Document 8R2YQZzwbDmxwyeZdZOn91bxa
AR226-3030
CCR PROJECT 509800 GENE MUTATION ASSAY IN CHINESE HAMSTER V79 CELLS
IN VITRO ( V79/ HPRT)
WITH
REPORT Study Completion Date: October 06,1995
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Test Report C C R Project 509800
COPY OF GLP CERTIFICATE
HFUAMEMSWSIILESILCETHU, EENSNDMEGRINEGSIISEUT,NEJDURHGIUEEMNITDF,R
GLP-Bescheinigung
Bescheinigung
C ertific a te
Hiermit wird besttigt, dafi die Prfcinnchtunj(cn| CCR C ytotest Ceil Research GmbH t Co. KG in 64380 Rodorf, 2a den Lcppsteinswie3Eul9
(Ort, Anschrift) der RCC/CCR Holding Verwaltung GmbH
(Firma) am 05./06./07. April 1995
(Datum)
It is hereby certified that the test faeiiityfies) CCR Cytotesl Cell Research GmbH & Co. KG in 64380 Ro&dorf. In den Leppstetnswiesen 19
(location, address) or RCC/CCR Holding Verwaltungs GmbH
(company name) an 05./06./07. April 1995
(date)
von der fr die berwachung rustAndigen Behrden ber die der Grundstze der Guten Laborpraxis inspiziert worden ist (sind).
was (were) inspected by the competent authority regarding compliance with the Principles of 'Good Laboratory Practice.
Bs wird hierm it besttigt, dafi folgende Prfungen in dieser Prfeinrichtung nach den Grundstzen der Guten Laborpraxis durchgefhrt werden.
It is hereby certified that studies in this test facility are conducted in compliance with the Principles of Good Laboratory Practice.
Prdfktt o r l. nach 3 19 d A ta. 3 C fc .m lta U .n l, in der Fassung vom 29.Juli 1994 (BGBL I S. J703|. zutetzt gendert u 27. September 1994 (BGBl. I 3. 2705) in Verbindung m it der Allgemeinen Verwaltungsvorsctirift zum Verfohreiyder behrdlichen Obererechung der Einhaltung der Grundstze der Guten Lnborprazis vom 21. Oktober 1990 (BAnz. 204 am 31.10.1990):
Tozikoiogisehe Eigenschaften
Toxicological properties
PrU ltategorle g m it OECD Panel an Onad Laboratory Practice (January 1992)
Prfungen auf tozikoiogisehe Eigenschaften Prfungen auf m utagene Eigenschaften (in vitro, in vivo)
Toxicity studies Mutagenicity studies
Im Auftrag
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Company
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Test Report CCR Project 509800
CONTENTS
COPY OF GLP CERTIFICATE
PREFACE General Project Staff Schedule Project Staff Signatures Good Laboratory Practice Guidelines Archiving
*
STATEMENT OF COMPLIANCE
QUALITY ASSURANCE UNIT
SUMMARY - ' Conclusion
OBJECTIVE Aims of the Study Relevance of the Test System
MATERIALS AND METHODS Test Article Controls Test System Mammalian Microsomal Fraction S9 Mix Pre-Test on Toxicity Dose Selection Experimental Performance Data Recording Acceptability of the Assay f Evaluation of Results
RESULTS Pre-Test on Toxicity
RESULTS AND DISCUSSION
REFERENCES
DISTRIBUTION OF THE REPORT Annex: Tables of Results Experiment I Tables of Results: Experiment II
DEVIATIONS TO THE PROTOCOL.
BIOMETRY
7
8
9 9
111000 1111 1122
13 14 14 16 18 18 19
2200
21 22 22
23 26 29
29
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Test Report C CR Project 509800
PREFACE
General
Sponsor:
Study Monitor: Testing Facility:
CCR Project No.: Test Article: Title:
UISnEteGrAsucFhournscghsugensgesl-lsucnhdaft mbH Zeppelinstrae 3 D-63741 Aschaffenburg Dr. Derra CCYCTROTEST CELL RESEARCH GMBH & Co. KG In den Leppsteinswiesen 19 D-64380 Rodorf, F.R.G. 509800
Gene Mutation Assay in Chinese Hamster V79 Cells in vitro (V79/HPRT) witj
Project Staff
Study Director: Management: Quality Assurance Unit
Dr. Hans-Eric Wollny Markus Arenz Frauke Hermann
/
Schedule
Date ofProtocol Start of Pre-Test: End of Pre-Test: Start of Experiments : End of Experiments : Date of Draft: Date of Final Report:
'
April 13, 1995
July 27, 1995 July 31, 1995 August 01, 1995 Sept. 11, 1995 Sept. 13, 1995
Oct. 06, 1995
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Company Sanitised. C
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> Project Staff Signatures
Study Director
Dr. Hans-Eric Woilny
Management
Date: October 06, 1995
Good Laboratory Practice
The study was performed in compliance with: Chemikaliengesetz ("Chemicals Act") o f the Federal Republic o f Germany, Anlage 1 ("Annex 1"), dated July 25, 1994 (BGBL. 1 1994 S. 1703)." "The OECD Principles of Good Laboratory Practice", Paris 1981.
