Document LK0YBd05R7qnLp9DBKmZY584X
AR226-2996
. STUDY TITLE: CHROMOSOME ABERRATION ASSAY
IN CHINESE HAMSTER V79 CELLS IN VITRO
REPORT
DATA REQUIREMENTS: OECD Guideline 473 EEC Directive 92/69
EPA 40 CFR Ch. 1 (7-1-86 ed. 798.5385) AUTHOR:
Dr. Wolfgang Volkner STUDY COMPLETION:
July 06, 1993 PERFORMING LABORATORY: CCR - Cytotest Cell Research GmbH & Co. KG P.O. Box 1252, D-6101 Rossdorf, Germany .LABORATORY PROJECT ID:
CCR 326430
SPONSOR: ISEGA Forschungs- und Untersuchungsgesellschaft mbH
D-8750 Aschaffenburg
Study Monitor Heike Kramer
Page 1 of 32
RCC
Group
Co.P.nyB-zed.Don,-.co-,,a,TSCACBI
COPY OF GLP CERTIFICATE
^^S^
HESSISCHES MIN1STER1UM . . FOR UN-WELT, ENERGIE UND BUNDESANGELEGENHE1TEN
GLP-Bescheinigung
Beachelnlgung
Hiennit wird bestaligt, da6 die Priifungseinrichlung(an)
Cytotest Cell Research GmbH & Co KG In den Leppst'einswiesen TS
in.610-1.^0.'?.!0!:.^..--....-........__--.-,,......-,,
(On. /reoirifi)
(jgr.RCC_Holding Verwaltung 6nbH_
tFinTii)
03-08., 04.08., 05.08.- undj6.08.92
(Datum)
von der fur die Uberwachung zusiandigan BahOrde uber die Einhaltung der Grundsitze dar Gutan Labcrprsuds , Inspia'ert worden ist (sind).
Es wird hiermit bestSL'gt, daO (olgenda Priifungan in
dieser Priifeinrichlung nach den Grundsitzan dar Gutan
Laborpraxis durchgetuhn werden.
'
.
Certincata
It is hereby csnitied lhat Ihe lest ladlity(ies)
Cytotest Cell Research GmbH & Co KG
In den Lepps'teTnswi'e'sen \'5~"~~"
610'1 RoBdorf
.
Oocalicn, iddrttu)
RCC Holding Venfaltung GmbH
of (company nuna)
03.08., 04.08., 05.08. and.06.08.92
(ctab,)
was (wara) inspedBd by the compatent authority regar ding conipliancB with lha Pririciples o( Good Laboratory Practica.
It is hareby ceni(ied lhat studies in this test (acility ara conducted in comptianca with tfia Prindples of Good Laboratory Practica._
Toxikologiscne Eigenschaf'
Toxicological. proDerties
Im Auftrag
' .
CF^.</Gtt^L-r
(Or .Heeler)
Wiesbaden, den ^S-
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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 Fraction S9 Mix
Pre-Test for Toxicity Dose Selection
Experimental Performance Data Recording
Acceptability of the Assay Evaluation of Results
BIOMETRY
RESULTS
Tables of Results
Pre-Test for Toxicity
EXPERIMENT I
Number of Polyploid Cells
and Mitotic Index Structural Chromosomal Aberrations
EXPERIMENT II
Number of Polyploid Cells
and Mitotic Index Structural Chromosomal Aberrations
PAGE
2
10 10 10 11 11 11 13 14 15 15 16 18 18 19 20 21 21 21 22 22 23 26 26 27
DISCUSSION
30
REFERENCES
32
1
DISTRIBUTION
32
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PREFACE
GENERAL
Sponsor:
Study Monitor;
Testing Facility;
CCR Project No,
Test Article: Title:
ISEGA Forschungs- und Untersuchungsgesellschaft mbH D-8750 Aschaffenburg
He ike Kramer
CCR
CYTOTEST CELL RESEARCH GMBH & CO. KG
D-6101 RoMorf
326430
Chromosome Aberration Assay in
Chinese Hams terV7 9 Cells in vitro
withj------------------i
PROJECT STAFF
Management:
Study Director; Author: Head of Quality Assurance Unit: Technical Coordination:
Dr. Hans-Emil Knoell Dr. Albrecht Heidemann Dr. Wolfgang Volkner
Dr. Christiane Helmrich
Frauke Klihner
SCHEDULE
Date of Protocol: Start of Pre-Test:
Start of Experiment I:
End of Experiment I:'
Start of Experiment II;
End of Experiment II:
Date of Draft:
Date of Report:
November 11, 1992 December Ol/ 1992
March May March May
01, 1993 24, 1993
29, 1993 05, 1993
May
26, 1993
July
06, 1993
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'
PROJECT STAFF SIGNATURES
Study Director:
Author:
Management:
Dr. Albrecht Heideman
CA<sA-?oC4 . Date: July 06, 1993
Dr. Wolfgang Volkner
L<). Ucll^)
Date: July 06, 1993 Dr. Hans^Emil Knoell
Date:
^- l
i
y
,
^
06
.
,
.
.1
.9.9.3
QUALITY ASSURANCE
;
\1
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)."
"The OECD Principles of Good Laboratory Practice", Paris 1981.
GUIDELINES
''
This study was conducted according to the procedures indicated
by the following internationally accepted guidelines and recom
mendations :
First Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 473,-adopted May 26, 1983, vitro Mammalian Cyto-
genetic Test"
EEC Directive 92/69, L 383 A, Annex V, B 10, dated December 29,
1992.
