Document x1Z7akaJp0qpreGOddMX8kKRb
AR226-2985
^a^BS^. CCR Cytotest Cell Research GmbH & Co. KG
CCR PROJECT 326417
SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY
WITH
Study Completion Date:
February 17,1993
REPORT
^^.------nTSCACB'
CCR In den Leppsteinswiesen 19 D-6101 Ropdorf F.R.G. Telephone: 0 61 54 - 80 7-0 Telefax O 61 54 - 8 33 99
COPY OF GLP CERTIFICATE
HESSISCHES MINISTERIUM FOR UMWELT, ENERGIE
UNO BUNDESANGELEGENHE1TEN
GLP-Bescheinigung
Bescheinigung
Hiermit wird besiStigt. daQ dig Pru(ungsainrichtun9(en)
Cytotest Call ReseaY-ch GmbH & Co KG
In den Leppsteinswiesen 19
i- 6101
RoBdorf
(On. Ajiidwn)
dar.RCCHolding Verwaltung 1-inbH____
(Rron)
am 3-08-' P4-08.-?..05--08-""!:.06:08-92
{Dalinn)
von der fur die Qberwachung zustandigen Behfirda ubef dia Snhaltung der GfundsAtzB dar Gutan Laborpraxn
.
inspizsen wordan ist (sind).
Es wird hiennit bestiligt. daS tolgenda Prufungen in dieser Priifeinrichtung nach den GrundsitZBn der Gutan Laborpraxis durchgefOhn warden.
Toxikologische Eigenschaftga-^ .
^SiST'E^
Certincsto
It is haraby cerUfied that the test laciitypes)
Cytotest Cell Research enbH & Co KG In den Leppstiinswiessn ~\'t.
610'1 Rofldorf
.
(toalicn. tOinoa)
RCC HoldingVerwaltung GinbH
(eanipan^ nxnMi)
03.08., 04.08., 05.08. and 06.08.92
on---------------------------------------------
(Ott.)
was (ware) inspected by lha csnipatant authority regar ding coniplianca with lha Principles <rf Good I-ahoralcry Practics. It is hereby cai-tilied (hat studies in this test (adBty ara conducted in complianca with (he Principles of Good Laboratory Pracliea.
Toxicological.properties
Im Auffrag
.
\y^. jCifJi^r
(Or..Hecicer)
Wiesbaden, den ^S.
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Company S.n.*."--'"""
CONTENTS
COPY OF GLP CERTIFICATE
^PREFACE
General
Project Staff
Schedule
Project Staff Signatures Quality Assurance
Guidelines Archiving
STATEMENT OF COMPLIANCE
QUALITY ASSURANCE UNIT Statement
SUMMARY
Conclusion
OBJECTIVE
Aims of the Study
Reasons for the Study
MATERIALS AND METHODS
The Test Article
The Controls The Test System Mammalian Microsomal Fraction
S9 Mix
Pre-Experiment for Toxicity
Dose Selection Experimental Performance Data Recording Evaluation of Results
BIOMETRY
RESULTS CONCLUSIONS
II Pre-Experiment for Toxicity
Tables of Results Experiment I
Tables of Results Experiment Summary of Results
REFERENCES
DISTRIBUTION OF THE REPORT
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PREFACE
GENERAL
Sponsor:
Monitoring;
Testing Facility:
CCR Project No.:
Test Article: Title;
I S E GA
Forschungs- u. UntersuchungsGesellschaft mbH
Zeppelinstr. 3-5
D-8750 Aschaffenburg Heike KrMmer
CCR CYTOTEST CELL RESEARCH GMBH & CO. KG
D-6101 Rofldorf, F.R.G.
326417
Salmonella typhimurium Reverse MutatxomAs s ay with!
PROJECT STAFF Management:
Study Directors Quality Assurance Unit:
Dr. H.- E. Knoell
Dr. Albrecht Poth Dr. Ch. Helmrich
SCHEDULB
Date of Protocol;
'' November 10, 1992
Date of 1st Amendment to Protocol:
January 18, 1993
Start of Pre-Experiment: November 27, 1992
End of Pre-Experiment! December 04, 1992
Start of Experiment I: End of Experiment I;
Start of Experiment II;
End of Experiment .II:
Date of Draft:
December 15, 1992 December 18, 1992
January 05, January 15,
1993 1993
January 18, 1993
Date of Report:
February 17, 1993
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4 of 35 ,tc^nTSCAC^
Cowpa"^ SanW^d-Pa0^
PROJECT STAFF SIGM&TDRES
Study Director:
Dr. Albrecht Poth
Management:
Date: February 17, 1993
Dr. H.- E. Knoell
Date: February 17, 1993
OnaLITY ASSURANCE
l/
The study was performed in compliance with: Chemikaliengesetz ("Chemicals Act") of the Federal Republic of
Germany, Aniage 1 ("Annex I"), dated March 14, 1990 (BGBL. I S.
521).
