Document jkRdaV02LZZ4rL2547ML2Ko2
AR226-3030
r 'j
CCR PROJECT 509800
GENE MUTATION ASSAY IN CHINESE HAMSTER V79 CELLS
IN VITRO
(V79/HPRT)
WITH
REPORT Study Completion Date: October 06,1995
RCC
Company"-8'1-'308
Test Report CCR Project 509800 COPY OF GLP CERTIFICATE
HESSISCHES MINISTERIUM FOR
UMWELT. EMERGE, JUOEND, FAMDJE UND GESUNDHEIT
GLP-Bescheinigung
BMeaeialgug
Hicrmit mrd beatatigt. difi die Pnlfemrichtune(cn) CCR Cftotest CeU Research OmbH & Co. KG in 64380 Rotdorf, la dea Leppneinmrie3eal9
(Ort. Anschrift)
del RCC/CCa Holding Vmmltangi OmbH
(Finna)
am 05./06./07. April 1995
(Datum)
It ii hereby certified that the test hcility(ie;| CCR Cytatest CeU Renjrch GmbH & Co. Ka in 64380 RBfidorf, la den Leppsteinswiesen 19
(iocxciaa, dilreaa| of RCC/CCR Holding VeriTOltunf GmbH
(company onae) on 05./06./07. April 1995
Watel
voa der Xttr die Oberwachung zustAndigen Behftrden titber die Eulbaltunf der Gmadsitze der Guten Laborpfxu
fanpizien worien at (find).
m (infre) iaipeeted by the competent uthority
regitfding compiimce wita the Ptinciplea of
Good l^botssoty PrRCtxce.
Es wird hiermit beatltlgt, dafi folgende PriifUBgen ia dieser PrilfeinrichtunE nacli den Qnxndsatzea der Guten
Laborprodi duichRefahrt wcrden.
It ii hereby certified Out stadies in Chil
test facility ale conducted in compliance with
the Principlea of Oood Laboratory PraiiBcc-
PrOflntegari* aach 3 19 d Aba. 3 CheaaxUeagCEtx in der Fassung vam 29,Juli 1994 (BGBL I S. 1703). zaietzt geindcrt am 27. September 1994 (BCBI. I 3. 3705) in VerhindunK mit der AUgcmcioen Verwltangorchrift xum VerhhteivdBr behordlichen Qbennichung der Einhaltung der Grundaitxe der Guten
Labarpnads Tom 31. Oktaber 1990 (BAnx. 204 vom 31.10.1990):
Tazikolagixcbe Eigenicauten
Toxicological properties
Prafkatecorif ceiaaK OECD Panal oa Oaod Laboratory Practice (January 1993)
Prttungen auf toxikologlsche Elgenichaften PriitunEcn .ufmuugene Eigenaehaften (in jtro. in nTD)
Toiddty studies Mutagenicirr studiex
Im Auitrag Wiesbaden, den .."...Aupiat 1995
notc'sniain TSCACBt Company Sanitized. Does
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 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
4 4 4 4 5 5 6 6
10 10 10 11
11 12 12 13 14 14 16 18 18 19
20 20 21
22 22 23 26 29 29
.^ROtc3"ta'nTBCACBI
Compaq Sa.n. ^-C-3
Test Report CCR Project 509800
PREFACE
General
Sponsor:
Study Monitor: Testing Facility:
CCR Project No.
Test Article: Title:
ISEGA Forschungs- und Untersuchungsgesellschaft mbH
ZeppelinstraBe 3 D-63741 Aschaffenburg
Dr. Derra
CCR
CYTOTEST CELL RESEARCH GMBH & In den Leppsteinswiesen 19 D-64380 RoBdorf, F.R.G.
