Document bJmV36VeBN6NBzVwOvQNDa8y
AR226-2997
STUDY TITLE:
CHROMOSOME ABERRATION ASSAY IN CHINESE HAMSTER V79 CELLS IN VITRO WITH
REPORT
DATA REQUIREMENTS: OECD Guideline 473 EEC Directive 92/69
EPA 40 CFR Ch. 1 (7-1-86 ed. 798.5385)
AUTHOR: Dr. Wolfgang Vlkner
STUDY COMPLETION: July 06,1993
PERFORMING LABORATORY: CCR - Cytotest Ceil 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 Krmer
Page 1 of 32
RCC
Group
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COPY OF G LP CERTIFICATE
HESSISCHES MINISTERIUM . . FOR UMWELT. ENERGIE UND BUNDESANGELEGENHEITEN
GLP-Bescheinlgung
Bescheinigung
.' '
Hiermit wird besttigt, daB die Prfungsainrichtung(an) Cytotest Cell Research GmbH 4 Co KG
In den Leppsteinswiesen 19
ln 6101 Rodorf ' ~_________ ;_________
(Ort, Anschrift)
.
dar_RCC Holding Verwaltung GmbH________
(Firma)
-
03.08., 04.08., 05.08.- und.06.08.92
a m r-r- - .....T -irf ,- r.r--- ...r.....!-..---- -,------------------- ---------
(Datum)
von dar fr dia berwachung zustndigen Behnde ber
die Einhaltung der Grundstze dar Guten Laborpraxis ,
Inspiziert worden b t (sind).
. /*
Es wird hiermit besttigt, daB folgende Prfungen In
dieser Prfeinrichtung nach den Grundstzen der Guten
Laborpraxis durchgefhrt werden. - '
-
Certificata
It b hereby certified that the test iadCty(ies) Cytotest Cell Research GmbH & Co KG
In den Leppsteinswiesen 19
'
.in __6_1__0_1_____R_o____d__o_r__f_____`___ __
.'
(locacion, addrau)
______________
4 RCC Holding Verwaltung GmbH
Of - -----
............-- - .........
(company name)
03.08., 04.08., 05.08. and.06.08.92
was (were) inspected by the competent authority regar ding compliance with the Principles of Good Laboratory Practice.
It b hereby certified! that studies in thus test facility, are
conducted in compliance with the Principles of Good
Laboratory Practice. ' . .
Toxikologische Eigenschaf
Toxicological, properties
Im Auftrag .
GV. jtl.'Jus
(O r Hecker) '
Wiesbaden, den
v p v ijV
V?T- * ^
X!4ksi'5
O l . /t1
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UUNIfcNlb
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
DISCUSSION REFERENCES DISTRIBUTION .
PAGE
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 N o .:
Test Article:
Title:
"
ISEGA Forschungs- und Untersuchungsgesellschaft mbH D-8750 Aschaffenburg
Heike Krmer
CCR CYTOTEST CELL RESEARCH GMBH & CO. KG D-6101 Rodorf
Chromosome Aberration Assay in Chi^nese HHaammststeerr Vv79_ Cells in vitro witi
PROJECT STAFF
Management: Study Director: Author: Head of Quality Assurance Unit: Technical Coordination:
Dr..Hans-Emil Knoell Dr. Albrecht Heidemann Dr. Wolfgang Vlkner
Dr. Christiane Helmrich Frauke Khner
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 01, 1992
March May March May
01, 1993 24, 1993 29, 1993 05, 1993
May 26, 1993
July
06, 1993
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Study Director:
Author: Management :
Date: July 06, 1993
Dr. Wolfgang Vlknr
La). Oclku^j
Date: July 06, 1993
Dr. F ' ` Knoell
Date:
06, 1993
nALITY ASSURANCE
The study was performed in compliance with:
"Chemikaliengesetz ("Chemicals Act") of the Federal Republic of Germany, Anlage 1 ("Annex 1"), dated March 14, 1990 (B G B L . 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 M a y 26, 1983, vitrq_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 Rodorf will archive the following data for 30 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
Z
y
Dr. Albrecht Heidemann
Chromosome Aberration Assay in Chinese Hamster V79 Cells in vitro wit
To the best of m y knowledge and belief, this study performed in the testing" facility of CCR was conducted in compliance with the Good Laboratory. Practice Regulations :
Chemikaliengesetz ("Chemicals A c t ") of the Federal Republic of Germany, Anlage 1 ("Annex 1"), dated March 14, 1990 (BGB L . 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 Rodorf
STATEMENT
Project Number: Test Article: Study Director: Title:
326430
Dr. Albrecht Heidemann Chromosome Aberration Assay in Chinese Hamster V79 Cells in vitro w i t h H | H ^ ^ ^ p |
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 11, 1992
March
02, 1993
April
02, 1993
April
21, 1993
June
28 ,1993
,
Dates of Reports to the Study Director and to Management
November 11, 1992
March
02, 1993
April
02, 1993
April
21, 1993
June
28 ,1993
Head of
Quality Assurance Unit
Dr. Christiane Helmrich
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The test a r t i c l e ^ j H ^ B H ^ ^ ^ J was assessed for its potential to induce structural chromosomal 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 ug/ml 28 h: 300 pg/ml
