assay had normal morphology and appeared
to be in a healthy state. The initial viability was excellent (99.4%)
and the survival of viable cells in the negative control was 98.9%. The
average cellular ATP content of the negative controls 29.0 x 108 fg ATP
per 106 total cells, which wash within the historical range for acceptable
cultures. These results achieved the assay acceptance criteria and
provided confidence in the assumption that the collected data represented
typical responses to the test material.
B. Tables and Figures
This report is based on the data provided in Table 1 and Figures 1 and
2.
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TABLE 1
RABBIT ALVEOLAR MACROPHAGE (RAM) CYTOTOXICITY ASSAY DATA
en
i
ro
CO
LBI Assay No.: 5883
Test Material Identity: A81-05-030-672 (EA-2 10+3)
Test Date: October 22. 1981
Vehicle:
Sample
NCC
TEST
TEST
TEST
TEST
TEST
TEST
EMEM
Concentration8
ug/ml
__.
30
60
100
300
600
1000
Initial Cell Viability: 99.4%
Viable Macrophage Seeded/Flask: 1.03 x 106
Macrophage Population Percentage: >90%
Survival of Negative Control
Macrophage Over Treatment Time: 98.9%
Average Values
Viable Cells
106 Units
0.89
0.90
0.97
0.86
0.63
0.49
0.54
per Culture
Total Cells
106 Units
0.90
0.91
0.98
0.90
0.67
0.54
0.61
Flask
ATP .
108fgb
26.1
26.1
25.7
24.5
22.5
17.4
14.4
ATP Per
106 Cells
10s fg
29.0
28.7
26.2
27.2
33.6
32.2
23.6
Viability
%
98.9
98.9
99.0
95.6
94.0
90.7
88.5
Expressed
Viability
100.0
100.0
100.1
96.7
95.0
91.7
89.5
as Percent of Negative Control
Viability
Index ATP
100.0 100.0
101.1 100.0
109.0 98.5
96.6 93.9
70.8 86.2
55.1 66.7
60.7 55.2
ATP Per
106 Cells
100.0
99.0
90.3
93.8
115.9
111.0
81.4
apH change in culture medium: None observed
bfg = Femtogram (10-15 gram).
CNC = Negative Control, EMEM culture medium.
Determined from data plots in Figures 1 and 2.
VALUES:
>1000
1000 >1000
Toxidty
Classification: Low/Nondetectable Borderline
>1000
-------
120
FIGURE 1
EC50 DETERMINATION FOR
PERCENT VIABILITY (0) AND VIABILITY INDEX (•)
A81-05-030-672
(EA-2 10+3)
CONCENTRATION, JJG/ML
5-285
n
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FIGURE 2
EC50 DETERMINATION FOR
ATP/FLASK (0) AND ATP/106 CELLS (I)
A81-05-030-672
(EA-2 10+3)
10 100
CONCENTRATION, JJG/ML
1000
5-286
12
-------
VIII. ASSAY EVALUATION CRITERIA
The EC50 value represents the concentration of test material that reduces
the most sensitive parameter of the RAM assay to 50% of the vehicle or
negative control value. EC50 values are determined graphically by fitting
a curve by eye through relative toxicity data plotted as a function of
the logarithm of the applied concentration. Each data point normally
represents the average of three culture dishes. Statistical analysis is
unnecessary in most cases for evaluation.
The toxicity of the test material is evaluated as high, moderate, low,
or nondetectable according to the range of EC50 values defined in the
following table.
Solids Aqueous Liquids Nonaqueous Liquids
Toxicity (EC50 in ug/ml) (ECSO in ul/ml) (EC50 in ul/ml)
High
Moderate
Low
Not Detectable
<10
10 to 100
100 to 1000
>1000
<6
6 to 60
60 to 600
>600
<0.2
0.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests1.
Criteria for nonaqueous liquids are tentative and under evaluation. If
the organic or solid content is known, the solid evaluation criteria
are applied.
Another evaluation scheme is proposed for extracts obtained from SASS
train gas volumes. The proportion of the total gas volume corresponding
to the volume of extract used in the bioassay is calculated and expressed
as L/ml of culture medium (or DSCF/ml of culture medium). A criterion
of 1000 L/ml is set as the limit for nondetectable toxicity. This gas
volume corresponds to the average volume breathed by humans over a 2-hour
period. The subsequent toxicity ranges are defined by 10-fold dilution
steps to conform to standard procedure. The toxicity ranges are defined
in the following table for liter and dry standard cubic feet units:
~~' EC50 In EC50 In
Toxicity Liters/ml (L/ml) Dry Standard Cubic Feet/ml (DSCF/ml)
High:<10<0.35 DSCF
Moderate 10-100 0.35-3.5
Low 100-1000 3.5-35
Nondetectable >1000 >35
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BIONETICS 13
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IX.
1.
REFERENCES
Brusick, D.J., et a_K: IERL-RTP Procedures Manual:
mental Assessment Biological TestsT
Level 1 Environ-
2.
3.
EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington,
MD, September 1980, 177 pp. In press.
Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc., Kensington, MD, April 1980,
100 pp.
Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD, August
1981, pp. 52.
ffl
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GENETICS ASSAY NO.: 5884
LBI SAFETY NO.: 7168
MUTAGENICITY EVALUATION OF
A81-05-030-674
IEF2 1+FILTER)
TNTHE
EPFLIVEL 1
AMES SATJ^NELTATMTCROSOME
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 20895
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
5-289
BIONETICS
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PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Ames
Salmonella/microsome mutagenesis assay as described in "IERL-RTP Proce-
dures Manual: Level 1 Environmental Assessment Biological Tests"1. The
data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting"2.
The Ames Salmonella/microsome mutagenesis assay has been shown to be a
sensitive method for detecting mutagenic activity for a variety of chemi-
cals representing various chemical classes3. This assay is one of several
recommended by EPA to identify, categorize and rank the pollutant potential
of influent and effluent streams from industrial and energy-producing pro-
cesses. This assay has been well validated with a wide range of positive
and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good Labora-
tory Practices Regulations of 1979. Deviations from standard procedure
shall be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives, of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
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TABLE OF CONTENTS
Page No.
PREFACE i
I. ASSAY SUMMARY 1
II. OBJECTIVE 2
III. TEST MATERIAL 3
A. Description 3
B. Handling and Preparation 3
IV. MATERIALS 4
A. Indicator Microorganisms 4
B. Media 4
C. Activation System 5
1. S9 Homogenate 5
2. S9 Mix 5
V. EXPERIMENTAL DESIGN 6
A. Dose Selection 6
B. Mutagenicity Test 6
1. Nonactivation Assay 6
2. Activation Assay 6
C. Control Compounds 7
D. Recording and Presenting Data 7
VI. RESULTS 9
A. Interpretation 9
B. Tables 9
VII. EVALUATION CRITERIA 11
A. Surviving Populations 11
B. Dose-Response Phenomena 11
C. Control Tests 11
D. Evaluation Criteria 'for Ames Assay 12
1. Strains TA-1535 and TA-1537 12
2. Strains TA-98 and TA-100 12
3. Pattern 12
4. Reproducibility 12
E. Relation Between Mutagenicity and
Carcinogenicity 13
F. Criteria for Ranking Samples in the Ames Assay . . 13
VIII. REFERENCES 14
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I. ASSAY SUMMARY
A. Sponsor: Acurex Corporation
B. Material (Test Compound): Genetics Assay Number: 5884
1. Identification: A81-05-030-674 (EA-2 1+Filter)
2. Date Received: August 26, 1981
3. Physical Description: Fine, gray/black powder and
fiberglass filter with embedded
particles.
C. Type of Assay: EPA Level 1 Ames Salmonella/Microsome Plate
Test
D. Assay Design Number: 401 (EPA Level 1)
E. Study Dates:
1. Initiation: October 1, 1981
2. Completion: October 29, 1981
F. Supervisory Personnel:
A. Study Director: D.R. Jagannath, Ph.D.
G. Evaluation:
The test material, A81-05-030-674 (EA-2 1+filter), was tested
for activity in the Ames Salmonella mutagenicity assay over a
concentration range of 0.05 mg/plate to 5.0 mg/plate. The
test was performed in duplicate under nonactivation and acti-
vation test conditions with strains TA-1535, TA-1537, TA-98,
and TA-100.
The sample was not mutagenic under the test conditions employed
and was ranked as having nondetectable (ND) mutagenic activity
as defined by the IERL-EPA Level 1 criteria for the Ames bio-
assay1.
Submitted by:
Study Director
D.R. Jagannath, Ph.D.
Section Chief,
Submammalian Genetics,
Department of Molecular
Toxicology
Litton
BIONETICS
Date
5. 292
Reviewed by:
David J. Brusick, Ph.D.
Director,
Department of Molecular
Toxicology
-------
II. OBJECTIVE
The objective of this study was to determine the genetic activity of
A81-05-030-674 (EA-2 1+Filter) in the Salmonella/ microsome assay with
and without the addition of mammalian metabolic activation preparations.
The genetic activity of a sample is measured in these assays by its ability
to revert the Salmonella indicator strains from histidine dependence to
histidine independence. The degree of genetic activity of a sample is
reflected in the number of revertants that are observed on the histidine-
free medium.
Litton
5-293
BIONETICS
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ffl
Litton
III. TEST MATERIAL
A. Description
The test material, as received, was comprised to two separate components.
The first component, a fine, gray/black powder, was the 1 urn SASS train
participate catch. The second component was a fiberglass filter with
embedded participate material. This gray/black participate material
represented participates less than 1 urn collected io the SASS train sample.
Both components were supplied together in a Nalgene screw-top bottle.
B. Handling and Preparation
The test material was received at LBI on August 26, 1981. The sample
was assigned LBI safety number 7168 and LBI assay number 5884. The sample
was stored at +4°C in the dark.
The filter portion of the sample required removal of the embedded parti-
culates before testing could begin. The uncut filter was sonicated in
cyclohexane as recommended by current IERL-EPA pretest sample preparation
procedures1. The decanted particulate suspension from three successive
sonication treatments were combined and evaporated to dryness. The parti-
culate material was weighed and combined with the 1 urn particulate catch
portion of the sample. A total of 264.42 mg of the combined test material
available for testing was comprised of 70.28 mg (26.6%) of <1 urn particu-
• lates removed from the filter and 194.14 mg (73.4%) o:f 1 urn particulates.
Approximately 220 mg of the test material were used for the trial in the
Ames Salmonella Assay. The test material was suspended at 100 mg/ml in
dimethylsulfoxide (DMSO) and incubated overnight at 37°C on a rotary
shaker. This stock suspension was used to make dilutions in DMSO to be
used for dosing in the EPA Level 1 Ames Salmonella Assay.
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BIONETICS
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IV. MATERIALS
A. Indicator Microorganisms
The Salmonella typhimurium strains used in this assay were obtained from
Dr. Bruce Ames, University of California at Berkeley.4-8 The following
four strains were used.
Litton
Strain Gene Additional Mutations Mutation Type
Designation Affected Repair LPS R Factor Detected
TA-1535 his G A uvr B rfa - Base-pair
substitution
TA-1537 his C A uvr B rfa - Frameshift
TA-98 his D A uvr B rfa pKMlOl Frameshift
TA-100 his G A uvr B rfa pKMlOl Base-pair
substitution
All the above strains have, in addition to the mutation in the histidine
operon, mutation (rfa-) that leads to defective lipopolysaccharide coat,
a deletion that covers genes involved in the synthesis of vitamin biotin
(bio-) and in the repair of ultraviolet (uv) - induced DNA damage (uvrB-).
The rfa- mutation makes the strains more permeable to many large moTecules.
The uvrB- mutation decreases repair of some types of chemically or physi-
cally 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 mutagens.8
In addition, plasmid pKMlOl confers resistance to the antibiotic ampi-
cillin, 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 supplemented
with a trace of biotin and an excess of histidine. In addition, the
plates with plasmid-carrying strains contain ampicillin (25 ug/ml) to
ensure stable maintenance of plasmid pKMlOl. New stock culture plates
are made as often as necessary from the frozen master cultures or from
single colony reisolates that were checked for their genotypic character-
istics (his, rfa uvrB, bio) and for the presence of plasmid. For each
experiment, an moculunfTrom the stock culture plates is grown overnight
at 37°C in nutrient broth (Oxoid CM67) and used.
B. Media
The bacterial strains were cultured in Oxoid Media #2 (Nutrient Broth).
The selective medium was Vogen Bonner Medium E with 2% glucose.10 The
5-295
BIONETICS 4
-------
overlay agar consisted of 0.6% purified agar with 0.05 mM histidine,
0.05 mM biotin and 0.1M NaCl according to the methods of Ames et £[.
C.
1.
Activation System
59 Homogenate
A 9,000 x 2 supernatant prepared from Sprague-Dawley adult male rat liver
induced by Aroclor 1254 (Ames et ark9) was purchased commercially and
used in these assays.
2. S9 Mix
S9 mix used in these assays consisted of the following components:
Components
Concentration per Milliliter
S9 Mix
NADP (sodium salt)
D-glucose-6-phosphate
MgCl2
KC1
Sodium phosphate buffer
pH 7.4
Organ homogenate from rat
liver (S9 fraction)
4 umoles
5 umoles
8 umoles
33 umoles
100 umoles
100 uliters
5-296
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BIONETICS
-------
Utton
V. EXPERIMENTAL DESIGN
A. Dosage Selection
Test strategy and dose selection depend upon sample type and sample avail-
ability. The Level 1 manual1 recommends solids to be initially tested
at the maximum applicable dose (MAD) of 5 mg per plate and at lower con-
centrations of 2.5, 1, 0.5, 0.1 and 0.05 mg per plate. Liquids are tested
initially at the MAD of 200 ul per plate, and at lower concentrations of
100, 50 and 10 ul per plate. Samples are retested over a narrower range
of concentrations with strains showing positive results initially. Alter-
nate dose are employed if sample size is limiting or at the direction of
the sponsor.
Doses selected to test this sample covered the recommended dose range
for solids. The highest dose was at the MAD level of 5 mg per plate and
included five lower dose levels of 2.5, 1, 0.5, 0.1 and 0.05 mg per plate.
B. Mutagem'city Testing
The procedure used was based on the paper published by Ames et. a_L9 and
was performed as follows:
1. Nonactivation Assay
To a sterile 13 x 100 mm test tube placed in a 43°C water bath the fol-
lowing was added in order:
2.00 ml of 0.6% agar containing 0.05 mM histidine and
0.05 mM biotin.
0.05 ml of a suspension of the test chemical to give the
appropriate dose.
0.1 ml to 0.2 ml of indicator organism(s).
0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured onto minimal agar plates
(see IV B, Media). After the top agar had set, the plates were incubated
at 37°C for approximately 2 days. The number of his+ revertant colonies
growing on the plates were counted with an automatic colony counter and
recorded.
2. Activation Assay
The activation assay was run concurrently with the nonactivation assay.
The only difference was the addition of 0.5 ml of S9 mix (see IV C, Acti-
vation System) to the tubes in place of 0.5 ml of phosphate buffer which
was added in nonactivation assays. All other details were similar to
the procedure for nonactivation assays.
5-297
BIONETICS
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A detailed flow diagram for the plate incorporation assay is provided in
Figure 1.
C.
Control Compounds
A negative control consisting of the solvent used for the test material
was also assayed concurrently with the test material. For negative con-
trols, step 'b' of Nonactivation Assays was replaced by 0.05 ml of the
solvent. The negative controls were employed for each indicator strain
and were 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 were
made using this solvent. The amount of solvent used was equal to the
maximum volume used to give the appropriate test dose.
Specific positive control compounds known to revert each strain were also
used and assayed concurrently with the test material. The concentrations
and specificities of these compounds to specific strains are given in
the following table:
Assay
Nonactivation
Chemical
Sodium azide
2-Nitrofluorene
(NF)
9-aminoacridine
(9AA)
Concentratio
per plate
Solvent (ug)
Water
Di methyl -
sulfoxide
Ethanol
10.0
10.0
50.0
n
Salmonella
Strains
TA-1535,
TA-98
TA-1537
TA-100
Activation
2-anthramine
(ANTH)
Dimethyl-
sulfoxide
2.5
For all strains
D.
Recording and Presenting Data
The number of colonies on each plate were counted and recorded on printed
forms. These raw data were analyzed in a computer program and reported
on a printout. The results are presented as revertants per plate for
each indicator strain employed in the assay. The positive and solvent
controls are provided as reference points.
ffl
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5-298
BIONETICS
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Aliquot of
saline
AMES ASSAY (PLATE INCORPORATION METHOD)
Molten [45'C] overlay agar
appropriately supplemented
Test, positive or solvent
control chemical
0.1 ml
Aliquot of an overnight culture
of bacterial 1Q9 cells/ml]
0.5 ml
-S-9
0.5 ml S-9 mix [hepatic
S«9—— homogenate from PC6
pretreated rat plus
necessary cofactors
Overlay poured on selective
bottom agar medium
Plated incubated at 37*C for 46 hours
The numbers of revertants/plate counted
Data analyzed
Interpretation/Conclusion
Figure 1 AMES SALMONELLA/MICROSOME MUTAGENESIS ASSAY
5-299
-------
VI. RESULTS
A. Interpretations
The test material, A81-05-030-674 (EA-2 1+Filter), was dissolved in DMSO
at a stock concentration of 100 mg/ml and leached overnight on a shaker
at 37°C. Additional dilutions were prepared in DMSO for testing. The
maximum test level was 5.0 mg/plate. There was no evidence of toxicity
at this level.
Reverse mutation was measured in strains TA-1535, TA-1537, TA-98 and
TA-100. The test was conducted in duplicate both with and without rat
liver S9 mix for metabolic activation.
There was no mutagenic activity associated with the test material treat-
ment and the sample was considered nonmutagenic and non toxic. The sample
was ranked as having nondetectable (ND) mutagenic activity using the
IERL-EPA Level 1 evaluation criteria for the Ames Assay1.
Solvent control and positive control values were within acceptable ranges.
These results achieved assay acceptance criteria and provided confidence
in the assumptions that the recorded data represented typical responses
to the test material.
B. Tables
This report is based on the data provided in Table 1.
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5-300
BIONETICS
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RESULTS
TAHLf I
A. NAME Oft CODE DESIGNATION OF THE TEST COMPOUND: A-81-05-03G-674UA-2 1»FILTER>
6. SOLVENT: DMSO
C. T<:ST INITIATION DATES: 10/26/H1
0. TEST COMPLETION DATE: 10/29/81
E. s-s LOTH: REFOSO
NOTE: CONCENTRATIONS ARE GIVEN IN MILLIGRAMS PER PLATE
REVtRTANTS PER PLATE
TEST SPECIES TISSUE TA-1535 TA-153T TA-98
123 123 123
NONACTI VAUGN
TA-100
1 2
U1
I
CO
o
SOLVENT CONTROL
POSITIVE CONTROL**
TEST COMPOUND
0.050000 MG
0.100000 MG
0.500000 MG
1.333000 MG
2.5000CO MG
S.OOOQjO MG
ACTIVAT ION
16 19
123? 1032
11
10
22
II
21
23
10
H
15
15
23
23
733 650
ft
•J
ID
13
11
ia
6
16
21
21
B6P
26
C
116
118
1080
34
20
32
35
48
0
24
21
42
29
46
34
IOC
125
116
131
life
142
105
113
138
119
135
132
SOLVENT CONTROL RAT
POSITIVE CONTROL*** RAT
TEST COMPOUND
0.0 500 CO MG RAT
0.100000 MG RAT
0.500000 MG RAT
1 .COOOOO MG RAT
2.SOOOOO MG RAT
5.000000 MG RAT
T4-1035 SODIUM A/IDE
TA-1537 V-AHINOACRIDINC
TA-J8 2-NITROFLUORf NE
T4-1C1 SODIUM A^IOE
SOLVENT 50 UL/PLATE
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
15
479
10
11
11
14
11
16
11
509
>>
17
A
13
17
17
6
45r-
17
9
P
13
16
15
7
445
5
11
14
14
10
21
41
645
36
39
39
44
46
40
. * >
10 UG/PLATE
50 US/PLATE
37
1991
21'
40
3-j
30
34
45
TA-1535
TA-1537
10 UU/PLATE TA-98
10 UG/PLATE
TA-10C
92 101
2371 1861
89 108
103 100
124 114
119 113
148 116
124 114
2-ANTHRAMINE
2-ANTHRAMINE
k'-ANTHRAMINE
2-ANTHRAMINcT
2.5 UG/PlAt^
2.5 UU/PLATf:
2.5 UG/PLAT:
2.5 UG/PLAT1:
C INDICATES CONTAMINATION
-------
VII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA
Statistical methods are not currently used, and evaluation is based on
the criteria included in this protocol.
