CODING FORM FOR SRC INDEXING
INDEXING
REVISED 10/15/86
REVISED
10/15/86
OTS0510914
OTSO510914
42073 B2-8
40-8472010
Date Produced
Date Received
TSCA section
4
Submitting
Submitting OrganiZ2llion
Organization
CHEi1 MFGS ASSN
CHEMO
Contractor
Contractor
PHARMAKON RES INTL
Document Title
PLATE TEST
SALMONELLA/MICROSOME
AMES SALMONELLA/MICROSOME
TEST
Chemical Category
2-MERCAPTOBENZOTHIAZOLE
2-MERCAPTOBENZOTHIAZOLE
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PHARMAKON RESEARCH
RES£ARCH INTERNATIONAL,
INTERNATIONAL, INC.
INC.
PHARMAKON
IffAVE"LY. 'ENNSYLVANIA '''7~
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Ames Salmonella/Microsome
Ames
Salmonella/Microsome
Test
Plate Test
PH 301-CMA-001-83
301-CMA-OOI-B3
PH 301-CMA-001-83A
301-C~~-001-B3A
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~ercaptobenzothiazole
ercaptobenzothiazole
Lot #
139-148
Lot
39-14B
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Submitted
Submitted to
to
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Manufacturers Association
Association
Chemical Ma~ufacturers
washington,
D.C.
Washington, D.C.
:
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Edmund G.
G. Goaek,
Godek, B.S.
Study
Study Director
~W~·4.~'
Robert W. Naismith, Ph.D.
~aismith,
Ph.D.
Richard J.
President
Director
Director of Toxicology
Toxicology
Matthews,
Ph.D.
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February 3,
3, 1984
February
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'l'ABLE OF CO~lTENTS
TABLE
CONTENTS
PAGE
I
SOMMARY
•••••••••••••••••
~ •••••••••••••••••••••••••••••••••••
~ •••• l1
SUMMARY..........
....................
.....
..........
II
II
STUDY DESCRIPTIVE
••••••••••••••••••••••••••••••••••••••••••••••••
DESCRIPTIVE ...................
.....................
22
III
II!
IV
IV
PURPOSE.........................
PURPOSE ••••••••••••••••••••••••••••••••••••••••••••••••
.................
......... ~
••••••••• 22
TEST SYSTEM..........................................2
SySTEM •••••••••••••••••••••••••••••••••••••••••••••••••••••• 2
Test
2
Test Organism••••••••••••••••••••••••••••••••••••••••••••••••••
Organism........................................2
Strains
Strains. ••••••••••••••••••••••••••••••••••••••••••••••••••••••••
..................................
22
Source
Source.•••••••••••••••••••••••••••••••••••••••••••••••••••••••••
.
...................................
.
2. 2
Rationale
••••••••••••••••••••••••••••••••••••••
22
Rationale for
for Test
Test System
System.............
......
[
V
MATER.I.AI.S AN'D
I£THODS ••••••••••••••••••••••••••••••••••••••••••••
V MATERIALS
AND METHODS
...................
...................
......
33
Test
maintena~ce...................
••••••••••••••••••••••••••
Test organism
organism storage
storage and
and maintenance
.......
33
Negative and
Positive controls................................3-4
controls ••••••••••••••••••••••••••••••••• 3-4
and Positive
Negative
Preliminary Toxicity
Toxicity Screen
4
Preliminary
Screen.••••••••••••••••••••••••••••••••••••
...................
........
4
Selection.......................................4
••••••••••••••••••••••••••••••••••••••••••••••••• 4
Dose Selection
Plate Incorporation
•••••••••••••••••••••••••••••••••••••• 4
Incorporation Assay
Assay.....................4.
Plate
Top
Agar ••••••••••••••••••••••••••••••••••••••••••
~ ••••••••••••55
................
.................
Top Agar...................
I~
l;
Metabolic
System•••••••••••••••••••••••••••••••••••• 5
Activation System...................5
Metabolic Activation
Data
Reporting •••••••••••••••••••••••••••••••••••••••••••••••••
55
Data Reporting.
............................
.....
5-6
Evaluation Criteria.
Criteria ••••••••••••••••••••••••••••••••••••••••••••
S-6
...................
...................
Evaluation
Records Maintained......................................6
Maintained •••••••••••••• ~ •••••••••••••••••••••••••••••• 6
Good L3Doratory
Statement •••••••••••••••••••••••••••• 6
Practices Statement..................6.
Laboratory Practic~s
Good
Test
•••••••••••••••••••••••••••••••••••
~ •••••••• 7
Purity
.....................................
Test Article
Article Purity
Test
Stability ••••••••••••••••••••••••••••••••••••••••• ,
Article Stability.....................7
Test Article
VI
RESULTS
RESULTS AND DISCUSSION
DISCUSSION...............
.........
.........
77
VII
CONCLUSION
••••••••••••••••••••••••••••••••••••••••••••••••••••••• 7
CONCLUSION...........................................7
VIII
VIII
BIBLIOGRAPHY
•••••••••••••••••••••••••••••••••••••••••••••••••••••
................
77
.................
BIBLIOGRAPHY...................
IX
IX
SUMMARY DATA TABLES ••••••••••••••••••••••••••••••••••••••••••••••
8-9S-9
........
...................
...................
XX
QUALITY
10
10
.............
...................
