Document 1gJ9eEDMvm2dMXMYwnqkjwJwm
AR226-2997
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 Untersuchungsgesellscnaft mbH
D-8750 Aschaffenburg
y
Study Monitor Heike Kramer
Page 1 of 32
RCC
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COPY OF GLP CERTIFICATE
HESSISCHES MINISTE.R1UM . . FOR UNWELT, ENERGIE UNO BUNDESANGELEGENHEITEN
GLP-Bescheinigung
Beachelnlgung
1-Sennit wird bestStigt. daB die Prijfungsainrichtung(an)
Cytotest Cell Research GmbH & Co KG
In den Leppsteinswiesen 19
;,, 6101
RoBdorf
(Ort AroOlrift)
dar ,RCC Holding yerwaltung GmbH____
(Finn*)
03.08., 04.08., 05.08.- und.06.08-92
(D.lum)
von dar (ur dia Ubefwactiung zustSndigen Bahflrtia uber
dia Enhallung der Grundsitza dar Gulan Laborpraxis , inspiziert wordan ist (a'nd}.
/
*
Es wrd hiemiit bestSligt. dafi tolgenda Prufungan in
diaser Prufeinrichtung nach den GrundSSttan dac Guten
Laborpraxis durchgaluhrt warden.
.
Certincata
II Is haraby csniGed that the test tadGty(ies)
Cytotest Cell Research GmbH & Co KG
' " In den Leppi'te'inswiesen 19 610'1 RoBdorf
.
(loation. xddreaa)
RCC Holding Verwaltung GmbH
_
(ampuir niun)_
03-08., 04.08-, 05.08.and.06.08.92
en.......,...--- (dm)
was (ware) inspectsd by the conipatant authority regar ding complianca wilh lha PrinciplBS o( Good Laboralory Prectics.
It is hareby cartiCed that studias in this test (acility. ara
conducted in complianca with lha Principles of Gocd
'
Laboratory Practica.
-
Toxikologische Eigensc^h-$alfltliasa7-^B.^
lexicological, properties
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C?<<^.iA<j-
(Or.-.Hecter)
Wiesbaden, den ^S-
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UUNltNIb
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
II Number of Polyploid Cells
and Mitotic Index Structural Chromosomal Aberrations
EXPERIMENT
Number of Polyploid Cells
and Mitotic Index Structural Chromosomal Aberrations
DISCUSSION REFERENCES DISTRIBUTION
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
30 32 32
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GENERAL
Sponsor:
Study Monitor;
Testing Facility;
CCR Project No.:
Test Article: Title:
ISEGA Forschungs- und Untersuchungsgesellschaft mbH D-8750 Aschaffenburg
Heike Kramer
CCR CYTOTEST CELL RESEARCH GMBH & CO. KG
D-6101 RoJidorf
326430
L
>1
Chromosome Aberration Assay in
Chinese Hamster 3 '779 Cells in yitro
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 Kiihner
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:
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November 11, 1992 December 01, 1992
March May March May
01, 1993 24, 1993
29, 1993 05, 1993
May
26, 1993
July
06, 1993
T^SCACBt notcon^in
.San^-0085
page 4 of 32
Study Director;
Author:
Management:
Dr. Albrecht Heidema
(^.^Q^ c Date: July 06, 1993
Dr. Wolfgang Volkner
(A OclkLj)
Date: July 06, 1993 Dr. Hans^Emil Knoell
06, 1993
QUALITY ASSURANCE
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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ARCHIVIHG
C C R, D-6101 RoBdorf 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.