"OECD Principles of Good Laboratory Practice", Paris, 1981
GUIDELIMES
This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: First Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Salmonella typhimurium. Reverse Mutation
Assay", adopted May 26, 1983 and
EEC Directive 79/831, Annex V, B 14.
/
ARCHIVTHG
C C R, D-6101 RoBdorf/F.R.G. will archive the following data for
30 years;
Raw data, protocol, and copy of report.
The following sample will be archived for at least 12 years: Sample of test article. No raw data or material relating to the study will be discarded without the sponsor's prior consent.
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Company Sanitized. Doss not contain TSCA CBI
STATEMENT OF COMPLIANCE
Project Number;
326417
Test Article ;
Study Director;
Title
:
Dr. Albrecht Poth
Salmonella typhimurium Reverse Mutation Assay with
To the best-of my knowledge and belief, this study performed in the testing facility of CCR was conducted in compliance with Good
Laboratory Practice Regulations:
Chemikaliengesetz ("Chemicals Act") of the Federal Republic
of Germany, Aniage 1 ("Annex I"), dated March 14, 1990
"OECD Principles of Good Laboratory Practice, Paris, 1981
There were no circumstances that may have affected the quality or integrity of the study.
Study Director
CCR
Dr. Albrecht Poth
cy...!'^
Date: '~&^WLM ^9 ./?<?3
6
(
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Compaq Sanded. Does not contain TSCACBl
QUALITY ASSURANCE UNIT
C C R, Cytotest Cell Research GmbH & Co. KG,
.:
In den Leppsteinswiesen 19, D-6101 RoBdorf, F.R.G.
S'SS3"i^rKST
Project Numbers
Test Article :
Study Director:
Title
-
:
326417
Dr. Albrecht Poth Salmonella typhimurium Reverse Mutation^s say with
This report was audited by the Quality Assurance Unit and the
study and/or testing facility were inspected on the following
dates.
Dates of QAU Inspections/ Audits
Dates of Reports to the Study Director and to Management
November 11, 1992
December 15, 1992
/
January 21, 1993
November 11, December 15, January 21,
1992 1992 1993
Head of Quality Assurance Unit
Dr. Ch. Helmrich
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page 7 of 35
Company Ssanaintizwe"d-. Does not contain TSCACBI
SUMMARY
,s study was performed to |to induce gene mutations
iancvceosrtdiignagt etot hteh ep optleant et i ailncoof r|p_o_ra_ti_o_n"
,est (experiment I) and the pre-incubation test (experiment II)
using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98,
TA 100, and TA 102.
The assay was performed in two independent experiments both with
and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test arti
cle was tested at the following concentrations:
33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 ug/plate
No relevant" toxic effects occurred in the test groups with and
without metabolic activation in experiment I and II in all
strains used.
The plates incubated with the test article showed normal back
ground growth up to 5000.0 ng/p'late with and without S3 mix in
all strains used.
Mo substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with jHB----H|H.at any dose level, either in the presence or absence of metabolic activation (S9 mix) . There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.
/
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
CONCLUSION
In conclusion, it can be stated that during the described mutage-
nicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes
or frameshifts in the genome of the strains used.
Therefore, |----IB^^H^ is considered to be non-mutagenic in this
Salmonella typhimurTum reverse mutation assay.
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C.m^yS.nl.."-."08'"''"0"'-"'50"6"
OBJECTIVE
AIMS OF THE STUDY
II The experiments were performed to assess the potential of the
test article to induce gene mutations by means of two independent
Salmonella typhimurium reverse mutation assays. Experiment I was
performed as a plate incorporation assay. As a negative result
was obtained in this experiment, experiment
was "performed as
a pre-incubation assay.
REASONS FOR THE STUDY
The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to
perform, and give reliable data on the ability of an agent to
interact with DNA and produce mutations.
However, the Hacteria most commonly used in these assays do not possess the enzyme systems which, in mammals, are known to con vert promutagens into active DMA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
In spite of great differences between bacterial and eucaryofcic
cells with respect to structure and function there is an associa
tion between mutagenicity in bacteria and carcinogenicity in
mammals described in literature (7,8).
Reverse mutation assays determine the frequency at which an agent
abolishes or suppresses the effect of the forward mutation. The
genetic target presented to an agent is therefore small, specific
and selective. Several bacterial strains, or a single strain with
multiple markers are necessary to overcome the effects of mutagen
specificity. The reversions of bacteria from growth-dependence on
a particular amino acid to growth in the absence of that amino
acid (reversion from auxothrophy to prototrophy) is the most
widely used marker.
''
The Salmonella typhimurium histidine (his) reversion system
measures his" --> his"*" reversions. The S. typhimurium strains
are constructed to differentiate between base pair (TA 1535, TA
100, TA 102) and frameshift (TA 1537, TA 98) mutations.
According to the direct plate incorporation and the pre-
incubation method the bacteria were exposed to the test article
with and without metabolic activation and plated on selective medium. After a suitable period of 'incubation, revertant colonies
were counted.