Co. KG
509800
Gene Mutation Assay in Chinese Hamster V79 Cells in vitro (V79/HPRT) wit
Project Staff
Study Director: Management: Quality Assurance Unit;
Schedule
Date of Protocol Start ofPre-Test: End ofPre-Test: Start of Experiments: End of Experiments: Date of Draft: Date of Final Report:
Dr. Hans-Eric Wollny Markus Arenz Frauke Hermann
April 13, 1995
July July
27, 1995 31, 1995
August 01. 1995
Sept. 11.I995
Sept. 13, 1995
Oct. 06. 1995
not contain TSCACBi
Compa"r?aSan'fc-^-0^3
i C3L rwpun v^^rv rii/j^i-i sjuouuu
Project Staff Signatures
Study Director
Dr. Hans-Eric Wollny
Management
Date: Octobers, 1995
J
Markus Arenz
r- {
^
M?ft L\J^ci^^(J
Date: October 06, 1995
Good Laboratory Practice
The study was performed in compliance with:
Chemikaliengesetz ("Chemicals Act") of the Federal Republic of Germany, Aniage 1
("Annex I"), dated July 25, 1994 (BGBL. 11994 S. 1703)."
"The OECD Principles of Good Laboratory Practice", Paris 1981.
c^ainTSCACBl
Company .S^aut-itzEezedo. D" oes not
-Pnfff 'inf?') -
Test Report CCR Project 509800
Guidelines
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 RofidorW.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 2 years following the date on which the report is audited by the Quality Assurance Unit:
sample of the test article No raw data or material relating to the study will be discarded without the sponsor's prior
consent.
r^rrnt^- Compaq Sanitized^ . Does contain TSCACBi
Test Report CCR Project 509800
STATEMENT OF COMPLIANCE
Project Number:
509800
Study Director:
Dr. Hans-Eric Wollny
Title:
Gene Mutation Assay in Chinese Hamster V79 Cells
in vitro (V79/HPRT) witf
This study performed in the testing facility of C C R was conducted in compliance with
Good Laboratory Practice Regulations.
Chemikaliengesetz ("Chemicals Act") of the Federal Republic of Germany, Aniage 1 ("Annex I"), dated July 25, 1994 (BGBL. 11994 S. 1703)."
"The 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. Hans-Eric Wollny
Date: W^^, IW
Company Sban.di.e^d". Coes not c^nTSCACSl
n-,.- " ..nn
Test Report CCR project auaauu
QUALITY ASSURANCE UNIT
;C C R, Cytotest Cell Research GmbH & Co. KG, In den Leppsteinswiesen 19, D-64380 RoBdorf, F.R.G.
Statement
Project Number:
509800
Test Material:
Study Director: Title:
Dr. Hans-Eric Wbllny
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 ofQAU Inspections/
Audits
Protocol Audit: Process Inspection Draft Audit:
April 18, 1995
August 11, 1995 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
..........^..-....L^..^^1^...
Date: tc^s^ pfc.JW
Compa^ ny Sanitized. Does net contain TSCA CB! n-,.--. o -^r in
Test Report CCR Project 509800
SUMMARY
^^^i-^^ft The study was performed to investigate the potential
ohiSHmUS^leUrJ| tations at the HPRT locus in V79 cells of the Chinese
to induce gene mu
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 ng/ml with S9 mix: 1.0; 10.0*; 100.0; 200.0; 250.0* and 300.0 ug/ml
Experiment n:
without S9 mix: 1.0; 100.0; 200.0; 250.0* and 300.0 ug/ml with S9 mix: 1.0; 100.0; 200.0; 250.0* and 300.0 ug/ml
No relevant toxic effects occurred up to the limit of solubility and above. The highest con centration (300 ug/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 of 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. ,
ThereforeMHBHB|is considered to be non-mutagenic in this HPRT assay.
* not evaluated, culture not continued
Company Sanitized. Doe's ''."I e-'.nfa'n TSCA CSS
I esi nepon L,L,K t-rojeci auaauu
OBJECTIVE
Aims of the 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 of the treated original
population are monitored for the loss of functional HPRT enzyme. HPRT (hypoxanthine-guanine phosphoribosyitransferase) catalyzes the conversion of the nontoxic 6TG (6-thi6guaiune) to its toxic ribophdsphorylatedderivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6TG. These cells are able to proliferate in the presence of 6TG whereas the non-mutated cells die. However, the mutant phenotype requires a period of 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 of mutant colonies corrected for cell survival.