Plate test data consists of direct revertant colony counts obtained from
a set of selective agar plates seeded with populations of ^utant cells
suspended in a semisolid 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 semiquanti-
tative in nature. Although these features of the assay reduce the quanti-
tation of results, they provide certain advantages not contained in a
quantitative suspension test:
The small number of cell divisions permits potential
mutagens to act on replication DNA, which is often more
sensitive than nonreplicating 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 quantisation 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 concen-
trations, the highest of these doses being selected to show slight toxicity
as determined by subjective criteria.
B. Dose-Response Phenomena
The demonstration of dose-related increased in mutant counts is an impor-
tant criterion in establishing metagenicity. A factor that might modify
dose-response results for a mutagen would be the selection of doses that
are too low (usually mutagenicity and toxicity are related). If the
highest dose is far lower than a toxic concentration, 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.
C. Control Tests
Positive_and negative control assays were conducted with each experiment
and consisted of direct-acting mutagens for nonactivation assays and
mutagens that require metabolic biotransformation in activation assays.
ffl
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5-302
BIONETICS 11
-------
Negative controls consisted of the test material solvent in the overlay
agar together with the other essential components. The negative control
plate for each strain gave a reference point to which the test data was
compared. The positive control assay was conducted to demonstrate that
the test systems were functional with known mutagens.
The following normal range of revertants for solvent controls are generally
considered acceptable.
TA-1535: 8-30
TA-1537: 4-30
TA-98: 20-75
TA-100: 80-250
D. Evaluation Criteria for Ames Assay
Because the procedures to be used to evaluate the mutagenicity of the
test material are semi quantitative, the criteria to be used to determine
positive effects are inherently subjective and are based primarily on a
historical data base. Most data sets will be evaluated using the following
criteria.
1. Strains TA-1535 and TA-1537
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to three times the solvent control value will
be considered to be mutagenic.
2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to twice the solvent control value for TA-98
and TA-100 will be considered to be mutagenic.
3. Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain
(G-46), 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 that cannot be
reproduced in additional runs, the initial positive test data lose signi-
ficance.
The preceding criteria are not absolute, and other extenuating factors
may enter into a final evaluation decision. However, these criteria
will be applied to the majority of situations and are presented to aid
those individuals not familar with this procedure. As the data base is
increased, the criteria for evaluation can be more firmly established.
5-303
BIONETICS 12
Litton
-------
E. 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, however,
that correlative and functional relations have been demonstrated between
these two endpoints. The results of comparative tests on 300 chemicals
by McCann et aj.4 show an extremely good correlation between results of
microbial mutagenesis tests and jn 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 muta-
genic activity.
F. Criteria for Ranking Samples in the Ames Assay
The goal of EPA Level 1 Ames testing is to rank source' streams by relative
degree of genetic toxicfty (mutagenicity). Samples are first identified
as mutagenic or nonmutagenic by the criteria in Section D above and
then ranked using the mutagenicity categories presented in the table
below. The lowest concentration giving a positive response in any strain,
with or without metabolic activation, is identified as the minimum effec-
tive concentration (MEC) for that sample. The mutagenicity of the sample
is evaluated as high (H), moderate (M), low (L), or nondetectable (ND)
according to the evaluation criteria developed in the Level 1 manual1
and summarized below. Samples with no detectable activity at the maximum
applicable dose (MAD) are ranked nondetectable (ND).
Ames Assay Mutagenicity Ranking Criteria1
Mutagenic
Activity
High (H)
Moderate (M)
Low (L)
Not Detectable (ND)
Solids
(MEC in ug/plate)
<50
50-500
500-5000
>5000
Liquids3
(MEC in ul/plate)
<2
2-20
20-200
>200
Concentration of organic extracts is based upon organic content (pg
orgamcs per plate) and not volume (ul extract per plate) of sample
tested.
Litton
5-304
BIONETICS
13
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VIII. REFERENCES
1. Brusick, D.J., et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment ITological Tests.EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD, August
1981, 52 pp.
4. 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.
5. Ames, B.N., Gurney, E.G., Miller, J.A. and Bartsch, H.: Carcinogens
as frameshift mutagens: Metabolites and derivatives of 2-acetylamino-
fluorene and other aromatic amine carcinogens. Proc. Nat. Acad.
Sci., USA 69:3128-3132, 1972.
6. 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.
7. 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.
8. 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.
9. 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.
10. Vogel, H.J. and Bonner, D.M.: Acetylornithinase of E. coli partial
purification and some properties. J. Biol. Chem., 218:9T:I06, 1966.
Litton
5-305
BIONETICS 14
-------
GENETICS ASSAY NO.:
LBI SAFETY NO.:
CYTOTOXIC EVALUATION OF
A8P05-030-674
(EA-2 1+FILTER)
TFTHE RABBIT
ALVEOLA! MATROPHAGE (RAM)
CTfOTOXICITY ASSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
m
Utton
5-306
BIONETICS
-------
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the rabbit
alveolar macrophage (RAM) cytotoxicity assay as described in "IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests" (1).
The data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting" (2).
The RAM cytotoxicity assay has been shown to be a sensitive method for
detecting cytotoxic activity for a variety of chemicals representing
various chemical classes (3). This assay is one of several recommended
by EPA to identify, categorize and rank the pollutant potential of influent
and effluent streams from industrial and energy-producing processes.
This assay has been well validated with a wide range of positive and
negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepara-
tion, testing and evaluation of the test material shall conform to Litton
Bionetics, Inc. standard operating procedures and the Good Laboratory
Practices Regulations of 1979. Deviations from standard procedure shall
be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
Litton
5-307
BIONETICS
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TABLE OF CONTENTS
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
PREFACE . .
ASSAY SUMMARY
OBJECTIVE
TEST MATERIAL
A. Description
B. Handling and Preparation . . .
MATERIALS
A. Indicator Cells
B. Media
C. Negative Controls
EXPERIMENTAL DESIGN
A. Procurement of Cells
B. Sample Forms
C. Dose Selection
D. Treatment
E. Cell Viability Assay
F. ATP Assay
ASSAY ACCEPTANCE CRITERIA
RESULTS
A. Interpretation
B. Tables and Figures
ASSAY EVALUATION CRITERIA
REFERENCES
Page No.
i
1
2
. . „ 3
3
3
4
4
4
4
5
5
5
6
6
6
7
8
9
9
9
13
14
m
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5-308
BIONETICS . ii
-------
I. ASSAY SUMMARY
A. SPONSOR: Acurex Corporation
B. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5884
1. Identification: A81-05-030-674 (EA-2 1+Filter)
2. Date Received: August 26, 1981
3. Physical Description: Fine, gray/black powder and fiberglass
filter with embedded particulate material.
C.
D.
E.
F.
G.
TYPE OF ASSAY: Rabbit Alveolar Macrophage (RAM) Cytotoxicity Assay
ASSAY DESIGN NUMBER: 443
STUDY DATES:
1. Initiation: October 1, 1981
2. Completion: October 14, 1981
SUPERVISORY PERSONNEL:
1. Study Director: Brian Myhr, Ph.D.
2. Laboratory Supervisor: Robert Young, M.S.
EVALUATION:
The combined particulate material from the filter and 1 micron catch
caused a dose-related increase in toxicity for applied concentrations
greater than approximately 20 ug/ml. All four assay parameters
were responsive, but the primary effect was the reduction in cellular
ATP content. The EC50 for the ATP content was 77 ug/ml, which
resulted in an evaluation of moderate (M) toxicity for the combined
particulate catch, using the toxicity categories defined for the
IERL-EPA Level 1 RAM Cytotoxicity Assay.
Submitted by:
Study Director
P f M/JJA
, Ph.U).
M
BrTan Myhr, Ph.ID. Date
Associate Director,
Department of Molecular
Toxicology
5-309
j; Brusick, Ph.D.
Director,
Department of Molecular
Toxicology
Date
Utton
BIONETICS
-------
II. OBJECTIVE
The objective of this study was to determine and rank the cytotoxicity
of A81-05-030-674 (EA-2 1+filter) to cultured rabbit alveolar macrophage
(RAM) cells. The measure of cytotoxicity was the reduction in cell
viability and adenosine triphosphate (ATP) content of the cultures after
a 20 hour exposure to the test material. At the conclusion of the exposure
period, the number of viable cells and total ATP content in the treated
cultures were compared to the corresponding values in unexposed control
cultures. The concentration of test material that reduced each experi-
mental parameter by 50% was estimated graphically and referred to as the
EC50 value. Standard EPA Level 1 toxicity evaluation criteria for the
RAM cytotoxicity assay were used to rank the toxicity potential of the
test material based upon the most sensitive parameter.
m
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5-310
BIONETICS
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III. TEST MATERIAL
A. Description
The test material, as received, was comprised of two separate components.
The first component, a fine black/gray powder, was the 1 Mi" SASS train
particulate catch. The second component was a fiberglass filter with
embedded participate material. This dark material represented particu-
lates less than 1 urn collected in the SASS train sample. Both components
were supplied together in a Nalgene screw-top bottle.
B. Handling and Preparation
The test material was received on August 26, 1981, and was assigned LBI
assay number 5884 and LBI safety number 7168. The sample was stored at
+4°C in the dark.
The filter portion of the sample required removal of the embedded particu-
lates before testing could begin. The uncut filter was sonicated in
cyclohexane as recommended by current IERL-EPA pretest sample preparation
procedures1. The decanted particulate suspensions from three successive
sonication treatments were combined and evaporated to dryness. The parti-
culate residue was weighed and combined with the 1 urn particulate catch
portion of the sample. A total of 264.42 mg of combined test material
was available for testing and was comprised of 70.28,mg (26.6%) of <1 pm
particulates removed from the filter and 194.14 mg (73.4%) of the 1 urn
catch.
Approximately 34.4 mg of test material was used for the assay. The test
material was suspended in serum-free EMEM culture medium at a concentration
of 2000 ug/ml and incubated at 37°C on a roller drum for 8 hours. A
fine suspension was formed that settled on standing. No pH changes were
noted. The suspension was serially diluted with EMEM (serum-free) and
applied to the cultures at a maximum concentration of 1000 jjg/ml in the
presence of 10% serum.
Litton
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BIONETICS
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m
Utton
IV. MATERIALS
A. Indicator Cells
The assay employed short-term primary cultures of alveolar macrophage
cells obtained by lung lavage of a male New Zealand white rabbit (2.25 kg).
The rabbit was maintained on Purina Lab Rabbit Chow 5321 and water ad
libitum and was examined for the absence of respiratory illnesses prior
to use.
B. Media
The cells were maintained and treated in Eagle's Minimum Essential Medium
(EMEM) with Earle's salts and supplemented with 10% fetal bovine serum
(heat-inactivated), 100 units/ml penicillin, 100 ug/ml streptomycin,
17.6 M9/ml kanamycin, and 0.4 ug/ml amphotericin B.
C. Negative Controls
The negative control consisted of three untreated cultures carried through
the same experimental time period as the treated cells. The average
viability and ATP content of the negative control provided the reference
points for determining the effects of different concentrations of the
test material on the assay parameters.
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Litton
V. EXPERIMENTAL DESIGN
A. Procurement of Cells
®
A rabbit was sacrificed by injection of Nembutal (60 mg/ml) into the
marginal ear vein, and sterile operating techniques were used to perform
a tracheostomy. Prewarmed normal saline (30 ml) was then introduced
into the lungs via a catheter and allowed to stand for 15 minutes. This
lavage fluid was removed and placed into a 50-ml sterile centrifuge tube
on ice. Nine additional lavages were similarly performed and collected,
except the saline was removed shortly after its introduction into the
lungs. Any lavage fluid containing blood or mucous was discarded. The
lavages were centrifuged at 365 x g for 15 minutes and the cells resus-
pended in cold 0.85% saline. After two washes in saline by centrifugation,
the cell pellets were resuspended in cold EMEM containing 20% serum and
then combined. A cell count was obtained by hemocytometer and the suspen-
sion diluted to 5.02 x 105cells/ml. Viability was determined by trypan
blue staining and the cells were not used if less than 95% viable. Also,
a differential cell count from Wright-stained smears was performed to
verify that the macrophage content was above 90%.
B. Sample Forms
The usual sample form for application to the cells is a suspension of
particulate material. Solid samples are ground to fine particles and a
weighed portion is suspended in a known volume of EMEM (0% FBS) for about
eight hours to help leach any water-soluble material. Finely-divided
test material may be suspended directly in culture medium without further
grinding. Aqueous liquids, suspensions, or slurries containing less
than 0.5% organic solvent are added by volume to culture medium.
Samples supplied as solutions in organic solvents are usually solvent-
exchanged into DMSO before testing. Original sample volumes may first
be reduced a maximum of 10-fold in a Kuderna-Danish concentrator, and
the concentrative factor is used to convert assayed volumes into equi-
valent original sample volumes in the absence of information about solute
concentration. An aliquot of the reduced volume is exchanged into OMSO
by repeated, partial evaporation under a stream of nitrogen in a warm
water bath (50°C); the evaporated volumes are replaced with equal volumes
of DMSO.
Samples adsorbed on XAD-2 resin are extracted with methylene chloride
or acetone in a Soxhlet apparatus for 24 hours. The extract is then
concentrated and solvent-exchanged into DMSO. Alternatively, acetone
extracts can be assayed directly at concentrations up to 2% by volume in
the culture medium.
Samples impregnated on fiber glass or teflon filters are repeatedly soni-
cated in cyclohexane to remove particulates. The resulting cyclohexane
particulate suspension is then evaporated to dryness and the particulates
resuspended in EMEM culture medium at the desired concentration.
5-313
BIONET1CS
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Sponsor-specified handling of sample materials will be followed if the
above procedures are not applicable or a specific procedure is desired.
C. Dose Selection
Unless the approximate toxicity is already known or the sample size is
limiting, the following usual dose ranges are tested for different sample
forms. Dry, particulate material is tested at six dose levels from
1000 ug/ml to 3 ug/ml. Aqueous samples, suspensions, or slurries are
tested from 600 ul to 3 ul/ml in six dose steps. Samples that are solvent-
exchanged into DMSO are tested from 20 ul/ml (2% DMSO in growth medium)
to 0.2 pi/ml, also in six dose steps. A second dose study is performed
with an adjusted dose range if the EC50 was not located properly in the
initial test. However, EC50 values greater than 1000 ul/ml for particu-
late material, 600 ul/ml for aqueous samples, or 20 ul/ml for organic
solutions will not be determined.
This test material, A81-05-030-674 (EA-2 1+filter), was tested at 6 dose
levels, starting at the maximum applicable dose (MAD) of 1000 ug/ml and
including 600, 300, 100, 60 and 30 ug/ml.
D. Treatment
A series of 25 cm2 culture flasks were prepared, each containing 2.0 ml
of serum-free medium at 37°C and the test material at, twice the desired
final concentration. Three flasks were prepared for each test concen-
tration. Aliquots of cell suspension (2 ml) were then added; each flask,
therefore, contained 1 x 106 viable cells in a 4-ml volume of media con-
taining 10% serum. The flasks were placed on a rocker platform in a
37°C incubator with a humidified atmosphere containing 5% C02. After
sitting for about 30 minutes, the flasks were slowly rocked for the
remainder of a 20-hour exposure period.
If the test substance causes a color change in the growth medium, the pH
is determined in additional treated flasks. After the exposure period,
the pH of the medium in the experimental flasks is again recorded.
E. Cell Viability Assay
At the end of the treatment period, the medium containing unattached
cells was decanted into a centrifuge tube on ice. The attached cells
were rinsed with 1 ml of 0.1% trypsin/0.01% versene and then incubated
with 2 ml of the trypsin/versene solution for about 5 minutes at 37°C.
The trypsinates and decanted media were combined for each culture to
yield a 7-ml cell suspension for subsequent analysis.
A 1.0 ml aliquot of the cell suspension was removed for cell count and
viability determination. The aliquot was combined with 1.0 ml of 0.4%
trypan blue and counted by hemocytometer about 5 to 15 minutes later.
The total number of cells counted per culture was the sum of the numbers
found in five squares for each chamber of the hemocytometer (1 ul total
volume). The numbers of live (colorless) and dead (blue) cells were
recorded.
5-314 •
BIONETICS c
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F. ATP Assay
ATP was immediately analyzed by extraction of a 0.1-ml sample of cell
suspension with 0.9 ml of 90% DMSO. After 2 minutes at room temperature
5.0 ml cold MOPS buffer (0.01 M morpholinopropane sulfonic acid) at pH 7.4
was added and the extract mixed well and placed on ice. Aliquots of
10 pi were injected into a cuvette containing a luciferin-luciferase
reaction mixture in a DuPont Model 760 Luminescence Biometer. The Biometer
was calibrated daily with standard ATP solutions to provide- a direct
read-out of the ATP content. Each test sample was assayed at least twice
to obtain repeatable readings.
Litton
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VI. ASSAY ACCEPTANCE CRITERIA
The assay will be considered acceptable for evaluation of the test results
if the following criteria are met:
1. The macrophage population is 90% or greater of the total nucleated
cells collected by lung lavage.
2. The percent viability of the macrophages used to initiate the assay
is 95% or greater.
3. The survival of viable macrophages in the negative control cultures
over the 20 hour treatment priod is 70% or greater.
4. A sufficient number of data points (for five test concentrations or
less) are available to clearly locate the EC50 of the most sensitive
test parameter within a toxicity region as defined under Assay Eval-
uation Criteria.
5. The data points critical to the location of the EC50 for the most
sensitive parameter are the averages of at least two treated cultures.
6. If all the test parameters yield EC50 values greater than 1000 ug/ml,
600 pi/ml for aqueous solutions, or 20 u]/ml for organic solutions,
the plotted curves for ATP content and viability index parameters
do not exceed 120% of the negative control.
IJtton
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BIONETICS 8
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VII. RESULTS
A. Interpretation
The test material was ingested by the macrophages and caused a decline
in their viability when the applied concentration exceeded approximately
20 |jg/ml. Absolute values for the assay parameters are given in Table 1,
as well as the parameter values relative to the negative control cultures,
and the relative values are plotted in Figures 1 and 2.
The most sensitive assay parameter was the culture ATP content, which
yielded an EC50 of 77 ug/ml. This reduction in ATP was also reflected in
the ATP/106 total cells parameter, which paralled the culture ATP curve
but was somewhat less sensitive (EC50 = 140 ug/ml). The ATP/106 cells
measurement normally lags the ATP measurement because cellular disruption
reduces the denominator of this parameter. In order for ATP/106 cells
to be very responsive, the percent viability must decrease and the
viability index (which measures the total number of viable cells) must
not decrease as rapidly as the total ATP. As shown in Figure 1, the
percent viability did decrease (EC50 just above the MAD of 1000 ug/ml)
and the viability index declined with a more shallow slope than the ATP
and leveled off near 40% of the negative control. Therefore, the primary
effect of the combined particulate sample was to cause a drop in cellular
ATP content and secondarily, a disruption of the macrophages. This toxi-
city was clearly evident in the low toxicity range of 100-1000 ug/ml, as
defined for the IERL-EPA Level 1 RAM assay1. However, the inhibition
began in the moderate region of 10-100 ug/ml for these parameters, and
the ATP EC50 of 77 ug/ml resulted in an evaluation of moderate (M) toxi-
city for the test material. Although this response by the RAM cells
closely approached the moderate/low toxicity borderline, the ATP EC50
would be expected to usually remain in the moderate region for repeated
trials.