To establish a dose response effect five dose levels with ade
quately spaced intervals were tested. The maximum dose level was
5000.0 ag/plate, unless limited by toxicity or solubility of the test article. To validate the test, reference mutagens were tested in parallel to the test article.
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Company Sani-,t-esdrf. nDoes not contain TSCACB!
MATERIALS AND METHODS
THE TEST ARTICLE
The test article and the information concerning the. test article
were provided by the sponsor.
Name;
Batch No. ;
Aggregate State at RT:
Colour: Analysis: Purity:
Stability;
liquid
Storage;
4C
Expiration Date; not indicated by the sponsor
On the day of the experiment, the test article ZONYL RP 18 was dissolved in Ethanol. The solvent was chosen because of its solubility properties and its relative nontoxicity for the bacte ria.
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notconialnTSCACBl Co^paw Sanded. Does
rest Kepor-c CCK Jb'rojec-c j^oij./
THE COMTROLS
The Negative Con-trols Concurrent untreated and solvent controls were performed.
The Positive Control Substances
Without: metabolic activation
Strains:
Name:
Supplier: Catalogue No.; Purity: Dissolved ins
Concentration;.
TA 1535, TA 100
sodium azide, NaNg SERVA, D-6900 Heidelberg,
30175
at least 99 % aqua dest. 10 ng/plate
F.R.G.
Strains:
Name:
Supplier: Catalogue No.: Purity:
Dissolved in:
Concentration:
TA 1537, TA 98 4-nitro-o-phenylene-diamine, 4-NOPD SIGMA, D-8024 Deisenhofen, F.R.G.
N 9504
> 99.9 %
DMSO
50 ug/plate
Strain:
Name:
Supplier: Catalogue No.: Purity: Dissolved in: Concentration;
TA 102
methyl methane sulfonate, MMS
MERCK-SCHUCHARDT, D-8011 Hohenbrunn, 820775
> 99.0 %
aqua dest.
1.0, nl/plate
F.R.G.
With metabolic activation
Strains:
Name:
Supplier: Catalogue No.: Purity: Dissolved in: Concentration:
TA 1535, TA 1537, TA 98, TA 100, TA 102 2-aminoanthracene, 2-AA SIGMA, D-8024 Deisenhofen, F.R.G.
A 1381 97.5 %
.DMSO
2.5 ng/plate
The stability of the positive control substances in solution was
unknown but a mutagenic response in the expected range is sufficient evidence of biological stability.
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SanIB"'1- -- Company
Test Report- CCR Project 326417
THE TEST SYSTEM
Characterisation of the Salmonella typhi-nmrium Strains
The strains are derived from S. typhimurium strain LT2 and 'due to a mutation in the histidine locus are histidine depen
dent. Additionally due to the "deep rough" (rfa-minus) mutation
they possess a faulty lipopolysaccharide envelope which en ables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an exci sion repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". .In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101
carries the_ampicillin resistance marker. The strain TA 102 does not contain the uvrB'-mutation. Additionally TA 102 contains the multicopy plasmid pAQI, which carries the hisG428 mutation and a
tetracyclin resistance gene. TA 102 contains the ochre mutation in hisG gene.
In summary, the mutations of the study can be described as follows;
TA strains used in this
Salmonella typhimurium
TA 1537; his C 3076; rfa"; uvrB";
; frame shift mutations
TA 98: his D 3052; rfa"; uvrB"; R-factor:
"
"
TA 15 35": his G 46; rfa"; uvrB'";
: base-pair substitutions
TA 100: his G 46; ra~; uvrB"; R-factor:
TA 102: his G 428; rfa"; uvrB'*'? R-factor;
"
"
Regular checking of the properties of the strains with regard to membrane permeability, ampicillin- and tetracyclin-resistance as well as normal spontaneous mutation rates is performed in the
it laboratory of C C R according to Aroes et al. (1). In this way
was ensured that the experimental conditions set down by Ames
were fulfilled.
The bacterial strains were obtained from Dr. Heinz Trager, Knoll
AG, D-6700 Ludwigshafen, F.R.G.
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TSCACB1
Compare San..,te,,ed. .cDooeess not contain
Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
Precultures
From the thawed ampoules of the strains 0.5 ml bacterial suspen sion was transferred to 250 ml Erienmeyer flasks containing 20 ml
nutrient medium. This nutrient medium contains per litre:
8 g Merck Nutrient Broth 5 g Mad
The bacterial "culture was incubated in a shaking water bath for 10 hours at 37 C.
Selective Aq-ar
2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective
agar. Each petri dish was filled with 20 ml of this nutrient
medium. Sterilisations were performed at 121 C in an autoclave.
Overlay Acrar
/
The overlay agar contains per 6.0 g Merck Agar Agar 6.0 g NaCI
10.5 mg L-histidine x HC1 x 12.2 mg biotin
litres
H^O
Sterilisations were performed at 121 C in an autoclave.