with S9 mix:
18 h: 30; 100; 300 ng/ml
28 h: 300 fig/ml
Experiment II without S9 mix: 18 h: 30; 100; 200 ug/ml 28 h: 200 ug/ml
with S9 mix: 18 h: 30; 100; 200 jig/ml 28 h: 200 pg/ml
The concentration range of the test article was determined in pre-experiments. In experiment II the highest concentration was 200 ug/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 ug/ml of the test article (with and without S9 m i x ) . 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 af er
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 and under the experimental conditions r e p o r t e d t h e test article di not induce structural chromosomal aberrations as determined y the chromosomal aberration test in the V79 Chinese hamster ce 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 V79.
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 d amage that is_. compatible with_cell .divisi o n .-- 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 V79 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 m a d e . 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.
Aggregate State at R T :
Colour:
Analysis : Molecular Weight:
liquid not indicated by the sponsor
a) 335 b) 257
Storage: Expiration Date:
4 0C 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 c e l l s. 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 (etha nol) were performed.
The Positive Control Substances
Without metabolic activation
Name :
EMS; Ethylmethanesulfonate
Supplier:
Merck-Schuchardt, D-8000 Mnchen
Catalogue n o .:
820774 (purity > 98 %)
Dissolved in:
nutrient medium
Final Concentration: 0.4 mg/ml = 3.25 mM
Solution prepared on 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 > 99.5 %) nutrient medium 0.93 ng/ml = 3.3 pM (in deviation to protocpl where 2.8 pg/ml = 10.0 pM was 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 77.3.
The V79 cell line has been used successfully for many years in in 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 V7 9 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 cm2 plastic flasks (GREINER, D-74'43 Frickenhausen) . About 5 x 10 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 fPreparation by C C R^
The S9 liver microsomal fraction was obtained from the livers of 8 - 1 2 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 mg/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 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 u s e . The protein content was determined using the analysis kit of Bio-Rad Laboratories, D-8000 Mnchen: 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 M g C l2 3 3 mM K C l
5 mM glucose-6-phosphate 4 mM NADP in 100 m M 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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PR E-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 w ith 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 pg/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 jig/nil 28 h: 10.0; 30.0; 100.0; 300.0 ng/ml
with S9 mix 18 h: 1.0; 3.0; 10.0'; 30.0; 100.0; 300 (ig/ml 28 h: 10.0; 30.0; 100.0; 300.0 (ig/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.
Experiment II 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 (ig/ml
200 ug/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 ng/ml
However, 200 pg/ml were not completely dissolved too, so the concentrations used in each experiment cannot be given accu
rately.
The treatment interval was 4 h with metabolic activation. 18 h
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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, 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 jig/ml)
in both fixation intervals, but in experiment II no-reduction of
the mitotic index was found at the highest concentration used
(200 jig/ml) . Therefore, in cytogenetic experiment I, in the
absence and. presence of S9 mix cultures after treatment with
300.0 ug/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 jig/ml at each fixation interval
with and without S9 mix.
EXPERIMENTAI. 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--M g -- free salt solution (Trypsin: Difco Laboratories, Detroit, USA).
The Ca-Mg-free salt solution was composed as follows
NaCl KC1 Glucose NaHC03
8000 mg 400 mg
1000 mg 350 mg
(per litre):
Prior to the trypsin treatment the cells were rinsed with Ca-Mgfree 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 10 - l x 10 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):
NaCl - '
8000 mg
KC1 400 mg
Glucose
1100 mg
N a 2H P 0 4 .7H20 290 mg
K H 2P04
150
pH is adjusted to 7.2
mg.