not contain TSCACB Com pany Sanitized. Does
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Test Report C C R Project 509800
G u id e lin e s
. This study followed the procedures indicated by the following internationally accepted ' guidelines and recommendations: Second Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 476, adopted April 4, 1984, "In vitro Mammalian Cell Gene Mutation Tests" EEC Directive 87/302, L 133, p. 61 - 63
Archiving
C C-R, D-64380 RoBdorfTF.RG. will archive the following data for 30 years: Raw data, protocol and copy of report. The following sample will be archived for at least 2 years following the date on which the report is audited by the Quality Assurance Unit: sample o f the test article No raw data or material relating to the study will be discarded without the sponsor's prior consent.
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Test Report C C R Project 509800
STATEMENT OF COMPLIANCE
` Project Number:
509800
Test Material :
Study Director:
Dr. Hans-Eric Wollny
Title: Gene Mutation Assay in Chinese Hamster V79 Cells in vitro (V79/HPRT) wit
This study performed in the testing facility o f C C R was conducted in compliance with Good Laboratory Practice Regulations.
Chemikaliengestz ("Chemicals Act") o f the Federal Republic o f Germany, Anlage 1 ("Annex 1"), dated July 25, 1994 (BGBL. 1 1994 S. 1703)."
"The OECD Principles of Good Laboratory Practice", Paris 1981."
There were no circumstances that may have affected the quality or integrity o f the study.
Study Director
Dr. HanCs-CErRic Wollny
Date: 0 G, I f f f '
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Test Report C C R Project suauuu
QUALITY ASSURANCE UNIT :C C R, Cytotest Cell Research GmbH & Co. KG,
In den Leppsteinswiesen 19, D-64380 Rodorf, F.R.G.
Statement
Project Number:
509800
Test Material : Study Director: Title:
L Dr. Hans-Eric Wollny Gene Mutation Assay in Chinese Hamster V79 Cells in vitro (V79/HPRT) wit
This report was audited by the Quality Assurance Unit and the conduct of this study was inspected on the following dates.
Phases and Dates of QAU Inspections/ Audits
Protocol Audit: April 18, 1995
Process Inspection August 11, 1995
Draft Audit:
Sept. 22, 1995
Dates of Reports to the Study Director and to Management
April 18, 1995 August 11,1995 Sept. 22, 1995
Head of Quality Assurance Unit
Frauke Hermann
.................\ ^
Date: f e k W j p ( o {
.........
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Test Report C C R Project 509800
SUMMARY
The study was performed to investigate the potential ( ' tations at the HPRT locus in V79 cells of the Chinese
The assay was performed in two independent experiments, using identical procedures, both
with and without liver microsomal activation.
The test article was tested with the following concentrations:
Experiment I:
without S9 mix: 1.0;10.0*; 100.0; 200.0; 250.0* and 300.0 pg/ml with S9 mix: 1.0;10.0*; 100.0; 200.0; 250.0* and 300.0 pg/ml
Experiment II:
without S9 mix: 1.0;100.0;200.0; 250.0* and 300.0 pg/ml with S9 mix: 1.0;100.0;200.0; 250.0* and 300.0 pg/ml
No relevant toxic effects occurred up to the limit of solubility and above. The highest con centration (300 pg/ml) resulted in a slight perturbation of the medium due to formation of small undisolved droplets. Survival at the lowest concentration was approximately in the range o f the negative control. Up to the highest investigated concentration no relevant increase in mutant colony numbers was observed in both independent experiments.
Appropriate reference mutagens were used as positive controls and showed a distinct in crease in induced mutant colonies.
Conclusion
/
In conclusion it can be stated that during the described mutagenicity test and under the ex
perimental conditions reported the test article did not induce gene mutations at the HPRT
locus in V79 cells. _
T h e r e fo r ^ Q H ^ m tf> s considered to be non-mutagenic in this HPRT assay.
* not evaluated, culture not continued
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I est Kepon u u k rroject auaouu
OBJECTIVE
Aims o f th Study
This in vitro assay is performed to assess the potential of the test article to induce gene mutations by means of two independent HPRT experiments using the Chinese hamster cell line V79.
Relevance of the Test System
In vitro, methods are valuable when it is desirable to accurately control the concentration and exposure time of cells to the test article under study. However, due to the limited ca pacity for metabolic activation of potential mutagens an exogenous metabolic activation system is necessary.