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cS,apn,ifteizeed". D-oes not contain TSCACBl
Company
i'est Report. CCK Project ^2b41/
MAMMaLIAH MICROSOMAL FRACTION S9 MIX
S9 fPreparation by C C R)
The S9 liver microsomal fraction was obtained from the liver of
8-12 weeks old male Wistar rats, strain WU (SAVO-Ivanovas, med.
Versuchstierzuchten GmbH, D-7964 Kisslegg, F.R.G.; weight approx.
150 - 200 g) which received a single i.p. injection of 500 mg/kg
b.w. Aroclor- 1254 (Antechnika, D-7500 Karlsruhe, F.R.G.) in olive
oil 5 days previously.
After cervical dislocation the livers of the animals were re moved, washed in 150 mM KC1 and homogenised. The homogenate, diluted 1+3 in KC1 was centrifuged cold at 9,000 g for 10
minutes. A stock of the supernatant containing the microsomes was
frozen in ampoules of 2, 3 or 5 ml and stored at -70 C. Small numbers of the ampoules are kept at -20 C for only several weeks before use. The standardisation of the protein content was made
using the analysis kit of Bio-Rad Laboratories, D-8000 Miinchen:
Bio-Rad protein assay. Catalogue 500 000 6 (6).
The protein concentration in the S9 preparation was 31.6 mg/ml (lot 060792).
S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cof actor solution. The amount of S9 supernatant was 15% v/v. The composition of the cof actor solution was concentrated to yield the following concentrations in the S9
mix:
8 mM MgCl2 33 HIM KC1
5 mM glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The
S9 mix preparation was performed according'to Ames et al.(2).
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TSCACBI
S^ed..0n"oee.. "not contain
ConiP^
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PRE-EXPERIMEHT FOR TOXICITY
To evaluate the toxicity of the test article a prestudy was
performed with strains TA 98 and TA 100. 8 concentrations were
tested for toxicity and mutation induction with each 3 plates.
The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test article may be evidenced by a reduction in
the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.
DOSE SELECTION
According to the results of the pre-experiment the concentrations applied in the main experiments were chosen.
The maximum concentration was 5000.0 ug/plate. The concentration range included two logarithmic decades. In this study six ade quately spaced concentrations were tested. Two independent exper iments were performed.
As the results of the pre-experiment are in accordance with the criteria described above, these data are reported as a part of
the main experiment I,
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Company
rest Kepori: CCK -freject j^ti4J./
EXPERIMEHTM. PERFORMANCE
For each strain and dose level, including the controls, a minimum
of three plates were used.
: The following materials were mixed in a test tube and poured onto the selective agar plates;
100 nl 500 nl
100 ^1
2000 nl
Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
Bacteria suspension (cf. test system, pre-culture of the strains),
Overlay agar
In the pre-inc-ubation assay 100 nl test solution, 500 p.1 S9 mix / S9 mix substitution buffer and 100 ul bacteria suspension were mixed in a test tube and incubated at 37 C for '60 minutes. After pre-incubation 2.0 ml overlay agar was added to each tube. The mixture was poured on minimal agar plates.
After solidifi cation the plates were incubated upside down for at
least 48 hours at 37 (2 in the dark.
DATA RECORDING
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GMbH, D-6367 Karben; F.R.G.). The counter was
connected to an IBM AT compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard deviations and enhancement
factors as compared to the spontaneous reversion rates (see
tables of results). If precipitation of the test article preclud
ed automatic counting the revertant colonies were counted by
hand.
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Sanitized. Dc."s not co-rtain TSCA CBi Company
Test Report: OCR Project j2b41/
EVALUATION OF RESULTS
The generally accepted conditions for the evaluation of the
results are: corresponding background growth on both negative control and
- test plates - normal range of spontaneous reversion rates.
Range of spontaneous reversion frequencies (5/9)*
1535
1537
98
100
102 (+)
3-37 4-31 15-60 75 - 200
* These values refer co the negative control without metabolic activation (+) The range of strain TA 102 Has dacaroined from our hl.3Corl.cal control iatas
120 - 300
Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical proce
dure can be recommended for analysis of data from the bacterial assays at this time (5).
A test article is considered as positive if either a dose related
and reproducible increase in the number of revertants or a sig nificant and reproducible increase for at least one test concen tration is induced.
A test article producing neither a dose related and reproducible
increase in the number of revertants nor a significant and repro ducible positive response at any one of the test points is con sidered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered as mutagenic if in strain TA 100 and
TA 102 the number of reversions is at least twice as high and in
it strains TA 1535, TA 1537, and TA 98
is at least three times
higher as compared tp the spontaneous reversion rate (4).
Also, a dose-dependent and reproducible increase in the number of
revertants is regarded as an indication of possibly existing
mutagenic potential of the test article regardless whether the
highest dose induced the above described enhancement factors or
not.