The macrophages collected for this assay had normal morphology and appeared
to be in a healthy state. The initial viability was excellent (99.3%)
and the survival of viable cells in the negative control was 96.0%. The
average cellular ATP content of the negative controls was 25.1 x 108 fg
ATP per 10s total cells which was within the historical range for accept-
able cultures. These results achieved the assay acceptance criteria and
provided confidence in the assumption that the collected data represented
typical responses to the test material.
B. Tables and Figures
This report is based on the data provided in Table 1 and Figures 1 and
2.
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TABLE 1
RABBIT ALVEOLAR MACROPHAGE (RAM) CYTOTOXICITY ASSAY DATA
LBI Assay No. : 5884
Test Material Identity: A81-05-030-674 (EA-2 1+Filter)
Test Date: October 13. 1981
Vehicle:
Sample
en
£ NCC
CO
TEST
TEST
TEST
TEST
TEST
TEST
EMEM
Concentration9
(jg/ml
30
60
100
300
600
1000
Initial Cell Viability: 99.3%
Viable Macrophage Seeded/Flask: 1.0 x 106 cells/flask
Macrophage Population Percentage: >90.0%
Survival of Negative Control
Macrophage Over Treatment Time: 96.0%
Average Values
Viable Cells
106 Units
0.97
0.85
0.67
0.60
0.44
0.44
0.35
per Culture
Total Cells
10B Units
1.01
0.91
0.72
0.68
0.58
0.70
0.61
Flask
ATP .
108fgb
25.4
18.7
14.7
10.7
3.7
2.7
2.1
ATP Per
106 Cells
108 fg
25.1
20.5
20.4
15.7
6.4
3.9
3.4
Viability
96.0
93.4
93.1
88.2
75.9
62.9
57.4
Expressed
Viability
100.0
97.3
97.0
91.9
79.1
65.5
59.8
as Percent
Viability
Index
100.0
87.6
69.1
61.9
45.4
45.4
36.1
of Negative Control
ATP
100.0
73.6
57.9
42.1
14.6
10.6
8.3
ATP Per
106 Cells
100.0
81.7
81.3
62.5
25.5
15.5
13.5
apH change in culture medium: None observed
fg = Femtogram (10-15 gram).
CNC = Negative Control. EMEM culture medium.
Determined from data plots in Figures 1 and 2.
EC50 VALUES:
>1000
210
77
Toxiclty
Classification: Moderate
140
-------
FIGURE 1
EC50 DETERMINATION FOR
PERCENT VIABILITY (0) AND VIABILITY INDEX (•)
A81-05-030-674
(EA-2 1+FILTER)
1000
CONCENTRATION, JJG/ML
5-319
11
-------
FIGURE 2
EC50 DETERMINATION FOR
ATP/FLASK (0) AND ATP/106 CELLS (•)
AST-05-030-674
(EA-2 1+FILTER)
3
LU
10 100
CONCENTRATION, JJG/ML
1000
5-320
12
-------
VIII. ASSAY EVALUATION CRITERIA
The EC50 value represents the concentration of test material that reduces
the most sensitive parameter of the RAM assay to 50% of the vehicle or
negative control value. EC50 values are determined graphically by fitting
a curve by eye through relative toxicity data plotted as a function of
the logarithm of the applied concentration. Each data point normally
represents the average of three culture dishes. Statistical analysis is
unnecessary in most cases for evaluation.
The toxicity of the test material is evaluated as high, moderate, low,
or nondetectable according to the range of EC50 values defined in the
following table.
Solids
Toxicity (EC50 in ug/ml)
High <10
Moderate 10 to 100
Low 100 to 1000
Not Detectable >1000
Aqueous Liquids
(EC50 in Ml/ml)
<6
6 to 60
60 to 600
>600
Nonaqueous Liquids"
(EC50 in Ml/ml)
<0.2
0.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Test?1.
Criteria for nonaqueous liquids are tentative and under evaluation. If
the organic or solid content is known, the solid evaluation criteria
are applied.
Another evaluation scheme is proposed for extracts obtained from SASS
train gas volumes. The proportion of the total gas volume corresponding
to the volume of extract used in the bioassay is calculated and expressed
as I/ml of culture medium (or DSCF/ml of culture medium). A criterion
of 1000 L/ml is set as the limit for nondetectable toxicity. This gas
volume corresponds to the average volume breathed by humans over a 2-hour
period. The subsequent toxicity ranges are defined by 10-fold dilution
steps to conform to standard procedure. The toxicity ranges are defined
in the following table for liter and dry standard cubic feet units:
EC50 In : EC50 In
Toxicity Liters/ml (L/ml) Dry Standard Cubic Feet/ml (DSCF/ml)
igh < <0.35 DSCF
Moderate 10-100 0.35-3.5
Low 100-1000 3.5-35
Nondetectable >1000 >35
Utton
5-321
BIONETICS 13
-------
IX.
1.
REFERENCES
Brusick, D.J., et a_K: IERL-RTP Procedures Manual:
mental Assessment BiologTcaT
Level 1 Environ-
2.
3.
rests. EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington,
MD, September 1980, 177 pp. In press.
Brusick, D.J.: Level 1 Bjoassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc., Kensington, MD, April 1980,
100 pp.
Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD, August
1981, pp. 52.
ffl
Litton
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BIONETICS
14
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GENETICS ASSAY NO.: 5880
LBI SAFETY NO.: 7164
MUTAGENICITY EVALUATION OF
A81-05-030-676
(EA-2 XAD EXTRACT)
IFTHl
EPA~LEVEL 1
AMES SAlfTONFLTATMTCROSOME
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 20895
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
5-323
, BIONETICS
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ffi
Litton
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Ames
Salmonella/microsome mutagenesis assay as described in "IERL-RTP Proce-
dures Manual: Level 1 Environmental Assessment Biological Tests"1. The
data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting"2.
The Ames Salmonella/microsome mutagenesis assay has been shown to be a
sensitive method for detecting mutagenic activity for a variety of chemi-
cals representing various chemical classes3. This assay is one of several
recommended by EPA to identify, categorize and rank the pollutant potential
of influent and effluent streams from industrial and energy-producing pro-
cesses. This assay has been well validated with a wide range of positive
and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good Labora-
tory Practices Regulations of 1979. Deviations from standard procedure
shall be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
5-324
BIONETICS
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Litton
TABLE OF CONTENTS
Page No.
PREFACE i
I. ASSAY SUMMARY 1
II. OBJECTIVE 2
III. TEST MATERIAL 3
A. Description 3
B. Handling and Preparation 3
IV. MATERIALS 4
A. Indicator Microorganisms 4
B. Media 4
C. Activation System 5
1. S9 Homogenate 5
2. S9 Mix 5
V. EXPERIMENTAL DESIGN 6
A. Dose Selection 6
B. Mutagenicity Test 6
1. Nonactiyation Assay . 6
2. Activation Assay 6
C. Control Compounds 7
D. Recording and Presenting Data 7
VI. RESULTS 9
A. . Interpretation 9
B. Tables 9
VII. EVALUATION CRITERIA 11
A. Surviving Populations 11
B. Dose-Response Phenomena 11
C. Control Tests 11
D. Evaluation Criteria for Ames Assay 12
1. Strains TA-1535 and TA-1537 12
2. Strains TA-98 and TA-100 12
3. Pattern 12
4. Reproducibility 12
E. Relation Between Mutagenicity and
Carcinogenicity 13
F. Criteria for Ranking Samples in the Ames Assay . . 13
VIII. REFERENCES 14
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BIONETICS 11
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I. ASSAY SUMMARY
A. Sponsor: Acurex Corporation
B. Material (Test Compound): Genetics Assay Number: 5880
1. Identification: A81-05-030-676 (EA-2 XAD Extract)
2. Date Received: August 26, 1981
3. Physical Description: Clear, gold liquid.
C. Type of Assay: EPA Level 1 Ames Salmonella/Microsome Plate Test
D. Assay Design Number: 401 (EPA Level 1)
E. Study Dates:
1. Initiation: September 23, 1981
2. Completion: October 5, 1981
F. Supervisory Personnel:
A. Study Director: D.R. Jagannath, Ph.D.
G. Evaluation:
The test material, A81-05-030-676 (EA-2 XAD extract), contained
2.5 mg organics per ml after solvent exchange into di methyl -
sulfoxide (DMSO). The solvent exchanged sample was evaluated
for its genetic activity in the EPA Level 1 Ames assay, directly
and in the presence of a metabolic activation system. The
test sample exhibited mutagenic activity with TA-98 and TA-100
in the presence and absence of S9 mix. The minimum effective
concentration at which the mutagenic activity was observed was
at 10 ul per plate (or 25 ug organics per plate) with TA-98 in
the nonactivation assay. These tests indicate that the test
material contains both frame shift and base-pair type mutagens.
The mutagenic activity of the sample was classified as high (H)
according to the IERL-EPA Level 1 evaluation criteria1.
Submitted by:
Study Director
Reviewed by:
Jagannath, P+rrD.
Section Chief,
Submammalian Genetics,
Department of Molecular
Toxicology
ate
David J.
Director,
Department of Molecular
Toxicology
m
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BIONETICS
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II. OBJECTIVE
The objective of this study was to determine the .genetic activity of
A81-05-030-676 (EA-2 XAD extract) in the Salmonella/ microsome assay
with and without the addition of mammalian metabolic activation prepara-
tions. The genetic activity of a sample is measured in these assays by
its ability to revert the Salmonella indicator strains from histidine
dependence to histidine independence. The degree of genetic activity of
a sample is reflected in the number of revertants that are observed on
the histidine-free medium.
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III. TEST MATERIAL
A. Description
The test material was received as a clear, gold solution in methylene
chloride. The sample contained 9.0 milligrams of organic material in an
undetermined volume of methylene chloride. No information on the sampling
parameters (such as the equivalent volume of stack gas represented by
the sample) was provided.
B. Handling and Preparation
The test material was received at LBI on August 26, 1981. The sample
was assigned LBI safety number 7164 and LBI assay number 5880. The sample
was stored at +4°C in the dark.
Pretest sample preparation consisted of solvent exchanging the sample
into dimethylsulfoxide (DMSO). The sample was transferred with methylene
chloride rinses into a graduated conical tube. The methylene chloride
was gradually evaporated (50°C under a stream of nitrogen) and DMSO was
sequentially added. The sample was brought to volume in 3.6 ml of DMSO,
giving a sample concentration of 2.5 mg organics per ml DMSO. The sample
was transferred to a glass vial and sealed with a teflon-coated rubber
septum.
Approximately 2.6 ml of test material was used for testing. Varying
aliquots of the test material were added directly to the test mixtures
to give the desired concentration.
LJtton
5-328
BIONETICS
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IV.
MATERIALS
A.
Indicator Microorganisms
The Salmonella typhimurium strains used in this assay were obtained from
Dr. Bruce Ames, University of California at Berkeley.4-8 The following
four strains were used.
Strai n
Designation
Gene Additional Mutations
Affected RepairLP5R Factor
Mutation Type
Detected
TA-1535
TA-1537
TA-98
TA-100
his G A uvr B rfa
his C A uvr B rfa
his D A uvr B rfa pKMlOl
his G A uvr B rfa pKMlOl
Base-pair
substitution
Frameshift
Frameshi ft
Base-pair
substitution
All the above strains have, in addition to the mutation in the histidine
operon, mutation (rfa-) that leads to defective lipopblysaccharide coat,
a deletion that covers genes involved in the synthesis of vitamin biotin
(bio-) and in the repair of ultraviolet (uv) - induced 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 physi-
cally 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 mutagens.8
In addition, plasmid pKMlOl confers resistance to the antibiotic ampi-
cillin, 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 supplemented
with a trace of biotin and an excess of histidine. In addition, the
plates with plasmid-carrying strains contain ampicillin (25 ug/ml) to
ensure stable maintenance of plasmid pKMlOl. New stock culture plates
are made as often as necessary from the frozen master cultures or from
single colony reisolates that were checked for their genotypic character-
istics (his, rfa uvrB, bio) and for the presence of plasmid. For each
experiment, an inoculunfTrom the stock culture plates is grown overnight
at 37°C in nutrient broth (Oxoid CM67) and used.
B.
Media
The bacterial strains were cultured in Oxoid Media #2 (Nutrient Broth).
The selective medium was Vogen Bonner Medium E with 2% glucose.10 The
5-329
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BIONETICS
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overlay agar consisted of 0.6% purified agar with 0.05 mM histidine,
0.05 mM biotin and 0.1M NaCl according to the methods of Ames et a±,
C.
1.
Activation System
S9 Homogenate
A 9,000 x 2 supernatant prepared from Sprague-Dawley adult male rat liver
induced by Aroclor 1254 (Ames et ah9) was purchased commercially and
used in these assays.
2. S9 Mix
S9 mix used in these assays consisted of the following components:
Components
Concentration per Milliliter
S9 Mix
NADP (sodium salt)
D-glucose-6-phosphate
MgCl2
KC1
Sodium phosphate buffer
pH 7.4
Organ homogenate from rat
liver (S9 fraction)
4 umoles
5 umoles
8 umoles
33 umoles
100 umoles
100 Milters
CH
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BIONETICS
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Litton
V. EXPERIMENTAL DESIGN
A. Dosage Selection
Test strategy and dose selection depend upon sample type and sample avail-
ability. The Level 1 manual1 recommends solids to be initially tested
at the maximum applicable dose (MAD) of 5 mg per plate and at lower con-
centrations of 2.5, 1, 0.5, 0.1 and 0.05 mg per plate. Liquids are tested
initially at the MAD of 200 ul per plate, and at lower concentrations of
100, 50 and 10 ul per plate. Samples are retested over a narrower range
of concentrations with strains showing positive results initially. Alter-
nate dose are employed if sample size is limiting or at the direction of
the sponsor.
Doses selected to test this sample covered the recommended dose range
for liquids. The highest dose was at the MAD level of 200 ul per plate
and included three lower dose levels of 100, 50 and 10 ul per plate.
These dose levels corresponded to 500, 250, 125, and 25 ug orgaincs per
plate.
B. Mutagenicity Testing
The procedure used was based on the paper published by Ames et. aj.9 and
was performed as follows:
1. Nonactivation Assay
To a sterile 13 x 100 mm test tube placed in a 43°C water bath the fol-
lowing was added in order:
2.00 ml of 0.6% agar containing 0.05 mM histidine and
0.05 mM biotin.
0.01 ml to 0.2 ml of a solution of the test chemical to
give the appropriate dose.
0.1 ml to 0.2 ml of indicator organism(s).
0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured onto minimal agar plates
(see IV B, Media). After the top agar had set, the plates were incubated
at 37°C for approximately 2 days. The number of his+ revertant colonies
growing on the plates were counted with an automatic colony counter and
recorded.
2. Activation Assay
The activation assay was run concurrently with the nonactivation assay.
The only difference was the addition of 0.5 ml of S9 mix (see IV C, Acti-
vation System) to the tubes in place of 0.5 ml of phosphate buffer which
was added in nonactivation assays. All other details were similar to
the procedure for nonactivation assays.
5-331
BIONETICS 6
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A detailed flow diagram for the plate incorporation assay is provided in
Figure 1.
C. Control Compounds
A negative control consisting of the solvent used for the test material
was also assayed concurrently with the test material. For negative con-
trols, step V of Nonactivation Assays was replaced by 0.05 ml of the
solvent. The negative controls were employed for each indicator strain
and were 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 were
made using this solvent. The amount of solvent used was equal to the
maximum volume used to give the appropriate test dose.
Specific positive control compounds known to revert each strain were also
used and assayed concurrently with the test material. The concentrations
and specificities of these compounds to specific strains are given in
the following table:
Assay
Nonactivation
Chemical
Sodium azide
2-Nitrofluorene
(NF)
9-aminoacridine
(9AA)
Concentratio
per plate
Solvent (ug)
Water
D i methyl -
sulf oxide
Ethanol
10.0'
10.0
50.0
n
Salmonella
Strains
TA-1535,
TA-98
TA-1537
TA-100
Activation
2-anthramine
(ANTH)
Dimethyl-
sulfoxide
2.5
For all strains
0.
Recording and Presenting Data
The number of colonies on each plate were counted and recorded on printed
forms. These raw data were analyzed in a computer program and reported
on a printout. The results are presented as revertants per plate for
each indicator strain employed in the assay. The positive and solvent
controls are provided as reference points.
ffi
Litton
5-332
BIONETICS
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AMES ASSAY [PLATE INCORPORATION METHOD]
Molten [45*C] overlay agar
appropriately supplemented
Test, positive or solvent
control chemical
0.1 ml
Aliquot of an overnight culture
of bacterial 10$ cells/ml]
Aliquot of
saline
0.5 ml
-S-9
0.5 ml S-9 mix [hepatic
S-9 —— homogenate from PCB
pretreated rat plus
necessary cofactors
Overlay poured on selective
bottom agar medium
Plated incubated at 37*C for 48 hours
The numbers of revertants/plate counted
Data analyzed
Interpretation/Conclusion
Figure 1 AMES SALMONELLA/MICROSOME MUTAOENESIS ASSAY
5-333
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Litton
VI. RESULTS
A. Interpretations
The test material, A81-05-030-676 (EA-2 XAD extract), in methylene chloride
was solvent exchanged to DMSO before conducting the EPA Level 1 Ames
assays. The solvent exchanged material was tested directly and in the
presence of liver microsomal enzymes from Aroclor induced rats. Due to
the limited amount of the test sample, only TA-98 and TA-100 were used
in the assays. Tests were conducted in duplicate except for TA-100 with
activation, where only one plate per dose was used.
The results of the tests conducted on the sample in the absence of a
metabolic activation were positive with both TA-98 and TA-100.
The results of the tests conducted on the sample in the presence of a
rat liver activation system were positive with TA-98 and TA-100.
These results indicate that the test sample contains direct acting frame
shift and base-pair type of mutagens. The minimum effective concentration
(MEC) that exhibited mutagenic activity was at 10 pi per plate (or 25 ug
organics per plate) with TA-98 in the nonactivation assays. This response
was categorized as high (H) mutagenic activity using the IERL-EPA Level 1
evaluation critiera for the Ames Assay1.
Solvent control and positive control values were within acceptable ranges.
These results achieved assay acceptance criteria and provided confidence
in the assumptions that the recorded data represented typical responses
to the test material.
B. Tables
This report is based on the data provided in Table 1.
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BIONETICS
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RESULTS
TABLE 1
A. NAME OR CODE DESIGNATION OF THE TEST CONPCUNCt A81-05-030-676 1EA-2 KAO EXTRACT!
B. SOLVENT: OHSO
C. TEST INITIATION DATES: 10/01/81
0. TEST COMPLETION DATE: 10/05/81
E. S-9 LOT*: REF050
NOTE: CONCENTRATIONS ARE GIVEN IN NICROLITERS FER PLATE
TEST
NONACTIVATION
REVERTANTS FER
SPECIES TISSUE TA-98 TA-100
12! 123
F L A T E
cn
I
to
co
cn
SOLVENT CONTROL
POSITIVE CONTROL"
TEST COMPOUND
10.00 UL
90.00 UL
100.00 UL
200.00 UL
ACTIVATION
30 20
760 814
£8
174
217 186
301 260
134 1
-------
VII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA
Statistical methods are not currently used, and evaluation is based on
the criteria included in this protocol.
Plate test data consists of direct revertant colony counts obtained from
a set of selective agar plates seeded with populations of mutant cells
suspended in a semisolid 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 semiquanti-
tative in nature. Although these features of the assay reduce the quanti-
tation of results, they provide certain advantages not contained in a
quantitative suspension test:
The small number of cell divisions permits potential
mutagens to act on replication DNA, which is often more
sensitive than nonreplicating 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 quantisation 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 concen-
trations, the highest of these doses being selected to show slight toxicity
as determined by subjective criteria.