In order to establish a concentration response effect of 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.
cwtalnT.SC.ACBl
Compaq Sam-.-S-A^n---D-n-"<--1t "
Qnm. in ^r in
Test Report CCR Project 509800
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:
liquid yellow
Analysis:
Purity:
Stability Stability
pure: In solvent:
not indicated by the sponsor not indicated by the sponsor
Storage:
4C
Expiration Date:
not indicated by the sponsor
On the day of 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^a, nd its non-toxicity for the cells. The final concentration
ofDMSO in the culture medium did not exceed 1 % v/v.
Wft Compan,y,S,l,<,d.D..,. ,..""
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 Munchen, 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, D 3254; (Purity: approx. 95%) DMSO, Dimetnylsulfoxide;
final concentration in nutrient medium 1 %
3.85 ug/ml =15.0 uM
F.R.G.
The stability of both positive control substances in solution is unknown, but a mutagenic response in the expected mutation range is sufficient evidence of biological stability. The
dilutions of 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 cells have a stable karyotype with a modal chromosome number of 22 (3).
C^^.d.o^..c...,TS^r.B,
-Page 12 of 29-
Test Report CCR Project 509800
Cell Cultures
Large stocks of 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
CCR allowing the repeated use of the same cell culture batch in experiments. Before free
zing, the level of 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 of 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 5xl05 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 subcultured-twice 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 ug/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 Fullinsdorf weight approx. 220 - 320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Ettlingen. 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 was diluted 1+3 in KC1 and centrifiiged at 9,000 g for 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 ofBio-Rad Laboratories, D-80939 Miinchen: Bio-Rad protein assay. Catalogue 500 000 6 (6).
The protein concentration in the S9 preparation was 33.2 mg/ml (lot 220595).
,^S,d.Do..n~.con,..nTSCAca
-Pilge 13 of 29-
Test Report CCR Project 509800
59 Mix
. An appropriate quantity ofS9 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:
8mM 33 mM
5 mM
4mM
MgClz KC1 glucose-6-phosphate 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 mutagenicity 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 ofXTT-labelling 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.
-^^nTS^51
sempa^ny n"-"^ed-O^^'
-Page 14 of 29-
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 concentrationrelated 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 of 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 nig/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 nm) was not significantly reduced after treatment with concentrations up to the limit of solubility at 300 ug/ml with and without metabolic activation (see table PRE-TEST OF TOXICITY).
Experiment I and II were performed with four concentrations ranging from 1.0 to 300.0
ug/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 ug/ml with S9 mix: 1.0; 100.0; 200.0 and 300.0 ug/ml
Experiment II:
without S9 mix: 1.0; 100.0; 200.0 and 300.0 ug/ml with S9 mix: 1.0; 100.0; 200 J3 and 300.0 ug/ml
c,^,-,..-.'".1-------'"^3"81
-Pnge 15 of 29 -
Test Report CCR 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):
NaCI
. K C 1Glucose
NaHCO^
8000 mg 400 mg 1000 mg 350 mg
Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing
200 mg/1EDTA (ethylene diamine tetraacetic acid).
The cell suspension was seeded into plastic culture flasks (Greiner, D-72632 Frickenhausen). Approximately 1.5xl06 (single culture) and 5xl02 cells (in duplicate) were seeded in
MEM with 10 % PCS (complete medium) for the determination of 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 pi/mi 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):
NaCI KC1
Glucose Na2HP04x7H20 KHzP04
8000 mg 400 mg 1100 mg
290 mg 150 mg
pH is adjusted to 7.2
Experimental Scheme:
Segment a): Procedure for determination oftoxicity
Segment b): Procedure for determination of mutation rates
C
o
m
^S
paq
aJ
n
W
^
-
0
0
'
-
3
^^TSCACBl
-Page 16 of 29-
Test Report CCR Project 509800
Day 1: Subculturing of a log-phase culture which showed an initial spontaneous mutation rate at the beginning of the experiment of 14.0 (experiment I) and 2.9 (experiment II) mutants per 106 cells.
