In Vitro Chromosomal Aberrations
Transcript of In Vitro Chromosomal Aberrations
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Final Report
Study Title Screening Assay for Chromosomal Aberrations in
Chinese Hamster Ovary (CHO) Cells with
Argentyn 23
Author(s) Hemalatha Murli, PhD
Sponsor Natural Immunogenics Corporation7440 S.W. 50th Terrace, Unit 107
Miami, Florida 33155
Test Facility Covance Laboratories Inc.
9200 Leesburg Pike
Vienna, Virginia 22182-1699
Covance Study Number 24742-0-437SC
Report Issued 08 April 2003
Page Number 1 of 15
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STUDY INFORMATION
1. Sponsor: Natural Immunogenics Corporation
2. Test Article: Argentyn 23, A Professional Colloidal Silver Formulation
23ppm concentration Ultra-fine Ag+dispersion,Lot No.:86HT
Date Received: 30 January 2003
Physical Description: Transparent, colorless liquidStorage Conditions: Room temperature
3. Type of Assay: Screening Assay for Chromosomal Aberrationsin Chinese Hamster Ovary (CHO) Cells
Protocol Number: 437SC, Edition 1, (+/-S9)
Covance Study No.: 24742-0-437SC
4. Study Dates:
Study Initiation Date: 27 January 2003
Experimental Start Date: 04 February 2003Experimental Termination Date: 20 March 2003
INTRODUCTION
At the request of Natural Immunogenics Corporation, Covance investigated the ability of
Argentyn 23 to induce chromosomal aberrations in Chinese hamster ovary (CHO) cellswith and without exogenous metabolic activation. The assay was initiated both in the
presence and absence of an exogenous metabolic activation system of mammalian
microsomal enzymes derived from AroclorTM
-induced rat liver (S9).
Most known chemical clastogens (chromosome-breaking agents) require a period of
DNA synthesis to convert initial DNA damage into chromosome alterations visible at
mitosis. At predetermined intervals after exposure to the test article, the cells were
treated with a metaphase-arresting substance, Colcemid , then harvested and stained, and
metaphase cells were analyzed microscopically for the presence of chromosomal
aberrations.
Many mutagenic chemicals do not act directly on DNA but do so after being converted to
active inter-mediates by enzymes found in liver. CHO cells have little or no capacity to
metabolize test articles, so an exogenous metabolic activation system (rat liver S9homogenate) was included with a series of treatments to enhance the degree of
conversion and the ability of the assay to detect clastogenic, metabolic intermediates.
This study evaluated structural chromosomal aberrations (defined as structural
chromosome damage expressed as breakage, or breakage followed by reunion, of both
sister chromatids at an identical site). Numerical aberrations (a change in the number of
chromosomes from the modal number of 21 for the CHO cell line used in this assay) are
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not determined by this protocol. However, the occurrence of polyploidy or
endoreduplication, which was scored, may indicate that the test article has the potential toinduce numerical aberrations.
The in vitro metabolic activation system (Maron and Ames, 1983) consisted of S9 and an
energy-producing system (NADP plus isocitric acid). Various hepatic P450 isoenzymelevels were increased by treatment of the rats with AroclorTM
1254 (single concentration
of 500 mg/kg) and sacrificed 5 days later (Molecular Toxicology, Inc., Lot No. 1393).
The S9 fraction, prepared in potassium chloride, was retained frozen at -60C until use.Aliquots of S9 were thawed immediately before use and added to the other components
to form the activation system described as follows:
S9 Activation System
Component Concentration in Cultures
NADP (sodium salt) 1.5 mg/mL (1.8 mM)Isocitric acid 2.7 mg/mL (10.5 mM)
Homogenate (S9 fraction) 15.0 L/mL* (1.5%)*This concentration of rat S9, obtained from Molecular Toxicology Inc., Boone,
NC, has consistently caused cyclophosphamide to be highly clastogenic.
The CHO cell line was derived from an ovarian biopsy of a female Chinese hamster. TheChinese hamster ovary cells (CHO-WBL) used in this assay were from a permanent cell
line and were originally obtained from the laboratory of Dr. S. Wolff, University of
California, San Francisco. The cells were subsequently subcloned in this laboratory, andstock cultures stored in liquid nitrogen. The CHO-WBL subclone is a permanent cell line
with an average cycle time of 12 to 14 hours and a modal chromosome number of 21.
The CHO cells were grown in McCoys 5a culture medium which was supplementedwith .10% heat-inactivated fetal bovine serum (FBS), L-glutamine (2mM), penicillin G
(100 units/mL), and streptomycin (100 g/mL). Single cultures were used for each dose
of the test article. Cultures were incubated with loose caps in a humidified incubator at
37C 2C in an atmosphere of 5% 1.5% CO2 in air.
