Document KROaOQyw69Q05BNvB5GoXRgbX
CCR Cytotest Cell Research GmbH & Co. KG
AR226-2983
CCR PROJECT 326417
SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY
WITH
Study Completion Date: February 17,1993
REPORT
CCR In den Leppsteinswiesen 19 D-6101 Ropdorf F.R.G. Telephone: 0 61 54-80 7-0- Telefax 0 61 54- 8 33 99Company Sanitized. Does not contain TSCA CBI
COPY OF GLP CERTIFICATE
HESSISCHES MINISTER1UM FORUN-WELT, ENERG1E UNO BUNDESANGELEGENHEITEN
GLP-Bescheinigung
Bescheinlgung
Hiermit wird bestaiigt, dafi dia Prufungs9inrichtung(en)
Cytotest Cell Research GmbH & Co KG In den Leppsteinswiesen 19 in ^lP.1.,,Rol^^orT
(On, Araicftnn)
dar^0. holding Verwaltung GmbH
(Funu)
03.08., 04.08., 05.08.- und.06.08.92
am....--,__...,,_-.,,.,,,,'....-- __,,,,, i, ,,,,,,, ,,,,,,,,, i, .,..,,,.
(Datum)
von der ffir dia Oberwachung zustandigen BehSrda Qbec dia Hnhaltung der Gmndsatza der Gutan Laborpraxis
.
inspizien wordan ist (sind). Es wirrf hiermit bestaBgt. daB folgenda Priiftjngen in dieser PrOteinrichtung nach den GnjndsteBn der Gutsn Laborpraaa's durchgafuhrt werden.
^^S^ Toxikologische giggnsc-haftpn,
Im Auf'h~ag .
'3^. ^JLt-^L-r
(Dr.-Hecicer)
Wiesbaden, den JS-
CertlHcatB
It is hereby certified lhat the lest (adlity(ies)
Cytotest Cell Research GmbH & Co KG l1?~den"reppst'ernsw'iesen~~'t'9''~'-'"
SlO'l RoGdorf -
.
pOdlion. tOdiBM)
RCC Holding Verwaltung GmbH
of (ccniplny ntina)
on 03.08., 04.08., 05.08. and.06.08.92
was (were) inspected by lha campetant authority regar ding conipfiancs with Itia Pririciplas of Good Laboratory Practlea. It is hareby canified Inat studiss in Ihis test lacilily ara conducted in compBanea wilh he Principles of Good Laboratory Pracliea.
Toxicological.properties
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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 S9 Mix
Fraction
Pre-Experiment for Toxicity Dose Selection
Experimental Performance
Data Recording
Evaluation of Results
BIOMETRY
RESULTS CONCLUSIONS
Pre-Experiment for Toxicity
Tables of Results Experiment I
Tables of Results Experiment II
Summary of Results
.REFERENCES
DISTRIBUTION OF THE REPORT
PAGE
10 10 11 12 14 15 15 16 1617 18 19 19 20 26 32 33 34 35
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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 Kramer
CCR CYTOTEST CELL RESEARCH GMBH S CO. KG
D-6101 Rofidorf, F.R.G.
326417
Salmonella typhimurium Reverse Mutatioi^As say with
PROJECT STAFF Management:
Study Director; Quality Assurance Unit:
Dr. H.- E. Knoell
Dr. Albrecht Poth Dr. Ch. Helmrich
SCHEDULE
Date of Protocol:
'' November 10, 1992
Date of 1st Amendment to Protocol:
January 18, 1993
Start of Pre-Experimerit; 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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PROJECT STAFF SIGNATURES
Study Director:
'&x0i 7 ^ Dr. Albrecht Poth
Date: February 17, 1993
Management:
QUALITY ASSURANCE
i/
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
GUIDELINES
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, Mo. 471, "Salmonella typhimurium. Reverse Mutation
Assay", adopted May 26, 1983 and
EEC Directive 79/831, Annex V, B 14.
