United States
             Environmental Protection
             Agency
                Office of Mobile Source Air Pollution Control
                Emission Control Technology Division
                2565 Plymouth Road
                Ann Arbor, Michigan 48105
EPA 460/3-84-006
August 1984
&EPA
             Air
Filter Extraction and Ames
Bioassay  Results for
EPA Particulate Samples

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                                      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

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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
                                      11

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                              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.
                                    111

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                               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

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                      TABLE OF CONTENTS






                                                        Page




FOREWORD                                               iii




ABSTRACT                                                iv




LIST OF TABLES                                           vi




I.    SUMMARY                                            1




n.   INTRODUCTION                                        4




m.   ANALYTICAL PROCEDURES                             8




IV.   RESULTS                                              9




REFERENCES                                             10




APPENDIX
                                  v

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                            LIST OF TABLES
Table                                                           Page

  1        Summary of Ames Bioassay of Extracts, Dose-Response    2
           Relationship Calculated by Linear Regression

  2        Summary of Ames Bioassay of Extracts, Dose-Response    3
           Relationship Calculated by EPA-RTP Non-Linear Model

  3        Results of Methylene Chloride Extraction on Filters        5
           Provided by EPA

  4        Results of Individual Ames Tests of Extracts, Dose-        6
           Response Relationship Calculated by Linear Regression

  5        Results of Individual Ames Tests of Extracts,.Dose-        7
           Response Relationship Calculated by EPA-RTP Non-Linear
           Model
                                       VI

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                              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 with a
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-
DNP6).

     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/l,8-DNP6.

     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.

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                                            TABLE  1.  SUMMARY OF AMES BIOASSAY OF EXTRACTS, DOSE-RESPONSE
                                                   RELATIONSHIP CALCULATED BY LINEAR REGRESSION
                                                                                        rev/Ug
                             TA  1535     TA  1537
                                                      TA  1538
                                                                    TA 98
                                                                                 TA 100
 Sample Identification
TAEB-81-0781
VW LA-4 baseline
TAEB-82-0191b
VW LA-4 JM-13 trap
TAEB-82-0101b
VW LA-4 JM-13 trap
TAEB-82-0131b
VW Regen JM-13 trap
TAEB-82-0221
Toyota LA-4 BS2-1 trap
TAEB-82-0161
Toyota LA-4 BS2-1 trap
TAEB-84-OOlO
Mercedes LA-4 baseline
TAEB-84-0020
Mercedes LA-4 NGK-2 trap
                                                                                                                 TA 98/1,8-a
                                                                   TA 97a      TA 102a     TA 104a    TA 98NRa      DNPfi
                            -S9    +S9   -S9   +S9   -S9    +S9   -S9    +S9   -S9    +S9    -S9   +S9   -S9   +S9   -S9   +S9   -S9   +S9   -S9   +S9
-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

-0.05  1.65  5,53  5.16  15.53  9.73   9.56  7.53   4.57   2.98


-0.11  0.40  1.82  1.87   5.29  4.55   3.90  2.75   3.19   2.39

  —c   —c   —c   —c    —c   —c   2.00  7.64   1.52   2.99

 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

-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

 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

-0.03  0.36  4.98  2.10   3.52  3.17  12.22  3.20  14.06  12.15
bNo repeat analysis
 Insufficient sample to perform repeat analysis
Insufficient sample to perform analysis in TA 1535, TA 1537  and TA 98

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 Sample  Identification
TAEB-81-0781
VW LA-4  baseline
TAEB-82-0191b
VW LA-4  JM-13 trap
TAEB-82-0101b
VW LA-4  JM-13 trap
TAEB-82-0131b
VW Regen JM-13 trap
TAEB-82-0221
Toyota LA-4 BS2-1  trap
TAEB-82-0161
Toyota LA-4 BS2-1  trap
TAEB-84-0010
Mercedes LA-4 baseline
TAEB-84-0020
Mercedes LA-4 NGK-2  trap
                                               TABLE 2.  SUMMARY OF AMES BIOASSAY OF EXTRACTS,  DOSE-RESPONSE
                                                    RELATIONSHIP CALCULATED BY EPA-RTP NON-LINEAR MODEL
                                                                                         rev/lig
                              TA 1535
                                          TA 1537
                                                       TA 1538
                                                                     TA 98
                                                                                  TA 100
                             -S9
        +S9    -S9    +S9   -S9    +S9   -S9    +S9   -S9
                                                                                       +S9
                                               TA 98/1,8-a
                                                  DNP6
-S9   +S9   -S9   +S9   -S9   +S9   -S9   +S9   -S9   +S9
                                                                                               TA 97a
                                                                               TA 102a     TA 104a    TA 98NRa
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

