Document ZnRNpxjed8dLRE3E70xjRo5D8
AR226-3174
TRADE SECRET FINAL REPORT
Study Title
H-24616; In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells
Authors Ramadevi Gudi, Ph.D.
Caren Brown, M.S.
Report Completion Date March 12,2001
PerformingLaboratory
BioReliance 9630 Medical Center Drive
RockviUe.MD 20850 for
E. I. du Font de Nemours and Company Haskell Laboratoryfor Toxicology and Industrial Medicine
P.O. Box 50, Elkton Road
Newark, DE 19714-0050 Performing Laboratory Study Number
AA37AZ.331.BTL
DuPont Project ID
DuPont-5235
Page 1 of 35
Company Sanfd'zed. DOBS nof confam TSCA CBI
H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells____
CERTIFICATION
DuPont-5235
We, the undersigned, declare that this report provides an accurate evaluation of data obtained
from this study.
Reviewed by:
^
Richard San, Ph.D. BioReliance Study Management
/-? A^a^t, 3<?P/
Date
Issued by Study Director:
^JLtA^d^G^'' Ramadevi Gudi BioReliance StudyDirector
MA^ 12, Date
Approved by Study Monitor
\ \ OsA^~ o^. vljS^' t-^-- Maria Donner, Ph.D. Senior Research Scientist
01 HAt2too
Date
BioReliance Statty No. AA37AZ.331.BTL
Company Sanftized. Does not conia.n TSCA CBf
H-24616: // F/fro Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells
DuPont-5235
STUDY INFORMATION
Substance Tested: Synonym/Codes:
Haskell Number: Purity: Known Impurities: Physical Characteristics: Stability:
Solubility:
Sponsor:
Study Initiated/Completed: In-Life Initiated/Completed:
The test substance appeared to be stable
under the conditions of the study; no evidence of instability was observed.
Aquatics: Dispersible in water All Others: Dimethyl sulfoxide
E.I. du Font de Nemours and Company Wilmington, Delaware 19898 U.SA.
November 15,2000/(see report cover page) November 28,2000/November 29,2000
BioReliance Study No. AA37AZ.331 .BTL
Company Sanitized. Does not contain TSCA CB1
H-24616: In Vitro Chromosome Aberration Test in Chinese Hamster Ovmy (CHO) Cells
DuPonfr5235
TABLE OF CONTENTS
Page
CERTIFICATION.....----...............----------------....------------------------,,,,.....--------------..........2
STUDY INFORMATION ~tt.~..........~^........i.................,.,,,,............,.....^^w,.M,....,........,.....^^^.^, 3 TABLE OF CONTENTS .................^..,.....,.----.....-^^----.,,----...^----...._^__^........... 4
S.VSVT'VOSEi "^i*a*Bcr**c****Brf*MB^**B*fri*fHf***r----r^nmi***uff**afffBfa^H 7
CHARACTERIZATION OF TEST SUBSTANCE AND CONTROL ARTICLES _____......____ 7
MATERIALS AND METHODS--------------------...................................------....___________ 7
KdutjJuKO AJNJU J^lOVo Ui&UflV'l f***4f*iH----***----**t*f**cw***n*****BfMia----if* Xl
mr'PB'RirKidrs _
i A
TABLE 1: CONCURRENT TOXICITY TEST USING H-24616 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; 4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD...................... 15
TABLE 2: CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH H-24616 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; 4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD............................................................................................................................... 16
TABLE 3: CONCURRENT TOXICITY TEST USING H-24616 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION; 4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD...................... 17
TABLE 4: CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH H-24616 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION; 4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD................................................................................................................................... 18
TABLE 5: CONCURRENT TOXICITY TEST USING H-24616 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; 20 HOUR CONTINUOUS TREATMENT.................................................. 19
TABLE 6: CYTOGENETIC ANALYSIS OP CHO CELLS TREATED WITH H-24616 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; 20 HOUR CONTINUOUS TREATMENT. 20
TABLE 7: SUMMARY.................................................................................................................................. 21 APPENDIX A HISTORICAL CONTROL DATA...........................--..--------------------...............---- 22
BioReliance Study No. AA37AZ.331.BTL
4
Company Sanitized. Does not contain TSCA CBS
H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHQ) Cells____________________________DnPont-5235
urJrJEu^Jt^JL& Sf 0& vift rj&^JUl \J\^%flj ----^o-->* >------ ><> w
BioReliance Study No. AA37AZ.331.BTL
5
Company S,ntod.D^ no, conlalnTSCACB,
H-246S6: In Vitro Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells
DiiPont-5235
SUMMARY
H-24616UUR|was The test substance,
tested in the chromosome aberration assay
using Chinese hamster ovary (CHO) cells in both the absence and presence of an
Aroclor-induced 89 activation system in cider to evaluate the clastogenic potential of the test
substance.
Water was determined to be the solvent of choice based on information provided by the Sponsor and compatibility with the target cells. The test substance was soluble in water at a concentration of 50 mg/mL, Hie maximum concentration tested.
In the chromosome aberration assay, the maximum dose tested was 5000 (Ag/mL. The cells were Heated for 4 and 20 hours in me non-activated test system and for 4 hours in the S9 activated test system, and all cells were harvested at 20 hours after treatment initiation. Visible precipitate was observed in treatment medium at 5000 pg/mL in the non-activated 4 hour group. Dose levels S 1500 ug/mL were soluble in treatment medium in the non-activated 4
hour treatment group. Visible precipitate was observed in treatment medium at concentrations >. 1500 ug/mL in me S9 activated 4 hour group and in the non-activated 20 hour exposure group. Dose levels S 500 ug/mL were soluble in treatment medium in Ihe S9 activated 4 hour group and in the non-activated 20 hour exposure group.
Selection of dose levels for the cytogeneticstest was based upon post-treatment cell growth inhibition relative to the solvent control and solubility of me test substance.
Treatment Time (hours)
Recovery Time (hours)
Harvest S9 Cell growth
Tune
inhibition at
(hours)
highest d o s e
Mitotic Index Reduction
LED (lowest
effective dose) for
scored
*
Structural
(ug/mL)
Aberrations
4
16
20
15%at 5000 25%
None
20
0
20
-
36%at 5000 23%
None
4
16
20
+ 40%at 2500 52%
None
* relative to solvent control at high dose evaluated for chromosome aberrations
LED for
Numerical Aberrations
None None None
The positive and solvent controls fulfilled the requirements for a valid test. Based on the
jUU|was findingsofthis study
concluded to be negative for the induction of structural
and numerical chromosome aberrations in Chinese hamster ovary (CHO) cells in the presence
and absence of Aroclor-induced rat liver S9.
BioReliance Study No. AA37AZ.331.BTL
Company Sanitized. Does not contain TSCA CBI
H-24616; In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells___________________________DoPont-5235
PURPOSE
The purpose of this study was to evaluate the clastogenic potential of a test substance based upon its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) celts.
CHARACTERIZATION OF TEST SUBSTANCE AND CONTROL ARTICLES
The test substance, H-24616, was received by BioReliance on November 6,2000 and was
"BHBQ^ assignedthe code number AA37AZ. The test substance was characterized by the Sponsor as an ^ould be stored at ambient temperature. An expiration date was not I by the Sponsor. Upon receipt, the test substance was described as w was stored at room temperature, protected from exposure to light,.
The solvent used to deliver H-24616 to me test system was sterile water (CAS 7732-18-5), from the Life Technologies Company. Mitomycin C (MMC; CAS No.: 50-07-7), was obtained from the Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations of 1 and 2 ug/mL for use as me positive control in the non-activated test system. Cyclophosphamide (CP; CAS No.: 6055-19-2), was obtained from Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations of 100 and 200 ug/mL for use as the positive control in the S9 activated test system. For each positive
control one dose with sufficient scorable metaphase cells was selected for analysis. The solvent for the test substance was used as the solvent control at the same concentration as that found in the test substance-treated groups.
Test System
MATERIALS AND METHODS
Chinese hamster ovary (CHO-K,) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA, on May 29,1997. In order to assure the karyotypic stability of the ceil line, working cell stocks were not used beyond passage 20. The freeze lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens(Preston et al., 1981).
Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was
prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254,500 mg/kg, five days prior to sacrifice. The S9 was batch prepared and stored at
BioReliance Study No. AA37AZ.331 .BTL
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H-24616: In Vitro Chromosome Aberration Test in Chinese Hamster Ovary (CHO)
Cells_________________________
DuPoat-5235
^-70C until used. Each bulk preparation of S9 was assayed for sterility and its ability to
metabolize 2-aminoanthracene and 7,12-dimethylbenz(cx)anthracene to forms mutagenic to Salmonella typhxmwiwn TA100.
Immediately prior to use, the S9 was thawed and mixed with a cofactor pool to contain 2
mM magnesium chloride, 6 roM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotmamide adenine dinucleotide phosphate(NADP) and 20 uL S9 per milliliter medium (McCoy's 5A serum-free medium supplemented with, 100 units penicillin and 100 pg streptomycin/mL, and 2 mM L-glutaniine).
Chromosome Aberration Assay
Selection of dose levels for the cytogenetics test was based upon post-treatment cell growth inhibition relative to the solvent control and solubility of the test substance. Whenever possible,
the high dose to evaluate chromosome aberrations was selected to give at least 50% reduction
in cell growth. The chromosome aberration assay was performed using standard procedures (Evans, 1976), by exposing duplicate cultures of CHO cells to the test substance as well as positive and solvent controls. For the chromosome aberration assay, CHO cells were seeded at approximately 5 x 105 cells/25 cm2 flask and were incubated at 371C in a humidified
atmosphere of 51% CO; in air for 16-24 hours. Treatment was earned out by refeeding duplicate flasks with 4.5 mL complete medium (McCoy's 5A medium supplemented with 10% FBS, 100 units penicillin and 100 ug streptomycin/mL, and 2 mM L-glutamine) for the non-
activated study or 4.5 mL S9 reaction mixture for me S9 activated study, to which was added
500 uL of dosing solution of test or control article in solvent or solvent alone. The osmolality of the highestconcentration of dosing solution in me treatment medium was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using
testtape.
In me non-activated study, the cells were exposed to the test substance for 4 hours or
continuously for 20 hours up to me cell harvest at 371C in a humidified atmosphereof5l% CO; in air (Swierenga et al., 1991). In the 4 hour exposure group, after the exposure period, the
treatment medium was removed, the cells washed with CMF-PBS, refed with complete
medium and returned to the incubator. Two hours prior to the scheduled cell harvest,
Colcemid* was added to duplicateflasks for each treatment condition at a final concentration of
0.1 (ig/mL and me flasks returned to the incubator until cell collection.
to the S9 activated study, the cells were exposed for 4 hours at 371C in a humidified
atmosphere of 5=bl% CO; in air (Swierenga et al., 1991). After the exposure period, the
treatment medium was removed, the cells washed with CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid was added to duplicate flasks for each treatment condition at a final concentration of
BioReliance Study No. AA37AZ.331.BTL
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H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells____________________________DuPont-5235
0.1 ug/mL and the flasks were returned to tine incubator until cell collection.
A concurrent toxieity test was conducted in both the non-activated and the S9 activated test
systems. After cell harvest an aliquot of the cell suspension was removed from each culture and counted using a Coulter counter. The presence of test substance precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent
control.
Collection ofMetaphase Cells
Two hours after the addition of Colcemid11,metaphase cells were harvested for both the non-activated and S9 activated studies by trypsinization. Cells were collected approximately 20 hours after initiation of treatment (Galloway et al., 1994). The cells were collected by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspendedin 2-4 mL 0.075 M potassium chloride (KC1) and allowed to stand at room temperature for 4-8 minutes. The cells were collected by centrifugation, the supernatant aspirated and the cells fixed with two washes of approximately 2 mL Camoy's fixative (methanol:glacial acetic acid, 3:1, v/v). The cells were stored overnight or longer in fixative at approximately 2-8C.
Slide Preparation
To prepare slides, the fixed cells were centrifuged at approximately 800 rpm for 5 minutes, the supernatant was aspirated, and 1 mL fresh fixative was added. After additional centrifagation(at approximately 800 rpm for 5 minutes) the supernatant fluid was decanted and the cells resuspended to opalescence in fresh fixative. A sufficient amount of cell suspension was dropped onto the center of a glass slide and allowed to air dry. Slides were identified by the study number, date prepared and the treatment condition. The dried slides were stained with 5% Giemsa, air dried and permanently mounted.
Evaluation ofMetaphase Cells
Slides were coded using random numbers by an individual not involved with the scoring process. To ensure that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 202 centoomeres were examined undei oil immersion without prior knowledge of treatment groups. Initially, the non-activated and S9 activated 4
hour exposure groups were evaluated for chromosome aberrations and if a positive result was
obtained in me non-activated 4 hour exposure group, we non-activated 20 hour continuous exposure group was not evaluated for chromosome aberrations. Whenever possible, a minimum of 200 metaphase spreads(100 per duplicate flask) were examined and scored for chromatid-lype and chromosome-type aberrations (Scott et al., 1990). The number of
_______St_ud_y ________________9 ____Company Sanitized. Does not contain TSCA CBI
BioReliance
No. AA37AZ.331.BTL
H-24616: / FTft-o Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells____________________________DuPont-5235
metaphase spreads that are examined and scored per duplicate flask may be reduced if the percentage of aberrant cells reaches a statistically significant level before 100 cells are scored.
Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in me absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (S10 aberrations) were also recorded. Chromatid and isochromatid gaps were
recorded but not included in me analysis. The XY coordinates for each cell with chromosomal
aberrations were recorded using a calibrated microscope stage. Polyploidand endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored.
Controls
Mitomycin C was used as me positive control in me non-activated study at final
concentrations of 0.1 and 0.2 ug/mL. Cyclophosphamide was used as the positive control in the S9 activated study at final concentrations of 10 and 20 ug/mL. For both positive controls the dose level exhibiting a sufficient number of scorable metaphase cells was selected for
analysis. The solvent vehicle for the test substance was used as the solvent control at the same concentration as that found in die test substance-treated groups.
Evaluation of Test Results
The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the aberration studies. The number and types of aberrations round, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the tote! population of cells examined, and the mean aberrations per cell was calculated and reported for each group. Chromatid and isochromatid gaps are presented in the data but are not included in me total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.
Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test substance dose
level, the Cochran-Annitage test was used to measure dose-responsiveness.
All conclusions were based on sound scientific basis; however, as a guide to interpretation of me data, the test substance was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more
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BioReliance Study No. AA37AZ.331.BTL10
H-24616: In Vitro Chromosome Aberration Test
m Chinese Hamster Ovaiy (CHO) Cells____
______________PuPont-5235
concentrations being statisticallysignificant (pSO.05). If the test substance did not demonstrate
a statisticallysignificant increase in aberrations it will be concluded to be negative.
Criteria for a Valid Test
Hie frequency of cells with structural chromosome aberrations in the solvent control must be within the range of the historical solvent control. The percentage of cells with chromosome
aberrations in the positive control must be statistically increased (p<0.05, Fisher's exact test) relative to the solvent control.
Deviations
No known deviations from the protocol or assay method SOPs occurred during the conduct of this study.
Archives
All raw data, the protocol, all reports, andstamedand coded slides will be maintained
according to Standard Operating ProcedurejI--------nbmye BioReliance RAQA unit headquartered at BioReGance, 14920 BroschartKoad'RoclvilleM, D 20850. Paper records
will be retained for at least three years after which time the Sponsor will be contacted for a decision as to the final dispositionof the materials. All study materials returned to the Sponsor or destroyed will first be copied and (he copy will be retained in me BioReliance archives for a minimum of 10 years.
Solubility
RESULTS AND DISCUSSION
Water was determined to he me solvent of choice based on information provided by the
Sponsor and compatibility with the target cells. The test substance was soluble in water at a
concentration of 50 mg/mL, the maximum concentration tested.
Chromosome Aberration Assay
Selection of the dose levels for the cytogenetics test was based upon post-treatment cell growth inhibition relative to the solvent control and solubility of me test substance. Whenever possible, the high dose to evaluate chromosome aberrations was selected to give 50% toxicity.
