Methods of Procedure

Preparation of Minimal Medium. Vogel-Bonner (VB) medium is prepared in

three steps. First, a 50X concentrated VB salt solution is made by serial
addition to 670 ml warm (45°C), glass distilled water of 10 g of magnesium
sulfate (MgSO 4°7H20), 100 g of citric acid monohydrate, 500 g potassium phos-
phate (dibasic), and 175 g sodium ammonium phosphate (NaNH ,HPO,°4H20). Spe-
cial attention is given to the requirement that each chemical is added and
dissolved successively. The final 50X salt concentrate is then aliquoted into
40 and 80 ml portions into storage bottles are are loosely capped and auto-
claved for 15 min at 250°F at 15 p.s.i. The second step in medium formulation
involves preparation of a 40% glucose solution. Four hundred grams of dex-
trose is dissolved in warm (45°C), glass distilled water and the volume is
adjusted to 1 liter. One-hundred milliliter aliquots are transferred into
storage bottles that are loosely capped and autoclaved as above. In the final
step of medium formulation, 30 g of Difco Agar is dissolved in 1800 ml glass
distilled water and autoclaved. The sterile agar is then allowed to cool to
70°C in a constant temperature bath, and 40 ml of the 50X salt solution and
100 ml of the 40% glucose solution are added. The final medium is thoroughly
mixed and 25 ml portions are poured into sterile 100 x 15 mm petri dishes.

The plates are allowed to gel at room temperature on a level surface prior to

storage, after inversion, at 4°C.

Preparation of Top Agar

The procedure used for preparation of the top agar is similar to that

described above. The top agar is prepared by adding 6 g of Bactoagar and 5 g


TABLE 5.

RESULTS OF INDIVIDUAL AMES TESTS OF EXTRACTS, DOSE~RESPONSE
RELATIONSHIP CALCULATED BY EPA-RTP NON-LINEAR MODEL

rev/ug _
TA98/1, 6-9
Sample TA 1535 TA_1537 A 1538 TA 98 TA 100 TA 972 TA 1022 TA 194% —TA_98NR? DNPG
Identification Test =s9_ +59 -S9 489 «~-S9_+4S9 «-S9 459 "-S9 459 “-S9 489 “-S9 459 “-S9 489 “-S9 489 =s9 +89
‘TAEB-81-0781 initial 0.01 0.42 5.38 2.68 15.1 5.71 8.13 5.20 8.66 2.29 5.00 0.75 2.86 2.34 17.2 6.77 2.63 3.71 8.96 0.89
\WLA-4 baseline repeat 0-07 0.51 0.84 4,45 . 6.77 4.2217.6 4.86 6.30 4.85
avg. 0.04 0.47 3.11 3.57 11.0 4,97 12.8 5.03 7.48 3.57
‘raep-82-0191 ?7?
VW LA-4 JM-13 initial 1.43 4.93 15.8 43.6 143 37.7 28.5 19.4 22.5 8.92
trap
bra
TAEB-82-0101
VW LA-4 JM-13 initial 0.76 2.81 8.24 15.9 27.9 36.0 14.9 7.66 13.9 6.64
trap
aep-82-0131 ?’* coe coo c
VW Regen. JM-13| initiah 9 ~~ == --© 4.54 24.7 4.47 9.36
trap
‘TAEB-82-0221 initial 0.00 0.35 1.55 0.33 1.70 0.96 1.79 1.47 1.58 1.17 1.99 1.06 2.54 1,82 3.41 2.48 0.30 5.28 0.95 0.46
Toyota LA~4 BS2-1 repeat 0-00 0.36 0.71 0.32 1.60 0.76 2.72 1.05 0.79 0.88
trap avg. 0.00 0.36 1.13 0.32 1.65 0.86 2.26 1.26 1.19 1.03
TAEB-83-0161 initial 0.09 0.04 0.43 0.45 0.28 0.88 1.76 0.86 1.79 1.42 0.41 0,88 0.98 0.34 3.45 1.38 0.26 0.77 0.56 0.13
Toyota LA-4 BS2-1 repeat 0.01 0,04 0.44 0.24 0.76 1.22 1.62 1.80 1.36 0.96
trap avg. 0.05 0.04 0.44 0.35 0.52 1.05 1.69 1.33 1.58 1.19
‘TAEB-84-0010 initial 0.00 0.45.12.9 1.17 4.48 2.00 5.66 3.35 4.91 7.07 2.18 1.93 20.6 1.34 7.19 2.60 1.26 2.92 - 9.25 0.16
Mercedes LA-4 repeat 0.00 0.47 1.73 0.84 6.52 1.98 5.98 2,12 11.0 8.08
baseline avg. 0.00 0.46 7.32 1.01 5.50 1.99 5.84 2.74 7.96 7.58
TAEB-84-0020 initial 0.00 0.79 2.48 2.12 3.40 29.1 25.2 6.57 7.51 3.70
Mercedes LA-4 repeat , 0.00 0.93 -- 12.6 4,93 28.4 41.8 10.2 1.13 2.72
NGK-2 ‘trap avg. 0.00 0.86 2.48 7.36 4.17 28.8 33.5 8.39 60.3" 3.21

3No repeat analysis
Pinsufficient sample to perform repeat analysis
Insufficient sample to perform analysis in TA 1535, TA 1537, TA 98


8:30 a.m. the following morning, when the assay is to be conducted, the bac-
terial strains are removed from the incubator and placed on ice while the
other necessary preparations for the assay are completed. Occasionally, an
aliquot is removed to determine the bacterial concentration spectrophoto-

metrically at 625 nm..

