EPA-600/1-76-022
April 1976
RELIABILITY OF BACTERIAL MUTAGENESIS TECHNIQUES TO DISTINGUISH
CARCINOGENIC AND NONCARCINOGENIC CHEMICALS
By
Barry Commoner
Center for the Biology of Natural Systems
Washington University
Box 1126
St. Louis, Missouri 63130
Contract No. 68-01-2471
Project Officer
Robert E. McGaughey
Office of Health and Ecological Effects
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
OTECTION A6BPCY
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
WASHINGTON, D.C. 20460
-------
DISCLAIMER
This report has been reviewed by the Office of Health and Ecological
Effects, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views
and policies of the U.S. Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or recommendation
for use.
ii
-------
CONTENTS
Page
List of Figures iv
List of Tables v
Acknowledgments vi
I Introduction 1
II The Salmonella Test System 2
III Results 11
IV Discussion 43
V References 47
VI Appendices 48
111
-------
FIGURES
Figure Page
1. Dose Response Curves of Mutagenically Active
Compounds 12-19
2. Frequency Distribution of Noncarcinogens and
Control Plates (TA 1535; Microsomes from
Various Tissues) 30
3. Frequency Distribution of Noncarcinogens and
Control Plates (TA 1537; Microsomes from
Various Tissues) 31
4. Frequency Distribution of Noncarcinogens and
Control Plates (TA 1538; Microsomes from
Various Tissues) 32
5. Frequency Distribution of Mutagenic Activity
Ratios of Presumptive Carcinogens and
Noncarcinogens 33
6. Cumulative Percentage of Compounds as a
Function of Mutagenic Activity Ratio
(For Optimal Strain, Microsome Preparation
and Substance Concentration) 34
7. Cumulative Percemtage of Compounds as a
Function of Mutcigenic Activity Ratio
(For Each Microsomal Preparation with
Optimal Strain and Concentration) 40
8. Cumulative Percentage of Compounds as a
Function of Mutagenic Activity Ratio
(For Each Substance Concentration with
Optimal Strain and Microsome Preparation) 41
iv
-------
TABLES
Table Page
1. Protein Content of Microsome Preparations
from Different Tissues 5
2-1. Detailed Results of Typical Control Runs
in Absence of Test Compound 7
2-2. Detailed Results of a Typical Experimental
Run 8
3. Average Number of Control Values (CAv) for
Different Microsome Preparations and
Salmonella Strains 21
4-1. Mutagenic Activity Ratios for Noncarcinogens:
With Optimal Strain, Microsome Preparation
and Substance Concentration 23-25
4-2. Mutagenic Activity Ratios for Presumptive
Carcinogens: With Optimal Strain,
Microsome Preparation and Substance
Concentration 26-28
5. Probabilities of Correctly Classifying
Presumptive Carcinogens and Noncarcinogens
at Various Mutagenic Activity Ratios 36
6. List of "False Negatives" 37
7. Probabilities of Correctly Classifying
Presumptive Carcinogens and Noncarcinogens
(When These are Equal) at Given Cutoff
Mutagenic Activity Ratios - As a Function of
Substance Concentration and Microsome
Preparation 39
8. Application of Bayes1 Theorem 44
-------
ACKNOWLEDGMENTS
We would like to express our appreciation to our colleagues
Marcy Hartstein and Ghada Shams for their help in the
preparation of this report and Howard Hirsch for assistance
in the statistical analysis of the data.
We also wish to acknowledge, with our thanks, the technical
and administrative guidance provided by the Environmental
Protection Agency through the Project Officer Robert E.
McGaughy, which was very helpful in the successful completion
of this project.
We also wish to thank Dr. Bruce Ames for supplying the
strains of Salmonella used in this work.
vi
-------
I. INTRODUCTION
The purpose of these investigations was to determine the
reliability with which an expanded Salmonella mutagenesis
test system can distinguish between those organic chemical
substances that cause cancer in laboratory animals (presumptive
carcinogens*) and those that do not (noncarcinogens). As
required by the contract, we have completed such tests of 100
organic compounds (50 presumptive carcinogens and 50 non-
carcinogens) . The results are described in this report.
In general, the results indicate that the Salmonella
mutagenesis system that has been employed in these tests
can be used to distinguish, with a high degree of reliability,
between presumptive carcinogens and noncarcinogens, in pop-
ulations of test samples in which these two classes of
compounds occur in approximately equal proportions. The
results also show that the statistical reliability of the test
system needs to be improved for the purpose of applying it to
those populations of environmental samples in which the pro-
portion of active substances (i.e., presumptive carcinogens)
may be relatively low. Certain of the results indicate what
steps need to be taken to achieve this improvement.
*We reserve the use of the word "carcinogens" for those
substances which are known to cause cancer in people; a
"presumptive carcinogen" is a substance known to cause
cancer in laboratory animals.
-------
II. THE SALMONELLA TEST SYSTEM
GENERAL FEATURES
The mutagenesis test system which we have employed is based
on procedures originally developed by Ames, et al., and makes
use of Ames1 special strains of Salmonella typhimurium.
These strains have a histidine-negative genome (and there-
fore are unable to grow unless histidine is supplied) and
include other genetic factors that render them specially
sensitive to chemical mutagenesis. One of Ames1 strains
(TA 1535^ is designed to detect mutations due to base-pair
substitutions and therefore tends to respond selectively to
mutagens such as alkylating agents. Two strains (TA 1538
and TA 1537) detect frameshift mutations; TA 1538 responds
particularly well to a number of carcinogens such as 2-nitroso-
fluorene; TA 1537 responds to carcinogens such as 9-araino-
acridine. Each strain also includes in its genome mutations
that greatly increase its overall sensitivity to mutagens:
one of these causes loss of the DNA excission repair system
and the other the loss of the lipopolysaccharide barrier that
coats the surface of the bacteria (thus enhancing the pen-
etration of large molecules). Strain TA 100, derived from
TA 1535, contains an R Factor plasmid, which increases ix_s
response to certain mutagens; this strain has a rather high
rate of spontaneous mutation.
Mutation causes the bacteria to revert to a histidine-
positive genome (i.e. the mutant bacteria can now synthesize
histidine) and can therefore be detected by the growth of
colonies when they are inoculated on a nutrient medium that
lacks histidine. Most carcinogenic organic compounds are
not mutagenic toward Salmonella unless they are converted to
an active metabolite, for example, an hydroxylated derivative.
In the original Ames method this was accomplished by incor-
porating a preparation of rat liver microsomes in the test
culture plate. We have extended the system by testing each
sample with microsome preparations from seven different rat
tissues, in the expectation that some presumptive carcinogens
might be more effectively activated by the microsomes from
some tissue other than liver. It was also known from the
work of Ames and others that the dose-response curve of the
Salmonella test is not linear; the mutation rate often
exhibits a maximum at a particular mutagen concentration,
with higher concentrations tending to be toxic. Accordingly,
it is necessary to test a presumptive carcinogen over a rather
wide range of concentrations.
-------
It was our purpose to design an extended version of the
original Salmonella test that, based on a suitable combi-
nation of the foregoing parameters, would optimally distin-
guish between presumptive carcinogens and noncarcinogens.
Accordingly, each substance was tested against four different
Salmonella strains (TA 1535, TA 1537, TA 1538 and TA 1536 or
TA 100) at three different concentrations, with microsome
preparations from seven different rat tissues (and in the
absence of any microsome preparation). From these data, and
suitable controls, it was possible to determine how the results
obtained from the two classes of substances were affected by
these parameters and to determine what combinations of
conditions optimized the distinction between the two classes.
METHODS
Tester Strains
The tester strains, upon receipt from Dr. Ames1 laboratory,
were grown in nutrient broth and 0.8 ml. of each of the
resultant cultures was mixed with .07 ml. of dimethyl-
sulfoxide, in a 2 ml. sterile glass screw-capped vial and
stored at -80°C. This served as the master copy of the strain.
The inocula were prepared from streak plates on nutrient agar,
incubated at 37°C for 48 hours (and then stored at 4°C). A
single colony from the plate was inoculated into 5 ml. of
nutrient broth and incubated overnight at 37°C, with shaking.
Aliquots of this culture were used for the test plates.
Fresh cultures for the test were made at least once a week
and kept in the refrigerator until ready to use. A streak
plate was prepared from each liquid culture as a means of
propagating the strain.
Microsome Preparations
Male Wistar rats (200-250 g.) were maintained on Purina
laboratory chow. Five days before sacrifice their drinking
water was made to 0.1% in sodium phenobarbital, to induce
microsome production. The rats were sacrificed by decap-
itation and the tissues were immediately removed and placed
in a sterile, ice-cold beaker. The tissue microsome (S-9)
fraction was prepared according to the procedure of Garner,
et al., ^ using a Potter-Elvehjem apparatus with a Teflon
pestle for homogenation and a Lourdes centrifuge (LRA-1) for
centrifugation of the homogenate (10 minutes at 9,000 x g).
Aliquots of the resultant supernatant (the S-9 fraction)
were distributed in 2-3 ml. portions in small plastic tubes,
quickly frozen in dry ice, and stored at -80° C in a Revco
freezer. The preparation was thawed as required; it was
-------
maintained at 0°C during preparation of the plates and any
unused portion discarded. Protein determinations of typical
S-9 preparations from different tissues are reported in
Table 1.
For use on the test plates, the S-9 mixture was prepared to
contain, per ml., the following: 0.3 ml. S-9 fraction, 8 p.
moles MgCl?/ 33 p. moles KC1, 5 jo. moles glucose-6-phosphate,
4/i moles NADP, and 100 ju moles sodium phosphate (pH 7.4).
The various stock solutions were either prepared in sterile
water or autoclaved, as appropriate. The biochemical agents
were stored at -20°C and inorganic solutions at refrigerator
temperature, The S-9 mixture was freshly prepared each day
and kept at 0°C before use.
Stock Solutions of the Test Compound
Ten mg. of the test compound was dissolved in 10 ml. of
dimethylsulfoxide (DMSO). Two successive 10-fold serial
dilutions in DMSO were prepared, so that 0.1 ml. of each of
the solutions contained 100, 10 and 1 jag of the compound
respectively. Stock solutions were coded, so that the
substances were tested blind, in that the personnel who
counted the plates were unaware of the classification of the
compound being tested.
The Mutagenesis Test
The general procedures are those reported originally by Ames,
et. ajl. Petri plates (Lab Tek 88 mm. sterile, disposable)
were prepared with approximately 30 ml. of sterilized minimal
glucose agar medium. (One liter of the minimal glucose agar
medium contains 13 gin. Sigma No. A-7002 agar, 20 gin. glucose,
13.12 gnu K~HP043H2O, 3.5 gm. NaNt^HPO* 4H2O, 2 gm. citric
acid monohydrate, and 0.2 gm. MgSO,.7H2O.) The plates were
stored at room temperature until used (usually for no more
than a few days),
Top agar (0.6% Difco agar, 0.5% NaCl) was prepared, auto-
claved and stored in 100 ml. bottles at room temperature.
Before use the agar was melted and 10 ml. of a sterile
solution of 0.5 mM L-histidine-HCl and 0.5 mM biotin added.
(The trace of histidine in the top agar supports several
bacterial divisions, usually a necessary prerequisite for
mutagenesis.)
To prepare the mutagenesis plate, there is added to 2 ml. of
molten top agar (45°C): 0.1 ml. of the nutrient broth culture
of the bacterial tester strain, the test sample, and if
-------
Table 1
PROTEIN CONTENT OF MICROSOME PREPARATIONS
FROM DIFFERENT TISSUES
Tissue
Liver
Kidney
Brain
Spleen
Lung
Stomach
Blood
Protein Concentration
(mg/ml)
15.8
10.2
4.1
10.3
7.8
2.5
67.2
-------
necessary 0.5 ml. of a given S-9 mixture (in that order).
The contents are mixed in a 5 ml. tube by means of a Vertex
mixer, and poured on a minimal-glucose-agar plate. The
covered plate is then tilted and rotated to spread the top
agar and allowed to harden for several minutes. Within an
hour thereafter the plates are incubated at 37°C in the dark
for 48 hours. The mutant colonies are counted by means of
a Quebec colony counter. For very high counts (over 1,000),
an indirect count is made. Four 1 cm.2 sections of the plate
are counted and the total number of colonies on the plate
computed from the ratio of the surface area of the plate
(60.8 cm.2) to 4 cm.2.
SELECTION OF TEST COMPOUNDS
Selection of the 100 compounds to be tested was based on the
following criteria: Substances were regarded as suitable for
inclusion in the list of presumptive carcinogens if published
reports showed that they produced a statistically significant
elevated incidence of tumors in treated groups of one or more
species of laboratory animals (usually mouse or rat) when
compared to control groups of animals maintained under iden-
tical conditions but not given the test compound. The final
list was selected to include representatives of different
classes of organic compounds; it was to some degree affected
by the availability of the compounds. A list of noncarc-^nogens
was assembled in the same way, based on reports in the
literature that a given substance, when examined under the
foregoing conditions,, had yielded no statistically significant
increase in tumor incidence in one or more species of labor-
atory animals. Two substances, bromobenzene and e-caprolactone,
were included in the list of noncarcinogens on the basis of
their classification in a National Cancer Institute list
(see Appendix A, reference 4). The compounds to be tested
were obtained from commercial sources or were provided through
the courtesy of the National Cancer Institute. Appendix A
lists the compounds eind the references that provided the
basis for classifying them.
SPECIFIC PROCEDURES
In what follows we describe the procedures used in testing
the 100 compounds as they were exemplified by a specific
substance, the presumptive carcinogen, 2-aminofluorene.
Controls
Three types of controls were run concurrently with each exper-
imental test. In the example given in Table 2-1 the controls
showed that in the absence of any other constituents, the
-------
r(
1
CN
0)
XI
r
E^
O
u
H
VI
H
0
Z
M
in
z.
2
,J
§
H
Z
O
CJ
<
CJ
a,
H
O
to
H
_i
3
VI
u
06
Q
U
iJ
<
S
u
Q
(U
C
01
r-l
O
3
^4
O
c
H
1
CM
@"
O
(^
s:
8
H
CO
H
o
to
§
H
Z
J
g
NUMBER OF REVERTANT COLONIES PER PLATE ]
i SAMPLE
cO
H
0)
4_>
CJ
s
o
z
00
f"l
in
r-i
«s
r^»
ro
m
*-t
g
^D
CO
in
i i
<
H
in
m
m
i i
£
5
ca
<
5
pa
<
3
CQ
<
5
CO
<
>
<
CO
<
00
a>
COMPOUND
TISSUE
BACTERIA
a^
t-t
ON
r-t
.0->
T |
C*
(
1
C6
U
1
+
3
j
S
c*-
00
o
o
u-
1
o
Ul
z
1
CN
i
+
<^
in
-i
m
o
m
Pw
S
i
+
0
SB
00
§
s
-a
VO
C
\f
u
Liver
4-
suaHoutoaeo
paepue^s q 3 -p M
SUTEJ^S jE|aa:jDBq
uo suna loaquoo
O
O
0
1
Liver
i
c
o
o
1
a
c
T
i
o
o
o
c
f-
c
L.
CC
O
o
a
i
c
0)
0)
r-
O
C/5
1
C
C
o
1
o
c
1
e
0
o
i
.£
U
0
E
O
4J
cn
1
sarjeusSouioq
uo suna
c
a
o
i
-c
o
c
C£
1
o
o
o
Cofactors
i
i
c
o
o
V
c.
E
<-
^
u
d
t-
1
I
anssf q
i;oaquoD
oc
vC
O
CN)
r^.
en
3
O
O
0
Cvl
o
,
QJ
C
a
&:
+
00
1-1
r-t
CM
m
iH
vO
0
H
CN
O
O
O
m
o
CM
Ch
'
c
Cl
CQ
+
c^
f-x
Ch
m
vO
3-
^
rH
-
m
xO
CO
1
G
CD
C
to
+
IN
m
es
r^-
0
00
3
'H
0
r^
m
-*
^o
i
0
C
+
-
CT
c*"
f-
r-s
s£)
O
0
o
CsJ
Cs)
-J
CM
a
r-
|
£.
U
n
1
to
+
en
CN
1^
o>
CM
r^
m
00
o
0
o
iO
00
f-H
m
t-H
1
a
o
o
CQ
+
-±
rH
00
ON
f-H
&
1
-------
CM
I
CN
(0
U
CLl
a
H
rJ
e»| 3
w £
£3 §
g
Id
Q
S O
CM O
i
O J3
W
CO r4
W O
E-<
_J
Cu
EU
o-
en
bJ
1
o
u
H
Z
<
H
a:
>
w
od
tK
o
a:
W
PQ
Z
u:
fe
§
to
m
m
iH
<
ro
in
<
ro
in
rH
JS
in
CO
m
<
H
>
<
PQ
<
M
<
>
<
QQ
<
>
00
3«
COMPOUND
u
:z>
en
to
H
BACTERIA
ot
3
c
r-
*~
vO
u"
O
0
o
vO
\0
in
O
0
+
1
+
tf
r-
CM
+
1
+
in
\o
00
r-v
O
m
so
0
O
00
rH
rH
CM
-£>
rH
O
o
0
CM
~T
r-~
0
O
+
M
U
>
J
+
O
vO
O
in
m
rH
<±
in
0
vO
in
00
m
-1
+
m
iH
0
CN
O
0-
CD
cs
O
0
O
0\
in
CM
+
u
0)
-1
+
n
CM
o
o>
n
m
CO
CM
^r
r^
m
rH
r-t
0
O
O
r**
p^
i
O
O
+
,S
V
c
T>
$6
' +
00
rH
O
0
rH
in
ON
rH
CM
CM
rH
O
O
o
o
o
o
o
+
>,
(LI
C
T3
u:
+
r^
en
rH
rH
rH
O
o
o
o
m
>
QJ
C
-^
+
CM
-J-
0
m
m
m
t t
m
CNJ
in
O
O
o
o
C*l
CO
+
c
n
ca
f
r^
oo
vO
SO
CO
CO
en
o
o
o
CO
CO
1
o
o
+
c
0)
a
C/)
+
r-~
rH
VO
00
-1
->
-H
o
o
O
O
+
C
aj
D
^H
U5
+
^D
r^
O
O
CM
-1
( 1
CN
,_H
r
4-
C
O
O
o
m
m
O
O
0
+
T3
O
O
CQ
+
-------
four strains produced numbers of colonies ranging from 0-19
per plate (averages of two duplicate plates, A and B). This
set of controls also determined the number of colonies
produced by each bacterial strain in the presence of a
standard mutagen (not requiring activation by a tissue
extract) to which it should respond. As shown in Table 2-1,
in each case more than 1,000 colonies per plate were observed,
indicating that each strain was appropriately active. Finally,
this set of controls shows that strain 1538 responds
positively, as it should, when the presumptive carcinogen AAF,
is activated by a liver preparation. Thus, these controls
determine whether all four bacterial cultures exhibit both
their expected spontaneous rates of mutation and their
appropriate activities toward standard mutagens.