1.43   4.93 15.8  43.6   143   37.7   28.5  19.4    22.5     8.92


0.76   2.81  8.24 15.9   27.9  36.0   14.9   7.66   13.9     6.64

 — °    — °   —°   —C    —°   —C   4.54 24.7     4.47    9.36

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

0.05   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   3.45 1.38  0.26  0.77


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


0.00   0.86  2.48  7.36   4.17 28.8   33.5   8.39   60.3     3.21
                                                                                                                 8.96  0.89
                                               0.95  0.46

                                               0.56  0.13


                                               9.25  0.16
bNo repeat analysis
 Insufficient sample to perform repeat analysis
clnsufficient sample to perform analysis  in TA 1535, TA 1537  and  TA 98

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                            E. INTRODUCTION
     This project provided for the extraction of 23  filters to produce eight
samples  for Ames bio assay 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/l,8-DNP6.  TA 97 is
responsive to  aridine compounds, and  is recommended  as a replacement for
TA 1537.M 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-DNPe.

     Ames results were processed by two models, linear regression and a non-
linear  model.   SFBR calculated the slope (rev/Ug)  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
a non-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.

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         TABLE 3.  RESULTS OF METHYLENE CHLORIDE EXTRACTIONS ON FILTERS PROVIDED BY EPA
Filter Number

TAEB-81-0780
TAEB-81-0800
   Sample
   Extract
   Number
    Sample        Particulate    Extract      Percent
Identification      Wt.  (g)      Wt. (g)    Extractables
TAEB-81-0781    VW LA-4baseline      3.53
                                  0.8793
                          24.9%
TAEB-82-0190
TAEB-82-0200
TAEB-82-0210
TAEB-82-0191    VW LA-4 JM-13 trap   1.41
                                  0.0784
                           5.6
TAEB-82-0100
TAEB-82-0110
TAEB-82-0120
TAEB-82-0101    VW LA-4 JM-13 trap    1.35
                                  0.0778
                           5.8
TAEB-82-0130
TAEB-82-0140
TAEB-82-0150
TAEB-82-0131    VW Regen. JM-13
                   trap
                    11.35
             0.0188
              0.2
TAEB-82-0220
TAEB-82-0230
TAEB-82-0240

TAEB-82-0160
TAEB-82-0170
TAEB-82-0180

83-0640
83-0650
83-0660
TAEB-82-0221
TAEB-82-0161
Toyota LA-4 BS2-1
   trap
Toyota LA-4 BS2-1
   trap
TAEB-84-0010    Mercedes LA-4
                   baseline
1.82
2.45
                     3.58
0.5508
0.6395
             0.3198
30.3
26.1
              8.9
810
820
830
TAEB-84-0020    Mercedes LA-4 NGK-2  1.28
                   trap
                                  0.1233
                           9.6

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                                            TABLE 4.   RESULTS OF INDIVIDUAL AMES TESTS OF EXTRACTS,  DOSE-RESPONSE
                                                       RELATIONSHIP CALCULATED BY LINEAR REGRESSION
                                                                                rev/yg
Sample
Identification
TAEB-81-0781
VW LA-4 baseline

TAEB-82-0191
VW LA-4 JM-13
trap
TAEB-82-0101
VW LA-4 JM-13
trap
TAEB-82-0131
trap
TAEB-82-0221
Toyota LA-4 BS2-1
trap
TAEB-82-0161-
Toyota LA-4 BS2-1
trap
TAEB-84-0010
Mercedes LA-4
baseline
TAEB-84-00^0
Mercedes LA-4
NGK-2 trap

Test
initial
repeat
avg.

initial


initial



initial
repeat
avg.
initial
repeat
avg.
initial
repeat
avg.
initial
repeat
avg.
TA 1
-S9
-0.02
-0.03
-0.02

-0.05


-0.11

c

0.00
0.00
0.00
-0.02
0.00
-0.01
0.06
0.01
0.04
-0.03
-0.03
-0.03
535
+S9
0.04
0.08
0.06

1.65


0.40

c

0.09
0.04
0.06
0.04
0.03
0.04
0.40
0.11
0.26
0.39
0.34
0.36
TA
-S9
1.23
o.ir
0.67