Environmental Protection Agency, Code of Federal Regulations,
Title 40, Subpart F-Genetic Toxicology, Revision July 1, 1986, "In vitro mammalian cytogenetics"
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ARCHIVING
C C R, D-6101 Rofidorf will archive the following data for
3 0 years:
raw data, protocol and copy of report.
The following specimen and samples will be archived for at least
12 years:
sample of test article, microscopic slides.
No raw data or material relating to the study will be discarded
without the sponsor's prior consent.
0
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STATEMENT OF COMPLIANCE
Project Number;
Test Article:
Study Director;
Title:
326430
Dr. Albrecht Heidemann
Chromosome Aberration Assay jji Chinese
Hamster V79 Cells in vitro wit}
To the best of my knowledge and belief, this study performed in
the testing facility of CCR was conducted in compliance with the
Good Laboratory. Practice Regulations:
Chemikaliengesetz ("Chemicals Act") of the Federal Republic of
Germany, Aniage 1 ("Annex I"), dated March 14, 1990 (BGBL. I
S. 521)."
"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. Albrecht Heidemann
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QUALITY ASSURANCE UNIT
C C R, Cytotest Cell Research GmbH & Co KG, In den Leppsteinswiesen 19, D-6101 RoBdorf
STATEMENT
Project Number;
Test Article:
Study Director;
Title:
326430
Dr. Albrecht Heidemann Chromosome Aberration Assay ^p Chines (
Hamster V79 Cells in vitro with^
This report was audited by the Quality Assurance Unit and the
study and/or testing facility were inspected on the following
dates.
Dates of QAU Inspections / Audits
November
March
April April
June
II/ 1992
02, 1993 02, 1993 21, 1993 28 ,1993
Dates of Reports to the Study Director and to Management
November
March
April April
June
11, 1992 02, 1993 02, 1993 21, 1993 28 ,1993
Head of Quality Assurance Unit
Dr. Christiane Helmrich
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Date: ^A r^, -W^
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SUMMARY
The test article|JHB----^B9| was assessed for its potential to induce structuraj^chromosomSrf aberrations in V79 cells of the
Chinese hamster in vitro in two independent experiments.
The chromosomes were prepared 18 h and 28 h after start of treat ment with the test article. The treatment interval was 4 h with /metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal
aberrations.
The following concentrations* were evaluated (18 h: 3 concentra tions; 28 h: highest concentration):
Experiment I
.'
without S9 mix: 18 h: 30; 100; 300 ng/ml
28 h: 300 ug/ml
with S9 mix:
18 h: 30; 100; 300 ng/ml 28 h: 300 ng/ml
Experiment .II -
without S9 mix: 18 h: 30; 100; 200 ng/ml 28 h: 200 ng/ml
with S9 mix:
^
18 h: 30; 100; 200 'ug/ml
28 h: 200 ng/ml
The concentration range of the test article was determined in
pre-experiments. In experiment II the highest concentration was
200 ng/ml due to the limited solubility of the test article.
The colony forming ability of V79 cells as indicator for a toxic-
ity response was not affected by treatment with concentrations up
to 300 ng/ml of the test article (with and without S9 mix).
In the absence of S9 mix, in experiment I the mitotic index was
slightly reduced after treatment with the test article. However,
this effect could not be confirmed in experiment II.
In the presence of S9 mix in both experiments the mitotic index
'
was
not reduced.
In both independent experiments, there were no statistically relevant increases in'cells with structural aberrations after treatment with the test article. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test article as compared to the controls.
Appropriate reference mutagens were used as positive controls and
showed distinct increases in cells with structural chromosomal
aberrations.
CONCLUSION
In conclusion, it can be stated-that in the study described
under the experimental conditions reported, the test article
and
did
not induce structural chromosomal aberrations as determined by
the chromosomal aberration test in the V79 Chinese hamster cell
line.
* cannot be given accurately; see page 15
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OBJECTIVE
AIMS OF THE STUDY
This in vitro assay was performed to assess the potential of the test article to induce structural chromosomal aberrations by
means of two independent chromosomal aberration experiments in .the Chinese hamster cell line V^9.
REASONS FOR THE STUDY
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 capacity for
metabolic activation of potential mutagens an exogenous metabolic activation system is necessary.
This in vitro test is an assay for the detection of structural
chromosomal aberrations. These aberrations are frequently lethal to the damaged cells. However, cytogenetic damage in somatic cells is an indicator of a potential to induce more subtle chro mosomal damage that is compatible with cell division. Similar damage induced in germinal cells may lead to heritable cyto genetic abnormalities. Heritable cytogenetic abnormalities are known to have deleterious effects in man, e.g. induction of neoplastic events or birth'defects.
The VPS cells were exposed to the test article both with and without exogenous metabolic activation. The cells were then
harvested at sequential intervals and chromosome preparations were made. The stained preparations were examined and metaphase cells were scored for chromosomal aberrations.
Chromosomal aberrations are generally evaluated in first post
treatment mitoses. With the majority of chemical mutagens, in duced aberrations are of the chromatid type, but chromosome type aberrations also occur.
/
The time at which the aberration frequency is at the maximum va
ries from agent to agent. Because different chemicals have ef fects at different parts of the cell cycle and VPS cultures are
asynchronous, multiple post-treatment sample times are necessary to precisely define the response. Due to mitotic delay or meta
bolic and pharmacokinetic effects the appearance of the first
post-treatment mitosis can be considerably delayed. Therefore
samples taken at 18 h and 28 h after beginning of treatment cover the intervals in which maximum aberration frequency is expected. For the assessment of clastogenic activity three concentrations were evaluated at the central sampling time of 18 h and one con centration at 28 h. The highest concentration should exhibit a
cytotoxic effect, if possible.