The second set of controls examines the bacterial content of
the various constituents of the test system, which should be
zero. The data reported in Table 2-1 show that the expected
results were obtained; all of these preparations produced no
colonies on the culture plates.
The third set of controls determined the effects of the
various microsome preparations and of the cofactor prepara-
tion on the numbers of mutant colonies produced by the
several strains. These values represent the spontaneous
mutation rate plus the influence of any traces of histidine
(which would stimulate the growth of nonmutant bacteria)
that might be present in the microsome preparations. The
control value used to compute the mutagenic activity ratio
(see below) is the number of revertant colonies on the
control plates (average of two plates) obtained on the same
day, with the same strain and tissue preparation. As can be
seen from Table 2-1 none of these control colony counts are
significantly different from the background of spontaneous
mutations exhibited by the bacteria alone.
Experimental Data
Table 2-2 reports the complete results of experimental runs
with 2-aminofluorene. Three concentrations of the substance
were tested, in the absence of any microsome preparation and
in"the presence of microsomal preparations from the seven
standard tissue preparations. In the absence of a microsome
preparation only background counts are obtained, except for
100 jigm. of 2-aminofluorene tested with strain TA 1538. This
yielded a significant count, 98 colonies per plate, suggesting
that the preparation of 2-aminofluorene may contain a trace
amount of some directly active component, possibly the
active intermediate that is formed when this substance is
-------
incubated with a microsome preparation. The table also
shows that: (a) The substance (2-aminof luorene) is
mutagenic only toward strain TA 1538; all other strains
yield colony counts that are not significantly different
from the comparable control counts. (b) Preparations of
microsomes from liver, kidney, and brain are most effective
in activating the substance; less activity is obtained with
the stomach preparation and marginal activity with spleen
and lung preparations. Blood appears to be inactive. These
data are typical of a substance which is an activatable
mutagen--i. e. , it enhances the mutation rate only when
activated by a suitable microsome preparation. According
to Table 2-2, the mutagenic activity of 2-aminof luorene
increases progressively at concentrations of 1, 10 and 100
Data such as those reported in Tables 2-1 and 2-2 were obtained
for each of the 100 substances that we have tested. For each
compound 294 plates were counted, or a total of 29,400
plates for the total list of 100 compounds.
10
-------
III. RESULTS
GENERAL
The experimental results obtained for the 50 presumptive
carcinogens and the 50 noncarcinogens respectively are
summarized in Appendix B. In keeping with the contract
requirements, dose-response determinations were carried out
for each of the 41 presumptive carcinogens and 5 noncarcinogens
which showed an optimal mutagenic activity ratio of 2.0 or
higher (see Table 4) in the initial test. The dose-response
curves were carried out using the bacterial strain and
microsomal preparation that yielded optimal activity. The
results are shown in Figure 1.
For the 41 presumptive carcinogens, each of the dose-response
curves shown in Figure 1-1 is indicative of a mutagenically
active substance. Among the noncarcinogens Figure 1-2 shows
that p-aminoazotoluene is clearly positive and benzylmercaptan,
2-methyl-N, N-dimethylaminoazobenzene and sulfaguanidine are
clearly negative. Although the shape of the curve for
naphthalene is somewhat similar to that yielded by an active
compound, it also has to be considered negative since the
number of colonies formed at the optimal concentration (30
colonies at 500 ;ugm) is not significantly higher than that
of the control.
In the course of the tests it was observed that strain TA 1536
gave uniformly negative results with all substances. Accord-
ingly, after consultation with the EPA project officer, it
was agreed that TA 100 would be substituted for TA 1536.
This action in effect removed TA 1536 from the comprehensive
comparison of the parameters that influence the response of
the Salmonella system to different substances. Hence, in
what follows we report only the data based on strains TA 1535,
TA 1537 and TA 1538. The results obtained with TA 1536 (or
TA 100) are, however, reported in Appendix B.
For the purpose of quantitative evaluation of the data, the
original values were entered into computer punch cards, and
all numerical computations and preparations of graphs that
are reported in what follows were then made by means of suit-
able computer programs. The raw data reported in Appendix B
were also recorded by means of a computer printout.
11
-------
Figure I-I
Dose Response Curves of Presumptive Carcinogens
2-Ac«tamidofluor*n«
i
0.
o
£
(0
3000 -
2OOO -
1000 -
2OO
150
100
0 .5 1
160-
120-
2000 -
1500
1OOO
5OO
75
1OO
Aflatoxin
IA 15 IK, I wer
B.H tiTia alone 10 in
Bai CITI.I + I WIT S-u
en i i' i/p 1,11 c>
2OOO -
15OO -
10OO
5OO
10
p-Amino-azo-benzene
IA 1538, 1 iver S-9
Bacteria alone 13 olonies/p1 ate
Bartend + Liver S-9 23 colonies/plate
400
8OO
1200
1600 20OO
500 -
400 -
300 -
200 -
100
lA 15)8, Liver S-9
BaitLTi.i alone 20 colonies/plate
U.uterid + I.IVIT b-9 i colonies/plate
N-Ac«to«y-2-Fluof«nyl Ac«t«n>Mi
TOO
200
30O 4OO 5OO
2- Amineanthr»c0r
1A I5]8, Lung S-9
li.uteria alone 16 L o 1 on ics/pla te
Bacteria + l-ung S-9 i6 i o Ionics/plate
50
1OO
150 2O
4-Aminobiphao^
1A 1538, Liver S-9
Bacteria alone 11 colonies/plate
Bacteria + Liver S-9 19 colonies/plate
100
200
3OO
400
500
Microg rams/Plate
12
-------
Figure I-1, continued
Dose Response Curves of Presumptive Carcinogens
2000
1500
WOO
500
6-Aminochry««ne
2-Aininofluor«M
°o
4OO -
300 -
200 -
250
200
150
100
i A I r> IK, I i vt r S-9
K.H ten.i l.ilnni' I 1 i ..Ionic s/|>l.ite
B.lcteri.i -f ] IVCT S-9 19 i iilnnifs
10000
eooo
6000
4000
2000
20
40
60
80 100
1-Aminopyrene
TA 1538, Liver S-9
Bacteria alone 16 colonies/plate
Bacteria + Liver S-9 31 colonies/plate
300 -
200
100
15O -
10O -
50 -
1A 1538, ] wcr S-9
Bacteria alone 11 colonies/plate
Bacteria + Liver S-9 19 colonies/plate
_J I I
50
TOO 150 200
1,2-Betuanthraceft*
1A L538, Liver S-9
Bacteria alone 15 c_olonies/p la te
Bacteria + Liver S-9 27 colonies/plate
10O 200 300
100
200
3OO
400
500
4OO 5OO 6OO TOO
1,12- B«nzop«ryl«ne
TA 1538, Liver S-9
Bacteria alone 12 colonies/plate
Bacteria + Liver S-9 26 colonies/plate
1OO 2OO
3OO
400 500
6OO
Micrograms/Plate
13
-------
Figure I-1, continued
Dose Response Curves of Presumptive Carcinogens
Benzo(a)pyr»ne
150 -
100-
I
Q.
0
+*
CO
h. SO
150 -
1OO -
150
100
50
800 -
600 -
1OO 200 300 400 5OO 6OO
200 300 400
7H- Dibenzo(c. g)csrba;
1A 1538, Liver S-9
Battena dlonf 9 ( o 1 onies/plat e
Bacteria + livr ',-9 4 ^nloni es/p late
4OO
20O-
50
10O
Dibanzo(a,i)pyrene
150 200 i
3,3-Dichlorob«nzi<
TA Ib38, 1 iver S-9
BatCeria alono !7 i ol on, es/p 1,1 tt-
Bacteria + Liver S-9 21 ioIonles/plate
XX) 200 300 400 500 600
5OO
400
3OO
200
100
1A 1538, Luns; S-9
Bacteria alone 14 colonies/plate
Bacteria + Lune S-9 42 colonies/plate
50
100
150
2C
Micrograms/Plate
14
-------
Figure I-1, continued
Dose Response Curves of Presumptive Carcinogens
1000
8OO
600
4OO
200
3,3-Dimalhoxybenzldlne
0)
S
Q.
150
100
o
S
(0
°0L
45OO
3750
3000
2250
1500
750
°0
IA ] 51H, I Ivor S-9
U.litC'ria .llone 16 ( olmilos/p l.lto
B.ictcri.i + I IVIT S-9 2H i n 1 onli-s/pl.iU'
-O .
240
180-
4-Dln»thylamlne«tHban«
1OO 20O 300 400 500
7,9- Dimethyl Benz(c)acridine
IA IMS, [ Ivor S-9
H.H ttTi.i .tlotu1 7 i o 1 on U's/ p ] .1 to
UTi.i + I,Ivor S-9 J2 t o Innli's/n lat
250
200
150 -
100 -
50
TOO 2OO 3OO
400 500 600
4,4-Dinitrobiphenyl
200
400
600
800
1000
200 400 600
9,10- Dimethyl -1,2- benzanthracene
100
200
3OO
400 500
Ethylenimine
10OO 20OO 3000 40OO
5OOO
Micrograms/Plate
15
-------
Figure I-1, continued
Dose Response Curves of Presumptive Carcinogens
i
2400
1800
1200 -
6OO
too
80
"
20
36,000
27,000 -
18,000 -
9OOO
EthyhEthylnitroso Carbamate
i 1 1 1 8400
7000-
1400
K>0 200 300 400 500
3- Methylcholanthr en«
i i i tr
IA 153«, I iv(?r S-9
Ha< tena alone II i o 1 on i t-s/p 1 at c
Battena -t- Liver S-9 19 ( o 1 on les/p 1 a 11'
2OO
400
75 -
60
45
30
15
600
800 1000
MNNG
FA 1535 No S-9
Bacteria alone H (o1onies/p1 ate
6000 r
25
50
75
1OO
N-Hydroxy AAF
TA 1538, I iver S-9
3a(teria alone 1^ .olonies/p]ate
bacteria + liver S-9 16
-------
Figure I-1, continued
Dose Response Curves of Presumptive Carcinogens
KX>
75
50
25
500
400
O
O
300
200
100
600
4OO -
200 -
fl-N«phthy lamina
so
100
150
200
150
100
50
Natulan HydrochlorM*
4-Nitrobiphenyl
4000-
3000 -
2OOO -
10OO -
100 200 300 400 5OO
Nitrogen Mustard Hydrochloride
1A 1535, Livpr S-9
Bai.Cerl,i alone 8 ( o 1 onies /p lat e
Bacteria + liver S-9 12 i olomes/platc
1000
BOO
600
400
200
250
500
750
100O
TA I5i8, I iver S-9
K.utena alnne 6 i u lonies/p 1 dtc?
Bacteria + liver S-9 16 cc» lonieb/pla t e
100 2OO 300 400 5OO 600
2-Nitrofluorww
I'A 15-18, Stomach S-9
Bacteria alone 11 iolonies/plate
Bacteria + Stomach S-9 15 colonies/plate
50
100
150
200
4- Nitroquinoline-N-oxide
'lA 1538, Kidney S-9
Bacteria alone 16 colonies/plate
Bacteria + Kidney S-9 29 colonies/plate
25
50
75
100
Micrograms/Plate
17
-------
Figure I-1, continued
Dose Response Curves of Presumptive Carcinogens
I
8
o
s
12,000
9000i-
8000 h
3000 r-
Propane Sultone
100
1000*"
7501-
500r-
250 h
250
500
750
1000
o-Tolidine
T"
1A 15)8, I iver s-9
B,uteri.i alone 14 t o! on U'p/p 1 at e
B.uterl.J + 1 IVCT b-9 '] . olonles/p
100
200
300
4OO
500
Uracil Mustard
1000
750
500
250
1OOJ-
fi-Proptotoctooo
[A 15]1;, Spleen S-9
B.jiteri.] .iione 1 ft t o lonlos/pla te
B.uteri.i -f Spleen S-9 27 c olonies/plate
100 200 300 4OO 500
4-(o-Tolylazo)-o-Toluidln«
1A 15J8, l.iver S-9 J
Baiterid alone II colonies/plate i
Bdttena + Liver S-9 19 colonies/plate j
250
500
750
1000
250
500
750
1000
Micrograms/ Plate
18
-------
Figure 1-2
Dose Response Curves of Noncarcinogens
240
180 -
120 -
*
KX>-
75
o
o
f 50
(0
25 h
°6=
100
75
50
25-
p-Aminoaiotolu«ne
1OO 200 300 400 500
2-Methyl-N,N-dimethylaminoazobenzene
TA 1537, Kidney S-9
Bacteria alone 5 colonies/plate
Bacteria + Kidney S-9 5 colonies/plate
1OO
200
300
400 500
Sulfaguanidine
TA 1537, Lung S-9
Bacteria alone 7 colonies/plate
Bacteria + Lung S-9 9 colonies/plate
100
75-
50-
25
°0
100
75
50
100
Benzylnwcaptan
TA 1537, Blood S-9
Bacteria alone 5 colonies/plate
Bacteria + Blood S-9 9 colonies/plate
200
300
400 500
Naphthalene
TA 1537, Lung S-9
Bacteria alone 7 colonies/plate
Bacteria + Lung S-9 13 colonies/plate
250
500
750
1000
100
200
300
400
500
Microg rams/Plate
19
-------
EVALUATION OF THE RESULTS
General
The main purpose of these investigations was to determine
the reliability with which test results, such as these
described above, can be used to distinguish between .-
presumptive c.arcinoger. and a noncarcinogen. Accordingly,-
i'c was necessary to derive some objective, numerical
expression <~,i nrctagenin activity thai: would enaMo a
statistical comparison of the. results obtained from tnc.
V.-JG classes of compounds. In part: miar , such a raeasure
~.vust -rak:G into ar.count the relationship between the results
of s-periraerital anc control runs. Two types of considerations
;iro involved- First,, the control should account for ^ariatiOB
Jrcm day to day, in the spontaneous mutation rate of a giver;
3alajo.v:ella strain. This effect suggests that the- eol&.iy
counts (per plate) of the controls should be subtracted fr^iti
.-.he experimental counts obtained on that day. Secondly r ,-,a
:onvpari;ig the mutagenic activity of different compounds, it
:.s often necessary to compare these activities against
different Salmonella si-rains. Th i s suggests that the mutc.-
genic activity should be expressed as the ratio between the
experimental counts and the control counts,
Muta genie Activity Rat lo
In order to obtain a comparison of the nratacenic activity
of different compounds that taker: the forayoing relatiou-
shirjc into account we ha^^e computed a rAutaganic cctivj ty
rat:'.o - This is defined as (P: - n)/cv,"f whore"
exp».rxra,~n-cal number of revertant co-.oniejs ^average of twr?
plr-tcfc) ; C is the number o? rex'ertanc colonies on th~ C7oat.r.-..l
plates (average of tv7o plates) obtained on the same day with
the same strain and tissue preparation; and Cgv is the; overall
ave"a»ie of the same controls for r.J 1 2 Of! t.= st "plates ;J-,v;o
p'.a'-^s e.-..nh for 3.00 tests). TItf valuer; of C:vr for coirsbinfetior
of different strains .-ind tissue pre, .-.'-.raticna nre shot-jr- i:.
Table 3. The differences in the spontaneous mutation rates
of the three strains is evident. The differences among the
control t'fiiues of different micro .coro,-il preparations , for a.
given strain, do net appear to be significant.
Statistical Analsis of Results
We have developed the following 3ta "-.rjtical procedure to
estimate the reliability with which the exanded a
^
test can distinguish between presumptive carcinogens end
20
-------
Table 3
AVERAGE NUMBER OF CONTROL COLONIES (CAv)
FOR DIFFERENT MICROSOME PREPARATIONS AND SALMONELLA STRAINS
(Average of 200 Test Plates Each)
Microsome Preparation
None
Liver
Kidney
Brain
Spleen
Lung
Stomach
Blood
Salmonella Strain
TA 1535
9
11
15
12
13
13
12
9
TA 1537
6
9
12
7
9
10
7
7
TA 1538
13
22
25
19
21
19
19
17
21
-------
The procedure is based on the s-:l oct:lo,~
from the full array of data obtained on each compc-uncl, r>\!
the combination of strain, tissue microsome preparctfcion,
and substance concentration which yields the highest nrntd-
genic activity ratio, (E - C)/C, . In practice, aa a p-^rt
of the computer program used to analyse the data, the
computer selected from the entire array of results that
cor^binatir-u of parair.cters whJcu yielded tha highest muta-
c£nio activity ratio '-?r«d printed o\it iDOth the ratj.c .snd -'
optinal ooinbinatior/ of parerrT/tf-rr: , This proca'U'Tr- was fa
ot; t for bot1; presumptive carcinogens and noncarc.i run-ens .
The: ..oii-J;iviat.-ic>!; o<'" parameters (I.e., of strain
ions, and trs^ua from which the microaome prepara-
cJ.o.x wa3 raacl-l that -/tolled the highest mutagenic ^ctiT-Titv"
ratio, for aa.-;b of the substance? tested? is showr> in Tables
4-1 and 4-2. It should be noted that thin procedure r.electi?
the-, iTiaxirrjut; \~3 U:-*- - TTr.
j. i, w.i.i. j. £."2 j)«.)i_C J ij. tJil '-.'l-i.- ',S 'i /. uiiat. -.', t-ii'_ .., *>' p- 'i.STi.upi,-'- *'r'
carcinogens.. three exhibited roaxiroal rnutagenic activity
ratios in the ^l/sencri of a Tnicrosnme preparatlcr.- - tbsse are
r~ ".i.uta.7C!~r» tV-t do not ?:oq:i;;.r'-= ar-'tiv.T ion. Ir
cf sivch -r:--,3c:; - tba presence of a in'U:~oS'.--Mi" c.vi'jn-er' i.ir-ii
ir a roductxor in mutagenj c activ5 A -;r apparently- >o^.<;sr-3^ -'-l',e
prcximatf: nruh^gcn is ;^-.sel.c metabo] ic.ally ."' raativst :-;<"";
(Three additional pre.-uinpt.l'^e carcinog-ars exhibit their
highest nrtagenic activity ratio when no rn.Icresume prepara-
tion is PresGnt: however, in theFK c'^so" c'".e ir.^tio ',- so low
usual ly leer. J''".f',"' ".-tbat. Lue .7nbsl-ancoo ar- ob'-.-.'-clj
inac hive '' f alBC ;.