The Plate Incorporation Assay

While the tester strains are on ice, the top agar is melted, supplemented
with histidine and biotin, and then distributed in 2 ml aliquots into 13 x
100 mm test tubes which are placed in a multiblock heater maintained at a
temperature of 45°C. The activated $9 mix is freshly prepared as described
above and chilled on ice. The chemicals are weighed out, Placed in solution,
and diluted to different concentrations as previously described. The minimal
medium plates are properly labeled in triplicate and brought into the hood in
groups of ten. “The refrigerated tester strains and the S9 mix which are kept
chilled in ice are also brought inside the vented hood. The usual procedure
is to first inoculate each 13 x 100 mm text tube containing molten top agar
with 0.1 ml of organism, followed by 0.1 ml of chemical and, if required,

0.5 ml of activated S9 mix. The test tube is then removed from the multiblock
heater and vortexed for 2 sec; the tube contents are then poured into the
appropriate prelabeled plate, which is gently tilted to spread the top agar
over the entire surface of the minimal medium plate. The plates are allowed
to stand in the hood at room temperature for gelling while the other plates
are poured in the same manner. After all the plates have been poured and the

top agar overlay has been allowed to gel for a minimum of 30 min, the plates


ABSTRACT

This report describes filter extractions and Ames bioassay of. filter
extracts performed for the Emission Control Technology Division of the
Environmental Protection Agency. Eight sets of particulate-loaded filters were
provided to SwRI by the sponsor. The filters were soxhlet-extracted in
methylene chloride, and the extracts were dried and weighed. The organic
extracts were analyzed using the Ames bioassay at Southwest Foundation for
Biomedical Research (SFBR), formerly Southwest Foundation for Research and
Education. The data were analyzed using linear and non-linear regression
methods,

iv


of NaCl to 1 liter of distilled water in a 2-liter flask. The final agar con-
centration is 0.6% and the final NaCl concentration is 0.5%. The contents of
the flask are brought to boiling on a heated magnetic stirrer. Aliquots of
100 ml are then transferred into milk dilution bottles and loosely capped;
they are then autoclaved for 15 min at 250°F and 15 psi. The caps are tight-
ened prior to removal of the bottles from the autoclave. The bottles are
allowed to stand at room temperature until the agar gels, and they are stored

at room temperature prior to usage.

Por each experiment, bottles containing an appropriate amount of top agar
are reheated in the autoclave for 6 min at the same temperature and pressure
as described above. The bottles are then placed in a 45°C oven and are
allowed to temperature equilibrate for approximately 2 hr. The melted top
agar is supplemented with 10 ml of a sterile 0.5 mM L-histidine HC1-0.5 mTM
biotin solution for each 100 ml of top agar, and 2-ml aliquots of the supple-

mented top agar, are distributed into sterile capped 13 x 100 mm test tubes.

Preparation of Chemicals for the Plate Incorporation Assay

All chemicals, whether known or unknown, are carefully weighed in a
vented glove box housing a five-place Mettler balance. The chemicals are
weighed in individual sterile 13 x 100 mm capped test tubes. The chemicals
are transferred from the glove box into the vented hood and placed into
solution using the proper amount of a sterile solvent (usually DMSO). The
stock chemicals are further diluted into sterile capped tubes containing the

appropriate volumes of sterile solvent.


To determine UV sensitivity, nutrient agar plates are sectioned into
eight areas and all 15 samples are streaked across the sectioned plates in
parallel strips. A piece of cardboard is placed over ‘the uncovered plate so
that half of each bacterial streak is covered. A UV lamp, warmed and func-
tioning for a minimum of 15 min, is placed approximately 33 cm from the top of
the plates and the covers of the plates are removed prior to exposure. Non-
R-factor strains (i.e., TA 1538) are irradiated for 6 sec and R-factor strains
(i.e., TA 98) are irradiated for 8 sec. TA 102 is used on the same plates as
a control for the UV exposure. After exposure, the plates are incubated over-
night at 37°C, and examined for evidence of sensitivity to UV by the presence

or absence of growth.

To test for the other diagnostic requirements, such as histidine and bio-
tin, or spontaneous mutation rates, minimal medium agar plates are employed.
For spontaneous reversion rates, each sample is assayed in triplicate; there-
fore, 45 minimal medium plates plus 45 test tubes (13 x 100) with 2 ml of top
agar (supplemented with biotin-histidine) are used. To 2 ml of top agar,

0.1 ml of the bacterial culture is added, vortexed, and poured onto a properly
labeled petri dish containing minimal medium. The plates are incubated at
37°C for 48 hr, after which time the revertant colonies are counted. Accept-
able levels of spontaneous reversion in our laboratory are TA 1538: 15-35;

TA 97: 90~180; TA 98: 20-50; TA 100: 100-200; TA 102: 240-320, and TA 104:
275-425. When the number of spontaneous revertants per plate fails to fall
within the acceptable range, new stocks are prepared by selection of clones
containing the desired activity. The new stocks are aliquoted and stored

frozen at -80°C.