STATEMENT.••••••••••••••••••••••••••••••••••••••
ASSURANCE STATEMENT
QUALITY ASSURANCE
XI
AMENDMENT
••••••••••••••••••••••••••••••••••••••••••••••••••••••••
11
11
....................................
AMENDMENT.......
.............
XII
XII
APPENDIX
APPF.NDIX
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PHARMAKON
PHARMAKON RESEARCH
RESEARCH INTERNATIONAL.
INTERNATIONAL, INC.
INC.
WAVIRL
y. PENNSYLVANIA
"INNIY~VANIA1M'1
WAVERLY,
l8471
Ames
Salmonella/Hicr~some
Salmonella/Microsome
Plate Test
PHONE
'HON[
C7nlll.2.'1
(717)886-2411
301-CMA-001-B3
PH 301-CMA-OOI-B3
301-CMA-001-B3A
PH 301-eMA-oOl-8JA
Hercaptobenzothia~ole
Mercaptobenzothiazole
#39-148
Lot t39-14B
SUMMARY
SUHHARY
Test article,
article, HercaptobcnzothiAzole,
Mercaptobenzothiazole , Lot '39-14B,
#39-14B, was soluble
soluble in
in DI".50.
DMSO.
DQse
Dose levels in
in the Preliminary Tbxicity
Toxicity Screen wer~
were100,
100, 333,
333, 1000,
1000, 3333 and
10,000 ug/plate. Strains TAlS38
TA1538 and 'rAlOO
TA100 showed
showed complet~
complete inhibition of
bacterial growth at the 10.000
to
10,000 and 3333 ug/p1ate
ug/plate levels and excessive
excessive
to
moderate inhibition of bacterial growth
growth as deterJllined
determined by the presence of a
ug/plate. Dose levels for
dense lawn of small pindot colonies at 1000 and 333 ug/plate.
assay were 3,
test article Hercaptobenzothiazole,
Mercaptobenzothiazole, Lot 139-14B,
#39-14B, in
in the plate assay
3,
10,
(42 JIlg
10, 30, 100 and 300 ug/plate.
ug/plate. There were 0.07 ml of S-9 supernatant (42
mg
protein per JIll)
ml) per 1.0 ml
ml of 5-9
S-9 IDi.x
mix used in
in the rat liver IIlicrosomal
microsomal
activation system.
Results obtained for strains TA1538
TA1S38 and TA98 in
in the initial experiment at
at
300 ug/plate with JIletabolic
activation were greater than one standard
metabolic activation
deviation of the historical mean (TA1538 =
• 30 %
% 10'.
A
+- 9,
9, TA9S
TA98 .40
= 40 +10).
A second
study was conducted in
in strains
TAl538 and TA98 with metahol:'o:
metabolic activation
strains TA1538
preparation at dose levels of 100,
300, 450 and 6ro
600 ug/plate.
ug/plate. The 450
100, 250,
250, 300,
preparation
ug/plate doses were included
included to determine if
similar results
results would be
and 600 ug/plate
if similar
obtained at higher
higher doses.
doses.
article Mercaptobenzothiazole
Mercaptobenzothiazole,, Lot '39-14B,
were
The resul~s
results for test article
#39-14B, were
negative in
TA1538,
~A98 and TAlOO
Salmonella
TA1537,
TA1538, TA98
TA100 of 5alDlonella
negative
in strains 'I'A1S35,
TA1535, TA1537,
typhimuriUJll
JDetabolie activation preparation
preparation at 3,
3, 10,
typhimurium both with and without metabolic
10,
30, 100 and 300 ug/plate.
ug/plate. In addition,
Mercaptobenzothiazole, Lot 139-14Il,
30,
addition, Mercaptobenzothiazole,
#39-14B,
was evaluated at higher dose levels of <50
ug/plate with metabolic
450 snd
and 600 ug/plate
activation and found to be negative in
in strains TA1538
TAlS38 and TA98 of Salmonella
SalJllOnella
~himurium.
in the evaluation of the
typhimurium.
All solvent and positive controls used in
test article were within the acceptable
Accep~ablc range of mean historical data.
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STUDY DESCJUPTIVE
DESCRIPTIVE
Sponsor:
Sponsor:
Chemical
Manufa~~~ers
Association
Manufacturers Association
Chemical
2501 M
M Street,
Street, NW
2501
NW
W~shington,
D.C. 20037
20037
Washington, D.C.
Study
Number:
Study Number:
PH 301-CHA-OO.-B3
301-CMA-001-83
PH 301-CMA-001-83A
30l-CHA-OOl-B~~
Study
Description:
Study Description:
Ames
J.a!Microsome Assay
Assay with
without
with anc5
and without
Ames S&lmonel
Salmonella/Microsome
metabolic
preparation
according to
to Standard
metabolic act:r,;ation
activation
preparation according
Standard
Operating
on:
PH-301 on:
Procedure PH-30l
Operating Procee5ure
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Mercaptobenzothiazole
Mercaptobenzothiazole
#39-14B
Lot '39-10
Date
Initiated:
Date Initiated:
February
February 5,
5, 1983
1983
Date
Date Completed:
Completed:
February
28,1983.
February 28,
1983
Pharmakon
Pharmakon Reference:
Reference:
No~ebook
Notebook '374,
#374,
Study
Study Director:
Director:
Edmund C.
B.S., Pharmakon
PhaI'1llakon R,.search
Research
Edmund
G. Godek,
Godek, B.S.,
International, Inc.
International,
Inc.
Technical
Technical
Performance:
Performance:
F.dmund C.