/
ARCHIVIHG
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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STATEMENT OF COMPLIANCE
Project Number;
326417
Test Article
Study Director;
Dr. Albrecht Poth
Title
Salmonella typhimurium
Reverse
T^
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
^ y . j ^ D.ta= -&^M JK /?(?3
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QUALITY ASSURANCE UNIT
C C R, Cytotest Cell Research GmbH & Co. KG,
;
In den Leppsteinswiesen 19, D-6101 Rofidorf, F.R.G,
STATEMENT
Project Number;
Test Article
Study Director;
Title
326417
Dr. Albrecht Poth Salmonella typhimurium
wReitvherrse Mutation As say
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
^.^^^^
Date; ^^^^^^
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Company Samfeed. uo.. - - ^ ------^
SUMMARY
^This study was performed -to
JUto induce gene mutations
investigate the potential of according to the plate incorporation
test (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 micrpsomal 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/plate with and without S9 mix in
all strains used.
No substantial increases in revertant colony numbers of any of
^mm|at ^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.
c
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,UUUm is considered to be non-mutagenic in this
Salmonella typhimurium reverse mutation assay.
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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 typhimurimn 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.
REASOMS 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 b'acteria 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 eucaryotic
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 typhimurimn histidine (his) reversion system
measures his" --> his4" 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 preincubation 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 ug/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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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 ^^^lelfl^------l 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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1'est Report CCK. project j^bti/
THE CONTROLS
The Negative Con-fcrols Concurrent untreated and solvent controls were performed.
The Positive Control Substances
Without metabolic activation
Strains;
Name:
Supplier: Catalogue No.: Purity:
Dissolved in:
Concentration:.
TA 1535, TA 100
sodium azide, NaN3 SERVA, D-6900 Heidelberg,
30175
at least 99 % aqua dest. 10 ng/plate
F.R.G.
Strains:
Maine:
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. N9504 > 99.9 %
DMSO
50 ng/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, ul/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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Company Sand. ed,
Test Heport OCR Project j^bli/
THE TEST SYSTEM
Characterisation of the Salmonella tvphimurium 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-f actor plasmid pKM 101 carries the..ampicillin resistance marker. The strain TA 102 does not contain the uvrB" -mutation. Additionally TA 102 contains the multicopy piasmid 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 1535*: his G 46; rfa~; uvrB";
:base-pair substitutions
TA 100: his G 46; rfa"; uvrB";R-factor;
"
TA 102; his G 428; rfa"; uvrB4'; R-f actor;
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 Ames 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, P.R.G.
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.Lteia i- xwpoj-1, ^.^.R. ^j-ujtrfi-i- j^ot^./
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 Nad The bacterial culture was incubated in a shaking water bath for 10 hours at 37 C.
Selective Aqar
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 Aqar
/
The overlay agar contains per litre:
6.0 g Merck Agar Agar 6.0 g NaCi
10.5 mg L-histidine x HC1 x H^O 12.2 mg biotin
Sterilisations were performed at 121 C in an autoclave,
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resT- K-eporr. CCK ATOJBCT. j^btJL/
MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 (Preparation 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 cofactor solution. The amount of S9 supernatant was 15% v/v. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9
mix:
8 mM MgCl2
33 mM 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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Company Sani-t..'zsdrf. pDO^^--pSR^-c--"C"<2i"TSCACr!S
Xfaital- tWfJU^.L. l.L.K. .f-LUJtal-l. O^OtX/
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 (ig/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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.LC^L. i\,i=p>->jL i, i^^xv jr^-<->jte;ll- j^Otj./
EXPERIMEHTAL 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 jj.1