10


Preparation of Cofactors

The cofactor solution consists of D-glucose-6-phosphate, NADP monosodium
salt, magnesium chloride, potassium chloride, monobasic and dibasic sodium
Phosphate. For each 100 ml of cofactor solution, 0.157 g D-glucose-6~P04,
0.350 g NADP, 0.180 g MgCl., 0.273 g KCl, 1.278 g dibasic sodium phosphate,
and 0.278 g monobasic sodium phosphate are added to glass distilled water.
When all the chemicals are in solution, the volume is adjusted to 100 ml and
the solution is sterile-filtered with a disposable 100 ml Nalgene filter
unit. The sterile cofactor solution is dispensed into sterile screw-capped
tissue culture tubes in 9-ml aliquots, quickly frozen, and stored at -20°C.
Upon determination of the amount of S9 mix needed for a given-assay, appropri-
ate amounts of S9 homogenate and cofactor solutions are thawed and mixed at a

ratio at 1 ml S9 homogenate to 9 ml of cofactor solution.

Preparation of the Bacterial Culture

The procedures described herein apply to all S. typhimurium tester
strains, The bacterial tester strains are removed from the Revco freezer and
inoculated into separate 16 x 180 mm Kim-capped test tubes, each containing
5 ml of Oxoid nutrient broth. Inoculation is performed by transferring 20 ul
of a thawed bacterial culture into the Oxoid nutrient broth. The Oxoid broth
is prepared by dissolving 25 g of Oxoid nutrient broth No. 2 in 1 liter of

distilled water and autoclaving.

The tester strains are inoculated into the Oxoid nutrient broth at’ 4:30

Pem. and incubated overnight at 37°C, with agitation, in an incubator. At


APPENDIX

DETERMINATION OF THE IN VITRO GENOTOXICITY
OF CHEMICALS BY THE

SOUTHWEST FOUNDATION FOR RESEARCH AND EDUCATION

S. typhimurium Histidine Reversion Assay

Protocol 101


TABLE 2.

SUMMARY OF AMES BIOASSAY CF EXTRACTS, DOSE-RESPONSE

RELATIONSHIP CALCULATED BY EPA-RTP NON-LINEAR MODEL

rev/ug
TA 98/1,8-8
TA 1535 TA 1537 TA 1538 TA 98 TA 100 ta 979 TA 1023 __TA 1042 _TA_98NR@ DNPG
Sample Identification =89_ +89 “-s9 #89 ““s9 459 “SO 489 _-S9_+4S9.=«C=-S9.«==#S9 «SO. 489 ~-S9 +89 «-S9 +89 ~~-59 459
TAEB-81-0761 0.04 0.47 3.11 3.57 11.0 4.97 12.8 5.03 -7.48 3.57 5.00 0.75 2.86 2.34 17.2 6.77 2.63 3.71 8.96 0.89
VW LA-4 baseline
‘TAEB-82-01915 1.43 4,93 15.8 43.6 143 37.7 28.5 19.4 22.5 8.92
vw LA-4 JM-13 trap
82-0101)

TAEB~62-0101 0.76 2.81 8.2415.9 27.9 36.0 14.9 7.66 13.9 6.64
VW LA-4 JM-13 trap
‘TAEB-82-0131> FF EEE 54 24.7 4.47 9.36
VW Regen JM-13 trap
TAEB-82-0221 0.00 0.36 1.13 0.32 1.65 0.86 2.26 1.26 1.19 1.03 1.09 1.06 2.54 1.82 3.41 2.48 0.30 5.28 0.95 0.46
Toyota LA-4 BS2-1 trap
‘TAEB-82-0161 0.05 0,04 0.44 0.35 0.52 1.05 1.69 1.33 1.58 1.19 0.41 0.88 0.98 0.34 3,451.38 0.26 0.77 0.56 0.13
Toyota LA-4 BS2-1 trap
TAEB-84-0010 0.00 0.46 7.32 1.01 5:50 1.99 5.82 2.74 7.96 7.58 2.18 1,93 20.6 1.34 7.19 2.60 1.26 2.92 9.25 0.16
Mercedes LA-~4 baseline
TAEB- 84-0020 .00 0:86 2.48 7.36 4.17 28.8 33.5 8.39 60.3 3.22

Mercedes LA~4 NGK-2 trap

a ;
No repeat analysis

Insufficient sample to perform repeat analysis .
“Insufficient sample to perform analysis in TA 1535, 1A 1537 and TA 98


TECHNICAL REPORT DATA

{Please read Instructions on the reverse before completing)

i. REPORT NO, 2.
EPA 460/3-84-006

3. RECIPIENT'S ACCESSIONNO.

4. TITLE AND SUBTITLE

Filter Extraction and Ames Bioassay Results for
EPA Particulate Samples

5. REPORT DATE
August 1984
6. PERFORMING ORGANIZATION CODE

7, AUTHOR(S)
Mary Ann Warner-Selph

i. PERFORMING ORGANIZATION REPORT NO.

9. PERFORMING ORGANIZATION NAME AND ADDRESS
Southwest Research Institute

6220 Culebra Road

San Antonio, Texas 78284

10. PROGRAM ELEMENT NO.

[ii CONTRACT/GRANT NO, SSS
68-03-3162 Work Assign. No, 2|

12, SPONSORING AGENCY NAME AND ADDRESS

Environmental Protection Agency
2565 Plymouth Road
Ann Arbor, Michigan 48105

13. TYPE OF REPORT AND PERIOD COVERED

Final (Sept. 83 =
14, SPONSORING AGENCY CODE

15, SUPPLEMENTARY NOTES

76. ABSTRACT

linear and non-linear regression methods.