Godek, Dino
Daniels,
Edmund
G. Godek,
Dino Mecca,
Mecca, Susan
Susan Daniels,
Susan Lucenti,
Ruth Sorg,
and Margaret
Kevish
Susan
Lucenti,
Ruth
Sorg, and
Margaret Kevish
page
page 44
44
Notebook 1375,
pages 31-32,
37-38
Notebook
#375, pages
31-32, 37-38
PURPOSE
PURPOSE
To
evaluate the
the te::it
chemical
range of
genetic
To evaluate
test
chemical over
over aa wide
wide range
of concentrations
concentrations for
for
genetic
activity using
using Salmonella
Salmonella typhimurium
typhimurium with
ancS without
activity
with and
without th~
the adc5ition
addition of
of aa
metabolic activation
activation system.
mammalian
mammalian metabolic
system.
SYSTEM
TEST SYSTEH
11
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Test
Organism:
Test Organism:
Salmonella typh1Jnurium
Salmonella
typhimurium
Strains:
'l'A153S,
TAlS37,
'l'A153S, TA9S
TA1535,
TA1537, TA1538,
TA98 and TAIOO
TA100
Source:
Source:
Dr.
N. Ames
Ames
Dr. Bruce
Bruce N.
Oniversity
of Califomia,
Biochemistry Department
Department
University of
Clifornia,
Biochemisty
Berkeley,
california 94720
Berkeley, California
Rationale for
Test System
Rationale
for Test
System
Chemicals
of inducing
been shown
shown to increase
~e
have been
increase the
capable of
inducing IIUtations
mutations have
Chemicals capable
reversion
frequency
the histidine
histidine locus
selected tester
Cltrains of
tester
strains
of
the
locus in
in
selected
reversion
frequency at
at
Salmonella typhimurium
activation
salmonell!
typhimurium with and without the addition
ae5dition of a metabolic activation
system.
.
system.
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Ames
test
Salmonella/Microsome Plate test
Ames Salmonella/Microsome
PH 30l-CHA-00l-B3
301-CMA-001-83
PH
301-CMA-001-83A
PH 301-CMA-001-83A
~TERIAI.S
MATERIALS
AND METHODS
METHODS
AND
Test organism storage and maintenance
maintenance
The tester strains
were maintained
maintained in
quadruplicate at
at aa minimum
minimum of
of -60·C,
and
strains were
in quadruplicate
-60 degrees
Celsius,
and
served as a lIulster
culture.
master and stock culture.
In
surface thawing and re-freezing
re-freezing of frozen
f:cozen
In .order
order to avoid the effect of surface
permanent
master plates were employed
source of
of
employed as a source
stock, master
of bacterial
bacterial stock,
vials of
permanent vials
inoculum
Maater Plates were prepared
prepared by spreading
spreading
inoculum for B1utagenesis
mutagenesis testing. Master
O.l
glucose
mM biotin
biotin on the surface of minimal glucose
0.1 ml of 0.1 H
M histidine and 0.5 aM
agar
plates
using
a
sterile
glass
spreader.
For
R-factor
strains,
0.1 ml
strains,
R-factor
the
spreader.
agar plates using a sterile
of ampicillin
(8 mg/ml) was spread
spread in
in addition to the histidine
bistidine and
and biotin as
as
ampicillin
pressure
the pllllllDid.
'lhe plates
plates were prepared
prepared from
trolll frozen
frozen
The
plasmid.
retaining the
for retaining
pressure for
stock
plates. The
The
stock cultures and were then streaked onto the surface of the plates.
plates
were then refrigerated.
refrigerated. These
'lhese
Celsius
and were
37degrees
were incubated
incubated overnight at 37·C
plates were
plates
testing for 2-3
2-3
testing
mutagenesis
inoculm for mutagenesis
be used
used as a source of inoculum
can be
plates cu
months. New Master Plates were always made from frozen stock
stock cultures.
cultures.
Fresh cultures for lIIu~agenesis
prepared by inoculating
inoculating aa loop of
of
mutagenesis testing were prepared
inoculum
SO ml of oxoid
broth and grown
grown overnight
overnight at
at
oxoid broth
Master Plates into 50
inoculum frolll
from HasteI'
37·C
in
Brunswick Scientific Model
Hodel G24
G24 Environmental
Environmental Incubator
Incubator Shaker.
Shaker.
in aa New Brunswick
37degrees
Celsius
Tester strains were routinely cheeked
appropriate
checked for the presence of the appropriate
genetic
histidine requirement,
requirement,
The marke~s
markers routinely tested are for histidine
markers. The
genetic markers.
crystal
ampicillin resistant R factor
factor and avr
uv~ B
B deletion.
deletion.
violet sensitivity, ampicillin
crystal violet
Negative
Positive Controls
Negative and Positive
II
II
Tester strains TAl
5 35 , TAl537,
TA1538, 'lA98
'tAlOO were
vue plated in
in
TA98 and TA100
TA1537, TA1538,
TA1535,
triplicate with DHSO,
activation, to obtain
DMSO, both with and without metabolic activation,
background
negative solvent
solvent
formation to serve as negative
background lawn and revertant colony fOrJlllltion
controls. In
tester
In order to validate the integrity of the test system, all tester
strains were also run, in
knO~l positive
positive response chemicals.
chemicals.
in triplicate,
triplicate, with known
Positive
requiring metabolic
metabolic activation
activation were
were strain specific
specific and
Positive controls not requiring
were as follows:
were
TA1535
without activation
activation -- Sodium
azide (10ug/ml)
(lOug/ml) in
in H20.