Two additional dose levels, demonstrating lime or no toridty were also included. CHO cells were exposed to solvent alone and to nine concentrations of test substance ranging from 0.5 ug/mL to 5000 pg/mL in the absence of an S9 reaction mixture. CHO cells were exposed to
BioReliance Study No. AA37AZJ31.BTLTT
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H-24616:7n ^tft-o Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells____
DuPont-5235
solvent alone and to eight concentrations of test substance ranging from 500 ug/mL to 4000 (ig/mL in the presence of an S9 reaction mixture. Hie results of the evaluation of cell growth inhibition are presented in Tables 1, 3 and 5. Visible precipitate was observed in treatment medium at 5000 pg/roL in the non-activated 4 hour group. Dose levels < 1500 pg/mL were soluble in treatment medium in the non-activated 4 hour treatment group. Visible precipitate was observed in treatment medium at concentrations >. 1500 ug/mL in the S9 activated 4 hour group and in the non-acdvated 20 hour exposure group. Dose levels ^ 500 pg/mL were soluble in treatment medium in the S9 activated 4 hour group and in the non-activated 20 hour exposure
group. Based upon the results of the toxicity study, the dose levels selected for evaluation in the
chromosome aberration assay were as follows:
Treatment Condition
-S9 +S9
Treatment Time (hours)
4
Recovery Time (hours)
16
Dose levels
(pg/mL)
500,1500,5000
20
0
500,1500,5000
4
16
1500,2000,2500
Chromosome Aberration Assay
In the non-activated chromosome aberration assays the osmolality in treatment medium of the highestconcentration tested, 5000 pg/mL, was 259 mmol/kg. The osmolality in treatment medium of the lowest precipitating dose, 1500 ug/mL, was 267 mmol/kg. The osmolality in treatment medium of the highest soluble dose, 500 pg/mL, was 264 mmol/kg. The osmolality of the solvent (water) in treatment medium was 273 mmol/kg. The pH of we highest concentration of test substance in treatment medium was approximately 7. Changes of higher
than 50 mmol are not acceptable.
Cell growth inhibition relative to the solvent control of H-24616 in CHO cells when treated for 4 hours in the absence of S9 activation was 15% at 5000 pg/mL, the highest test concentration evaluated for chromosome aberrations (Table 1). The activity of H-24616 in (he induction of chromosome aberrations is presented by treatment flask in Table 2 and summarized by group in Table 7. The mitotic index at the highest dose level evaluated for chromosome aberrations, 5000 ug/mL, was 25% reduced relative to the solvent control. Toe dose levels selected for microscopic analysis were 500,1500 and 5000 pg/mL. The percentage of cells with structural and numerical aberrations in tile test substance-treated groups was not significantly increased above that of the solvent control (p>0.05. Fisher's exact test). The percentage of structurally damaged cells in the MMC group was found to be statistically
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BioReliance Study No. AA37AZ.331.BTL
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H-24616: In Vitro Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells
DuPont-5235
significant (11.5%).
There was no dose level with 50% toxidty (cell growth inhibition) in the S9 activated treatment group, so this portion of the assay was repeated. Results from die initial S9 activated
assay were recorded but are not reported. Toxicity of H-24616 (cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in the presence of S9 activation in
the repeat assay was 40% at 2500 ugtaL, the highest test concentration evaluated for chromosome aberrations (Table 3). The activity of H-24616 in me induction of chromosome aberrations is presented by treatment flask in Table 4 and summarized by group in Table 7. The mitotic index at the highestdose level evaluated for chromosome aberrations, 2500 ug/mL, was 52% reduced relative to me solvent control. The dose levels selected for microscopic analysis
were 1500,2000 and 2500 ug^nL. The percentage of cells with numerical aberrations in the test substance-treated groups was not statistically increased above that of the solvent control (p>0.05. Fisher's exact test). The percentage of cells with structural aberrations in me test substance-treated groups was statistically increased above mat of the solvent control at dose level 2000 ug/mL (p<0.05. Fisher's exact test). However, the percentage of cells with structural aberrations (4%) was within the historical solvent control range of 0% to 6.5%. Therefore, it is not considered biologically significant The percentage of structurally damaged
cells in the CP group was round to be statisticallysignificant(14.5%).
In the absence of a positive response in me non-activated 4 hour exposure group, slides
from me non-activated 20 hour exposure group were evaluated for chromosome aberrations.
Toxicity of H-24616 (cell growth inhibition relative to the solvent control) was 36% at 5000
ug/mL, the highest test concentration evaluated for chromosome aberrations in the non-
activated 20 hour continuous exposure group (Table 5). The activity of H-24616 in me induction of chromosome aberrations is presented by treatment flask in Table 6 and
summarized by group in Table 7. The mitotic index at (he highest dose level evaluated for chromosome aberrations, 5000 ug/mL, was 23% reduced relative to me solvent control. The dose levels selected for microscopicanalysis were 500,1500, and 5000 ug/mL. The percentage
of cells with structural or numerical aberrations in the test substance-treated groups was not significantly increased above that of me solvent control (p>0.05). The percentage of structurally damaged cells in the MMC group was round to be statistically significant (13.5%).
CONCLUSION
The positive and solvent controls fulfilled me requirements for a valid test
Based on the findings of this study'IHHHBv^ concluded to be negative for the
induction of structural and numerical chromosome aberrations in Chinese hamster ovary (CHO)
cells in the presence and absence ofAroclor-induced rat liver S9.
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H-24616:/ra Frtro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells_______________________DuPont-5235
REFERENCES
Evans, H.J. (1976) Cytological methods for detecting chemical mutagens, in: A. Hollaender (Ed.), Chemical Mutagens, Principlesand Methods for their Detection, vol 4. Plenum Press, New York.
Galloway, SM, MJ. Aardema, M. Ishidate Jr., J.L. Ivett, DJ. Kiridand, T. Morita, P.
Mosesso and T. Sofuni (1994) Report from working group on in vitro tests for chromosomal aberrations. Mutation Research 312(3):241-261.
International Conference on Hannonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 of me ICH process on July 19, 1995. Federal Register 61:18198-18202, April 24,1996.
International Conference on Hannonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. S2B document recommended for adoption at step 4 of the ICH process on July 16, 1997. Federal Register 62:16026-16030,
November 21,1997.
OECD Guideline for the Testing of Chemicals, Guideline 473 (In Vitro Mammalian Chromosome Aberration Test), July 1997.
Preston, RJ., W. Au, MA. Bender, J.G. Brewen, A.V. Canano, J.A. Heddle, A.F. McFee,
S. Wolffand J.S. Wassom (1981) Mammalian in vivo and in vitro cytogenetic assays: a report of the Gene-Tox Program, Mutation Research, 87:143-188.
Scott, D., NJ>. Danford, B.J. Dean and DJ. Kirkland. 1990. Metaphase Chromosome Aberration Assays In Vitro. In: Basic Mutagenicity Tests: UKEMS Recommended
Procedures. DJ Kirkland (ed). CambridgeUniversity Press, New York, NY.
Swierenga S.H.H., 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 a survey of current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister-
chromatid exchange in Chinese hamster ovary, V79 Chinese lung and human lymphocyte cultures. Mutation Research 246:301-322.
BioReliance Study No. AA37AZ.331.BTLIT
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TABLE 1 CONCURRENT TOXICITY TEST USING H.24616 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION
4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD
Treatment (uoAnL)
Flask
Cell Count Averages
(x10*)
Cell Viability
(%)
Mean Colls
per Flask (xlO")
CeB Growth
Index
(%)
Cell Growth Inhibition (%)
Water
A
2.62
B
2.59
H-24616 0.5
A
2.63
B
2.69
1.S
A
2.52
B
2.62
S
A
2.50
B
2.65
15
A
2.36
B
2.85
SO
A
2.53
B
2,51
150
A
2.54
B
2.84
500
A
2.21
B
1,89
1600
A
2.04
B
2.02
5000
A
2.22
B
2.22
MMC.0.1
A
2.37
B
2.77
MMC. 0.2
A
2.06
B
2.23
98% 97%
97% 96%
98% 95%
97% 98%
98% 96%
97% 98%
95% 96%
98% 98%
97% 99%
97% 98%
98% 97%
96% 95%
2.54
2.57 2.48 2.51 2.43 2.46 2.57 2.01 1.99 2.16 2.50 2.05
100
101
-1
98
2
99
1
96
4
97
3
101
.1
79
21
76
22
85
15
99
1
81
19
Treatment: CHO cells were boated In the absence of an exogenous source of metabolic activation
for4hoursat371C. Coll Viability datermined by trypan blue dye exclusion. Mean Cell* per Flack " cell count x % viable cells, reported as a mean of flask* A and B. Cell Growth Index (calls per flask In treated group/cells per flask In control group), expressed as a percentage.