B. Dose-Response Phenomena
The demonstration of dose-related increased in mutant counts is an impor-
tant criterion in establishing metagenicity. A factor that might modify
dose-response results for a mutagen would be the selection of doses that
are too low (usually mutagenicity and toxicity are related). If the
highest dose is far lower than a toxic concentration, 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.
C. Control Tests
Positive and negative control assays were conducted with each experiment
and consisted of direct-acting mutagens for nonactivation assays and
mutagens that require metabolic biotransformation in activation assays.
EH
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5-336
BIONETICS 11
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Negative controls consisted of the test material solvent in the overlay
agar together with the other essential components. The negative control
plate for each strain gave a reference point to which the test data was
compared. The positive control assay was conducted to demonstrate that
the test systems were functional with known mutagens.
The following normal range of revertants for solvent controls are generally
considered acceptable.
TA-1535: 8-30
TA-1537: 4-30
TA-98: 20-75
TA-100: 80-250
D. Evaluation Criteria for Ames Assay
Because the procedures to be used to evaluate the mutagenicity of the
test material are semiquantitative, the criteria to be used to determine
positive effects are inherently subjective and are based primarily on a
historical data base. Most data sets will be evaluated using the following
criteria.
1. Strains TA-1535 and TA-1537
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to three times the solvent'control value will
be considered to be mutagenic.
2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to twice the solvent control value for TA-98
and TA-100 will be considered to be mutagenic.
3. Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain
(G-46), 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 that cannot be
reproduced in additional runs, the initial positive test data lose signi-
ficance.
The preceding criteria are not absolute, and other extenuating factors
may enter into a final evaluation decision. However, these criteria
will be applied to the majority of situations and are presented to aid
those individuals not familar with this procedure. As the data base is
increased, the criteria for evaluation can be more firmly established.
5-337
BIONETICS 12
Lrtton
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CB
Litton
E. Relation Between Mutaqenicity and Carcinoqenicity
It must be emphasized that the Ames Salmonella/Microsome Plate Assay is
not a definitive test for chemical carcinogens. It is recognized, however,
that correlative and functional relations have been demonstrated between
these two endpoints. The results of comparative tests on 300 chemicals
by McCann et aj.4 show an extremely good correlation between results of
microbial mutagenesis tests and jn 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 muta-
genic activity.
F. Criteria for Ranking Samples in the Ames Assay
The goal of EPA Level 1 Ames testing is to rank source streams by relative
degree of genetic toxicity (mutagenicity). Samples are first identified
as mutagenic or nonmutagenic by the criteria in Section D above and
then ranked using the mutagenicity categories presented in the table
below. The lowest concentration giving a positive response in any strain,
with or without metabolic activation, is identified as the minimum effec-
tive concentration (MEC) for that sample. The mutagenicity of the sample
is evaluated as high (H), moderate (M), low (L), or nondetectable (ND)
according to the evaluation criteria developed in the Level 1 manual1
and summarized below. Samples with no detectable activity at the maximum
applicable dose (MAD) are ranked nondetectable (ND). .'
Ames Assay Mutagenicity Ranking Criteria1
Mutagenic
Activity
High (H)
Moderate (M)
Low (L)
Not Detectable (ND)
Solids
(MEC in jig/plate)
<50
50-500
500-5000
>5000
(MEC
<2
2-20
Liquids3
in (jl/plate)
20-200
>200
Concentration of organic extracts is based upon organic content (|jg
organics per plate) and not volume (ul extract per plate) of sample
tested.
5-338
BIONETICS 13
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VIII. REFERENCES
1. Brusick, D.J., et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment BTological Tests.EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics Inc., Kensington, MD, April 1980,
100 pp. ' •
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD, August
1981, 52 pp.
4. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N.: Detection of
carcinogens as mutagens in the SalmoneTIa/microsome test: Assay of
300 chemicals. Proc. Nat. Acad. Sci., USA 72:5135-5139, 1975.
5. Ames, B.N., Gurney, E.G., Miller, J.A. and Bartsch, H.: Carcinogens
as frameshift mutagens: Metabolites and derivatives of 2-acetylamino-
fluorene and other aromatic amine carcinogens. Proc. Nat. Acad.
Sci., USA 69:3128-3132, 1972.
6. 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.
7. 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.
8. 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.
9. 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.
10. Vogel, H.J. and Bonner, D.M.: Acetylornithinase of E. coli partial
purification and some properties. J. Biol. Chem., 2l8:WFlQ6t 1966.
Utton
5-339
BIONETICS
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GENETICS ASSAY NO.: 5880
LBI SAFETY NO.: 7164
CYTOTOXIC EVALUATION OF
A81-05-030-676
(EA-2 XAD EXTRACT)
IN THE
RODENT~ClLL (CHO)
CLONAL TOKTCTTTRSSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
Utton
5-340
BIONETICS
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PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Chinese
hamster ovary cell (CHO) clonal toxicity assay as described in "IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests" (1).
The data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting" (2).
The CHO clonal toxicity assay has been shown to be a sensitive method for
detecting cytotoxic activity for a variety of chemicals representing
various chemical classes (3). This assay is one of several recommended
by EPA to identify, categorize and rank the pollutant potential of
influent and effluent streams from industrial and energy-producing
processes. This assay has been well validated with a wide range of posi-
tive and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good
Laboratory Practices Regulations of 1979. Deviations from standard
procedure shall be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
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BIONETICS
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TABLE OF CONTENTS
Page No.
PREFACE
I. ASSAY SUMMARY
II. OBJECTIVE
III. TEST MATERIAL
A. Description
B. Handling and Preparation
IV. MATERIALS
A. Indicator Cells ....
B. Media
C. Controls ........
V. EXPERIMENTAL DESIGN
A. Dose Selection
B. Clonal Toxicity Assay .
VI. ASSAY ACCEPTANCE CRITERIA . .
VII. RESULTS
A. Interpretation
B. Tables and Figures . . .
VIII. ASSAY EVALUATION CRITERIA . .
IX. REFERENCES
i
1
2
3
3
3
4
4
4
5
5
7
8
8
8
11
12
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BIONETICS
5-342
n
-------
I. ASSAY SUMMARY
A. SPONSOR: Acurex Corporation
B. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5880
1. Identification: A81-05-030-676 (EA-2 XAD Extract)
2. Date Received: August 26, 1981
3. Physical Description: Clear, gold liquid
C. TYPE OF ASSAY: Rodent Cell (CHO) Clonal Toxicity Assay
D. ASSAY DESIGN NUMBER: 442
E. STUDY DATES:
1. Initiation: September 23, 1981
2. Completion: October 6, 1981
F. SUPERVISORY PERSONNEL:
1. Study Director: Brian C. Myhr, Ph.D.
2. Laboratory Supervisor: Robert Young, M.S.
G. EVALUATION:
The test material caused a slight increase in toxicity with
increasing concentrations up to 1.0 ul/ml. The relative survival
dropped to nearly zero at 3 ul/ml and was zero for doses of
6 ul/ml and above. The EC50 was estimated graphically to be
1.72 ul/ml which was equivalent to 4.3 ug of organics per ml.
This sample was therefore evaluated to be in the high (H) toxicity
category defined for the IERL-EPA Level 1 CHO clonal toxicity
bioassay.*
Submitted by:
Study Director
fo/vu^flt
Brian Myhr,
Associate Director,
Department of Molecular
Toxicology
Reviewed by:
ate
Javid J Brusick,
Director,
Department of Molecular
Toxicology
5-343
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BIONETICS
-------
II. OBJECTIVE
The objective of this study was to determine and rank the cytotoxicity
of A81-05-030-676 (EA-2 XAD extract) to cultured Chinese hamster cells
(CHO-K1 cell line). The measure of cytotoxicity was the reduction in
colony-forming ability after a 24-hour exposure to the test material.
After a period of recovery and growth, the number of colonies that
developed in the treated cultures was compared to the colony number in
unexposed vehicle control cultures. The concentration of test material
that reduced the colony number by 50% was estimated graphically and
referred to as the EC50 value. Standard EPA Level 1 toxicity evaluation
criteria for the CHO clonal toxicity assay were used to rank the toxicity
potential of the test material.
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BIONETICS
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III. TEST MATERIAL
A. Description
The test material was received as a clear, gold solution in methylene
chloride. The sample contained 9.0 milligrams of organic material in
1.0 ml of methylene chloride. No information on the sampling parameters
(such as the equivalent volume of stack gas represented by the sample)
was provided.
B. Handling and Preparation
The test material was received at LBI on August 26, 1981. The sample was
assigned LBI safety number 7164 and LBI assay number 5880. The sample was
stored at +4°C in the dark.
Pretest sample preparation consisted of solvent-exchanging the sample
into dimethylsulfoxide (DMSO). The sample was transferred with methylene
chloride rinses into a graduated conical tube. The methylene chloride
was gradually evaporated (50°C under a stream of nitrogen) and DMSO was
sequentially added. The sample was brought to volume in 3.6 ml of DMSO,
giving a sample concentration of 2.5 mg organics per ml DMSO. The sample
was then transferred to a glass vial and sealed with a teflon-coated rubber
septum.
A total volume of 0.45 ml of test sample was used in:the CHO assay. The
maximum concentration of 20 pi/ml was obtained by adding 0.12 ml of sample
to 5.88 ml of F12 medium; this resulted in 2% (v/v) DMSO in the medium
and effectively limited the concentration of test material that could be
assayed. Only two plates were dosed at the top dose in order to conserve
sample. Another 0.12 ml aliquot of sample was used to prepare the 10 ul/ml
test concentration. An additional 0.21 ml of test sample was used to
prepare a series of dilutions in DMSO from which 1:100 dilutions into
growth medium were performed to obtain the lower assayed concentrations.
Thus, except for the 20 ul/ml test concentration, the final DMSO concen-
tration was constant at 1% (v/v).
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BIONETICS
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IV. MATERIALS
A. Indicator Cells
The indicator cells for this study were Chinese hamster CHO-K1 cells
(ATCC No. CCL 61) obtained from Flow Laboratories, Inc., Rockville, MD.
This cell type was derived from ovarian tissue and has spontaneously
transformed to a stable, hypodiploid line of rounded, fibroblastic cells
with unlimited 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. Laboratory stock are maintained by
routine serial subpassage. Cells are cultivated in Ham's F-12 nutrient
medium at 37°C in 5 percent C02 with saturated humidity. Stocks are
continually observed macroscopically and microscopically for possible
microbial contamination. Laboratory cultures are periodically checked
by culturing and staining methods for the absence of mycoplasma contami-
nation. Laboratory cultures are discarded every three months and new
cultures started from mycoplasma-free, long-term frozen cultures.
B. Media
The CHO-K1 cell line has an absolute requirement for proline and therefore
must be maintained in culture medium containing sufficient amounts of
this amino acid. Ham's F12 medium, which contains 3 x 10-4 M L-proline
was used, supplemented with 10% fetal bovine serum, 2mM L-glutamine,
100 units/ml of penicillin, 100 ug/ml of streptomycin, and 0.9 pi/ml of
amphotericin B. A 10X formulation of Ham's F10 is available commercially
and was used for the testing of aqueous test samples in order to avoid
the dilution of medium components. This medium contains 1 x 10-4 L-proline
and was supplemented in the same manner as F12, except that kanamycin at
40 |jg/ml is included for additional protection against bacterial contami-
nation. Both media formulations support the growth and cloning of CHO
cells equally well.
C. Controls
The negative control consisted of three untreated cultures carried through
the same experimental time period as the treated cells. Since the test
material was tested as a solution in an organic vehicle (DMSO) and was
diluted into the medium to provide each test concentration, two sets of
vehicle control cultures containing the organic solvent at 13 and 2% by
volume were prepared in triplicate.
The average number of colonies in the negative control established the
cloning efficiency of the CHO cells used in the assay, and the appropriate
vehicle control provided the reference points for determining, the effects
of different concentrations of the test material on cell survival.
Litton
5-346
BIONETICS
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Utton
V. EXPERIMENTAL DESIGN
A- Dose Selection
Unless the approximate toxicity is already known or the sample size is
limiting, the following dose ranges are usually tested for different
sample forms. Aqueous samples, suspensions, or slurries are tested from
600 ul/ml to 3 pi/ml, usually in six dose steps. Eight doses are often
used when the amount of test sample is limited to provide a more precise
description of toxicity in the event of sharp dose-response curves. Dry,
particulate material is dissolved or suspended in DMSO, diluted into
growth medium, and tested at six dose levels from 1000 ug/ml to 3 ug/ml.
Samples that are solvent-exchanged into DMSO are tested from 20 ul/ml
(2% DMSO in growth medium) to 0.2 ul/ml, also in six dose steps. A
second dose study is performed with an adjusted dose range if the EC50
was not located properly in the initial test. However, EC50 values
greater than 1000 ug/ml for particulate material, 600 pi/ml for aqueous
samples, or 20 ul/ml for organic solutions will not be determined.
This sample, A81-05-030-676 (EA-2 XAD extract), was tested at eight dose
levels. The concentrations started with the maximum applicable dose (MAD)
of 20 Ml/ml and included 10, 6, 3, 1, 0.6, 0.3, and 0.1 pi/ml. The
corresponding concentration of organics at the MAD level was 50 ug/ml;
the lower doses were equivalent to 25, 15, 7.5, 2.5, 1.5, 0.75, and
0.25 ug orgam'cs/ml.
B. Clonal Toxicity Assay
Cells from monolayer stock cultures in logarithmic growth phase were tryp-
sinized with 0.1% trypsin plus 0.0155 versene for 4 minutes and the density
of the resulting cell suspension determined by hemocytometer. A number
of 60-mm culture dishes were then seeded with 200 cells and 4 ml of culture
medium per dish. The cultures were incubated for approximately 6 hours
at 37°C in a humidified atmosphere containing 5% C02 to allow attachment
of the cells. The 6-hour attachment period was used in order to avoid
cell division and the subsequent formation of two-cell colonies prior to
treatment.
The medium was aspirated from the cultures arid 4 ml medium containing
the test material applied. Three cultures were exposed to each test con-
centration. After an exposure time of 24 hours at 37°C, the medium was
removed by aspiration and each culture washed three times with approxi-
mately 4 ml aliquots of Dulbecco's phosphate buffered saline (pre-warmed
to 37°C). Fresh culture medium (5 ml) was placed in each dish and incuba-
tion at 37°C is continued for an additional 6 days to allow colony develop-
ment.
The test material caused a color change in the culture medium, the pH of
the medium containing the high dose would be determined at the time of
treatment. The pH at the lowest dose that results in a slight color change
would also recorded. At the end of the treatment period, the pH values
of the discarded media from the two described treatments would be recorded
again. No sample related pH effects were noted.
5-347
BIONETICS 5
-------
After the incubation period, the medium was drained from the cultures
and the surviving colonies fixed with 100% ethanol and stained with
Giemsa. Colonies were counted by eye; tiny colonies of approximately
50 cells or less were arbitrarily excluded from the counts.
ffi
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BIONETICS
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VI. 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
negative 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 location 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 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.
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5-349
BIONETICS
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VII. RESULTS
A. Interpretation
The application of sample A81-05-030-676 (EA-2 XAD extract) to the CHO
cell cultures caused a rapid lowering of the number of cells able to form
colonies as the concentration increased above 1.0 ul/ml. Relative sur-
vival values were calculated as the ratio of colonies formed in treated
cultures to the colonies formed in the appropriate vehicle control, and
these relative survival values were plotted against the concentration of
test material. As shown in Figure 1, the relative survival decreased
gradually in the 0.1 to 1.0 ul/ml range and dropped to nearly zero at
the 3.0 ul/ml dose level.
The concentration expected to kill 50 percent of the cells (EC50) was
found to be 1.72 ul of test material per ml of culture medium. This con-
centration was equivalent to 4.3 ug of organic material per ml of
culture medium. This value placed the test material in high (H)
toxicity range defined for the IERL-EPA CHO clonal toxicity bioassay.
The cells used for the assay were in logarithmic growth phase and were
98.9 percent viable. About 89 percent of the seeded cells formed colonies
in the negative control. Colony growth was normal and well distributed
on the culture dishes. The combined results were considered to achieve
assay acceptance criteria and provided confidence in the assumption that
the recorded data represented typical responses to the test material.
B. Tables and Figures
This report is based on the data provided in Table 1 and Figure 1.
Litton
5-350
BIONETICS
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TABLE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
Sample Identity: A81-05-030-676
(EA-2 XAD Extract")
EC50 Value: 1.72 pi/ml (4.3 M9
organics/ml)
Description of Sample:
gold liquid
Clear.
High (H)
5880
LBI Assay No.:
Date Received: August 26. 1981
Test Date: September 29. 1981
Vehicle: DMSO
Cell Type: CHO-K1
Toxicity
Classification:
pH Alterations:
Comments on
Treatment: Sample prepared in
None
DMSO at a concentration of
2.5 ug organics/ul
Cells Seeded per Dish: 200
COLONY COUNTS
Sample
NCb r
VC, 1ST
VC, 2%
TEST
TEST
TEST
TEST
TEST
TEST
TEST
TEST
Applied
Concentration
ul /ml
...
10
20
0.1
0.3
0.6
1.0
3.0
6.0
10.0
20.0
Dish
#1
170
157
146
145
136
125
133
0
0
0
0
Dish
#2
183
158
153
168
153
134
132
0
0
0
Q
Dish
#3
178
164
137
158
157
140
132
3
0
°d
Sd
Average
Count
177.0
159.7
145.3
157.0
148.7
133.0
132.3
1.0
0
0
0
Relative
Survival
*
W mm
100.0
100.0
98.3
93.1
83.3
82.8
0.6
0
0
0
Cloning
Efficiency
%
88.5
79.9
72.7
^Relative to 2% VC for 20 ul/ml treatment and to 1% VC for other treatments.
NC = Negative Control, F12 medium
5VC = Vehicle Control, percent DMSO given
S = Plate not set up to conserve limited test sample.
5-351
Litton
BIONETICS
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FIGURE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
EC5Q DETERMINATION
A81-05-030-676
(EA-2 XAD EXTRACT)
HO r^i
1 10
CONCENTRATION, ul/ml
100
5-352
10
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VIII. ASSAY EVALUATION CRITERIA
The EC50 value represents the concentrations of test material that reduces
the colony-forming ability of CHO cells to 50% of the vehicle or negative
control value. EC50 values are determined graphically by fitting a curve
by eye through relative survival data plotted as a function of the loga-
rithm of the applied concentration. Each data point normally represents
the average of three culture dishes. In order to indicate the variability
of the data, the high and low colony counts for each concentration are
used to calculate the relative survivals, and the range is shown by a
bar at the position of the plotted average. If no bar is shown, the
variability was within the size of the symbol. Statistical analysis is
unnecessary in most cases for evaluation.
The toxicity of the test material is evaluated as high, moderate, low,
or nondetectable according to the range of EC50 values defined in the
following table.
Solids
Toxicity3 (EC50 in ug/ml)
High <10
Moderate 10 to 100
Low 100 to 1000
Not Detectable >1000
Aqueous Liquids
(EC50 in pi/ml)
<6
6 to 60
60 to 600
>600
Nonaqueous Liquids
(EC50 in ul/ml)
<0.2
0.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level I Environmental Assessment Biological Tests.
Criteria for nonaqueous liquids are tentative and under evaluation.
If the organic or solids content is known, the sample is evaluated under
the solids criteria.