H 0.
TA1535 -- without
Sodium azide
2
[Sigma
S2002]
[Sigma - 52002)
rH
TAl537 - without activation --9-aminoacridine
TA1537
9-aminoacridine
(1500
1n DMSO.
DMSO. (Sigma
A-OSlO)
(Sigma - A-0510)
(100 ug~l)
ug/ml) in
2-nitrofluorene
TA1538 - without activation
'lAl538
activation - 2-nitrofluorene
(50 ug/ml) in
DMSO.
in DMSO.
TA98
'lAgS
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2-nitrofluorene
- without activation - 2-nitrofluorene
ug/ml) in
DMSO.
in DMSO.
(50 ug/ml)
'lA100
TA100
(Aldrich - N1,
Wl, 675-A]
675-A)
[Aldrich
(Aldrich
- N1,
.1, 675-4]
675-4)
[Aldrich
without activation
(10 ug/ml)
ug/ml) in
in H20.
H20 •
- without
activation -- Sodium
Sodium azide
azide (10
S2002]
[Sigma - 52002)
[5i9llla
3
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Salmonella/Microsome Plate
Plate test
test
Ames Salmonella/Microsome
PH 301-CMA-001-83
301-CMA-00I-B3
PH
301-CMA-00I-B3A
PH 301-CMA-001-83A
positive control requiring
requ1r1nq metabolic
metabolic activation
activation was 2-anthramine
2-ant.~riUlline
The positive
The
(2-Aminoanthraccne) (50
(SO ug/ml)
uq/ml) (Aldrich
A3,880-0l in
strains. The purity
in all strains.
[Aldrich A3,888-0]
(2-Aminoanthracene)
control articles
articles was supplied
supplied on the manufacturer's
manufacturer's label.
label.
of the control
Preliminary Toxicity
Toxicity Screen
Preliminary
The preliminary
preliminary toxicity screen for the Ames
Ame. Assay used two of the histidina
histidina
The
auxotrophs of Salmonella
SallDOnella typhimurium
typh1Jnurium TA1538
'1'1.1538 and
~d TA100.
'1'AlOO.
'1'he preliminary
preliJllinary
The
auxotrophs
toxici ty screen was designed to determine
detemin. at which levels
levels the compound
compound
toxicity
exhibits toxic effects
effects to the Salmonella
SallllOnella typhimurium
~hiJlluriWll tester
tester strains.
strains. The test
test
exhibits
compound was
was prepared
prepar..d to a concentration
dilutions
Logarithmic
mg/ml. Loqarithmic
concentration of 100 eq/ml.
compound
ot
~hi!l stock solution were made
IUde in
in DMSO
DKSO to give
qive the following
follCl¥iDq concentrations:
concentrations:
of this
1.0,
3.3, 10
~O and 33.33 mg/m1.
IIIq/ml. Top agar,
aq&r, used
used as
a~ an overlay,
ove~l.y, was reconstituted
reconstituted
1.0, 3.3,
into a molten
molt~n state and supplemented
supplementea with 0.5mM histidine - 0.5mM biotin
biotin at a
volume
of
0.1
1IIl/1II1
of
aqar,
and
maintained
at
4S·C
until
used.
iterile
glass
Sterile glass
Celsius
until used.
volume of 0.1 ml/ml of agar, and maintained at 45degrees
tubes with k~puts
at
Dry Bath at
Isotemp
kaputs were labeled and placed into a Fisher I80temp
4S·C.
All control
control and treated tubes and plates were done in
in duplicate.
duplicate. Using
45
degrees
Celsius.
sterile
technique, the
the following ~ere
added to each tube:
tube: 2 ml aliquots of
of
were added
sterile technique,
top agar
solution, 0.1 ml
IIIl of tester strain and 0.1 ml
JIll of the appropriate
appropriate
agar solution,
top
concentration of the test compound.
compound. The tubes were vortexed
vortexed and poured onto
miniQal
plates. The sample
~ample was evenly distributed
distributed on the plate,
plate, and
glucose plates.
minimal qlucose
the top agar
aqar overlay was allowed to harden.
harden. The same
s~e procedure was repeated
repeat~d
for each tester strain. Within an hour the plates were inverted and placed
placed in
L~
incubator. The plates were incubated for 48 hours
bours followinq
following which
37 degrees
Celsius
incubator.
a dark 37·C
backqround lawn and spontaneous
spontaneous revertants
revertants were observed and scored as
the background
normal growth, inhibited growth or no growth. Inhibition
Icored by the
Inhibition was scored
presence
of pindot
and the absence of a confluent
bacteria.
confluent lawn of bacteria.
colonies and
pindot colonies
presence of
Dose Selection
.II ,•~
I •
I•
~
.
l:
Test article Mercaptobenzothiazole
bacterial
Mercaptobenzothiazole produced complete inhibition of bacterial
qrov~
at
lo,cno
and 3333
3333 ug/plate
ug/plate and excessive inhibition of bacterial lawn
growth at 10,000 and
as determined
pindot colonies at 1000
1000
of small pindot
presence of a dense lawn ofamall
determned by the presence
and 333 u9/pla~e.
The high dose chosen
cho~En for test article
Article Mercaptobenzothiazole
Hercaptobenzothiazole
ug/plate.
was 300 uq/plate.
ug/plate.
and 100 ug/plate.