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vSa.il^.Doeanotco^nTSCACBl
Company Sann.^"
Test; Hepqrr. CCK. Project; .i2b41/
BIOMETRY
:No appropriate statistical method is available (5)
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Company Sanded. Does not contain TSCACB1
rest. Kepori; CI-A jrrujyui. .a^otj-/ RESULTS
PRE-EXPERIMBNT FOR TOXICITY
.To evaluate the toxicity of the test article a pre-study was
performed with strains TA 98 and TA 100.
The results are given in the following table;
test groups
concentration
per plate
4g
Negative control Solvent control
4-NOPD
Sodium azide 2 -aminoanthracene
50.0 10.0
2.5 3.3 10.0 33.3 100.0 333.3 1000.0 2500.0
5000.0
reve rtants
TA 98
-
+
37
42
30
40
2542
/
/
/
/ 575
22
33
34
23
25
35
34
43
33
39
25
24
28
32
30
25
per plate
TA 100
+*
76 104
82 106
/
/
925
/
/ 1119
73 108
72
98
76
84
72
85
64
88
50
84
50
76
57
64
* - - tri-Efaouc S9 mx; + -
/ not: performed
tri-th S9 mix
The plates with the test article showed normal background growth
up to 5000.0 ng/plate in strain TA 98 and TA 100, respectively.
According to the dose selection criteria, the test article was
tested at the following concentrations:
33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 ng/plate
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TSCACB1
^mpanySanifeed.Do3snot contain
Test Report CCR Project 326417 TABLES OF RESULTS EXPERIMENT I
PLATE INCORPORATION TEST
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co.p.nys.^.o...----.""^06'
Test article:
39 mix from : rat liver (Batch R 060792) Test strain : TA 1535
Dose
ug/plate
Plati3
1
2
3
without S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
-
Positive control Sodium azide (lOng/plate)
14
15
13
20
15
15
19
22
23
18
16
14
11
13
17
17
14
13
17
7
15
12
15
19
1142 1178 1135
with S9 nn'y
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
Positive control
2-Aminoanthracene
(2.5Hg/plate)
enhancement factor
14
15
19
21
22
27
26
21
21
27
26
22
22
26
20
19
21
19
21
30
28
24
21
21
135
153
190
E revertants/concentr. test article revertants/solvent control
Revertants/'pla.te '
mean s.d. fa.ctor+
14
1.0
17
2.9
1 .0
21
2.1
1 .3
16
2.0
1 .0
14
3.1
0 .8
15
2.1
0 .9
13
5.3
0 .8
15
3.5
0 .9
1152 23.1 69 .1
16
2.6
23 3.2 1.0
23
2.9
1.0
25
2.6
1. 1
23
3.1
1. 0
20
1.2
0. 8
26
4.7
1. 1
22
1.7
0. 9
159 28.0
6. 8
str5
21 of 35
Company Sanded. Does not contain TSCA CBl
Test article:
S9 mix from : rat liver (Batch R 060792) Test strain : TA 1537
Dose
(ig/plate
Plate
1
2
3
without S9 wly
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
-
8
10
5
4
8
7
7
8
7
6
8
11
4
6
6
5
4
6
8
7
5
9
4
5
Positive control
4-Mitro-o-
307
311
350
phenylene-diamine
(50)ig/plate)
with S9 imx
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
12
15
11
14
11
12
13
19
8
10
13
13
8
12
12
15
11
11
6
14
6
8
11
10
Positive control
2-Aminoanthracene
115
110
93
(2.5ng/plate)
+ enhancement factor
revertants/concentr. test article
revertants/sol vent control
Revi5-rtants/'plate mean s.d. factor+
8
2.5
6
2.1
1.0
7
0.6
1.2
8
2.5
1.3
5
1.2
0.8
5
1.0
0.8
7
1.5
1.1
6
2.6
0.9
323 23.8 50.9
13
2.1
12 1.5 1.0
13 5.5 1.1
12
1.7
1.0
11
2.3
0.9
12 2.3 1.0
9
4.6
0.7
10 1.5 0.8
106 11.5
8.6
sir 5
22 of 35
Company Sanitized. Does not contain TSCA CB8
Test article:||U
a
S3 mix from : rat liver ( Batch R 060792) Test strain : TA c?8
Dose
ug/plate
Plate
1
2
3
without S9 nn.TC
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
-
Positive control 4-nitro-ophenylene-diamine (50 (il/plate)
42
35
35
27
33
31
28
24
24
31
35
36
24
37
37
32
26
17
19
26
39
28
28
33
2464 2501 2660
with S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
Positive control
2-Aminoanthracene
(2.5ng/plate)
38
/
47 29 43 42 25 30 29
J & U
44 34 30 40 41 26 34 23
454 ~f ij '.&
43 40 45 45 33 21 31 24
7r 5i 1j.