Exposure time 18 and 28 hours (without S9 m i x ) :
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 % C02 (95.5 % air).
Preparation of the Cultures
15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 jig/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).
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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 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 reproducibly 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 nonmu tagenic 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 M-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 chiI square 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
negative control _ solvent control ethanol
0.1 |ig 0.3 iig . 1.0 p.g 3.0 p.g 10.0 |ig 30.0 jig 100.0 jig 300.0 p.g
negative control solvent control ethanol
0.1 jig 0.3 jig 1.0 p,g 3.0 jxg 10.0 (ig --- 30.0 p,g 100.0 p,g 300.0 p.g
S9 colonies counted mix flask I flask II
302 355
251 282 277 243 251 264 320 284
306 315
294 296 312 295
254 309 - 294 274
+
+
+
+
+
+
+
/
+ +
--
+
288 303 252 263 304 281 284
317 265 297
279 252 255 287 231 266 267 310 301 238
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 materia 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 change; cx = chromosome type exchange; cd = chromosomal d i s m e gration (= pulverization)
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Experiment I
Table 2: Number of Polyploid Cells and Mitotic Index n experiment I; fixation intervals 18 h and 28 h; without and with metabolic activation pj
VO
ro NJ H
cone, S9 fixation per ml mix interval
polyploid cells* culture total mean
mitotic index (% )**
culture
abs. rei.***
12
12
Negative control
Solvent control ethanol Positive control EMS Test article
1.0 % 0.4 mg 30.0 P-9 100.0 p-g 300.0 p-g
-
-, -
18 h
18 h 18 h
18 h 18 h 18 h
347
3.5
20.0 22.9
21.5 100.0
134
2.0
17.1 20.9
19.0 100.0
369
4.5
10.3 17.3
13.8 64.3
909
4.5
11.4 10.2
10.8 56.8
404
2.0
14.1 14.3
14.2 74.7
437
3.5
13.1 12.4
12.8 67.1
Negative control Solvent control ethanol
Positive control CPA Test article
1.0 % 0.93 p,g 30.0 p-g 100.0 p -g1 300.0 p-g
+ + + +
+
+
18 h 18 h
18 h
18 h 18 h 18 h
32 5
2.5
16.8 16.4
16.6 100.0
5 5 10
5.0
11.1 16.0
13.6 100.0
325
2.5
14.3 12.5
13.4 80.7
123
1.5
14.7 16.5
15.6 115.1
2 13
1.5
17.0 16.1
16.6 122.1
4 59
4.5
15.1 18.0
16.6 122.1
Solvent control ethanol 1.0 %
Test article
300.0 p-g
-
-
28 h 28 h
527
3.5
13.5 14.9
14.2 100.0
112
1.0
10.6 10.5
10.6 74.3
Solvent control ethanol
1.0 %
+
Test article
300.0 p-g +
28 h 28 h
022
1.0
20.4 17.2
18.8 100.0
404
2.0
17.3 20.7
19.0 101.1
* The number of polyploid cells was determined in 100 cells per culture of each test group. ** 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 the negative controls; for the test article treatment groups the values are related to the solvent controls
notcontainT 8 C A 6
iQ (D
to to
O hh
U>
to
O Table 3: Structural chromosomal aberrations
pi -J
experiment I; fixation interval 18 h; without metabolic activation
IO
to
to
M
cone. S9
cells aberrant cells (%mean) types of aberrations found
per ml mix
scored incl. excl. exchan- -gaps- -chromatid type- -chromosome type- -other-
gaps gaps ges
g ig b f d ex ib if id cx ma cd
Negative control
- cult. 1 100
cult. 2 100 -total- 200
5.0 2.0 0.0
30
50 80
0 10
200 2 10
0
0 0
0000
0200 0200
00
00 00
Solvent
control ethanol
1.0 % - cult. 1 100
cult. 2 100 -total- 200' 5.5 3.0 0.5
30
30 60
2 10
1 10 320
1 0 1
0000
0000 000 0
00 00 00
Positive control EMS
0.4 mg -
cult. 1 100 cult. 2 100 -total- 200
13.5 11.5
7.0
30
40 70
500 6 310 8 8 1 0 14
000 1
000 1 000 2
00
10 10
Test article
30.0 p,g -
cult. 1 100 cult. 2 100 -total- 200
7.5 2.5 0.0
31 70 10 1
2 10 0 10 220
0 0 0
100 0 000 0 10 0 0
00 00 00
II 100.0 ng - cult. 1 100
30 300 0 0000 00
cult. 2 100
10 0 1 1 0 0 0 1 0 0 0 0
-total- 200
9.0 3.0 0.0
13 0 4 1 0 0 0 1 0 0 0 0
II 300. 0 p,g - cult. 1 100
50 110 0 0000 00
cult. 2 100
70 310 0 0000 00
-total- 200
8.0 2.5 0.0
12 0 4 2 0 0 0 0 0 0 0 0
PO>
iQ (D
For abbreviations see RESULTS/ABBREVIATONS
to
to
O
H.
