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-------
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-------
ANALYSES
SAFETY SPECIALISTS, Inc.
3284 F Edward Avanua. Stnta Ctira. California 96060
ASSAY REPORT
Taiephon* (408) 988-1111
Acurex Corporation
485 Clyde Avenue
Attn: Ms. Linda Bohannon, M/S 0-1212
Mountain View, California 94042
Date: June 3, 1981
Date Samples Received: 4/30/81
Customer Order No.: RB59185A, Rel. 10
SSI No.
Client Description
Activity
Gross Alpha
pCi/gram
Gross Beta
pCi/gram
C
D
E
Adelphi - 1, ly + filter
Adelphi - 1, 3y + 10y
Adelphi - 2, filter rinse +
10y + 3u
Adelphi - 2, lu + filter
224.1 ± 47.7 128.8 ± 38.6
196.7 ± 20.0 79.3 ± 11.3
93.7 ± 8.5
24.8 ± 4.8
40.5 ± 14.7
16.3 ± 2.9
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
w * i w i *
The £ values are equal to or less than three sigma of the counting error.
5-72
-------
SAFETY SPECIALISTS, Inc.
3284F Edward Avenue, Santa Clara, California 95050 • Teieohone (403) 988-11'
ASSAY REPORT
Acurex Corporation
Attn: Mr. Larry Waterland
485 Clyde Avenue
Mountain View, California 94042
Date: August 7, 1981
Date Samples Received: 4/30/81
Customer Order No.: RB59185A. Rel. 1C
SSI No.
Client Description
Activity
Gross Gamma
p Ci/o
C
D
E
Adelphi - 1, IP and 1, filter
Adelphi - 1, 3u and 1, 10y
Adelphi 2, filter rinse; 2, 10y
and 2, 3u
Adelphi 2, ly and 2, filter
553
183
219
300
292
293
100 ± 290
Analyst: Pamela S. Snreve
Approved: T. C. Noble, Director
Safety and Health Services
*The ± values are the two sioma Poisson standard deviation of the counting
error.
The < values are equal to or less than three sigma of the counting error.
5-73
-------
GENETICS ASSAY NO. 5806
SAFETY NO. 7032
MUTAGENICITY EVALUATION OF
424 (ADELPHI 1 XAD EXTRACT)
IN THE
EPA LEVEL 1
AMES SALMONETCA7MICROSOME
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: SEPTEMBER 1981
BIONETICS 5-74
-------
ffl
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Ames
Salmone11 a/microsome mutagenesis assay as described in "IERL-RTP Proce-
dures Manual: Level 1 Environmental Assessment Biological Tests"1. Tne
data was 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 cnenr-
cals representing various chemical classes3. This assay is one of severa:
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 Act 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.
BIONETICS
Utton 5-75
-------
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
8. Mutagenicity Test . 6
1. Nonactivation Assay '. . . 6
2. Activation Assay 6
C. Control Compounds 8
D. Recording and Presenting Data 8
VI. RESULTS 9
A. Interpretation 9
B. Tables . . . . 9
VII. ASSAY ACCEPTANCE AND 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
m
Utton
BIONETICS 5_76
-------
I. ASSAY SUMMARY
A. Sponsor: Acurex Corporation
B. Material (Test Compound): Genetics Assay Number: 5806
1. Identification: 424 (Adelphi 1 XAD extract)
2. Date Received: June 28, 1981
3. Physical Description: Clear, yellow 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: August 22, 1981
2. Completion: September 7, 1981
F. Supervi sory Personnel:
A. Study Director: D.R. Jagannath, Ph.D.
G. Evaluation:
The test material, 424 (Adelphi 1 XAD extract), was evaluated
for mutagenic activity over a concentration range of 10 ul/plate
to 200 ul/plate in the Ames Salmonella/microsome assay. With-
out metabolic activation, the test results were negative for
all test strains. With metabolic activation, strains TA-1537,
TA-98 and TA-100 were all positive. TA-1535 was negative but
was evaluated at only one test concentration because of limited
sample. The minimum effective concentration (MEC) was found
to be 10 MVplate in strain TA-98 with activation. This was
equivalent to 49 ug organics per plate. This was evaluated
as essentially on the borderline between high and moderate
(H/M) mutagenicity categories defined for the Level 1 Ames
assay1.
Submitted by:
Study Director
Reviewed by:
m
Litton
jfar : D.R. Jagannat
Section Chief,
Submammalian Genetics,
Department of Molecular
Toxicology
BIONETICS
'Date
DavT9 J. Brusick, Pt^D.
Director,
Department of Molecular
Toxicology
ate'
5-77
-------
II. OBJECTIVE
The objective of this study was to determine the genetic activity of 424
(Adelphi 1 XAD extract) 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.
CH
BIONETICS 5-78
Litton
-------
m
Utton
III. TEST MATERIAL
A. Description
The test material was received as a clear, yellow solution in methylene
chloride. The sample contained 17.0 milligrams of organic material in ar
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 June 28, 1981. The sample was
assigned LBI safety number 7032 and LBI assay number 5806. 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 3.5 ml of DMSO,
giving a sample concentration of 4.9 mg organics per ml DMSO. The sample
was transferred to a glass vial and sealed with a teflon-coated rubber
septum.
A total volume of 2.56 ml of test material was used between three trials.
In all trials, varying sized aliquots of the test material were added
directly to the mixtures to give the desired concentration. The amount
of sample used was 0.36 ml for Trial 1, 1.08 ml for Trial 2 and 1.12 ml
for Trial 3.
BIONETICS 5"79
-------
IV.
A.