revertants/concentr. test article
+
enhancement factor
"
----------------------------------------
t revertants/solvent control
Revertantsi/plate mean s.d. factor+
37
4.0
30
3.1
1.0
. 25
2,3
0.8
34
2.6
1.1
33
7.5
1.1
25
7.5
0.8
28 10.1 0.9
30
2.9
1.0
2542 104.1 .83.8
42 3.2
40
6.5
1.0
35 9.0 0.9
43 2.5 1.1
39 4.9 1.0
24
2.6
0.6
32 2.1 0.8
25
3.2
0.6
w57**5^ 156 >K j w . 0u
1.& 4A. - -!^
str5
23 of 35
Company Sanitized. Does nol contain TSCACBfe
Test article:
S9 mix from ; rat liver (Batch R 060792)
Test strain : TA 100
Dose
pg/plate
Plati3
1
2
3
without S9 im'x
Negative Solvent
33.3
locr.o-
333.3 1000.0 2500.0 5000.0
control control
-
Positive control Sodium azide (lOng/plate)
78
82
68
80
76
89
63
86
80
63
79
75
55
66
70
50
50
51
38
53
58
45
63
64
946
942
886
with S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
103
111
99
8,9
114
116
90
82
81
94
89
73
96
71
98
93
102
58
77
80
71
56
67
68
Positive control
2-Aminoanthracene 1356 1083
919
(2.5ng/plate)
enhancement factor
Z revertants/concentr. test article revertants/solvent control
Re-yertcints/plate meaja si.d. factor+
76
7 .2
82
6 .7
1.0
76 11 .9
0.9
72
8 .3
0.9
64
7.8
0.8
50
0 .6
0.6
50 10 .4
0.6
57 10 .7
0.7
925 33 .5 11.3
104
6.1
106 15.0 1.0
84
4. 9
0.8
85 11. 0
0.8
88 15. 0
0.8
84 23. 2
0.8
76
4. 6
0.7
64
6. 7
0.6
1119 220. 8 10.5 .
str5
24 of 35
Company Sanitized. Does not contain TSCA CBI
Test article:
S9 mix from : rat liver (Batch R 060792) Test strain : TA 102
Dose
^.g/plate
Plate
1
2
3
without S9 im^c
negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
-
Positive control
methyl methan sulfonat
(l.Onl/plate)
200
207
208
206
225
211
181
178
154
179
244
199
109
179
188
171
151
190
237
228
212
199
201
209
827 1107 1010
with S9 mix
Negative control Solvent control
280
296
243
253
318
357
33.3
252
312
343
100.0
318
262
324
333.3
364
291
292
1000.0
204
289
251
2500.0
284
272
289
5000.0
. 214
284
295
Positive control
2-Aniinoanthracene
752
861
864
(2.5|ig/plate)
enhancement factor
X revertants/concentr. test article t revertants/sol vent control
Revisrtants/'plate mean 's.d. factor+
205
4.4
214
9.8
1.0
171 14.8 0.8
207 33.3
1.0
159 43.2
0.7
171 19.5
0.8
226 12.7
1.1
203
5.3
0.9
981 142.2 4.6
273 27.2
309 52.5
1.0
302 46.3
1.0
301 34.2 1.0
316 41.9 . 1.0
248 42.6 0.8
282 8.7 0.9
264 43.9
0.9
826 63.8 2.7
str5
25 of 35
Compaq S^ti^Does^c^nTSCACBI
TABLES OF RESULTS EXPERIMENT II
PRE-INCUBATION TEST
page
26 of 35
TSCACB1
Company S1ze-.D... ".>.-'"
Test article:
S9 mix from : rat liver (Batch R 060792) Test strain : TA 1535
Dose
jig/plate
Plate
1
2
3
without S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
" -
Positive control Sodium azide (lOng/plate)
19
20
22
21
18
21
23
18
17
15
13
24
17
14
23
10
12
12
13
9
15
16
16
17
1044 1096 1091
with S9 mix
Negative Solvent
33.3 100.0
33,3.3
1000.0 2500.0 5000.0
control control
Positive control
2-Aminoanthracene
(2.5Hg/plate)
24
17
13
3-1
24
27
24
26
18
21
24
25
31
26
31
28
28
29
28
27
30
22
20
17
163
187
167
enhancement factor
2 revertants/concentr. test article revertants/solvent control
Reveirtants/plate mean 's.d. fact<
20
1.5
20
1.7
1.0
19
3.2
1.0
17
5.9
0.9
18
4.6
0.9
11
1.2
0.6
12
3.1
0.6
16
0.6
0.8
1077 28.7 53.9
18
5.6
27
3.5
1.0
23
4.2
0.8
23
2.1
0.9
29
2.9
1.1
28
0.6
1.0
28
1.5
1.0
20
2.5
0.7
172 12.9
6.3
str5
27 of 35 ^,ze-.C.no<c.n..,nTSCACBi
CoRipa^y
Test
M M . article:|H|
^
S3 mix from : rat: liver (IBatch R (360792)
Test strain ; TA 1537
Dose
ug/piate
Plate
1
2
3
without S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
4
4
7-,
8
7
9
9
6
11
8
9
12
6
11
9
11
9
6
13
7
9
11
8
8
Positive control
4-Nitro-o-
212
245
176
phenylene-diamine
(50ng/plate)
with S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
7
13
10
13
14
13
1-2
15
13
14
13
9
11
18
13
12
12
7
10
8
10
11
11
9
Positive control .