This in vitro test is an assay for the detection of forward gene mutations in mammalian cells. Gene mutations are considered to be an initial step in the carcinogenic process (2).
The V79 cells are exposed to the test article both with and without exogenous metabolic activation. At a defined time interval after treatment the descendants o f the treated original population are monitored for the loss o f functional HPRT enzyme.
HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6TG. These cells are able to proliferate in the presence o f 6TG whereas the non-mutated cells die. However, the mutant phenotype requires a period o f tune before it is completely expressed. The phenotypic ex pression is achieved by allowing exponential growth of the cells for 7 - 9 days. The expres sion period is terminated by adding 6TG to the culture medium (3).
Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to deter mine the surviving cells. After a suitable period the colonies are counted. Mutant frequen cies are calculated from the number o f mutant colonies corrected for cell survival.
In order to establish a concentration response effect o f the test article at least four concen tration levels are tested. These concentration levels should yield a concentration related toxic effect. The highest concentration level should induce a reduced level of survival.
To demonstrate the sensitivity of the test system reference mutagens are tested in parallel to
the test article.
.
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MATERIALS AND METHODS
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 Stability
pure: not indicated by the sponsor In solvent: not indicated by the sponsor
Storage:
4 C
Expiration Date:
not indicated by the sponsor
On the day o f the experiment (immediately before treatment), the test article was dissolved in Ethanol (E. MERCK, D-64293 Darmstadt; purity 99.8 %). The solvent was chosen ac cording to its solubility properties^and its non-toxicity for the ceils. The final concentration o f DMSO in the culture medium did not exceed 1 % v/v.
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Test Report CCR Project 509800
Controls
Negative Controls
Concurrent negative and solvent controls were performed.
Positive Control Substances
Without metabolic activation
Name: Supplier: Catalogue no.: Dissolved in: ~ Final concentration:
EMS; Ethylmethanesulfonate Merck-Schuchardt, D-85662 Mnchen, F.R.G. 820774; (Purity: > 98%) nutrient medium 0.6 mg/ml - 4.8 mM
Solution prepared on day of experiment.
With metabolic activation
Name: Supplier: Catalogue no.: Dissolved in Final concentration:
DMBA; 7,12-dimethylbenz(a)anthracene SIGMA CHEMIE GMBH, D-82041 Deisenhofen, F.R.G. D 3254; (Purity: approx. 95%) DMSO, Dimethylsulfoxide; final concentration in nutrient medium 1 % 3.85 pg/ml = 15.0 pM
Trehseposntasbeiliintythoef ebxopthecpteodsitmivuetactoionntroral nsguebsitsansucfefsiciinenstoleuvtiidoenncise uonfkbnioowlong,icbaultstaabmiluittya.geTnhiec dilutions o f the stock solutions were prepared on the day of the experiment and used im mediately.
Test System
Reasons for the Choice of the Cell Line V79
The V79 cell line has been used successfully in in vitro experiments for many years. Espe cially the high proliferation rate (doubling time' 12 - 16 h in stock cultures) and a good cloning efficiency of negative control cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The ceils have a stable karyotype with a modal chromosome number o f 22 (3).
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Test Report C C R Project 509800
Cell Cultures
Large stocks o f the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni` cal University; D-64293 Darmstadt; F.R.G.) are stored in liquid nitrogen in the cell bank of C C R allowing the repeated use of the same cell culture batch in experiments. Before free zing, the level o f spontaneous mutants was depressed by treatment with HAT-medium as described in [3], Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters o f the experiments remain similar because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 C in 80 cm2 plastic flasks (GREINER, D72632 Frickenhausen, F.R.G.). About 5x10s cells are seeded into each flask with 15 ml of MEM (minimal essential medium; SEROMED, D -12247 Berlin, F.R.G.) supplemented with 10 % foetal calf serum (FCS; Boehringer Mannheim, 68261-Mannheim, F.R.G.). The cells are subculturedJwice weekly. The cell cultures are incubated at 37 C in a 4.5 % carbon dioxide atmosphere (95.5% air). For the selection of mutants the medium is supplemented with 11 gg/ml thioguanine (6TG, SIGMA GmbH, D-82041 Deisenhofen, F.R.G.).
Mammalian Microsomal Fraction S9 Mix
Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays with cell cultures as many chemicals only develop a mutagenic potential after metabolisation by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with mammalian liver microsome prepara tions (S9 mix).
S9 (Preparation by C C R)
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats, strain Hanlbm (BRL, CH-4414 Fiillinsdorf weight approx. 220 - 320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Et tlingen, F.R.G.) in olive oil 5 days previously. Aanfdterhocmerovgiceanlisdeidsl.oTcahteiohnotmhoegleivneartseowfatshedialuntimedal1s+w3eirne KreC-1maonvdedc,enwtarsifhuegdedinat1590,0m00MgKfCo1r 10 minutes at 4 C. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80 C. Small numbers of the ampoules are kept at 20 C for up to several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 Mnchen: Bio-Rad protein assay, Catalogue 500 000 6 (6). The protein concentration in the S9 preparation was 33.2 mg/ml (lot 220595).