Table 4-2 shows that Thile the microsome preparation *:hat
yields tli3 highest mutaganic acti-."it.v is rnort cft.f.n
derived from liver, this .is not always the case. Of the
50 presumptive carcinogens tested, 11 e:«i-iil>ited a mavinal
mutagenic act.Jvity ratio with a r.-.i^roGotne pxeparatir" fx"ora
some tissue other than liver (excluding "false negatives").
Table 4-2 also shovs that the maximum ratio usually occurs
with 100 pgm, of the test substance- The role of the
microsomal tissue and the concentration of the test
substance in governing the reliability of the test system
is discussed in more detail below.
22
-------
I
>
CJ -H U
to > o >
C3 -H -H 1 <
3 O cQ W
K < o^
rH
M
C
u CJ
C tu B ^
O 4J -H W
AJ CO LJ "-^
op, ex
> X
O J-j F'l
pi w
CX
O tl) -O
«) *-* C
Vj *H U 3
(DC ^- O
^D O IX
E -1 -HE
3O O O
Z U MO
c o
O K
u ^
c
o
H
r-l . t
4J (X
c
at &o
y a.
c
o
CJ
a) o
B -H
O u
tn co
O M
CJ Cl,
P-I
a
rH C
01 -H
C n)
B "
^-< c/i
n)
en
o
c
3
O
CX
o
o
(ft
0
u-,0 KtOr-r-rs.vC^^^rHcoov
O rH 0(D(uoc
J>O I>OCOOC(13>CC.>O'O
^pQ(jeQrjcQ«.-Jcq.-JKJ,z;^-icQ^
sss^ssss"ss?;"ss
o
H
-H <
QJ 0
G < u sr-. -Hffl C O
»-H -H C >^. O I*-" -iH C
Q^O)OOO(J >ooc:..XrH aoutn HU>.
XCNCi,-.Hr3 CC--H -O, o -H -H i -M ^
CO O *H -iH -iH -r-l --H v, tX U CX N O O O
^*JCCE.= nrHX"HW.Or3 NNN
out \ . iccci4J*Jd,oo
<< a.cxfM
CN rH
CO ^^
CM rH
Q^ , "o
c o
a* ^
23
-------
d)
3
C
H
4J
fl
O
CJ
fH
I
^P
0)
IS
EH
o
H fs
a w
B -H
4-J 4->
3 0
s <:
4J
£ OJ
a *j
4J cfl
H i-H
CJ IX,
a) M
BS OJ
ex
U-l
o in
aj
u -1-1
S c
& 0
£ rH
§ 0
S5 O
(J
0 >
,H 1 <
U o
n w
Dd
rH
ra
4-*
e
CJ
s ^^
H tJ
CJ
a.
S
13
O 3
^ O
0.
rH E
O O
(-1 O
C O
0 K
0 ^
c
o
H CJ
oj ra
I* rH
-u O.
c .
01 00
" 3.
c
0
u
c
OJ O
E -H
O J-J
W) C3
O >-i
U a
0 CX,
H OJ
y t.
51 £
BJ
r-H
rH C
OJ -H
C C3
O )-<
e -u
rH) CO
03
V5
C
3
0
O.
E
O
CJ
M
CJ
H
CN O O to rH
rH rH rH rH rH
m ^ oo CN r^
rH rH CN CO CN
CN r^ CN vj- rn
O rH O O rH
rH rH rH
C >,
CJ *O (U CJ
GJ o c -H aj
rH O -O CO C
CX rH -H u O
c/i co x en 2
r-* r^- oo in oo
co m r*i m m
m m m m m
T3
H
u
< CJ
T)
a >.
4J CJ
o -a
U rH
0) Cfl
^ N
X C
O OJ
C ,0
a* o
^
OJ
c
T3
*H
X
m
CO
in
rH
o
c
CJ
.c
a
nitro
H
a
i
5
*
}
i
^OLn-^-oa~*r-.%>, 1
*-* J-i -O CJ CJ O
eUCUGJCUOOOaJCCC-DO-O
>d>e:corH"ciT3^u
H O -H O 3 rH CX -H -H ^ 3 J
r-J^^-trZl-jmCrj^i^.J^J |
1
i
mmr-.cor^r^coinr--.oor^
<*o m co c*n ro co m m n n r*> i
ininmmLTiinininmtni")
?j
c
OJ
N
C
OJ
J=
c
o
o
c
G
?3
OJ r~^
"D >,
H jz
E u
nj « < < e i -j
rH H CO X rj]
>sQ UOJ ^5-f-;oJ
^WC -HC l-'JP'rjC
" n) rHOcrai^ni
OJEI4-, rH-HO(J-, rj
rt -H 3ElJWj:rHJ= « j;
O"Otf)P30JrtAJXii>*j
I ooj3jDua-^o^;j-;
iiIn-Ol.J3prH»JS4-la
H c CJ -H r-t r; CJ y r:
^QU^uOZErMZi;
24
-------
(U
3
H
-P
C
o
u
XI
td
E-"
o
H X
0) -rH
6Q >
nj *H
4J ij
3 0
2: <:
4-1
nj 4J
V-l r-l
<1) (14
>
01 M
OS 01
u-t
O 0)
CD
J-l *r*
H 1 <
iJ O
co u
r-H
0)
c
01
r-l U
V
&
W
"O
U 3
*-s O
H E
0 0
Vj c_)
C 0
o z
O v^
c
o
H 01
tO CO
M r-H
c --
oo o in
CN -^ i i mc^J rHi-Hr-H
Q^ r-4 ^-| m C") rH O fO O i 1 "-J **O
CN cO
ininminminininmin*nin
O OJ
iH rH C
o o -H
cu < C E
C cu d -o
H t-H CJ X --H -H
E O *H O Du T) cj
to C C O % .C _C *H O tU *H C U
X£ D. 4-» rH i t t-H (U >-.H'H o
a.cu*JCuo o
0 C C O
C/i >J S^ OQ
in r^» r-. in
m in *n m
QJ
O *H QJ
C 13 C C
C) *
-------
(N
I
0)
(0
EH
c
o
iH
4-1
O
c
GJ
0
c
CO O
8 S
5= C
M co
U 4-»
cd w
O 3
W
w
> T3
M C
( * CO
c/} O
W *H
O-< 01
Jj
tu C3
o a
o a.
H - 0
M U
> -H
M £
U
< C
H
U (U
U W
U
H 'ej
O.
0
U
-H
3
^**
O
H >,
C 4-1
CJ -H O
to > o >
to -H -H 1 <
Son) w
X < K
rH
a
a
4J Q)
e o g *~.
rt -u -H w
4-> C3 l-i ^-
M rH V
O fL, p,
> X
O M W
Di CJ
UJ rf^N
o en -x)
OJ ^ C
U -H U 3
O C ^ o
j3 o a.
E >H i-l E
30 o o
Z O H (_>
J-l
C 0
o z
o ^^
c
o
rt (!)
4-1 4-1
rt cj
Vj t 1
4-1 (X
CJ 00
O 3
C
o
QJ O
E -H
O i-i
O ^-i
>-i rt
o e.
H CJ
,H
H C
QJ -<
C CO
O M
E ^
«
*p
3
O
O.
E
O
^
U
H
rH CT> 0 ^ CN
rHenenr^oo-.eMr^,-H rHin^r*- i r- * CN
en m cs en CN
co *n i i \D o ^D o> r~* in co o ^T CM in --j1 < ) co
encoco\£)inrHCMinsocMrHcMmr~.r^eNCN
OO^VOrHrHrnrHCOvO COeMrHrHCl
CO \D i-H <} rH f~- in
co^cor-^-Jt-Hcoocoin -T cM^jr^-CNeoco
rHOOrHOOOrHOOOr-HOOOOO
OO OOO OOO OOOrHO
""HrH r-HrHrH rHrHrH rHrHrH rH
a
OOajQJQJW)QJQJQJQJQJTHE>>C>>>>>e)O,>>.^ >>
3 -H -H -H 3 -H «iH -H *iH -H ^4 4-J -rt -H -H 1 *H
rJ|JrJrJl-3rJrJrJrJ»-lrflC.O>JPJr4rJrJ
r-.cocococococococor--r^cococococotn
l~i in in in in in in *n in in in in in in in in in
a>
c
QJ
)-j
O
rH
U-, OJ
O rH
u o
GJ -H N
C E (0 O QJ
QJ TO QJ aj -H c C
Vj U c C ki CJ QJ
O QJ QJQJrHQJQJ 4J (J rH
30 UNXCC 1 n ^QJ
rHOJ nlcCCQJQJCJ^ ij SJC
**-<( rHkjGJQJ(n»jC»Q>J^ OOJ
oescajc^j^^oflJcMCi-' ci,*-.
T)l 1 u O O. 1^ 3 l-i -OC OX
Q* -H >sC C (^-HX:rH>,rH^: O CJ N O.
*OEX-ri*C3rJjDO**-«CX( J-JNCC^-v
'HraOXOOOOOOOQ>C-HQj(0
E*J*-»OCC CCCCCeOTJOQ^'C
(0 QJ OJ *J -H -rJ -H -H -H -rH -rH 5s C3 -H | On)
^uucaEEEEEEEXi NCNIN*J
OJ<
-------
0)
.3
C
-H
O
u
CN
I
Q)
rH
.a
(0
EH
o
r-l X
O >
C) -rl -rl 1 <
4J 4-J i-* O
son w
rC *£ O*
r-l
CJ
C
4-1 CJ
co e X-N
o ex
6 rH tH E
DO O O
55 CJ rl 0
c o
o la
CJ ^
C
o
H CJ
JJ 4J
a ts
H tH
u ex
C ""^-
CJ U)
c
0
CJ
c
01 O
E *rl
O 4-1
W CJ
O rl
rl CJ
u ex
H CJ
£
C3
rH
O *H
O rl
45«
tc
£
£
g
0
4-1
en
CJ
H
< CN
LA - rH O
CN
-d- MJ CO
in rH CN]
m
r^. vo co
O O rH
O rH
Vj )\-< QJ
QJ a) C
> > Td
H -H -H
,-J r-l W
r^ r-« oo
in m m
CJ 01
C rH
CJ O
0 N
OJ U J3
C X rl
H 4J 13
a c o
U N 60
0 C
O CJ U
*~v X ^"^
r-l >H O
Q N
n i c
N r. XI
c: m -H
H a:
Q rH I-.
co
CO
rH
O
rH
O"»
CM
O
O
1-1
CJ
>
H
,-]
CO
CO
in
CJ
c
CJ
ex
H
O
N
C
,O
H
O
,0
CO vO
-a-
m m
O rH
VO CO
rH CM
o o
o o
bo CJ
c >
P -rl
rJ rJ
CO CO
CO CO
in in
CJ
o> c
C -rl
H -o
a -H
H N
N C
C CJ
CJ XI
o y.
r. O
o x
rH 4J
X CJ
u E
H 'rl/
Q O
1 1
fO CO
CO CO
r^ r^ m tH
o co -d- a>
CO *d" MT> CO
CO O ^ O>
rH tH
m > a
H -H >H in
^- ^J » J P-l
OO OO f~- CO
CO CO CO CO
in m in m
CJ
C
CJ
u
ra
CJ U V^
c c x
CJ -H 4J
N -a c:
C CJ -H «
CJ C rl N
Xt dJ u C
O X E3 O
N rH ^> XI
(fl -H U 1
0 4-1 ^ CM
C (t> N
rl O C rH
E C CJ 1
O -rt -D rH
rH g r-l X
x ta x x
X rH X iJ
4J X *J O
CJ X CJ E
E ^ e -H
v-f ft) *H Q
Q e Q i
t -H | O
2; Q Ch rH
1 »
m
o
o
CO
Ol
r 1
O
o
M
0)
H
i_l
OO
CO
U")
.-H
>^
C
a.
x:
a.
o
H
C
-H
I
rH
O
rH
U~l
Ot
O
O
T3
O
O
rH
PQ
in
m
tn
,
X
QJ
r-H
X
X
u
rH
rH
ro
00
(NJ
V0
CM
rH
O
rH
QJ
H
rJ
CO
m
m
OJ
0)
o
3
rH
M~*
O
T3
H
e
OJ
o
n]
X
X
o
Tl
X
1
-a-
-J
rH
rH
O
O
rH
OJ
^
r-t
rJ
^
m
m
OJ
threni
c
r-l
o
U
rH
.>,
*TJ
o
1*
r">
r-
CO
S
^
rH
O
O
rH
X
4->
rH
o
x:
o
>,
x
a
i
cs
rsi
tn
CO
in
in
o
o
o
o
m
cs
0
o
i i
QJ
o
in
m
m
(U
c
rH
*O
iH
C
rt
t-0
o
[/)
E
-w
H
C
rH
jj
SI
27
-------
0)
3
C
H
-P
c
o
u
CN
I
0)
rH
XI
(0
u
rl >,
C 4J
CU -H O
t>0 > 0 >
CTJ -H -H I <
4-1 4J 4J U
3 CJ « W
S < rtf
r-H
cd
c
u CU
C cu 6 x-,
nj 4J -H w
4-» CJ rl *-^
rl rH CU
CJ C, O.
> X
0 rl W
05 o
Cl.
UH ^^
o co *a
tu ^ C
rl -rl U 3
cu c ^ o
.DO P.
B rH rH e
30 o o
"Z. C_> rl U
U
c o
o a
u
f.
o
IH a
CO (0
rl rH
4-1 a.
c ~^
cu to
y s-
(^
o
o
c
OJ O
§-H
4-1
to co
O rl
r* CO
o a.
*£
CTJ
rH
fH C
flj -H
c d
o M
f= *-*
rH CO
rt
t/1
-a
c
p
o
CL
g
O
(_j
w
o
H
O p- oO r- vo o->
O*>rnrOvlDomrHOOLn-^j-OOcMv£i
ooo^ .-^fv^fsj^, r>io^ ,-H
rH m vo in
rHCTimcTtcococOiHcoinroooc^om
vomao^comOrH.-Hovo ooso
mO mcncNjrsj SD^XJ oominooo^inrOrHrHco'.ncofO
rHtN-HrHrH r-.r-^C^J ,HCNJ
OOOOOOOOOOOOOOO
OrHOOrHOrHrHOOOrHOOO
"f ^~* *~t t-H rH rH rH rH rH fH
-c ,c .c
O U C rj
n)lHlH.UfO^| QJ 03JHV-I
ECJQJ4J£(UoOiU(lJC.J(..J<.UEQJQJ
O>>>o>CCCC'HCO>>
WrJt-qPjr/ihjHja5K^wg5cnH-l.-J
minoooocomcOinr-minr^'^-ooco
rnro<^cO{^rocornrornrOrococ^tf*~,
ininminininmmininininininin
rHrHrHrHrHrHrHrHrHrHrH
OJ
*T3
-H
r-l
O (U
H C
.C » | .H g O
U >- .E 2 6 (tj AJ
*~" O | CTJ rH |
D ^H TD QJ fH >* O
o,c«.Hxtlcc A
O-HOC(Ui-».-H±J.DOO (U O
.^ei-<(Ul-(WO<.U6AJ4rJ -O N
AJta'Ojco.3c-HTH--Hcj -H rj
"~<'HXC.i.DX-HT3T.)3« GCJr-H
C X X -H .-H 3OOtfirH C3C>,
rH,c .-Oi*-C/crc/;w o AJ -H » i
XAJCOOOO o O £AJ.r£..->;AJ;25»;jr OO.*4--THHN-'
1 1 O 1 1 -H | | | !w 1 O JT 1 t
^Ca^^^^^TSrCCUf^cnH o
-------
Figures 2, 3, and 4 show the frequency distributions of the
mutagenic activity ratios of noncarcinogenic substances,
tested with TA 1535, TA 1537 and TA 1538 and of the con-
current control plates. It is evident that in each case the
frequency distribution for the noncarcinogenic substances is
very similar to that of the controls. This indicates that
the presence of noncarcinogens has no systematic effect on
the spontaneous mutation rates of the several strains of
bacteria. The differences among the spontaneous mutation
rates (TA 1538, TA 1535, TA 1537) is evident in both the
control plates and the experimental plates.
Figure 5 compares the frequency distribution of the mutagenic
activity ratio for noncarcinogens and presumptive carcinogens,
at the optimal combination of bacterial strain, microsomal
tissue and substance concentration, for each substance.
(This figure is a plot of the frequency distribution of the
mutagenic activity ratios shown in Tables 4-1 and 4-2.) Of
the 50 noncarcinogens tested, one (p-aminoazotoluene) exhibits
a ratio (12) that is clearly outside the range of the ratios
exhibited by the remaining noncarcinogens. Of the remaining
noncarcinogens, one shows a ratio of 2.8, and one each of
2.5, 2.3 and 2.1 respectively. All the remaining ratios are
2.0 or less. As indicated by Figure 5 the preponderance of
ratios lies between .5 and 2.0.
The lower panel of Figure 5 shows the frequency distribution
of the mutagenic activity ratios of the 50 presumptive
carcinogens. Of these substances, nine yield ratios that
are below 2.0. Of the remaining 41 presumptive carcinogens
2 yielded ratios between 2.0 and 3.0 (2.9 and 3). However,
the dose-response curves obtained from these substances
(o-tolidine and natulan hydrochloride) which are shown in
Figure 1, clearly indicate their positive mutagenic activity.
The relationship between the mutagenic activity ratios of
presumptive carcinogens and of noncarcinogens is shown in
more detail in Figure 6. This figure, which is based on the
data of Tables 4-1 and 4-2, shows the variation of the
cumulative percentage of compounds (average of two duplicate
plates per compound) with mutagenic activity ratio. The
cumulative curves of Figure 6 establish, quantitatively, the
relationship between a given value of the mutagenic activity
ratio and the reliability with which that ratio distinguishes
between presumptive carcinogens and noncarcinogens. Thus the
curves (triangular points) representative of the results for
all 100 compounds, show that 82 percent of the presumptive
carcinogens yield ratios above 1.5 and that the same per-
centage of noncarcinogens yield ratios below that value.