Cell Growth Inhibition " 100% - cell growthindex; not calculated for negative controls.
BioReliance Study No. AA37AZ.33LBTL
15
Company Sanitized. Does not contain TSCA CBt
TABLE2
CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH H-24616 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION
4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD
Treatment (pg/mL) Water
H-24616 500
1500
5000
MMC0.2
Flask
MItoUc
Index
(%)
Cells Scored
% Aberrant Cells Numerical Structural
6.4
100
8.2
100
6.0
100
5.6
100
7.6
100
7.8
100
A
6.0
100
6.4
100
5.6
100
12
5.2
100
11
Total Number of Structural Aberrations
Caps
ChnxnaHd
Br
Ex
Chromosome Br Die Ring
01 0
000
0 1 0 0
0 1 0 1
0 1 0 1
2 1 1 0
Severely Damaged
CeKs
Average Aberrations
PerCen
0.010 0.000
0.010 0.010
0.010 0.010
0.010 0.010
0.160 0.130
Treatment; CHO cells were (mated for 4 hours at 371C In the absence of an exogenous source of metabolic activation. Additional dose levels of 0.5,1.5, 5,15, 50 and 150 ug/mL were tested as a safeguard against excessive toxicity at higher dose levels but were not required for microscopic examination. MltoUclndex= number of milotic figures x1(XV500 celts counted. % Aberrant Cdte: numerical Includes polyploldand endoredupllcated cells; structural excludes cells with only gaps. ChromtUd break* Include chromatic! and teochromaud breaks and fragments (Br); chromatid exchange figures (Ex) Induce quadriradlals, triradiats and complex rearrangements.
Chromosome breaks include breaks and acentric fragments (Br); dteentric chromosome (Die). Severely damaged cells Includes ceHa with one or more pulverized chromosomes and oete with 10 or more aberrations. Average Aberrations Per Cell: severely damaged cells and pulverizations were counted as 10 aberrations.
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BioReliance Study No. AA37AZ.331.BTL
16
TABLES
CONCURRENT TOX1CITY TEST USING H-24616 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION
4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD
Treatment (PSAml.)
Flask
Cell Count Averages
(x1<?)
Cell Viability
(%)
Mean Cells
perFtetk (xlO")
eel Growth
Index (%)
CeC Growth Inhibition
(%)
Water H-24616 500 1000 1500 2000 2500 3000 3500 4000 CP.10 CP.20
A
1.80
B
1.90
A
2.02
B
2.09
A
1.90
B
1.93
A
2.12
B
1.69
A
1.42
B
1.43
A
1.12
B
1.1S
A
0.33
B
0.48
A
0.10
B
0.10
A
0.12
B
0.11
A
1.25
B
1.37
A
1.15
B
1.11
99% 100%
99% 99%
98% 100%
99% 98%
97% 98%
96% 98%
96% 97%
0% 0%
0% 0%
98% 99%
97% 98%
1.84
2.04 1.89 1.88 1.39 1.10 0.39 0.00 0.00 1.29 1.10
100
111
-11
103
-3
102
-2
75
25
60
40
21
79
0
100
0
100
70
30
80
40
Treatment CHO cells were treated In the absence of an exogenous source of metaboito activation for4housat371*C. Cell VlabilHy determined by trypan blue dye exclusion. Mean Cell* per Flask cell count x % viable cells, reported as a mean for flasks A and B. Cell Growth Index " (ceNsper flask In treated group/cells par flask In control group), expressed as
a percentage. Call Growth tnhlbitfon 100% - cell flrowth index; not calculated for negative controls.
BioReliance Study No. AA37AZ.331.BTL
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TABLE 4
CYTOGENEDC ANALYSIS OF CHO CELLS TREATED WITH N-24818 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION
4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD
Treatment (W/mL) Water
H-24616 1500
2000
2500
CP.10
Flask
MItotto
Index (%)
Cells !Scored
%Abemint Calls INumerical Structural
A
9.4
100
9
1
B
8.8
100
7
0
A
7.8
100
8
1
B
8.8
100
8
2
A
7.2
100
11
4
B
8.8
100
10
4
A
4.4
100
4
2
B
4.2
100
5
2
A
8.8
100
4
18
B
7.8
100
3
13
Total I
ofStrud
rfnns
Gaps
0 1
Chrotnatk)
Br
Ex
0
0
0
0
ChiWOOIIOJTIO Br Dto Ring
01 0 00 0
Severely Damaged
Cell*
0 0
2
1
0
00 0
0
1
1
0
0 0
1
0
2
4
1
00 0
0
0
4
0
00 0
0
1
2
0
00 0
0
2
1
1
00 0
0
5
12
9
21 0
0
2
8
8
11 0
0
Average Aberrations
Per Cell
0.010 0.000
0.010 0.020
0.050 0.040
0.020 0.020
0.240 0.180
Treatment: CHO cells were treated for 4 hours at 37A1-C hi the presence of an exogenous source of metabolic activation. Additional dose levete of 500 and 1000 \iylmL were tested as a safeguard against excessive Soxicftyat higher dose levetobut were not required for microscopic examination. Dose levels 3000,3500 and 4000 |igAL were not analyzed due to excessive toxtelty.
MItoths Index a number of rnltotic figures x 100/500 cells counted. % Aberrant Cells: numerical Includes polyploldand endoredupltoated cells; structural excludes cells with only gaps. Chromattd br--to Include chromatid and Isochromatid breaks and fragments (Br); chromattd exchange figures (Ex) include quadriradlate, triradlals and complex rearrangements. Chromosome breaks Include breaks and acentric fragments (Br); dteentric chromosome (Die).
Severely damaged cells includes cells with one or more pulverized chromosomes and cells with 10 or more aberrations. Average Aberrations Per Cell; severely damaged cells and pulverizations were counted as 10 aberrations.
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TABLES
CONCURRENT TOXICITY TEST USING H-24616 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION
20 HOUR CONTINUOUS TREATMENT
Treatment (MC/mL)
Flask
Call Count Averages
(xlO")
Cell Viability
(%)
Moan Cells
perHaak (x101)
Cell Growth Index (%)
Cell Growth Inhibition (%)
Water
A
2.08
B
2.19
H-24616 0.5
A
2.19
B
2.25
1.5
A
2.22
B
2.17
5
A
2.31
B
2.33
15
A
2.34
B
2.25
50
A
2,28
B
2.37
150
A
1.99
B
1.98
500
A
1.71
B
1.89
1500
A
2.04
B
2.11
5000
A
1.45
B
1.44
MMC.0.1
A
1.82
B
1.90
MMC.0.2
A
1.56
B
1.48
100% 88%
99% 100%
98% 99%
97% 98%
98% 99%
97% 97%
98% 99%
95% 94%
94% 93%
93% 95%
97% 98%
98% 99%
2.13
2.21 2.16 2.26 2.26 2.25 1.96 1.70 1.94 1.36 1.81 1.50
100
104
-4
102
-2
106
-6
106
-6
106
-6
92
8
80
20
91
9
64
36
85
15
70
30
Treatment: CHO cells were treated In the absence of an exogenous source of metabolic activation for 4 hours at 371 "a. Cell Viability determined by trypan blue dye exclusion.
Mean Calls per Flak" call count x% viable calls, reported as a mean for ffiaste A and B.
Cell Growth Indttx B (cells per flask in treated group/cells per flask In control g.oup), expressed as a percentage.
Celt Growth Inhibition -100% -call growth Index; not calculated for negative controls.