The dose rangefinding assay was conducted with a ~3.0-hour treatment period in the
presence of S9, and ~20.0-hour treatment period without S9. All cultures were harvested~20.0 hours from the initiation of treatment. This harvest time corresponds to 1.5 times
the cell cycle time of approximately 13 hours (Galloway et al., 1994).
The chromosomal aberrations assay was conducted with a ~3.0-hour treatment period inthe presence of S9, and ~20.0-hour treatment period without S9. All cultures were
harvested ~20.0 hours from the initiation of treatment. This harvest time corresponds to
1.5 times the cell cycle time of approximately 13 hours (Galloway et al., 1994).
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RESULTS
Test Article HandlingThe dosing solutions were prepared in dimethylsulfoxide (DMSO; Acros Organics,
Lot No. A017325001). Argentyn 23 was solubilized in DMSO at a stock concentration
100-fold higher than the dose in tissue culture medium. Lower doses were obtained byserial dilutions of the stocks with DMSO. A dose volume of 10.0 L/mL was used.
A summary of the treatment times is given below.
Summary of Rangefinding/Chromosomal Aberrations Assay Treatment Schedule in
Hours (Approximate)
Activation
Condition
Test Article
Added
Wash Colcemid
Added
Harvest
Started
S9 0 --- 18.0 20.0
+ S9 0 3.0 18.0 20.0
In the dose rangefinding assay, concentrations of 7.81, 15.6, 31.3, 62.5, 125, 250, 500,
and 1000g/mL were tested with and without S9. No toxicity was observed in the
cultures treated with 1000g/mL tested with and without S9 (Tables 1 and 2). The
chromosomal aberrations assay was conducted with concentrations of 2000, 3500, and
5000g/mL with and without S9.
In the chromosomal aberrations assay without S9, no toxicity was observed in any of the
test cultures (Table 3). Structural chromosomal aberrations were evaluated at
5000g/mL (Table 2). No significant increase in the number of cells with structural
chromosomal aberrations, polyploidy, or endoreduplication was observed.
In the chromosomal aberrations assay with S9, no toxicity was observed in any of the test
cultures (Table 5). Structural chromosomal aberrations were evaluated at 5000g/mL
(Table 6). No significant increases in the number of cells with structural chromosomalaberrations, polyploidy, or endoreduplication was observed.
CONCLUSION
Argentyn 23 was considered negative for inducing structural chromosomal aberrations,
polyploidy, or endoreduplication with and without metabolic activation.
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REFERENCES
Evans, H.J., Chromosomal aberrations produced by ionizing radiation. International
Review of Cytology, 13:221-321 (1962).
Evans, H.J., Cytological Methods for Detecting Chemical Mutagens. ChemicalMutagens, Principles and Methods for their Detection, Hollaender, A. (ed.), Vol. 4, pp. 1-
29, Plenum Press: New York and London (1976).
Galloway, S.M., Aardema, M.J., Ishidate, M., Jr., Ivett, J.L., Kirkland, D.J., Morita, T.,
Mosesso, P., and Sofuni, T., Report from working group on in vitro tests forchromosomal aberrations. Mutation Research, 312(3):241-261 (1994) .
Maron, D.M., and Ames, B.N., Revised methods for the Salmonella mutagenicity test.
Mutation Research, 113:173-215 (1983).
Thakur, A.J., Berry, K.J., and Mielke, P.W., Jr., A FORTRAN program for testing trendand homogeneity in proportions. Computer Programs in Biomedicine, 19:229-233(1985).