29
-------
Figure 2
Frequency Distribution of Non Carcinogen and Control Plates
(TA 1535; Microsomes from Various Tissues)
1000
800
600
400
o
a
O
200
w
o
« 0
a
CM 800
10
a
|
600
400
200
1 I I I 7 T
Non Carcinogenic Substances
1 I i i 1 1 r
Controls
' 1 I I I I 1 1
0 5 10 15 2O 25 30 35 40 45 50 55 60 65 70 75
Number of Colonies per Plate
30
-------
Figure 3
Frequency Distribution of Non Carcinogen and Control Plates
(TA 1537; Microsomes from Various Tissues)
1000
800
600
~ 400
O 200
i
in
0
«) °
Z
CM
^ 1000
0
ra
E
o
i. 800
600
400
200
Non Carcinogenic Substances
i | I r i
Controls
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
Number of Colonies per Plate
31
-------
Figure 4
800
Frequency Distribution of Non Carcinogen and Control Plates
(TA 1538; Microsomes from Various Tissues)
Non Carcinogenic Substances
iiiiiiiiiiiiiiiii
- 600(-
400 -
200
Controls
tb*.
. . . 1 I ) l iiiiiiiI//ii
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 285 290
Number of Colonies per Plate
32
-------
Figure 5
|
Frequency Distribution of Mutagenic Activity Ratios
of Presumptive Carcinogens and Non Carcinogens
40
30
20
10
o
o
"O 40
30
20
10
0
.01
I III
Non Carcinogenic Substances
I I f"""f I
| 1 ! I
i I II
Carcinogenic Substances
I i""r
.3 .5 1 10 100
Mutagenic Activity Ratio
1000 10,000
33
-------
suaBoupjBQ aAjidwnsajd -spunodiuoo jo a6e)uaajad
(£>
o>
o
<
o
'£ -
SL ?
- o O) i
O n
§
sua6ou;ojeo UON - spunodiuoo jo
aAi)e|nuino
34
-------
In other words, if a mutagenic activity ratio of 1.5 were
chosen as the cutoff point between the two classes of
compounds, 82 percent of both groups would be correctly
classified. At a cutoff ratio of 2.5 the reliability with
which a noncarcinogen is correctly classified is improved;
about 95 percent of the noncarcinogens, but only about 81
percent of the presumptive carcinogens, would then be correctly
classified. Conversely, at a cutoff ratio of 0.7 the prob-
ability of correctly classifying a presumptive carcinogen is
increased to about 95 percent, while the probability of
correctly classifying a noncarcinogen is reduced to about
20 percent. (See Table 5.)
It is evident from the cumulative curve for presumptive
carcinogens shown in Figure 6 that of the 50 compounds tested
9 are to be considered "false negatives." That is, if the
mutagenic activity ratio is chosen at a value (1.5) that
maximizes the percentage of thetwo groups (82 percent) that is
correctly classified, 9 presumptive carcinogens would be
classified in the noncarcinogenic group. In the case of
these compounds (listed in Table 6), the conditions of the
Salmonella test employed do not elicit a significant mutagenic
response. However, it has been noted^ that by slight changes
in the test system (for example, by replacing DMSO with water
as the solvent, and preincubation with PCB-induced liver
microsome preparation) it is possible to obtain mutagenic
activity for three of the false negatives (N,N-dimethylamino-
azobenzene, N-nitrosodimethylamine and N-nitrosodiethylamine).
Thus, by finding suitable ways of altering the test conditions
it may be possible to reduce or eliminate the number of
"false negatives." In this connection it is of interest to
determine what overlap between the mutagenic activity ratios
exhibited by the two classes of compounds would occur if it
were possible, eventually, to avoid the occurrence of "false
negatives" (and of the 1 in 50 "false positive"). This
would indicate the inherent overlap between the results
yielded by the two classes of compounds, and thus set a limit
on the maximum reliability achievable. In Figure 6, the
dotted lines report the cumulative results if the 9 "false
negatives" and the 1 "false positive" are not considered.
The two lines cross at a mutagenic activity ratio of 2.8,
with a probability of correct classification of about 98
percent.
It is of interest to analyse the influence of variations in
the concentration of the test substance and of the tissue
from which the S-9 microsome preparation is made on the
reliability of the test system. From the raw data it is
possible to obtain a computer printout of the variation in
35
-------
Table 5
PROBABILITIES OF CORRECTLY CLASSIFYING PRESUMPTIVE CARCINOGENS
AND NONCARCINOGENS AT VARIOUS MUTAGENIC ACTIVITY RATIOS
Mutagenic Activity
Ratio
E - C
CAv
I: Probability of
Correctly
Classifying a
Presumptive
Carcinogen
II: Probability of
Correctly
Classifying a
Noncarcinogen
Probability (percent) for Different Policy Alternatives*
From Present Experimental
Data
Set Prob-
ability I
to =
Probabil Lty
II
1.5
82
82
Set Prob-
ability I
to = 95%
0.7
95
20
Set Prob-
ability II
to = 95%
2.5
81
95
With "false positives" and
"false negatives" eliminated
Set Prob-
ability I
to =
Probability
II
2.8
98
98
Set Prob-
ability I
to = 957,
3.2
95
99
Set Prob- j
ability II j
to = 95%
1
I
1
2.4
99
95
*For optimal combination of tester strain, microsome preparation and
substance concentration.
36
-------
Table 6
PRESUMPTIVE CARCINOGENS WHICH
SHOWED NEGATIVE RESPONSE* IN THE SALMONELLA TEST
("False Negatives")
1. Acetamide
2. 3-Amino-l,2,4-triazole
3. Azoxymethane
4. 1,2,5,6-Dibenzanthracene
5. N,N-Dimethylaminoazobenzene
6. N-Nitrosodiethylamine
7. N-Nitrosodimethylamine
8. Safrole
9. Thioacetamide
Mutagenic activity ratio less than 1.5 under
optimal conditions.
37
-------
the cumulative percentage of test compounds with the
mutagenic activity ratio separately for each of the
combinations of substance concentrations and microsomal
tissue, and for combinations which optimize the ratio. By
superimposing the curves for presumptive carcinogens and
for noncarcinogens, in the manner shown in Figure 6, one can
determine the cutoff ratio at which the probabilities of
correctly classifying each type of compound are equal, as
well as the value of the probability of that ratio. The
resultant data are shown in Table 7. Curves for different
tissues, at optimal concentration (with the optimal
Salmonella strain) are shown in Figure 7. Curves for the
different concentrations, for optimal strain and tissue
preparation are shown in Figure 8. (Figures 7 and 8 are
intended to show only the region of the cutoff ratio and
unlike Figure 6 do not extend over the full range of
observed mutagenic activity ratios.) In both figures the
curves for the optimal combination of parameters are also
shown for purposes of comparison.
Table 7 leads to certain relevant generalizations about
the influence of substance concentration and microsomal
tissue preparation on the probability of correctly
classifying the two types of compounds.
(a) In general the probability of correct classification
at a mutagenic activity ratio which yields equal probabilities
for the two classes of compounds increases with substance
concentration. However, with the exception of kidney, lung
and blood, the probability of correct classification is
further enhancer! if it is based on the optimal concentration--
i.e., the one thai; yjelds the highest mutagenic activity ratio,
This reflects the toxicity effect, which in some instances
reduces the apparent mutagenic rate at high concentrations.
(b) For tests with a microsomal preparation from any single
tissue, liver clearly yjelds the highest probability of
correct c3assification. Of the remaining tissues, blood
is essentially inactive, while kidney, lung and stomach
microsome preparations yield probabilities that most
closely approach that of liver alone. At concentrations
of 1 jugm.. and 10 jugia. per plate the liver preparation is
not always optimal, so that if the test is based on the
tissue preparation that yields the optimal mutagenic activity
ratio, the probability of correct classification is improved
somewhat. At a concentration of 100 jugm. per plate this
advantage is not evident.
38
-------
CO
Z
W
o
o
z
Pi
CJ
z
o
z
C/3
o
o
z
M
o
CO
w
PQ
§
P-I
CO
o
H
o
U
r-
0)
, i
XI
03
f-4
(^
^
u
w
HH
H
PM
p^
CO
W
P-,
o
z
b-(
fj^
h- 1
CO
CO
o
>-
I-J
H
o
W
O
U
Z
w
o
H
^
fa
0
H
O
Z
w
M
o
H
^^
^
cx
w
fjj
^
w
CO
y*\
C
H
CO
4-1
CO
rH
Cfl
H
4-1
p,
O
,c
4J
H
^~s
01
4J
cfl
^H
e
00
^
z
o
M
H
H
Z
w
z
o
o
w
o
z
H
CO
CO
c
o
H
^H -tJ
6 >-i
*rH 4J
4-1 C
Ou cu
o a
C
o
u
o
o
0
r \
4-1
i i
H x-^
O gs°
CO ^
0
)-i
14-1
u-i o
O -H
1 1 1 1
3 cfl
o pi
H
*rH /"""v
,0 B-S
CO ^
O
^ J
M-l O
0 -H
4J 4J
3 cfl
U Pi
>,
4J
,_J
H /-v
,0 S-S
J3
0
^_j
PM
LW
14-1 O
O -H
3 CO
U Pi
4J
i (
H -.
O gvg
cfl -^
O
M
P-i
U-l
U-i O
0 -H
3 cfl
U Pi
z
0
W M
2 H
O <
co PS
O <
Pi PH
O W
M Pi
S 0,
CNICNCSIOOOCNU->O> CS
r^cor^vor^r^r-iri oo
r^oovovom^ou-iLO m
r 1
\^ r H CN] CN1 00 CN1 f^ ON C"~l
vD 00 r^» vo vO r*^- r*^ u^ 00
^r^roroco^ro<± O
I
Sf^SvoSSSin ^
-H
E 4J
JC ^H O Cfl
>> C 0 CO CO t-l
C1J CU C'H CU GOg O-HVJD.
c >-a cflrH c o o 4-iooj
O'H'i-l )-l CX3 4-1 r 1 CX -H M
Z >-J ii CO 00 _1 CO CQ OSD-,
39
-------
(O) suaBoupjeQ aAjidwnsaJd-spunodiuoo jo eBejuaojad aAj
a*
«- Q.
51
< §
y u
I
(N) suaBoupjeo uoN-spunodwoo jo a6e)uaojad aAi
40
-------
tf)
'g
<3
(A
o
Figure 8
Cumulative Percentage of Compounds
as a Function of Mutagenic Activity Ratio
(For Optimal Strain and Microsome Preparation)
0-,
10-
20
30
40
50
60
O>
£
0)
o
I
2
~ 80
70
90
100
Optimal Concentration
11 i i i 11 i i i i
100
90
80
70
60
50
40
20
10
O
(A
0)
I
'5
3
I
Q.
3
o>
30 £
I
3
O
Mutagenic Activity Ratio
Experimental Control Colonies Plate
Average Control Colonies Plate
(Ex
41
-------
(c) If one chooses an optimal combination of both
concentration and microsomal tissue, the probability of
correctly classifying a compound (at the mutagenic activity
ratio at which both probabilities are equal) is maximal,
82 percent. This same probability can be achieved if only
the liver S-9 preparation is used in the test system.
However, Figure 7 shows that the optimal combination yields
more favorable results than liver alone when a cutoff ratio
is chosen which either maximizes, separately, the probability
of correctly classifying a presumptive carcinogen or a non-
carcinogen. Thus if a cutoff ratio of 3.0 is chosen in
order to maximize the probability that any substance yielding
a lower ratio will be correctly classified as a noncarcinogen,
then the probability of correctly classifying a presumptive
carcinogen is somewhat better with the optimal combination
than it is with liver alone. This effect, and the tendency
for the choice of an optimal microsomal tissue to yield
somewhat higher probabilities at certain test substance
concentrations indicates that there would be some advantage
in including in the test at least several of the more active
tissue preparations in addition to liver itself.
42
-------
IV. DISCUSSION: APPLICABILITY OF THE SALMONELLA SYSTEM
FOR TESTING UNKNOWN COMPOUNDS
We have thus far considered the reliability with which the
extended Salmonella test system distinguishes between
presumptive carcinogens and noncarcinogens in the actual
test that we have carried out on 50 compounds of each type.
In other words, the probabilities of correct classification
cited above are retrospective. In effect, they describe
the probability of correctly classifying any given compound,
on the basis of a stated mutagenic activity ratio, if the
foregoing tests were to be repeated on the same 100 compounds.
If the test were to be carried out on a different group of
compounds, equally divided between presumptive carcinogens
and noncarcinogens, we would expect similar results provided
that the number of "false negatives" in the former group
were of the order of 18 percent or less.
We consider now the expected probability of correctly
classifying a compound if the test were carried out with a
different group of compounds in which the proportion of pre-
sumptive carcinogens is not 50 percent, but considerably less.
This situation is of interest since in applying the test to
a population of unknown environmental samples, the proportion
of presumptive carcinogens is likely to be significantly
lower than 50 percent. In such a situation it is expected
that the probabilities of correctly classifying the two types
of compounds will be altered in accordance with Bayes1
theorem. In Table 8 we compute, from the data of Table 5
and Bayes' formula, for a population of samples in which only
10 percent are presumptive carcinogens, the probability that
a sample testing positive (i.e., mutagenic activity ratio is
greater than the indicated cutoff ratio) is actually positive,
and that a sample testing negative is actually negative. It
can be seen from Table 8 that while the latter probability
is 97.3 - 97.8 percent (depending on the mutagenic activity
ratio chosen), the former probability ranges from 11.7 - 64.3
percent. Table 8 shows that these relatively low probabilities
could be elevated to 68.8 - 91.3 percent, provided that it
were possible to eliminate the "false negatives" from the
test.
We conclude therefore, that the extended Salmonella test,
carried out as described above, can satisfactorily
distinguish between presumptive carcinogens and noncarcinogens
in test populations in which the two groups of compounds
43
-------
Table 8
APPLICATION OF BAYES' THEOREM
(Where Probability of Positive Sample in Test Population is 0.1)
'
Mutagenic Activity
Ratio
E - C
CAv
Probability that a
Sample Testing
Positive is
Actually Positive
Probability that a
Sample Testing
Negative is
Actually Negative
Probability (percent) for Different Policy Alternatives*
From Present Experimental
Data
Set Prob-
ability I
to =
Probability
II
1.5
33.6
97.6
Set Prob-
ability I
to = 95%
.7
11.7
97.3
Set Prob-
ability II
to = 95%
2.5
64.3
97.8
With "false positives" and
"false negatives" eliminated
Set Prob-
ability I
to =
Probability
II
2.8
84.5
99.8
Set Prob-
ability I
to = 95%
3.2
91.3
99.4
Set Prob-
ability II
to = 95%
2.4
68.8
99.9
Bayes' Formula:
Pr(P/+) = _
Probability I = Probability of Correctly Classifying a Presumptive
Carcinogen in Mutagenesis Tests.
Probability II = Probability of Correctly Classifying a
Noncarcinogen in Mutagenesis Tests.
Pr(+/P)Pr(P)
Pr(+/P)Pr(P) + (1 - Pr(-/N))Pr(N)
Pr(N/-) = Pr(-/N)Pr(N)
Pr(-/N)Pr(N) + (1 - Pr(+/P))Pr(P)
Where:
Pr(P/+) = Probability that a sample testing positive is actually positive.
Pr(N/-) = Probability that a sample testing negative is actually negative.
Pr(-tVP) = Probability of correctly classifying a positive sample.
Pr(-/N) = Probability of correctly classifying a negative sample.
Pr(P) = Probability of a positive sample in test population.
Pr(N) = Probability of a negative sample in test population.
*For optimal combination of tester strain, microsome preparation and
substance concentration.
44
-------
occur in approximately equal proportions. However, if the
test is to be equally reliable in analysing populations of
samples in which the proportion of presumptive carcinogens
is relatively low (of the order of 10 percent) it is important
that the test procedure be modified in ways that either
eliminate, or sharply reduce, the occurrence of "false
negatives."
Certain results which we have obtained during studies carried
out in parallel with the above suggest how the necessary
improvements can be achieved. We have found that 2 of the 9
"false negatives" (N,N-dimethylaminoazobenzene and 1,2,5,6-
dibenzanthracene), when fed to rats result in the appearance
of mutagens in the urine that can be readily detected by means
of the Salmonella test (see reference 5 for a description of
the method and results obtained with one of these substances,
DAB). Thus, if tested compounds were fed to rats, and the
urine analysed for mutagens, at least two of the "false
negatives" could be eliminated. Such an in vivo procedure
is, of course, more cumbersome than the in vitro Salmonella
test. However, by means of suitable investigations it might
be possible to determine where, in the rat, the presumptive
carcinogens are activated, and on that basis develop new
in vitro procedures that respond to these substances.
One reason why a presumptive carcinogen may not yield a
positive result in the Salmonella test system, as it is
presently designed, is that its active metabolites may be
further metabolized to an inactive compound on the test
plate. It is now known that the crude S-9 microsome
preparations that are used in the Salmonella test are
extremely heterogeneous. Although the overall system
oxidizes compounds including several presumptive carcinogens
by way of the microsomal cytochrome P-450 system, it is clear
that various types of cytochromes occur which differ in their
substrate specificities. Accordingly, it is likely that the
metabolic transformations that occur in the present Salmonella
system involve more than a simple activation of the presumptive
carcinogen and that additional processes may in some cases
interfere with the detection of mutagenic metabolites.
Analysis of this problem and the resultant refinement of the
activation system, is likely to further reduce the occurrence
of "false negatives" in the Salmonella test.
Another problem that requires further investigation is the
possibility that DMSO may interfere with the activity of
certain presumptive carcinogens. Preliminary studies suggest
that this effect may account for at least one of the "false
negatives" (N,N-dimethylaminoazobenzene).
45
-------
We believe that given the results of such studies, the
Salmonella test is likely to become capable of detecting
presumptive carcinogens, as they occur in low frequency in
populations of environmental samples, with the same, or
better, reliability that it exhibits in testing populations
in which presumptive carcinogens and noncarcinogens occur in
equal proportions.
46
-------
REFERENCES
1. Ames, B. N., Durston, W. E., Yamasaki, E., and
Lee, F. D., Proc. Nat. Acad. Sci., 7£, 2281 (1973).