This report describes filter extractions and Ames bioassay of filter extracts
performed for the Emission Control Technology Division of Environmental Protection
Agency. Eight sets of particulate-loaded filters were provided to SwRI by the
sponsor. The filters were soxhlet-extracted in methylene chloride, and the
extracts were dried and weighed. The organic extracts were analyzed using the
Ames bioassay at Southwest Foundation for Biomedical Research (SFBR), formerly
Southwest Foundation for Research and Education. The data were analyzed using

Motor Vehicles

ka KEY WORDS AND DOCUMENT ANALYSIS

a. DESCRIPTORS Ib. IDENTIFIERS/OPEN ENDED TERMS |c. COSAT! Field/Group
Air Pollution Organic Extractables
Exhaust Emissions Ames Bioassay

18. DISTRIBUTION STATEMENT. 19, SECURITY CLASS (This Report) 21, NO. OF PAGES
Unclassified 28
Release Unlimited 20. SECURITY CLASS (This page) 22. PRICE
Unclassified

EPA Form 2220-1 (9-73)


Table

LIST OF TABLES

Page
Summary of Ames Bioassay of Extracts, Dose-Response 2
Relationship Calculated by Linear Regression
Summary of Ames Bioassay of Extracts, Dose-Response 3
Relationship Calculated by EPA-RTP Non-Linear Model
Results of Methylene Chloride Extraction on Filters 5
Provided by EPA
Results of Individual Ames Tests of Extracts, Dose- 6
Response Relationship Calculated by Linear Regression
Results of Individual Ames Tests of Extracts,.Dose- 7

Response Relationship Calculated by EPA-RTP Non-Linear
Model

vi


TABLE OF CONTENTS

FOREWORD

ABSTRACT

LIST OF TABLES

I SUMMARY

I. INTRODUCTION

Il, ANALYTICAL PROCEDURES
Iv. RESULTS

REFERENCES

APPENDIX

10


The Ames Salmonella typhimurium Histidine Reversion Assay

The Ames Samonella typhimurium histidine reversion assay is utilized on a
regular basis at the Southwest Foundation for Research and Education. Mr.
Arnaldo Noyola, who performs the bacterial mutagenesis procedure, received his
training in conducting the assay in the laboratories of Dr. Thomas Matney at
The University of Texas Health Science Center in Houston, Tx, in 1977, and Dr.

Vincent Simmon at SRI in Palo Alto, CA, in 1979.

The tester strains we are using have been obtained from the laboratories
of Dr. Bruce Ames and Dr. Herbert Rosenkranz. These strains, as indicated in
the Methods. of Procedure section, are examined regularly using the diagnostic
tests described by Ames et al. (1975), Levin et al. (1982), and Maron and Ames
(1983) to insure the stability and integrity of the individual tester

strains. The spontaneous mutation rate is regularly monitored.

For each sample, a minimum of five concentrations are tested in tripli-
cate with each tester strain for evaluation of mutagenicity, both with and
without in vitro metabolic activation. Tester strains TA 97, TA 98, TA 100,
TA 102, and if requested, TA 1538, are routinely employed. Other tester
strains that are available for testing include TA 1535, TA 1537, TA 98NR,

TA 98/1 ,8-DNP,, and TA 104. Also if requested (and at additional cost), a
cytotoxicity assay will be performed at each concentration to determine bac-
terial concentration by a dilution procedure, so that revertants per surviving

bacterium can be quantitated, in addition to revertants per plate.


TABLE 1.

RELATIONSHIP CALCULATED BY LINEAR REGRESSION

SUMMARY OF AMES BIOASSAY OF EXTRACTS, DOSE-RESPONSE

rev/Ug
TA 98/1,8-8
TA 1535 TA_1537 TA 1538 TA_98 TA 100 TA 974 TA 1022 TA 1044 TA_98NR@ DNP6

Sample Identification =s9 489 -S9 489 “-s9 #59 _-59 459 _-S9 489 =S9 459 “-S9 489 -S9 +489 _-S9 459 “-S9 489
TAEB-81-0781
vw LA-4 baseline -0.02 0.06 0.67 1.12, 2.30 1:96 3.32 2.01 3.07 1.82 1.52 0.74 1.13 1.55 7.32 2.51 0.88 1.01 1.99 0.66
TAEB-82-0191>
VW LA-4 JM-13 trap -0.05 1.65 5,53 5.16 15.53 9.73 9,56 7.53 4.57 2.98
TAEB-82-0101>
VW LA-4 JM-13 trap -0.11 0.40 1.82 1.87 5.29 4.55 3.90 2.75 3,19 2.39
TAEB-82-0131>
VW Regen JM-13 trap w-F  --€ EEF 2.007064 1.52 2.99
TAEB-82-0221
Toyota LA~4 BS2-1 trap 0.00 0.06 0.27 0.32 0.58 0.74 0.72 1.14 0,89 0.85 0,43 0.54 0.50 0.55 1.30 1.05 0.30 0.56 0.41 0.19
TAEB-82-0161
Toyota LA-4 BS2-1 trap -0.01 0.04 0.20 0.28 0.26 0.68 0.74 0.72 0.96 0.54 0.33 0.87 0.58 0.33 1.62 1.27 0.26 0.56 0.38 0.09
TAEB-84-0010
Mercedes LA-4 baseline 0.04 0.26 0.54 0.60 1,18 1.12 1.70 1.32 2,84 3.44 0.46 0.67 1.04 0.83 2.61 1.32 0.81 1.00 1.35 0.19
‘TAEB-84-0020
Mercedes LA-4 NGK-2 trap -0.03 0.36 4.98 2.10 3.52 3.17 12.22 3.20 14.06 12.15