10, 30 and
were 3, 10,
ug/plate. Additional doses w~re~,
Plate Incorporation
Incorporation Assay
Assay
mutagenesis
plate incor,poration
TheThe
auxotrophs
used the five histidine auxotrophs
incorporation assay us.d
mutagenesis
of
Salmonella
typhimurium
TA153S,
TA1S37,
TA1538,
TA9S and TAlOO.
TA100. The genetic
TA98
TA1538,
TA1537,
TA1535,
of Salmonella typhimurium
4escription
table.
in the following table.
description of these strains are found in
Strain
Des1qnation
Designation
Gene
Affected
Affected
R
Factor
LPS Pactor
~
Repair
Repair
.\~a.
A uv.\
ILLEGIBLE
uvrB rfa
TEXT
Mutation 'l'ype
Type
Detected
'1'1.1535
TA1535 his
IJ.UGG
T.Al537
TA1537
It.i.6c
.\~a. uvr B rfa
A uVlt B TEXT
hisC ILLEGIBLE
Prameshitt
Frameshift
1:
TAl538
TA1538 his
hi6D
1L~a. uvr B- rfa
D ILLEGIBLE
A uv.\ B TEXT
Prameshift
Frameshift
, t
TA9S
TA98
pKM101
hi6D
AuvIL B
rfa
B TEXT
1L~a. uvrpJCH10l
his D ILLEGIBLE
Frameshift
Frameshift
TAlOO
TA100
hi6G
his G
1'ase-pair
Base-pair
Substitution
Substitution
I
Balle-pair
Base-pair
Substitution
Substitution·
I'.
~
I:
i
~
• I
uv.\ B
1L~a.
A ILLEGIBLE
TEXT
B rfa
uvr
.
pJCMlOl
pKM101
• : 0000·09
••
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--.-
:,~
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J
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Ames
test
Ames Salncnella/Microsome
Salmonella/Microsome Plate test
301-CMA-001-83
PH 301-CMA-001-83
301-CMA-001-83A
PH 301-CMA-001-83A
Top Agar
Agar
Top agar,
reconstituted into a molten state
.tate and---agar, u~ed
used as an overlay,
overlay, was reconstituted
and
.upplemented
0.5 mM
~ histidine
histidine and 0.5
0.5 aM
biotin
at
a
volume
of 0.1
ml per
per
supplemented with 0.5
mM biotin at a volume of
0.1 ml
maintained at
"S·C
W1til
used. Steri:'e
kaputs were
were
ml of agar, anlS
and maintained
at 45
degress
Celsius
until R
used.
Sterile tubes
tubes with
with kaputs
labeled
placed into a Fisher
D%y Bath
Bath at "S"C.
All negative
negative and
labeled and placed
Fisher Iaotemp
Isotamp R Dry
45 degrees
Celsius.
~lates were eSone
positive control tubes and plates
done in
in triplicate. All
All
compound-treatelS
plates were eSone
duplicate. Using sterile
compound-treated tubes and plates
done in
in duplicate.
technique,
each tube in
technique, tho!
the following were addelS
added to each
in the following r:lrder:
order: 2 ml
aliquots
atrain, anlS
aliquots of top agar
agar solution,
solution, 0.1 .1
ml of te.ter
tester strain,
and 0.1 al
ml of the
appropriate concentration
concentration of the test cOlllpound.
vortexed and
compound. The tubes vere
were vortexed
pl.tes. The aample
w•• evenly distributed
distributed on the
poured onto minimal glucose plates.
sample was
plate, and the top agar overlay was allowed to harden.
Metabolic Activation
Activation System
Metabolic
Tubes requiring metabolic activation have, in
in addition to
agar ingredients,
ingredients, .n
an 5-9
S-9 fraction of rat liver homogenate
Aroclor 1254-treatcd
Aroclor
1254-treated Sprague Dawley
Dawley rats. The .ctivation
activation
contained the following per 1Il1:
ml:
0.4 M
H MgC12'
M XCI
MgCl2; 1.65
1.65 M
KC1
1
Phosphate
1 M Glucose
Glucose - 6 - Phosphate
0.1 H
M NADP
L
H
. 1.:
L
l~
U
20
20 ul
ul
5S ul
ul
ul
40 ul
0.2 M
H Phosphate buffer pH 7.4
0.2
t~
the preceding
preceding top
top
obtained from
from
system (S-9 mix)
Sterile
distilled H
0
Sterile distilled
H20
2
S-9 Fraction
Fraction
ul
500 ul
365
ul
365 ul
70 ul
u1
The S-9 fraction
fraction was th.wed
0.5 ml of
thawed on the day of use and kept colIS
cold on ice. 0.5
the S-9 mix was added to the tubes which were then vortexed
vortexed and IlOured
poured onto
onto
minimal glucose plates. The plates
pl.tes were allowelS
for sf!Veral
minimal
allowed to harden for
several
repeated for e.ch
.train. Within an
minutes. Tne
The aame
same procelSure
procedure vas
was repeated
each tester strain.
37·C
incubatc,r.
hour the plates were inverted and pl.ced
placed in
in a dark 37
Celsius
degrees
incubator. The plates
vere incubate<!
checkecS for unifor1ll
background lawn,
lawn, anlS
were
incubated for 48 - 72 houra,
hours, checked
uniform background
and
.cored
scored by counting revert.nt
revertant colonies on an electronic
electronic colony counter
counter
interfaced with a computer.
computer.
interfaced
Data Reporting
Reporting
aa.ay, the positive
positive anc!