UJ to
n Table 4: Structural chromosomal aberrations
pj ~j
experiment I; fixation interval 18 h; with metabolic activation
1X3
N3
to
h
cone. S9
cells aberrant cells (%mean) types of aberrations found
per ml mix
scored incl. excl. exchan- -gaps- -chromtid type-
gaps gaps ges
9 ig b f d ex
Negative control
+ cult. 1 100 cult. 2 100 - t o t a l- 200
6.5 4.0 0 . 0
30 20 50
2 10 3' 0 0 5 10
0 0 0
Solvent control ethanol
1.0 % + cult. 1 100 cult. 2 100 - t o t a l- 200x 8.0 2.5 0.5
50 60 n0
000 2 10 2 10
0 0 0
ss
o
3 % O a
e3.
o O >T3 B (H iQ (D
fO
Positive control CPA Test article
II
It
0.93 mg + cult. 1 100 cult. 2 100 - t o t a l- 200
30.0 fig +
cult. 1 100 cult. 2 100
- t o t a l- 200
100.0 p,g + cult. 1 100 cult. 2 100 - t o t a l- 200
300.0 p.g +
cult. 1 100 cult. 2 100 - t o t a l- 200
22.0 2.0 3.0 3.5
For abbreviations see RESULTS/ABBREVIATONS
18.0
0 .0
1.5 2.0
8.0
0 .0 0 .0 0 .0
51 631 8 12 0 6 9 0 9 17 1 12 12 1 17
20 20 40
000 000 000
0 0 0
20 20 40
0 20 0 10 030
0 0 0
10 30 40
030 100 130
0 0 0
-chromosome typeib if id cx
0 0' 0 0 0200 0200
0000 u00 1 000 1
1 1- 0 0 030 2 14 0 2
0000 0000 0000
0000 0000 0000
0000 0000 0000
-otherma cd
00 00 00
10 00 10
10 00 10
00 00 00
00 00 00
00 00 00
O
Hi
Lo ro
O Table 5: Structural chromosomal aberrations *P)0 experiment I; fixation interval 28 h; without and with metabolic activation
tv)
to
h
cone. S9
cells aberrant cells (%mean) types of aberrations found
per ml mix
scored incl. excl. exchan- -gaps- -chromatid type- -chromosome type- -other-
gaps gaps ges
9 19 b f d ex ib if id cx ma cd
Solvent
control ethanol
1.0 % -
cult. 1 100
cult. 2 100 -total- 200
5.5 2.0 0.5
30 31 61
1. 0 0 1 10 2 10
0 1 1
0000 0000 0000
00
00 00
Test
300.0 p.g - cult. 1 100
article
cult. 2 100
-total- 200v 3.5 1.5 0.0
10 40 50
010
200 2 10
0 0 0
0000 0000 0000
00 00 00
Solvent
control ethanol
1.0 % + cult. 1 100
cult. 2 100 -total- 200
3.0 1.0 0.0
f Test
300.0 p,g + cult. 1 100
article
cult. 2 100
o o(0
-total- 200
2.0 0.5 0.0
u>
3
O<*`6> O
For abbreviations see RESULTS/ABBREVIATONS
o
3
2.
