Introduction

Final Report
Screening Assay for Chromosomal Aberrations inChinese Hamster Ovary (CHO) Cells withArgentyn 23 Natural Immunogenics Corporation7440 S.W. 50th Terrace, Unit 107Miami, Florida 33155 Covance Laboratories Inc.
9200 Leesburg PikeVienna, Virginia 22182-1699 Covance 24742-0-437SC
STUDY INFORMATION
Argentyn 23, A Professional Colloidal Silver Formulation23ppm concentration Ultra-fine Ag+dispersion,Lot No.:86HT Screening Assay for Chromosomal Aberrationsin Chinese Hamster Ovary (CHO) Cells Experimental Termination Date: 20 March 2003 INTRODUCTION
At the request of Natural Immunogenics Corporation, Covance investigated the ability ofArgentyn 23 to induce chromosomal aberrations in Chinese hamster ovary (CHO) cellswith and without exogenous metabolic activation. The assay was initiated both in thepresence and absence of an exogenous metabolic activation system of mammalianmicrosomal enzymes derived from AroclorTM-induced rat liver (S9).
Most known chemical clastogens (chromosome-breaking agents) require a period ofDNA synthesis to convert initial DNA damage into chromosome alterations visible atmitosis. At predetermined intervals after exposure to the test article, the cells weretreated with a metaphase-arresting substance, Colcemid , then harvested and stained, andmetaphase cells were analyzed microscopically for the presence of chromosomalaberrations.
Many mutagenic chemicals do not act directly on DNA but do so after being converted toactive inter-mediates by enzymes found in liver. CHO cells have little or no capacity tometabolize test articles, so an exogenous metabolic activation system (rat liver S9homogenate) was included with a series of treatments to enhance the degree ofconversion and the ability of the assay to detect clastogenic, metabolic intermediates.
This study evaluated structural chromosomal aberrations (defined as structuralchromosome damage expressed as breakage, or breakage followed by reunion, of bothsister chromatids at an identical site). Numerical aberrations (a change in the number ofchromosomes from the modal number of 21 for the CHO cell line used in this assay) are Covance 24742-0-437SC
not determined by this protocol. However, the occurrence of polyploidy orendoreduplication, 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 anenergy-producing system (NADP plus isocitric acid). Various hepatic P450 isoenzymelevels were increased by treatment of the rats with AroclorTM 1254 (single concentrationof 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 ≤-60°C until use.
Aliquots of S9 were thawed immediately before use and added to the other componentsto form the activation system described as follows: S9 Activation System
*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 cellline and were originally obtained from the laboratory of Dr. S. Wolff, University ofCalifornia, San Francisco. The cells were subsequently subcloned in this laboratory, andstock cultures stored in liquid nitrogen. The CHO-WBL subclone is a permanent cell linewith an average cycle time of 12 to 14 hours and a modal chromosome number of 21.
The CHO cells were grown in McCoy’s 5a culture medium which was supplementedwith .10% heat-inactivated fetal bovine serum (FBS), L-glutamine (2mM), penicillin G(100 units/mL), and streptomycin (100 µg/mL). Single cultures were used for each doseof the test article. Cultures were incubated with loose caps in a humidified incubator at37°C ± 2°C in an atmosphere of 5% ± 1.5% CO2 in air.
The dose rangefinding assay was conducted with a ~3.0-hour treatment period in thepresence 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 timesthe 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 wereharvested ~20.0 hours from the initiation of treatment. This harvest time corresponds to1.5 times the cell cycle time of approximately 13 hours (Galloway et al., 1994).
Covance 24742-0-437SC
Test Article Handling
The 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 by
serial dilutions of the stocks with DMSO. A dose volume of 10.0 µL/mL was used.
A summary of the treatment times is given below.
Summary of Rangefinding/Chromosomal Aberrations Assay Treatment Schedule in
Hours (Approximate)
In the dose rangefinding assay, concentrations of 7.81, 15.6, 31.3, 62.5, 125, 250, 500,and 1000 µg/mL were tested with and without S9. No toxicity was observed in thecultures treated with 1000 µg/mL tested with and without S9 (Tables 1 and 2). Thechromosomal aberrations assay was conducted with concentrations of 2000, 3500, and5000 µg/mL with and without S9.