2. Garner, R. C., Miller, E. C., and Miller, J. A.,
Cancer Research, 32, 2058 (1972).
3. Sugimura, T., Paper presented at the Fourth
Environmental Research Conference of the U.S.-
Japanese Cooperative Medical Science Program,
Seattle, Washington, July, 1975.
4. Bayes, T., Philosophical Transactions of the Royal
Society of London (1763)., Reprinted in Biometrika,
j45_, 296 (1958),
Brawnlee, K. A., Statistical Theory and Methodology
in Science and Engineering, John Wiley and Sons,
New York, N.Y. (1965).
5. Commoner, B., Vithayathil, A. J., and Henry, J. I.,
Nature, 249, 850 (1974).
47
-------
APPENDIX A
The noncarcinogens and presumptive carcinogens tested are
listed in Appendices A-l and A-2 respectively.
The references that provided the basis for classification
are given by numbers following the name of the compound as
follows:
1. "Survey of Compounds Which Have Been Tested for Carcinogenic
Activity," 7 Volumes, U.S. Dept. of Health, Education and
Welfare, Publication No. 149, 1951-73.
2. "IARC Monographs on the Evaluation of Carcinogenic Risk
of Chemicals to Man," Volumes 1-9, Published by International
Agency for Research on Cancer, Geneva, Switzerland, 1973-75.
3. Weisburger, J. H. and Weisburger, E. K., Chemical and
Engineering News, 44, 124 (Feb. 1966).
4. "List of Compounds Selected for Mutagenicity Program,"
National Cancer Institute, 1972.
5. Miller, James A., Cancer Research, 30, 559 (1970).
6. Druckrey, H. , and Lange, A., Federation Proceedings, 3JU
(1972).
7. Rosenkranz, H. S., Department of Microbiology, New York
University, Valhalla, New York; personal communication.
8. Report on Trifluralin, EPA/OPP.
48
-------
APPENDIX A-l
LIST OF NONCARCINOGENS TESTED
Substance Reference
Acetaldehyde 1
Acetophenone 1
p-Aminoazotoluene 1
p-Aminobenzoic Acid 1
2-Aminobiphenyl 3
4-Aminosalicylic Acid 1
Aniline' 1
Anthracene 1
Antipyrene 1
L-Ascorbic Acid
Atrazine 1
Atropine Sulfate 1
Benzo-p-dioxin 1
Benzoic Acid 1
Benzo(e)pyrene 1
Benzylmercaptan 1
Bromobenzene 4
Caffeine 1
e-Caprolactone 4
Coumarin 1
2,4-Dichlorophenoxyacetic Acid 1
p-Dimethylaminobenzaldehyde 1
N,N-Dimethylformamide 1
c<. -Dinitrophenol 1
Disodium EDTA 1
Endosulfan 1
Ferbam 1
Gibberellic Acid 1
Glutathione
Malathion 1
Methyl Carbamate 1
2-Methy1-N,N-dimethylaminoazobenzene ^
Methyl Orange 1
Naphthalene 1
2-Naphthol 1
O^-Naphthylamine 1/2
p-Nitrophenol 1
d-Pantothenic Acid
Penicillin G !
Pentachlorophenol 1
o-Phenylenediamine 1
Pyridine 1
Riboflavin
49
-------
APPENDIX A-l
Substance Reference
Saccharin 1
Salicylic Acid 1
cis-Stilbene 1
Simazine 1
Sulfaguanidine 1
Tetracycline 1
Trifluralin 8
50
-------
APPENDIX A-2
LIST OF PRESUMPTIVE CARCINOGENS TESTED
Substance Reference
Acetamide 1
2-Acetamidofluorene 1
N-Acetoxy-2-acetamidofluorene 5
Aflatoxin-BI 1
2-Aminoanthracene 1
p-Aminoazobenzene 1
4-Aminobiphenyl 1
6-Arainochrysene 1
2-Aminofluorene 1
1-Aminopyrene 1
3-Amino-1,2,4-triazole 1,2
Azoxymethane 6
1,2-Benzanthracene 1
Benzidine 1
1,12-Benzoperylene 1
Benzo(a)pyrene 1
Captan 7
Dibenz(a,j)acridine 1,2
1,2,5,6-Dibenzanthracene 1,2
7H-Dibenzo(c,g)carbazole 1/2
Dibenzo(a,i)pyrene 1/2
3,3'-Dichlorobenzidine 1/2
3,3'-Dimethoxybenzidine 1/2
N,N-Dimethylaminoazobenzene 1/2
4-Dimethylaininostilbene 1
7,9-Dimethyl-benz(c)acridine 1
9/10-Dimethyl-l,2-benzanthracene 1
4,4'-Dinitrobiphenyl 1
Ethylenimine 1
Ethylethylnitrosocarbamate 1
*N-Hydroxyacetamidofluorene 5
3-Methylcholanthrene 1
22-Methylcholanthrene 1
N-Methylnitrosoguanidine 1
N-Methylnitrosourea 1
p> -Naphthylamine 1 / 2
Natulan Hydrochloride 1
4-Nitrobiphenyl 1/2
2-Nitrofluorene 1
Nitrogen Mustard Hydrochloride 1/2
4-Nitroquinoline-N-oxide 1
N-Nitrosodiethylamine 1/2
N-Nitrosodimethylamine 1/2
51
-------
APPENDIX A-2
Substance Reference
Propane Sultone 1,2
yB-Propiolactone 1,2
Safrole 1,2
Thioacetamide 1,2
o-Tolidine 1,2
4-(o-Tolylazo)-o-toluidine 1,2
Uracil Mustard 2
52
-------
APPENDIX B
Appendices B-l and B-2 show the results of the Salmonella
test on noncarcinogens (NC) and presumptive carcinogens (C)
respectively with microsome preparations from different
tissues. In each class the compounds are listed alphabetically
followed by a computer code number (e.g.; C-10 stands for
presumptive carcinogen No. 10) which we have used to identify
each compound. Concentrations are given in pgm. per test
plate (0, lf 10, and 100); duplicate values are shown for
each of the four indicated tester strains (TA 1535, TA 1537,
TA 1538, TA 1536 [or TA 100]).
53
-------
X
M
O
2
w
p<
a
CO
CO CD
C 4J
O Px
C
H Q)
CJ -P
H «3
nJ O
O -H
C rH
O Pu
o
O -
CU
-p -p
co nJ
0) rH
EH P4
(1)
O4
O
fd
CO
o
^
o
u
CO
p
co a)
0) >
Pi CU
ro
\s> :
O
S
Q)
I
4J
Q)
O 3
i?
in
ii
O « O
CO
!3
Q)
I
cy
cu
O 5
O-*OO O O O O O O 9-*OO O-*OO O-«C3 O OO O
"* """ *" "" **
54
-------
(J
w
c
rl
4J
fj
O
0
^ '
rH
1
CQ
X
D
S3
W
n.
PM
(0
W C)
C -P
VT* . ...j
o A<
f4
H 0)
O -P
M cd
td o
C rH
O ft
S3 3
Q
O -
Q)
-p -p
en cd
fl) rH
id ?-i
rH 0)
0)
f4 W
O
PH 0)
K
H
H
rH
^, ,,, * °*"*Z "^"~ -^^^ .-«.,* .^^« ,,..^« »*«->
rj :
CO
o
5 *
L z
o J «l i
^
CN 5 " ~-~~ -->.». _«.-_ --^^ ~-
* : .... , o.
^ -^^^ --^^ ^^^^ ^« ^ -
N^X
CU - - ----- - 2- 2 2"*
(^
p^ to «N«"t «**<>« r«- O * * O>P-«Jr^ ^^O«V «) 0- -* m «1 m .* ttO*-*^
^\ "^
o
H
^d
1
CN
o <- o o o oo o -- o o o o o o oo o -- o o o e> o o o o
-.«.,
at
> X 0
W> tw y « iu O X. o
55
-------
(Continued)
rH
i
CQ
H
D
W
CM
w
w Q)
tTlrH
Test on Noncarcino
Plate; Duplicate P
rH Q)
r-l fit
c w
O Q)
^H t*
(0 O
10 rH
O
*M O
4J
W C
rH 4->
W G
(U >
« 0
CO
Q)
C 0- ^. ^^..v- ,- ,
O -
c(} - »''^; --^ j£
4-1
<-^ H
O"OO O"OO O -* O O O^*OO O-*OO O-'OO Oo«OO O"OO
z
«i« £ ar >u o x o
0 --f.~ ~~-« ~^~,~ --^^ ^N-,-, ^,^^,>, «~-.~ *«*.".
» ~ ~ ~
CO ,
Is ------
Q>
H 2
rH «
*l~l "* «-»« « *-M «.-.« >d . |M ». .-
56
-------
Tl
(Continue
rH
1
CQ
X
H
APPEND
w
Ul Q)
P
QJ (tf
carcino
icate P
C rH
O di
p
c
0 -
QJ
p -p
W ftf
<1J rH
r-l QJ
rH ft
C W
O Q)
E -H
rH C
fO O
CO rH
0
MH O
O
-P
w £
I I rri
rH 4J
S M
QJ
Q) >
(^ 03
CO
CM
O
QJ
+J
cd
<4H
rH
CO
tropine
in
1
O
0
H
N
tti
P
« ** "°*~ i^^S «CHi0S S*i!C *25^ frmCM iS M - ^ -
o
* o - e o o o o o o
*~z'j £ « £ j vi «
« * * * * *' »
» » r
**»^* * *
2 «*»i* "**B* * %« . .... .».. »..
g
-o -o <> ° -o ° "2 °"°2 °"'2S ""2S
' - ~ r 5 5
1 H | | 5-5 I \
57
-------
Tl
(Continue
r-l
X
Q
W
Pu
w
W CD
C -P
0 fO
carcino
icate P
o a
c
o -
P -P
w (d
Q)
c w
O (D
JT2
H C
nj O
CO rH
o
o
4J
w C
P (0
.H 4J
(0 0)
Q) >
PH (U
P^
u -
^
o -
o
-H
os * " *"*" """* *
g :
Q)
JQ
§
H '
^ -2 --S -2 -3 -2 -S -2 -2
1
X
*" * ** r^
ar > o * -* a: *->,
>- Z >J 1C VI «J M >
^
1 = " 2~ """
5
Q) ^
O
4* ^ ^\ »M * r- «S( (^i o ff- ?\ 4 ~* >fi o*9-
-------
O * c- ry o + i* -O in fsi O
(Continued
H
i
X
H
(-1
52
W
CM
W
in m
:arcinogen
.cate Plat<
O D4
Q
C
0 -
0)
4J 43
W (0
0) rS
EH pi|
H Q)
iH CU
Q)
C W
O
& Q)
^"
3 -
H 5
O
0
(0
< I
\ z
t" * £ f O O -*O ** O **O O **O - W
o
§
X
«, > z *>
5 «C *" Hf "* to
s "
o
u =
0)
C S Z-°~* '">' ^~2~ Z,.~- '^°" "2i~ »--f ~5-*
£ 7 " < o S
~ g j « S 5> 3 S e
59
-------
T3
(Continue
H
i
«
X
H
Q
Z
W
P^
P-i
<
CO
QJ
Cn
^v
carcinc
c
o
2
a
o
4J
W
0)
EH
fd
rH
rH
(!)
«
<>D
1
O
*-Tf
T2
H
O
CO
1
O
55
C
H
X
o
»_i
*o
1
Benzo-p
t
tft
5 - - ~°'- ~ss^ -s=s ~s== -2
KN ~*
-
S - -
i
~O "" O -* O * O -JO O ~* O O
X
> * °
« M Z «M * O
tU Mf Z . M< O X O
giSSiiES
»- 3t _» K « V> -" * «^
O »,-»* «s* .* r* >% r« «>M 1-1 >st ^»»^»^»i» <*J«^» f>w*tfnf^
***»» MnifMrst ^> ni f>«n« IN «n « ej i« ^i r» «n
' * *
^ - « ^, ^ ^ ^ _ M(v «^«_* ^, tv**-* »^«-*_
rfs i- **
M* «- - *«. . _4
ar
a
<
« o-«oo e> o o o o o o-*oo o oo ooo o oo o o o
*- " O -*O "* O O -'O -"O « O "O
«J -*»-* -**
u
£
O
S £ « £ 5
*rt«J lu z ' u> o x 0
** 5 > « * - z o o
-o -_ tt ^ 3 ^ _,
-* ~» w « *si ~t *« e>
60
-------
ro
C
H
4J
C
O
»
rH
1
X
Q
W
(X
rtj
tA
U) Q)
C -P
Q) rd
O IX
C
H Q)
0 -P
M nJ
rt o
C! r- 1
O ft
D
£
0 -
Q)
-P -P
(A rd
0> rH
EH IX
fO V-l
rH Q)
rH ft
O
O Q)
rH C
(0 0
CO rH
O
tw cj
O
-P
W C
rH -P
W Q)
O >
« (!)
* ^^^^ »O<^IA OA-r'M *n^m<»« w-o-%0 **-»^ »-o«^* "^n^*
9M^^ ~ r* ~ -. IM « ^ ^ .» _ _ « r. _« M ^ rw -« N - IM «-»*r»
5 ! !"] !!". !"! "" !!" !!" "II Il!i]
o
ex
O -
CD - 2"2~ 2- 22 S --
g
t 1
r5^
N S
d s
§
X
M^ > *-* * -* Z ° °
^S -< * « j *^ *
« * *
0 » *k* « »V «BO4 »^*f"«* ff1 * O * »*-*O^ «»^*» ^.^^^ * «D «S 0
^^ m «MW« N«r%N tn**^ ---M t*,^-»«< «*-<>*- r,-rN«^ *,-.-.
r-l ^
U| *~ «!Q^!C «M^H^<*t »M ^ IM
-------
(Continued
rH
m
X
i-i
APPEND
10
CO 0)
£ -P
v fd
rni-l
arcino
cate P
u -^
C rH
o a
S 3
Q
C
O -
0)
4J -P
CO 03
(1) rH
f-f CL,
rtJ M
r-i 0)
rH ft
<1)
rt rn
s of Salmoi
nt Colonie
Result
Reverta
/~>
00
1
o
&
v»^
a;
c:
H
"* **
iri
^o -*o -«o o o o o o
X
*- r *»
«e ui x «> « o
«j«wr»iuoco
X >O«_JZ O O
o ** x a- ^ »- -*
^. f ^ K 0 ** -* *A 0
>«
n
* *. . . . . * «... .... ».«»
** ^ «M * *-3.»» 0 « « - £»-.- *»0.0^ ^«^2 5 * ££
**
*, o*~-">- * ** »***< oww'** «> *»o« *!!'"''" ^"^lfc£
^M_> «*«*» IN* IN 'wfs «^*n'wi^ r» *wf% ^ *^ ** *» * ^. n» «*
v«
. Vtf?OCD »fc»w^ »^ "^ » « O*rf»** OO«» O ^O ^irffc. ^ « m ^
.^-4*^ .«_- _r>*_*A4 nt«Hf^.> *->^ -- ». -^ -» - -. »^_«
r< R.N *Si *. !«. « - »M»MWJ»* **
*n
* » ^ *^ * +&£* « « <4 « O i^ « ^- > 'w «» " « *X rf* " O ^ + O *» rf» »
«M »s, tsi r* *- r^ ^ M__^ fx -« ^- -* *«- -*
I
', 0-00 0-00 0 00 0 00 0 00 O 00 0 00 000
^- o -*o -*o o o -«o o v
X
> * V
* uj f a* « O
w5*£So«o
7>u*_lZOO
o *6.r>*-='
»-* ^ K
-------
jr*^
TJ
a)
£3
c
H
4J
C
O
o
H
1
X
Q
53
W
P-i
w -
W Q)
C -P
o (d
tpH
O (^
C
-H O
O -P
j-i (d
«d u
O -H
C .H
o cu
S3 0
D
C
0 -
O
1 i 1 1
w (d
0) r-l
«d J-i
iH Q)
O
C w
O OJ
e -H
^ c
CO .H
O
o
4J
(0 C
P fd
M 4J
W Q)
Q) >
(X 0)
PS
~
t^j
1
^
H
cd
I
o
u
m
in
I
o
53
0)
o
4J
o
rH
Q
ctf
U
1
0)
£ ^<*0--> «^*° --0-^ ^-0-* *««* O-^^O- ^**^ «.«--
^ --
1-1 -* >.
"2 "*2 "S ""2 ~*2 "S *"2 "2
i iu «u * ~ »* ** * O
0 * «c fc D *- ^
*" * "*
O O "^ -* O1 r«v%«o> F-^nr> «*» O « 0- »HP.«^ fun«r« _ m ^> ^ | « 4-
^* S^^Xi ^»S£ ^« * ^ «OC« oS" S i^-^ ^2»? 2*SJ
"* *"**** «» »* «M **^ *-«.*««* »v^^
^ **
5 -*-<»Ni -* «
--.*"' *°"%* "-
tTl
g
O-*OO O O 0 O-^OO O O O O>«OO O O O O-*OO O O O
O O 0 *- O - O ~ O <-> O O
X
63
-------
Tl
(Continue
H
1
CO
X
M
a
APPEN
to o
C -1-1
CTlrH
carcino
icate P
C rH
O PJ
G
O -
OJ
-P JM
w fd
P4 o
o
1
CJ
X)
U « J T 0 O
5
= «=£ ziii ziii -=£: ~£d£ =s£s i£ass ££=i
: js.v*- .j^;,- ,:^£ .-^^^ v^^o ~-,j££ o^«^ ^,w
jj -_n,_. ____ »_. -mr» -r«- - ~^-<" _«._
§
<
£ o oo o o o o -. o o o -* o 3 o-*oo o-^oo o o o o o o
tu *
u
§
i 5
S^SSESO: s
1/1 Jt > (j d . » Q O
-0 - ^ * 0. » - g
64
-------
to
10 0)
C -P
0) rtf
O (X
C
H 0)
0 -P
>-l n3
m o
L (Continuec
Test on None
Plate; Duplj
i
pb nJ M
rH 0)
X (U
H C W
Q 0 0
§3c
PU *0 O
Pj CO rH
< 0
ILJ r )
o
-p
W C
rH -P
0) >
«
CO ~
1
O
**-* j. _ « __^.- ___ _
o - -..«,-_.