5.53


1.82

c

0.29
0.25-
0.27
0.18
0.21
0.20
0.67
0.41
0.54
4.37
5.60
4.98
1537
+S9
1.01
1.23
1.12

5.16


1.87

c

0.33
0.32
0.32
0.30
0.25
0.28
0.60
0.59
0.60
1.59
2.60
2.10
A 1
-S9
3.15
1.45
2.30

15.53


5.29

c

0.70
0.46
0.58
0.24
0.27
0.26
1.22
1.15
1.18
2.69
4.34
3.52
538
+S9
2.36
1.55
1.96

9.73


4.55

c

0.73
0.76
0.74
0.86
0.49
0.68
1.03
1.20
1.12
3.03
3.31
3.17
TA
-S9
3.40
3.23
3.32

9.56


3.90



0.53
0.90
0.72
0.74
0.74
0.74
1.79
1.60
1.70
12.47
11.97
12.22
98
+S9
2.07
1.95
2.01

7.53


2.75



1.23
1.05
1.14
0.86
0.57
0.72
1.33
1.30
1.32
2.54
3.86
3.20
TA
-S9
3.20
2.94
3.07

4.57


3.19



1.05
0.73
0.89
1.05
0.86
0.96
2.18
3.49
2.84
14.50
13.63
14.06
100 TA 97a
+S9 -S9 +S9
2.23 1.52 0.74
1.41
1.82

2.98


2.39



0.84 0.43 0.54
0.86
0.85
0.60 0.33 0.87
0.47
0.54
3.49 0.46 0.67
3.38
3.44
12.21
12.09
12.15
TA98/l,8-a
TA 102a TA 10 4a TA 98NRa DNPfi
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
1.13 1.55 7.32 2.51 0.88 1.01 1.99 0.66










0.50 0.55 1.30 1.05 0.30 0.56 0.41 0.19


0.58 0.33 1.62 1.27 0.26 0.56 0.38 0.09


1.04 0.83 2.61 1.32 0.81 1.00 1.35 0.19





 No repeat analysis
 Insufficient sample to perform repeat analysis
CInsufficient sample to perform analysis in TA 1535, TA 1537, TA

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                                            TABLE 5.  RESULTS OF INDIVIDUAL AMES TESTS OF EXTRACTS, DOSE-RESPONSE

                                                    RELATIONSHIP CALCULATED BY EPA-RTP NON-LINEAR MODEL
                                                                                rev/gg
Sample
Identification
TAEB-81-0781
VWLA-4 baseline

b, a
TAEB-82-0191
VW LA-4 JM-13
trap
b,a
TAEB-82-0101
VW LA-4 JM-13
trap
b,a
TAEB-82-0131

VW Regen. JM-13
trap
TAEB-82-0221
Toyota LA-4 BS2-1
trap
TAEB-83-0161
Toyota LA-4 BS2-1
trap
TAEB-84-OOIO
Mercedes LA-4
baseline
TAEB-84-0020
Mercedes LA-4
NGK-2 trap

Test
initial
repeat
avg.


initial



initial



. .
initial

initial
repeat
avg.
initial
repeat
avg.
initial
repeat
avg.
initial
repeat
avg.
TA 1
-S9
0.01
0.07
0.04


1.43



0.76



c


0.00
0.00
0.00
0.09
0.01
0.05
0.00
0.00
0.00
0.00
0.00
0.00
535
+S9
0.42
0.51
0.47


4.93



2.81



c


0.35
0.36
0.36
0.04
0.04
0.04
0.45
0.47
0.46
0.79
0.93
0.86

-
5.
0.
3.


15.



8.






1.
0.
1.
0.
0.
0.
12.
1.
7.
2.
_
2.
TA
S9
38
84
11


8



24



c


55
71
13
43
44
44
9
73
32
48
_
48
1537
+S9
2.68
4.45
3.57


43.6



15.9



c


0.33
0.32
0.32
0.45
0.24
0.35
1.17
0.84
1.01
2.12
12.6
7.36
A 1
-S9
15.1
6.77
11.0


143



27.9






1.70
1.60
1.65
0.28
0.76
0.52
4.48
6.52
5.50
3.40
4.93
4.17
538
+S9
5.71
4.22
4.97


37.7



36.0



c


0.96
0.76
0.86
0.88
1.22
1.05
2.00
1.98
1.99
29.1
28.4
28.8


8
17
12


28



14






1
2
2
1
1
1
5
5
5
25
41
33
TA
-S9
.13
.6
.8


.5



.9






.79
.72
.26
.76
.62
.69
.66
.98
.82
.2
.8
.5
98

5
4
5


19



7






1
1
1
0
1
1
3
2
2
6
10
8
t
+S9
.20
.86
.03


.4



.66






.47
.05
.26
.86
.80
.33
.35
.12
.74
.57
.2
.39

_C
8.
6.
7.


22.



13.