Another evaluation scheme is proposed for extracts obtained from SASS
train gas volumes. The proportion of the total gas volume corresponding
to the volume of extract used in the bioassay is calculated and expressed
as L/ml of culture medium (or DSCF/ml of culture medium). A criterion
of 1000 L/ml is set as the limit for nondetectable toxicity. This gas
volume corresponds to the average volume breathed by humans over a 2-hour
period. The subsequent toxicity ranges are defined by 10-fold dilution
steps to conform to standard procedure. The toxicity ranges are defined
in the following table for liter and dry standard cubic feet units:
EC50 In EC50 In
Toxicity Liters/ml (L/ml) Dry Standard Cubic Feet/ml (DSCF/ml)
Litton
HTg < <0.35 DSCF
Moderate 10-100 0.35-3.5
Low 100-1000 3.5-35
Nondetectable >1000 >35-
5-353
BIONETICS 11
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IX. REFERENCES
1. Brusick, D.J., et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment Biological Tests. EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc. , Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD,
August 1981, pp 52.
ffl
Litton
5-354
BIONETICS
12
-------
GENETICS ASSAY NO.: 5887
LBI SAFETY NO.: 7171
MUTAGENICITY EVALUATION OF
A81-05-030-744
"TlA-2 FLYASH)
~~!fi THE
EPFlEWL 1
AMES SAIRQNEIJA7MICROSOME
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 20895
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
5-355
, BIONETICS
Litton
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ffl
Litton
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Ames
Salmonella/microsome mutagenesis assay as described in "IERL-RTP Proce-
dures Manual: Level 1 Environmental Assessment Biological Tests"1. The
data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting"2.
The Ames Salmonella/microsome mutagenesis assay has been shown to be a
sensitive method for detecting mutagenic activity for a variety of chemi-
cals representing various chemical classes3. This assay is one of several
recommended by EPA to identify, categorize and rank the pollutant potential
of influent and effluent streams from industrial and energy-producing pro-
cesses. This assay has been well validated with a wide range of positive
and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good Labora-
tory Practices Regulations of 1979. Deviations from standard procedure
shall be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
5-356
BIONETICS
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TABLE OF CONTENTS
Page No.
PREFACE i
I. ASSAY SUMMARY 1
II. OBJECTIVE 2
III. TEST MATERIAL 3
A. Description 3
B. Handling and Preparation 3
IV. MATERIALS 4
A. Indicator Microorganisms 4
B. Media 4
C. Activation System 5
1. S9 Homogenate 5
2. S9 Mix 5
V. EXPERIMENTAL DESIGN 6
A. Dose Selection 6
B. Mutagenicity Test 6
I. Nonactiyation Assay 6
2. Activation Assay 6
C. Control Compounds 7
D. Recording and Presenting Data 7
VI. RESULTS 9
A. Interpretation 9
B. Tables 9
VII. EVALUATION CRITERIA 11
A. Surviving Populations 11
B. Dose-Response Phenomena 11
C. Control Tests 11
D. Evaluation Criteria for Ames Assay 12
1. Strains TA-1535 and TA-1537 12
2. Strains TA-98 and TA-100 12
3. Pattern 12
4. Reproducibility 12
E. Relation Between Mutagenicity and
Carcinogenicity 13
F. Criteria for Ranking Samples in the Ames Assay . . 13
VIII. REFERENCES . 14
5-357
_ BIONETICS
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I. ASSAY SUMMARY
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Litton
A. Sponsor: Acurex Corporation
B. Material (Test Compound): Genetics Assay Number: 5887
1. Identification: A81-05-030-744 (EA-2 Flyash)
2. Date Received: August 26, 1981
3. Physical Description: Gray and white particles with larger
(long and thin) black chunks.
C. Type of Assay: EPA Level 1 Ames Sal monell a/Mi crosome Plate Test
D. Assay Design Number: 401 (EPA Level 1)
E. Study Dates:
1. Initiation: September 23, 1981
2. Completion: September 28, 1981
F. Supervisory Personnel:
A. Study Director: D.R. Jagannath, Ph.D.
G. Evaluation:
The test material, A81-05-030-744 (EA-2 flyash), was tested
for activity in the Ames Salmonella mutagenicity assay over a
concentration range of 0.05 mg/plate to 5.0 mg/plate. The
test was performed in duplicate under nonactivation and acti-
vation test conditions with strains TA-1535, TA-1537, TA-98,
and TA-100.
The sample was not mutagenic under the test conditions employed
and was ranked as having nondetectable (ND) mutagenic activity
as defined by the IERL-EPA Level 1 criteria for the Ames bio-
assay1.
Submitted by:
Study Director
Reviewed by:
D.R. Jagahnath, Ph.D.
Section Chief,
Submammalian Genetics,
Department of Molecular
Toxicology
Date
-
David J. Brusick, Ph.DT
Director,
Department of Molecular
Toxicology
Date
5-358
BIONETICS
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II. OBJECTIVE
The objective of this study was to determine the genetic activity of
A81-05-030-744 (EA-2 flyash) in the Salmonella/ microsome assay with and
without the addition of mammalian metabolic activation preparations.
The genetic activity of a sample is measured in these assays by its ability
to revert the Salmonella indicator strains from histidine dependence to
histidine independence. The degree of genetic activity of a sample is
reflected in the number of revertants that are observed on the histidine-
free medium.
Litton
5-359
BIONETICS
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ffl
Litton
III. TEST MATERIAL
A. Description
The test material was received as gray and white particles with larger
black chunks (15 gm) and was used without further preparation. No infor-
mation on actual particle size distribution or on sampling parameters
was received.
B. Handling and Preparation
The test material was received at LBI on August 26, 1981. The sample
was assigned LBI safety number 7171 and LBI assay number 5887. The sample
was stored at +4°C in the dark.
A total of 242.89 mg of test material was weighed and suspended in 24.3 ml
of dimethylsulfoxide. The sample formed an opaque suspension after
vortexing that settled upon standing. The suspension was incubated at
37°C on a shaker overnight to help leach material out of the particulates.
Serial dilutions were made in DMSO such that 50 ul aliquots of each dilu-
tion give the desired concentration. The suspension was well mixed when
aliquots were removed for dosing.
5-360
BIONETICS
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IV.
MATERIALS
A.
Indicator Microorganisms
The Salmonella typhimurium strains used in this assay were obtained from
Dr. Bruce Ames, University of California at Berkeley.4-8 The following
four strains were used.
Strain
Designation
Gene Additional Mutations
Affected Repair IPS R Factor
Mutation Type
Detected
TA-1535
TA-1537
TA-98
TA-100
his G A uvr B rfa
his C A uvr B rfa
his D A uvr B rfa pKMlOl
his G A uvr B rfa pKMlOl
Base-pair
substitution
Frameshi ft
Frameshift
Base-pair
substitution
All the above strains have, in addition to the mutation.in the histidine
operon, mutation (rfa-) that leads to defective lipopolysaccharide coat,
a deletion that covers genes involved in the synthesis of vitamin biotin
(bio-) and in the repair of ultraviolet (uv) - induced 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 physi-
cally 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 mutagens.8
In addition, plasmid pKMlOl confers resistance to the antibiotic ampi-
cillin, 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 supplemented
with a trace of biotin and an excess of histidine. In addition, the
plates with plasmid-carrying strains contain ampicillin (25 ug/ml) to
ensure stable maintenance of plasmid pKMlOl. New stock culture plates
are made as often as necessary from the frozen master cultures or from
single colony reisolates that were checked for their genotypic character-
istics (his, rfa uvrB. bio) and for the presence of plasmid. For each
experiment, arTTnoculum from the stock culture plates is grown overnight
at 37°C in nutrient broth (Oxoid CM67) and used.
B.
Media
The bacterial strains were cultured in Oxoid Media #2 (Nutrient Broth).
The selective medium was Vogen Bonner Medium E with 2% glucose.10 The
5-361
Litton
BIONETICS
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overlay agar consisted of 0.6% purified agar with 0.05 mM histidine,
0.05 mM biotin and 0.1M NaCl according to the methods of Ames et aj.9
C. Activation System
1. S9 Homogenate
A 9,000 x £ supernatant prepared from Sprague-Dawley adult male rat liver
induced by Aroclor 1254 (Ames et a_L9) was purchased commercially and
used in these assays.
2. 59 Mix
S9 mix used in these assays consisted of the following components:
Components
Concentration per Milliliter
S9 Mix
NADP (sodium salt)
D-glucose-6-phosphate
MgCl2
KC1
Sodium phosphate buffer
pH 7.4
Organ homogenate from rat
liver (S9 fraction)
4 umoles
5 umoles
8 umoles
33 umoles
100 umoles
100 Milters
5-362
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BIONETICS
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Litton
V. EXPERIMENTAL DESIGN
A. Dosage Selection
Test strategy and dose selection depend upon sample type and sample avail-
ability. The Level 1 manual1 recommends solids to be initially tested
at the maximum applicable dose (MAD) of 5 mg per plate and at lower con-
centrations of 2.5, 1, 0.5, 0.1 and 0.05 mg per plate. Liquids are tested
initially at the MAD of 200 ul per plate, and at lower concentrations of
100, 50 and 10 ul per plate. Samples are retested over a narrower range
of concentrations with strains showing positive results initially. Alter-
nate dose are employed if sample size is limiting or at the direction of
the sponsor.
Doses selected to test this sample covered the recommended dose range
for solids. The highest dose was at the MAD level of 5 mg per plate
and included five lower dose levels of 2.5, 1, 0.5, 0.1, and 0.05 mg per
plate.
B. Mutagem'city Testing
The procedure used was based on the paper published by Ames et. al_.9 and
was performed as follows:
1. Nonactivation Assay
To a sterile 13 x 100 mm test tube placed in a 43°C water bath the fol-
lowing was added in order:
2.00 ml of 0.6% agar containing 0.05 mM histidine and
0.05 mM biotin.
0.05 ml of a suspension of the test chemical to give the
appropriate dose.
0.1 ml to 0.2 ml of indicator organism(s).
0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured onto minimal agar plates
(see IV B, Media). After the top agar had set, the plates were incubated
at 37°C for approximately 2 days. The number of his+ revertant colonies
growing on the plates were counted with an automatic colony counter and
recorded.
2. Activation Assay
The activation assay was run concurrently with the nonactivation assay.
The only difference was the addition of 0.5 ml of S9 mix (see IV C, Acti-
vation System) to the tubes in place of 0.5 ml of phosphate buffer which
was added in nonactivation assays. All other details were similar to
the procedure for nonactivation assays.
5-363
BIONETICS 6
-------
A detailed flow diagram for the plate incorporation assay is provided in
Figure 1.
C.
Control Compounds
A negative control consisting of the solvent used for the test material
was also assayed concurrently with the test material. For negative con-
trols, step 'b1 of Nonactivation Assays was replaced by 0.05 ml of the
solvent. The negative controls were employed for each indicator strain
and were 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 were
made using this solvent. The amount of solvent used was equal to the
maximum volume used to give the appropriate test dose.
Specific positive control compounds known to revert each strain were also
used and assayed concurrently with the test material. The concentrations
and specificities of these compounds to specific strains are given in
the following table:
Concentration
per plate Salmonella
Assay
Nonactivation
Chemical
Sodium azide
2-Nitrofluorene
(NF)
9-aminoacridine
(9AA)
Solvent (ug)
Water
Di methyl -
sulf oxide
Ethanol
10. 0
10.0
50.0
Strains
TA-1535, TA-100
TA-98
TA-1537
Activation
2-anthramine
(ANTH)
Dimethyl-
sulfoxide
2.5
For all strains
D.
Recording and Presenting Data
The number of colonies on each plate were counted and recorded on printed
forms. These raw data were analyzed in a computer program and reported
on a printout. The results are presented as revertants per plate for
each indicator strain employed in the assay. The positive and solvent
controls are provided as reference points.
ffl
Litton
5-364
BIONETICS
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AMES ASSAY [PLATE INCORPORATION METHOD]
Aliquot of
saline
0.5 ml
—S-9
Molten |45*C] overlay egar
appropriately supplemented
0.1 ml
Test, positive or solvent
control chemical
Aliquot of an overnight culture
of bacterial 109 cells/ml]
0.5 ml S-9 mix [hepatic
S-9—— homogenate from PCB
pretreated rat plus
necessary cofactors
Overlay poured on selective
bottom agar medium
Plated incubated at 37*C for 48 hours
The numbers of revertants/plate counted
Data analyzed
Interpretation/Conclusion
Figure 1 AMES SALMQNELLA/MICROSOME MUTAGENESIS ASSAY
5-365
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ffl
Litton
VI. RESULTS
A. Interpretations
The test material, A81-05-030-744 (EA-2 flyash), was dissolved in DMSO
at a stock concentration of 100 mg/ml and leached overnight on a shaker
at 37°C. Additional dilutions were prepared in DMSO for testing. The
maximum test level was 5.0 mg/plate. There was no evidence of toxicity
at this level.
Reverse mutation was measured in strains TA-1535, TA-1537, TA-98 and
TA-100. The test was conducted in duplicate both with and without rat
liver S9 mix for metabolic activation.
There was no mutagenic activity associated with the test material treatment
and the sample was considered nonmutagenic and non toxic. The sample
was ranked as having nondetectable (ND) mutagenic activity using the
IERL-EPA Level 1 evaluation criteria for the Ames assay1.
Solvent control and positive control values were within acceptable ranges.
These results achieved assay acceptance criteria and provided confidence
in the assumptions that the recorded data represented typical responses
to the test material.
B. Tables . .
This report is based on the data provided in Table 1.
5-366
BIONETICS
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RESULTS
TABLE 1
A. NAME OR CODE DESIGNATION OF THE TEST COMPOUND: A81-05-030-744 (EA-2 FYLASH)
H. SOLVENT: ONSO
C. TEST INITIATION DATES: 09/24/R1
0. TEST COMPLETION DATE: 09/2d/Bl
E. s-9 LOTH: s-9 n
NOTE: CONCENTRATIONS ARE GIVEN IN MILLIGRAMS PER PLATE
co
TEST
SPECIES TISSUE
NONACTIVATION
SOLVENT CONTROL
POSITIVE CONTROL** —
TA-1535
1
12
1076
2
17
961
TA-1537
3 1
9
621
TA-98
231
12
628
745
2
38
811
TA-100
3 1
132
1308
2
106
1359
3
TEST CONFOUND
0.050
0.100
0.500
1.000
2.500
5.000
ACTIVATION
MG
MG
MG
MG
MG
MG
SOLVENT CONTROL RAT
POSITIVE CONTROL*** RAT
...
__-
— _
— -
LIVER
LIVER
10
10
11
12
11
9
17
308
17
15
12
10
9
21
11
294
11
9
11
11
12
17
13
339
11
8
5
14
16
13
8
372
49
47
53
29
34
26
45
1562
39
40
45
44
31
31
34
1600
162
153
130
151
140
141
101
2065
149
139
124
135
140
131
123
1832
TEST COMPOUND
0.050
0.100
0.500
1.000
2.500
5.000
TA-1535
IA-1537
T4-98
TA-100
SOLVENT
MG RAT .
MG RAT
MG RAT
MG RAT
MG RAT
MG RAT
SODIUM A/ IDE
9-AMINOACKIOINE
2-NITROFLUORENE
SODIUM A/IDE
50 UL/PLATE
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
15
T
14
13
2
8
13
12
11
•t
8
7
11
12
11
13
9
13
10
20
10
12
7
10
54
46
62
49
37
47
10 UG/PLATE
50 UG/PLATE
10 UG/PLATE
10 UG/PLATE
46
34
54
44
45
48
TA-1535
TA-1537
TA-98
TA-100
128
137
149
134
140
125
163
140
111
127
136
142
2-ANTHRAHINr
2-ANTHRAHCNE
2-ANTHRAKINE
2-ANTHRAHINE
2.5 UG/PLATE
2.5 UG/PLATE
2.5 UG/PLATC
2.5 UG/PLATE
-------
VII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA
Statistical methods are not currently used, and evaluation is based on
the criteria included in this protocol.
Plate test data consists of direct revertant colony counts obtained from
a set of selective agar plates seeded with populations of^mutant cells
suspended in a semi sol 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 semiquanti-
tative in nature. Although these features of the assay reduce the quanti-
tation of results, they provide certain advantages not contained in a
quantitative suspension test:
The small number of cell divisions permits potential
mutagens to act on replication DNA, which is often more
sensitive than nonreplicating 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 quantisation 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 concen-
trations, the highest of these doses being selected to show slight toxicity
as determined by subjective criteria.
B. Dose-Response Phenomena
The demonstration of dose-related increased in mutant counts is an impor-
tant criterion in establishing metagenicity. A factor that might modify
dose-response results for a mutagen would be the selection of doses that
are too low (usually mutagenicity and toxicity are related). If the
highest dose is far lower than a toxic concentration, 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.
C. Control Tests
Positive and negative control assays were conducted with each experiment
and consisted of direct-acting mutagens for nonactivation assays and
mutagens that require metabolic biotransformation in activation assays.
m 5-368
BIONETICS 11
Utton
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Negative controls consisted of the test material solvent in the overlay
agar together with the other essential components. The negative control
plate for each strain gave a reference point to which the test data was
compared. The positive control assay was conducted to demonstrate that
the test systems were functional with known mutagens.
The following normal range of revertants for solvent controls are generally
considered acceptable.
TA-1535: 8-30
TA-1537: 4-30
TA-98: 20-75
TA-100: 80-250
D. Evaluation Criteria for Ames Assay
- Because the procedures to be used to evaluate the mutagenicity of the
test material are semi quantitative, the criteria to be used to determine
positive effects are inherently subjective and are based primarily on a
historical data base. Most data sets will be evaluated using the following
criteria.
1. Strains TA-1535 and TA-1537
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to three times the solvent control value will
be considered to be mutagenic.
2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to twice the solvent control value for TA-98
and TA-100 will be considered to be mutagenic.
3. Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain
(G-46), 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 that cannot be
reproduced in additional runs, the initial positive test data lose signi-
ficance.
The preceding criteria are not absolute, and other extenuating factors
may enter into a final evaluation decision. However, these criteria
will be applied to the majority of situations and are presented to aid
those individuals not familar with this procedure. As the data base is
increased, the criteria for evaluation can be more firmly established.
5-369
, __ BIONETICS 12
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CB
Litton
E. Relation Between Mutaqenicity and Carcinogenicity
It must be emphasized that the Ames Salmonella/Microsome Plate Assay is
not a definitive test for chemical carcinogens. It is recognized, however,
that correlative and functional relations have been demonstrated between
these two endpoints. The results of comparative tests on 300 chemicals
by McCann et a_L4 show an extremely good correlation between results of
microbial mutagenesis tests and in 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 muta-
genic activity.
F. Criteria for Ranking Samples in the Ames Assay
The goal -of EPA Level 1 Ames testing is to rank source streams by relative
degree of genetic toxicity (mutagenicity). Samples are first identified
as mutagenic or nonmutagenic by the criteria in Section D above and
then ranked using the mutagenicity categories presented in the table
below. The lowest concentration giving a positive response in any strain,
with or without metabolic activation, is identified as the minimum effec-
tive concentration (MEC) for that sample. The mutagenicity of the sample
is evaluated as high (H), moderate (M), low (L), or nondetectable (ND)
according to the evaluation criteria developed in the Level 1 manual1
and summarized below. Samples with no detectable activity at the maximum
applicable dose (MAD) are ranked nondetectable (ND).
Ames Assay Mutagenicity Ranking Criteria1
Mutagenic
Activity
High (H)
Moderate (M)
Low (L)
Not Detectable (ND)
Solids
(MEC in ug/plate)
<50
50-500
500-5000
>5000
(MEC
<2
2-20
Liquids3
in pi/plate)
20-200
>200
Concentration of organic extracts is based upon organic content (ug
organics per plate) and not volume (pi extract per plate) of sample
tested.
5-370
BIONETICS 13
-------
VIII. REFERENCES
1. Brusick, D.J., et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment Biological Tests. EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data formatting.
EPA-600/7-80-079, Litton Bionetics Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD, August
1981, 52 pp.
4. 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.
5. Ames, B.N., Gurney, E.G., Miller, J.A. and Bartsch, H.: Carcinogens
as frameshift mutagens: Metabolites and derivatives of 2-acetylamino-
fluorene and other aromatic amine carcinogens. Proc. Nat. Acad.
Sci., USA 69:3128-3132, 1972.
6. 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.
7. 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.
8. 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.
9. 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.