In acoring
scoring the assay,
and
If
negative control
control values
valu•• did
eSid not
If the negative
Man
valuea, the remaining plates were
mean values,
A RDIIWlry
in
summary of the eS.ta
data are presented
presented in
this report.
negative controls
contro18 were first evaluated.
ev.luated.
negative
fall within the acceptable
historical
accptable historical
Dot
acorecS aDeS
wa. rpeated.
repeated.
not scored
and the ••••y
assay was
the SWIIIIa%y
cont.inelS in
summary data .heet
sheet contained
in
II
Evaluation Criteria
Evaluation
Criteria
I.~
In most
lDO.t tests
teat. with the SalJllC)nella/Microsome
Asaay, results are either
In
Salmonella/Microsome Assay,
either clearly
poaitive result is
ia defined a.
reprolSucible,
as a reproducible,
positive or clearly negative. A positive
do.e-related
colonies.
This
dose-related increa.e
increase in
in the number
number of hiati4ine-in~epeneSent
histidine-independent
colonies. This
5
)~
000010
•
00009
_. ... .
--
_.-
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"
r
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Ames Salmonella/Microsome
Salmonella/Microsome Plate test
test
PH 301-~~-JOl-8~
301-CMA-301-83
PH
PH 301-CHA-OOl-83A
301-CMA-001-83A
dose-response relationship
modification of
of
relationship occasionally necessitates
necessitates slight modification
the original doses in
in a repeat .ssay.
is within one
assay. If
If the solvent control is
d~viation of the historical mean (See Historical
D&ta) for control
control
standard deviation
Historical Data)
values and the t~st
produces the highest increase
increase equai'
values
test chemical produces
equal to or greater
greater
than three
three times the solvent control value, the test chemical is
is considered
positive. A negative
negati~e result is
reproducible
as the absence of •a reproducible
is defined .s
positive.
increase in
histidine-independent colonies.
increase
in the number of histidin-independent
HISTORICAL DATA
Spontaneous
Revertants
Spontaneous Revertants
Salmonella
Salmonella typhimurium
typhimurium
Ames Tester Strains
Alnes
Strains
Strain:
Strain:
TA1535
TA1535
++
TA1537
"1'11.1537
-
+
TA1538
':1\1538
+
J.
Trials
1~
Mean
Mean
L
Std. Dev.
Dev.
J~
Strain:'
Strain:
I:
II
11..
r
H
H
I:
a
Trials
Mean
Dev.
Std. Dev.
131
126
126
124
122
122
128
12S
128
12
13
14
17
19
30
4
5
6
9
334
3
TA98
TA9S
- +
TA100
TAl00
+
127
125
136
126
28
40
157
139
66
10
40
42
Records
Maintained
Records Maintained
correspondence pertinent
pertinent to the study between
All correspondence
between the sponsor and Phamalton
Pharmakon
Inc.,, protocol,
protocol, amenc!IIDents
protocol, raw data,
Research International,
Research
International, Inc.
amendments to the protocol,
test chemical
chemical "eight
a.surance
eight or volWlle,
volume, dispensation reports,
reports, quaUty
quality assurance
reports
Pharlukon Archives.
Archive••
in the Pharmakon
maintained in
is 1Il&1ntained
reports and the final report is
Good Laboratory Practices Statement
Statement
This study was conducted in
in complianc.
compliance with the Good Laborctory
Laboratory Practice
Practice
Regulations.
Regulations.
6
",
0000
•• 11
00010
r
r
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[
Ames Salmonella/Microsome
Salmonella/Microsome Plate
PI~te test
test
Ames
301-CM.'.-·'001-83
PH 301-CMA-001-83
301-CMA-001-S3A
PH 301-CMA-001-83A
Test Article
Article Purity
Purity
The identity,
identity. purity,
purity, quality.
strength of the test
test article
article is
is the
quality, and strength
responsibility of the sponsor.
Documentation of the test article
article analysis
sponsor. Documentation
responsibility
supplied by
by the
the sponsor
sponsor is
is appended
appended to
to this
this report.
report.
supplied
Test Article
Article Stability
Stability
Test
apparent change in
in the physical
physical state
.tate of the test or control
control
There was no apparent
articles during the assay.
RESULTS
RESULTS AND DSCUSSON
DISCUSSION
[
r
I~
L
r;
II
The results for test article Mercaptobenzothiazole
MercaptobenzothilLzole can be found in
in the data
sumrna.ry
tables. A
A second study was conducted
conducted in strains
.trai~s TA1538
TAl538 and TA98
TAgS with
summary tables.
metabolic activa~ion
preparation .t
100. 250,
250. 300, 450 and
ane
at dose levels of 100,
activation preparation
600 ug/plate.
was conducted
conducted to determine
deteflllL"\e if
if the values obtained in
in
ug/plate. This study was
strains TA1538
TA1538 and TA98 were
were reproducible
reproducible at the original
levels te&ted
tested as
original levels
strains
a~higher
doses.
well as at
higher doses.
CONCLUSION
CONCLUSION
The results for test article Mercaptobenzothiazole
Mercaptobenzothiazole,, Lot
Lot '39-l4B,
were
#39-14B,
negative in
in strains
strains TA1535,
TAl535, TA1537,
TAl537. TAIS38,
TAgS and TA100
TAlOO of Salmonella
Salmonella
TA1538, TA98
typhimurium
with and
without metabolic
metabolic activation
10,
at 3,
3, 10,
preparation at
activation preparation
and without
both with
typhimurium both
30, 100 and 300 ug/plate.