To validate the test, reference mutagens were tested in parallel
to the test article.
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.Company Sanfnzstf. DSBS nsf eonSsm TSCA CB(
MATERIALS AND METHODS
THE TEST ARTICLE
The test article and the information concerning the test article
were provided by the sponsor.
Name:
Batch No. :
not indicated by the sponsor
Chemical name:
Aggregate State at RT:
Colour:
Analysis: Molecular Weight;
liquid not indicated by the sponsor not indicated by the sponsor
Purity:
Stability:
Storage: Expiration Date:
35% in Isopropanol and H^O
/
a) Pure:
not indicated by the sponsor
In solvent: not indicated by the sponsor
4 C
not indicated by the sponsor
On the day of the experiment (immediately before treatment), the
test article was dissolved in ethanol (E. Merck, D-6100 Darm stadt; purity S 99 % ). The solvent was chosen according to its solubility properties and its non-toxicity to the cells. The final concentration of ethanol in the culture medium did not
exceed 1 % v/v.
D
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THE CONTROLS
The Negative Controls Concurrent negative (culture medium) and solvent controls (ethanol) were performed.
The Positive Control Substances
Without metabolic activation
i
Name:
Supplier: Catalogue no.: Dissolved in: Final Concentration: Solution prepared on
EMS; Ethylmethanesulfonate
Merck-Schuchardt, D-8000 Munchen
820774 (purity > 98 %) Nutrient medium
0.4 mg/ml = 3.25 mM
day of experiment.
4
. j,
. /'
; '/
^' ,
f$- '
The stability of the positive control substance in solution was
proven by the mutagenic response in the expected range.
With metabolic activation
Name:
Supplier: Catalogue no.: Dissolved in: Final Concentration:
^.GPA^Cyclophosphamide
'
SERVA, D-6900 Heidelberg
17681 (purity >. 99.5 %)
nutrient medium 0.93 ng/ml = 3.3 oM (in deviation
cpl where 2.8 pg/ml = 10.0 uM was
to protogiven)
The stability of CPA at room temperature is good. At 20 C only
1 % of CPA is hydrolysed per day in aqueous solution.
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THE TEST SYSTEM
Reasons for the Choice of the Cell Line V79
The V79 cell line has been used successfully for many years in ^n vitro experiments. Especially the high proliferation rate
(doubling time of clone V79/T5 in stock cultures: 12 h, deter mined on October 22, 1992) and a high plating efficiency of untreated 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.
Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays with cell cultures as many chemicals only develop mutagenic potential when they are metabolized by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with liver microsome preparations (S9 mix).
Cell Cultures
Large stocks of the V79 cell line (supplied by LMP, Technical University Darmstadt, D-6100 Darmstadt) were 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 freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experi ments remained similar because of the reproducible characteris tics of the cells.
"
)
Thawed stock cultures were propagated at 37 C in 80 cm-- plastic flasks (GREINER, D-7443 Frickenhausen). About 5 x 105 cells per flask were seeded in 15 ml of MEM (minimal essential medium;
Biochrom KG? D-1000 Berlin 46)' supplemented with 10 % fetal calf serum (FCS; Biochrom). The cells were subcultured twice weekly. The cell cultures were incubated at 37 C in an atmosphere with 4.5 % carbon dioxide- (95.5 % air).
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MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 (Preparation by C C R)
8-12 The S9 liver microsomal fraction was obtained from the livers of
weeks old male rats, strain Wistar/WU (SAVO, med. Ver-
suchstierzuchten GmbH, D-7964 Kisslegg; weight approx. 150 - 200
g) which received a single i.p. injection of 500' ing/kg b.w. Aroclor 1254 (Antechnika, D-7500 Karlsruhe) in olive oil 5 days
previously.
After cervical dislocation the livers of the animals were re
moved, washed in 150 mM KC1 and homogenized. The homogenate,
diluted 1+3 with KC1 was centrifuged twice at 9.000 g for 10 minutes (4 C). A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 3 ml and stored at -70 C. Small numbers -of the ampoules were kept at -20 C for only sever al weeks before use. The protein content was determined using
the analysis kit of Bio-Rad Laboratories, D-8000 Munchen: Bio-Rad
protein assay. Catalogue No. 500 000 6.
The protein concentration in the S9 preparation is usually be
tween 20 and 45 ing/ml. In experiment I and II the protein concen
tration was 38.6 mg/ml (Lot. No.: 071292).
S9 Mix
An appropriate'quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentra tion of 0.75 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl^ 33 mM KC1
5 mM glucose-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. (1).
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PRE-TEST FOR TOXICITY
A pre-test was performed in order to determine the toxicity of the test article. The general culturing and experimental condi tions in this pre-test were the same as described below for the
mutagenicity experiment. In this pre-test the colony forming
ability of approximately 500 single cells (duplicate cultures per /concentration) after treatment with the test article was observed and compared to the controls. Toxicity of the test article was evidenced by a reduction in colony forming ability. Concurrent with the pre-test high density cultures (approx. 200,000 cells/slide) were treated with the test article to simu late the conditions of the main experiment. Cell number and morphology were examined qualitatively 4 h and 24 h after the start of treatment (with metabolic activation: after washing with
Saline G) .