MATERIALS
Indicator Microoraanisms
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
TA-1537
TA-98
TA-100
Gene
Affected
his G
his C
his D
his G
Additional Mutations
Repair
A uvr B
A uvr B
A uvr B
A uvr B
IPS
rfa
rfa
rfa
rfa
R Factor
-
pKMlOl
pKMlOl
Mutation Type
Detected
Base-pair
substitution
Frameshift
Frames hi 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
Litton
BIONETICS
5-80
-------
overlay agar consisted of 0.6% purified agar with 0.05 niM rvstidine,
0.05 mM biotin and 0.1M NaCl according to the methods of Ames et z\_/-
C.
1.
Activation System
59 Homogenate
A 9,000 x cj supernatant prepared from Sprague-Dawley adult male rat live-
induced by Aroclor 1254 (Ames et al_.9) was purchased from Bionetics Labora-
tory Products, Litton Bionetics, Inc. 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
m
Litton
5-81
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 (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 pi 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.
Because the amount of test material was potentially limiting, a preliminary
range-finding trial was run using a single plate per dose with strain TA-98
without metabolic activation. This trial, as did the two subsequent trials,
used the recommended dose levels of 200, 100, 50, and 10 ul 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 follow-
ing 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 £9 mix (see IV C,
Activation 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.
Ltd BIONETICS 5-82 6
Litton
-------
Aliquot of
saline
AMES ASSAY [PLATE INCORPORATION METHOD]
Molten [45°C] overlay agar
appropriately supplemented
0.5 ml
0.01 - 0.2 ml
Test, positive or solvent
control chemical
0.1 mi
Aliquot of an overnight culture
of bacteria! 109 cells I 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
5-83
-------
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 Salmonel"
Assay
Nonactivation
Chemical
Sodium azide
2-Nitrofluorene
(NF)
9-aminoacridine
(9AA)
Solvent (pg)
Water
D i methyl -
sulf oxide
Ethanol
10.0
10.0
50. D
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.
ffl
Litton
5-84
BIONETICS
-------
E
Utton
VI. RESULTS
A. Interpretation
The test material, sample 424, (Adelphi 1 XAD extract), was initially
tested in strain TA-98 without activation. This trial served as a range-
finding study to help select doses for subsequent testing with all four
strains. The dose range for all trials was 200, 100, 50 and 10 ul per
plate using a single plate per dose. Trial 2 was with strain TA-98 with
activation and strain TA-100 both with and without activation. Trial 3
employed strains TA-1535 and TA-1537 both with and without activation.
The nonactivation results for all strains tested were negative. Slight
increases were observed at 100 ul/plate with TA-1537, TA-98 and TA-100
but they were not close to the criteria set for designating a positive
response.
The activation test results for strains TA-1537, TA-98, and TA-100 were
all positive by established criteria. The mutagenic response was generally
dose-related over the range of concentration tested. TA-1535 was not
active at the single dose of 200 ul/plate that was tested.
The minimum effective concentration (MEC) was found to be 10 ul per plate
with strain TA-98 with activation. This concentration was equivalent to
49 ug or organics per plate. This value was essentially on the borderline
between high and moderate (H/M) mutagenicity using IERL-EPA Level 1
criteria for the Ames bioassay1.
Solvent control and positive control values were within acceptable ranges.
These results achieved the assay acceptance criteria and provided confi-
dence in the assumption that the recorded data represented typical respon-
ses to the test material.
B. Tables
This report is based on the data provided in Tables 1 and 2.
5-85
BIONETICS
-------
RrSULTS
TAMLE t
A. NAME OR CODE DESIGNATION OF THE IEST COMPOUND: 424IADELPHI 1 XAD EXTRACT)
R. SOLVENT: OHSO
c. TEST INITIATION DATES: 08/22/01 on/25/ni
0. TtST COMPLETION DAU: 09/01/81
E. S-9 L01»: S9-11
NOTE: CONCENIRATIONS ARE GIVEN IN MICROLITEHS PER PLATE
en
oo
TEST
NONACTIVATION
SOLVENT CONTROL
SOLVENT CONTROL
POSITIVE CONTROL**
POSITIVE CONTROL**
TEST CO WOUND
10.000 UL
50.000 UL
100*000 UL
200.000 UL
ACTIVATION
SOLVENT CONTROL
SOLVENT CONTROL
POSITIVE CONTROL**
SPECIES
...
___
-_-
— -.
RAT
RAT
* RAT
POSITIVE CONTROL*** RAT
TEST COMPOUND
10.000 UL
50.000 UL
100.000 UL
200.000 UL
» • i
RAT
RAT
RAT
RAT
TISSUE
___
...