C
QJ
CX S
O
4J
H
H g
Q 5
tu
S
*J
> x £
trtUJ U» X W i» X O
W17 > U « ~* Z O O
C^ t* (r- OO- nortw AIISI MW r4 « A O- IM *M *^ o «* ^ o «N» **»f-O OO>»-N
^O
1
a S
0)
e 5 -
6
O
rH 2
4J
-------
o
0)
3
d
-H
-p
d
o
u
w
W 0)
d -P
O (0
tPrH
O CM
d
H Q)
O -P
H (0
to u
O -H
d rH
O Pi
D
d
o -
-P -P
to nj
B Oj
H
VD
I
O
fd
r-l
P
w
o
na
a
w
r:5
rH
1
X
H
w
CM
CM
rH
rH DJ
0)
d w
o o
rH d
ftf O
CO rH
O
«M U
o
4J
to d
rH -P
3 M
CO (U
Q) >
PS OJ
P<
1
c_>
H
O
| :
H
13
O
W
H
66
-------
(Continued)
rH
1
CQ
X
H
Q
W
<
C
O
Test on Noncarcinog
rH
rH
0)
c
o
&
rH
to
M-l
O
w
-P
rH
W
Q)
to
0
-P
Plate; Duplicate PI
0)
a
w
(U
-H
a
rH
o
u
4J
c
M
0)
Di
1
O
H
O
O
H
Gibber ell
£T
vo
1
u
e
(d
2
5
TISSUE CONCENTRATION T
o
5
*«
irt
.- _ _ M»_^^ « ^.
O -< O O O-*OO O O O O O O O O O O-« OO O O O O O O
«- O O O O -* O O O O
; S 1 ! § ; 1 i
** **
Q)
PM
§
H
67
-------
,^ N
rt
0)
fj
H
-P
c
o
o
x^
7
m
H
Q
5s
w
PL,
CM
<
CO
tO Q)
a -P
0) ra
tJIrH
O CM
H Q)
0 -P
M fd
fd o
0-H
C rH
o a
53 3
Q
o -
Q)
-P -P
co nl
Q) rH
EH CM
id M
rH Q)
rH Dj
Q)
c to
O
cc; Q)
- *
s
*«I
r>
W
r«
r>^_ *"
LO
1
U U
o
H :;
& "
nJ
S 1
5
IU
a:
«
3
*r>
«^
*"
«
^
f>\
.^x .
CM
rH
1
S I
(U
a
H R
U> <-
cc
4-1
O
O^N-^ r^o*r-» «-n<^*I r>*jrt»^ in * *- -o c-r>jf->i»> *-»- mrsi*vio 009*4 »o«3-* *+ + r> tt -^f-or^ or*.*-r ««iowo
V*,* « »«>4^r4 -4^« f* r+ +4 -* f* f» f-t ft f^ r-t -* *4 ,*^^^*,* 14-4 "^
N^^O- fj -« O C- *ae*\(*M frWOrt rjrtiNim rfO-^ t^M/sjrt ^'"SC
. * » *« ** »
frfr^O »slM*«o ^t_4rjv« «vj<^f-*V ^^^J« fsir-Of-* *s*-*O(^ 2 *
'
"
°^o - K O
O.«-« *t * ^ *" i
X -* V «0 4A -*
*
- - - * *
- *- -*- - - - -
O<-*OO O -« O O O -^ O O O -* O O O -* O O O -^ O O O O O O"OO
X
* > £*7 tl z o o
z r» ** ** *D«»«
68
-------
(Continued)
rH
i
«
X
H
Q
J3
W
P-i
Cn
<
W
W CD
£ -P
cu fd
CniH
Test on Noncarcino
Plate; Duplicate P
rd M
H CD
i i til
0)
C (/)
O tt)
g -H
r-H C
ID O
W /H
0
l(_| QJ
O
4J
to c
P fd
rH 4J
0 ^1
W V
0) >
« Q)
&
0)
0
0) 2
N
C
Q)
JQ
O_
N 5
cd
O
c
H
E
fd "
rH
FN '^
,G ^D
4J fO
CU 1
go s
i *-y ^
H ^i
T3 v-x «
2-Methyl-N,N-
TISSUE CONCENTRATION
O
o
9
s,-.
-^_ -.-*.-- «^« « r^r,.-.-, -- « -
O O O O O O O OO O O O O O O O -* => O O O O O O O
o - s> o - o «> o «> «-o
£ r £ So
_ __ -~-- _.**. «.--~ ~-_~ ---_ ~._-_ ~-~_
-"«> -s;-5 is;; _s22 222S ~s;s 2^22 s^gs
---- - -
*-o -*o -*o o o o o o
y.
wS^-SoIo
O«"* »D»- _j
69
-------
0
0)
jj
H
-P
C
O
0
*~^
^
1
X
H
CM
a
CO
CO
O
p
to £
P ?d
rH -P
3 M
CO Q)
a) >
& (U
5-
rH
1
_ 5*2
* * * > * * *«
fc O"-*«* ^- * O * « > -A O * » * * »-i^»-C> *-%*»O ^ i^O .*^^»*
MM^». t^ «»-. _. r* _« <\, v*rt«--i -» - ^-»«-« n«n*.'fv .-..*.
0 0 ° 20 0 0 0 -«0 0
X.
*" * ^t O
w 2 z.- w o r o
Z > «J « _* -X 0 0
-0 - K . 3 »- ^
70
-------
*"*
1 - (Continue
i
PQ
X
M
Q
JS
w
Pk
CM
"w
cn o
C -P
QJ fO
icarcinc
icate I
&-i i i
o a
D
0 -
Q)
-P -P
w to
OJ rH
rH 0
rH &,
a w
O (1)
rT2
f3 O
0
m o
°.P
w C
-P (0
H J->
r-J It
(L) ^"
(^ (U
/^^>
z w
a: u 7 u> ^ o
tutu jr w o i. o
z>u« >*zoo
o « a. r> - _i
fc- Z _l M B *A _» *» «
tfi
m
if
^ ** *
CN
71
-------
(Continue
rH
i
X
M
U
53
W
2!
CO
CO CU
p
CD ftf
carcino
icate P
tn f*i
O Gi
Q
c
o -
Q)
rj 1 .Jj
to re
CU rH
EH fl|
rH Q)
O
C to
O (U
r^'^
to O
CO rH
O
O
4J
CO C
rH -U
^J It
co CD
0) >
*^-»
H
rH ° ° °°° ° °00° 00° ° °
CN
1
% -
H
S =
U " " "" "
H
.§ =
O
P
a
rtf §
W »j
o
X
*»> uj iu x " ' *" V * O
t« Z > u « _f Z C i"i
-
0 -^-- .^.^ _ _ .
1
CJ
1 1 W-.K.IVI - - -._-.
o
a ^
M
4J
H
^y
1
fl. O -J O O O O O O -* O O O O O O O O O O O O O O O O O
1 * «- O *-O -*O -*O "*O -"O "*₯
> , « u
u,**"1**'^* °
IBilllsl
72
-------
APPENDIX B-l (Continued)
Results of Salmonella Test on Noncarcinogens
(Revertant Colonies per Plate; Duplicate Plates)
oo -
CJ>
0)
S4 s - ~
rH
O
CO
M
f~t O -* O O O O O O OO O + O O O OO O OO O«OO O OO
f^l o O ' O 0 O O O O
0) -__--_-_
P-I
£ * £ 5 o
o i " * £ 5 £ 5
»-Z ^ tf « Irt _, «A X
s
»- O OO O fM O -* "^ O « O O O O O O O*-*OO OOOO -* O O O O O O O
s
rH
1
B = "
*- *VtrtO -**» » < »N O Vr^^O 4-^r^O 9«t O O * V* 3 O
o -
c
rH S
.H *
o
H
QJ o
PM S
*
r S
73
-------
4 O - M> (
O & ~« it r> tt r*i +
*~,
IB
c
H
4J
c
o
o
" '
1
w
H
Q
ks
w
(II
a
w
w o
C -P
O (0
O P^
H 0)
O -P
(C 0
O -H
O (X
D
C
0 -
P 4J
W fd
(L) rH
H O<
(d M
rH Q)
rH CL,
QJ
C w
O
PH (U
5
in
I
g a - - - -
(1)
A?
"*
JJ" ~ * *" » ? o
« »
j, 00 000 00 00
c\i s ~2-r S-s;^ gtxs ^^2S °^-- -5*^: s;«° s-^nz
I
^ - sr:*^ 2rsi ss-2 r*s^ ^^ss -*c^ srii** 2**^
H ^
f-j *~ «rntniO Wi/\iOp> »M OJ O O «> 7S f~- ts* «^aof^ Kt C> ^ rf» 1*1 ifi f\ 03 -f -f ^ >^
i
74
-------
*a
0)
j3
a
H
p
a
0
u
* -
rH
T
X
M
^
w
a.
p^
rtj
to
to o>
C -P
Q) fd
CnrH
O ft
C
H QJ
0 -P
M fd
ftJ O
C rH
O fit
X 0
P .
c
o -
Q)
-P -P
to nj
0) rH
E~* Oi
rH Q)
a)
C W
O 1)
e-H
rH C
fd o
to rH
O
m o
o
jj
W C
P (d
rH -P
PH "° * = ^^ "
= - ~-
E
g
«
»- -*O -«o *"O o -*o --O -"O O
0
s
** > 3r u
7 K tu 7 u* « o
S3? > £ i j r S 2
-g - - « «j 3 ^ j
« 00 00 or. 0 0000 OOOO - O O - O ~ O O O ~ O O
«
. ... .«..
»--o 2"2z: 222 -^S* 2"2" i"!;"' °S2° i"°~
22»° £2~2 "~22 £-~-' SSi-1 "'"'i- 2*'° -""'
**
5- -______^ - _ __ "
«
"* ** * ** "* * -*-*^* ^. ^. rw ^. M ^( _i
g
x
iu«|«£oSo
-------
co
d
o
p
a
H
p
C
o
CJ
i
«
X
D
W
to
CO -l
rH
rt o
(V
v_J
d
(U
rO
iH
H
4-1
CO
1
W
H
f^
rH
0
^
H
0
0
""-t - -- - - *-
^
O
sSsi^Ils
*" J«*^-^vt»
.
' " 2C~_ K-i2 S"" i- 2-
2
-.-
tft
O
H
rH
cd
co
76
-------
(Continued)
rH
i
PENDIX
<
to
W Q)
C -P
>
PS a)
CO ~
rH - ,;;,; ^
U
0 ~
f«
rH x
»-t °
^^ «c o**oo o--oo o-'oo o-*oo o o o a o o o oo o oo
f- - O -* 0 -* 0 - O - O O O 0
w ""
§
X
UJ > Z U
^iwS^SIu » 0
* O * * « « . 3 ^ ^
O S-«-« -4-<-4 rl*4r« -(,-».- rl^^r* ^ ^ ^ _ _* ^ ^* _ rf
^ * *
( * *»
^f^n-f + & + ift 04^%^ r- o * -* « ^> o > <^ tt o «^ -o » r>- »»^ r^ o o ^
CO
in
U
^;
^ »^» »^
H -
fd
a
H
W
5
*tf Ul X lU * 0
MI u« y ** tu O «: o
77
-------
13
Q)
3
c
-rH
4J
d
o
U
»
1
m
J.
M
a
Z:
w
A<
EM
rti
w
Q)
CP
arcinc
o
a
o
a
0
4J
to
0)
CO
rH
rH
Q)
a
o
rH
fO
in
IH
0
to
rH
10
o
.-,
to
o
(0
rH
0)
-P
fC
u
H
rH
Du
D
>
(U
-P
fO
rH
M
0)
DJ
w
OJ
H
o
o
U
4J
P
f.
Q)
Q)
5
i
o s
c
H
r-H ^ »*»* -0-1"0 "iSH ""-S -2"'0 ~2i- S'2' «2*»
^
rH
UH
H
5H g
I 'I
o> «^a **o o - -< " *
1
o
^-4 *- O*ttf*O ^ IM B 0 ^ tf% a _ « r- « O »* O £-O«DO O***O -*««*0
H
^ =
JH
4J
0)
FH <
O OO O O O O O O O - O O O OO O "- O O O O O O - O O
^. o * o **u **o *- o * o -- o o
X
>. jr «-»
« ku Jc w * U
JC > 0>4 -I X 0 k>
78
-------
+J QJ
rH K
to
QJ
CM
O
QJ
QJ
in
0
rH
O
VJ
H
6
M
QJ
O
1
CM
rH
CO
|
QJ
H
&
cti
4J
CJ
O
>* ° -* "
5 *ZSS aS|| SSSS «S= SSSS 7 = ££ 2 = 25 R-«
^ O IM -^ r^ -. O
Mt
S * " " "
z
o
O**OO O OO O OO O O O O-*OO O--OO O -* O 0 O O O
1> o -- o - o o -*o o o
X
> JT O
ffl
- --- ^r,«
j, _-_ _ «, n *t
-" " . -.-r.- «_*._ ^- ^ ___
79
-------
(Continued)
CN
1
PQ
X
H
Q
13
W
Oi
0«
<
ID
(U
en
O
H
O
on Presumptive Car
-P
W
tt
%n
t>
(?
c
t-
r\
mi
*ff
«
f
c
:t
(D
4
u
-.e
-C
0
«J
\a
3
tf
O O O O rvo-^O O O O OOOO OO>^-* O O O O O O O O ** O O
-«O O O «M O O -* OOOO OOOO 0 - O O O O O 0 O O O OOOO
o-
2
" -. -
f»l rw*^« «MS^ ^» -r »»rv i-i«Mrt rt
O <-* O O 0 0 O O O O O O O O -* O O O O O O O O O O O
-*O «O -«O -«O «O "«O -*O *O
X
> X <-»
e tu f uj * o
IMUJ z i-> w« O L O
Z'>C*-<_IXOO
*J~- «eo.3*-^
X. ~t «uM_jtA«>
«si « m «*> o«o>*> «nr~o« « o urtn-^r^ B»«*tr> ^ «>« **.*r4*i _ isi « 9- ^> r^ (*> r^ , «^ ^4 x r^ «r>g(nM^ _r^m«
* t «
O> r- 4> 4 -^ -O O « AN^o r- o *>* O «»*>* ' rfl in «o «^'v
**«^i* ngp,f\ O "* ^OO'^
-^ (M _ rt o*- *x r- -* o^ * ^ « rfv
^ «n r1- -* *v
- *o M 4 « «^- rw » ^ «t «
*w«n « ^m .
-0.0. -^, --- _ -~r- 1 _;;
" ""
^ -.ftj _-. _*-^ ^ -^
o o o o -* o o o o o o-*oo o oo o-»oo o-*oo o o a
0 0 0 0 « 0 0 0 0
J.
> X **
C W X «* « 0
UkJKM(uo * O
2t > 0 ^ _t 3T O O
o -* tt. a. a»-_»
3Ewac«ut_j(M«
80
-------
(Continued)
CN
1
W
X
H
D
2
W
P<
P-.
<
C
on Presumpti
-p
en
(l)
H
nj
rH
H
Q)
C
£
rH
rO
01
<+-!
O
W
-P
rH
3
CO
o
«
to
(1)
-p
nj
iH
A<
0)
-P
n5
O
M
iH
D4
3
0
Q)
-P
(0
rH
A,
H
(1J
ft
w
onie
rH
o
o
-P
C!
ro
-P
f-t
o
>
o>
«
s-^
GO
rH
I
O
***s
Q)
C
(U
CO
>>
£
O
o
c
H
|
vO
y^N
rH
1
O
v^x
1
&
(1)
,C
CX
H
^3
0
H
><« o » O O « (^ -* »*
p% >4>4N WO-*'-* rtOO^ «*«v*fr ^i«^- M o tf* *4 ~t -r *4 + «iNf^rf< O*V«D-« I ^) ^ -^ »«^*-*o »w»r-o ««**
M**«*^t ,^ . > ^ rs< « CD ^ «v -- r» ^ »s, u^ *« *« *w «x * - **
" -" -"
m
S
*^O i o ' O -* O -* O "O -* O O
X
> JC «J
C «u Z lu « Q
ttu tu z oj O X O
Z>U4t^ZOO
o « a. 3 t- j
-JE J «C a *A _J M «
m
m
e\ -** -*
_«
A
-
V*
i O -- O O O -* O O O -» O 0 O*^OO O O 0 O O O O O O O O O
o -* o o o -* o -» o -* o o
s
e *« z *u «t u
UJtUZHWUXD
se >U4_t«e o o
-*O cC & 3 »- j
fr-Z -J M «> w» _J v> *
81
-------
(Continued)
CN]
1
«
X
H
Q
2
W
CM
<
(/)
O OJ
C -P
H fd
0 iH
M CX|
(0
U QJ
4J
Q) HJ
*> ri
m Presumpti^
'late; Duplic
VJ l-M
-P M
W Q)
-p 0
r-l ft!
rt N_-^
to
GJ
«
m
u)
0)
a
Q)
N
c
Q)
A
O
N
/r<
O
£
H
|
a
lO
1
Q)
Pi
QJ
O
03
M
,C
C
CO
o
a
H
1
CNl
m
r>-
m
£
m
«n
ft
»-
X
O
TtSSUe COSCSNT
«
w»
*^
w\
^
r
o
^
-«-«
«- -*
.M ^^
X
* * ₯ o
.£ ?! 2 £ *» * o
r > o - -*5£3
K^W«M-l^ «
oooo oooo oooo oooo oooo oooo oooo o o o o
*^O ^-Nrt .o-« ^«>«3O fr^-*«3 § ^ «* -*
*rd_4<«i IM o o cr ^ 1-1 o <^«^'1-'-- «-w«O «-^-^r- r% 01 « »».-,aiM
xm« »M KI o - * "* -
*\ * M rt «« * *
tf'^tO-' e> ifs* <^ *M r^CM«i^ » *«4 (" -r -r- ^ o r* *- O- -- o ^ *^ ^ v O «^
ISJN ««'/* 1-1 «o is* /^ «s« «^i«*a) M^(Mf>«
W l^ -* N **
n m r«N *«
rsj r« rsi ^ f-j^i^n. «_>rw^ «> rw « a3 **--f-* f«r-K*r> o«*-i/* Oi^-^*«
«^ IA rv - * ^ o r* - * in r *J
C U> V M4 4O
UJMfcv«M/OX O
X >U<_iZDO
o--«rft.r>--'
Z »* »C » Wl -J V* »
82
-------
(Continued)
CN
|
M
x
M
Q
W
CO
0) ^
tr> co
O Q)
C -P
-H (t!