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Test Report C C R Project 509800
S9 Mix
. An appropriate quantity o f S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures. Cofactors were ad ded to the S9 mix to reach following concentrations:
8 mM MgCb 335 mmMM gKlCu1cose-6-phosphate 4 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.(l).
Pre-Test on Toxicity
A pre-test was performed in order to determine the concentration range for the mutageni city experiments. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment. The following method was used in the pre-test: XTT-Assay: The XTT-assay is based on the cleavage of the yellow tetrazolium salt XTT to form an or ange formazan dye by hydrogenase activity in active mitochondria. 18 - 20 h after treatment with the test article the XTT-assay was initiated by adding a mixture of XTT-labeliing rea gent with an electron coupling reagent (PMS). After 4 h of incubation the absorption was determined at 450 nm (690 nm reference) using an ELISA reader (SLT, Labinstruments Austria, A-5082 Grodig). The viabilities of the cells are calculated as percentages of the solvent controls and reported as tables.
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Test Report CCR Project 509800
Dose Selection
. According to the recommendations of the guidelines (see page 6), several concentrations ' (usually at least four) of the test article should be used. These should yield a concentration-
related toxic effect. The highest concentration should produce a low level of survival and the survival in the lowest concentration should approximate the negative control. Relatively insoluble substances should be tested up to their limit o f solubility under culture conditions. For freely-soluble nontoxic substances the maximum concentration should be as recommen ded in the in vitro cytogenetic assay, namely 5 mg/ml or 10 mM. If the maximum concen tration is based on cytotoxicity the cloning efficiency should be reduced to less than 50 % and/or culture growth at subcultivation should be at least 20% of the corresponding solvent control. In the pre-test of toxicity (XTT-assay) the. extinction (measured at 450/690 nrn) was not
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Experiment I and II were performed with four concentrations ranging from 1.0 to 300.0 pg/ml with and without metabolic activation. Therefore, the test article was tested with the following concentrations: Experiment I: without S9 mix: 1.0; 100.0; 200.0 and 300.0 pg/ml with S9 mix: 1.0; 100.0; 200.0 and 300.0 pg/ml Experiment II: without S9 mix: 1.0; 100.0; 200.0 and 300.0 pg/ml with S9 mix: 1.0; 100.0; 200.0 and 300.0 pg/ml
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Test Report C C R Project 509800
Experimental Performance
Seeding
Three days old exponentially growing stock cultures (more than 50 % confluent) were trypsinized at 37 C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentra tion for all subculturing steps was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Labo ratories, Detroit, USA). The Ca-Mg-free salt solution was composed as follows (per litre):
-. _ .
NaCl 8000 mg KC1 400 mg Glucose 1000 mg NaHC03 350 mg
Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/1 EDTA (ethylene diamine tetraacetic acid). The ceil suspension was seeded into plastic culture flasks (Greiner, D-72632 Frickenhau sen). Approximately 1.5x10 (single culture) and 5xl02 cells (in duplicate) were seeded in MEM with 10 % FCS (complete medium) for the determination o f mutation rate and toxi city, respectively (see experimental scheme).
Treatment
After 24 h the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 50 il/ml S9 mix. After 4 h this medium was replaced with complete medium after two washing steps with "saline G".
The "saline G" solution was composed as follows (per litre):
NaCl KC1 Glucose Na2HP04x7H20 KH2PO4
8000 mg 400 mg 1100 mg 290 mg 150 mg
pH is adjusted to 7.2
Experimental Scheme:
Segment a): Procedure for determination of toxicity Segment b): Procedure for determination of mutation rates
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Test Report C C R Project 509800
Day 1: Subcuituring o f a log-phase culture which showed an initial spontaneous mutation rate at the beginning o f the experiment o f 14.0 (experiment I) and 2.9 (experiment II) mutants per IO6 ceils.
a) About 500 cells in 5 ml medium/25 cmz-plastic-flask for cloning efficiency; in duplicate
per experimental point
b) lxlO6 cells in 30 ml medium/175 cm2-plastic-flask for the mutagenicity test, 1flask per experimental point
Day 2: Treatment o f a) and b)
experiment I
Day'5: Subculturing o f b) in 175 cm2-plastic-flasks 1.5xl0cells in 30 ml medium/175 cm2plastic-flasks
experiment II
Day 6: see day 5
Day 8: Fdeixteartmioninaantidonstoaifncionngcoenf tcroaltoinonie-sreinlaate)d-flcalsoknsing efficiency
Day 9: Subculturing o f b) in five 80 cm2-plastic-flasks containing selective medium: mutant ssuelbeccutilotunri(nagboouftb3)-i5nxtlwOos c2e5llcsm/fl2a-sfkla);sks for cloning efficiency (about 500 cells/flask)
Day 16: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (cloning ef ficiency).