a) About 500 cells in 5 ml medium/25 cn^-plastic-flask for cloning efficiency; in duplicate per experimental point
b) IxlO6 cells in 30 ml medium/175 cn^-plastic-flaskfor the mutagenicity test, 1 flask per
experimental point
Day 2: Treatment of a) and b)
experiment I
Day'5: Subculturing ofb) in 175 cm^plastic-flasks1.5X106 cells in 30 ml medium/175 cm2plastic-flasks
experiment II
Day 6: see day 5
Day 8: Fixation and staining of colonies in a)-flasks determination of concentration-related cloning efficiency
Day 9: Subculturing ofb) in five 80 cn^-plastic-flaskscontaining selective medium: mutant selection (about 3-5xl05 cells/flask); Subculturing ofb) in two 25 cn^-flasks 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).
f
Day 18: Fixation and staining of colonies in b) -
selection).
derived flasks seeded on day 9 (mutant
The cultures were incubated at 37 C in a humidified atmosphere with 4.5 % COz. The co
lonies were stained with 10 % methylene blue in 0.01 % KOH solution (E. MERCK, D-
64293 Darmstadt, F.E..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 Dusseldorf, F.R.G.).
TSCACBI
^ ^ Q ^..,....,contain -Page 17 of 29-
Test Report CCR 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 106 cells found in the negative and/or solvent con
trols fall within the laboratory historical control data range: 0-45 mutants/tO6 cells.
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. of the
untreated cultures].
Compsr.y "-a,-z.-^-.--'e-^d- CL----aJs net conSasn TSCA CSi
-P-IUP lit nOO -
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 of 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.
Comply Sani- tze^ dn.D^o.-.'---S.tc^ontain TSCACS8
-Page 19 of 29-
Test Report CCR Project 509800
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) mean standard deviation
% of the corresponding control*
0.18 1.22 1.17 1.19 1.21
0.00 0.03 0.02 0.04 . 0.07
0.00 105.57 100.00 102.35 104.17
1.16
0.03
99.44
1.14 1.08 1.12 1.10 1.13
0.04 0.05 0.06 0.06 0.07
97.16 91.36 95.14 93.33 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 (JQ/ml
^ / I 1 0.300 1.000 3.000
10.000 30.000 100.000 200.000 300.000
extinction (450/690 nm)
mean
standard deviation
0.178
0.007
1.009
0.054
0.966
0.048
0.926
0.074
0.914
0.069
0.872
0.038
0.852
0.032
0.829
0.030
0.775
0.024
0.716
0.032
0.688
0.063
% of the corresponding control*
0.000 105.361 100.000 94.893 93.306 87.977 85.534 82.568 75.763 68.181 64.724
* corrected with the blanc
Co.?.^,^.--------------"""63'
-p-iup 70 r>no
Test Report CCR Project 509800
RESULTS AND DISCUSSION
articljgHmH^vas : The test
assessed for its potential to induce gene mutations at
the HPRT locus using V79 ceMS*ofthe Chinese hamster.
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 of 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 of a concentration depend increase of mutant colonies.
nibOthexperiinenfsoffhis study (with andwithout S9 mix) the range of the n'egative 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 106 cells.
EMS (0.6 mg/ml) and DMBA (3.85 ug/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
conditions reported the test article did not induce gene mutations at the HPRT locus in V79
cells.