In addition,
addition, Mercaptobenzothiazole
He~captobenzothiazole.
#39-14B,
, Lot .39-l4B,
ug/plate. In
30,
was evaluated
evaluated at higher dose levels of 4Sa
.nd 600 ug/plate v_~th_~et~~ic
with metabolic
450 and
activation and found to be
be' negative in
in strains TAlS38
Salmonella
TA1538 and TA98 of Salmonella
typhimurium. All
positive controls
controls used
uaed in
evaluation of the
in the evaluation
and positive
solvent and
All solvent
typhimurium.
test article
article were within
with~n the acceptable
acceptable range of mean historical data.
d~ta.
BIBLIOGRAPHY:
AllIes, Bruce N.,
N., Joyce McCann,
Yamasaki
Edith Yamasaki
McCann, and Edith
Ames,
Methods for Detecting
Detecting Carcinogens
Mutagens w~th
with
Carcinogens and Mutagens
Methods
the Salmonella/Mammalian-Microsome
Mutagenicity Test.
Salmonella/Mammalian-Microsome Mutagenicity
Mutation Research
347-364.
347-364.
(1975)
Research 31: (1975)
Mutation
1...1
GTI083
7
•
000012
00011
./
,III
t.
I
PHARMAKON
INTERNATIONAL, INC.
INC.
PHARMAKON RESEARCH INTERNATIONAL,
".7'
WAVE"LY.
"ENNSYLYANIA
WAVERLY,
PENNSYLVANIA
ILLEGIBLE TEXT
f
"HONE
17nl 111.2."
AJDe s '1'es
Ames
Testt
Salmonella
typhimurium
Salmonella typhimurium
J
r
r
SUMMARY DATA
SUMMARY
Client: Chemical
Chemical Manufacture~s
Manufacturers
Association
Client:
Associaticn
Date
nate Initiated:
Materials:
Mercaptobenzothiazole
Materials: Hercaptobenzothiazole
Description: pale yellow powder
powder
1983
Completed: February
Date COllpleted:
February 28,
2B, 19B)
Date
Notebook
t3 74 ~ page
page 44
Notebook 174;
Phazmakon
Reference: #375;
'375~ pgs.
pqs. 31-3~,
Pharmakon Reference:
31-32, 37-3
37-3
Solvent:
DMSO
Solvent: DHSO
Study No.:
No.:
Study
February
5, 1983
February 5,
1983
~-
~
PH
301-aA-OOl-83
PH 301-CMA-001-83
CONTROLS
CONTROLS
1.
f
Spontaneous Revertants(x)
I;
1.
Neqative Controls
L
Positive Controls
Sodium Azide
1ug/plete
(-)
1~
2-NitrQfluorene
5ug/plate
(-)
9-Aminoacridine
lS0ug/plate
(-)
5ug/plate
(+ )
II
S-9
2-Anthramine
'1'A1535
TAl537
TA1538
TA98
T~.lOO
(_)1
12
14
20
28
135
(+ )2
13
20
26
42
137
836
8e9
1379
1112
589
2356
1479
188
207
1950
'!'EST COMPOUND
11
Revertant Colonies/Plate (x)
r
S-9
"Al~3S
'rAIS]7
,.111538
TA9B
'1'1.100
3
-/+
16/13
21/26
17/31
43/51
140/124
10
-/+
18/10
15/22
18/29
39/51
143/11~
30
-/+
20/6
13/15
24/29
38/49
146/117
Ii
100
-/+
13/8
15/16
15/34
34/52
179/128
g
300
-/+
16/16
13/48
23/57
170/107
l;
I~
.\
Dose Levels (ug/p1ate)
9/13
1<_) Without S-9 Aroclor-induced rat liver .etabolic activation.
.
~:: W';::. I.;1\-1
•
;......._....:....-=----::.......;...........:....;:.......;=---
•
• s ••4y· Director
B
•CL..J..<l.A."tL
PHARMAKON RESEARCH
RESEARCH INTERNATIONAL,
INTERNATIONAL. INC.
INC.
PHARMAKON
WAVE'lL".
"E~NSYLYANIA' ' ' 7 '
"HONE
17171 51&-2."
Ame:!s Test
Test
Ames
Salmcnella ,mhimurium
Salmonella
typhimurium
SUMMARY DATA
SUMMARY
Client: Chemical Manufacturers
Association
Manufacturers Association
Date Initiated:
Initiated:
Date
Materials': Mercaptobenzothiazole
Materials:
Mercaptobenzothiazole
Description:
Description: pale yellow powder
Date Completed:
February
Date
Completed:
February
1374,
Notebook #374,
Notebook
Reference:'375,
Pharmakon Reference:#375,
Pharmakon
Solvent: DMSO
DMSO
Study No.:
February 5,
1983
February
, 1983
28, 1983
1983
28,
page 44
44
page
pgs. 31-32,
31-32, 37-31
pgs.