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TABLE 6
CYTOGENET1C ANALYSIS OF CHO CELLS TREATED WITH ?-24816 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION
20 HOUR CONTINUOUS TREATMENT
Treatment (ugAnL)
Water
Flask
MItotic
Index
<%)
Colls Scored
%Abemint Cells
Numerical Structural
A
7,2
100
3
0
B
8.4
100
4
1
Total Number of Structural Abeirattons
Gaps
Chroirotid
Br
Ex
Chromasome Br Die Ring
1
0
0
1
0
0
00 0 01 0
Severely Damaged
Cell*
0 0
Average AbemUons
Par Cell
0.000 0.010
500
A
5.8
100
4
0
1
0
0
00 0
0
B
6.8
100
4
2
1
1
0
01 0
0
1500
A
6.0
100
2
1
0
0
0
02 0
0
B
6.6
100
2
1
0
0
0
01 0
0
5000
A
6.2
100
4
1
1
1
0
00 0
0
B
5.8
100
5
2
1
1
0
01 0
0
MMC. 0.1
A
6.6
100
4
13
1
8
3
50 0
0
B
7.4
100
4
14
3
14
3
11 0
0
0.000 0.020
0.020 0.010
0.010 0.020
0.160 0.190
Trwhnenfc CHO calls ware treated tar 20 houre at 371"C In the absence of an exogenous source of metabolc activation. Additional dose levels of 0.5.1.5, 5.15. 50 and 150 ug/mL were tasted as a safeguard against excessive toxidty at higher dose levels but were not required (or microscopic examination. MItoUc Index " number of rnltotic figures x 100/500 cells counted. % Aberrant Cells: numerical includes polypioldand endoredupHcated cells; structural excludes cells with only gaps. ChromaHd breaks inciuda chromatid and Isochromatid breaks and fragments (Br); chromaUd exchange figures (Ex) Include
quadriradlala, Mradlais and complex reanangements.
Chromoeome br--ka Include breaks and acentric fragments (Br); dteentric chromosome (CHc).
Sevw^damafled cells IndiKtewitet^ one or more pulverized chroffiosomeaarKlcetevrith 10 or rnoreabenate Average AbemUoiw Per Cell: severely damaged oeRs and pulverizations were counted as 10 aberratjons.
BioReKance Study No. AA37AZ.331.BTL
20
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TABLE 7 SUMMARY
Treatment (Mfl/mL)
Water
H-24616 500 1500 5000
MMC 0.2
S9 j activation
Treatment Time
Mean Mitottc
Index
Cells Scored
Abenations Per Cell
(Mean +/-SD)
C;ellsWrth;^befraUons
tNumerical Structural
(%)
(%)
4
8.3 200 0.005 0.071
3.5
0.5
4
5.8 200 0.010 0.100
7.5
1.0
4
7.7 200 0.010 0.100
3.5
1.0
4
6.2 200 0.015 0.122
6.0
1.0
4
5.4 200 0.145 0.442
4.0
11.5"
Water
+
4
9.0 200 0.005 0.071
8.0
0.5
H-24616
1500
+
2000
+
2500
+
4
7.3 200 0.015 0.122
6.0
1.5
4
6.9 200 0.045 0.271
10.5
4.0-
4
4.3 200 0.020 0.140
4.5
2.0
CP
+
4
7.3 200 0.210 0.563
3.5
14.5"
10
Water
H-24616 500 1500 5000
MMC 0.1
20
20 20 20
20
7.8 200 0.005 0.071
3.5
6.3 200 0.010 0.100
4.0
6.3 200 0.015 0.158
2.0
6.0 200 0.015 0.122
4.5
7.0 200 0.175 0.475
4.0
0.5
1.0 1.0 1.5 13.5"
Treatment: cells from an treatment conditions were haivested at 20 hours after the Initiation of the treatments. AbemiHoro Par Cell: A severely damaged cell was counted as 10 aberrations. * psO.05; **, p^O.01; Rshsrs exact test.
BioReliance Study No. AA37AZ.331.BTL
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21
H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells____
DuPont-5235
APPENDIX A Historical Control Data
BioRelianceStudyNo.AA37AZ.331.BTL
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22
H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells____
DuPont-5235
IN VITRO MAMMALIAN CYTOGENETIC TEST USING CHINESE HAMSTER OVARY (CHO) CELLS
HISTORICAL CONTROL VALUES STRUCTURAL ABERRATIONS
1997-1999
Historical Values
Mean SD* Range
Untreated/ Medium
(%)
1.5
1.4
0.0.6.0
Water
(%)
1.3 1.4 0.0-5.5
NON-ACTIVATED TEST SYSTEM
Saline
(%)
DMSO (%)
Acetone
(%)
EthanoS
(%)
1.7 1.8
0.0-6.0
1.6 1.3 0.0-5.5
1.4 1.6 0.0-4.5
1.3 1.3 0.0-4.0
Other* (/.)
1.5 1.3 0,0-5.0
All
Solvents
(%)
1.5
1.4
0.0-6.0
Positive coatroP
(%)
24.0
17.7
6.5-100.0
Historical Values
Mean SD' Range
Untreated/ Medium
(%)
1.8
1.3
0.0-6.0
Water
(%)
1.8 1.6 0.0-6.5
S9-ACTIVATED TEST SYSTEM
Saline
(%)
DMSO (%)
Acetone
(%)
Ethanol
(%)
1.9 1.4 0.0-4.5
1.8 1.5 0.0-5.5
1.9 2.2 0.0-5.0
1.5 1.4 0.0-4.0
Other' (%)
2.1 1.3 0,0-4.5
All
Solvents
(%)
1.8
1.5
0.0-6.5
Positive Control3
(%) 32.5
16.1
6.5-94.5
SD = standard deviation. Positive control for non-activated studies, N-mefeyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.75-2 ugtel), and Mitomycin C (MMC, 0.08-0.15 ug/ml). Positive control for S9-activated studies, cyclophosphamide(CP, 10-50 ug/ml), and benzo(a)pyrene, (B[a]P, 30 ugtel). Other solvents include non-standard solvents and Sponsor supplied vehicles.
BioReliance Study No.: AA37AZ.331 .BTL
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H-24616: In Vitro Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells
DuPont-5235
IN VITRO MAMMALIAN CYTOGENETIC TEST USING CHINESE HAMSTER OVARY (CHO) CELLS
HISTORICAL CONTROL VALUES COMBINED NUMERICAL ABERRATIONS (POLYPLOID AND ENDOREDUPLICATED CELLS)
1997-1999
Historical Values
Mean SD' Range
Untreated/ Medium
(%) 2.5
1.6
0.0-9.5
Water
(%)
2.3
12
0.0-7.5
NON-ACTIVATED TEST SYSTEM
Saline
(%)
DMSO (%)
Acetone
(%)
Ethaaol
(%)
2.4 1.3 0.0-6.0
2.4 1.4 0.0-8.0
3.0 1.7 1.5-6.0
2.0 1.4 0.0-6.0
Other'
(%)
2.3 1.4 0.0-5.5
All
Solvents
(%) 2.3
1.3
0.0-8.0
Positive Control2
(%)
3.2
1.8
0.0-9.5
Historical Values
Mean SD' Range
Untreated/ Medium
(%)
2.9
1.8
0.0-9.5
Water
(%)
4.0 2.4 0.0-11.5
S9-ACTIVATED TEST SYSTEM
Saline
(%)
DMSO (%)
Acetone
(%)
Ethanol
(%)
3.6 1.4 0.5-5.5
2.6
1.3 0.0-6.0
3.2 1.2 2.5-4.5
3.3 1.5 0.5-5.5
Other'
(%)
2.9 1.2 1.5-6.5
All
Solvents
(%)
32
1.8
0.0-11.5
Positive Control3
(%)
3.7
2.0 0.0-10.5
SD = standard deviation. Positive control for non-activated studies, N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG, 0.75-2 (ig/ml), and Mitomycin C (MMC, 0.08-0.15 ug/ml). Positive control for S9-activated studies, cyclophosphamide(CP, 10-50 jig/mi), ai benzo(a)pyrene, (B[a]P, 30 pg/ml). Other solvents include non-standard solvents and Sponsor suppliedvehicles.