O tH
rd
U Q)
-P
QJ cd
> 0
Dn Presumpti
Plate; Dupli
1 1 ll
CO OJ
QJ DJ
H
CO
rd QJ
rH -H
QJ O
C rH
O O
E O
rH
rt -P
CO C
rd
t|^^[ 4-)
o ^
QJ
CO >
-P QJ
rH CZ
r~J s
CO
QJ
.__ o r* n m o O- O O -* *- oa r-« » ^ in o » o * *- *«>» o * ^ -« «B rt » -« -o
^ j; -..o --P.- ,..* _,, ,
t- fv *«i r- o tr>Or*».r r^miniA P-^r-o rvK-r-o- o ^- » «st^*>,o gam^~<
I -^fttO tv *o & « «- fy r*i ntNi/v^ r*» * r44
0
v_v
Q_) *^ -< o e- m *4 ^
c :
i^ «*^ *- «^ "*
O *%
g :
1 '
r-l
- O - 0 ** O " O - O - O «- O «- 0
X
>- Z U
« Hi x *w « O
uivJz:**M«O)co
Z>U4^ZOU
«0 « tC A. ^ »_ J
»- a: ^ w o «n - v» «
« OOOO OOOO OOOO OOOO *<^~'O OOOO OOOO OOOO
o
rM ^^
o - 2SS =s ~g" " -'
OJ '=S ~~ "°~
a
o
r-^ * ^* r« .* - « ^. ^* M n (M _ -*
l[ 1
.g = ~ ~ "
1
1
-*o *- o -« o o - o * o - o o
-slsrojg
gs^S:;?!!
»-X _< K «> « 3 5 S
83
-------
(Continued)
CM
H
Q
S3
0*
W
C
tn tn
O 0)
£ -P
H tO
O rH
>n Presumptive Car
'late; Duplicate P
W l-H
CO Q)
0 O4
fS
P 0)
rH PJ
W
q
1 k^
0) ?1
a -
crt 5
cu
X ^
Azo
TISSUE CONCENTRATION 1
fl
1
U
Q) "-
O
N
Cg ».
H 2
4J =
^ =
rH t
1
O
a
H
g 0
^Q *
i I
fr\ wj
X
D
*
_ - - --
-* « ~ -* « -* «
O - o O O O O O O O O-«OC' Cl O O O-*OO O-*OO O-«OO
X
> X U
r u* ar v* « o
WN^-
n^«M ««« ^ **^*
-^.Mr,. - m^,^«
"* "*"* *«-*ni - rtNr-n*! -« -*
,:«5^ J^^ ^^ JJW jJ^-J ^s-~ =2'- ~S"-
X
> X »J
e M* ac u* ^o
84
-------
(Continued)
CN
1
w
X
M
Q
55
w
Pu
P4
<
10
C
0) ^
tn w
O
C -P
-H (0
on Presumptive Care
Plate; Duplicate PI
P M
CO 0}
0) Hi
E-«
to
rt
P 0)
iH Pi
^ ^-^
W
«
rH
I
O
O
a
H
3
N
a
o
w
,* \
m
CO
l
o
o o o
f- «-"o >a -*o **o -*o o -*o * o
u>
u
2T
O
\J
3.
U. > X U
W» Vj tu Z t" O X O
**^r > LJ «-_j 7 o u
o - « o. o *- ^
*- Z j *: U J» ^ *« *j
r^^^in o (o O O «j_jr»tn *^OCB« O *M * m w « ft 4> tM^ic3^ r^a}n a«^ri_* <, M rv m -vi*».«
> o » ^- * »« »M IA /v ri>m4)x>o
rt *, ^«>, UM-H-- M «M fsi - »N. r» rM «^_^ ^ ^ _ Z 2
ir> ~ rn
M «
Ki
s
2
O
« O-*OO O-*OO O-^OO O--OO O^OO O"*OD O^OO O O C
»_ t> O - t» O ~ O O O O
o
Jt
0
V)
w > ar o
3 «c *" Z w -it*
«^t tu u* * *" *a Jt o
«*»X > iJ * J z o O
0 - tf ft. 3 J_ j
~z _* it « wt j U ^
85
-------
(Continued)
CN
1
X
H
O
a
o<
PM
<;
in
£
w
Op)
a -p
-rH Cd
O rH
H PM
(d
O G)
-P
OJ td
> 0
on Presumpti
Plate; Dupli
-P M
W QJ
H
w
(d QJ
rH -H
rH C
C rH
o o
£ U
rH
fd -P
CO C
m jJ
O M
QJ
W >
^J QJ
rH tf
W
QJ
«
m
CNJ
1
o
\^
QJ
C
QJ
M
!>>
Pu
cd
XwX
0
N
d
QJ
PQ
/~^
CO
ro
C_>
>^^»
Q)
C!
(U
rH
>>
«j
QJ
CU
O
c
Q)
PQ
1
CNJ
rH
rv
rH
A -
Z r-o--* s;ss -5S2 CrSS ti2^ 5~~2 iCX^ "^~S
_^.^ .
-* f> p^ »
tfl
2 -~-° s^SS SSSS Z~Z* -*"" =~ = ~ -::'::: - = = -
1
O -*O *-O * O «" O Jl .i *
s s I 5 | I 1 !
rs^;£-3--
g SSS- =SS: 5S5S SSSS KSSS ~- = 2 S~r,~ 2~~.,
£=st Sss~ Ssss s=s^ ssss: s-s?: sx=^ ss~.
2^22 KCSI ssss ssss ~sss s»:s~ s~-s
5s^5£3
rS^« £«. ^v,«
86
-------
I
o
(U
H
T3
0
3
c
-H
-P
c
o
o
w
c
tr> W
O O
C -P
H nj
O rH
^ PM
«
U o
H -H
4J ^
04 &
e 3
3 Q
(0
OJ .-
>-l O « O "* 1» -*O -«O ""O -*O -*O
87
-------
(Continued)
CN
1
APPENDIX
w
CJ
Cn
C
H
O
fd
CJ
CJ
H
4J
e
(0
0)
O
JJ
w
CJ
EH
Salmonella
UJ
(0
-P
rH
W
P4
Ifl
4J
-P
O
r-H
cu
P
GJ
(d
PJ
M
OJ
0,
it Colonies
>-«
nJ
P
CJ
CJ
*^s
CD
O
5
M
cd
0
60
O
N
a
H
w
x N
CM
1
C
O
£
a
cd
N
a
QJ
H
1
vD
CM
»\
rH
K\
'A
<
«s
<
«
m
<
*-
r»
n
4
n
m
«
^>
m
«
«n
«
Z
O
«
«<
z
«*J
u
z
O
u
r>
i^
M> * W «* - -.
ITS * * « i
~*~ - -~ ~ ~ --
-
~ -
-O O O « O O 0 -* O O
X
» X «J
AC *J f ui * O
twiu x iu o X o
Z>U. «c . D - ^
* *J KAVI^«rl«>
=s=£ s~s£ ~ss^ =~£ £=-= ss-= s=i^ st£=
"" -"
---- -~ - - --- ^
G~*OO O -^ O O O -J O O O -* O O O -J O O O -* O O O -* O O O O O
- O - O - O -* O «- O " O *- O 0
X
>- r o
tf w X w * O
UltU^HMt^KO
Z >U« _l X O D
88
-------
^-^
rd
O
jj
c
rH
-P
c
o
O
--'
CM
1
CQ
X
H
0
w
CU
w
0)
o
c
H
0
flj
u
0)
J>
H
-P
p^
fi
to
c
H
H
N
Q)
,£>
o
0
^
O
H
n
i
CO
^
co
vD
"^J"
1
O
(U
c
QJ
J>->
H
&
O
N
a
o
tQ
H
o
w% M - o- ^* "^ r* -* *^ r« ^ ^ w>
fi »- * »
^ ° ~* ** -«5- -"^SJ Si*S -*«*
,
"".- - - - -
2 «**« ~« ^-*^- "*"< -
mmw»* nnrs.« ^^^_ ,-_*, -_- _^-^ ^
,
, O«*OO O O O O O O O**OO O -* O O O ^ O O O-*OO O -^ O O
0 0 0 0 -* 0 0 0 0
tu 5 Z *" ^0
O «*« r\«- «> .«p,,-r^ ^9«O * V* *- 'OV'9* OP-** O> tft « fs«
* »
is, rx. - _ r. r, ^ ^ *, r, - ^ -^n,^ m«M--. r» - - «s, ^, - r, - r^r-»»-.
* **^^ #\i*«!2* jf^«f*»-^» *^i o -o p- * -* «D "i »f» rs» rt ^ o^~*'h> » O ^ «3
1C "*
4 «* *> t * * »*
~-~ - - - - ---
O
£
of O O O O "* O O O -* O O O -* O O O -* O O O -* O O O -* O O O O O
_ »-. o O O -* O O «- O O - U
o
s
VtM'lU Z « w 0 x o
*«z > w < ^ x o o
0 « »-. « c. ;» J; «
»- * -* « » *«1 _* *t «
89
-------
(Continued)
CM
1
m
X
H
Q
di
U)
Q) ^
tP W
o o
C -P
H fd
U r \
rd
O QJ
-P
O fd
> O
on Presumpti
Plate; Dupli
P \-\
w
-P Q)
rH K
W
Q)
rH
1
CJ)
QJ
a
QJ
N
C!
QJ
0
nS
O
H
a
rH
QJ
E
H
Q
S3
^
1
C
H
H
N
C
QJ
O
,c
4-1
6
H
Q
1
CO
*~ o^j**r- -**&< >r n o *~ «^ a fM * o «> ^ * w- o -* *r> o fr (r
* -« «s» «s« IM FI .* ** m PI 4^'rv ».««.* « pt _ . w »«4 * . ^j ^, .- ^j
j; - - -
X
o
££ -. O O O O -* O ' O O 0
3E «»*« -*-*-"
O
g
U
S « & « £ 5
1 i 1 ! 1 i " 1 . 1 i
E
1 ==ss =S2S ss=; ^:ss zssx ssss ss^^ s===
=K2= 25SS SSSJ ^S=S 2=2= ~S?S SiS- 2~-=
E -- - " - " -
£ " ~~ "~~" '" "
z
o
o ^ o e> o -^ o o o ^ o o o -* o o o -- o o o
-------
(_^s
rt
fl)
c
H
4J
a
o
H
CM
1
PQ
X
H
O
2
W
Cu
Cu
rH
-P
Pi
e
CO
0)
M
Cu
C
O
-P
CO
0)
etf
rH
rH
0)
£
0
e
rH
CO
o
CO
-p
rH
CO
CU
W
^^
CO
cu
4J
Ctf
rH
CU
CU
p
(0
U
H
H
CU
Q
^
0)
-p
rH
CU
M
cu
cu
CO
CU
H
c
o
rH
o
o
p
(Tj
4J
M
CU
v£> g 2S-2 -X^j ssss ~"^: r:2S- s-jr. ss~s sx'^
O") ^
1 "" o £ £ £ 7»-£ " i « ? ? T ^ S o S ~ " S?'sS S^2" EJST
Q^ ^ ~ Mr*~ ^r-«*«.
w «* "^ ^ * "*M t**i m<* »*-.»*> nMmM ^N^rM
H
fi »+ « -J ^ -* .* ,^
rd S
o --* - - *- . '
N
cu 5 ------- .
«
rH
r*1
,G
I-1 5
cu r
Cj * o o o
*r-\ 5 "*2 "2 ""2 ^2 "2 "2 "* ° "" °
r^ **
i §
CTN
r^ 3 t x * "
Uz > £ < ** ° * °
rS"SSi5^1
r » «t f^ -ON^!* * » o' o-* <£*v'«« ^f-*«» N « m « «^"' ,^J**
-
X^^ l~ »»^*<4« *«O*-* * -* » O w\»v.rfpta -^ « 10 « d*v«D*> B (h « m 'tftO
__ tst r-v n r- «» F- m -^ e rn tn *'«n^o ^i^».»^ m » r> o n«r^<^ «M««
CO
£> « > 0- o ID *f^r-« trtf-nr- O- O- e- « -* »--»«>- *4^*t«.
^ ' 2
< * * * »>
CU
__! = .---_
H
CO
o
rj
| - ],]" """ ". "."" ".".. ...'. .". '
rH " --_-«.-
4J
S 5
9 1
I °
<
3 " « £ * £
v»»" "J * «u o I §
^£2^5^§2§
>- « J «»«3^B
91
-------
MV) O VS O -
»- V O > O O «<
(Continued)
i
w
H
Q
O.
W
d
Q) ^
tP C/l
O Q)
d -P
-H nj
O, i
(^5
CJ Q)
JL>
0) re!
> 0
m Presumpt:
'late; Dupli
1 j ^_J
W Q)
0) O<
rH -H
rH C
Q) O
d rH
O O
g O
rH
crt JJ
m
IM .iJ
O SH
w >
4J X «J
3 * *" *""" w ^ o
+ x I» it «> *« -* "» *
92
-------
(Continued)
CN
1
«
X
H
Q
js
H
Oj
&t
<
w
c;
(U ^~.
tn w
Ofli
c -P
H (d
?n Presumptive Care
3late; Duplicate P3
-p M
W (U
-P 0)
rH «
*-* " '
W
(11
cr;
f\J - ^» -* r* r*, f\ -. i"» ** IN
-. . M ej p~ vt n* r« *
0) £ *
4-J -
e
nJ
^ 2
rt ,
O
to *
vJ «fi rn » o r^-o *s.e- - --C- r^
f. -»^ CW^*"^»^l»^
"^ *n f«- W f«-t fs,r- MiM i^_ «
^ * nM^t*-i^r*
P
rH
^
rC
JJ
O O 0 O-*OO O O O O O O O-*OO O O O 0 O O O 00
QJ «0 0 O O O O O 0
T1
£
t J > ar «
*" K *" X 4U *. O
r_n uftuz«M(OXO
P-I z>o«_,Zoo
O - ec a. ^ >_ ^
»~Z J X E> «rt _« VI 0
f>F*frn O « m «» «j O «- 4P -r O * O «-«-- r«O«u«' *^ ^- O *^
*4 rt^t H^. ^ ^ .^^. ^^ ^rt ^^,
^ fsj INI ^ & -t r~- *\ rt r\«ni*\o »s« » *-j -o » ^ o O O
- fr * »* * IM t a> trt ^ o p. «4 *>n M *4 -»*»
r*. *j 10 ^> ^ «-i -a n «oo»-*
r^. m«o ^ f- *n * t» B> -r C- a O- O n «n O *st_O « ^ A
rt v4«jn »* .- wa M o r- ^« ^ o *s* *» ^. o -> ,- ri *^ »w^
f"* M "^* * rt -* «* * * « " * *
-P ' .
W
-O -^O «O -<«> -»O -*O *«O - j « w
93
-------
^
rrj
Q)
£3
£
H
-P
a
0
0
CM
1
CQ
X
H
Q
W
P^
a
to
(L)
£
O
fO
o
CD
£>
-H
-P
QJ
E
w
QJ
JH
PM
C
o
-P
W
Q)
EH
«J
rH
rH
0)
c
0
g
rH
CO
M-l
0
CO
-P
rH
P"
CO
CD
rv*
M-i
W
QJ
-P
(0
rH
Q)
-p
n3
O
H
rH
CU
£3
Q
*.
CD
-P
(0
rH
(1,
S-l
Jc3
4-1
a)
*J^1
|
CO
'"^
in
I
O
CU
P!
(D
O
rH
»H
O
»a
.4
n
e
ro
fl)
O
ftf
&
O
ffi
i
^^4
-
10
.
**
2 V-or^O^ »o»^! u?nr^^ OOBN tsJoo -^(^^^ ^ o *? J^rCw!
fM r**,^ «N«v( -. _ r*~
x
O
-* o -* o - o o o - «* - »*o -* o
"*"*"***""""
£ « £ 5 o
o-"2»52S
^X _, ^ C w* J V» »
* * *»*r_t
oof^m > a* »^ ^- »v w ^<^/^-- wci«*«« iz,S»I_
§WVrt*r» M>-OO-« **O tftMV** * **»*" «* ^*tn» r»*»^P- «-»o«r-,r-
% -* -* * o
t- p>r«M?4 ruO*J>O ^OO'** r- - » «rt »<-j^» Orj*-* *«n"*Irl
«n««v»««\o^-mm« « ^ ^ «
« » ..- *-
W N fW «r OW*»O 4>»O O**-* r»*^O« <**-***+ ^^-rtirv O^O***
w> "***"< "*>«%> "-"*« -** ***'*' * * *** *
^ .»* .- «... ...» .«.. «...
M « r--ff v v *. o»o^ £±f£ !22S»
,«^n^ ^.n ^ o ^i«n^« «* * - ^- n « ---.^o- "^^ il
-*+ -* O- trt 0 - *« O -* " * "
Mt *
4
» ^ ^ _*««
^ ^^^
*- ""S* *22^ "22^ -»«- ««^* ^2-"*
X
>l >. ^ V>
sy5l's^?sn
S5 2 2 2 ^ f> - ^
t-ar ^ « *> « - ^* *
94
-------
r-.