a i
No repeat analysis

Insufficient sample to perform repeat analysis
“Insufficient sample to perform analysis in TA1535, TA1537 and TA 98


are inverted in groups of ten, removed from the vented hood, and placed in a

vented Forma reach~in incubator set at 37°C for 48 hr incubation.

After 48 hr, the plates are removed from the incubator and the revertants
on each plate are counted using an electronic counter (NBS Model C111). In
our laboratory, the spontaneous mutation rates of solvent controls in all
tester strains is similar to those reported by Maron and ames (1983). With
the activated S9 system present, 2-aminofluorene is regularly used as a
positive control. NaN, (TA 100, TA 1535), methyl methane sulfonate (TA 102),
and 2-nitrofluorene (TA 98, TA 1538), crotonaldehyde (TA 104), ICR-191

(TA 97), and 9-aminoacridine (TA 1537) are positive controls for testing

without added S9.

Testing of Bacterial Strain Sensitivity for Isolation of Desirable Tester

Strain Stock

Diagostic tests to confirm the stability and integrity of our tester
strains are performed regularly. For this purpose, samples of the tester
strains are inoculated into 5 ml of Oxoid nutrient broth and allowed to grow
for approximately 16 hr in a 37°C incubator. The following day, the bacteria
are streaked on sterile 100 mm petri plates with 25 ml of nutrient agar and
incubated for 24 hr at 37°C to recover single cell isolates of each of the
tester strains. After 24 hr, three single cell isolates of each tester strain
are inoculated into three separate 18 x 150 mm test tubes containing 5 ml of
Oxoid nutrient broth then incubated overnight. Three samples of each tester

strain are then available for sensitivity testing.


United States Office of Mobile Source Air Pollution Control EPA 460/3-84-006
Environmental Protection Emission Control Technology Division August 1984
Agency 2565 Plymouth Road

Ann Arbor, Michigan 48105

Air

GEPA Filter Extraction and Ames
Bioassay Results for
EPA Particulate Samples


TABLE 3.

RESULTS OF METHYLENE CHLORIDE EXTRACTIONS ON FILTERS PROVIDED BY EPA

Sample

Extract Sample Particulate Extract Percent
Filter Number Number Identification Wt. (g) Wt. (g) Extractables
TAEB-81-0780 .
TAEB-81-0800 TAEB-81-0781 Vw LA-4baseline 3.53 0.8793 24.9%
TAEB-82-0190
TAEB-82-0200 TAEB-82-0191 VW LA-4 JM-13 trap 1.41 0.0784 5.6
TAEB-82-0210
TAEB-82-0100
TAEB-82-0110 TAEB-82-0101 VW LA-4 JM-13 trap 1.35 0.0778 5.8
TAEB-82-0120
TAEB-82-0130
TAEB-82-0140 TAEB-82-0131 VW Regen. JM-13 11.35 0.0188 0.2
TAEB-82-0150 trap
TAEB-82-0220
TAEB-82-0230 TAEB-82-0221 Toyota LA-4 BS2-1 1.82 0.5508 30.3
TAEB-82-0240 trap
TAEB-82-0160
TAEB-82-0170 TAEB-82-0161 Toyota LA-4 BS2-1 2.45 0.6395 26.1
TAEB-82-0180 trap
83-0640
83-0650 TAEB-84-0010 Mercedes LA-4 3.58 0.3198 8.9
83-0660 baseline
810
820 TAEB-84-0020 Mercedes LA-4 NGK-2 1.28 0.1233 9.6
830 trap


Il. INTRODUCTION

This project provided for the extraction of 23 filters to produce eight
samples for Ames bioassay analysis. The filters were generated by EPA and
shipped to SwRI for methylene chloride extraction. A list of all filters
extracted by SwRI is shown in Table 3. Extraction results, in mass and percent
of particulate mass, are also listed in the Table.

The program initially called for a full Ames analysis of the methylene
chloride extractables. This plan included analysis in five tester strains in
triplicate, with and without metabolic activation, with a repeat. The test
procedure required a minimum of 145 mg of extract. Four of the samples
contained insufficient extract to perform the full bioassay. Samples -0191 and
-0101 contained only enough extract to perform initial analyses with no repeat.
Sample -0131 was tested in only 2 tester strains, TA 98 and TA 100, without a
repeat, and at reduced dosage levels. Sample -0020 was tested at reduced
dosage, but a repeat analysis was included. -

With the approval of Craig Harvey, EPA Branch Technical
Representative, additional analyses of the four largest samples (having more
than minimum required mass) were performed. These tests were performed to
bring the level of effort up to that in the original test plan. Samples +0781,
-0221, -0161, and -0010 were analyzed in five additional tester strains without a
repeat: TA 97, TA 102, TA 104, TA 98NR, and TA 98/1,8-DNPg¢. TA 97 is
responsive to aridine compounds, and is recommended as a replacement for
TA 1537.(1) Tester strains TA 102 and TA 104 are especially sensitive to nitro-
PAH compounds. TA 98NR is insensitive to 1-nitro-PAH compounds, but
sensitive to dinitro compounds. The reverse response pattern occurs with tester
strain TA 98/1,8-DNP¢.