___
---
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
R C V C R
IA-98
1 2
43
44
053
732
64
63
70
2B
40
39
1772
1R31
H3
260
316
437
TA-98 2-NITROFLUORENE
TA-100 SODIUM
A/ IDE
TA-100
3123
139
123
IB55
1748
162
1R8
221
121)
122
126
2271
2161
142
214
255
216
* * *
10 UC/PLATE TA-98 2-ANTHRAHINE 2.5 UG/PLATt
10 UG/PLATE TA-100 2-ANTHRAHINE 2.5 UG/PLATE
-------
RCSUITS
TABLE 2
Ol
00
-a
A. NAME OR CODE DESIGNATION OF THE IEST COMPOUND: 424 CADELPMI 1 KAD EXTRACT)
it. SOLVENT: OMSO
C. TEST INITIATION DATES! 09/03/81
0. TEST COMPLETION DATE: 09/01/81
r. s-9 LOT*: s9-n
NOTE: CONCENTRATIONS ARC GIVEN IN NICROLITCRS FER PLATE
REVERTANTS PER PLATF
TEST SPF.CICS
NONACTI VAIION
SOLVENT CONTROL
SOLVENT CONTROL
POSITIVE CONTROL'*
POSITIVE CONTROL**
TEST COMPOUND
10.000 UL
50.000 UL
100.000 UL
200.000 UL
ACTIVATION
SOLVENT CONTROL RAT
SOLVENT CONTROL RAT
POSITIVE CONTROL*" RAT
POSITIVE CONTROL*** RAT
TEST COMPOUND
10.000 UL RAI
50.000 UL RAT
100.000 UL RAT
200.000 UL RAT
* • i
TA-133S SODIUM AZIOE
TISSUE
— -
LIVER
LIVER
LIVCR
LIVER
LIVER
LIVER
LIVER
LIVER
TA-1535
1 2
18
2fl
1041
954
-
-
-
41
15
14
5B6
369
-
-
-
15
TA-13J? 9-AMINOACR10INE
TA-153T
3 123
7
12
R23
349
11
IT
20
•»
n
10
29T
1 13
41
37
46
26
• • •
10 DC/PLATE IA-1535 2-ANINR AMINE 2.5 UG/PLATT
50 UC/PLATF IA-1537 2-ANTIIRAMINF 2.5 UG/PLAU
SOLVENT 50 UL/PLAIE
- INDICATES ICSI WAS NOT RONE
-------
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 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 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. Surv iving Popu1at1ons
Plate test procedures do not permit exact quantitation of the number of
cells surviving chemical treatment. At low concentrations of the test
material, the surviving population on the treatment plates is essentially
the same as that on the negative control plate. At high concentrations,
the surviving population is usually reduced by some fraction. Our protocol
will normally employ several doses ranging over two or three log 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.
LD BIONET1CS 5-88 12
Litton
-------
Negative controls consisted of the test material solvent in the over",ay
agar together with the other essential components. The negative contro"
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.
E5-89 13
BIONETICS
Litton
-------
E.
Relation Between Mutagenlclty and Carcinogem'clty
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 j_n vivo rodent carcinogenesis assays.
All evaluations and interpretation of the data to be presented in the
final report will be based only on the demonstration, or lack, of 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.
ffl
Litton
BIONETICS
5-90
14
-------
VIII. REFERENCES
1. Brusick, D.J., et ah: IERL-RTP Procedures Manual: Level 1 Envl^or-
mentalI Assessment Biological Tests. EPA Contract No. 66-02-2581,
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 Environmental Assessment:
Limits of Bioassay Resolution and Detectability. EPA Contract
No. 68-02-2681, Technical Directive No. 301, Litton Bionetics,
Inc., Kensington, MD, February 1981, 56 pp. In press.
4. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N.: Detection of
carcinogens as mutagens in the Salmonel1 a/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
plasnrids. 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 Salmonel1 a/mammalian-microsome
mutagenicity test. Mutation Res., 31:347-364, 1975.
10. Vogel, H.J. and Bonner, D.M.: Acetylornithinase off. coli partial
purification and some properties. J. Biol. Chem., 218:97-106, 1966.
BIONETICS 5-91 15
Litton
-------
Ltd BIONETICS
Litton
GENETICS ASSAY NO. 5806
LBI SAFETY NO. 7032
CYTOTOXIC EVALUATION OF
424 (ADELPHI 1 XAD EXTRACT)
IN THE
RODENT CELL (CHO)
CLONAL TOXTclTY ASSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94092
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO. 22064
REPORT DATE: SEPTEMBER 1981
5-92
-------
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Chinese
hamster ovary cell (CHO) clonal toxicity assay as described in "lER^-RT^
Procedures Manual: Level 1 Environmental Assessment Biological Tests" (1).
The data was 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 Act 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.
Ltj BIONETICS 5-93
Litton
-------
TABLE OF CONTENTS
PREFACE .
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
ASSAY SUMMARY
OBJECTIVE
TEST MATERIAL
A. Description
EL Handling and Preparation
MATERIALS
A. Indicator Cells
B. Media
C. Controls
EXPERIMENTAL DESIGN
A. Dose Selection
B. Clonal Toxicity 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
.... 7
.... 8
.... 8
.... 8
11
.... 12
m
Litton
BIONETICS
5-94
ii
-------
I. ASSAY SUMMARY
A. SPONSOR: Acurex Corporation
B. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5806
1. Identification: 424 (Adelphi 1 XAD extract)
2. Date Received: June 28, 1981
3. Physical Description: Transparent liquid with a yellow
tint.
C. TYPE OF ASSAY: Rodent Cell (CHO) Clonal Toxicity Assay
D. ASSAY DESIGN NUMBER: 442
E. STUDY DATES:
A. Initiation: August 20, 1981
B. Completion: August 27, 1981
F. SUPERVISORY PERSONNEL:
A. Study Director: Brian C. Myhr, Ph.D.
B. Laboratory Supervisor: Robert Young, M.S.
G. EVALUATION:
The test material caused a slight increase in toxicity with
increasing concentrations up to 10 ul/ml. The material was
completely lethal to the cells at the maximum applicable dose
of 20 Ml/ml. The EC50 was estimated graphically to be 13 jjl/ml
which was equivalent to 63.2 ug of organics per ml. This sample
was therefore evaluated to be in the moderate (M) toxicity
category defined for the IERL-EPA Level 1 CHO clonal toxicity
bioassay.1
CB
Litton
Submitted by:
Study Director
Brian Myhr, Ph.!
Associate Director,
Department of Molecular
Toxicology
BIONETICS
Date
5-95
Reviewed by:
Javid J. Brusick, Ph.D.