Compar^-y ^^aswl''wwt
Test Report CCR Project 509800
REFERENCES
: 1. B.N. Ajmes, J. McCann, and E. Yamasaki 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, 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 of the gene-tox program Mutation Research 87, 81-142, 1981
4. S. Rettig Modellierung, Simulation und statistische Analyse des HGPRT-Mutagenitatstests, 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 Study Director
2x (original, copy) 1x (copy)
Comp^S^ed..oesnotc^nTB--
-P;iee22of29-
Test Report CCR Project 509800
Annex: Tables of Results Experiment 1
Table I: Toxicity data; experiment I
cone. per ml
S9
number of cells per flask*
mix seeded
found
mean
l/ll
1
II
CE%**
absolute
/L^/c*** relative
cells/ml at 1st subcultivation
cell den sity; % of control
column
1
2
3
4
5
6
7
8
9
10
Negative control (Untreated cells)
Negative control
0.00 ug -
523 306 523 236
301
303.5
274 255.0
Solvent control with Ethanol
0.00 ug -
523 214
266 240.0
Positive control with EMS
Test article
0.6 mg 1.00 ug -
523 189 523 253
201
195.0
245 249.0
Test article
10.00 ug -
523 203
244 223.5
Test article
1 oo.oo MQ -
523 234
214 224.0
Test article
200.00 ug -
523 197
184 190.5
Test article
250.00 ug "
523 193
183 188.0
Test article Negative control
300.00 ug 0.00 ug +
523 175 52'3l 269
204 189.5 214 241.5
Solvent control with DMSO
0.00 ug +
523 252
214 233.0
Solvent control with Ethanol
0.00 ug +
523 224
255 239.5
Positive control with DMBA
3.85 jjg +
523 158
179 168.5
Test article
1.00)jg +
523 200
195 197.5
Test article
10.00 ug +
523 186
211
198.5
Test article
100.00 pg +
523 207
226 216.5
Test article
200.00 ug +
523 241
223 232.0
Test article
250.00 ug +
523 237
222 229.5
Test article
300.00 pg +
523 229
233 231.0
only colonies with more than 50 cells 7 days after seeding were scored
CE absolute (value column 6 / value column 3 .\ 100 CE relative (value column 6 / value column 6 of corresponding control x
58.0
48.8 45.9
37.3 47.6 42.7 42.8 36.4 35.9 36.2 46.2 44.6
45.8
32.2 37.8 38.0 41.4 44.4 43.9 44.2
100)
100 2011333 100 2098000
76.5
103.8 93.1 93.3 79.4 78.3 79.0 100.0
100.0
1779333
2258667 2312000 1848667 1880667 1876667 1875333 2190000
2272000
100.0 2468000
72.3
82.5 82.9 90.4 96.9 95.8 96.5
1542667
2346667 2476667 2646000 2497333 2060667 2444667
100
100
88.5
107.7 110.2
88.1 89.6 89.5 89.4 100.0 100.0
100.0
67:9
95.1 100.4 107.2 101.2
83.5 99.1
-Page 23 of 29-
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
I
II
factor" cells calculated seeded
cells*** survived
column
1
2
3
4
5
6
7
8
9
Negative control
0.00 ug -
505 296
301
298.5
0.59
435000
257124
Solvent control with DMSO
0.00 ug -
513 303
353
328.0
0.64
414000
264702
^sitive control with EMS
0.6 mg -
504 318
325
321.5
0.64
414000
264089
Test article
I.OOpg -
504 347
365
356.0
0.71
450000
317857
Test article
10.00pg -
culture was not continued"
Test article
100.00 pg -
500 325
360
342.5
0.69
408000
279480
Test article
200.00 pg -
511
398
406
402.0
0.79
402000
316250
Test article
250.00 ijg -
culture was not continued"
Test article
300.00 ug -
503 380
410
395.0
0.79
426000
334533
Negative control Solvent control with DMSO
0.00 pg + 0.00 pg +
503 381
409
395.0
500 336
328
332.0
0.79 0.66
414000 478500
325109 317724
Solvent control with Ethanol
0.00 pg +
509 362
372
367.0
0.72
396000
285525
Positive control with DMBA
3.85 jjg +
502 291
302
296.5
0.59
432000
255155
Test article
1-OOug +
507 345
342
343.5
0.68
463500
314028
Test article Test article
10.00 pg + 100.00 pg +
culture was not continued"
511
366
349
357.5
0.70
420000
293836
Test article
200.00 pg +
502 375
362
368.5
0.73
486000
356755
Test article Test article
250.00 pg + 300.00 pg +
507'|
culture was not continued*
358
317)
337.5 I
0.67
522000
347485
only colonies with more than 50 cells 7 days after seeding were scored factor calculated (value column 6 / value column 3)
cells survived after plating in TG containing medium (value column 8 / value column 7)
concerning concentration range and loxicity four concentrations were selected to be evaluated at the end of the experiment
-Page 24 of 29-
Test Report CCR Project 509800
Table III: Mutagenicity data; experiment I (part 2: mutation rates)
cone.