Ames results were processed by two models, linear regression and a non-
linear model. SFBR calculated the slope (rev/lg) using linear regression. For
samples in which the extract dose became toxic to the bacteria, the dosages
were not included in the slope calculation. The non-linear model, however,
includes all data points, regardless of toxicity. Ames data were processed using
anon-linear computer program at EPA-RTP.

Ames results calculated by linear regression are listed by individual test
in Table 4. Data calculated by the non-linear method are listed by individual
tests in Table 5. As mentioned previously, summaries of results calculated by
linear regression and by the non-linear method are reported in Tables 1 and 2,
respectively.


I. SUMMARY

Results from the Ames bioassay are reported in this section. The data
that were analyzed using linear regression are reported in Table 1. Samples
TAEB-82-0191 and TAEB-~83-0020 caused the greatest Ames response of the
eight samples. TAEB-82-0191, produced by a VW during the LA-4 cycle witha
JM-13 trap, was most mutagenic in TA 1537, TA 1538, and TA 98. Sample
TAEB-83-0020, produced by a Mercedes equipped with an NGK-2 trap and
operated over the LA-4, was most mutagenic in TA 98 and TA 100. The
greatest Ames response was observed in TA 104 for samples analyzed in the
five additional tester strains (TA 97, TA 102, TA 104, TA 98NR, and TA-98/1,8-
DNP¢),

Ames data analyzed with the non-linear model are summarized in Table 2.
The highest Ames response was observed with samples TAEB-82-0191 and TAEB-
84-0020, similar to results calculated by linear regression. Sample -0191
produced the highest revertants per microgram of extract-in tester strains
TA 1537, TA 1538, and TA 98, and sample -0020 had the highest response in
TA 1538, TA 98, and TA 100. TAEB-82-0221 and TAEB-82-0161 produced
consistently low Ames results in most of the strains. The Ames response of the
samples tested in the five extra tester strains did not show marked differences
between strains. The average response in TA 104 was somewhat higher,
however, than responses in TA 97, TA 102, TA 98NR, and TA 98/1,8-DNP¢.

Ames dose-response data calculated by the non-linear model: were
generally higher than those determined by linear regression. As discussed
above, however, the same trends occurred regardless of the method chosen for
data analysis.


The dilutions are arranged so that the amount of chemical to be added per
plate will be contained in 0.1 ml. The compounds are generally tested for
mutagenicity at levels between 0.01 and 1000 ug per plate. Sufficient
material is placed in solution so that triplicate plates can be run at each

concentration, with and without S9 present for metabolic activation.

Preparation of the S9 Mix (metabolic activation system)

Male Sprague-Dawley rats, weighing approximately 200 g, are given an i.p.
injection of Aroclor 1254 (a polychlorinated biphenyl mixture), diluted in
corn oil to a concentration of 200 mg/ml, at a dosage of 500 mg/kg of rat
weight 5 days prior to sacrifice. Twelve hours prior to sacrifice, the food
is. removed and, on the fifth day, the rats are stunned by a blow to the head
and decapitated. Using sterile techniques, the liver is removed and weighed
in a preweighed sterile beaker containing ice cold 0.15 M KCl. The livers are
subsequently washed with one volume of cold, sterile 0.15 M KCl per weight of
wet liver, then placed in three volumes of cold sterile 0.15 M KCl per weight
of liver. The liver is minced with sterile scissors, transferred into a
sterile tissue homogenizer (Tissuemizer), and homogenized. The homogenate is
then centrifuged in sterile capped tubes at 9000 g (8700 rpm) for 10 min in a
Sorval RC2-B centrifuge. The supernatant (S9) fraction is aliquoted in 3 or
8.5 ml volumes into sterile capped polypropylene tubes that are immediately
frozen in dry ice. The tubes are stored until needed in a -80°C Revco

freezer.


To test for histidine and biotin requirement, sterile stock solutions of
histidine (1 9/100 ml glass distilled water) and biotin (10 mg/100 ml water)
are used. To three minimal medium agar plates, 0.1 ml of the histidine solu-
tion is spread throughout the surface of the plates with an L-shaped glass rod
until they are dry. To a second set of plates, 0.1 ml of the biotin solution
is similarly added... To the third set of plates, 0.1 ml of the histidine solu-
tion is spread: until dry, followed by 0.1 ml of the biotin solution that is
also spread until dry. The plates are divided into eight sections, streaked
with the cultures as described above, and incubated overnight at 37°C to

determine the presence of normal growth.