Director,
Department of Molecular
Toxicology
-------
II. OBJECTIVE
The objective of this study was to determine and rank the cytotoxicity
of sample 424 (Adelphi 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
Litton
BIONETICS 5"96
-------
III. TEST MATERIAL
A. Description
The test material was received as a clear yellow tinted solution in
methylene chloride. The sample contained 17.0 milligrams of organic
material in an undetermined volume of methylene chloride. No infor-
mation 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 June 28, 1981. The sample was
assigned LBI safety number 7032 and LBI assay number 5806. 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 3.5 ml of DMSO,
giving a sample concentration of 4.86 nog 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 Ml/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 pi/ml test concentration, the final DMSO concen-
tration was constant at 1% (v/v).
E5-97
B1ONETICS
Litton
-------
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 contamination. 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
ami no 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 sup-
plemented in the same manner as F12, except that kanamycin at 40 ug/ml
is included for additional protection against bacterial contamination.
Both media formulations support the growth and cloning of CHO cells
equally well.
C. Controls
The negative control consists of three untreated cultures carried through
the same experimental time period as the treated cells. The test material
was supplied as a solution in an organic vehicle (usually DMSO) and was
diluted into the medium to provide each test concentration, three vehicle
control cultures containing the organic solvent at 1% or 2% by volume are
prepared. Particulate test samples were suspended in small amounts of
DMSO, but DMSO was not included in the negative control because the maxi-
mum content in the treated cultures is only 0.42%. The average number of
colonies in the negative control establishes the cloning efficiency of
the CHO cells used in the assay, and the appropriate negative or vehicle
controls provide the reference points for determining the effects of dif-
ferent concentrations of the test material on cell survival.
LU BIONETICS 5-98
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 pi/ml to 3 ul/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 (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 ug/ml
for particulate material, 600 ul/ml for aqueous samples, or 20 ul/ml for
organic solutions will not be determined. .
This sample, number 424 (Adelphi 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 ul of test material
per ml of culture medium. The corresponding concentration of organics
at the MAD level was 97.2 ug/ml; the lower doses were equivalent to 48.6,
29.2, 14.6, 4.9, 2.9, 1.5, and 0.5 ug/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.
If 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.
If 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
Ltd BIONETICS 5~"
Lrtton
-------
would also be 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.
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.
CB
Litton
BIONET1CS 5"100
-------
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.
HB B.ONET.CS
Litton
-------
VII. RESULTS
A. Interpretation
The application of sample 424 (Adelphi 1 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 10 pi/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 10 ul/ml range and dropped to zero at the 20 ul/ml
dose level.
The concentration expected to kill 50 percent of the cells (ECs0) was
found to be 13 pi of test material per ml of culture medium. This con-
centration was equivalent to 63.2 M9 of organic material per ml of
culture medium. This value placed the test material in the moderate (M)
toxicity range defined for the IERL-EPA CHO clonal toxicity bioassay.
The cells used for the assay were in logarithmic growth phase and were
100% viable. About 77% of the seeded cells formed colonies and in both
the 1* and 2% DMSO vehicle control, slightly less than 72% of the cells
formed colonies. Colony growth was normal and well distributed on the
culture dishes. These results achieved the 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 Table 1 and Figure 1.
LJj BIONETJCS 5-102
Litton 8
-------
TABLE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
Sample Identity: 424 (Adelphi 1 XAD
Extract
Description of Sample: Transparent
liquid with a yellow tint
LBI Assay No.: 5806
EC50 Value: 63.2 ug organics/ml
(13.0 Ml/ml)
Toxicity
Classification: Moderate (M)
pH Alterations: None
Date Received: June 28. 1981
Test Date: August 20, 1981
Vehicle: DMSO
Comments on
Treatment: Sample prepared in DMSO
at a concentration of 4.86 ug
organics/pl
Cell Type: CHO-K1
Cells Seeded per Dish: 200
COLONY COUNTS
Sample
NCb „
VC,1*C
VC,2%
Test
Test
Test
Test
Test
Test
Testx
Testd
Applied
'Concentration
Ml /ml
— —
10
20
0.1
0.3
0.6
1.0
3.0
6.0
10.0
20.0
Dish
#1
157
154
145
145
119
122
132
123
129
117
0
Dish
#2
160
131
140
130
131
137
130
119
121
106
0
Dish
#3
144
145
144
133
134
130
110
120
115
121
Average
Count
153.7
143.3
143.0
136.0
128.0
129.7
124.0
120.7
121.7
114.7
0
Relative
Survival
_»•
100.0
100.0
94.9
89.3
90.5
86.5
84.2
84.9
80.0
0
Cloning
Efficiency
76.9
71.7
71.5
Relative to 2% VC for 20 ul/ml treatment and to 2% VC for other treatments.
bNC = Negative Control, F12 medium.
SfC = Vehicle Control, percent DMSO given.
dOnly two plates dosed to conserve limited test material.
m
Utton
BIONETICS
5-103
-------
HO r
130
r 70
FIGURE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
EC50 DETERMINATION
424 (ADELPHI 1 XAD EXTRACT)
100
CONCENTRATION, yl/ml
5-104
10
-------
VIII. ASSAY EVALUATION CRITERIA
The EC50 value represents the concentrations of test material that -ecuces
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
Toxicity (ECSO in ug/ml)
High <10
Moderate 10 to 100
Low 100 to 1000
Not Detectable >1000
Aqueous Liquids
(EC50 in ul/ml)
<6
6 to 60
60 to 600
>600
Nonaqueous Liquias
(EC50 in pi/ml)
<.2
.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level 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:
Toxicity
High
Moderate
Low
Nondetectable
EC50 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
5-105
BIONET1CS ..