S9
per ml
mix
column
Negative control Solvent control with DMSO Positive control with EMS Test article
Test article
Test article
Test article
Test article
Test article Negative control Solvent control with DMSO Solvent control with Ethanol Positive control with DMBA Test article Test article Test article Test article Test article Test article
1
2
0.00 ug " 0.00 ug -
0.6 mg -
1.00 ug 10.00 ug 100.00 ug 200.00 ug 250.00 ug 300.00 ug -
o.oo ug +
0.00 ug +
0.00 ug +
3.85 ug +
I.OOug + 10.00 ug + 100.00 ug + 200.00 Mg + 250.00 ug + 300.00 yg +
number of mutant colonies per flask*
found after plating in TG medium
I
11
III
IV
V
3
4
5
6
7
6
7
2
5
5
2
4
3
mean
a
standard mutant** deviation colonies
per 106 cells
9
10
3
4.6
3
3.4
2.1 < 1.1
17.9 12.8
137
5
6 3
7 2 9
118
117
151
7
8
4
culture was not continued"
3
5
6
5
6
11
culture was not continued"
5
4
5
6
3
4
8
1
6
136
4
i i
9 4 4
131.8 5.6
4.6 5.6
6.0 3.8 5.6
14.3 1.8
1.5 3.3
2.0 1.5 3.2
499.1 17.6
16.5 17.7
17.9 11.7 17.6
4
4
5
2
5
4.0
1.2
14.0
107 5
1 7
4|
117
103
103
4
-a
3
culture was not continued"
4
4
3
10
6
6
culture was not continued*
1|
6
5
89 103.8
5
4.0
2
2.8
7
7.2
3
3.8
10.1 1.0
1.3 1.6
1.9
406.8 12.7
9.5 20.2
10.9
only colonies with more than 50 cells 7 days after seeding were scored
value column 8 (this page) x 106 / value column 9 of table II
concerning concentration range and toxicity four concentrations were selected to be evaluated at the end of the experiment
-Page 25 of 29-
Test Report CCR Project 509800
Tables of Results: Experiment II
Table I: Toxicity data; experiment II
cone. per ml
39
number of cells per flask*
mix seeded
found
mean
l/ll
1
II
CE%*-* absolute
^C*n-*
relative
cells/mi at 1st subcultivation
cell den sity; % of
control
column
1
2
3
4
5
6
7
8
9
10
Negative control (Untreated cells) Negative control Solvent control with Ethanol Positive control with EMS Test article
Test article
Test article
Test article
Test article
Negative control Solvent control with DMSO Solvent control with Ethanol Positive control with DMBA Test article Test article Test article Test article Test article
0.00 pg -
0.00 pg -
0.6 mg I.OOpg 100.00 pg 200.00 pg 250.00 pg 300.00 |jg 0.00 ijg + 0.00 pg +
0.00 pg +
3.85 pg + 1-OOpg + 100.00 pg + 200.00 pg + 250.00 pg + 300.00 pg +
552
552
552
552 552 552 552 552 552 501 501
501
501 501 501 501 501 501
377
308
334
263 326 283 297 256 293 244 201
207
146 194 215 217 195 214
402 304
261
229 310 251 281 334 291 196 197
213
152 208 200 222 219 226
389.5
306.0
297.5
246.0 318.0 267.0 289.0 295.0 292.0 220.0 199.0
210.0
149.0 201.0 207.5 219.5 207.0 220.0
70.6
55.4
53.9
44.6 57.6 48.4 52.4 53.4 52.9 43.9 39.7
41.9
29.7 40.1 41.4 43.8 41.3 43.9
100
100
80.4 106.9
89.7 97.1 99.2 98.2 100.0 100.0
100.0
74.9 95.7 98.8 104.5 98.6 104.8
2329333
2437333
2336667 2626667 2175333 1870000 2176000 2710667 2995333 3155333
3060000
2293333 2958000 2173333 1767333 2904667 3184667
100
100
100.3
107.8 89.3 76.7 89.3
111.2 100.0 100.0
100.0
72.7 96.7 71.0 57.8 94.9 104.1
only colonies with more than 50 cells 7 days after seeding were scored CE absolute (value column 6 / value column 3 x 100 CE relative (value column 6 / value column 6 of corresponding control x
100)
-Page 26 of 29-