10. Vogel, H.J. and Bonner, D.M.: Acetylornithinase of E. coli partial
purification and some properties. J. Biol. Chem., 218:97-106. 1966.
Utton
5-371
BIONETICS 14
-------
GENETICS ASSAY NO.: 5887
LBI SAFETY NO.: 7171
CYTOTOXIC EVALUATION OF
A81-05-030-744
"TIF2 FLYASH)
INTHE RABBIT
ALVEOLAE MATROPHAGE (RAM)
CTTOTOXICITY ASSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
E
Litton
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
5-372
BIONETICS
-------
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the rabbit
alveolar macrophage (RAM) cytotoxicity assay as described in "IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests" (1).
The data were evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting" (2).
The RAM cytotoxicity assay has been shown to be a sensitive method for
detecting cytotoxic activity for a variety of chemicals representing
various chemical classes (3). This assay is one of several recommended
by EPA to identify, categorize and rank the pollutant potential of influent
and effluent streams from industrial and energy-producing processes.
This assay has been well validated with a wide range of positive and
negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepara-
tion, testing and evaluation of the test material shall conform to Litton
Bionetics, Inc. standard operating procedures and the Good Laboratory
Practices Regulations of 1979. Deviations from standard procedure shall
be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
Utton
5-373
BIONETICS
-------
TABLE OF CONTENTS
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
PREFACE
ASSAY SUMMARY
OBJECTIVE
TEST MATERIAL
A. Description
B. Handling and Preparation
MATERIALS ....
A. Indicator Cells
B. Media
C. Negative Controls
EXPERIMENTAL DESIGN
A. Procurement of Cells
B. Sample Forms .
C. Dose Selection
D. Treatment .
E. Cell Viability Assay
F. ATP Assay
ASSAY ACCEPTANCE CRITERIA
RESULTS
A. Interpretation
B. Tables and Figures
ASSAY EVALUATION CRITERIA
REFERENCES
Page No.
. . . . . i
1
2
3
3
3
4
4
4
4
5
5
. . . . . 5
6
6
6
7
8
9
9
9
16
17
ffl
Litton
5-374
BIONETICS ii
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I. ASSAY SUMMARY
A. SPONSOR: Acurex Corporation
B. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5887
1. Identification: A81-05-030-744 (EA-2 Flyash)
2. Date Received: August 26, 1981
3. Physical Description: Fine, gray and white particles with shreds
of black material.
C. TYPE OF ASSAY: Rabbit Alveolar Macrophage (RAM) Cytotoxicity Assay
D. ASSAY DESIGN NUMBER: 443
STUDY DATES:
1. Initiation: September 23, 1981
2. Completion: October 14, 1981
SUPERVISORY PERSONNEL:
1. Study Director: Brian Myhr, Ph.D.
2. Laboratory Supervisor: Robert Young, M.S.
EVALUATION:
E.
F.
G.
The test material was tested as supplied and after puliverization
to a very fine powder. Both forms of the material caused only slight
toxicity at concentrations above 500 ug/ml. The most sensitive
parameters, ATP content and viability index, indicated ECSO values
above the MAD level of 1000 ug/ml. Therefore, the results were
evaluated as showing nondetectable (ND) toxicity for this test
material, according to the IERL-EPA Level 1 toxicity categories in
the RAM Cytotoxicity Assay.
Litton
BIONETICS
Submitted by:
Study Director
Brian Myhr, Ph.(0. Date
Associate Director,
Department of Molecular
Toxicology
5-375
David J. Brusick, Ph.D.
Director,
Department of Molecular
Toxicology
Dat
LSL
-------
II. OBJECTIVE
The objective of this study was to determine and rank the cytotoxicity
of A81-05-030-744 (EA-2 Flyash) to cultured rabbit alveolar macrophage
(RAM) cells. The measure of cytotoxicity was the reduction in cell
viability and adenosine triphosphate (ATP) content of the cultures after
a 20 hour exposure to the test material. At the conclusion of the exposure
period, the number of viable cells and total ATP content in the treated
cultures were compared to the corresponding values in unexposed control
cultures. The concentration of test material that reduced each experi-
mental parameter by 50% was estimated graphically and referred to as the
EC50 value. Standard EPA Level 1 toxicity evaluation criteria for the
RAM cytotoxicity assay were used to rank the toxicity potential of the
test material based upon the most sensitive parameter.
EH
Litton
5-376
BIONETICS
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III. TEST MATERIAL
A. Description
The test material was received as a gray and white participate containing
thin shreds of black material. The amount of sample supplied was 15 grams.
No information on the sampling parameters was provided.
B. Handling and Preparation
The test material was received on August 26, 1981, and was assigned LBI
assay number 5887 and LBI safety number 7171. The sample was stored at
+4°C in the dark.
Approximately 34 mg of the test material was tested as supplied. Then
on October 1, 1981, the remaining sample was ground in a mortar and pestle
to fine black powder. Approximately 2.5 grams of the ground sample was
further pulverized on October 8, 1981, to a very fine, black powder of
which 30 mg was used in the second trial of the assay. For both trials,
the test material was suspended in serum-free EMEM culture medium at a
concentration of 2000 ug/ml and incubated at 37°C on a roller drum for
8 hours. The original material settled quickly on standing, but the
suspension formed from the pulverized powder remained well-dispersed for
dilutions. No pH changes were observed. The suspensions were serially
diluted with EMEM (serum-free) and applied to the cultures at a maximum
concentration of 1000 ug/ml in the presence of 10% serum.
Litton
5-377
BIONETICS
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m
Litton
IV. MATERIALS
A. Indicator Cells
The two trials employed short-term primary cultures of alveolar macrophage
cells obtained by lung lavages of male New Zealand white rabbits (2.0-
2.5 kg). The rabbits were maintained on Purina Lab Rabbit Chow 5321 and
water ad libitum and were examined for the absence of respiratory illnesses
prior to use.
B. Media
The cells were maintained and treated in Eagle's Minimum Essential Medium
(EMEM) with Earle's salts and supplemented with 10% fetal bovine serum
(heat-inactivated), 100 units/ml penicillin, 100 ug/ml streptomycin,
17.6 ug/ml kanamycin, and 0.4 ug/ml amphotericin B.
C. Negative Controls
The negative control for the first trial consisted of six untreated
cultures carried through the same experimental time period as the treated
cells. _Six cultures were used because a large number of cells was obtained
by pooling the yield from two rabbits in order to run two concurrent
assays. Only one animal was used for the second trial, and the usual
three untreated cultures were prepared. The average viability and ATP
content of the negative controls provided the reference points for deter-
mining the effects of. different concentrations of the test material on
the assay parameters.
5-378
BIONETICS
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Litton
V. EXPERIMENTAL DESIGN
A- Procurement of Cells
The rabbits were sacrificed by injection of Nembutal (60 mg/ml) into the
marginal ear vein, and sterile operating techniques were used to perform
a tracheostomy. Prewarmed normal saline (30 ml) was then introduced
into the lungs via a catheter and allowed to stand for 15 minutes. This
lavage fluid was removed and placed into a 50-ml sterile centrifuge tube
on ice. Nine additional lavages were similarly performed and collected,
except the saline was removed shortly after its introduction into the
lungs. Any lavage fluid containing blood or mucous was discarded. The
lavages were centrifuged at 365 x g for 15 minutes and the cells resus-
pended in cold 0.85% saline. After two washes in saline by centrifugation,
the cell pellets were resuspended in cold EMEM containing 20% serum and
then combined. A cell count was obtained by hemocytometer and the suspen-
sion diluted to between 5 x 10s and 106 cells/ml. Viability was determined
by trypan blue staining and the cells were not used if less than 95%
viable. Also, a differential cell count from Wright-stained smears was
performed to verify that the macrophage content was above 90%.
B. Sample Forms
The usual sample form for application to the cells is a suspension of
particulate material. Solid samples are ground to fine particles and a
weighed portion is suspended in a known volume of EMEM (0% FBS^for about
eight hours to help leach any water-soluble material. Finely-divided
test material may be suspended directly in culture medium without further
grinding. Aqueous liquids, suspensions, or slurries containing less
than 0.5% organic solvent are added by volume to culture medium.
Samples supplied as solutions in organic solvents are usually solvent-
exchanged into DMSO before testing. Original sample volumes may first
be reduced a maximum of 10-fold in a Kuderna-Danish concentrator, and
the concentrative factor is used to convert assayed volumes into equi-
valent original sample volumes in the absence of information about solute
concentration. An aliquot of the reduced volume is exchanged into DMSO
by repeated, partial evaporation under a stream of nitrogen in a warm
water bath (50°C); the evaporated volumes are replaced with equal volumes
of DMSO.
Samples adsorbed on XAD-2 resin are extracted with methylene chloride
or acetone in a Soxhlet apparatus for 24 hours. The extract is then
concentrated and solvent-exchanged into DMSO. Alternatively, acetone
extracts can be assayed directly at concentrations up to 2% by volume in
the culture medium.
Samples impregnated on fiber glass or teflon filters are repeatedly soni-
cated in cyclohexane to remove particulates. The resulting cyclohexane
particulate suspension is then evaporated to dryness and the particulates
resuspended in EMEM culture medium at the desired concentration.
5-379
BIONETICS
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ffi
Litton
Sponsor-specified handling of sample materials will be followed if the
above procedures are not applicable or a specific procedure is desired.
C. Dose Selection
Unless the approximate toxicity is already known or the sample size is
limiting, the following usual dose ranges are tested for different sample
forms. Dry, particulate material is tested at six dose levels from
1000 ug/ml to 3 ug/ml. Aqueous samples, suspensions, or slurries are
tested from 600 ul to 3 ul/ml in six dose steps. Samples that are solvent-
exchanged into DMSO are tested from 20 ul/ml (2% DMSO in growth medium)
to 0.2 (jl/ml, also in six dose steps. A second dose study is performed
with an adjusted dose range if the EC50 was not located properly in the
initial test. However, EC50 values greater than 1000 ul/ml for particulate
material, 600 ul/ml for aqueous samples, or 20 ul/ml for organic solutions
will not be determined.
This test material, A81-05-030-744 (EA-2 flyash), was tested as supplied
at 6 dose levels in the first trial, starting at the maximum applicable
dose (MAD) of 1000 ug/ml and including 600, 300, 100, 60 and 30 ug/ml.
The second trial was performed with only three doses of the finely ground
test material: 1000, 600 and 300 ug/ml.
D. Treatment
A series of 25 cm2 culture flasks were prepared, each containing 2.0 ml
of serum-free medium at 37°C and the test material at twice the desired
final concentration. Three flasks were prepared for each test concen-
tration. Aliquots of cell suspension (2 ml) were then added; each flask,
therefore, contained from 1 to 2 x 10s viable cells in a 4-ml volume of
media containing 10% serum. The flasks were placed on a rocker platform
in a 37°C incubator with a humidified atmosphere containing 5% C02.
After sitting for about 30 minutes, the flasks were slowly rocked for
the remainder of a 20-hour exposure period.
If the test substance causes a color change in the growth medium, the pH
is determined in additional treated flasks. After the exposure period,
the pH of the medium in the experimental flasks is again recorded.
E- Cell Viability Assay
At the end of the treatment period, the medium containing unattached
cells was decanted into a centrifuge tube on ice. The attached cells
were rinsed with 1 ml of 0.1% trypsin/0.01% versene and then incubated
with 2 ml of the trypsin/versene solution for about 5 minutes at 37°C.
The trypsinates and decanted media were combined for each culture to
yield a 7-ml cell suspension for subsequent analysis.
A 0.5 ml or 1.0 ml aliquot of the cell suspension was removed for cell
count and viability determination. The aliquot was combined with 1.0 ml
of 0.4% trypan blue and counted by hemocytometer about 5 to 15 minutes
later. The total number of cells counted per culture was the sum of the
5-380
BIONETICS c
-------
numbers found in five squares for each chamber of the hemocytometer (1 ul
total volume). The numbers of live (colorless) and dead (blue) cells
were recorded.
F. ATP Assay
ATP was immediately analyzed by extraction of a 0.1-ml sample of cell
suspension with 0.9 ml of 90% DMSO. After 2 minutes at room temperature
5.0 ml cold MOPS buffer (0.01 M morpholinopropane sulfonic acid) at pH 7.4
was added and the extract mixed well and placed on ice. Aliquots of
10 ul were injected into a cuvette containing a luciferin-luciferase
reaction mixture in a DuPont Model 760 Luminescence Biometer. The Biometer
was calibrated daily with standard ATP solutions to provide a direct
read-out of the ATP content. Each test sample was assayed at least twice
to obtain repeatable readings.
Litton
5-381
BIONETICS
-------
VI. ASSAY ACCEPTANCE CRITERIA
The assay will be considered acceptable for evaluation of the test results
if the following criteria are met:
1. The macrophage population is 90% or greater of the total nucleated
cells collected by lung lavage.
2. The percent viability of the macrophages used to initiate the assay
is 95% or greater.
3. The survival of viable macrophages in the negative control cultures
over the 20 hour treatment priod is 70% or greater.
4. A sufficient number of data points (for five test concentrations or
less) are available to clearly locate the EC50 of the most sensitive
test parameter within a toxicity region as defined under Assay Eval-
uation Criteria.
5. The data points critical to the location of the EC50 for the most
sensitive parameter are the averages of at least two treated cultures.
6. If all the test parameters yield EC50 values greater than 1000 ug/ml,
600 ul/ml for aqueous solutions, or 20 ul/ml for organic solutions,
the plotted curves for ATP content and viability index parameters
do not exceed 120% of the negative control.
m
Litton
5-382
BIONETICS 8
-------
VII. RESULTS
A. Interpretation
Two trials were performed to test the effect of puliverization on the
toxicity of the test material to the RAM cells. The original test material
consisted of a fine powder and long, thin shreds of black material, and
the test results for this material are presented in Table 1 and Figures 1
and 2. When the test material was puliverized to a very fine powder,
the results shown in Table 2 and Figures 3 and 4 were obtained. Absolute
and relative assay parameters are provided in the tables, whereas the
relative values are plotted in the figures to determine EC50 positions.
In both assays, the test parameters remained above 70% of the negative
control values for all applied dosed up to the MAD level of 1000 ug/ml.
Some toxicity was indicated in the 100-1000 ug/ml concentration range by
the viability index and the ATP content, but the decreased in these para-
meters were insufficient to ascribe toxic properties to the test material.
Pulverization of the test material appeared to slightly reduce the toxi-
city, if it did anything, perhaps by eliminating the long thin strands
of material that could pierce the cells after being englufed. Since the
most sensitive assay parameters (ATP content and viability index) indicated
ECSO values above 1000 ug/ml, the test material was evaluated as having
nondetectable (NO) toxicity, according to the toxicity categories defined
for the IERL-EPA Level 1 RAM assay1.
The macrophages collected for both assays had normal morphology and
appeared to be in a healthy state. The initial viability was approxi-
mately 99% and the survival of viable cells in the negative controls for
both trials was at least 96 percent. The average cellular ATP content
of the negative control (ATP/106 total cells) of the negative controls
was within the historical range for acceptable cultures in both assays.
These results achieved the assay acceptance criteria and provided confi-
dence in the assumption that the collected data represented typical
responses to the test material.
B. Tables and Figures
This report is based on the data provided in Tables 1 and 2 and Figures 1
to 4.
Litton
5-383
BIONETICS
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TABLE 1
RABBIT ALVEOLAR MACROPHAGE (RAM) CYTOTOXICITY ASSAY DATA
tn
i
CO
LBI Assay No.: 5887 (Trial I, Unground sample)
Test Material Identity: A81-05-030-744 (EA-2 Flyash)
Test Date: September 23, 1981
Vehicle: EMEM
Sample
NCC
TEST
TEST
TEST
TEST
TEST
TEST
apH change
h
Concentration3
ug/ml
...
30
60
100
300
600
1000
In culture medium:
Average Values per Culture Flask
Viable Cells
106 Units
2.14
1.91
1.96
1.83
1.91
1.53
1.62
None observed
Total Cells
10s Units
2.16
1.92
2.00
1.86
2.04
1.63
1.89
ATP .
108fgD
66.4
67.6
65.5
65.9
64.7
56.7
47.0
Initial Cell Viability: 98.8%
Viable Macrophage Seeded/Flask: 2.0 x 10s cells/flask
Macrophage Population Percentage: >90.0%
Survival of Negative Control
Macrophage Over Treatment Time: 99. IX
ATP Per
106 Cells
10« fg
30.7
35.2
32.8
35.4
31.7
34.8
24.9
dEC50 VALUES:
ug/ml:
Viability
%
99.1
99.5
98.0
98.4
93.6
93.9
85.7
Expressed
Viability
100.0
100.4
98.9
99.3
94.5
94.8
86.5
>1000
as Percent
Viability
Index
100.0
89.3
91.6
85.5
89.3
71.5
75.7
>1000
of Negative Control
ATP
100.0
101.8
98.6
99.2
97.4
85.4
70.8
>1000
ATP Per
106 Cells
100.0
114.7
106.8
115.3
103.3
113.4
81.1
>1000
fg = Femtogram (10-1S gram).
CNC = Negative Control, EMEM culture medium.
Determined from data plots in Figures 1 and 2.
Toxicity
Classification: Nondetectable
-------
TABLE 2
RABBIT ALVEOLAR MACROPHAGE (RAH) CYTOTOX1CITY ASSAY DATA
LBI Assay No.: 5887 (Trial II Ground sample)
Test Material Identity: A81-05-030-744 (EA-2 Flyash)
Test Date: October 13, 1981
Vehicle: EMEM
en
i
CO
CO
en
Sample
NCC
TEST
TEST
TEST
apH change
h_
Concentration3
ug/ml
...
300
600
1000
in culture medium:
Average Values
Viable Cells
108 Units
0.97
0.90
0.83
0.75
None observed
per Culture
Total Cells
106 Units
1.01
0.95
0.86
0.80
Flask
ATP K
108fgD
25.4
22.4
22.7
21.1
Initial Cell Viability: 99.3X
Viable Macrophage Seeded/Flask: 1 x 106 cells/flask
Macrophage Population Percentage: >90.0%
Survival of Negative Control
Macrophage Over Treatment Time: 96. OX
ATP Per
106 Cells Viability
108 fg %
25.1 96.0
23.6 94.7
26.4 96.5
26.4 93.8
dEC50 VALUES:
ug/ml:
Expressed
Viability
100.0
98.6
100.5
97.7
>1000
as Percent of Negative Control
Viability
* Index • ATP
100.0 100.0
92.8 88.2
85.6 89.4
77.3 83.1
>1000 >1000
ATP Per
106 Cells
100.0
94.0
105.2
105.2
>1000
fg = Femtogram (10-1S gram).
CNC = Negative Control, EMEM culture medium.
Determined from data plots in Figures 3 and 4.
Toxicity
Classification: Nondetectable
-------
FIGURE V
EC50 DETERMINATION FOR
PERCENT VIABILITY (0) AND VIABILITY INDEX (I)
A81-05-030-744
(EA-2 FLYASH)
TRIAL 1
10 100
CONCENTRATION, JJG/ML
1000
5-386
12
-------
§ 60
FIGURE 2
EC50 DETERMINATION FOR
ATP/FLASK (0) AND ATP/106 CELLS (I)
A81-05-030-744
(EA-2 FLYASH)
TRIAL 1
1000
CONCENTRATION, JIG/ML
5-387
13
-------
FIGURE 3
EC50 DETERMINATION FOR
PERCENT VIABILITY (0) AND VIABILITY INDEX (i)
A81-05-030-744
(EA-2 FLYASH)
TRIAL 2
1000
CONCENTRATION, JJ6/ML
5-388
14
-------
FIGURE 4
EC50 DETERMINATION FOR
ATP/FLASK (0) AND ATP/106 CELLS (•)
A81-05-030-744
(EA-2 FLYASH)
TRIAL 2
3 60 "—I-
o
£
10 100
CONCENTRATION, JJG/ML
1000
5-389
15
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ffl
Utton
VIII. ASSAY EVALUATION CRITERIA
The EC50 value represents the concentration of test material that reduces
the most sensitive parameter of the RAM assay to 50% of the vehicle or
negative control value. EC50 values are determined graphically by fitting
a curve by eye through relative toxicity data plotted as a function of
the logarithm of the applied concentration. Each data point normally
represents the average of three culture dishes. Statistical analysis is
unnecessary in most cases for evaluation.