Utton n
-------
IX. REFERENCES
1. Brusick, O.J., et a_L: IERL-RTP Procedures Manual: Environmental
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 Environmental Assessment:
Limits of Bioassay Resolution and Detectability.EPA Contract
No. 68-02-268, Technical Directive No. 301, Litton Bionetics,
Inc., Kensington, MD, February 1981, 56 pp. In press.
CH 5-106
BIONET.CS
Utton
-------
GENETICS ASSAY NO.
LBI SAFETY NC.
MUTAGENICITY EVALUATION OF
436 (ADELPHI 2 XAD EXTRACT)
IN THE
EPA LEVEL 1
AMES SALMONELTA7MTCROSOME
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: SEPTEMBER 1981
LU BIONETICS 5-107
Utton
-------
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Ames
Salflionella/microsome mutagenesis assay as described in "IERL-RTP Proce-
dures Manual: Level 1 Environmental Assessment Biological Tests"1. The
data was 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 Act 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.
LU BIONETICS 5-108
Litton
-------
TABLE OF CONTENTS
Page No.
PREFACE -;
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
I. 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. ASSAY ACCEPTANCE AND 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
_ BIONETICS 5-109
Litton
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I. ASSAY SUMMARY
A. Sponsor: Acurex Corporation
B. Material (Test Compound): Genetics Assay Number: 5809
1. Identification: 436 (Adelphi 2 XAD Extract)
2. Date Received: June 28, 1981
3. Physical Description: Clear, colorless liquid
C. Type of Assay: EPA Level 1 Ames Sal monell a/Mi crosome Plate Test
Assay Design Number: 401 (EPA Level 1)
Study Dates:
1. Initiation: August 22, 1981
2. Completion: September 7. 1981
Supervi spry Personne 1 :
A. Study Director: D.R. Jagannath, Ph.D.
Evaluation:
D.
E-
G.
The test sample, 436, was mutagenic in the Ames Salmonella/
mi crosome assay. The sample was mutagenic for frameshift tester
strains TA-1537 and TA-98 under activation (+S9) conditions.
Strain TA-100 was reverted by the sample but the response did
not meet the established criteria for a positive effort. Strain
TA-1535 was not affected by sample 436 at the single concentra-
tion evaluated. The minimum effective concentration (MEC)
was 10 pi/plate with strain TA-98. This was equivalent to
13 ug organics/plate. This response was evaluated to be in
the high (H) mutagenicity range defined for the IERL-EPA Level 1
Ames bioassay1.
Submitted by:
Study Director
Reviewed by:
. . _
D.R. Jagannattf, Ph.D.
Section Chief,
Submammalian Genetics,
Department of Molecular
Toxicology
ate
David J. Brusick, Ph.D.
Director,
Department of Molecular
Toxicology
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Litton
BIONETICS
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II. OBJECTIVE
The objective of this study was to determine the genetic activity of
sample 436 (Adelphi 2 XAD extract) in the Salmonel1 a/microsome assay
with and without the addition of mammalian metabolic activation prepara-
tions. The genetic activity of a sample is measured in these assays oy
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.
5-111
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III. TEST MATERIAL
A. Description
The test material was received as a clear, colorless solution in methylene
chloride. The sample contained 4.7 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. Hand!ing and Preparation
The test material was received at LBI on June 28, 1981. The sample was
assigned LBI safety number 7033 and LBI assay number 5809. 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 3.5 ml of DMSO,
giving a sample concentration of 1.3 mg organics per ml DMSO. The sample
was transferred to a glass vial and sealed with a teflon-coated rubber
septum.
A total volume of 2.56 ml of test material was used between three trials.
In the trials, varying aliquots of the test material were added directly
to the test mixtures to give the desired concentration. The amount of
sample used was 0.36 ml for Trial 1, 1.08 ml for Trial 2 and 1.12 ml for
Trial 3.
BIONETICS 5"U2
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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
TA-1537
TA-98
TA-100
Gene
Affected
his G
Ms C
Mi D
Mi G
Additional Mutations
Repair
A uvr B
A uvr B
A uvr B
A uvr B
LPS R Factor
rfa
rfa
rfa pKMlOl
rfa pKMlOl
Mutation Type
Detected
Base-pair
substitution
Frameshift
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 \sg/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
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BIONETICS
5-113
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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 al_.9
C.
1.
Activation System
S9 Homogenate
A 9,000 x 3 supernatant prepared from Sprague-Dawley adult male rat liver
induced by Aroclor 1254 (Ames et a_K9) was purchased from Bionetics Labora-
tory Products, Litton Bionetics, Inc. and used in these assays.
2.
S9 Mix
59 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 M
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BIONETICS
5-U4
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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 testec
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
A preliminary range-finding test was run with strain TA-98 using the
recommended doses of 200, 100, 50, and 10 ul per plate. Subsequent testing
with the other strains utilized these dos6 levels. These doses were
equivalent to 260, 130, 65, and 13 ug of organics 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.
0.01 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.
CH BIONETICS 5~U5
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A detailed flow diagram for the plate incorporation assay is provided in
Figure 1.
C.
ContrQL_Cgmp_p_unds
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 concentra-
tions 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 (yg)
Water
Dimethyl-
sulfoxide
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]
Molten [45*C] overlay agar
appropriately supplemented
.01 - .2 ml
Test, positive or solvent
control chemical
0.1 ml
Aliquot of an overnight culture
of bacteria [ 109 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/piate counted
Data analyzed
Interpretation/ Conclusion
Figure 1 AMES SALMONELLA/MICROSOME MUTAQENESIS ASSAY
5-117
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VI. RESULTS
A. Interpretations
The test material, sample 436, was tested in strains TA-98, TA-100, TA-1535
and TA-1537 both with and without the addition of metabolic activation.