For each of the 15 isolates tested for crystal violet, UV,: ampicillin,
tetracycline, histidine and biotin sensitivities, and spontaneous reversion
rate, a data chart is constructed. The zones of inhibition around the crystal
violet disk are measured and the values are entered into the data chart. All
tester strains should be sensitive to UV, except TA 102, and they should be
able to grow in the combination of histidine and biotin. TA 1538 should be
sensitive to ampicillin; TA 98 and TA 100 should be resistant to ampicillin.
TA 102 is the only strain which is resistant to tetracycline. From the infor~
mation in the chart, the single cell isolates which are most representative of
published and our own historically acceptable values are used to prepare stock
cultures for storage. Isolates are inoculated into culture tubes with 5 ml
nutrient broth, then incubated overnight at 37°C. After 16 hr growth, the
tester strain cultures are diluted with 0.09 ml of DMSO for each ml of cul-

ture, quickly frozen in dry ice, and stored in a Revco freezer at -80°C.

1


FOREWORD

This project was conducted for the U.S. Environmental Protection Agency
by the Department of Emissions Research, Southwest Research Institute. The
work was carried out between September 1983 and May 1984 under EPA
Contract No. 68-03-3162, Work Assignment Number 2. It was identified within
Southwest Research Institute as Project 03-7338-002. The EPA Project
Officers were Mr. Robert J. Garbe and Mr. Craig A. Harvey, and the Branch
Technical Representative was Mr. Craig A. Harvey, both of the
Characterization and Technical Applications Branch, Emission Control
Technology Division, Environmental Protection Agency, 2565 Plymouth Road,
Ann Arbor, Michigan. The Southwest Research Institute Project Manager was
Charles T. Hare, and the Project Leader and Principal Investigator was Mary
Ann Warner-Selph.

iii


IL ANALYTICAL PROCEDURES

The particulate filters provided by EPA were extracted by SwRI, and
Ames bioassay of the organic extractables was performed by SFBR. Filter and
extract processing was carried out under yellow lights. Each filter was
extracted in a soxhlet apparatus with methylene chloride, at 4 cycles per hour
for 8 hours, to provide a total of approximately 32 cycles per filter. The
resulting organic extractables were filtered and concentrated under vacuum on
a roto-evaporator. The extracts were further concentrated under a dry
nitrogen purge and transferred to preweighed vials. The samples were weighed
when dry, and percentages of extractables were calculated relative to
particulate loading. The completed samples were then delivered to SFBR for
Ames bioassay. The Ames bioassay procedure is included as an Appendix.


REFERENCES

1. Maron, D.M. and Ames, B.N., Revised Methods for the Salmonella
Mutagenicity Test, Mutation Research, Vol. 113, (173-215) 1983.

10


This report is issued by the Environmental Protection Agency to report
technical data of interest to a limited number of readers. Copies are available
free of charge to Federal employees, current ‘contractors and grantees, and
nonprofit organizations - in limited quantities - from 2565 Plymouth Road,-Ann
Arbor, Michigan 48105.

This report was furnished to the Environmental Protection Agency by Southwest
Research Insitute, 6220 Culebra Road, San Antonio, Texas, in fullfillment of
Work Assignment No. 2 of Contract No. 68-03-3162. The contents of this report
are reproduced herein as received from Southwest Research Institute. The
opinions, findings, and conclusions expressed are those of the author and not
necessarily those of the Environmental Protection Agency. Mention of
company or product names is not to be considered as an endorsement by the
Environmental Protection Agency.

Publication No. EPA 460/3-84-006

di


Nutrient agar plates are used to assay for crystal violet, ampicillin,
and UV sensitivity. An aliquot (0.1 ml) of each culture is added to 2.0 ml of
nutrient top agar, mixed, and poured onto a prelabeled nutrient agar plate.
With sterile forceps, a crystal violet disc (0.25 inch with 10 ul of a 1 mg/ml
crystal violet solution) is placed in the center of each plate. The plates
are then incubated overnight, and the zone of inhibition around the disc is
determined. A strain is acceptable (i.e., has a good rfa) if the zone of

inhibition is 14 mm or greater.

To determine ampicillin resistance, 0.1 ml of a sterile solution of
8° mg/ml. ampicillin in 0.02 N NaOH is spread to dryness on two nutrient agar
plates. Two plates without ampicillin serve as the controls for ampicillin
resistance and UV sensitivity test as described below. Each of the four
plates is divided into eight sections,‘ and three samples of each of the five
strains are streaked on ampicillin plus and ampicillin minus sections. The
plates are incubated overnight at’ 37°C to determine the ability to grow in the

presence of ampicillin.

To determine the presence of the tetracycline resistance gene in TA 102,
plates containing ampicillin and 2 ug/ml of an 8 mg/ml tetracycline solution
in 0.02 N HCl are used. The bacterial cultures are streaked in the same
manner as the ampicillin test plates and an R~factor strain (i.e., TA 100) is
used as a control. The plates are placed in an incubator overnight at 37°C,
and the tetracycline resistance gene is confirmed by the growth capability.
TA 100, which lacks the gene, is sensitive to tetracycline, while TA 102 is

resistant.


EPA 460/3-84-006

Filter Extraction and Ames Bioassay
Results for EPA Particulate Samples

by
Mary Ann Warner-Selph

Southwest Research Institute
6220 Culebra Road
San Antonio, Texas 78284

Contract No. 68-03-3162
Work Assignment No. 2

EPA Project Officers: Robert J. Garbe
Craig A. Harvey
Branch Technical Representative: Craig A. Harvey

Prepared for

ENVIRONMENTAL PROTECTION AGENCY
Office of Mobile Source Air Pollution Control
Emission Control Technology Division
2565 Plymouth Road
Ann Arbor, Michigan 48105

August 1984


REFERENCES

Ames, B. N., McCann, J., and Yamasaki, E. (1975). Methods for, detecting car-
cinogens and mutagens with the Salmonella/mammalian microsome mutagenic-
ity test. Mutat. Res. 31:347-364.