2-Aminoanthracene
113
94
91
(2.5ng/plate)
+
enhancement factor
I revertairts/concentr. test article 1 revertants/solvent control
Revertants /plate mean s .d. factor+
5
1.7
8
1.0
1.0
9
2.5
1.1
10
2.1
1.2
9
2.5
1.1
9
2.5
1.1
10
3.1
1.2
9
1.7
1.1
211 34.5 26-. 4
10
3.0
13
0.6
1.0
13
1.5
1.0
12
2.6
0.9
14
3.6
1.1
10
2.9
0.8
9
1.2
0.7
10
1.2
0.8
99 11.9
7.5
str5
28 of C3o5mpany Sanitized. Ooes not contain TSCA CBB
Hk 3 Test article:
S9 mix from : rat liver ( Batch R 060792) Test strain : TA !38
Dose
ug/plate
Plate
1
2
3
without S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
Positive control
4-nitro-o-
phenylene-diamine
(50 ng/plate) with S3 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
Positive control
2-Aminoanthracene
(2.5Hg/plate)
22
30
25
23
27
26
18
29
31
19
23
25
19
18
20
20
25
20
17
19
19
17
13
26
1856 1874 1940
42
39
33
39
45
43
43
37
45
39
44
42
33
46
45
38
41
33
37
41
33
45
33
44
448
352
335
+
enhancement factor
"
2 revertants/concentr. test article
--- -
--------------
X revertants/solvent control
Rev<srtants/plate
mean s . d. factor-f
26
4'.0
25
2.1
1.0
26
7.0
1.0
22
3.1
0.9
19
1.0
0.8
22
2.9
0.9
18
1.2
0.7
19
6.7
0.7
1890 44.2 74.6
38
4.6
42
3.1
1.0
42 4.2 1.0
42 2.5 1.0
41
7.2
1.0
37
4.0
0.9
37
4.0
0.9
41 6.7 1.0
378 60.9
8.9
str5
29 of 35
Qowsasy .^.oo----""'""^"'
Test article: f|------.3
S9 mix from : rat: liver (Elatch R 060792)
Test strain : TA 100
Dose
(ig/plate
Plat<s
1
2
3
without S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
Positive control Sodium aside (lOng/plate)
82
95
81
79
93
86
89
99
79
79
86
91
72
87
76
82
86
78
70
89
78
98
86
78
1133 1214 1196
with S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
Positive control
2 -Aminoanthracene
(2.5ng/plate)
+
pnhanfwn^n'f" Fa/'+nr'
as
106
91
90
109
97
89
84
92
80
103
98
97
87
87
105
78
75
74
92 . 88 84
78
84
78
1190
845 1089
X revertants/concentr. test article
Z revertants/solvent control
Revertants /plate mean s. d. factor+
86
7.8
86
7.0
1.0
89 10.0 1.0
85
6.0
1.0
78
7.8
0.9
82
4.0
1.0
79
9.5
0.9
87 10.1 1.0
1181 42.5 13.7
96
9.0
98 10.1
1.0
85
6.1
0.9
99
3.2
1.0
93 10.4
0.9
76
2.1
0.8
88
4.0
0.9
80
3.5
0.8
1041 177.4 10.6
Str5
30 0 C3o5mp. any ,S,a,.nf^a.ezd"-Duo"es not contain TSCACBt
Test article:!----
< 1
S9 mix from : rat liver (]3atch R 060792) Test strain ; TA 102
Dose
tig/plate
Plate
1
2
3
withou-fc S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
Positive control
methyl methan sulfonat
(1.0 til/plate)
with S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
Positive control
2-Aminoanthracene
(2.5ng/plate)
151 158
178 188 198 188 190 159
171 175 166 175 177 179 156 167
179 167 169 176 187 189 178 189
998 1034.
897
-^ - .
260
219
270
197
218
245
233
210
233
217
219
245
194
201
206
224
199
213
240
216
230
237
256
174
811
924
702
revertants/concentr. test article
+
enhancement factor
t revertants/solvent control
Revertants^'plate mean s . d. factor+
167 14.4
167
8.5
1.0
171
6.2
1.0
180
7.2
1.1
187 10.5
1.1
185
5.5
1.1
175 17.2
1.0
172 15.5
1.0
976 71.0
5.9
250 27.0
220 24.1 1.0
225 13.3
1.0
227 15.6
1.0
200
6.0
0.9
212 12.5
1.0
229 12.1
1.0
222 42.9
1.0
812 111.0
3.7
str5
31 of 35
Company Sanitized. Does noi c^fa'n TSCA CB?
sromaRY OF RESULTS
59 mix from : rat liver (Batch R 060792)
without S9 mix
Dose
tig/plate
Neg. contr. Solv . contr.
33 .3 . 100 .0
333 .3
1-000 -.0 .