1.
0.
1.
1.
1.
1.
4.
11.
7.
7.
1.
60.
PA :
59
66
30
48


5



9






58
79
19
79
36
58
91
0
96
51
13
.3'
LOC
H
2
4
3


8



6






1
0
1
1
0
1
7
8
7
3
2
3
) TA 97a TA 102a TA 194a TA 98NRa
hS9 -S9 4-S9 -S9 +S9 -S9 +S9 -S9 +S9
.29 5.00 0.75 2.86 2.34 17.2 6.77 2.63 3.71
.85
.57


.92



.64






.17 1.09 1.06 2.54 1.82 3.41 2.48 0.30 5.28
.88
.03
.42 0.41 0.88 0.98 0.34 3.45 1.38 0.26 0.77
.96
.19
.07 2.18 1.93 20.6 1.34 7.19 2.60 1.26* 2.92
.08
.58
.70
.72
.21
TA98/l,8-a
DNP6
-S9 +S9
8.96 0.89















0.95 0.46


0.56 0.13


• 9.25 0.16





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

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                         ANALYTICAL PROCEDURES
     The participate  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.

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                              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 participate 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.

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                              REFERENCES
1.    Maron, D.M. and Ames,  B.N.,  Revised  Methods  for the  Salmonella
     Mutagenicity Test, Mutation Research, Vol. 113, (173-215) 1983.
                                      10

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                    APPENDIX
   DETERMINATION OF THE IN VITRO GENOTOXICITY




              OF CHEMICALS BY THE




SOUTHWEST FOUNDATION FOR RESEARCH AND EDUCATION









   _S_.  typhimurium Histidine  Reversion Assay




                  Protocol 101

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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-DNPg, 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.

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Methods of Procedure








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



three steps.  First,  a  SOX concentrated VB salt solution is made by serial



addition to 670 ml warm (45°C),  glass distilled water of 10 g of magnesium




sulfate (MgS04«7H20), 100 g  of citric acid raonohydrate, 500 g potassium phos-



phate (dibasic), and  175 g sodium ammonium phosphate  (NaNH4HP04•4H20).  Spe-




cial attention is given to the requirement that each chemical is added and



dissolved successively.  The final SOX salt concentrate is then aliquoted into



40 and 80 ml portions into storage bottles are are loosely capped and auto-




claved for 15 rain 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 SOX 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

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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°P 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.








     For 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 raM L-histidine HC1-0.5 mM




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.

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     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 pg 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  rag/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 KC1.   The livers are



subsequently washed  with one  volume of cold, sterile 0.15 M KC1 per weight of




wet liver, then placed  in three  volumes  of  cold sterile 0.15 M KC1 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 rain 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.

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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,  Oil57 g D-glucose-6-PO.,



0.350 g NADP, 0.180 g MgClj,  0.273 g KC1,  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



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

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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  S9 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 89 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

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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 59 system present, 2-aminofluorene is regularly used as a



positive control.  NaN3  (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-arainoacridine  (TA 1537)  are  positive controls for testing




without added 59.








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  Qxoid 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.

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     Nutrient  agar  plates  ace  used to assay foe crystal violet, arapicillin,



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 rag/ral




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" rag/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 pg/ml of an 8 rag/ml tetracycline solution



in 0.02 N HC1  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.

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     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 0V lamp, warmed and func-




tioning for a minimum of 15 rain,  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

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     To test  foe histidine  and  biotin  requirement,  sterile stock solutions of




histidine  (1  g/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 378C 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.  Prom 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.

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REFERENCES








Ames, B. N., McCann, J.,  and Yamasaki,  E.  (1975).   Methods foe. 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, 0. and Ames, B. N.  (1983).  Revised  methods for the Salmonella




     mutagenicity test.   Mutat.  Res.  113:173-215.
                                       12

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                                    TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.
  EPA 460/3-84-006
                                                             3. RECIPIENT'S ACCESSION»NO.
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
                                                             8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORG "VNIZATION NAME AND ADDRESS
  Southwest Research Institute
  6220 Culebra Road
  San Antonio,  Texas  78284
                                                             10. PROGRAM ELEMENT NO.
              11. CONTRACT/GRANT NO.

                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. 1983  - May 1984)
              14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT

  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
  linear and non-linear regression  methods.
17.
                                 KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                               b.lDENTIFIERS/OPEN ENDED TERMS
                           c.  COSATI Field/Group
    Air  Pollution
    Exhaust Emissions
    Motor  Vehicles
   Organic  Extractables
   Ames Bioassay
13. DISTRIBUTION STATEMENT

  Release  Unlimited
19. SECURITY CLASS (This Report)
     Unclassified
21. NO. OF PAGES
      28
20. SECURITY CLASS (Thispage)
     Unclassified
                            22. PRICE
EPA Form 2220-1 (9-73)

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