Levin, D. E., Yamasaki, E., and Ames, B. N. (1982). A new Salmonella tester
strain, TA 97, for the detection of frameshift mutations. Mutat. Res.
94:315~330.

Maron, D. and Ames, B. N. (1983). Revised methods for the Salmonella

mutagenicity test. Mutat. Res. 113:173-215.

12


TABLE 4, RESULTS OF INDIVIDUAL AMES TESTS OF EXTRACTS, DOSE-RESPONSE.
RELATIONSHIP CALCULATED BY LINEAR REGRESSION

xev/ug =
7 TA98/1, 8-3
Sample TA 1535 TA_1537 A_1538. TA 98 TA 100 TA 97 TA 1024 TA 104° TA 98NR@ DNPS
Identification Test ~-s9 +89 -s9 +89 -s9 +89 -s9 +89 -s9 +59 -s9 +89 -s9 +89 -s9 +89 -s9 +59 ~s9 +89
‘TAEB-81-0781 initial -0.02 0.04 1.23 1.01 3.15 2.36 3.40 2.07 3.20 2.23 1.52 0.74 1.13 1.55 7.32 2.51 0.88 1.01 1.99 0.66
WW LA-4 baseline repeat -9.03 0.08 0.11 1.23 1.45 1.55 3.23 1.95 2.94 1.41
avg. -0.02 0.06 0.67 1.12 2.30 1.96 3.32 2.01 3.07 1.82
TAEB-82-0191
VW LA-4 JM~13 initial -0.05 1.65 5.53 5.16 15.53 9.73 9.56 7.53 4.57 2.98
trap
‘TAEB-82-0101
VW LA-4 JM-13 initial -0.11 0.40 1,82 1.87 5.29 4.55 3,90 2.75 3.19 2.39
trap .
TAEB-82-0131
WW Regen. JM-13. initial -- --©  --S --© —~-E | --© 2.00 7.64 1.52 2.99
trap
‘TAEB-82-0221 initial 0.00 0,09 0.29 0.33 0.70 0,73 0.53 1,23 1.05 0.84 0.43 0.54 0,50 0.55 1.30 1,05 0.30 0.56 0.41 0.19
Toyota LA-4 BS2-1 repeat 0.00 0.04 0.25' 0.32 0.46 0.76 0.90 1.05 0.73 0.86
trap avg. 0.00 0.06 0,27 0.32 0.58 0.74 0.72 1.14 0.89 0.85
TAEB-82-0161 initial -0.02 0.04 0,18 0.30 0.24 0.86 0.74 0.86 1.05 0.60 0.33 0.87 0.58 0.33 1.62 1.27 0.26 0.56 0.38 0.09
Toyota LA-4 BS2-1 repeat 0.00 0.03 0.21 0.25 0.27 0.49 0.74 0.57 0.86 0.47
trap avg. -0.01 0.04 0.20 0.28 0.26 0.68 0.74 0.72 0.96 0.54
‘TAEB-84-0010 initial 0.06 0.40 0.67 0.60 1.22 1.03 1.79 1.33 2.18 3.49 0.46 0.67 1.04 0.83 2.61 1.32 0.81 1.00 1.35 0.19
Mercedes LA-4 repeat 0,01 0.11 0.41° 0.59 1,15 1.20 1.60 1.30 3.49 3.38
baseline avg. 0.04 0.26 0.54 0,60 1.18 1.12 1.70 1.32 2.84 3.44
TAEB-84-0020 initial -0.03 0.39 4.37 1.59 2.69 3.03 12.47 2.54 14.50 12.21
Mercedes LA-4 repeat -0.03 0.34 5.60 2.60 4.34 3.31 11.97 3.86 13.63 12.09
NGK-2 trap avg. -0.03 0.36 4.98 2.10 3.52 3,1712.22 3,20 14.06 12.15

@No repeat analysis
Insufficient sample to perform repeat analysis
“Insufficient sample to perform analysis in TA 1535, TA 1537, TA 98


IV. RESULTS

Four of the extracts were produced by a Volkswagen Rabbit (VW), two
were from a Toyota, and two from a Mercedes. As seen in Table 3, a relatively
large fraction of the VW baseline filter sample was composed of organic
extractables, compared to the trap and regeneration filters. Although the
regeneration filter had relatively high particulate loading, less than one percent
was organic soluble material. The two Toyota samples which were taken with a
trap on the vehicle produced 30 and 26 percent extractables. The Mercedes
baseline sample produced higher particulate levels than the Mercedes with trap,
but.a slightly lower fraction of extractables.

The results of the Ames bioassay by the non-linear model (in revertants
per microgram of extract) generally exceeded the values determined by linear
regression. The largest discrepancy between the two sets of data was with
sample -0010, from the Mercedes baseline. Several of the non-linear model
values were up to 20 times higher than linear values, as shown in Tables 1 and 2.
For other samples, the non-linear values were up to five times higher than
values determined by linear regression.