Day 18: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (mutant selection).
The cultures were incubated lonies were stained with 10
at %
37 C in a methylene
humidified atmosphere blue in 0.01 % KOH
with 4.5 solution
% (E.
CMOE2R. TChKe,
co D-
64293 Darmstadt, F.R.G.).
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope (Nikon, D-40407 Dsseldorf, F.R.G.).
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Test Report C C R Project 509800
Data Recording
-. The data generated were recorded in the raw data. The results are presented in tabular form, including experimental groups with the test article, negative and positive controls.
Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria: a) the numbers of mutant colonies per 10s ceils found in the negative and/or solvent con
trols fall within the laboratory historical control data range: 0 -4 5 mutants/tO6 ceils. b) the positive control substances must produce a significant increase in mutant colony fre
quencies. c) the cloning efficiency (absolute value) of the negative and/or solvent controls must ex
ceed 50 %. The data of this study comply with the above mentioned criteria [a) and b) see mutation rate, tables III and VI, c) see tables II and V, factor calculated referring to the C.E. o f the untreated cultures].
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Test Report CCR Project 509800
Evaluation of Results
;A test article is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response for one of the test points. A test article producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system. A significant response is described as follows: The test article is classified as mutagenic if it induces a reproducible mutation frequency that is at least three times higher than the spontaneous mutation frequency in the experiment at one or more o f the concentrations. The test article is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the correspon ding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0 - 45 mutants per 10^ cells) a concentration-related increase of the mutations within this range has to be discussed.
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RESULTS
Pre-Test on Toxicity
Without S9 mix:
Blanc Negative control Solvent control test article ~ test article test article test article test article test article test article test article
concentration pg/ml
/ / /
0.3
1.0
3.0
10.0
30.0
100.0 200.0
300.0
extinction (450/690 nm) % of the
mean standard corresponding
deviation control* 0.18 0.00
0.00
1.22 0.03
105.57
1.17 0.02
100.00
1.19 0.04
102.35
1.21 . 0.07
104.17
1.16 0.03
99.44
1.14 0.04
97.16
1.08 0.05
91.36
1.12 0.06
95.14
1.10 0.06
93.33
1.13 0.07
96.36
With S9 mix:
Blanc Negative control Solvent control test article test article test article test article test article test article test article test article
concentration pg/ml
'/ / /
0.300
1.000
3.000
10.000
30.000
100.000 200.000
300.000
extinction (450/690 nm) % of the
mean standard corresponding
deviation control*
0.178
0.007
0.000
1.009
0.054
105.361
0.966
0.048
100.000
0.926
0.074
94.893
0.914
0.069
93.306
0.872
0.038
87.977
0.852
0.032
85.534
0.829
0.030
82.568
0.775
0.024
75.763
0.716
0.032
68.181
0.688
0.063
.
64.724
* corrected with the blanc
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Test Report CCR Project 509800
RESULTS AND DISCUSSION : tThheeHtPesRt T locussing V79 cdM?oftheasCsehsisneedsefhoarmitsstepro. tential to induce gene mutations at
The study was performed in two independent main experiments, using identical procedures, both with and without liver microsomal activation. No relevant toxic effects occurred up to the limit o f solubility and above. In both experiments the numbers of mutant colonies per 106 cells did not exceed the values of the corresponding controls substantially and remained well within the historical range. Furthermore, there was no indication o f a concentration depend increase o f mutant colonies. In Both' ex p erim en ts of this study (with and without S9 mix) th e range of the negative con trols was from 3.0 up to 17.9 mutant colonies per 106 cells; the range of the groups treated with the test article was from 5.7 up to 20.2 mutant colonies per 10ceils. EMS (0.6 mg/ml) and DMBA (3.85 pg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, it can be stated that in this mutagenicity assay and under the experimental cceolnlsd.itions reported the test article did not induce gene mutations at the HPRT locus in V79