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Table 1: Assessment of Toxicity for Chromosome Aberrations Assay - Without Metab
~20 Hour Treatment, ~20 Hour Harvest
Assay No.: 24742 Trial No.: A1 Date: 02/25/03 Lab No.Test Article: Argentyn 23
Confluencea
% %
% Vehicle Mitotic Mitotic
Control Index Reduction
Vehicle Control DMSO 10.0 L/mL 100 10.1 0
Test Article 1000 g/mL 100 10.8 0
Treatment
a This endpoint is based upon visual observations which are made prior to the harvest of the
metaphase cells. At the time of the confluence observation the flasks are also evaluated forthe appearance of floating mitotic cells and dead cells.DMSO = dimethylsulfoxide
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Table 2: Assessment of Toxicity for Chromosome Aberrations Assay - With Metabo
~3 Hour Treatment, ~20 Hour Harvest
Assay No.: 24742 Trial No.: A1 Date: 02/25/03 Lab No.Test Article: Argentyn 23
Confluencea
% %
% Vehicle Mitotic Mitotic
Control Index Reduction
Vehicle Control DMSO 10.0 L/mL 100 12.1 0
Test Article 1000 g/mL 100 12.6 0
Treatment
a This endpoint is based upon visual observations which are made prior to the harvest of the
metaphase cells. At the time of the confluence observation the flasks are also evaluated forthe appearance of floating mitotic cells and dead cells.DMSO = dimethylsulfoxide
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Table 3: Assessment of Toxicity for Chromosomal Aberrations Assay - Without Metab
~20 Hour Treatment, ~20 Hour Harvest
Assay No.: 24742 Trial No.: B1 Date: 02/25/03 Lab No.Test Article: Argentyn 23
Confluencea
% %
% Vehicle Mitotic Mitotic
Control Index Reduction
Vehicle Control DMSO 10.0 L/mL 100 10.4 0
Test Article 2000 g/mL 100 14.4 0
3500 g/mL 100 16.2 0
5000 g/mL 100 14.2 0
Treatment
a This endpoint is based upon visual observations which are made prior to the harvest of
the metaphase cells. At the time of the confluence observation the flasks are alsoevaluated for the appearance of floating mitotic cells and dead cells.DMSO = dimethylsulfoxide
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Table 4: Chromosome Aberrations in Chinese Hamster Ovary Cells - Without Metab
~20 Hour Treatment, ~20 Hour Harvest
Assay No.: 24742 Trial No.: B1 Date: 02/25/03 Lab No.: CY012803 T% #
MITOTIC ENDO- # NUMBERS AND PERCENTAG
INDEX REDUPLI- POLY- JUDGE- SHOWING STRUCTURAL CHROMOCELLS REDUC- CATED PLOID MENTSCORED TION a CELLS CELLS (+/-) b Gaps
SimpleBreaks chte chre
CONTROLSVEHICLE: DMSO 10.0L/mL A 100 0 2 2
B 100 0 7 2TOTAL 200 4
AVERAGE % 0 0.0 4.5 2.0
POSITIVE: MMC 0.400g/mL A 50 0 6 3 15 20B 50 0 4 2 15 20
TOTAL 100 5 30 40
AVERAGE % - 0.0 5.0 - 5.0 30.0 40.0
TEST ARTICLE 5000g/mL 100 0 5
AVERAGE % 0 0.0 5.0 -
chte: chromatid exchange chre: chromosome exchange mab: multiple aberrations, greater than 4 aberrationsa % Mitotic index reduction as compared to the vehicle control.b Significantly greater in % polyploidy and % endoreduplication than the vehicle control, p 0.01.c -g = # or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps.d Significantly greater in -g than the vehicle control, p 0.01. DMSO = Dimethylsulfoxide MMC = Mitomycin C
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Table 5: Assessment of Toxicity for Chromosomal Aberrations Assay - With Metabo
~3 Hour Treatment, ~20 Hour Harvest
Assay No.: 24742 Trial No.: B1 Date: 02/25/03 Lab No.Test Article: Argentyn 23
Confluencea
% %
% Vehicle Mitotic Mitotic
Control Index Reduction
Vehicle Control DMSO 10.0 L/mL 100 14.0 0
Test Article 2000 g/mL 100 14.1 0
3500 g/mL 100 15.4 0
5000 g/mL 100 15.2 0
Treatment
a This endpoint is based upon visual observations which are made prior to the harvest of
the metaphase cells. At the time of the confluence observation the flasks are alsoevaluated for the appearance of floating mitotic cells and dead cells.DMSO = dimethylsulfoxide
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HISTORICAL CONTROL DATA
CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS,
~20 HOUR HARVEST - 1/2002 THROUGH 06/2002
Activation%-g
%+g
%Polyploid
Cells
% Endore-duplicated
CellsNegative Control Without MIN 0.0 0.0 0.0 0.0
3 Hour Treatment MAX 2.0 5.0 8.0 1.0
AVG 0.8 2.0 2.4 0.1
SD () 0.69 1.38 2.23 0.27N 23 23 23 23
Vehicle Control
(Pooled) Without MIN 0.0 0.0 0.0 0.03 Hour Treatment MAX 1.5 6.5 7.0 1.0
AVG 0.4 2.0 2.2 0.1
SD () 0.43 1.71 2.19 0.31N 23 23 23 23