100 (il 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-incubation assay 100 pi test solution, 500 (il S9 mix / S9 mix substitution buffer and 100 p.1 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 solidification the plates were incubated upside down for at least 48 hours at 37 Q 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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Compaq ^Sarlnliiuzzeeda. Duwo- esnotco"^"1'^^
-l.'s.at- l.v^yw.1- - '-t^.l.v JC JL. U J Cl- L. J.U1JLI
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 to cha negative controX without metabolic activation (+) The range of strain TA 102 was dacarmined from our historical control dataa
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 to 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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BIOMETRY
No appropriate statistical method is available (5)
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JLC3 I- I-YC;^/--*- l- IL^^JLV JCJL'-^J'CS^ L f ^ U t -L /
RESULTS
PRE-EXPERIMENT 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
W
reve rtants
TA 98
-
+
per plate
TA 100
+*
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
37
42
30
40
2542
/
/
/
/ 575
22
33
34
23
25
35
34
43
33
39
25
24
28
32
30
25
76 104
82 106
/
/
925
/
/ 1119
73 108
72
98
76
84
72
85
64
88
50
84
50
76
57
64
* - " without S9 mix; + " with S9 nix / not performed
The plates with the test article showed normal background growth up to 5000.0 ug/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 ug/plate
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rest Keporr. L-CK i/ro^ecr. J^bix/ TABLES OF RESULTS EXPERIMENT I
PLATE INCORPORATION TEST
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Test article:
S9 mix from ; rat liver (Batch R 060792) Test strain : TA 1535
Dose
tig/plate
Plate
1
2
3
without S9 mix
Negative Solvent
33.3
10D.O
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 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
Positive control
2-Aminoanthracene
(2.5ng/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
revertants/concentr. test article S revertants/solvent control
Reverta:nts/plate '
mean s .d. facti
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
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Test article:
S9 mix from : rat liver (Batch R 060792) Test strain : TA 1537
Dose
ug/plate
Plate
1
2
3
without S9 mix
Negative Solvent
33.3 1-00.0 333.3 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-Nitro-o-
307
311
350
phenylene-diamine
(50ng/plate)
with S9 mix
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)
enhancemant factor
S revertants/concentr. test article revertants/solvent control
Revesrtants/'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
str5
22 of 35
.arulizecS. Goes "3s C3ntai" T3CA CBI
Company w
Test article:
S9 mix from : rat liver (Batch R 060792)
Test strain : TA 98
Dose
lig/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 Hl/plate)
42 27 28 31 24 32 19 28
2464
35
35
33
31
24
24
35
36
37
37
26
17
26
39
28
33
2501 2660
with S9 mix
Negative control Solvent control
33.3
38
44
43
f
47
34
40
29
30
45
100.0
43
40
45
333.3
42
41
33
1000.0
25
26
21
2500.0
30
34
31
5000.0
29
23
24
Positive control
2-Aminoanthracene
520
454
751
(2.5ng/plate)
+
enhancement factor
1 revertants/concentr. test article
Z revertants/solvent control
Revertants,/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
575 156.0 14.3
str5
23 of 35 s.^.'""-0'"""'"'3""'
Coniltaw"""
Test article:
S9 mix from : rat liver (Batch R 060792) Test strain : TA 100
Dose
ug/plate
Plate
1
2
3
without S9 mix
Negative Solvent
33.3
1-00'. 0
333.3 1000.0 2500.0 5000.0
control control
-
Positive control Sodium azide (10(ig/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
! revertants/concentr. test article
X revertants/solvent control
Revertants,opiate mean s ,d. actor+
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
^fe^W"ot 24 of 35 compiffly^0"
contain TSCA'
Test article:
S9 mix from : rat liver (Batch R 060792) Test strain : TA 102
Dose
tig/plate
Plate
1
2
3
without S9 mix
Negative Solvent
33.3
1-00.0
333.3 1000.0 2500.0 5000.0
control control
-
Positive control
methyl methan
sulfonat
(l.Onl/plate)
with S9 mix
Negative Solvent
33.3 100.0 333.3 1000.0 2500.0 5000.0
control control
Positive control
2-Aminoanthracene
(2.5)ig/plate)
200 206 181 179 109 171 237 199
827
280 253 252 318 364 204 284 . 214
752
207 225 178 244 179 151 228 201
1107
296 318 312 262 291, 289 272 284
861
208 211 154 199 188 190 212 209
1010
243 357 343 324 292 251 289 295
864
enhancement factor
X revertants/concentr. test article revertants/solvent control
Revisrta.nts/'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
,,S^,,,^.D.e,ncS..^TSCACB>
Company
TABLES OF RESULTS EXPERIMENT II
PRE-INCUBAT10N TEST
page 26 of 35
Company Sanitized. Does not contain TSCA CB5
Test article:
S9 mix from : rat liver (Batch R 060792) Test strain ; TA 1535
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 Sodium azide (lOng/plate)
19
20
22
21
18
21
23
18
17
15
13
24
n
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
24
17
13
33.