DOSE SELECTION
According to -the results from this pre-test 6 concentrations (18 h interval) were chosen to be applied in the chromosomal aberration assay. The highest concentration (300 ug/ml) used in the pre-test and in the main experiment I was limited by the solubility of the test article in ethanol and other appropriate solvents (for example aqua bidest. or DMSO). As no toxic effect could be observed in the pre-test (colony
forming ability) the cytogenetic experiments were performed with
the concentrations listed below.
Experiment I
.
without S9 mix:
18 h: 1.0; 3.0; 10.0; 30.0; 100.0; 300 ug/ml 28 h: 10.0; 30.0; 100.0; 300.0 ug/ml
with S9 mix: 18 h: 1.0; 3.0; 10.0'; 30.0; 100.0; 300 ug/ml 28 h: 10.0; 30.0; 100.0; 300.0 pg/ml
In experiment I the stock solution (30 mg/ml) for formulation of
the highest concentration was not completely dissolved. Addition
ally, a test article precipitation in the aqueous culture medium
was observed. Therefore, in experiment II the concentration range
was only set up to 200 ng/ml.
I I E x p e r i m e n t
without S9 mix: 18 h: 1.0; 3.0; 28 h: 10.0; 30.0;
10.0; 100.0;
30.0; 100.0; 200.0 ug/ml
200 ng/ml
with S9 mix: 18 h: 1.0; 3.0; 10.0; 30.0; 100.0; 200 ng/ml 28 h: 10.0; 30.0; 100.0; 200.0 pg/ml
However, 200 pg/ml were not completely dissolved too, so the concentrations used in each experiment cannot be given accu rately.
The treatment
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interval
was
4
h
with
metabolic
activpaagteion15,
18
of
h
32
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and 28 h without metabolic activation. Per concentration dupli cate cultures were used.
The preparations of all test groups in both experiments were
assessed qualitatively for possible toxic effects (low number of remaining/surviving cells, low metaphase number, met'aphase quali
ty) .
In both experiments, in presence of S9 mix no toxicity could be
/observed up to the highest concentrations used (see pages 22 and 26). In absence of S9 mix, in experiment I the mitotic index was
slightly reduced up to the highest concentration used (300 ug/ml)
. in both fixation intervals, but in experiment II no-reduction of
the mitotic index was found at the highest concentration used
(200 ng/ml). Therefore, in cytogenetic experiment I, in the
absence and presence of S9 mix cultures after treatment with
300.0 ng/ml (18 h and 28 h) as the highest concentration were
evaluated for cytogenetic damage; in experiment II, the highest
concentration evaluated was 200.0 ng/ml at each fixation interval with and without S9 mix.
EXPERIMENTAL PERFORMANCE
Seeding of the Cultures
Three or four days old exponentially growing stock cultures more than 50 % confluent were trypsinized at 37 C for approximately 5 minutes. Then the enzymatic digestion was stopped by adding
complete culture medium.and'a single cell suspension was pre pared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA).
The Ca-Mg-free salt
NaCI KCl
Glucose
NaHC03
solution was composed as
8000 mg 400 mg
1000 mg 350 mg
follows
(per
litre):
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 cells were seeded into Quadriperm dishes (Heraeus, D-6450 Hanau) which contained microscopic slides (at least 2 chambers per. dish and test group). In each chamber 1 x 104 - 1 x 10-' cells were seeded with regard to preparation time. The medium was MEM +
10 % PCS (complete medium).
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Treatment
Exposure time 4 hours (with S9 mix):
-In both independent experiments, after 48 h (28 h preparation interval) and 55 h (18 h preparation interval) the culture medium was replaced with serum-free medium containing different concen trations of the test article and 50 Hi/mi S9 mix.
After 4 h the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
The "Saline G" solution is composed as follows (per litre)':
'
NaCI -
8000 mg
KC1
400 mg
Glucose
1100 mg
NB2HP04.7H20
290 mg
KH2P04
150 mg
pH is adjusted to 7.2
Exposure time 18 and 28 hours (without S9 mix):
In both independent experiments, after 48 h (28 h preparation interval) and 55 h (18 h preparation interval) the culture medium
was replaced with complete medium (10 % PCS) containing different
concentrations of the test article without S9 mix. This medium was not changed until preparation of the cells.
f
All cultures were incubated at 37 C in a humidified atmosphere with 4.5 % CDs (95.5 % air). -
Preparation of the Cultures
15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 tig/mi culture medium) to the cultures. 2.5 h later, the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KC1) for 20 min at 37 C. After incubation
in the hypotonic solution the cells were fixed with 3+1 metha-
nol + glacial acetic acid. Per experiment both slides per group were prepared. After fixation the cells were stained with Giemsa
(E. Merck, D-6100 Darmstadt).
l^
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Analysis of Metaphase Cells
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" (4))
.using NIKON microscopes with lOOx oil immersion objectives.
Breaks, fragments, deletions, exchanges and chromosomal disinte grations were recorded as structural chromosomal aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides (deviation to protocol where for the positive controls only 50 metaphases should be scored). Only metaphases with characteristic chromosome numbers of 22 1 were included in the analysis. To
describe a cytotoxic effect the mitotic index (% cells in mito sis), was determined. In addition, the nuinber of polyploid cells was scored '(% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).
DATA RECORDING
The data generated were recorded in the raw data file. The re
sults are presented in tabular form, including experimental
groups with the test article, negative and positive controls.