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Test Report CCR Project 509800
REFERENCES 1. B.N. Ames, J. McCann, and E. Yamasaki
Methods for detecting carcinogens and mutagens with the Salmoneila/mammalian micro some mutagenicity test In: B.J. Kilbey et al. (Eds.) "Handbook o f Mutagenicity Test Procedures", Elsevier, Amsterdam, 1-17, 1977 2. P. Howard-Flanders Mutagenesis in mammalian cells Mutation Research 86, 307-327, 1981 3. M.O. Bradley, B. Bhuyan, M.C. Francis, R. Langenbach,. Peterson and E. Huberman Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis of the literature: A report o f the gene-tox program Mutation Research 87, 81-142, 1981 4. S. Rettig Modellierung, Simulation und statistische Analyse des HGPRT-Mutagenittstests, The sis, Technical University of Darmstadt, 1990 5. EEC Directive 92/69, L 383 A, Annex V, B 10, dated December 29, 1992
DISTRIBUTION OF THE REPORT
Sponsor
2x (original, copy)
Study Director
lx (copy)
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ATanbnleeI:xT: oTxiacbityledsatao; fexRpeersimuelntst IExperiment 1
cone. per ml
S9 number of cells per flask*
mix seeded
found
mean
l/ll I
II
CE%** CE*** absolute relative
cells/ml cell denat 1st sub- sity; % of cultivation control
column
Negative control (Untreated cells)
1
23 4 5
6
7
8
523 306
301 303.5
58.0
9 10 1
Negative control
Solvent control with Ethanol Positive control with EMS
0.00 pg 0.00 pg -
0.6 mg -
523 236 523 214 523 189
274 255.0 266 240.0 201 195.0
48.8 100 2011333 45.9 100 2098000 37.3 76.5 1779333
100 100 88.5
Test article Test article
Test article Test article
Test article
Test article Negative control Solvent control with DMSO Solvent control with Ethanol
1.00 pg 10.00 pg 100.00 pg 200.00 pg 250.00 pg 300.00 pg
0.00 pg
+
0.00 pg +
0.00 pg +
523 253 523 203 523 234 523 197 523 193 523 175 523 269
523 252
523 224
245 249.0 244 223.5 214 224.0 184 190.5 183 188.0 204 189.5 214 241.5
214 233.0
255 239.5
47.6 42.7 42.8 36.4 35.9 36.2 46.2
44.6
45.8
103.8 93.1 93.3 79.4 78.3 79.0 100.0
100.0
2258667 2312000 1848667 1880667 1876667 1875333 2190000
2272000
100.0 2468000
107.7 110.2
88.1 89.6 89.5 89.4 100.0
100.0
100.0
Positive control with DMBA
3.85 pg
523 158
179 168.5
32.2
Test article
1.00 pg +
523 200
195 197.5
37.8
Test article
10.00 pg Hb
523 186
211 198.5
38.0
test article
100.00 pg
523 207
226 216.5
41.4
Test article
200.00 pg +
523 241
223 232.0
44.4
Test article
250.00 pg f
523 237
222 229.5
43.9
Test
article
300.00 pg 4*
oCnElyacbosololuntiees(vwailtuhemcoolruemthna6n
/5v0acluele5lsc2o73ludmayns23a2fxt9er10se0edi2n3g3were
2sc3o1r.e0d
44.2
CE relative (value column 6 / value column 6 of corresponding control x 100)
72.3 1542667
82.5 2346667 82.9 2476667 90.4 2646000 96.9 2497333 95.8 2060667 96.5 2444667
67:9
95.1 100.4 107.2 101.2
83.5 99.1
-Page 23 of 29 -
itized. Doss not contain TSGA 031
Test Report CCR Project 509800
Table II: Mutagenicity data; experiment I (part 1: cell survival)
cone. per ml
S9 number of cells per flask*
mix seeded
found
mean
l/ll 1 II
factor** cells calculated seeded
cells*** survived
column
1 23 4
5
6
7
8
9
Negative control
Solvent control with DMSO
0.0G pg 0.00 pg -
505 296 301 298.5 513 303 353 328.0
0.59 435000 57124 0.64 414000 264702
Positive control with EMS
0.6 mg -
504 318
325 321.5
0.64 414000 264089
Test article Test article
1.00 pg 10.00 pg -
504 347 365 356.0
0.71
culture was not continued*
450000
317857
Test article
100.00 pg -
500 325 360 342.5
0.69 408000 279480
Test article Test article
200.00 pg 250.00 pg -
511 398 406 402.0
0.79
culture was not continued*
402000
316250
Test article
300.00 pg -
503 380 410 395.0
Negative control
0.00 pg +
503 381
409 395.0
Solvent control with DMSO
0.00 pg
500 336 328 332.0
0.79 426000 334533 0.79 414000 325109
0.66 478500 317724
Solvent control with Ethanol
0.00 pg +
509 362
372 367.0
0.72 396000 285525
Positive control with DMBA
3.85 pg +
502 291
302 296.5
0.59 432000 255155
1*5
Test article Test article
1.00 pg + 10.00 pg +
507 345 342 343.5
0.68
culture was not continued*
463500
314028
<a9
Test article Test article
aoCD
Test article Test article
100.00 pg 200.00 pg 250.00 pg 300.00 uo
+ + + +
511 366 349 357.5 502 375 362 368.5
0.70 0.73
culture was not continued*
507 358l 317l 337751
67
420000 486000
522000
293836 356755
347485
o oo3&1
5" .