PH 301-CMA-001-83
301-0!A-001-83
CON'I'IDLS
CONTROLS
Spontaneous Revertants (x)
5-9
TA1538
TA9S
Negative Controls
Posit~ve Controls
Sodium Azide
lug/plate
(-)
2-Nitrofluorene
Sug/plate
(-;
9-Aminoacridine
150ug/plate
(-)
Sug/plate
(+ )
2-Anti.ramine
TEST
21
36
10B7
2150
COMPOUND
Revertant Colonies/Plate
Dose Levels (ug/plate)
;<-)
TA153B
TA9S
100
+
27
37
250
+
23
26
300
+
12
2S
450
+
15
23
600
+
S
12
Without 5-9 Aroclor-induced rat
(+) With 5-9
Date
S-9
•
~ I, ,'r~
•
liv~r met~lic
•
• stUdy'
(x)
activation.
D~.ctO' dLJA .,J...tL,
... '" '..
• a·
9
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00013
11.
'::....
J
PHARMAKON RESEARCH
RESEARCH INTERNATIONAL,
INTERNATIONAL, INC.
INC.
PHARMAKON
WAVERLY. PENNSYLVANIA , ••"
r
PHO"'E
17"1586-2."
QUALITY ASSURANCE
ASSUiiANCE: UNIT
UNIT STATEMENT
STAT£W·-:;O
QUALITY
1
I
I
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This study was performd
perforued in
in accordance
~ccord~nce with
with the
the Good
Goo!! Laboratory
Laborato%)'
Practices Regulation
~egu1ation for non-clinical
non-clinical laboratory
1aborat~ry studies
s~udies as developed
deve1op~l
by the
U. S.
S. Food and Drug Administration,
Administration, as
£5
the U.
by
~ndicated in
in
indicated
the Federal
Federal
the
~e9ister,
Part II
II of
of December
December 22,
22, 1978;
1978; Part
Part 58,
58, Title.21.
~itle.21.
Register, Part
Study
No.
Study No.
PH 301-CMA-001-83
301-CMA-OOl-83
PH 301-CMA-001-83A
JOl-CMA-OOl-B;A
PH
l'
The following inspections
inspections were
were performed:
performed:
J.
Interval
Date
1.
Plating Phase (PH 301-CMA-001-83)
30l-CMA-OOl-B3)
Plating
2/18/83
Scoring
301-CMA-OOl-83)
Scoring Phase (PH 301-CMA-001-83)
2/21/83
L
i~
Reporting Phase
3/31/83
3/31/83
Results of the above inspections
Study
inspections were submitted to the Study
Director and
study.
and Management during the course of
of the study.
~"L;" ""1/oA/"l~
3/"/83
Date
Date
I
l
~
','
Qua
lity Assur~ce
Un,;,t
Assurance Unit
Quality
i
H
10
~
:-
.:. ..•. 00001 5~00014
"
r
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Amendment
Protocol
Protocol Amendment
Ames Salmonella/Microsome
Plate Test
Test
Ames
Salmonella/Microsome Plate
PH 301-CMA-OOl-83
301-CMA-001-83
PH
PH 301-CMA-001-83A
30l-CMA-001-83A
Pag~ 8
8 of
of protocol
protocol
Page
Additional Tester
Additional
Tester
Strain:
Strain:
Recently, Sal!llOnella
typhimurium tester
tester strain
strain TA97
TA97
Recently,
Salmonella
typhimurium
has been
been developed
developed by
by the
the laboratories
has
laboratories of
of Dr.
Dr. Eiruce
bruce
Until
Ames.
Until
such time
time as Pharmakon
Phamakon Research
Research
International, Inc.
historical data
data bank
International,
Inc. develops
develops a
a historical
bank on
on
tester strain
strain TA97,
TA9', the strain
strain will
be included as
as aa
will
tester
courtesy
to our
our spc:;nsors.
courtesy to
sponsors.
Reason
Amendment:
Reason for
for Amendment:
Was intended
as a courtesy to the
the sponsor
but due
due to
Was
intended as
sponsor but
to
erratic
response
of
tester strain,
it
was not
not
erratic
response
of
tester
strain,
it
was
conducted.
conducted.
I.
L
J
~
~M.JLL
Edmund G.
G. Godek,
B.S.
Godek, B.S.
Study Director
Director
Study
Pharmakon Research
Research International,
International, Inc.
Pharmakon
Inc.
l
December 1,
1, 1983
'
.'
1i
1~
,\
GTIOS3
1.1
000016
00015
J
,
l
J
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APPENDIX.
APPENDIX .
J.
J.
L
1~
II
--.:,"-<
000017
00016
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r
r
24:~[,1
12·75)
THE GOODYEAR TIRE & RUBBER COMPANY
CHEMICAL DIVISION
DIVISION
AKRON, OHIO
OHIO 44316
CERTIFICATE OF ANALYSIS
Product Cod.
Code
P,oduct
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Custom.,
Customer
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Date
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CERTIFICATE
AUTHENTICITY
CERTIFICATE OF AUTHENTICITY
THIS IS
the microimages
appearing on
on this
this microllche
are accurate
accUJ'atB
ISTO CERTIFY
CERTIFY that the
microimages appearing
microfiche are
and
Protection Agency
Agency
and complete
complete reproductions
reproductions of
of the
the rocords
records of
of U.S.
U.S. Environmental
Environmental Protection
courlie of
for microfilming.
microfilming.
documents
documents as delivered
delivered In
in the regular
regular course
of bUliiness
busines for
_--",5,-!_ _~J---,·3=--_----,",-cf_Z_
Data
Data produced
produced.....
(Month)
Place
Place
·
I
SyracuSft
Syracuse
(Day)
New York
(Year)
New York
----------(Cily)
(Slate)
yf~~
Camera Operator
Operator
Camera
a
AMTEK
corp
,
,