BioReliance Study No.: AA37AZ.331.BTL
24
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H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Celts____________________________DuPont-5235
APPENDIX B
Study Protocol
BioReliance Study No.: AA37AZ.331.BTL
25
Company Sanitized. Does not contain TSCA CBI
H-24616: Jn FI&-O Chromosome Aberration Test
to Chinese Hamster Ovary (CHO) CeBs__________________________DuPont-5235
SponsorProject Number: BioReliance Study Number
DuPom-5235 AA37AZ.331.BTL
1.0 PURPOSE
Vitro Mammalian Chromotome Aberration Test
The pmposc of this study is to evaluate the clastogenicpotential of a test substance based upon its ability to induce chromosome abeirarions in Chiacae hamster ovary (CHO) cells.
2.0 SPONSOR
2.1
Nfime:
2.2 Address:
2J Representative:
EJ.du Font deNeroouis and Company
Stine HaskeU Research Center DuPont HaskeU Laboratory P.O. Box 50 i 090 Elkton Road Newark, DE 19714-0050
Maria Donner.Ph-D. Phone: 302-366-5251 Fax: 302.235-7156 Email: inari&doncer@usa.dupontcom
2.4 SponsorProject #: DuPont-5235
2.5 WR#:
yuS\
2.6 HaskeU:
24616
2.7 Service Code: HH^
3.0 BDENTinCATIONOFTEST AND CONTROL SUBSTANCES
3.1 Test Substance:
32 Test Substaoce 1.0.: H.246I6 (to be used in the report text)
3.3 Controls:
Solvent: Positive:
Test Substance Solvent (or Vehicle)
MitomycinC(MMC) Cyclophosphamide(CP)
3.4 Determination of Strength,Purity, etc.
Unless alternate arrangements are made, the testing facility at BioReliance will not
perform analysis of the dosing solutions. The Sponsorwill be directly responsible
for determination and documentation of die analytical purity and composition of die
ProtocoiNo-SPCTO! 03-Nov-2000
I of 9
6 | BlORELIANCE- C_ oomm^SWppyaannfyawy*<swral\vinHMfi,A|iICCittzjz>j>keebiiqefaf4.l.|jttD<<DJjiloo4XtteeKo*src>i*ini>iot
BioReliance Study No.: AA37AZ.331.BTL
26
contafn
TSCA
CBI
H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (080) Cells_____
_____________ ________DuPont-5235
Sponsor Project Number: DuPont - 5235
BioReliance Study Number: AA37A2.331.BTL test substance, and the stability and strength of the test substance la the solvent (or vehicle).
3.4 Test Substance Retention Sample
The retention of a reserve sample of the test substance will be the responsibilityof
the Sponsor.
4.0 TESTING FACILITY AND KEY PERSONNEL
4.1 Name:
Toxicology Testing Facility BioReliance
4.2 Address:
9630 Medical Center Drive Rockville.MD 20850
4.3 StudyDirector
Ramadevi Gudi, Ph.D. Phone: (301)610.2169
Fax:
(301)738-2362
E-mail: rgudi@bioreliaace.com
5.0 TEST SCHEDULE
5.1 ProposedExperimental Initiation Date;
28Nov2000
52 ProposedExperimental Completion Date: 19 Jan 2001
5.3 ProposedReport Date:
31 Jan 2001
6.0 TEST SYSTEM
TteCHO-KiCeillineisaprolineauxotro^iwithamodalchromoso(aenumberof20anda population doublingtime of 10-14 hours. CHO-K, cells were obtained from die American
Type Culture Collection (repositorynumber CCL 61), Manassas, VA. The stabilityof the modal chromosome nmber of me cell line is routinely checked and the cell line is routinely
tested and determined to be free from mycoplasma contamination. This system has been
deaiooHnted to be sensitive to die clastogenicactivity of a variety of chemicals (Preston et al., 1981).
7.0 EXPERIMENTAL DESIGN AND METHODOLOGY
The chromosome aberration test will be conducted using standard procedures (Evans,
1976), by exposingcultures of CHO cells to a minimum of four concentrations of me test
substance as well as to positive and solvent controls. In the nog-activated test system,
treatment will be for 4 hours and for 20 hours; in die S9 activated test system, exposure will
be for 4 hours (Swierengaet al., 1991). To ensure evaluation of first division metaphase cells me dividing cells will be arrested in metaphase and harvested for microscopic
ProtocrtNo.SPGTMl 03-Nw-tWO
H,t9
----B--IORELIANCE"
^Sy PrRrrlvMict6kiolof>(l *ioftiti
BioReliance Study No.: AA37AZ331.BTL
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Company Sanitized. Does not contain
TSCA
CBI
H-24616; In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells___________________________DuPont-5235
SponsorProjectNumber DuPont-5235
BioReliaace StudyNumber: AA37AZ.331.BTL
evaluation of chromosome aberrations at approximately20 hours (1.5 normal cell cycles)
after the initiation oftreaanent (Galloway et al., 1994). The clastogenicpotential of the test
substance will be measured by its ability to increase stnicttnal chromosome aberrations in a
dosc-responaivemanner when compared to the solvent control group. In the event of a
positive response in me 4 how non-activated study, me prolongedexposure non-activated
if study may not be scored. The test substance will also be assessed for
numerical chromosome aberrations.
ability to induce
7.1 SolubilityDetermination
Unless tee Sponsor has indicated (he test substance solvent, a solubility
detenmnationwil! be conducted to determine the solvent and taeinaximum soluble
concentration up to a maximum of 500 nig/ml. Solvents compatible with this test system, in older of preference, include but are not limited to sterile water (CAS 7732-18-5), dimethylsulfbxide(CAS 67-<S8.5),ethanol (CAS 64.17-5), and acetone (CAS 67-64.1). The solvent will be the test substance solvent, selected in older of prefereace,that penmts preparationof me highestsoluble stock concentration, up to
500mg/ml.
72 PreliminBryToxicteyTest for Selection ofDose Levels
Selection of toe dose levels for die cytogeaeucs test will be based upon posttreatment toxkity (cell growth inhibition relative to toe solvent control) and solubilityof the test substance. CHO cells will be exposedto solvent alone and to at least nine concentrations of test substance. The highestconcentration tested will be 5 mg/ml or 10 mM whichever is lower for freely soluble-test substances, or the maximum concentration resulting in a workable suspensionfor pooriy soluble test substances not to exceed 5 mg/ml. The pH will be measured at the highest test substance malment condition and will be adjusted,if necesauy, in ocder to maintain a neutral pH in the tteatment medium. The osmoialityofthe highestdose level,
lowest precipitatingdose level (where applicable)and the highestsoluble dose level (where applicable)in treatment medium will also be measured. Cells seeded 16*24
hours eariier will be exposed for 4 hours in the absence and presence of S9 and for 20 hours in the absence of S9. Just prior to ttypsinizationthe cell cultures will be visually inspectedfor me extent of monolayer confluency relative to the solvent con&oL Twenty hows after treatment initiation the cells will be harvested by ttypsinizittionand counted using an automatic cell coumer and the cell viability will be assessed usingtrypan blue dye exclusion. The cell counts and percent viability will be used to determine celt growth inhibition relative to the solvent control.
Whenever possible, the high dose to evaluate chromosome aberrations will be
selected to give at least 50% loxicity (cell growth inhibition relative to the solvent
control) irrespectiveof solubility but not to exceed 5 mg/ml or 10 mM. At least two
additional dose levels, demonstratingminimal or no toxicity will be included. In the event the test substance cannot be dissolved at a high enough concentration in an appropriatesolvent to be toxic, then me highestdose to be tested in the chromosome
Protocol No. SPGT331 03.Nov.2000
BioReliance Study No.: AA37AZ.331.BTL
3 of 9 28
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H-24616: In Vitro Chromosome Abeiradon Test
in Chinese Hamster Ovary (CHO) Cells____
DuPont-5235
Sponsor Project Number:. DuPont-5235
BioReliance Study Number:, AA37A2.331.BTL aberration test will be the concentration resulting in minimum precipitationin test medium. Precipitationwil! be determined by direct visual inspection. Intheevcnt the test substance demonstrates a dose-responsive increase in toxteity at concentrations that exceed solubility in treatment medium, then the highestdose to be tested wili be the maximum concentration that resutts in at least 50% toxicity. In the event that neither eyiotoxicity nor insolubility is observed in the preliminary test, the highestdose in me chromosome abeoaoon test will be 5 mg/ml or 10 mM
whichever is lower. If excessive precipitationof die test substance-solvent solution occurs upon addition to treatment medium, or if the osmolality of tile treatment
medium is considered excessive, the Sponsorwill be consulted.