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STATEMENT OF COMPLIANCE
Project Number:
Test Article:
Study Director: Title:
326430
Dr. Albrecht Heidemann Chromosome Aberration Assay in Chinese
Hamster V79 Cells in vitro witi
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
I
/ ^
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Date: ^v ,( (^ ^.^3
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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 in Chinese
Hamster 779 Cells in vitro withj
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
11, 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
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Dr. Christiane Helmrich
iU.*.*'k?.**4^i-*v*f.*.<Y^t I*
Date: y^ C^, -W^
page 8 of 32
r? The test article^--------1^9 J^as assessed for its potential to
induce structural chromosomal aberrations in 779 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 ag/ml 28 h: 300 ag/ml
with S9 mix:
18 h: 30; 100; 300 ag/ml 28 h: 300 ag/ml
II Experiment
-
without S9 mix:
18 h: 30; 100; 200 tig/mi
28 h: 200 ag/ml
with S9 mix: 18 h: 30; 100; 200 ag/ml 28 h: 200 ag/ml
II The concentration range of the test article was determined in
pre-experiments. In experiment the highest concentration was
200 ag/mi due to the limited solubility of the test article. The colony forming ability of 779 cells as indicator for a toxicity response was not affected by treatment with concentrations up to 300 ag/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
it In conclusion,
can be stated that in the study described and
under the experimental conditions reported, the test article 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 VPS.
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 chromosomal damage that is., compatJAle with__cell .dryisj.on^_Siniilar 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 V79 ceils 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 V79 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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MATERIALS AND METHODS
THE TEST ARTICLE
The test article and the information concerning the test article
were provided by the sponsor.
Name:
Batch No.: Chemical name;
Aggregate State at RT;
Colour: Analysis; Molecular Weight:
Purity: Stability:
liquid not indicated by the sponsor
a) 335 b) 257
Storage: Expiration Date:
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 > 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.
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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
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.
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:
CPA; Cyclophosphamide
SERVA, D-6900 Heidelberg 17681 (purity S 99.5 %)
nutrient medium 0.93 ag/ml = 3.3 uM (in deviation
col where 2.8 ng/ml = 10.0 ^iM was
to proto given)
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 .yi. 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-74'43 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 (PCS; 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 fPreparation bv C C R)
The S9 liver microsomal fraction was obtained from the livers of
8-12 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 nig/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 KCl and homogenized. The homogenate, diluted 1+3 with KCl was centrifuged twice at 9.000 g for 10
minutes (4 C). A stock of the supernatant containing the micro-
somes 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 Miinchen: Bio-Rad
protein assay. Catalogue No. 500 000 6.
The protein concentration in the S9 preparation is usually be
tween 20 and 45 mg/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 KCl
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. Ceil 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 ug/ml
II 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
the concentration range
was only set up to 200 ug/ml.
Experiment II
without S9 mix: 18 h: 1.0; 3.0; 10.0; 30.0; 100.0; 200 ug/ml 28 h: .10.0; 30.0; 100.0; 200.0 ug/ml
with S9 mix: 18 h: 1.0; 3.0; 10.0; 30.0; 100.0; 200 ug/ml 28 h: 10.0; 30.0; 100.0; 200.0 ug/ml
However, 200 ug/ml were not concentrations used in each
rately.
The treatment
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was
4
completely dissolved too, so the experiment cannot be given accu
h
with
metabolic
actipvaagteion15,
18
of
h
32
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II, 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, metaphase 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 fig/mi). Therefore, in cytogenetic experiment I, in the
absence and presence of S9 mix cultures after treatment with
300.0 tig/mi (18 h and 28 h) as the highest concentration were
evaluated for cytogenetic damage; in experiment
the highest
concentration evaluated was 200.0 tig/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 solution was composed as
Nad
8000 mg
KC1
400 mg
Glucose NaHC03
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-S450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1 x 104 - 1 x 105 cells were seeded with regard to preparation time. The medium was MEM +
10 % FCS (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 nl/ml 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):
'
Nad -
KC1
8000 mg 400 mg
Glucose Na^HPO^.PH^O
1100 mg 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 % FCS) containing different concentrations of the test article without S9 mix. This medium was not changed until preparation of the cells.
/ ^
^
.
.
All cultures were incubated at 37 C in a humidified atmosphere with 4.5 % CC>2 (95.5 % air).
Preparation of the Cultures
15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 ng/ml culture medium) to the cultures. 2.5 h later/ the cells were treated on the slides in the chambers with hypo-
tonic 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).