ACCEPTABILITY OF THE ASSAY
The chromosomal aberration assay is considered acceptable if it
meets the following criteria:
a) The number of aberrations found in the- negative and/or solvent
controls falls within the range of historical laboratory
control data': 0.00 % - 4.00 %.
b) The positive control substances should produce significant increases of the number of cells with structural chromosomal aberrations.
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EVALUATION OF RESULTS
A test article is classified as mutagenic if it induces reproduc-
ibiy either a significant concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test
points.
A test article producing reproducibly neither a significant
concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducibly posi tive response at any one of the test points is considered nonmutagenic in this system.
This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered
together.
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Company Ssr^Sszed. Decs ".or contain TSCA CBt
BIOMETRY
A statistical evaluation of the results was not necessary to be performed. The mean aberration rates of the test groups after treatment with the test article did not exceed the control
values.
Exception was the test article group 200 p.g/ml at fixation inter
val 28h in experiment II.
Statistical significance at the five per cent level (p < 0.05) was evaluated for this test article group by means of the chisquare test. Evaluation was performed only for cells carrying
aberrations exclusive gaps.
Solvent control versus .
Test group
200.0 ng/ml
fixation interval
28 h
S9 mix
-
p-value
0.1>p>0.05
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RESULTS
TABLES OF RESULTS
Table 1: Pre-test for toxicity
In the pre-test the toxicity of the test article was examined determining the colony forming ability of cultures treated with the test article.
Colony forming ability without and with metabolic activation; .
Per flask approx. 500 cells were seeded.
cone. per ml
S9
colonies counted
mix flask I flask II
mean
relative
survival %
negative control solvent control
0.1 p.g 0.3 jig 1.0 |ig 3.0 p,g 10.0 iig 30.0 p,g 100.0 p,g
300.0 p.g
ethanol
302
355
261
282
277
243
251
264
320
284
306
315
294
296
312
295
254
309
294
274
328.5 271.5 260.0 257.5 302.0 310.5 295.0 303.5 281.5 284.0
100.0
95.8 94.8 111.2 114.4 108.7 111.8 103.7 104.6
negative control solvent control ethanol
0.1 M 0.3 )ig 1.0 \LQ
3.0 p-g . 10.0 p,g
30.0 fig 100.0 iig 300.0 p-g
288
279
303
252
252
255
263
287
304
231
281
266
284
267
317
310
265
301
297
238
283.5 277.5 253.5 275.0 267.5 273.5 275.5 313.5 283.0 267.5
100.0
91.4 99.1 96.4 98.6 99.3 113.0 102.0
96.4
ABBREVIATIONS
The following abbreviations of structural chromosomal aberrations are used in the following tables:
g = gap?
ig
=
iso-gap; type where
gaps are achromatic
no dislocation of
lesions of
chromosomal
chromatid
material
or
is
chromosome
visible (independent
of
the
size
of
the
achromatic
region).
b = break; ib = iso-break; f = fragment; if = iso-fragment; d
= deletion; id than 4 events
=
in
iso-deletion; ma = multiple
one cell [excluding gaps];
aberration (= more only exchanges are
rcehcaonrgdee; dexad= dcithiroomnoaslolyme intyptehesexechcaenlgles;); cedx = = chcrhormoomsaotmidal tdyipseinteex
gration (= pulverization)
ca7922r
page 21 of 32
Company Sarr'SEzed. Does naf csniaL-s TSCA CBI
Experiment I
Table 2: Number of Polyploid Cells and Mitotic Index experiment I; fixation intervals 18 h and 28 h; without and with metabolic activati
cone. per ml
S9 fixation mix interval
polyploid cells*
culture total mean
1
2
mit
culture
1
Negative control
Solvent control ethanol
Positive control EMS
Test article
-
Negative control Solvent control ethanol Positive control CPA
Test article
-
1.0 s
-
0.4 mg -
30.0 u,g -
100.0 |ig -
300.0 p.g -'
+
1.0 a
+
0.93 p-g +
30.0 p-g +
100.0 |ig' +
300.0 p-g +
18 h 18 h 18 h 18 h 18 h 18 h
18 h 18 h 18 h 18 h 18 h 18 h
3.5
20.0 22
2.0
17.1 20
347 4.5
10.3 17
134 9
0
9
4.5
11.4 10
369 2.0
14.1 14
3.5
13.1 12
440347 2.5
16.8 16
5
5
10
5.0
11.1 16
325 2.5
14.3 12