wo > o83
factor calculated (value column 6 / value column 3)-------- J ------------------- -- ----------- " - * ***" * * 'V * IU * w w j d W M i k t v w u u i g VTW1W S W U IW U cells survived after plating in TG containing medium (value column 8 / value column 7) concerning concentration range and toxicity four concentrations were selected to be evaluated at the end of the experiment
-Page 24 of 29-
Company
Test Report CCR Project 509800
Table III: Mutagenicity data; experiment I (part 2: mutation rates)
cone. per mi
S9 number of mutant colonies per flask* mean standard mutant**
mix Found after plating in TG medium
deviation colonies
I II
III
IV
V
oer 106 cells
column
1
23
4
5
6
7 a9
10
Negative control
0.00 pg -
67
2
5
3 4.6
2.1 ,
17.9
Solvent control
with DMSO
0.00 pg -
52
4
3
3 3.4
1.1
12.8
Positive control with EMS
0.6 mg -
137 118
117
151
136 131.8
14.3
499.1
Test article T est article Test article Test article Test article
1.00 pg 10.00 pg 100.00 pg 200.00 pg 250.00 pg
-
57
8
4
4 5.6
culture was not continued*
63
5
6
3 5 6 11
3 4.6 3 5.6
culture was not continued*
1.8
1.5 3.3
17.6
16.5 17.7
Test article
300.00 pg -
75
4
5
9 6.0
2.0
17.9
Negative control Solvent control with DMSO
0.00 pg + 0.00 pg +
26 98
3 1
4 6
4 3.8 4 5.6
1.5 3.2
11.7 17.6
Solvent control with Ethanol Positive control with DMBA Test article Test article Jest article Test article Test article Test article
0.00 pg +
3.85 pg +
1 00 pg 10.00 pg 100.00 pg 200.00 pg 250.00 pg 300.00 pg
+ + + + 4* +
44
5
2
107 117
103
103
54
3
3
culture was not continued*
14
4
3
7 10
6
6
culture was not continued*
4I 11
l
5|
5 4.0
89 103.8 5 4.0
2 2.8 7 7.2
3 3.8
1.2
10.1 1.0
1.3 1.6
1.9
14.0
406.8 12 7
95 20.2
10.9
voanlluyeccoololunmiesnw8i(tthhims opraegteh)axn 15006c/evlalslu7edcaoylusmafnter9soefetdaibnlge wIIere scored concerning concentration range and toxicity four concentrations were selected to be evaluated at the end of the experiment
Sanitized. Does not contain TSCA CBI
-Page 25 of 29-
J
Test Report CCR Project 509800
Tables of Results: Experiment II
Table I: Toxicity data; experiment II
cone. per ml
S9 number of cells per flask*
mix seeded
found
mean
l/ll 1 II
CE%** CE*** absolute relative
cells/ml cell denat 1st sub- sity; % of cultivation control
column
1 23 4 5
6
7
8
9
10
Negative control (Untreated cells)
552 377
402 389.5
70.6
Negative control
Solvent control with Ethanol
0.00 pg 0.00 pg -
552 308 552 334
304 306.0 261 297.5
55.4 53.9
100 2329333 100 2437333
100 100
Positive control with EMS
0.6 mg -
552 263
229 246.0
44.6 80.4 2336667 100.3
Test article Test article
1.00 pg 100.00 pg -
552 326 552 283
310 318.0 251 267.0
57.6 48.4
106.9 89.7
2626667 2175333
107.8 89.3
Test article
200.00 pg -
552 297
281 289.0
52.4 97.1 1870000
76.7
Test article
250.00 pg -
552 256
334 295.0
53.4
99.2 2176000
89.3
Test article
300.00 pg -
552 293
291 292.0
52.9 98.2 2710667 111.2
b'3
<p
Negative control Solvent control with DMSO Solvent control with Ethanol Positive control with DMBA
0.00 pg + 0.00 pg + 0.00 pg + 3.85 pg +
501 244 501 201 501 207 501 146
196 220.0 197 199.0 213 210.0 152 149.0
43.9 39.7 41.9 29.7
100.0 100.0
2995333 3155333
100.0 3060000
74.9 2293333
100 0 100.0 100.0
72.7
lzed. Does no! contain
Test article Test article Test article Test article
test article
1.00 pg 100.00 pg 200.00 pg 250.00 pg 300.00 pg
+ + + + +
501 194 501 215 501 217
501 195
501 214
208 201.0 200 207.5 222 219.5
219 207.0 226 220.0
40.1 41.4 43.8
41.3 43.9
95.7 98.8 104.5
98.6 104.8
2958000 2173333 1767333
2904667 3184667
96.7 71.0 57.8 94 9
104.1