The tests were conducted at concentrations of 10, 50, 100 and 200 yl/plate
except for strain TA-1535 which was tested only at 200 ul/plate. The
assay was conducted in three steps. Trial 1 was a range-finding assay
using strain TA-98 without activation. Trial 2 utilized strain TA-98
with activation and strain TA-100 both with and without activation.
Trial 3 was with strains TA-1535 and TA-1537 both with and without acti-
vation.
The response in all strains without metabolic activation was negative.
With activation, strains TA-98 and TA-1537 were mutagenic under the assay
conditions and both showed a dose-related increase in the number of revert-
ants. Strain TA-100 was reverted by the test material but the response
did not meet the criteria for a positive effect. Strain TA-1535 was
negative at the single dose tested. There was indication of sample related
toxicity at the maximum applicable dose of 200 pi/plate.
The minimum effective concentration (MEC) was observed to be 10 ul/plate
in TA-98 with activation. This was equivalent to 13 ug of organic material
per plate. This response was categorized as high (H) mutagenic activity
using the IERL-EPA Level 1 criteria for the Ames assay1.
Solvent and positive control values were within acceptable ranges. These
results achieve the assay acceptance criteria and provide 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.
LD BIONETICS 5"118
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RESULTS
T*HLC I
A. NAME OR CODE DESIGNATION OF THE TEST COMPOUND: 436
-------
RESOLIS
TABLE ?
NAME OR CODE DESIGNATION OF IMC ICST COMPOUND: 436IADELPHI 2 IAD EXTRACT}
SOLVENT: DHSO
TEST INITIATION DATES: 09/03S81
TEST COMPLETION DATE: 09/07/B1
S-9 LOIi: S9-11
CONCENTRATIONS ARC GIVEN IN MICROLIICRS PER PLATE
A.
B.
C.
D.
C.
NOTE:
TEST
NONACTIVATION
REVERTANTS FER PLATE
SPECIES TISSUE TA-1535 TA-1531
123 123
SOLVENT CONTROL
SOLVENT CONTROL
POSITIVE CONIROL**
POSITIVE CONTROL**
TEST COMPOUND
10.000
50.000
100.000
200.000
ACTIVATION
UL
UL
UL
UL
1R
28
1041
954
31
7
12
B23
349
13
19
23
25
SOLVENT CONTROL
SOLVENT CONTROL
POSITIVE CONTROL***
POSITIVE CONTROL***
TEST COMPOUND
10.000 UL
SO. 000 UL
100.000 UL
200.000 UL
RAT
RAT
RAT
RAI
RAT
RAT
RAT
RAT
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
15
14
5fl6
369
-
-
-
15
a
ID
29T
113
211
35
38
4A
TA-1335 SODIUM AZIOE
TA-15J7 9-AMINOACRIDINE
SOLVENT 50 UL/PLATE
- INDICATES TEST HAS NOT DONE
10 UC/PLATE
50 U6/PLATE
TA-1535
TA-153T
2-ANTHRAMINE
2-ANTHRAHINE
2.5 US/PLATE
2.5 US/PLATE
-------
vn- ASSAY ACCEPTANCE AND EVALUATION CRITERIA
Statistical methods are not currently used, and evaluation is based on
the criteria included in tnis 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.
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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
0. 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.
LLJ BIONETICS 5"122 13
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E- Relation Between Mutagenlcity and Carcinogeniclty
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 ni 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 tq rank source streams by relative
degree of genetic toxicity (mutagenicity). Samples are first identified
as mutagenic or nonmutagenic by the criteria in Section 0 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 (NO)
Solids
(MEC in pg/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 ((jg
organics per plate) and not volume (ul extract per plate) of sample
tested.
E5-123
BIONETICS
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VIII. REFERENCES
1. Brusick, D.J., et aj_.: IERL-RTP Procedures Manual: Level 1 Environ-
ment^ 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 Environmental Assessment:
Limits of Bioassay Resolution and Detectability.EPA Contract
No. 68-02-2681, Technical Directive No. 301, Litton Bionetics,
Inc., Kensington, MD, February 1981, 56 pp. In press.
4. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N.: Detection of
carcinogens as mutagens in the Salmone11 a/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., 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.
Ltd BIONETICS 5'124 15
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GENETICS ASSAY NO. 5809
LBI SAFETY NO. 7022
CYTOTOXIC EVALUATION OF
436 (ADELPHI 2 XAD EXTRACT)
IN THE
RODENT CELL (CHO)
CLONAL TOXICITY~AS'SAY
_ ^^^^ i^B^^—^^^^^^^IBBW ^
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94092
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO. 22064
REPORT DATE: SEPTEMBER 1981
E5-125
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 was 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 Act 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.
BIONETICS 5"126
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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 Cells 4
B. Media 4
C. Controls . 4
V. EXPERIMENTAL DESIGN 5
A. Dose Selection 5
B. Clonal Toxicity Assay 5
VI. . ASSAY ACCEPTANCE CRITERIA 7
VII. RESULTS 8
A. Interpretation . .
B. Tables and Figures
VIII. ASSAY EVALUATION CRITERIA 11
IX. REFERENCES 12
[B BIONETICS -
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I. ASSAY SUMMARY
A. SPONSOR: Acurex Corporation
B. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5809
1. Identification: 436 (Adelphi 2 XAD extract)
2. Date Received: June 28, 1981
3. Physical Description: Clear, transparent liquid
C. TYPE OF ASSAY: Rodent Cell (CHO) Clonal Toxicity Assay
D. ASSAY DESIGN NUMBER: 442
E. STUDY DATES:
1. Initiation: August 19, 1981
2. Completion: August 26, 1981
F. SUPERVISORY PERSONNEL:
A. Study Director: Brian C. Myhr, Ph.D.
B. Laboratory Supervisor: Robert Young, M.S.
G. EVALUATION:
The test material caused only a slight increase in toxicity at
the maximum applicable dose (MAD) of 20 ul/ml (26.8 u§ of
organics per ml). Since no tested dose produced 50 percent
killing of the cells, the EC50 could not be calculated. How-
ever, the sample was ranked by IERL-EPA evaluation criteria
as being excluded from the high toxicity category and as
having moderate (M) toxicity or less. The inability to test
higher concentrations was attributable to the extremely small
quantity of test material supplied (4.7 mg).