Positive Control - MMC Without MIN 41.0 44.0 0.0 0.0
3 Hour Treatment MAX 97.0 97.0 7.0 4.5AVG 70.9 74.4 2.3 0.5
SD () 14.14 13.11 2.18 0.98
N 23 23 23 23
Negative Control Without MIN 0.0 0.5 0.0 0.0
Continuous Treatment MAX 2.0 7.5 12.0 2.0
AVG 0.6 2.4 2.2 0.2
SD () 0.60 1.50 2.80 0.50
N 25 25 25 25
Vehicle Control(Pooled) Without MIN 0.0 0.0 0.0 0.0Continuous Treatment MAX 3.0 6.0 6.0 2.0
AVG 0.8 2.5 1.7 0.3
SD () 0.66 1.46 1.86 0.48
N 25 25 25 25
Positive Control - MMC Without MIN 30.0 33.0 0.0 0.0Continuous Treatment MAX 100.0 100.0 8.5 1.0
AVG 60.3 63.9 2.2 0.2
SD () 19.59 18.73 2.01 0.31
N 25 25 25 25
Negative Control With MIN 0.0 0.0 0.0 0.0
3 Hour Treatment MAX 3.0 8.0 12.0 2.5
AVG 0.8 2.4 2.2 0.5
SD () 0.87 1.99 2.82 0.78N 42 42 42 42
Vehicle Control
(Pooled) With MIN 0.0 0.0 0.0 0.0
3 Hour Treatment MAX 3.0 7.0 10.5 2.0AVG 0.6 2.0 2.2 0.3
SD () 0.80 1.64 2.31 0.46
N 43 43 43 43Positive Control - CP With MIN 15.6 19.4 0.0 0.03 Hour Treatment MAX 74.0 75.0 9.5 3.5
AVG 42.7 47.9 1.8 0.8
SD () 13.59 12.78 1.99 0.92N 43 43 43 43
N = Number of trials
MMC = Mitomycin CCP = Cyclophosphamide-g = % of cells with chromosome aberrations+g = % of cells with chromosome aberrations + % of cells with gaps
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DEFINITIONS OF CHROMOSOMAL ABERRATIONS FOR GIEMSA STAINED
CELLS
g (GAPS)
chtg Chromatid Gap: ("tid gap"). An achromatic (unstained) region in
one chromatid with clear discontinuity with no
visible connecting material, the size of which isequal to or less than the width of a chromatid.
chrg Chromosome Gap: ("isochromatid gap, IG"). Same as chromatid gapbut at the same locus in both sister chromatids.
SIMPLE BREAKS
chtb Chromatid Break: An achromatic region in one chromatid, larger thanthe width of a chromatid with clear partial or
complete displacement.
chrb Chromosome Break: Chromosome has a clear break, forming an
abnormal (deleted) chromosome with an acentricfragment that is dislocated.
ace Acentric Fragment: ace is different from the chrb only in that it is notapparently related to any specific chromosome.
COMPLEX EXCHANGES
chte (chromatid exchange)
id Interstitial Deletion: Length of chromatid "cut out" from midregion of a
chromatid resulting in a small fragment or ringlying beside a shortened chromatid or a gap in the
chromatid.
tr Triradial: An exchange between two chromosomes, or one
chromosome and an acentric fragment, which
results in a three-armed configuration.
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COMPLEX EXCHANGES (CONTINUED)
trf Triradial Fragment: A three-armed configuration with a fragment.
qr Quadriradial: As triradial, but resulting in a four-armed
configuration.
ci Chromosome Intrachange: Exchange within a chromosome; e.g., a ring thatdoes not include the entire chromosome.
su Sister Union: Intra-arm interchange with sister union of brokenends.
nud Non-union Distal: Intra-arm interchange with non-union of broken
ends distally.
nup Non-union Proximal: Intra-arm interchange with non-union of brokenends proximally.
cx Complex Rearrangement: An exchange among more than two chromosomes
or fragments, which is the result of several breaks.
chre (chromosome exchange)
dic Dicentric: An exchange between two chromosomes, which
results in a chromosome with two centromeres.This is often associated with an acentric fragment in
which case it is classified as dicf.
dicf Dicentric Fragment: Dicentric with fragment.
r Ring: A chromosome, which forms a circle, containing acentromere. This is often associated with an
acentric fragment in which case it is classed as RF.
rf Ring Fragment: Ring with associated acentric fragment.
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OTHER
ab Abnormal: Abnormal monocentric chromosome. This is a
chromosome whose morphology is abnormal for the
karyotype, and often the result of a translocation,
pericentric inversion, etc. Classification used ifabnormality cannot be ascribed (e.g., a reciprocal
translocation).
mab Multiple Aberrations: A cell, which contains more than 4 aberrations. A
heavily damaged cell should be analyzed to identifythe types of aberrations and may not actually have
>4 (e.g., multiple fragments such as those found
associated with a tricentric).
POLYPLOID CELLS
poly Polyploid cell: A cell containing multiple copies of the haploidnumber (n) of chromosomes.
endo Endoreduplication: 4n cell in which separation of chromosome pairshas failed.