The toxicity of the test material is evaluated as high, moderate, low,
or nondetectable according to the range of EC50 values defined in the
following table.
SolidsAqueous LiquidsNonaqueous Liquids
Toxicity3 (EC50 in \igM) (EC50 in Ml/ml) (EC50 in Ml/ml)
High
Moderate
Low
Not Detectable
<10
10 to 100
100 to 1000
>1000
<6
6 to 60
60 to 600
>600
<0.2
0.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests1.
Criteria for nonaqueous liquids are tentative and under evaluation. If
the organic or solid content is known, the solid evaluation criteria
are applied.
Another evaluation scheme is proposed for extracts obtained from SASS
train gas volumes. The proportion of the total gas volume corresponding
to the volume of extract used in the bioassay is calculated and expressed
as L/ml of culture medium (or DSCF/ml of culture medium). A criterion
of 1000 L/ml is set as the limit for nondetectable toxicity. This gas
volume corresponds to the average volume breathed by humans over a 2-hour
period. The subsequent toxicity ranges are defined by 10-fold dilution
steps to conform to standard procedure. The toxicity ranges are defined
in the following table for liter and dry standard cubic feet units:
In EC50 In
Toxicity Liters/ml (L/ml) Dry Standard Cubic Feet/ml (DSCF/ml)
High 1000 >35
5-390
BIONETICS 16
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IX. REFERENCES
1. Brusick, D.J., et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment ITological Tests.EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington,
MD, September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135, Litton Bionetics, Inc., Kensington, MD, August
1981, pp. 52.
BIONETICS
Lrtton
5-391
17
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GENETICS ASSAY NO.: 5887
LBI SAFETY NO.: 7171
TOXIC EVALUATION OF
A81-Q5-030-744
TEF2 FLYASH)
TN THE
EPA LEVEL~rAEDTE IN VIVO
RODENT TOXICITY ASSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MD 20795
LBI PROJECT NO.: 22064
REPORT DATE: NOVEMBER 1981
Utton
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PREFACE
This assay conforms to the standard EPA Level 1 procedure for the acute
™ vivo toxicity test in rodents as described in "IERL-RTP Procedures
Manual: Level 1 Environmental Assessment Biological Tests"1. The data
were evaluated and formatted as recommended in "Level 1 Biological Testing
Assessment and Data Formatting"2. The organisms used in this assay were
male and female weanling mice as recommended by the Level 1 Manual.1
The advantages of HI vivo toxicity assays are embodied mainly in the fact
that the toxicologTcal assessment is performed in whole animals. There
is a significant background of test data on a wide range of toxicants
for the rodent systems, thus supplying needed information for the reliable
interpretation of results with complex effluents3. The main disadvantage
of an acute rodent toxicity study is a possibly unsatisfactory prediction
of toxicity induced by long-term/ low-level exposures. An additional
consideration is the need for multi-gram quantities of test material
which may prohibit testing where small amounts of sample are available,
such as from source streams containing gaseous and particulate material.
Since the major objective of the Level 1 biological testing procedures
is to identify toxicological problems at minimal cost, a two-step approach
was developed for the initial acute j_n vivo toxicological evaluation of
unknown compounds. The first step is based on the quanta! (all-or-none)
response of dosing animals only at the maximum applicable dose. If no
animals die in the quantal test, further iji vivo testing is not initiated
and the sample toxicity is categorized as not detectable. If any animals
die in the quantal screening, a multiple dose quantitative test is initiated
to determine the dose that kills 50 percent of the animals (LD50)- The
toxicity potential of the test material is then ranked using standard
EPA Level 1 toxicity evaluation criteria for the acute iji vivo rodent
toxicity assay1.
All procedures and documents pertaining to the receipt, storage, prepara-
tion, testing and evaluation of the test material shall conform to Litton
Bionetics, Inc. standard operating procedures and the Good Laboratory
Practices Regulations of 1979. Deviations from standard procedure shall
be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology 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.
Litton
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TABLE OF CONTENTS
Page No.
PREFACE i
LIST OF TABLES iii
I. ASSAY SUMMARY 1
II. OBJECTIVES 2
III. TEST MATERIAL 3
A. Description 3
B. Handling and Preparation 3
IV. MATERIALS 4
A. Test Organisms 4
V. EXPERIMENTAL DESIGN 5
A. Quantal Test 5
B. Quantitative Test 5
VI. RESULTS
A. Interpretation 7
B. Tables 7
VII. EVALUATION CRITERIA 10
VIII. REFERENCES 11
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LIST OF TABLES
Table Title Page No.
1 Definition of Pharmacological Toxic Signs .... 6
2 Quantal Toxicity Data with Weanling Mice .... 8
3 Acute In Vivo Rodent Toxicity Assay
Evaluation Criteria 10
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I. ASSAY SUMMARY
A. SPONSOR: Acurex Corporation
B. MATERIAL (TEST COMPOUND): GENETICS ASSAY NO.: 5887
1. Identification: A81-05-030-744 (EA-2 Flyash)
2. Date Received: August 26, 1981
3. Physical Description: Gray and white particles with much
larger, long and thin black chunks.
C. TYPE OF ASSAY: EPA Level 1 Rodent Quanta! Toxicity Assay
D. STUDY DATES:
A. Initiation: October 5, 1981
B. Completion: October 23, 1981
E. SUPERVISORY PERSONNEL:
A. Study Director: David J. Brusick, Ph.D.
B. Senior Technician: Joan McGowan
F. EVALUATION:
The test substance, A81-05-030-744 (EA-2 Flyash), was not lethal
or toxic for weanling mice following an oral gavage dose of
5 gm/kg body weight. Although one female animal was found
dead, the death did not appear compound-related because of the
absence of toxic signs. Otherwise there were no unusual findings
upon necropsy that would suggest test substance related toxicity.
The LD50 of the test material was found to be higher than the
maximum applicable dose (MAD) of 5 gm/kg. The test sample
response was evaluated as being in the nondetectable range as
defined for the IERL-EPA Level 1 Rodent Quanta! Toxicity Assay1.
Submitted by:
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Litton
David J. Brusick, Ph.D. Uat
Director
Department of Molecular
Toxicology
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II. OBJECTIVES
The objective of this assay was to evaluate the acute toxicity of
sample A81-05-030-744 (EA-2 flyash) when administered by oral gavage to
male and female weanling mice. Test strategy involved initial testing
of the sample at the maximum applicable dose in the quantal assay. If
lethality was observed in the quantal assay, additional testing would be
initiated at lower doses to identify the LD50.
The assay consisted of recording any lethality and toxic signs that occur-
red initially and over a 14-day period following a single treatment.
Additional information was collected from necropsy observations on animals
that died during the course of the experiment or were killed at the end
of the 14-day observation period.
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III. TEST MATERIAL
A. Description
The test material, A81-05-030-744 (EA-2 flyash), was received as gray
and white particles with larger (long and thin) black chunks. The amount
of sample supplied was 15.0040 grams. No information on the sampling
parameter was provided.
B. Handling and Preparation
The test material was received at LBI on August 26, 1981. The sample
was assigned LBI safety number 7171 and LBI assay number 5887. The sample
was stored at +4°C in the dark.
On October 1, 1981, the test material was ground in a mortar and pestle
to a fine, black powder. The primary dosing suspension was prepared
24 hours in advance to permit water soluble materials in the flyash to
leach into the water at room temperature. A total of 1628.31 mg of test
material was suspended in 17.43 ml of sterile distilled water giving a
stock concentration of 93.6 mg/ml. This suspension would not pass freely
through a 246 gavage needle so it was discarded. On October 8, 1981,
approximately 2.5 gm of the previously ground sample was puliverized a
second time in a mortar and pestle. The suspension, prepared 24 hours
in advance of dosing, passed through the gavage needle without difficulty.
A total of 1411.04 mg of test material was suspended in 10.1 ml of sterile
water giving a stock concentration of 140 mg/ml.
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IV. MATERIALS
A- Test Organisms
The test organisms for this study were weanling Charles River CD-I mice.
Weanlings were used because they are likely to be more sensitive to toxic
effects of test materials than adult mice. In addition, significantly
less test material is required for dosing.
Eight nursing female Charles River CD-I mice with ten pups each (5 male
and 5 female) were obtained from Charles River Breeding Laboratories,
Inc., Wilmington, MA on September 30, 1981. The birth date of the pups
was September 13, 1981. The animals were quarantined for 5 days upon
receipt. The litters were individually housed on Ab-sorb-dri bedding in
polycarbonate cages and were cared for according to Litton Bionetics,
Inc., Department of Molecular Toxicology and LAMS Standard Operating
Procedures. Purina certified laboratory chow and water (pH 2.5) were
provided ad libitum. The pups were maintained with mothers until weaned.
The animals were identified by eartags and cage cards and were released
for study on October 9, 1981.
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V. EXPERIMENTAL DESIGN
A. Quanta! Test
Ten male and ten female weanling CD-I mice were used in the initial quanta!
screening test. The pups appeared to be in good health with no physical
or .behavioral problems noted. Pups that were selected were of similar
size. The pups were 26 days old at the time of dosing.
Prior to dosing, each animal was individually weighed and the mean weight
calculated for each sex. The volume of test material to be administered
was based on the mean weight if all animals were within plus or minus
15 percent of the average for the sex. If any animals were outside that
range, they were then excluded from the average, a new mean calculated
for the remaining animals and individual dosing volumes calculated for
each outlying animals.
The test material was administered by gavage to the pups at the rate of
5 gm/kg. The average weight of the males was 11.5 grams and that of the
females was 12.0 grams. The weight of one female, animal number 9058,
exceeded ±15 percent of the average of the females. This animal was
excluded, and the new average of 11.8 grams calculated for the females.
The test material, suspended at a concentration of 140 mg/ml, was applied
to the animals in two equal doses that totaled 0.41 ml for the males,
0.51 for the females, except animal number 9058 that received 0.42 ml.
Immediately following administration of the test substance and during
the first day, observations of the frequency and severity of all toxic
signs or pharmacological effects (Table 1) were recorded. Particular
attention was paid to time of onset and disappearance of signs. Observa-
tions were made and recorded on all animals through a 14-day period. At
termination of the observation period, all surviving animals were weighed,
killed, and then gross necropsies performed. Necropsies were also per-
formed on all animals that died during the course of this study.
B. Quantitative Test
Since no animals died during the preliminary quantal screening test, the
quantitative test to determine the LD50 was unneccessary.
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TABLE 1. DEFINITION OF PHARMACOLOGICAL TOXIC SIGNS
Organ System
Observation and
Examination
Common Signs of Toxicity
CNS and
somatomotor
Autonomic
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
Peri anal region
Color, turgor,
integrity
Conjunctiva, mouth
Eyeball
Transparency
Rectal or paw skin
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, flaccid skinfold,
eruptions, piloerection
Discharge, congestion,
hemorrhage cyanosis, jaundice
Exophthalmus, nystagmus
Opacities
Subnormal, increased temperature
Abnormal posture, emaciation
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VI. RESULTS
A. Interpretation
The test material, A81-05-030-744 (EA-2 flyash), was tested and evaluated
in the EPA Level 1 Acute In Vivo Rodent Toxicity Assay. The first phase
of testing was the quanta! toxicity test in which 10 male and 10 female
weanling CD-I mice were exposed to an oral dose of the test material.
This dose was at the maximum applicable dose (MAD) of 5 gm/kg as recom-
mended by the EPA Level 1 procedures manual1.
Nineteen animals survived the exposure with no evidence of any significant
compound-related behavioral or toxic signs (see Table 1 for definitions).
The animals seemed uncomfortable after dosing (slow moving, wiping mouth
and eyes half-shut) but animals appeared normal after 2 hours. There
was a small amount of test material on the muzzle of some animals after
dosing. One animal, female number 9053, was found dead on day 3 of the
study. The animal had been dead a number of hours; rigor mortis had set
in and the intestines were filled with gas. Necropsy of animal 9053
indicated necrosis of the liver but no other gross lesions. The death
of this animal did not appear directly attributable to the test material.
The test material was found to have an LD50 greater than the maximum
applicable dose of 5 gm/kg. Because of the lack of significant toxic
effects and because the death of animal number 9053 did not appear to be
compound-related, the quantitative study (LD50 determination) was unneces-
sary. The test material was evaluated as having nondetectable (ND)
toxicity based on EPA Level 1 evaluation criteria1.
B. Tables
This report is based on the data provided in Table 2.
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TABLE 2
QUANTAL TOXICITY DATA WTH WEANLING MICE
Quantal Toxicity: Weanling CD-I mice
Sponsor: Acurex Corporation
Test Article: A81-05-030-744 (EA-2 flyash)
Description: Gray and white particles with larger (long and thin) black
chunks. Sample pulverized to a fine, black powder.
Vehicle: Sterile, deionized water
Study Dates: October 8, 1981 to October 23, 1981
Animals: Charles River CD-I mice, P.O. 106949
Dose: 5 gm/kg administered by oral gavage
Animal No.
Males
9042
9043
9044
9045
9046
9047
9048
9049
9050
9051
Mean Body
Initial
Weight
gm
10.8
11.5
11.0
11.1
11.6
11.6
10.8
11.5
12.1
13.1
Weight: Initial
Final
Final
Weight
gm
19.1
21.2
20.8
23.9
22.4
22.3
20.7
21.0
23.0
25.8
= 11.5 ±
= 22.0 ±
Visible
Toxic
Signs Gross Necropsy Findings
NTSb
NTS
NTS
NTS
NTS
NTS
NTS
NTS
NTS
NTS
0.7 gm
1.9 gm
NSLC
NSL
NSL
NSL
NSL
Intestines yellow and
flaccid
NSL
NSL
NSL
. Large white mucous plug
in bladder and uretha.
(Standard Deviation)
(Standard Deviation)'
Animals observed over 14 days.
Note: Staining of the muzzle from the test material was noted in some
. animals after dosing. Animals seemed uncomfortable after dosing.
°NTS = No Toxic Signs.
CNSL = No Significant Lesions
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TABLE 2 (Continued)
QUANTAL TOXICITY DATA WTH WEANLING MICE
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Animal No.
Females
9052
9053
9054
9055
9056
9057
9058
9059
9060
9061
Mean Body
Initial
Weight
gm
11.3
11.3
11.1
13.1
11.3
11.6
14.3
11.9
12.4
12.0
Weight:
Initial
Final
Final
Weight
gm
18.4
9.5
19.6
20.3
18.4
19.4
19.4
19.3
18.9
20.3
= 12.0 ± 1.0
= 19.3 ± 0.7
Visible
Toxica
Signs
NTSb
Deathd
NTS
NTS
NTS
NTS
NTS
NTS
NTS
NTS
gm (Standard
gm (Standard
—.,. - '"•
Gross Necropsy Findings
NSLC
Animal had been dead
several hours, abdomen
bloated; intestines light
red and filled with gas.
Liver dark green colored
with apparent necrosis.
Lungs pale but normal.
No other gross abnormal-
ities noted.
NSL
NSL
NSL
NSL
NSL
NSL
NSL
NSL
Deviation)
Deviation)
Animals observed over 14 days.
Note: Staining of the muzzle from the test material was noted in some
. animals after dosing. Animals seemed uncomfortable after dosing.
°NTS = No Toxic Signs.
jNSL = No Significant Lesions
Animal found dead 8:00 a.m. 10-12-81 (day 3 of the study), last seen
alive 9:00 a.m. 10-11-81.
Animal 9053 excluded from average.
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VII. EVALUATION CRITERIA
If no mortality occurs in the quantal study, no further studies will be
performed with the test substance and the LD50 should be reported as
greater than 5 ml/kg or 5 g/kg. The test material is then ranked as
having nondectable 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 observations are
difficult to quantitate but provide insight into the sub!ethal.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 LD50 will be calculated
by the method of Litchfield and Wilcoxin4. If the data are not suitable
for calculation of a precise LD50, i.e., total mortality occurs for the
lowest dose, an estimate of the LD50 could be made or the LD50 could be
expressed as 0.05 ml/kg or 0.05 g/kg or less. Occasionally, it may be
necessary to use a different series of dosages in a repeat study to
accurately locate the LD50. The calculated LD50 value is used to rank
the toxicity of the test material according to the dose ranges presented
in Table 3.
Frequent observations are also made and recorded on all animals through
the 14-day period. As in the quantal phase, no attempt is made to quanti-
tate or rank 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 3
ACUTE IN VIVO RODENT TOXICITY ASSAY EVALUATION CRITERIA
Toxicity3
High
Moderate
Low
Not Detectable
Solids
(LD50 in g/kg)
<0.05
0.05 to 0.5
0.5 to 5
>5
Liquids
(LD50 in ml /kg)
<0.05
0.05 to 0.5
0.5 to 5
>5
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Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests.1
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VIII. REFERENCES
1. Brusick, D.J., et al.: IERL-RTP Procedures Manual: Level 1 Environ-
mental Assessment Biological Tests.EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington,
MD, September 1980, 177 pp., in press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc., Kensington, MD, April
1980, 100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Bioassay Sensitivity.
EPA-600/7-81-135 Litton Bionetics, Inc., Kensington, MD, August
1981, 52 pp.
4. Litchfield, J.T. and Wilcoxin, F.: "A Simplified Method of Evaluation
Dose-Effect Experiments." J. Pharmac. Exp. Ther., Vol. 96, 1949,
pp. 99-113.
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THE ACUTE TOXICITY OF FIVE
SAMPLES TO FRESHWATER ORGANISMS.
SUBMITTED TO
ACUREX CORPORATION
MOUNTAIN VIEW, CALIFORNIA
REPORT IBW-81-7-966
EG&G, Bionomics
Aquatic Toxicology Laboratory
790 Main Street
Wareham, Massachusetts
July, 1981
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TABLE OF CONTENTS .
PAGE
INTRODUCTION 1
MATERIALS AND METHODS 2
Test Organisms 2
Test Conditions ' 4
Water flea 4
Fathead minnow 6
Freshwater algae 7
Statistical Analysis 8
RESULTS 10
LITERATURE CITED 11
TABLES 12-30
APPENDIX A 31
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INTRODUCTION
The purpose of this study was to estimate the acute
toxicity of five samples received from the Acurex Corporation
to freshwater organisms. All five materials were tested with
the water flea (Daphnia magna) and the freshwater algae (Selenastrum
capricornutum) . Three of the samples were also tested with the
fathead minnow (Pimephales promelas). Results of tests performed
with water fleas and fathead minnows are reported as median lethal
concentrations (LCSO's) and corresponding 95% confidence intervals.
Results of the tests performed with the freshwater alga are re-
ported as the median effect concentration (EC50) and corresponding
95% confidence interval. Toxicity tests performed with water
fleas and fathead minnows were conducted at the Aquatic Toxicology
Laboratory of EG&G, Bionomics, Wareham, Massachusetts and the
tests performed with the alga were conducted at EG&G, Bionomics
Marine Research Laboratory (BMRL), Pensacola, Florida. All raw
data related to these tests are stored at the respective laboratory
at which they were performed.
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MATERIALS AND METHODS
Methods used in performing the acute toxicity tests followed
those described in "IERL-RTP Procedures Manual: Level I Environ-
mental Assessment Biological Tests" (1980) unless specified
otherwise.
The five samples were received at EG&G, Bionomics, Wareham,
Massachusetts on 24 June 1981. The samples were received at
ambient temperature (20-25°C) and were refrigerated (4°C) upon
receipt. On 25 June, a portion of each sample was shipped to
BMRL. Samples were received at BMRL on 26 June. At BMRL, the
four solid samples were stored at ambient room temperature, while
the liquid sample was stored at 4°C. The five samples are des-
cribed in Appendix A. Tests performed with D. magna and P. promelas
were limited to a high test concentration of 1000 mg/i. If
insufficient mortality was observed at this treatment level, the
LC50 was estimated to be >1000 mg/£.