2500 .0 5000 .0
T A IS535
I / II
14 20 17 20 21 19 16 17 14 18 1-5 11 13 12 15 16
Positive controls
Sodium azide 1152 (10|ig/p1ate)
4-Nitro-o-
phenylene-diann'ne (50ig/p1ate)
methyl methan
sulfonat (l.OiiL/plate)
1077
with S9 mix
Dose p.g/p1ate
Neg. contr. Solv. contr.
33.3 100.0 333.3 1000.0 2500.0 5000.0
TA 1535
I /, II
16 18 23 27 23 23 25 23 23 29 20 28 26 28 . 22 20
Positive control
2-Aminoanthracene (2.51-ig/plate)
159 172
Revertants/plate mean from three plates
TA 31537
I / II
8
5
6
8
7
9
8 10
5
9
5
9
7 10
6
9
TA 98
I / II
37 26 30 25 25 26 34 22
33 .19 25 22 28 18
30 19
TA 3100
I / II
76 86 82 86 76 89 72 85 64 78 50 82 50 79 57 87
323 211 2542 1890
925 1181
Revertants/plate mean from three plates
TA :1537
I -/ II
13 10
12 13
13 13
12 12
11 14
12 10
9
9
10 10
TA 98
r,f II
42 38 40 42 35 42 43 42 39 41 24 37 32 37 25 41
TA 100
I /' II
104 96 106 98
84 85 85 99 88 93 84 76 76 88 64 80
106 99
575 378 1119 1041
TA 102
I / II
205 167 214 167 171 171 207 180 159 187 171 185 226 175 203 172
981 976
JI\ 102
1 / II
273 250 309 220 302 225 301 227 316 200 248 212 282 229 264 222
826 812
str5
32 of 35
Co,^^.-."--"0"1"'"730"8'
CONCLUSIONS
The test article f||BIHHlwas assessed for its potential to
induce gene mutations according to the plate incorporation test
(experiment I) and the pre-incubation test (experiment II) using
Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100,
and TA 102.
The assay was performed in two independent experiments both with
and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test arti
cle was tested at the following concentrations:
33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 ag/plate
No relevant toxic effects occurred in the test groups with and.
without metabolic activation in experiment I and II in all
strains used.
The plates incubated with the test article showed normal back
ground growth up to 5000.0 ng/plate with and without S9 mix in
all strains used.
No substantial increases in revertant colony numbers of any of
HIIIUIpat ..the..,, five--tester strains were observed following treatment with
any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.
In conclusion, it can be stated that during the described mutage-
nicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes
or frameshifts in the genome of the strains used.
st5r
page 33 of 35
C,^,SIB^" -""""'" TSCACS!
REFERENCES
1. Antes/ B.N., W.E. Durston, E. Yamasaki, and F.D. Lee (1973)
Carcinogens are nnrtagens: a simple test system combining liver homogenates for activation and bacteria for detection Proc. Natl. Acad. Sci. (USA) 70, 2281-2285
2. Ames, B.N., J. McCann, and E. Yamasaki (1977) Methods for detecting carcinogens and mutagens with the
Salmonella/mammalian microsome mutagenicity test In; B.J. Kilbey et al. (Eds.)
"Handbook of Mutagenicity Test Procedures" Elsevier, Amsterdam, 1-17
3.
Claxton, Nestmann,
L.D., Alien, J.,
E. and Zeiger,
Auletta,
E. (1987)
A.,
Mortelmans,
K.,
Guide for the Salmonella typhimurium/mammalian microsome
tests for bacterial mutagenicity
Mutation Res. 189, 83-91
4. Hollstein, M., J. McCann, F.A. Angelosanto and
W.W. Nichols (1979)
Short-term tests for carcinogens and mutagens Mutation Res. 65, 133-226
5. Kier, L.E.., D.J. Brusick, A.E. Auletta, E.S. Von Halle, M.M. Brown, V.F. Sirnmon, V. Dunkei, J. McCann, K. Mortelmans, M. Prival, T.K. Rao and V. Ray (1986)
The Salmonella typhimurium/mammalian microsomal assay
A report of the U.S. Environmental Protection Agency Gene-Tox
Program Mutation Res. 138, 69-240
6. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent J. Biol. chem. 193,-265-275
7. McCann, J. and B.N. Ames (1976)
Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 Chemicals: Discussion. Proc. Natl. Acad. Sci. (USA) 73, 950-954
8.
McCann, J., E. Choi, E. Yamasaki and
Detection of carcinogens as mutagens
B.N. Ames (1975)
in the Salmonella/micro-
some test: Assay of 300 Chemicals.
Proc. Natl. Acad. Sci. (USA) 72, 5135-5139
9. de Serres F.J. and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay Mutation Res. 64, 159-165
st5r
page
34 of 35 Company Sanded. Does no-contain TSCACB1
DISTRIBUTION OF THE REPORT
Sponsor
Study Director
2x (original, copy) Ix (copy)
st5r
page 35 of 35
.Company Sanitized. Does not contain TSCA CSt