^^^------------------
___ ,, , 3.
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 number 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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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 ug/inl 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 ug/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
negative control solvent control ethanol
0.1 |ig 0.3 iig 1.0 p,g 3.0 p.g 10.0 [ig 30.0 |ig 100.0 u,g 300.0 u.g
negative control solvent control
0.1 M 0.3 (Jig 1.0 ^g 3.0 ij-g 10.0 (ig 30.0 u,g 100.0 p.g 300.0 u.g
ethanol
S9
colonies counted
mix flask I flask II
302
355
261
282
277
243
251
264
320
284
306
315
294
296
312
295
254
309
294
274
288
279
303
252
252
255
263
287
304
231
281
266
284
267
317
310
265
301
297
238
mean
328.5 271.5 260.0 257.5 302.0 310.5 295.0 303.5 281.5 284.0
283.5 277.5 253.5 275.0 267.5 273.5 275.5 313.5 283.0 267.5
relative survival %
100.0 95.8 94.8
111.2 114.4 108.7 111.8 103.7 104.6
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; gaps are achromatic lesions of chromatid or chromosome type where no dislocation of chromosomal material is visible (independent of the size of the achromatic region).
b = break; ib = iso-break; f = fragment; if = iso-fragment; d
= deletion; id = iso-deletion; ma = multiple aberration (= more than 4 events in one cell [excluding gaps]; only exchanges are
recorded additionally in these cells); ex = chromatid type ex
change; ex = chromosome type exchange; cd = chromosomal disinte gration (= pulverization)
ca7922r
Company SanKIzecL Does not contain TSCAC^S P^s 21 of 32
Experiment I
Table 2: Number of Polyploid Cells and Mitotic Index experiment I; fixation intervals 18 h and 28 h; without and with metabolic activatio
cone. per ml
S9 fixation mix interval
polyploid cells*
culture total mean
1
2
mito
culture
1
2
Negative control
-
Solvent control ethanol
3 4 7 Positive control EMS
1.0 s
.
0.4 mg -
3163 49 Test article
-
30.0 u.g 100.0 p.g -
9 0 9 300.0 p.g -v
18 h 18 h 18 h 18 h 18 h 18 h
3.5
20.0 22.
2.0
17.1 20.
4.5
10.3 17.
4.5
11.4 10.
2.0
14.1 14.
3.5
13.1 12.
44 03 47 Negative control
Solvent control ethanol
3 2 5 Positive control CPA 312253 Test article
+
1.0 %
+
0.93 p.g +
30.0 I-LQ +
100.0 ^g' +
300.0 p,g +
18 h 18 h 18 h 18 h 18 h 18 h
2.5
16.8 16.
5
5
10
5.0
11.1 16.
2.5
14.3 12.
1.5
14.7 16.
1.5
17.0 16.
4.5
15.1 18
24 15 39 Solvent control ethanol
1.0 %
-
Test article
300.0 p.g -
28 h 28 h
3.5
13.5 14
1.0
10.6 10
512172 Solvent control ethanol
1.0 s
+
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 to
controls; for the test article treatment groups the values are related to the solvent c
w
Table 3: Structural chromosomal aberrations
experiment I; fixation interval 18 h; without metabolic activation
Negative control
Solvent control ethanol Positive control
EMS
Test
article
n
cone. S9 per ml mix
-
1.0 % 0.4 mg 30.0 u,g 100.0 p-g 300.0 p.g -
cells aberrant cells (%mean) scored incl. excl. exchan-
gaps gaps ges
cult. 1 100 cult. 2 100
-total- 200
5.0 2.0 0.0
cult. 1 100 cult. 2 100 -total- 200s 5.5 3.0 0.5
cult. 1 100 cult. 2 100 -total- 200 13.5 11.5 7.0
cult. 1 100 cult. 2 100 -total- 200
7.5 2.5 0.0
cult. 1 100 cult. 2 100 -total- 200
9.0 3.0 0.0
cult. 1 100 cult. 2 100 -total- 200
8.0 2.5 0.0
For abbreviations see RESULTS/ABBREVIATONS
types of aberrations found