4 & O >
D> iQ (D
10 Ln
O Hi OJ to
11 30 41
20 10 30
100 0 10 1 10 030 000 030
0 0 0
0 0 0
0000 0000 00 0 0 0000 0000 0000
00 00 00
00 00 00
n Experiment II
VO Table 6: Number of Polyploid Cells and Mitotic Index
to to
experiment II; fixation intervals 18 h and 28 h; without and with metabolic activation
I
I
Oo CtfDi 3O#c* oo
s> 5'
"S
O
>o >
(1) iQ (I)
o g
M tn
o
Hi
J
cone, S9 fixation per ml mix interval
polyploid cells* culture total mean 12
mitotic index (% J**
culture
abs. rei.***
12
Negative control Solvent control ethanol Positive control EMS Test article
it
II
%1 . 0 0.4 mg
30.0 pg 100.0 pg
200.0 pg
-
-
v
-
18 h 18 h 18 h 18 h 18 h
18 h
246
3.0
18.3 13.0
15.7 100.0
123
1.5
13.2 13.1
13.2 100.0
4 6 10
5.0
8.1 7.7
7.9 50.5
0 22
1 .0
11.6 9.4
10.5 79.8
0 33
1.5
9.4 13.3
11.4 86.3
41
5
2.5
9.8 16.5
13.2 100.0
Negative control Solvent control ethanol Positive control CPA Test article
II
II
%1 . 0 0.93 pg 30.0 pg
100.0 pg
200.0 pg
+ + + + +
+
18 h 18 h 18 h 18 h
18 h
18 h
3 14
2.0
16.8 11.2
14.0 100.0
1 56
3.0
14.5 12.9
13.7 100.0
4 37
3.5
13.5 8.7
11.1 79.3
1 01
0.5
13.7 15.4
14.6 106.2
3 14
2.0
17.5 17.1
17.3 126.3
022
1 .0
13.7 15.9
14.8 108.0
Solvent control ethanol
%1 . 0
Test article
200.0 pg -
28 h 28 h
2 13 224
1.5 2.0
9.8 10.6 15.1 7.0
10.2 100.0 11.1 108.3
Solvent control ethanol
%1 . 0
+
Test article
200.0 pg +
28 h 28 h
0 11 325
0.5 2.5
12.3 14.3 14.2 15.8
13.3 100.0 15.0 112.8
* The number of polyploid cells was determined in 100 cells per culture of each test group. ** 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 the negative controls; for the test article treatment groups the values are related to the solvent controls
n cu -j
VO
N> to H
O OCVD)
8
s.
(ft
O
>
a
pj
iQ
o
h
(D
to
-j
o
H)
u> to
Table 7: Structural chromosomal aberrations experiment II; fixation interval 18 h; without metabolic activation
Negative control
Solvent control ethanol
Positive control EMS Test article
il
H
cone. S9 per ml mix
cells aberrant cells (%mean) scored incl. excl. exchan
gaps gaps ges
- cult. 1 100 cult. 2 100 - t o t a l - 200
4.0 1.5 0.5
1.0 % - cult. 1 100 cult. 2 100 - t o t a l - 200' 5.0 2.0 0.0
0.4 mg -
cult. 1 100
cult. 2 100 - t o t a l - 200
13.5 10.5
5.5
30.0 pg -
cult. 1 100 cult. 2 100 - t o t a l - 200
3.0 1.0 0.0
100.0 pg -
cult. 1 100 cult. 2 100 - t o t a l- 200
6.5 0.5 0.0
200. 0 pg -
cult. 1 100
cult. 2 100 - t o t a l- 200
4.5 2.0 0.0
types of aberrations found -gaps- -chromatid typeg ig b f d ex
-j--
50 1 10 0
0 0 O' 0 0 0 50 110 0
20 40
60
0 10
100 110
0
0 0
61
20 81
330 340 6 70
7
6 13
10
30 40
000 200 200
0 0 0
61 60
12 1
000 100 100
0 0
0
30 20 50
200
0 10 2 10
0
0 0
-chromosome typeib if id cx
000 1 00 0 0 000 1
0 100 0 100 0 20 0
0000 000 1 0001
0000 0000 0000
0000 0000 00 00
1000 0000 1000
-otherma cd
00 01 01
00 00 00
00 00 00
00 00 00
00 00 00
00 00 00
For abbreviations see RESULTS/ABBREVIATONS
o o -J vo to
to
H
(ft 01 3. 3* B to a
Oo
(D (0 3 O
Table 8: Structural chromosomal aberrations experiment II; fixation interval 18 h; with metabolic activation
Negative control
Solvent control ethanol
Positive control CPA Test article
H
li
cone, S9 per ml mix
cells aberrant cells (%mean) types of aberrations found
scored incl. excl. exchan -gaps- -chromatid type-
gaps gaps ges
g '9 b f d ex
-chromosome typeib if id cx
-otherma cd
+ cult. 1 100
cult. 2 100 -total- 200
4.0 1.5 0.0
10
40 50
310 000 3 10
0
0 0
0000
00 0 0 00 0 0
00
00 00
1.0 %- + cult. 1 100 cult. 2 100 -total- 200' 4.0 2.5 0.5
40 00 40
0 20 0 10 0 30