Test Organisms
The water flea used in this toxicity test were obtained from
laboratory stocks cultured at EG&G, Bionomics. The culture water
was prepared by reconstituting deionized water (U.S. EPA, 1975)
and filtering it through an Amberlite XAD-7 resin column to remove
any potential organic contaminants. This water had a total hardness
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and akalinity as calcium carbonate (CaC03) of 170 + 15 mg/i
and 120 + 10 mg/i, respectively, a pH range of 7.9-8.3, a temper-
ature of 22 + 1°C, a specific conductance of 400-600 micromhos
per centimeter (ymhos/cm), and a dissolved oxygen (DO) concentra-
tion of greater than 5.3 mg/i (60% of saturation).
The fathead minnows (Bionomics lot #81A6) were obtained from
cultures spawned and raised at EG&G, Bionomics, Wareham, Massachu-
setts. The fish were held in a 500-A fiberglass tank under a
photoperiod of 16 hours light and 8 hours darkness. All fish were
fed a dry, pelleted food, ad libitum, daily except during the
48 hours prior to testing. There was no mortality in the test
fish population during this 2 day period (Daily Record of Fish
Holding Conditions). The well water which flowed into this tank
was characterized as having total hardness and alkalinity ranges
as calcium carbonate (CaCO3) of 20-25 mg/i and 20-28 mg/i, respec-
tively, and a specific conductance range of 90-110 micromhos per
centimeter (ymhos/cro) (Weekly Gravity Feed Tank Water Quality
Analysis Logbook). Other parameters monitored in the holding
tank were a pH range of 6.2-6.9, a dissolved oxygen (DO) range of
80-92% of saturation and a flow rate range of 6-7 tank volume
replacements/day (Weekly Record of Fish Holding Water Characteris-
tics) . Test fish were maintained under these conditions for a .
minimum of 14 days. The temperature in the holding tank ranged
from 20-22°C during this 14 day period (Daily Record of Fish Holding
Conditions). The specific conductance was measured with a YSI
Model #33 conductivity meter, the pH was measured with an Instru-
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mentation Laboratory Model #175 pH meter and combination elec-
trode, the DO was measured with a YSI Model #57 dissolved oxygen
meter and probe and the temperature was measured with a Brooklyn
alcohol thermometer. Total hardness and alkalinity were measured
according to APHA et al. (1975). .
The freshwater alga were obtained from the U.S. Envir-
onmental Protection Agency's Environmental Research Laboratory,
Corvallis, Oregon and maintained in stock culture at BMRL. Culture
procedures used for the alga followed those described in "IERL-RTP
Procedures Manual: Level I Environmental Assessment Biological
Tests" dated September 1980.
Test Conditions
Water flea
The toxicity tests exposing D. magna to the samples were
conducted in 250 milliliter (m&) glass beakers. The dilution water
used during this study was prepared in the same manner as the
culture water except that the quantity of salts were reduced to
yield a total hardness and alkalinity of 107 mg/fc as CaC03 and
69 mg/i as CaC03, a pH of 8.0 and a specific conductance of 400
9
pmhos/cm. For each test concentration, the appropriate amount
of test material was added to dilution water to total 1000 m£,
then vigorously mixed on a magnetic stirrer. Eight hundred milli-
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liters of this test mixture were divided into four beakers to
provide replicate exposure treatments each containing 200 mi,
The remaining 200 mi of the control and the high, middle and low
test concentrations were used for 0-hour dissolved oxygen (DO) ,
pH and specific conductance determinations. Four control beakers
containing the same dilution water and maintained under the same
conditions as the exposure concentrations, but containing no
sample, were established. The ambient air temperature in the
laboratory was controlled in order to maintain test solution
temperatures at 21-22°C. Test solutions were not aerated. The
test area was illuminated with Durotest (Optima) fluorescent
lights at an intensity of 50-70 footcandles.
Twenty water flea, £24 hours old, were impartially distri-
buted to each concentration (5 water flea per replicate) within
30 minutes after the test solutions had been prepared. Mortalities
in replicate test solutions were recorded at 24 and 48 hour expo-
sures. Biological observations and observations of the physical
characteristics of each replicate test solution were also made
and recorded at 0, 24 and 48 hours. The pH, DO and specific con-
ductance were measured at 0 and 48 hours of exposure in the control
and the high, middle and low test solutions. The temperature of
the control and all test concentrations were measured at 0, 24 „
and 48 hours exposure.
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Fathead minnow
Toxicity tests performed with the fathead minnow were per-
formed in 19.6-£ glass jars which contained 15-* of test solution.
The dilution water used was hard water reconstituted from
deionized water according to recommended procedures (U.S. EPA,
1975). This water had a total hardness and alkalinity as CaC03
of 94 mg/£ and 68 mg/fc, respectively, a pH of 7.9 and a specific
conductance of 345 ymhos/cm (Reconstituted Water, Quality
Analysis).
Test solutions were prepared by adding appropriate amounts
of test material directly to test vessels containing a sufficient
quantity of dilution water to total 15 £. Solutions were mixed
by stirring with a glass rod. Each test concentration and controls
were replicated.
Two control jars containing the same dilution water as used in
the exposure jars, but containing no test material, were established.
All test solution temperatures were controlled by a system which
maintained temperatures at 21-22°C. Test solutions were not
aerated during the exposure period. The photoperiod during testing
was the same as that provided during acclimation.
Ten fathead minnows with a mean (range, N=30) wet weight and
total length of 0.29(0.13-0.47) grams and 33(25-38) millimeters,
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respectively (Fish Weights and Lengths Log) were randomly disti-
buted to each test jar within 10 minutes after the test solutions
had been prepared.
Mortalities were recorded and removed from each test jar
at 0, 24, 48, 72 and 96 hours exposure. Biological observations
of the fish and observations of the physical characteristics of
the test solutions were also made at each 24 hour interval. The
pH and DO concentrations of the control, high, middle and low test
concentrations were measured at 0, 24, 48 and 96 hours of the exposure
period. Specific conductance of the control, high, middle and
low test concentrations were measured at 0 hour. The temperature
was measured in the control jar every 24 hours during exposure.
Freshwater algae
The toxicity tests exposing the freshwater alga to the test
sample was conducted in 125 mJl flasks each of which contained
50 mi of test medium. Beginning cell numbers in the test flasks
were approximately 1.0 x 10* cells/mil. Triplicate cultures were
employed for each of the test concentrations and control. Cultures
were incubated at 24°C under approximately 2,400 lux illumination.
Cell counts were made at 0- and 120-hour exposures using a hema*-
cytometer and a Zeiss Standard 14 compound microscope. The pH of
all test solutions were measured at 0 and 120 hours of exposure.
5-415
-------
Statistical Analysis
The concentrations tested and corresponding mortality data
derived from the toxicity tests exposing water flea and fathead
minnows to the test materials were used to estimate median lethal
concentrations (LC50) and 95% confidence intervals. The LC50 is
defined as the concentration (nominal or measured) of the test
compound in dilution water which caused mortality of 50% of the
test animal population at the stated exposure interval. The compu-
ter program utilized (Stephan, 1978, personal communication) esti-
mated LC50 values using one of three statistical methods in the
following order of preference: moving average angle analysis,
probit analysis, binomial probability. The method selected was
determined by the characteristics of the data base (i.e. presence
or absence of test concentrations causing 100% mortality of the
test animal population, number of concentrations causing mortality
of a partial number of the test animal population). The computer
program scanned the data base, identified the most preferred
statistical method and performed the analysis. The no discernible
effect concentration was also determined for each effluent sample.
The no discernible effect concentration is defined as the highest
concentration tested .at which there were no mortalities or observed
behavioral and physical abnormalities (i.e. erratic swimming, .
flared carapace).
5-416
-------
The concentrations tested and corresponding percentage
decrease in cell numbers from the toxicity tests exposing the
freshwater alga to the test materials were used to estimate con-
centrations of each sample that caused a 50% and 95% decrease in
cell numbers of the exposed cultures, EC50 and EC95, respectively.
Each test concentration was converted to a logarithm and the
corresponding percentage decrease of cell numbers was converted
to a probit (Finney, 1971). The 120-hour ECSO's and EC95's and
their respective 95% confidence intervals were calculated by linear
regression.
5-417
-------
RESULTS
The estimated LC50 values, 95% confidence intervals and no
discernible effect concentrations for D. magna and P. promelas
exposed to the test samples are presented in Table 1. Table 2
presents the estimated 120-hour EC50 and EC95 values and corres-
ponding 95% confidence intervals for S_. capricornutum exposed
to the test samples. P. promelas was the least sensitive species
to the effects of the test materials. None of the three samples
(A80-09-023-5, A81-05-030-662, A81-05-031-765) tested with P.
promelas exhibited toxicity with this organism. S. capricornutum
was the most sensitive species to the toxic effects of the test
materials. All of the ash samples had 120-hour EC50 values less
than 400 mg/ji. The 48-hour LC50 values for the 5 materials tested
with D. magna ranged from 680 mg/£ to >1000 mg/£.
The nominal concentrations of the test materials and corres-
ponding effects for the three species tested are presented in
Tables 3-15. The water quality parameters measured during the
toxicity tests with D. magna and P. promelas are presented in
Tables 16 and 17, respectively. The pH of the test solutions
measured during the tests performed with S. capricornutum are
presented in Table 18.
5-418
-------
LITERATURE CITED
APHA, AWWA, WPCF. 1975. Standard methods for the examination
of water and wastewater. 14th Edition, Washington, D.C.
1193 pp.
Finney, D.J. 1971. Probit Analysis. Cambridge University
Press, London. 333 pp.
IERL-RTP Procedures Manual: Level 1 Environmental Asssessment
Biological Tests. 1980. 149 pp.
Stephan, Charles. 1978. U.S. EPA, Environmental Research
Laboratory, Duluty, Minnesota. Personal communication.
U.S. EPA. 1975. Methods for acute toxicity tests with fish,
macroinvertebrates, and amphibians. Ecological Research
Series (EPA-660/3-75-009) , 61 pp.
5-419
-------
TABLES*
*Results of samples other than those pertinent to this study (A81-05-030-662
and A81-05-030-744) have been purposely deleted from the original report.
5-420
-------
Table 1. Estimated LC50 values, confidence intervals and no discernible effect concentrations for D. magna
and P. proroelas exposed to Acurex samples.
Sample
Species
LC50 (95% confidence interval)*
24 hour
48 hour
72 hour
96 hour
No discernible
effect concentration
(mg/fc)
A81-05-030-662 D. magna
i
-F*
>1000
P_. promelas >1000
'-^ A81-05-030-744 D. magna >1000
-680
(570-830)
>1000
960
(830-1200)
>1000
>1000
220
1000
360
mg/i.
Estimated by the moving average angle method.
-------
Table 2. Calculated 5-day ECSO's and EC95's for Selenastruro capricornuturo
exposed to the five samples provided by the Acurex Corporation.
The EC values were based on decrease of cell numbers on exposed
cultures as compared to the control. (The 95% confidence limits
are in parentheses). Concentrations were based on
' milligrams
of the samples per liter of algal growth medium.
Sample EC50 EC95
A81-05-030-662 290(204-412) 853(534-1,362)
A81-05-030-744 347(328-367) 894(830-963)
5-422
-------
Table 6. Concentrations tested, corresponding percentage mortalities and observations made during the
48-hour exposure of the water flea (Daphnia magna) to the sample coded A81-05-030-662.
Nominal
concentration
en
IM
co
(mg/D
1000
600
360
220
130
control
A
0
0
0
0
0
0
B
20
0
0
0
0
0
24 hour
C
20
0
0
0
0
0
D
20
0
0
0
0
0
X
b
15
0
0
0
0
0
A
80
80
20
0
0
0
B
60
60
0
0
0
0
48 hour
C
80
20
0
0 •
0
0
D
80
20
0
0
0
0
X
b
75
45b
5
0
0
0
a
A dark gray colored particulate matter was present on the bottom of all mixtures of A81-05-030-662.
b
Several daphnids were lethargic.
-------
Table 7. Concentrations tested and corresponding percentage mortalities
of fathead minnows (Pimephales promelas) exposed to the sample
coded A81-05-030-662 for 24, 48, 72 and 96 hours.
Nominal
concentration* 24 hour
-------
Table 8. Results of a 5-day exposure of the freshwater algae Selenastrum
capricornutun to A81-05-030-662. Percentage change is decrease
of cell numbers in exposed cultures as compared to the control
at day 5.
Nominal concentration
(mg/fc) Percentage change
control
125 -7
250 -56
500 -84
1,000 -94
5-425
-------
Table 9. Concentrations tested, corresponding percentage mortalities and observations made during the
48-hour exposure of the water flea (Daphnia magna) to the sample coded A81-05-030-744.
Nominal
concentration
en
i
01
(mg/fc)
1000
600
360
220
130
control
A
0
0
0
0
0
0
B
0
0
0
0
0
0
24 .hour
C
0
0
0
0
0
0
D
0
0
0
0
0
0
X
ob
ob
0
0
0
0
A
20
0
0
0
0
0
B
40
20
0
0
0
0
48 hour
C
80
0
0
0
0
0
D
80
0
0
0
0
0
X
55b'C
5b
0
0
0
0
a
A gray-black colored particulate matter was present in all test mixtures of A81-05-030-744 ,
b
Particulate matter was adhering to many daphnids.
c
Several daphnids were lethargic.
-------
Table 10. Results of a 5-day exposure of the freshwater alga Selenastrum
capricornutum to A81-05-030-744. Percentage change is decrease
of cell numbers in exposed cultures as compared to the control
at day 5.
Nominal concentration
(mg/£) Percentage change
control
125 -4
250 -29
500 -?1
1,000 -9?
5-427
-------
Table 16. Water quality characterization of the test solutions measured
during the acute toxicity tests exposing the water flea (Daphnia
magna) to the Acurex test samples.
Nominal
con centration
Sample
Dissolved
oxygen
(ng/i)
Specific
conductance
(pmhos/cm)
A81-05-030-662
1000
360
130
control
6.1-8.2
8.1-8.3
8.0-7.9
7.5-7.6
10.1-9.2
9.2-8.8
8.9-8.5
8.1-8.1
430-420
370-400
360-370
350-360
A81-05-030-744
1000
360
130
control
8.2-8.1
8.2-8.0
8.3-8.0
7.5-7.6
10.2-9.0
9.5-8.7
8.9-8.3
8.1-8.1
380-380
370-380
350-360
350-360
0-48 hour measurements.
5-428
-------
Table 17. water quality parameters measured during 96-hour toxicity tests with Acurex
test samples and fathead minnow (Pimephales promelas).
Sample
Nominal
concentration
Parameter
0 hour 24 hour 48 hour 72 hour 96 hour
A81-05-030-662
pH
DO (mg/i)
specific
conductance
(ymhos/cm)
1000
360
130
control
1000
360
130
control
1000
600
360
220
130
control
9.9
9.4
8.5
8.0
8.7
8.6
8.6
8.5
380
370
360
350
345
340
10.0
9.2
8.6
7.5
6.9
7.6
7.5
7.7
-
-
-
_
9.8
9.1
8.4
7.3
5.8
6.8
6.1
7.4
-
-
-
^
9.6
8.9
8.3
7.3
5.8
6.8
6.7
5.5
—
-
—
—
9.4
8.8
8.2
7.3
5.6
6.2
6.6
4.6
-
-
_
5-429
-------
Table 18. pH's of test solutions during the acute toxicity tests exposing the
freshwater alga (Selenastrum capricornutum) to Acurex test samples.
Sample
Nominal
concentration
(ppm)
0 hour
120 hour
A81-05-030-662
A81-05-030-744
1000
500
250
125
control
1000
500
250
125
control
10.5
10.2
9.9
9.4
7.2
10.4
10.1
9.5
9.3
7.2
8.9
8.6
9.7
8.9
8.2
8.8
8.6
9.2
8.5
8.2
5-430
-------
Appendix A
Sample code
Sample name
Species
tested
Amount of
sample received
Sample description
A81-05-030-662
A81-05-030-744
co
EA-1
flyash
EA-2
flyash
D. magna
P_. promelas
S_. capricornutum
I), magna
IL- capricornutum
100 g
20 g
dark gray ash with white
flakes
dark gray ash
-------
D. magna and P. promelas tests
SUBMITTED BY:
EG&G, Bionomics
Aquatic Toxicology Laboratory
790 Main Street
Wareham, Massachusetts
August, 1981
PRINCIPAL INVESTIGATORS:
Donald C. Surprenant
m
Aquatic Biologist
Joseph V. Sousa
£j£jff/tf(rt
Aquatic Biologist
STUDY DIRECTOR:
Gerald A. LeBlanc
ft
Aquatic Toxieologist
DATA AUDITED BY:
Robert E. Bentley
Director, Quality Assurance Unit
5-432
-------
£. capricornuturo tests
PREPARED BY:
Terry A. Hollister
A.
Study (Director
v
Date
AUDITED BY:
Alan G. Miller
Quality Assurance Unit (/
Raw data audit: /<£ J*>&f
Date
Preliminary report audit: /(> .J&xsj
Final report audit: /£
/P
-------
TECHNICAL REPORT DATA .
(Please read Inunctions on the reverse before completing)
i. REPORT NO.
EPA-600/7-87-Ol2b
2.
3. RECIPIENT'S ACCESSIOf*NO.
4. TITLE AND SUBTITLE
Environmental Assessment of a Wood-Waste-Fired
5. REPORT DATE
March 1987
Industrial Watertube Boiler, Volume II.
Supplement
Data
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
C. Castaldini and L. R. Waterland
B. PERFORMING ORGANIZATION REPORT NO.
TR-82-98/EE
9. PERFORMING OROANIZATION NAME AND ADDRESS
Acurex Corporation
P. O. Box 7555
Mountain View, California 94039
10. PROGRAM ELEMENT NO."
11. CONTRACT/GRANT NO.
68-02-3188
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Office of Research and Development
Air and Energy Engineering Research Laboratory
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final: 3/81 - 3/84
14. SPONSORING AGENCY CODE
EPA/600/13
15. SUPPLEMENTARY NOTES AEERL project officer is Robert E. Hall, Mail Drop 65,
2477.
919/541-
16. ABSTRACT
two-volume report gives results from field tests of a wood-waste-fired
industrial watertube boiler. Two series of tests were performed: one firing dry (11%
moisture) wood waste, and the other firing green (34% moisture) wood waste. Emis-
sion measurements included: continuous monitoring of flue gas emissions; source
assessment sampling system (SASS) sampling of the flue gas with subsequent labor-
atory analysis of samples to give total flue gas organics in two boiling point ranges,
compound category information within these ranges, specific quantisation of the semi-
volatile organic priority pollutants, and flue gas concentrations of 73 trace elements?
Method 5 sampling for particulate; controlled condensation system sampling for SO2
and SO3; and grab sampling of boiler mechanical collector hopper ash for inorganic
and organic composition determinations. Flue gas CO emissions from the boiler
were quite high, attributed to the high excess air levels at which the unit operated.
NOx emissions were comparable with both fuels (175-200 ppm). SO2 and SO3 levels
were less than 10 ppm, in keeping with the low sulfur content of sboth fuels. Total
organic emissions decreased from 60-135 mg/dscm firing dry wood to 2-65 mg/
dscm firing green wood, in parallel with corresponding boiler CO emissions.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Croup
Pollution Sulfur Oxides
Wood Wastes Nitrogen Oxides
Water Tube Boilers
Flue Gases Trace Elements
Assessments Carbon Monoxide
Particles Organic Compounds
Polycyclin Compounds
Pollution Control
Stationary Sources
Particulate
Environmental Asses-
sment
07B
13B
11L
13 A
21B
14B
14G 07 C
06A
is. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (THis Report)
Unclassified
21. NO. OF PAGES"
469
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
5-434
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