1
(U
c
H
H
a
CO
0) -,
tn to
O Q)
C -P
H (d
O
CO
o
M
w
'Cl
cu
n
c
H
.p
c
o
u
* *
CNJ
1
X
H
Q
Z
W
ft
ft
ff
O rH
M ft
(d
U CU
-P
o
H -H
-P rH
a a
e 3
3 p
to
Q> .~
M CU
ft 4J
(0
C rH
FM
Jj ^1
10 QJ
W
fd 0)
rH -rH
P CU
rH (il
r^ «
to
CU
K
i * ^i^oo OIAOOJ ooo*" "*omin tf\«oo-r ^^vvo fv^Oi^ o o o
[ 1 -Jpgrviv^ r«j o int^« B««r-p~ >.Mnw r-r.o> »^rs>in .j.*^^
j-j "S" *>" S£~ -S *? 5 ""H £3
rH *
P-,
JG §
4J 5
fli eC O -- O 0 o O 0 O -* O O ' O - O O O -- O O o -* O O O OO O O O
JJJ J -0 -0 -0 -0 -0 -0 0 -0
^ 0 "* - - -
js S
r> > z «
rlEili^^i
J
:
1
" " " "* ~
c
(U
X! ~~ ""
-p
cj **^« -« --«
rH
rC S " '
u -
rH
Q)
I -to -*O -* O MO -*0 -« O -* O r«O
CM
CN
> K 0
t-X -I rf M V> .* *r» 0
95
-------
, .
r(3
Q)
pj
-rH
-P
C
0
CJ
(N
1
X
M
Q
W
PL,
to
O Q)
C -P
H rd
O rH
VH a.
id
O
P
QJ rd
> 0
-rH -rH
P rH
P, p,
6 3
3 Q
M
0)
rH 0)
(l( -M
rd
O C^
1 i ^1
CO Q)
QJ CU
H
to
rd Q)
rH -H
rH CI
QJ O
C rH
o o
6 0
rH
rd -P
cn c
O M
QJ
CO >
-P QJ
i I PH
CO
Q)
rl
CN|
i n
rH "
1
o ;
QJ
C S
H 2
B -
cd *
rH
rt.^ tr-
.u s
pi =
^
.-'
ca g
^
at
5
0
z
o
3
A
^
o
o
^
CO
1 ^
o s
<^"x "
CtJ
?i
d s
o 7
CO
o
4-)
H s
H :
s §
1 ^
1 *
0
0
3
I/I
i
OO-*O O O 's O *O'^O OOOO OOf«O Ocoo OOOO OOOO
Oo*"*O *^C*-"O OOO OOOO OOOO O O * OOO"3 OOOO
^*«s*nix p* r- * & »^ j o n ^r^i^iri ^r^^fs, ^ ^ f. ^ ^»tf^^ ^"*t>ir>
ssss sss: s:sj ^sjs ss^s SoSs 5t;ss x-ss
^o-n ^»^^ s!!5i °'-0'- -v*** __^ *
^o^o ^ > ^ » >:**,: ~ o « «: -<;,: r: J o o ^ ^ - ^ o j * ^ -;
0-*
*£s^i ^i^5 s ; 7 £ s^xn ^Srti sr:t?s rc£r.r" ^£^t:
*
O O 0 O O O O O 3 C O O C 00 0--OO 0 O 0 0 00
0 O W 0 0 0 W O
_ «
1
^*S;-i^Jo
Z > kJ < J Z O<_)
zj^iii.^^
* * * » »
«««>m w\^o.o «-M^ir- ««O r«a)oo T- « >" OO-«^ O-O^O
*- r*«._« tvt^f^r- o- m* o O «
« IS, *"
o-~**-<*g *
»! » r! *2 -^-00 0 » » ^ **0-^ rCo^4 -t»«-; *» *" * ^ ^ £ ^ ±
* . «
" -
« -.
"'"* . - .... ._... ^^^o j,*ffCJO -^*W^O ^ T" 0
" "i^ "~""""^ ** "* ^ "**J o f- *^ *
-~ tv *
"-*S -i^D ^^X.« *,-, - -»>.r>.«. -" * - ^^ - ^
0-00 0-00 000 0 00 0-00 0 00 0-00 000
X
""^-""sll
o -. ^ *. D t- j
96
-------
(Continued)
cs
i
X
0
w
%
c
CJ
Cn
c
H
on Presumptive Care
fl)
rH
rH
0)
c;
0
rH
CO
m
o
CO
-p
rH
QJ
w
4J
fO
Plate; Duplicate PI
QJ
10
H
c
o
rH
o
u
-p
c
JJ
o
Q)
*
f"~1 ^f
r i «
1
O
rH E
1 ;
r\ «
4-Nitrc
TISSUE CONCtNTRATION T
o
o
oo «
I
0) «
H
rH S
o
O
M
£ 5
Natulan
TISSUE COICCNTIUHON T
o o o o oooo o o r* o o o o o « o o o -*ooo o * o o o o o o
O O O O O M O O O O O O O Q O OOOO OOOO O O O O O O O
*" °,*° *££* i^s <; ££* j -«*^ "*l*i *^^EH ^-~s
^*^^ **'**' " "^ *° 1^ W *" i "* "2 r*'"^»O »s**irs»«n
O -* o o o o o o oo o oo o oo o o o o o o o oo
MO -* 0 - o O - O * O O O
£ * £ § o
tMiuac ««woxo
-. -.-.
o o o ooo o oo o oo ooo o-«oo o o o o oa
O -«O O "O * O O -"O O
X
u> 2 Z " 0 * °
O -**«». D ». _»
Z _* K ' H V1* mJt W% V
97
-------
(Continued
CN
1
m
X
[ 1
Q
S3
W
PH
(^
<
W
c
Q) ^
tr> to
O 0
£ -P
H nj
0 rH "
M O*
0
-_l 1
on Presumpt
Plate; Dupl.
P M
to ,
_i
rt
iJ
w
3
S
C!
o;
W)
o
M
+j
H
^
CO
1
U
Q)
c
n\
2-Nitrof lucre
^ »**!« _» r- p. O in O 4- * O » "> *-«^^»- *»«« Ci ft O «>«.»*
O««N «i r- «. * * i^. «i- ««.**» srr> ^ r- ^ > HN^<»«
r»f^f*»r» itr^nm niMm<^ *««»^*nM «.^^t«« «i^im*n »«» *v *>*"> r«^r««^
= * -. - ~~~~ - «- ~->- ~.,~~ - _-
*- CT^^V^K .-» -* ^ O iO0> W% »« fNl O«Omv -- fW ^ ^S tf\O^«
-----<-« O«^*y rtf»«*ntst mr*-t.~ *>>«>4vB.4.dv«.V
o
s
w
«" V Z 5
3 « UJ X *w ^ O
S₯S53^g§S
^S^2S53SJ!
n rt
^
M% «- » ro ****« « <^ 4 ^^l^rw rxa-« * M> « P»9O * *^*
-v »» n * -«ir «M »v "» ^ O tfiO'^'A ^ ^- m «^* * O M «/*
-* *B o i« -^ o * o- fMf^i^i^ n»«»ir -OO' n*w* -»oc-irt w-^^^
M^o * *w - « » * o »n »>**-» « cv 1-1 m v <^ r-
fSI * «M -* «n rt K*n r^N *** »« « **
<^ r* * tfN'^M ~t « o w-- t »^ * «no «*iO« - J* _j * *M "-i O ^t-»«"« «^-irt^>^- ^«-^O >-"* -3
-*iN^rf» MtnOr^ _ a> *»^r r>*-O *^O" »"" rf^O
~> (^ tf% r^ i»»3«»«-« O -O -^ it- *O-»-*^ Of^^« rf«O--* ^-»--* ^»M"»
^i -. . « rsi fvt v> r^ f^f^«^f" - rw>r _*r>j»^^ n ^*«^* -«pi*^\«n -- _> .«
4 » »«t» **
*- wr«M^ . o *si -^ * PJ «J ^ ^*wi^o rii^*^-* -* Z »*
3 ' « M* ar »** * «
^4u * z - ^ " K 0
«AZ > U < -1 7 O U
~O « « 3 " -J
*-X J V « *« J V» «
98
-------
(Continued)
CM
1
(A)
X
H
Q
W
W
tr> w
O (Ll
C 4J
-H rd
O Q)
-P
0) (tf
> 0
on Presumpti
Plate; Dupli
P M
W (P
EH
10
nj cu
rH -H
rH C
Q) O
C rH
O O
e u
rH
rc! -P
CO C
M-l -M
O to
-P
H «
W
» oo-o n»-o «oo- o-.«o oooo - - o o oooo J---
W\
O
CO
1 ....
QJ | '25-' °^ = * x^ss six= =£::;; =ij- is-= d~f*
H
cd s - - -
rH
4J
CO " " ~ " "
O
4-1 z
H £
|.-^>> U *""**«*»
I
O
I g i 1 1 1 I 1 I
^"at'*j * 5i 3 vi «
Wt
1
Cy "* * « *
^_^ ^ *OOO OfSOO *. « .* o W O O OMifO O * O O »#s.«o O*OO
X
1 JU
CD
a
rH m "^ "" ^ ^ "* "^ "* *^ ~* ^ "* *** ** ~ *
0
a<
4J
, [ ~ o o o -*o o o So °2o
1
5
K «w Z W » O
99
-------
(Continued)
CM
t
CQ
X
H
Q
$5
W
PM
PU
rtj
CO
c
-!
to >
4J GJ
rH OS
r-J s *
to
QJ
w^oo * o ooo w^-»o«» »»*oo ^^oo <^ «^ j\ jj» *"£
v> «^ *^ *»\ n * r4«a«\ * "^
si n n O w*ir>r-io « n r- v ^-wo< «»»v^o o * - " ** « o S
« - r« «» « ** * *
*vj ^i rNt-^rsifst >^A(f«^ % *M n «*^r^*- rv^»»v*^ ^\«-r«j-« ^*n«'1i«»N »»»>»
1 =
rj « -^
^^/ B«M.«ru ^ ^i ^ i»\ .* r»,^» r«^t. z " e>
WJ£Z«IMOZ;O
z>5^^zoo
i-i !^ w £ 2i 3 *« *
r\
t
r^
CNI
Q^ * i ^fs, m 21 2^ ££*£. *S *-»* w«« »» ^.ts4M» -MTMisir-* ^.f«
, Min^i^ rv^Mf «)*-»« «O« to »»- -» * » IA ,*K-«M»^ 221*^^
-> _ _ r« mr«i>\M rMM«nrM -*-<»^«M iMiM^rv -Nrnf* »S - t**n*-*f>*
d
H . . . , ,
to =
^K, «,_- nn - ^,,4-^ «,--- _-__. nr,--.. _ ,..-, __-_
JC
4-J
H ^
_
o
C/3
o
H
J25
100
-------
(Continued)
CM
1
X
H
D
W
<
to
CD
Cn
O
14
H
O
on Presumptive Car
m
cu
(0
H
cu
o
F,
1-1
o
to
-p
rH
CU
to
cu
4J
Plate; Duplicate P
Q)
Pu
W
onie
o
u
-p
rrt
CD
CD
O
CJ
QJ
rH
O
*4-i
c$
c/3
vo
Ov!
i
0
0)
rj
o
o
cd
rH
o
H
o
PM
I
£
5
r
X
z
o
i
s
2
s
TISSUE CONCENTRATION
~~~ ^--- ^ n. - ^ _..._.« M-.-M _ _ _-_._. _._
-.-. _
...
-- ^ -,*«,*----.«
O -- O O O OO 0 OO O OO O O O O " O O O OO O O O
5
« «u ar i*< * o
OOOO O O -* O OOOO O O O - OOOO O O -* O OOOO OOOO
OOOO OOO~* -J o -* -* _ OOOO OO"*O O O O -* OOOO OO O~O
"* -- .*M~.nt ^ _. -^ -*-*-, ^ -,
~S "P ~ 22 "S ~ =5 ~22 *-5 -5
O - O O O -* O 0 O -« O O O O O O O O O OO O OO O OO
5
M uJ Z IM -« o
101
-------
(Continued)
CN
I
cq
X
H
Q
2
W
PJ
CL,
ff,
W
£
Q) .
tn W
O
4J CD
rH tf
J-* ^.^rf.
CO
Q)
«
CTv
1
cu
a
H
n3
H
rH
o
H
1
o
CO
rxi
1
o
Q)
t>
H
6
cd
4-1
S
'1
*f
sr
«ij
i*
O
,3
a
;*
. X V
w M 2 MJ * rs
vuufXwu/tjXO
Z >tjdl_lZOW>
o oca.3»--»
X ,j * *> i* -> %* K
OOOO O *^l -- OQOO OOOO OOOO OOOO D O 0 O OOOO
IS.- -
"* - fy-cs..- --. - -_.-_ -,-«
*dr«»i>' -- rS) - ^^ oo
O -*O --O »^O -*O -"O <-"O O
5
«e *u Jc <« * o
VJiUJ>u*UOX.O
sr>o<-
-------
(Continued)
CN
1
w
X
H
Q
S3
W
PU
VI
C
0)
tn
O
C
-H
0
0)
>
on Presumptd
-P
in
EH
iH
iH
(L)
C
O
6
rH
rt
o
w
p
rH
(0
-
to
-p
0)
p
(tf
o
rH
a
0
D
Q)
P
(0
rH
P4
0)
W
Q)
o
rH
0
-P
rt
>
Q)
rH
1
O
-0
cd
w
S
H
-H
0
cd
^
CO
1
o
o
H
H
d
rH
O
U
. 1
O
1
o
N
cd
rH
O
H
1
O
1
o
0
r*
£
*»
in
O
TISSUE CONCCNTO
£
D
ft
V>
5
§
Z
UJ
O
IW
D
" " "" " ~~
- --- _- -
*.^<- ^^NV -^r^UN _«* -0- -,-r- ""S ***
£ x £ So
Iliiilll
"^2^ iC*°S MrCSlC «^iC!C2 ^^n^ SdSfH £»£M ^XS*
** « " ***** ** *!*!« * *" f« *^ -*
* -* - -
-
- *.- ^~ »V-N -.-^- -^ *...
o - o o o -« o «o ^* o o
> r 5
«<5x-2o0
103
-------
TECHNICAL REPORT DATA
//'lease read Instructions on the reverse he/ore comi>lelinnj
1 REPORT NO. |2.
EPA-600/1-76-022 J^
4. TITLE ANDSUBTITLE
Reliability of Bacterial Mutagenesis Techniques to
Distinguish Carcinogenic and Noncarcinogenic Chemicals
7 AUTHOR(S)
Marry Commoner
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Center for the Biology of Natural Systems
Washington University
Box 1126
St. Louis, Missouri 63130
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Health and Ecological Effects
Office of Research and Development
U.S. Environmental Protection Agency
Wash-ingl-nn, B.C. 20460
3. RECIPIENT'S ACCESSIONKNO.
5. REPORT DATF
April 1976
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
68-01-2471
13. TYPE OF REPORT AND PERIOD COVERED
Final June, 1974-Aug. , 1975
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This contract conducted investigations to determine the reliability with which
an expanded Salmonella mutagenesis test system can distinguish between those organic
chemical substances that cause cancer in laboratory animals (presumptive carcinogens*)
and those that do not (noncarcinogens) . The results of such tests of 100 organic
compounds (50 presumptive carcinogens and 50 noncarcinogens) are described.
In general, the results indicate that the Salmonella mutagenesis system that has
been employed in these tests can be used to distinguish, with a high degree of relia-
bility, between presumptive carcinogens and noncarcinogens, in populations of test
samples in which these two classes of compounds occur in approximately equal
proportions. The results also show that the statistical reliability of the test
system needs to be improved for the purpose of applying it to environmental samples
in which the proportion of active substances (i.e., presumptive carcinogens) may be
relatively low. Certain of the results indicate what steps need to be taken to
achieve this improvement.
* We reserve the use of the word "carcinogens" for those substances which are known
to cause cancer in people; a "presumptive carcinogen" is a substance known to cause
cancer in laboratory animals.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
carcinogens
mutagens
bioassay
Salmonella
IB. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
b. IDENTIFIERS/OPEN ENDED TERMS
19. SECURITY CLASS (This Report)
UNCLASSIFIED
20. SECURITY CLASS (This page)
UNCLASSIFIED
c. COSATI Field/Group
06 M, T
21. NO. OF PAGES
110
22. PRICE
IPA Form 2220-1 (9-73)
104
-------
INSTRUCTIONS
1. REPORT NUMBER
Insert the EPA report number as it appears on the cover of the publication.
2. LEAVE BLANK
3. RECIPIENTS ACCESSION NUMBER
Reserved for use by each report recipient.
4. TITLE AND SUBTITLE
Title should indicate cleaxly and briefly the subject coverage of the report, and be displayed prominently. Set subtitle, if used, in smaller
type or otherwise subordinate it to main title. When a report is prepared in more than one volume, repeat the primary title, add volume
number and include subtitle for the specific title.
5. REPORT DATE
Each report shall carry a date indicating at least month and year. Indicate the basis on which it was selected (e.g., date of issue, date of
approval, date of preparation, etc.j.
6. PERFORMING ORGANIZATION CODE
Leave blank.
7. AUTHOR(S)
Give name(s) in conventional order (John R. Doe, J. Robert Doe, etc.). List author's affiliation if it differs from the performing organi-
zation.
8. PERFORMING ORGANIZATION REPORT NUMBER
Insert if performing organization wishes to assign this number.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Give name, street, city, state, and ZIP code. List no more than two levels of an organizational hirearchy.
10. PROGRAM ELEMENT NUMBER
Use the program element number under which the report was prepared. Subordinate numbers may be included in parentheses.
11. CONTRACT/G RANT NUMBE R
Insert contract or grant number under which report was prepared.
12. SPONSORING AGENCY NAME AND ADDRESS
Include ZIP code.
13. TYPE OF REPORT AND PERIOD COVERED
Indicate interim final, etc., and if applicable, dates covered.
14. SPONSORING AGENCY CODE
Leave blank.
15. SUPPLEMENTARY NOTES
Enter information not included elsewhere but useful, such as: Prepared in cooperation with, Translation of, Presented at conference of,
To be published in, Supersedes, Supplements, etc.
16. ABSTRACT
Include a brief (200 words or less) factual summary of the most significant information contained in the report. If the report contains a
significant bibliography or literature survey, mention it here.
17. KEY WORDS AND DOCUMENT ANALYSIS
(a) DESCRIPTORS - Select from the Thesaurus of Engineering and Scientific Terms the proper authorized terms that identify the major
concept of the research and are sufficiently specific and precise to be used as index entries for cataloging.
(b) IDENTIFIERS AND OPEN-ENDED TERMS - Use identifiers for project names, code names, equipment designators, etc. Use open-
ended terms written in descriptor form for those subjects for which no descriptor exists.
(c) COSATI FIELD GROUP - Field and group assignments are to be taken from the 1965 COS ATI Subject Category List. Since the ma-
jority of documents are multidisciplinary in nature, the Primary Field/Group assignment(s) will be specific discipline, area of human
endeavor, or type of physical object. The application(s) will be cross-referenced with secondary Field/Group assignments that will follow
the primary posting(s).
18. DISTRIBUTION STATEMENT
Denote releasability to the public or limitation for reasons other than security for example "Release Unlimited." Cite any availability to
the public, with address and price.
19. &20. SECURITY CLASSIFICATION
DO NOT submit classified reports to the National Technical Information service.
21. NUMBER OF PAGES
Insert the total number of pages, including this one and unnumbered pages, but exclude distribution list, if any.
22. PRICE
Insert the price set by the National Technical Information Service or the Government Printing Office, if known.
EPA Form 2220-1 (9-73) (Reverse)
------- |