1 0 1
00 0 0 0100 0100
00 00 00
0.93 mg +
cult. 1 100 cult. 2 100 -total- 200
24.0 21.0 10.5
60 61 12 1
5 10 0 3 13 0 8 23 0
11
12 23
0100 0500
0600
00 00
00
30.0 p,g +
cult. 1 100 cult. 2 100 -total- 200
4.0 2.0 0.5
20
20 40
020 0 10 030
0
0 0
0000
000 1 00 0 1
00
10 10
100.0 p.g +
cult. 1 100
cult. 2 100 -total- 200
4.5 2.5 0.0
10 50 60
0 30 020 050
0
0 0
0000 0000 0000
00
00 00
200.0 ng +
cult. 1 100
cult. 2 100 -total- 200
2.0 0.5 0.0
20 10 30
0 10 000 0 10
0 0
0
0000 000 0
0000
00 00 00
contain T S C A
For abbreviations see RESULTS/ABBREVIATONS
o
hh Lo to
n Table 9: Structural chromosomal aberrations fu experiment II; fixation interval 28 h; without and with metabolic activation
U3
to
to
H
cone. S9
cells aberrant cells (%mean) types of aberrations found
per ml mix
scored incl. excl. exchan- -gaps- -chromatid type- -chromosome type- -other-
gaps gaps ges
S ig b f d ex ib if id cx ma cd
Solvent
control ethanol
1.0 % _ cult. 1 100 cult. 2 100 -total- 200
2.5 0.5 0.0
40 10 50
0, 0 0 000 000
0 0 0
100 0 0000 1000
00
00 00
Test
200.0 pg _ cult. 1 100
article
cult. 2 100
-total- 200' 3.5 2.5 0.5
00 20 20
020 10 0 120
0 0
0
0 100 000 1
0 10 1
00 10 10
Solvent control ethanol
Test article
1.0 % + 200.0 pg +
cult. 1 100 cult. 2 100 -total- 200
cult. 1 100 cult. 2 100 -total- 200
5.5 1.0 0.0 3.0 1.0 0.0
60 40 10 0
30 20 50
000 000 000
100 000 100
0 0 0
0 0 0
040 0 000 0 040 0
0000 10 0 0 100 0
00 00 00
00 00 00
For abbreviations see RESULTS/ABBREVIATONS
0) r iQ
fl)
O to
o
Hi
Ui to
i
DISCUSSION
The test a r t i c l e ^ H H l ^ ) was assessed for its potential to induce structural 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 S9 mix) and 18h and 28 h (without S9 m i x ) .
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 ng/ml 28 h: 300.0 jig/ml
with S9 mix: 18 h: 30.0; 100.0; 300 ng/ml 28 h: 300.0 (ig/ml
Experiment II
without S9 mix: 18 h: 30.0; 100.0; 200 ^g/ml 28 h: 200.0 ng/ml
with S9 mix: 18 h: 30.0; 100.0; 200 ng/ml 28 h: 200.0 ng/ml
The concentration range used in this study (300 ng/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 p r e sence of S9 mix after treatment with concentra
tions up to 300.0 jig/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 jig/ml) at both fixation intervals whereas in experiment II (highest concentration 200 |i.g/ml) ho reduction of the mitotic index was o b s e r v e d .'These effects are considered being biologi cally irrelevant since there was no dose relation in the test
groups used.
1
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
not contain TSCACBl g a i n e d , EKies
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 r a n g e : 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 jig/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 jucperimental conditions reported, the test article
not induce structural chromosomal aberrations in
"the V79 Chinese hamster cell line.
ca7922r
,,,,con*"'30* 08' Co
page 31 of 32
REFERENCES
1. B.N. A m e s , 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. H e d d l e , A.F. McFee, S. Wolff a n d J . S . Wassom (1981) Mammalian in vivo and in vitro cytogenetic as s a y s .. A report of the U.S. EPA's gene-tox program. Mutation Research 87, 143-188
4. Engelhardt G. , (1987)
_
"Arbeitsgruppe der Industrie, Cytogenetik"
Standard-Protokoll zur cytogenetisehen 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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ca7 922r
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