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Submitted by:
Study Director
D
Brian Myhr,
Associate Director,
Department of Molecular
Toxicology
ate
B1ONETICS
5-128
Reviewed by:
David J. Brusick, Ph.D.
Di rector,
Department of Molecular
Toxicology
Date
-------
II. OBJECTIVE
The objective of this study was to determine and rank the cytotoxicity
of sample 436 (Adelphi 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 devel-
oped 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.
E5-129
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III. TEST MATERIAL
A. Description
The test material was received as a clear, transparent solution in
methylene chloride. The sample contained 4.7 milligrams of organic
material in an undetermined volume of methylene chloride. No infor-
mation 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 June 28, 1981. The sample was
assigned LBI safety number 7033 and LBI assay number 5809. The sample
was stored at +4°C in the dark.
Pretest sample preparation consisted of solvent-exchanging the sample into
dimethylsulfoxide (DM50). The sample was transferred with methylene chloride
rinses into a graduated conical tube. The methylene chloride was gradually
evaporated (50°C under stream of nitrogen) and DMSO was sequentially added.
The sample was brought to volume in 3.5 ml of DMSO, giving a sample concen-
tration of 1.34 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 Ml/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).
[H B.ONET.CS
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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, MO. 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 contamination. 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
ami no 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 sup-
plemented in the same manner as F12, except that kanamycin at 40 ug/ml
is included for additional protection against bacterial contamination.
Both media formulations support the growth and cloning of CHO cells
equally well.
C. Controls
The negative control consists of three untreated cultures carried through
the same experimental time period as the treated cells. The test material
was supplied as a solution in an organic vehicle (usually DMSO) and was
diluted into the medium to provide each test concentration, three vehicle
control cultures containing the organic solvent at 1% or 2% by volume are
prepared. Particulate test samples were suspended in small amounts of
DMSO, but DMSO was not included in the negative control because the maxi-
mum content in the treated cultures is only 0.42%. The average number of
colonies in the negative control establishes the cloning efficiency of
the CHO cells used in the assay, and the appropriate negative or vehicle
controls provide the reference points for determining the effects of dif-
ferent concentrations of the test material on cell survival.
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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 pi/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 pg/ml. 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 pg/ml
for particulate material, 600 pi/ml for aqueous samples, or 20 ul/ml for
organic solutions will not be determined.
Sample 436 (Adelphi 2 XAD extract) was tested at eight concentrations.
The concentrations used started with the maximum applicable dose (MAD)
of 20 pi/ml and included 10, 6, 3, 1, 0.6, 0.3, and 0.1 pi of test
material per ml of culture medium. The MAD of 20 pi/ml was equivalent
to 26.8 pg of organics per ml of culture medium. The lower dose levels
were equivalent to 13.4, 8.04, 4.02, 1.34, 0.80, 0.40 and 0.13 pg of
organics per ml.
B. Clonal Toxicity Assay
Cells from monolayer stock cultures in logarithmic growth phase were tryp-
sinized with 0.1% trypsin plus 0.013 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.
If 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
EH BIONETICS 5"132
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change would also be recorded. At the end of the treatment period, tne
pH values of the discarded media from the two described treatments would
be recorded again. No sample related pH effects were noted.
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.
E5-133
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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.
LLJ BIONET1CS 5'134
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VII. RESULTS
A- Interpretation
The application of sample 436 (Adelphi 2 XAD extract) to the CHO cell
cultures caused only a small decrease in the number of cells able to
form colonies as the concentration increased to the maximum applicable
dose (MAD) of 20 ul/ml. Relative survival 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 only to about 88% for the 20 ul/ml treatment.
Since none of the tested doses caused killing approaching 50% of the cells,
an EC50 could not be calculated. However, a value greater than 20 ul/ml
(26.8 ug organics/ml) excluded the sample from the high toxicity category
and placed it in the moderate range or even less toxicity, based on the
IERL-RTP evaluation criteria.1 The plotted results suggested the possi-
bility of an ECSO less than 100 ul/ml (134 ug organics/ml), so the sample
could have been evaluated as moderately toxic or being on the moderate/low
toxicity borderline had sufficient sample been available. The toxicity
of the sample was therefore evaluated as undetermined but moderate (M)
or less.
Testing and evaluating materials such as this sample indicate the need
to supply sufficient quantities of test material to ensure adequate testing.
While minimum testable sample size is often a function of the biological
activity of the sample, efforts should be made to supply at least 100 mg
of SASS train organics for combined Ames and CHO testing.
The cells used for the assay were in logarithmic growth phase and were
99.4% viable. About 99% of the cells seeded and cultured in F12 medium
formed colonies. The cloning efficiency of the 1% vehicle control was
82.9% and the 2% vehicle control, 75.4%. Colony growth was normal and
well distributed on the culture dishes. These results achieved the 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.