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
2
3
4
5
6
7
8
9
Negative control
0.00 ug -
501
347
341
344.0
0.69
390000
Solvent control with DMSO
0.00 ug
519 400
423
411.5
0.79
414000
'ositive control with EMS
0.6 mg -
512 314
362
338.0
0.66
456000
Test article
I.OOug -
500 366
348
357.0
0.71
499000
Test article
100.00 ug -
512 355
370
362.5
0.71
390000
Test article
200.00 ug -
511
385
356
370.5
0.73
408000
Test article
250.00 ug -
culture was not continued*
Test article
300.00 |jg -
510 394
399
396.5
0.78
390000
Negative control Solvent control with DMSO
0.00 ug + 0.00 ug +
501 419
389
404.0
502 370
355
362.5
0.81 0.72
414000 385500
Solvent control with Ethanol
0.00 ug +
501
353
348
350.5
0.70
387000
Positive control with DMBA
3.85 [ig +
508 338
306
322.0
0.63
390000
Test article Test article Test article
I.OOug + 100.00 ug + 200.00 ug +
502 364
349
356.5
502 381
385
383.0
500 409
383
396.0
0.71 0.76 0.79
387000 414500 445000
Test article
250.00 ug +
culture was not continued*
Test article
300.00 ug| +
504^ 357
377
367.0
0.73
433500
only colonies with more than 50 cells 7 days after seeding were scored factor calculated (value column 6 / value column 3) cells survived after plating in TO containing medium (value column 8 / value column 7)
concerning concentration range and toxicity four concentrations were selected to be evaluated at the end
267784 828249
301031 356286 276123 295820
303206 333844 278374
270746
247205 274832 316242 352440
315664
Table III: Mutagenicity data; experiment II (part 2: mutation rates)
-Page 27 of 29.
Test Report OCR Project 509800
cone.
S9
per ml
mix
column
Negative control Solvent control with DMSO Positive control with EMS Test article
Test article
Test article
Test article
Test article Negative control Solvent control with DMSO Solvent control with Ethanol Positive control .with DMBA Test article Test article Test article Test article Test article
1
2
0.00 pg 0.00 [IQ "
0.6 mg -
1.00 ug 100.00 pg 200.00 pg 250.00 pg 300.00 ug -
0.00 ug +
0.00 ug +
0.00 ug +
3.85 ug +
1-OOpg + 100.00 Mg + 200.00 |jg + 250.00 ug + 300.00 ug +
number of mutant colonies per flask*
found after plating in TG medium
I
II
III
IV
V
3
4
5
6
7
mean
8
standard mutant** deviation colonies
per 10" cells
9
10
2 5
122 1 1 7
3 0 1
2
1
4
8
1
5
123
136
140
3
1
0
2
1
4
3
2
1
culture^was not continued*
4
3
2
1
1
1
1
2
1
4 4
142
3 1 2
4 2 1
2.6 4.6
132.6 1.6 1.8 3.0
3.2 1.0 1.2
1.3 2.5
i
9.5 1.3 1.3 2.3
0.8 0.7 0.4
9.7 14.0
440.5 4.5 6.5
10.1
10.6 3.0 4.3
2
2
1
2
t
1.8
0.4
6.6
119
0 5 1
5
106
116
130
133 120.8
1
3
2
t
1.6
6
6
4
4.6
i
3
1
<
2.0
culture was not continued"
4
2
5
6
4.4
10.9
1.1 1.7 1.0
1.5
488.7 5.8
14.5 5.7
13.9
only colonies with more than 50 cells 7 days after seeding were scored
value column 8 (this page) x 106 / value column 9 of table II
concerning concenu-ation range and toxicity four concentrations were selected to be evaluated at the end
-Page 28 of 29-
DEVIATIONS TO THE PROTOCOL
There were no deviations to the protocol.
BIOMETRY-
Since the distribution of mutant cells does not follow known statistical models, an adequate
statistical method is not available.
Die Ubereinstimmimg mit dem Original wird bestStigt.
,teolamTSCACBl ,,,S-rf.feed.Doesntf comply ^
-Pnge29of29-