24
27
24
26
18
21
24
25
31
26
31
28
28
29
28
27
30
22
20
17
Positive control
2 -Aminoanthracene
163
187
167
(2.5ng/plate)
X revertants/concentr. test article
+ enhancement factor
-
t revertants/solvent control
Reviertants/'plate mean s.d. factor+
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
TSCACB1
contain
Company Sa^6 -< us5'"'.5.t
Test article:
S9 mix from : rat liver (Batch R 060792) Test strain : TA 1537
Dose
i^g/plate
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/plate5
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
revertants/concentr. test article X revertants/solvent control
Revertants,Opiate 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 35
Csmipany SanTized. Doss no! con!a!n TSCA CBI
Test article:
S9 mix from : rat liver (Batch R 060792)
Test strain : TA 98
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 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)
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
X revertants/concentr. test article
2 revertants/solvent control
Revesrtants/'plate mean s.d. factor+
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
nQ-snotca^ainTSCACBI Compaq Sar..,-L2rsi d.DG-""
Test article:
S9 mix from : rat liver (Batch R 060792) Test strain : TA 100
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 Sodium azide (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)
+
enhancement factor
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
revertants/solvent control
Revertants,Opiate 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 of 35
.ntc-rtalnTSCACSi
c^-iized.C-oesnotc^^
Company sa-llze
Test article:
S9 mix from : rat liver (Batch R 060792) Test strain : TA 102
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 methyl methan sulfonat
(1.0 Hi/plate)
151
171
179
158
175
167
178
166
169
188
175
176
198
177
187
188
179
189
190
156
178
159
167
189
998 1034
897
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)
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
+
enhancement factor
revertants/concentr. test article X revertants/solvent control
Revertants/plate
mean s . d. facti
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
noSc^Sa-.nTSCACBt Company Sar^zeA E::;es
SUJMMARY OF RESULTS
Test article :lj^^^^kH. ^5g>
S9 mix from ; rat liver
(Batch R 060792)
without S9 mix
Dose
(jig/plate
Neg. contr. Solv . contr.
33 .3 100.0 333 .3 1000 .0 . 2500 .0 5000 .0
TA li535
I / II
14 20
17 20
21 19
16 17
14 18
15 11
~ 13
12
15 16
Positive controls
Sodium azide 1152 (lOfJ-g/plate)
4-Nitro-o-
phenylene-diaim'ne (50|jLg/p1ate) methyl methan
sulfonat (l.OnL/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-Ann'no-
159 172
anthracene
(2.5p,g/p1ate)
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 ]100
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
TA 98
I / II
13 10
12 13
13 13
12 12
11 14
12 10
9
9
10 10
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
TA 102
I / II
273 250 309 220 302 225 301 227 316 200 248 212 282 229 264 222
826 812
str5
32 of 35
cants'"TSCACB .^i
con^n,^"-008"'01"
CONCLUSIONS
The test article BB^HI^^D^'^5 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 ng/plate
Mo 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 ug/plate with and without S9 mix in
all strains used.
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with
HBHH^^f^t 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
^^^DW'm"a-"l^'aww'
REFERENCES
1. Ames, B.N., W.E. Durston, E. Yamasaki, and F.D. Lee (1973) Carcinogens are mutagens: a simple test system combining
liver homogenates for activation and bacteria for detection ,; Proc. Natl. Acad. Sci. (USA) 70, 2281-2285
2. Ames, B.M., 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, L.D., Alien, J., Auletta,
Nestmann,. E. and Zeiger, 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. Simmon, V. Dunkel, 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/micro-
some 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 CompanySan^d. Ooas^ contain TSC
DISTRIBUTION OF THE REPORT
Sponsor
Study Director
2x (original, copy) Ix (copy)
\v ^
^ s ^jr.
st5r
page 35 of 35
Company SanEEized. Does net co.'staSn TSCA CSI