1.5
14.7 16
325 1.5
17.0 16
122133 4
5
9
4.5
15.1 18
Solvent control ethano'1
1.0 a
-
Test article
300.0 iig -
28 h 28 h
3.5
13.5 14
1.0
10.6 10
512172 Solvent control ethanol
1.0 % +
Test article
300.0 p.g +
28 h 28 h
1.0
20.4 17
2.0
17.3 20
04 20 24 * The number of polyploid cells was determined in 100 cells per culture of each test grou
** The mitotic index was determined in 1000 cells per culture of each test group. *** For the positive control groups, the relative values of the mitotic index are related t
controls; for the test article treatment groups the values are related to the solvent
Table 3: Structural chromosomal aberrations
experiment I; fixation interval 18 h; without metabolic activation
Negative control
Solvent control ethanol Positive control
EMS
Test
article
ii
cone. S9 per ml mix
cells aberrant cells (%mean) scored incl. excL exchan-
gaps gaps ges
-
1.0 s 0.4 mg 30.0 (Jig 100.0 iig 300.0 ^ -
cult. 1 100 cult. 2 100
-total- 200
cult. 1 100 cult. 2 100
-total- 200" cult. 1 100
cult. 2 100
-total- 200 cult. 1 100
cult. 2 100
-total- 200
cult. 1 100 cult. 2 100 -total- 200 cult. 1 100 cult. 2 100 -total- 200
5.0 2.0 5.5 3.0 13.5 11.5 7.5 2.5 9.0 3.0 8.0 2.5
0.0 0.5 7.0 0.0 0.0 0.0
For abbreviations see RESULTS/ABBREVIATONS
types of
-gapsg <g
aberrations found
-chromatid type-
b
f
d
ex
3
0
0^ 1
0
0
5
0
2' 0
0
0
8
0
2100 3
0
3
0
2101 6
0
31210001 3
0
4
0
53010068 7
0
8
1
0
14
3 7 10
1 0 1
202211000000
3
0
10
0
13
0
314011000000
5 7 12
0 0 0
431211000000
-chr ib
000 000 000 011 000 000
'W
Table 4; Structural chromosomal aberrations
experiment I; fixation interval 18 hy with metabolic activation
cone. S9 per ml mix
cells aberrant cells (^mean) types of aberrations found
scored incl. excl. exchan- -gaps-
- chromatid type-
-ch
gaps gaps ges
g ig
b
f
d
ex
ib
Negative control
Solvent control ethanol
Positive control
CPA
+ cult. 1 100 cult. 2 100 -total- 200
6.5 4.0 0.0
1.0 s. +
cult. 1 100 cult. 2 100
-total- 200, 8.0 2.5 0.5
0.93 mg + cult. 1 100 cult. 2 100 -total- 200 22.0 18.0 8.0
3
0
2100 0 2
0
3' 0 0
0
5
0
5100 00 5
0
6
0
0000 0 11
0
22110000 00 5
1
12
0
6
6391089 0 17
1
12 12
1
17
Test
article
11
ii
30.0 (Jig + 100.0 u,g + 300.0 (zg +
cult. 1 100 cult. 2 100 -total- 200 cult. 1 100 cult. 2 100 -total- 200 cult. 1 100 cult. 2 100 -total- 200
2.0 0.0 0.0 3.0 1.5 0.0 3.5 2.0 0.0
For abbreviations see RESULTS/ABBREVIATONS
2 2 4
0 0 0
000000000000
2 2 4
0 0 0
000213000000
1 3 4
0 0 0
011303000000
Table 5: Structural chromosomal aberrations
experiment I; fixation interval 28 h; without and with metabolic ac
cone. S9 per ml mix
cells aberrant cells (%mean) types of aberrations found
scored incl. excL exchan- -gaps-
-chromatid type-
-c
gaps gaps ges
g ig
b
f
d
ex
ib
1
Solvent
control ethanol
1.0 % -
cult. 1 100 cult. 2 100 -total- 200
5.5 2.0 0.5
Test
300.0 iiQ - cult. 1 100
article
cult. 2 100
-total- 200' 3.5 1.5 0.0
Solvent control ethanol
Test
article
1.0 % + 300.0 (Jig +
cult. 1 100 cult. 2 100
-total- 200 cult. 1 100
cult. 2 100
-total- 200
3.0 1.0 0.0 2.0 0.5 0.0
For abbreviations see RESULTS/ABBREVIATONS
3
0
1, 0
0
0
3 6
1 1
21110011
000
1 4 5
0 0 0
022101000000
000
1 3 4
1 0 1
011101000000
2
0
1
0
3
0
000303000000
Experiment II
Table 6: Number of Polyploid Cells and Mitotic Index
experiment II; fixation intervals 18 h and 28 h; without and with metabolic activ
cone. per ml
S9 fixation mix interval
polyploid cells*
culture total rfiean
1
2
m cultu
1
Negative control
-
Solvent control ethanol
2 4 6 Positive control EMS
1.0 %
-
0.4 mg -
1 2 3 Test article
30.0 p.g -
-' 100.0 p-g
0 2 2 200.0 p.g -
18 h 18 h 18 h 18 h 18 h 18 h
3.0
18.3 1
1.5
13.2 1
4
6
10
5.0
8.1
1.0
11.6
1.5
9.4 1
2.5
9.8 1
043135 Negative control
Solvent control ethanol
3 1 4 Positive control CPA 41135016 Test article
+
1.0 % + 0.93 u,g + 30.0 p,g + 100.0 u.g + 200.0 |xg +
18 h 18 h 18 h 18 h 18 h 18 h
2.0
16.8 1
3.0
14.5 1
7
3.5
13.5
0.5
13.7 1
3
1
4
2.0
17.5 1
1.0
13.7 1
0 2 2 Solvent control ethanol
1,0 %
-
Test article
200.0 p-g -
28 h 28 h
1.5
9.8
2.0
15.1
22 12 34 Solvent control ethanol
1.0 % +
28 h
Test article
200.0 p-g
+
28 h
.