7.3 Frequency and Route of Administration
Target cells will be treated for 4 hours in the absence and presence of S9 and for 20 hours in the absence ofS9, by incorporation of me test substance-solvent mixture into the treatment medium. This techniquehas been demonstrated to be an effective method of detection of chemical clastogens in this test system (Evans, 1976).
If me Sponsor is aware of specificmetabolic requirements, then mis information
will be utilized in the preparation of the study design. Verification of a clear positive response is not required. Negative results will not be confirmed when justification can be provided. Equivocal results may be confirmed, upon consultation with me Sponsor, and may employ a modification of the studydesign. This guidance is based on the OECD Guideline 473 (1997) and ICH Guidance on SpecificAspectsof RegulatoryGenotoxieity Tests fix Phannaceuncals (1996).
7.4 Activation System
Aroctor 1254-ioduced rat liver S9 will be used as die metabolic activation system. The S9 will be prepared from male Sprague-Dawley rats induced with a single inaaperitoaeal injection of Aroclor 1254,500 mg/kg, five days prior to sacrifice. The S9 wall be batch preparedand stored fiozen at approximately"70<: until used. Each batch preparationofS9 will be tested for sterility and its ability to metabolize 2-afflinoantfaraceae and 7,12-dmicthylbeaz(a)anthracenc to forms mutagenic to Salmonella lyphimwiumXMOO.
Immediately priorto use, the S9 will be thawed and mixed with cofactois to contain 2 mM magnesium chloride (MgCl;,) 6 mM potassium chloride (KCI), ImM glucose-6-phosphate,)mM nicotinamide adeame dinucleodde phosphate(NADP) and 20 ulS9 per ml senim free medium.
Protocol No. SPGT331 Oi-Nov.2000
BioReliance StudyNo.: AA37AZ.331.BTL
4 019 29
Corn!
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'"Boes-wofcontam TSCA CBI
H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells____________________________DuPont-5235
7.5 Controls
Sponsor Project Number: BioReliance Study Number:
DuPent-5235 AA37AZ.331.BTL
7.5.1 Solvent (or Vehicle) Control
The solvent for the test substance will be used as the solvent control. For solvents other than water, physiological buffer, or medium, the final concentration sn treatment medium will not exceed 1%.
7.52 Positive Controls
Mitomycin C will be used at a conoeonxnon within 0.05-0.3 uf/ml as uw positive control in ihe lion-activated study. Cyclophospbanudewill be used at a concentration within 10-50 pg/ml as me positive control in the S9. activated study.
7.6 Preparationof Target Cells
Expoeentially growing CHO-K, cells will be seeded ia complete medium (McCo/s 5A medium containing 10% fetal bovine serum, 2 mM L-ghitamuie, 100 units penicillin/ml and 100 ug stieptomycin/ml) for each treatment condition at
approximately5 x 10s cells/25 cm3 flask. The flasks will be incubated at 37 iloC
matamudmedamK>sphcreof5l'/oCO;inairforl6-24hours.
7.7 Identification of Test System
Using a pemuoent marking pen, me treatment flasks will be identified by we BioReliance study number and a code system to designateme treatment condition and test phase.
7.8 Treatment of Target Cells
Treatment will be earned out in duplicate by refeeding me flasks with 5 ml completemedium for the non-activated exposure or 5 ml S9 reaction mixture for the S9-actvatcd exposure, to which will be added 50 ul of dosingsolution of test or control sulMtxace HI solvent or solvent alone. Larger volumes of dosing solution
may be used if water, physiologicalbuffer, or medium is used as me solvent
In me non-activated study, me cells will be treated for 4 hours and fix 20 hours; in the S9-activated study the cells will be treated for 4 hours. Treatment will be carried out at 37 1<>Cin a humidified atmosphere of 5 1% CO, in air. After the,4 hour treatment period in me non-activated and we S9-activated studies, me treatment medium will be aspirated, the cells washed with phosphatebuffered saline, refed with complete medium and returned to the incubator.
A (MncunTOt toxichy test to detemune cell growth inhibition relative to the solvent control will be conducted in both the non-activated and me S9-acnvated studies.
Protocol No. SPGTOt 03-Nov-2000
fof9
CompanlISnyiSiSslG^
BioReliance Study No,: AA37AZ.331.BTL
30
TSCA CBI
H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Ceils___________________________PnPont-5235
Sponsor Project Number: BioReliance Study Number:
DuPont-5235 AA37AZ.33t.BTL
7.9 Collection of Metaphase Cells
Cells will be collected approximately 20 hours after initiation of treatment This post-treatment harvest time represents approximately 1.5 nonnal cell cyclesand was selected to ensure that the cells are analyzed in the first division meiapbase after initiation of treatment Two hours prior to cell harvest, Colcanid" will be added to die cultures at a final concentration of O.I ug/ml.
Cells will be harvested by trypsinization, collected by centrifugationand an aliquot will be removed for counting using an automatic cell counter and trypan blue dye exclusion. The remainder of die cells will be swollen with 0.075M KC1, washed with two consecutive changes of fixative (methaaol:glacialacetic acid, 3:1 v/v),
cappedawl stored overnight or longer at approximately2-8<>C, The cell counts and percent viability will be used to determine ceil growth inhibition relative to die solvent control (*/ toxicity). To prepare slides, UK cells will be collected by centrifiqanon and resuspendedin fiesh fixative. 'Toe suspensionof fixed cells will be appliedto glass microscope slides and air-dried. The slides will be identified by
die experiment number, treatment condition and date. The slides will be stained with Giemsa and permanently mounted.
7.10 Scoringfor Metaphase Aberrations
To ensure that a sufficient number ofmetaphase cells are present on the slides, die pcrecntage of cells in mitosis per 500 cells cored(mitotic index) will be detennined and recorded for each coded treatment group selected for scoring chromosome abenations. Slides will be coded using nmdom numbers by an individual not involved with the scoringprocess. In the event of a positive response in the 4 hour non-activated study,the prolongedexposure non-activated study may siot be scored. Metaphase cells with 20 2 centtomeres will be examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads from each dose level (100 per duplicate flask) will be examined nd scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The number ofmetaphase spreadsthat will be examined and scored per
duplicate flask any be reduced if me percentage of abenant cells leaches a
statistically significant level before 100 cells are scofed. Chromatid-type aberrations include chromatid and isochroimtid breaks and exchange figuressuch as quadriradials(symmetricaland asymmetrical interchanges),triradials, and complex
resiTUigements. Chromosome-type aberrations include chromosome breaks and
exchange figures such as diccntries and rings. Fragments (chromatid or acentric) observed m the absence of any exchangefigure wUl be scored as a break (chromatid orchromosome). Fragmentsobserved with an exchange figure will not be scored as
an abenction but will be considered part of the incompleteexchange. Pulverized chromo3ome(s), pulverizedcells and severely damagedcells (2 10 aberrations)will
also be recorded. Chromatid and isochromatid gaps will be recorded but not
included in the analysis. The XY coordinates for each cell with o. structural
Protocol No. SPGT331 03.Nov-!OW
6 of 9
BioReliance Study No.: AA37AZ.33LBTL
31
Company aBltBedyOTOs-Bot'ttofrTttSeCjnA CB1
H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells__________________________DuPont-5235
Sponsor Project Number PuPont-5235
BioReliance Study Number: AA37AZ.331.BTL aberration will be recorded using a calibrated microscope stage. The percent polyploidand endoreduplicatedcells will be evaluated per 100 cells for each dose level analyzedfor structural aberrations.
8.0 CRITERIA FOR DETERMINATION OF A VALID TEST
8.1
Solvent Comrol
The frequencyof cells with structural chromosome aberrations in die solvent control must be within the range of die historical solvent control.