*** ConElyabcosololuntiees(vwailtuhemcoolruemthna6n/5v0acluelelsco7ludmayns 3afxter10se0eding were scored
0O>5
oro
*** CE relative (value column 6 / value column 6 of corresponding control x 100) -Page 26 of 29 -
"'I
Test Report CCR Project 509800
Table II: Mutagenicity data; experiment II (part 1: cell survival)
cone. per ml
S9 number of cells per flask*
mix seeded
found
mean
l/ll I
II
factor** cells calculated seeded
cells*** survived
column
1 23 4
5
6
7
8
9
Negative control
Solvent control with DMSO
0.00 pg 0.00 pg -
501 347 341 344.0 519 400 423 411.5
0.69 390000 0.79 414000
Positive control with EMS
0.6 mg -
512 314
362 338.0
0.66 456000
Test article Test article Test article Test article Test article
Solvent control with DMSO
1.00 pg 100.00 pg 200.00 pg 250.00 pg 300.00 pg
0.00 pg
+
0.00 pg +
500 366 348 357.0 512 355 370 362.5 511 385 356 370.5
0.71 0.71 0.73
culture was not continued*
510 394 399 396.5
0.78
501 419 389 404.0
0.81
502 370 355 362.5
0.72
499000 390000 408000
390000 414000 385500
Solvent control with Ethanol
0.00 pg +
501 353
348 350.5
0.70 387000
Positive control with DMBA
3.85 pg
508 338 306 322.0
0.63 390000
Test article Test article
,1.00 pg + 100.00 pg +
502 364 502 381
349 356.5 385 383.0
0.71 387000 0.76 414500
Test article
200.00 pg
500 409 383 396.0
0.79 445000
Test article
250.00 pg +
culture was not continued*
Test artfoiacnclletyorcoclaolncuielsat3we0dit0h(.v0ma0loupregectohlau+nm5n06c/elvl5sa0l7u4edi acyoslua3mf5tne7rl3s)eeding37w7ere score3d6L
0.73 433500
cells survived after plating in TG containing medium (value column 8 / value column 7)
concerning concentration range and toxicity four concentrations were selected to be evaluated at the end
267784 628249
301031 356286 276123 295820
303206 333844 278374
270746
247205 274832 316242 352440
315664
usdino
'SO i:'3I me;: dov:u r -.?3
Table III: Mutagenicity data; experiment II (part 2: mutation rates)
-Page 27 of 29 -
Test Report CCR Project 509800
cone. per ml
S9 number of mutant colonies per flask* mean standard mutant**
mix found after platine in TG medium
deviation colonies
I II
III
IV
V
per 106 cells
column
1 23 4
5
6
7 89
10
Negative control
0.00 pg -
22
1
4
4 2.6
1.3
9.7
Solvent control
with DMSO
0.00 pg -
58
1
5
4 4.6
2.5
14.0
Positive control with EMS
0.6 mg -
122 123
136
140
142 132.6
9.5
440.5
Test article Test article Test article
Test article Test article Negative control Solvent control with DMSO
1.00 pg 100.00 pg 200.00 pg 250.00 pg 300.00 pg
0.00 pg
-
-
+
0.00 pg +
13
1
0
3 1.6
1.3
12
1
4
1 1.8
1.3
73
2
1
2 3.0
2.3
culturewas not continued*
34
3
2
4 3.2
0.8
01
1
1
2 1.0
0.7
11
2
1
1 1.2
0.4
4.5 6.5 10.1
10.6 3.0 4.3
Solvent control with Ethanol
0.00 pg +
22
1
2
2 1.8
0.4
6.6
Positive control .with DMBA
3.85 pg +
119 106
116
130
133 120.8
10.9
488.7
Test article Test article Test article Test article Test article
100 pg
01
3
2
2 1.6
100.00 pg +
56
6
4
2 4.6
200.00 pg +
12
3
1
3 2.0
250.00 pg
culture was not continued*
300.00 pg + ____ SI....... 41
21
5
6 4.4
1.1 1.7 1.0
1.5
5.8 14.5
5.7
13.9
voanlluyeccoololunmiens w8i(tthhims opraegteh)axn 1500sc/evlalslu7edcaoylusmafnter9 soefetdaibnlge wIIere scored concerning concentration range and toxicity four concentrations were selected to be evaluated at the end
Company Sanrrzet. Coen re i contain TSCA CBI
-Page 28 of 29-
Test Report C C R Project 509800
DEVIATIONS TO THE PROTOCOL
There were no deviations to the protocol.
B IO M E T R Y Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.
Die bereinstimmung mit dem Original wird besttigt.
CompsBV Sanitized. Does ,,,,.corta-mfSCACB! -Page 29 of 29 -