In the chromosomal aberrations assay without S9, no toxicity was observed in any of thetest cultures (Table 3). Structural chromosomal aberrations were evaluated at5000 µg/mL (Table 2). No significant increase in the number of cells with structuralchromosomal aberrations, polyploidy, or endoreduplication was observed.
In the chromosomal aberrations assay with S9, no toxicity was observed in any of the testcultures (Table 5). Structural chromosomal aberrations were evaluated at 5000 µg/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. InternationalReview 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 Metabolic Activation -
~20 Hour Treatment, ~20 Hour Harvest
a This endpoint is based upon visual observations which are made prior to the harvest of themetaphase cells. At the time of the confluence observation the flasks are also evaluated forthe appearance of floating mitotic cells and dead cells.
DMSO = dimethylsulfoxide Covance 24742-0-437SC
Table 2: Assessment of Toxicity for Chromosome Aberrations Assay - With Metabolic Activation -
~3 Hour Treatment, ~20 Hour Harvest
a This endpoint is based upon visual observations which are made prior to the harvest of themetaphase cells. At the time of the confluence observation the flasks are also evaluated forthe appearance of floating mitotic cells and dead cells.
DMSO = dimethylsulfoxide Covance 24742-0-437SC
Table 3: Assessment of Toxicity for Chromosomal Aberrations Assay - Without Metabolic Activation -
~20 Hour Treatment, ~20 Hour Harvest
a This endpoint is based upon visual observations which are made prior to the harvest ofthe 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 Covance 24742-0-437SC
Table 4: Chromosome Aberrations in Chinese Hamster Ovary Cells - Without Metabolic Activation -
~20 Hour Treatment, ~20 Hour Harvest
SHOWING STRUCTURAL CHROMOSOME ABERRATIONS mab: multiple aberrations, greater than 4 aberrations a % 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.
Covance 24742-0-437SC
Table 5: Assessment of Toxicity for Chromosomal Aberrations Assay - With Metabolic Activation -
~3 Hour Treatment, ~20 Hour Harvest
a This endpoint is based upon visual observations which are made prior to the harvest ofthe 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 Covance 24742-0-437SC
Table 6: Chromosome Aberrations in Chinese Hamster Ovary Cells - With Metabolic Activation -
~3 Hour Treatment, ~20 Hour Harvest
SHOWING STRUCTURAL CHROMOSOME ABERRATIONS mab: multiple aberrations, greater than 4 aberrations a % 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.
Covance 24742-0-437SC
HISTORICAL CONTROL DATA
CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS, ~20 HOUR HARVEST - 1/2002 THROUGH 06/2002 N = Number of trialsMMC = Mitomycin CCP = Cyclophosphamide-g = % of cells with chromosome aberrations+g = % of cells with chromosome aberrations + % of cells with gaps Covance 24742-0-437SC
DEFINITIONS OF CHROMOSOMAL ABERRATIONS FOR GIEMSA STAINED
CELLS
chtg Chromatid Gap:
("tid gap"). An achromatic (unstained) region inone chromatid with clear discontinuity with novisible 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 orcomplete displacement.
chrb Chromosome Break:
Chromosome has a clear break, forming anabnormal (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 achromatid resulting in a small fragment or ringlying beside a shortened chromatid or a gap in thechromatid.
tr Triradial:
An exchange between two chromosomes, or onechromosome and an acentric fragment, whichresults 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-armedconfiguration.
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 brokenends distally.
nup Non-union Proximal:
Intra-arm interchange with non-union of brokenends proximally.
cx Complex Rearrangement:
An exchange among more than two chromosomesor fragments, which is the result of several breaks.
chre (chromosome exchange)
dic Dicentric:
An exchange between two chromosomes, whichresults in a chromosome with two centromeres.
This is often associated with an acentric fragment inwhich case it is classified as dicf.
dicf Dicentric Fragment:
A chromosome, which forms a circle, containing acentromere. This is often associated with anacentric fragment in which case it is classed as RF.
rf Ring Fragment:
Ring with associated acentric fragment.
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ab Abnormal:
Abnormal monocentric chromosome. This is achromosome whose morphology is abnormal for thekaryotype, and often the result of a translocation,pericentric inversion, etc. Classification used ifabnormality cannot be ascribed (e.g., a reciprocaltranslocation).
mab Multiple Aberrations:
A cell, which contains more than 4 aberrations. Aheavily damaged cell should be analyzed to identifythe types of aberrations and may not actually have>4 (e.g., multiple fragments such as those foundassociated with a tricentric).
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.

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