-gapsg ig
-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
1100 3
0
4
0
53010068 7
0
320 1
8
1
0
14
3
1
Z
1
0
0
7 10
0 1
02120000
3
0
10
0
13
0
314011000000
5
0
7
0
12
0
341121000000
-chr ib
000 000 000 011 000 000
Table 4; Structural chromosomal aberrations
experiment I; fixation interval 18 h; with metabolic activation
cone. S9 per ml mix
cells aberrant cells (%mean) scored incl. excL exchan-
gaps gaps ges
types of aberrations found
-gaps-
-chromat id type-
g 'g
b
f
d
ex
Negative control
Solvent control ethanol
Positive control
CPA
+ cult. 1 100 cult. 2 100 -total- 200
6.5 4.0 0.0
1.0 %. +
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 2
0
3' 0 0
0
5
0
5100 5
0
6
0
02010000 11
0
Z
1
0
0
669301.89 5
1
12
0
17
1
12 12
1
17
Test
article
30.0 (i.g + 100.0 u,g + 300.0 (ig +
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
-chr ib
0
00 000 011 000 000 000
Table 5: Structural chromosomal aberrations
experiment I; fixation interval 28 h; without and with metabolic act
cone. S9 per ml mix
cells aberrant cells (%mean) scored incl. excl. exchan-
gaps gaps ges
types of aberrations found
-gapsg 19
-chromatid type-
b
f
d ex
-chro 1b
Solvent
control ethanol
1.0 % -
cult. 1 100 cult. 2 100 -total- 200
5.5 2.0 0.5
Test
300.0 p.g - cult. 1 100
article
cuU. 2 100
-total- 200" 3.5 1.5 0.0
Solvent control ethanol
Test
article
1.0 % + 300.0 p.g +
cult. 1 100 cult. 2 100 -total- 200 cult. 1 100 cuU. 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
1
0 6
1
21110011 00 1
0
4
0
01 0
0
22010000 000 5
0
1 3 4
1 0 1
011101000000
000
2
0
1
0
3
0
000303000000
000
n
Experiment II
P)
-~-)
U3
Table 6: Number of Polyp "loid Cells and Mitotic Index
t\J 1\J
experiment II; fixation intervals 18 h and 28 h; without and with metabolic activat
h
cone. per ml
S9 fixation mix interval
polyploid cells*
culture
1
2
total
mean
mit
culture
1
&
0
s
w
B
3'
51
0
T3
S 0
iQ 01
(B
NJ (TI
0
Negative control
-
Solvent control ethanol Positive control EMS
Test article
1.0 %
-
0.4 mg -
30.0 p.g -
100.0 [ig -'
200.0 M
-
Negative control
Solvent control ethanol Positive control CPA
Test article
+
1.0 % + 0.93 p-g + 30.0 ^g + 100.0 [i,g + 200.0 \LQ +
Solvent control ethanol
1.0 %
-
Test article
200.0 ^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
28 h 28 h
3.0
18.3 13
1.5
13.2 13
246 4
6
10
5.0
8.1 7
123 1.0
11.6 9
1.5
9.4 13
022 2.5
9.8 16
043135 2.0
16.8 11
3.0
14.5 12
314 3.5
13.5 8
156 0.5
13.7 15
437 3
1
4
2.0
17.5 17
101 1.0
13.7 15
022 1.5
9.8 10
213 2
2
4
2.0
15.1 7
031215 Solvent control ethanol
1.0 % +
Test article
200.0 p.g *
28 h 28 h
0.5
12.3 14
2.5
14.2 15
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 c
ijj Ni
Table 7: Structural chromosomal aberrations
experiment II; fixation interval 18 h; without metabolic activation
Negative control
Solvent control ethanol Positive control
EMS
Test
article
11
cone. S9 per ml mix
-
1.0 % 0.4 mg 30.0 ^g 100.0 v.g 200.0 u,g -
cells aberrant cells (%mean)
scored incl. excl. exchan-
gaps gaps ges
cult. 1 100 cult. 2 100
-total- 200
4.0 1.5 0.5
cult. 1 100 cult. 2 100 -total- 200- 5.0 2.0 0.0
cult. 1 100 cult. 2 100
-total- 200 13.5 10.5 5.5
cult. 1 100 cult. 2 100 -total- 200
3.0 1.0 0.0
cult. 1 100 cult. 2 100 -total- 200
6.5 0.5 0.0
cult. 1 100 cult. 2 100 -total- 200
4.5 2.0 0.0
types of aberrations found
-gapsg <a
-chromatid type-
b
f
d
ex
5
0
0 0
0' 0
0
0
1100 5
0
1100 2
0
4