8.2 Positive Control
Hie percentage of cells with aberrations must be statistically increased (p^O.05,
Fisher's exact test) relative to the solvent control.
9.0 EVALUATION OF TEST RESULTS
The toxic effects of treatment are based upon cell growth inhibition relative to die solvent
control aiidwiU be presoited for the toxicity and abenation studies. The number and types
of aberraricms found, the percentage of stnienmulyaadnumericayydansaged cells (penxm aberrant cells) in the total populationof cells examined, and the mean aberrations per cell
will be calculated and reportedfor each treatment group. Chronutid and isochromatid gaps
are presented m die data but ace not included in die total peicenuge of cells with one or more aberraticms or u die feequencyof suwtundaberratrons per cell. Statistical analysisof the percentage of abcnant cells wiU be performed using the Fisher's exact test TheFisher's test wiU be used to conipBrepairv^ the percent abenam cells of each toeatment group with ttiat of the solvent control In the event of a positive Fisher's exact test at any test substance dose level, die Cochran-Armitage test will be used to measure dose-responsiveness. All conclusions will be based on sound scientific basis; however, as a guide to interpretationof
the data, die test substance will be considered to induce a positive response when die
percentage of cells with aberrations is increased in a dose-responsivemanner with one or
more concentrations being statistically significant (p$0.05). However, values that are statisticallysignificantbut do not exceed the range of historic negative or solvent controls may be judged as not biologically significant. Test substances not demonstrating a statisticallysignificantincrease in aberrations will be concluded to be negative.
10.0 REPORT
. A report of the results of BUS study will be prepared by BioReliance and will accurately describe all methods used for generation and analysis of die data.
Results presentedwill include, but not be limited to:
Protocol No. SPGTU1 03-Nov-2000
7 of 9
---- BlORELlANCF
^______________________Company _
BioReliance Study No.: AA37A2.331.BTL
32
SM^aynit_ilozfe--d*ir.*tuDroohefi)sfD|htcotAftcoocinatii|at i_ n --TSaC,Aa CBI RB&II
H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells____________________________DuPont-5235
Sponsor Project Number PuPont - 5235
BioRcUancc StudyNumber: AA37AZJ31.BTL Test substance: identification and CAS no., if known; physical nature and purity, if
known; physicoclsemicalproperties relevant to the conduct of die study, if known; stability of test substance, if known.
Solvent/Vehicle: justification for choice of vehicle; solubility and stability of test substance in solvent/vehicle, if known.
Source of cells, kaiyotypc features (modal chromosome number) and suitability of the cell
type used, absence ofmycoplasma, cell cycle length, passage number.
Test conditiofflf:a}mposition of medium; CO: coQcentrction;iacubatioatiin^ce^ density; solvent and solvent selection rationale; coacemnooa of test substance and concentration selection rationale; composition and acceptabilitycriteria fat die metabolic activation (S9) system; duration of treatment; duration of treatment with aad concentration of Colcemid*; type of metabolic activation system used; positive and solvent controls; methods of slide preparation;number of cell cultures; criteria fw scoring aberrations and criteria for consideringstudies positive, negative.
Results: descriptionof precipitation; pH and osmolality of the treatment medium; cell growth inhibition relative to me solvent control; mitotic index aad number ofmetaphaaes analyzed; type and number of aberration (structural and numerical) given separately for each treated and control culture; concentration-response relationship; statistical analysis;
historical control data.
11.0 RECORDS AND ARCHIVES
Upon completion of the final report, all raw data aad reports will be archived by BioReliance, RockviSle, MD for a periodof no less thaa I year from the study initiation
date.
12.0 REOULATORYREQUIREMENTMSOOD LABORATORY PRACTICE
Thu protocol has been written to comply with OECD Guideline 473 (& Mftv Mammalian Chromosome Aberration Test), July 1997 and with the IntenutioBal Conference on Hanoocdzation of Technical Requirements for RegistrationofPharmaceuocals for Human Use (1996 aad 1997).
1ms study will be performed using the ERA (TSCA) Good Laboratory Practice Regulations for Noacliaical Laboratory Studies as a guideline; however, the study will not meet GLP requirements.
Unless anxiigements are omde to the contrary, unused dosing solutions will be disposedof following administration to the test system and all residual test substance will be disposed of following finclizstion of we report.
ProtottttNo.SPGTOt 03-Mov-20M BioReliance Study No.: AA37AZ.331.BTL
Sor9 33
6l BlOREUANCF
ianHlTorf rings nnt nnntain TSCA CBI
H-24616: In Vitro Chromosome Aberration Test
in Chinese Hamster Ovary (CHO) Cells____
___ _____
____ DuPont-5235
13.0 REFERENCES
Sponsor ProjectNumber: BioReliance StudyNumber
DuPont-5235 AA37AZ.331.BTL
Evans, HJ. (1976) Cytological methods for detecting chemical nustagefis. in: A. Hollaender (Ed.), Chemical Musagens. Principles and Methods {their Detection, vol. 4. Plenum Pttas, New Yoik,NY.
Galloway, SM, MJ. Aaroema, M. Ishidate Jr.. J.L. Ivett, DJ. Kiddaad, T. Morita, P.
Moscsso aad T. Sofiaii (1994) Report fiwn working group on in vitro tests for chromosomal abemdoos. Mutation Research 312(3)^41-261.
IntenianoBai Conference on Hannomsatioa (ICH) of Technical Requiremenis for Registration of Pharmaceuticals for Human Use. Guidance oa Specific Aspects of Regulatory Genotoxicity Tests for PhannaceuQous. S2A document recommended for
adoption at step 4 of die ICH process on July 19,1995. Federal Register61:18198.18202, April 24,1996.
Intenixtiooci Conference OB Hannonisation (ICH) of Technical Requiiemeats for Registration of Phannaceuticcis for Human Use. Genotoxicity: A Stendud Batteiy for Genotoxicity Testing of Phannaceuticals. S2B document recommended far adoptionat step 4 of BM ICH proceas on July 16,1997. Federel Register 62:16026-16030, November 21,1997.
OECD Guideline {as the Testing of Chemicals, Guideline 473 {In Vitro Mammalian Chromosome Abezmion Test), July 1997.
Preston, RJ.. W. Au, MA. Bender, J.G. Brcwm, A.V. Carraoo, J.A. Heddte, A.F. McFee,
S. Wolffand J.S. Wcssom (1981) Mammaliaa m vivo and in vitro cytogeoeticassays: a repon of the Gcoe-ToK Program, Mutatioo Research, 87:143-188.
Scott, D., N.D. Danfoid, BJ. Dean and DJ. Kiddaod. 1990. Metaphase Chromosome Abemnoa Assay m Vitro, to: Basic Mutagenicity Tests: UKEMS Recommended Procedures. DJ Kmdaad (ed). CambridgeUniversity Press. New York, NY.
Swierenga SJEUL, J.A. Heddle, EA. Sigal, J.P.W. Oufflan, RL. Brillinger, G.R. Douglas and EJtNestmicm (1991)Recommended protocols based on a survey of cunentpnctice in geootoxicity testing labocBtories, IV. Chromosome aberration and sister-chromatid
exchaage ia Cliioese hamster ovaiy. V79 Chinese lung aad human lymphocyte cultures, Mutttkm Research 246:301-322.
BioReliance Smdy No.: AA37AZ.33iaTL
34
H-24616: In Vitro Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells
DuPont-5235
14.0 APPROVAL
Sponsor Project Number: BioReliioce Sway Number:
DiiPont-5235 AA37A2.331.BTL
"c^^r^
SPONSOR REPRESENTATIVE
cn f^ov 2.000 DATE
VAo^rCo^ Uoo^<-r
(Printor Type Nuae)
6,6U^mLff^<^' ^C<-^'
BIORELIANCE STUDY DIRECTOR
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DATE
ProtacotNo.SPGTMl g3.Nir.2aW
BioBfiKance Sta<tyNo.: AA37AZ.331.BTL
10 of 9 35
ftB^ORELIA BtORELIANCE-
Company y S aiffti[zzPfeeTdrrr.>lvDMMoiicecrsftemnrr<ofite<tliicAotnctiAatiin TSCA CB1