0.5
12.3
2.5
14.2
03 12 15 The number of polyploid cells was determined in 100 cells per culture of each test gr
** The mitotic index was determined in 1000 cells per culture of each test group. *** For the positive control groups, the relative values of the mitotic index are related
controls; for the test article treatment groups the values are related to the solvent
Table 7: Structural chromosomal aberrations
experiment II; fixation interval 18 h; without metabolic activation
cone. S9 per ml mix
cells aberrant cells (%mean) types of aberrations found
scored incl. excl. exchan- -gaps-
-chromatic! type-
-ch
gaps gaps ges
g ig
b
f
d
ex
1b
Negative control
Solvent control ethanol
Positive control
EMS
- cult. 1 100 cult. 2 100 -total- 200
4.0 1.5 0.5
1.0 % -
cult. 1 100 cult. 2 100
-total- 200'. 5.0 2.0 0.0
0.4 mg - cult. 1 100 cult. 2 100 -total- 200 13.5 10.5 5.5
5
0
1100 0 0
0
0' 0
0
0
0 5
0
1100 0 2
0
0
011101000000 00 1
0
6
0
6
1
2
0
33340076 0 8
1
6
7
0
13
Test
02000000 article
30.0 ng -
cult. 1 100 cult. 2 100 -total- 200
3.0 1.0 0.0
1
0
3
0
4
0
201000000000 100.0 p,g -
cult. 1 100 cult. 2 100 -total- 200
6.5 0.5 0.0
6
1
6
0
12
1
1 0 0 0 11
200.0 p.g -
cult. 1 100
3
0
2000 cult. 2 100
2
0
0100 -total- 200
4.5 2.0 0.0
5
0
2100 a
01
vQ (0
For abbreviations see RESULTS/ABBREVIATONS
(0
^j
0 h-h
LiJ t-J
Table 8: Structural chromosomal aberrations
experiment II; fixation interval 18 h; with metabolic activation
cone. S9 per ml mix
cells aberrant cells (%mean) types of aberrations found
scored incl. excl. exchan- -gaps-
-chromatid type-
-c
gaps gaps ges
g ig
b
f
d ex
i
Negative control
Solvent control ethanol
Positive control
CPA
Test
article
11
+
1.0 o- + 0.93 mg + 30.0 p,g + 100.0 (Jig + 200.0 p,g +
cult. 1 100 cult. 2 100
-total- ZOO
cult. 1 cult. 2 -total-
100 100 ZOO'
cult. 1 100 cult. 2 100 -total- ZOO
cult. 1 100 cult. 2 100 -total- ZOO
cult. 1 100 cult. 2 100 -total- 200
cult. 1 100 cult. 2 100 -total- 200
4.0 1.5 0.0 4.0 2.5 0.5 24.0 21.0 10.5 4.0 2.0 0.5 4.5 2.5 0.0 2.0 0.5 0.0
For abbreviations see RESULTS/ABBREVIATONS
1
0
4
0
5
0
4
0
0
0
4
0
6
0
6
1
12
1
3, 1 0
0
0' 0 0
0
3100
000213000101
5 10
0
11
3 13
0
12
8 23
0
23
2 2 4
0 0 0
000213000000
1
0
5
0
G
0
000325000000
2 1 3
0 0 0
000101000000
Table 9: Structural chromosomal aberrations
experiment II; fixation interval 28 h; without and with metabolic
cone. S9 per ml mix
cells aberrant cells (%mean) types of aberrations found
scored incl. excL exchan- -gaps-
-chromatid type-
-c
gaps gaps ges
g <g
b
f
d
ex
ib
Solvent control ethanol
1.0 % -
cult. 1 100 cult. 2 100 -total- 200
2.5 0.5 0.0
Test
200.0 iig - cult. 1 100
article
cult. 2 100
-total- 200- 3.5 2.5 0.5
Solvent
control ethanol
1.0 "a +
cult. 1 100 cult. 2 100 -total- 200
5.5 . 1.0 0.0
4
0
0, 0 0
0
1 5
0 0
00000000
0 2 2
0 0 0
011220000000
6
0
4
0
10
0
000000000000
Test
article
200.0 u.g +
cult. 1 100 cult. 2 100 -total- 200
3.0 1.0 0.0
For abbreviations see RESULTS/ABBREVIATONS
3 2 5
0 0 0
011000000000
DISCUSSION
The test article J3^BH|HUR was assessed for its potential to
induce structurar^chromosomal aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed.
The chromosomes were prepared 18 h and 28 h after start of treat ment with the test article. The treatment intervals were 4 h
(with 39 mix) and 18h and 28 h (without S9 mix).
In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal
aberrations.
The following concentrations were evaluated (18 h: 3 concentra tions; 28 h:. highest evaluable concentration):
Experiment I
without S9 mix: 18 h: 30.0; 100.0; 300 ng/ml 28 h: 300.0 u.g/ml
with S9 mix: 18 h: 30.0; 100.0; 300 ng/ml 28 h; 300.0 ug/ml
Experiment II
without S9 mix: 18 h: 30.0; 100.0; 200 ug/ml 28 h: 200.0 tig/mi
with S9 mix: 18 h: 30.0; 100.0; 200 ug/ml 28 h: 200.0 ug/ml
The concentration range used in this study (300 ug/ml as highest
concentration) was limited by the solubility of the test article
in ethanol and other appropriate solvents.
In the pre-test on toxicity (colony forming ability) in the absence and presence of S9 mix after treatment with concentra
tions up to 300.0 ug/ml (with and without S9 mix) the colony
forming ability was not reduced.
In the absence of S9 mix, in experiment I the mitotic indices
were reduced after treatment with the highest concentration used
(300 ng/ml) at both fixation intervals whereas in experiment II
(highest concentration 200 ng/ml) no reduction of the mitotic index was observed.'These effects are considered being biologi
cally irrelevant since there was no dose relation in the test
groups used.