0
0100 6
0
11010000 6
1
2
0
33340076 8
1
6
7
0
13
1
0
3
0
4
0
02Z 000 000 000
6
1
6
0
0 0 0 0 12
1
11000000 3
0
2
0
20010000 5
0
Z
1
0
0
For abbreviations see RESULTS/ABBREVIATONS
-chr ib
000 000 000 000 000 011
Table 8: Structural chromosomal aberrations
experiment II; fixation interval 18 h; with metabolic activation
Negative control
Solvent control ethanol Positive control
CPA
Test
article
11
cone. S9 per ml mix
+
1.0 % + 0.93 mg + 30.0 u,g + 100.0 |jLg + 200.0 [jig +
cells aberrant cells (%mean) scored incl. excl. exchan-
gaps gaps ges
cult. 1 100 cult. 2 100 -total- 200
cult. 1 cult. 2 -total-
100 100
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
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
types of aberrations found
-gaps-
-chromatid type-
g 19
b
f
d
ex
1
0
4
0
5
0
4
0
0
0
4
0
6
0
6
1
1Z
1
3, 1 0
0
030100
0
0
000213000101
5 10
0
11
3 13
0
12
8 23
0
23
2 2 4
0 0 0
000213000000
1 5 6
0 0 0
000325000000
2 1 3
0 0 0
000101000000
-chro 1b
000
00
0
0
00 000 000 000
Table 9; Structural chromosomal aberrations
experiment II; fixation interval 28 h; without and with metabolic a
cone. S9 per ml mix
cells aberrant cells (%mean) types of aberrations found
scored incl. excl. exchan- -gaps-
-chromatid type-
-chr
gaps gaps ges
g ig
b
f
d
ex
ib
i
Solvent
control ethanol
1.0 % -
cult. 1 100 cult. 2 100 -total- 200
2.5 0.5 0.0
Test
200.0 u,g - cult. 1 100
article
cult. 2 100
-total- 200" 3.5 2.5 0.5
4
0
0, 0 0
0
1 5
0 0
00000000
011
0 2 2
0 0 0
011220000000
000
Solvent
1.0 % + cult. 1 100
6
0
0000 0 control
cult. 2 100
4
0
0
000 0 ethanol
-total- 200
5.5 1.0 0.0
10
0
0000 0 Test 011000000000 011 article
200.0 u.g +
cult. 1 100 cult. 2 100 -total- 200
3.0 1.0 0.0
3
0
2
0
5
0
For abbreviations see RESULTS/ABBREVIATONS
DISCUSSION
The test articlewHUlBHU^B5 assessed for its potential to
induce structural chromosomal aberrations in VPS cells of the Chinese hamster in vitro in the absence and presence of metabolic
activation by 39 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 S9 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 hi highest evaluable concentration):
Experiment I
without S9 mix: 18 h: 30.0; 100.0; 300 ug/ml 28 h; 300.0 ug/ml
with S9 mix: 18 h: 30.0; 100.0; 28 h: 300.0 ug/ml
300 ug/ml
Experiment II
without S9 mix: 18 h: 30.0; 100.0; 200 ug/ml 28 h: 200.0 ug/ml
with S9 mix: 18 h: 30.0; 100.0; 200 tig/ail 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 jig/mi (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 ug/ml) at both fixation intervals whereas in experiment II
(highest concentration 200 ug/ml) ho 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
CoaapanS!
,n4alnTSCACBl
page 30 of 32
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 ing/ml) and CPA (0.93 tig/mi) were used as positive controls and showed distinct increases in cells with structural chromosomal aberrati'dns.
In conclusion, it can be stated that in the study described and
, under the ^cperimental conditions reported, the test article
m^^HHUJdid not induce structural chromosomal aberrations in
the V79 Chinese hamster cell line.
.^,
ca7922r
^con^nlSC^CBl
C^-^-00650
P3^ 31 of 32
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.
Peterson 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. 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
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