OB B.ONETICS 5-135
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TABLE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
Sample Identity: 436 (Adelphi 2 XAD
Extract
Description of Sample: Clear^,
transpa re nt]i qu id
LBI Assay No.: 5809
Date Received: June 28, 1981
Test Date: August 19. 1981
Vehicle: DMSO
Cell Type: CHO-K1
Cells Seeded per Dish: 200
EC50 Value: >26.8 ug organics/ml
(>20 pi/ml)
Toxicity
Classification: Undetermined, but
Moderate (M) orTess
pH Alterations: None
Comments on
Treatment: Sample prepared in DJ1SO
at a concentration of 1.34 pg
organics/ul.
COLONY COUNTS
Applied
'Concentration Dish
Sample Ml/ml #1
NCb f
vc.ir1
VC,2%
Test
Test
Test
Test
Test
Test
Test.
Testd
«•»•
10
20
0.1
0.3
0.6
1.0
3.0
6.0
10.0
20.0
184
164
157
160
155
166
160
147
154
166
135
Dish
#2
187
175
148
166
179
169
166
162
164
160
130
Dish
#3
182
158
147
158
145
150
156
171
143
153
"""*""""""
Average
Count
184.3
165.7
150.7
161.3
159.7
161.7
160.7
160.0
153.7
159.7
132.5
Relative
Survival
...
100.0
100.0
97.3
96.4
97.6
97.0
96.6
92.8
96.4
87.9
Cloning
Efficiency
92.2
82.9
75.4
Relative to 2% VC for 20 Ml/ml treatment and to 1% VC for other treatments.
NC = Negative Control, F12 medium.
= Vehicle Control, percent DMSO given.
Only two plates dosed to conserve limited test material.
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BIONETICS
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FIGURE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
EC50 DETERMINATION
436 (ADELPHI 2 XAD EXTRACT)
CONCENTRATION, pi/ml
5-137
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VIII. ASSAY EVALUATION CRITERIA
The EC50 value represents the concentrations of test material that reduces
the coTony-forming ability of CHO cells to 50% of the vehicle or negative
control value. EC50 values are determined graphically by fitting a curve
fay 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
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 ul/ml)
<.2
.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level Environmental Assessment Biological Tests.1
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 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 ECSO In
Toxicity Liters/ml (L/ml) Dry Standard Cubic Feet/ml (DSCF/ml)
High 1000 >35
m ,,
BIONETICS n
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IX. REFERENCES
1. Brusick, D.J. , et a_l_.: IERL-RTP Procedures Manual: Envi ronmenta'
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 Bi'oassay 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 Environmental Assessment:
Limits of Bioassay Resolution andDetectability.EPA Contract
No. 68-02-268, Technical Directive No. 301, Litton Bionetics,
Inc., Kensington, MD, February 1981, 56 pp. In press.
E 5-139 12
BIONETICS
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TECHNICAL REPORT DATA
(Please read Imirucnum on the re: era' before completing!
£pA-600''7-84-095b
3. RECIPIENT'S ACCESSION NO.
-:T_£ ANO SJBT.TLE
Environmental Assessment of a Firetube Boiler Firing
Coal/Oil/Water mixtures; Volume II. Data Supple-
ment
S. REPORT DATE
September 1984
6. PERFORMING ORGANIZATION CODE
? AUT«ORlS>
R. DeRosier
8. PERFORMING ORGANIZATION REPORT NO.
TR-81-89/EE
9 OERFOSMING ORGANIZATION NAME AND ADDRESS
.A cur ex Corporation
Energy and Environmental Division
P. O. Box 7555
Mountain View, California 94039.
10. PRC'GRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-02-3188
12 SPONSORING AGENCY NAME ANO ADDRESS
EPA, Office of Research and Development
Industrial Environmental Research Laboratory
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final; 2/81 - 11/83
14. SPONSORING AGENCY CODE
EPA/600/13
is. SUPPLEMENTARY NOTES IZRL-RTP project officer is Robert E. Hall. Mail Drop 65, 919/
541-2477. Volume I is the Technical Results.
16. ABSTRACT
volume is a compendium of detailed emission and test data from field
tests of a firetube industrial boiler burning a coal/ oil /water (COW) mixture. The
boiler was tested while burning COW fuel, and COW with soda ash added (COW+SA)
to serve as an SO2 sorbent. The test data include: preliminary equipment calibration
data, boiler operating data for both tests, fuel analysis results, and .complete flue
gas emission measurement and laboratory analysis results. Flue gas emission mea-
surements included: continuous monitoring for criteria gas pollutants; gas chroma-
tography (GC) of gas grab samples for volatile organics (C1-C6); EPA Method 5 for
particulate; controlled condensation system for SO2 emissions; and source assess-
ment sampling system (SASS) for total organics in two boiling point ranges (100 to
300 C and > 300 C), organic compound category information using infrared spectro-
metry (IR) and low resolution mass spectrometry (LRMS), specific quantitation of
the semivolatile organic priority pollutants using gas chromatography/mass spectro-
metry (GC/MS), liquid chromatography (LC) separation of organic extracts into
seven polarity fractions with total organic and IR analyses of eluted fractions, flue
gas concentrations of trace elements by spark source mass spectrometry (SSMS) and
atomic absorption spectroscopy (AAS), and biological assays of organic extracts.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSAT1 Field/Group
Pollution
Fire Tube Boilers
Coal
Fuel Oil
Water
Sodium Carbonates
Assessments
Flue Gases
Pollution Control
Stationary Sources
Coal/Oil/Water Mixture
Environmental Assess-
ment
13B
ISA
21D
07B
14 B
21B
3. OlSTfliBUTlON STATEMENT
Release to Public
19. SECURITY CLASS ITtta Report)
Unclassified
21. NO. OF PAGES
178
20. SECURITY CLASS fTnit page)
Unclassified
22. PRICE
EPA form 2220-t (9-73)
5-140
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