In the presence of S9 mix, in both experiments the mitotic in dices were not reduced after treatment with the test article at both fixation intervals.
ca7922r
page 30 of 32
Company Sa-.^ized. Dcss not contain TSCA CBI
In both experiments, in the absence and presence of S9 mix the
test article did not increase the frequency of cells with aberra tions (excl. gaps). The aberration rates of the cells after treat
ment with the test article (exp.I: 0.0 % - 3.0 %; exp. II: 0.5 %
- 2.5 %) were in the range of the solvent control values (exp. I:
-1.0 % - 3.0; exp. II: 0.5 % - 2.5 %) and within our historical
control range: 0.0 % - 4.0 %. Also, the number of cells carrying exchanges was not increased as compared to the solvent' controls.
Tables 2 and 6 show the occurrence of polyploid metaphases. In both experiments, no increase in the rate of polyploid metaphases (maximum 4.5 %) as compared to the rates of the controls (maximum
5.0 %) were found after treatment with the test article.
In both experiments, EMS (0.4 mg/ml) and CPA (0.93 ug/ml) were used as positive controls and showed distinct increases in cells with structural chromosomal aberrations.
In conclusion, it can be stated that in the study described and
under the experimental conditions reported, the test article
ZONYL RP 18 did not induce structural chromosomal aberrations in
the V79 Chinese hamster cell line.
ca7922r
page 31 of 32
CompaFry SanETszetf. Dc-as ntt contain TSCA CBf
REFERENCES
1. B.N. Ames, J. McCann, and E. Yamasaki (1977)
Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. B.J. Kilbey et al. (Eds.) "Handbook of Mutagenicity Test Procedures", Elsevier, Amsterdam, 1-17 2. M.O. Bradley, B. Bhuyan, M.C. Francis, R. Langenbach, A.
Peters on and E. Huberman (1981)
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 3. R.J. Preston, W. Au, M.A. Bender, J.G. Brewen, A.V. Carrano,
J.H. Heddle, A.F. McFee, S. Wolff and J.S. Wassom (1981) Mammalian in vivo and in vitro cytogenetic assays.. A report of the U.S. EPA^s gene-tox program. Mutation Research 87, 143-188
4. Engelha'rdt G. , (1987)
"Arbeitsgruppe der Industrie, Cytogenetik" Standard-Protokoll zur cytogenetischen Auswertung von Mitose- und Meiosechromosomen bei der Routineuntersuchung.
n 5. S.H.H. Swierenga, J.A. Heddle, E.A. Sigal, J.P.W. Gilman, R.L. Brillinger, G.R. Douglas and E.R. Nestmann (1991) Recommended protocols based on the a survey of current practice in genotoxicity testing laboratories, IV: Chromoso me aberration and sister chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocyte cultures. Mutation Research 246, 301-322 6. D.J. Kirkland (1992) Chromosomal aberration tests In vitro', problems with proto col design and interpretation of results. Mutagenesis 2, 95-106 7. C.L. Bean, M. J. Armstrong and S.M. Galloway (1992) Effect of sampling time on chromosome aberration yield for 7 chemicals in Chinese hamster ovary cells. Mutation Research 265, 31-44 8. M. J. Armstrong, C.L. Bean and S.M. Galloway (1992) A quantitative assessment of the cytotoxicity associated with chromosomal aberration detection in Chinese hamster ovary cells. Mutation Research 265, 45-60
DISTRIBUTION OF THE REPORT
Sponsor
Study Director
2x (Ix original, Ix copy)
Ix (copy)
ca7922r
page 32 of 32
Company Sar.'AizscL Doss net csrifain TSCA CB?
RCC
Research & Consulting Company Ltd Postfach
CH-4452 Itingen
Switzerland Phone: 41/61 975 11 11
Fax: 41/61 971 5284
RCC
Research & Consulting Company Ltd 1. route de Troinex CH-1227 Carouge Switzerland
Phone: 41/22 342 66 70 Fax: 41/22 342 66 43
RCC
Umweltchemie AG Postfach
''H-4452 Itingen
-' tzeriand Phone: 41/61 975 11 11
Fax: 41/61 971 5266
: BRL Biological Research Laboratories Ltd Wolferstrasse 4
CH-4414 Fullinsdorf
Switzerland
Phone: 41/61 9014242 Fax: 41/61 901 2565
RCC
Registration & Consulting Company AG Landstrasse 33 CH-4452 Itingen Switzerland
Phone: 41/61 971 5000 Fax: 41/61 971 5072
RCC NOTOX Research & Consulting Company B.V. .P.O. Box 3476 ML-5203 DL 's-Hertogenbosch
The Netherlands
Phone: 31/73 41 9575 Fax: 31/73 41 8543
RCC Umweltchemie GmbH & Co. KG In den Leppsteinswiesen 19
D-6101 Rossdorf Federal Republic of Germany Phone: 49/6154 80 70 Fax: 49/6154 8 33 99
CCR Cytotest Cell Research GmbH & Co. KG In den Leppsteinswiesen 19
D-6101 Rossdorf Federal -Republic of Germany. Phone:49/6154 80 70 Fax: 49/6154 8 33 99
RCC (UK) Ltd Research & Consulting Company Ltd Willow Court, Netherwood Road Hereford HR2 6JU United Kingdom
Phone: 44/432 35 40 52 Fax: 44/432 35 23 40
RCC
Group
^ ^ JZ^
^ y <>
Coa^sany Sa^iizedi. Dsss not captain TSCA CBi
