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FINAL REPORT Study Title
H-24616; In Vitro M am m alian Chromosome A berration Test in
Chinese H am ster O vary (CHO) Cells Authors
Ramadevi Gudi, Ph.D. Caren Brown, M.S.
Report Completion Date March 12,2001
Performing Laboratory BioReliance
9630 Medical Center Drive Rockville, MD 20850 for
E. I. du Pont de Nemours and Company Haskell Laboratory for 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
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C E R T IFIC A T IO N
We, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study.
Reviewed by:
Richard San, Ph.D. " " BioReliance Study Management
f % tS/Ja^X 2 OPf
Date
Issued by Study Director:
...fia u ia d k te *
R am adeviO udi
BioReliance Study Director
l2-MA&2Jxf
Date
Approved by Study Monitor:
OsA^-Maria Dormer, PhJD.
Senior Research Scientist
01 HAft to o
Date
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STUDY INFORM ATION
Substance Tested: Synonym/Codes:
Haskell Number: Purity: Known Impurities: Physical Characteristics: Stability:
Solubility:
Sponsor:
Study faitiated/Completed: In-Life Initiated/Completed:
H-24616
The test substance appeared to be stable under the conditions o f the study; no evidence o f instability was observed. Aquatics: Dispersible in water All Others: Dimethyl sulfoxide E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U .S A . November 1 5 ,2000/(see report cover page) November 2 8 ,2000/November 29,2000
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TABLE OF CONTENTS
CERTIFICATION.________ _____________ _____________________ ______ STUDY INFORMATION____________________________ _____ _________ TABLE OF CONTENTS....________ :____________ ____ _________ _______ SUMMARY__ ___ _________________________________________ _______
PURPOSE____ ________________ _______ _______________ ...___ ,________
CHARACTERIZATION OF TEST SUBSTANCE AND CONTROL ARTICLES MATERIALS AND METHODS________________________________ ______ RESULTS AND DISCUSSION____________ ____________________________ CONCLUSION______________________________________________________ REFERENCES__________________________ _____________________
D ATA T A B L E S___________________________________________________________________ _______...._____
TABLE 1: CONCURRENT TOXICITY TEST USING H -24616 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; 4 HOUR TREATMENT, 16 HOUR RECOVERY PER IO D ........................
TABLE 2: CYTOGENETIC A NA LY SIS OF CHO CELLS TREATED WITH H -24616 IN THE ABSENCE O F EXOGENOUS METABOLIC ACTIVATION; 4 HOUR TREATMENT, 16 HOUR RECOVERY PER IO D ......
16
TABLE 3: CONCURRENT TOXICITY TEST USING H -24616 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION; 4 HOUR TREATMENT, 16 HOUR RECOVERY PER IO D ........................ 17
TABLE 4: CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH H -24616 IN THE PRESENCE O F EXOGENOUS METABOLIC ACTIVATION; 4 HOUR TREATM ENT, 16 HOUR RECOVERY PE R IO D .......................... .................................... ....................................... ................................... . fg
TABLE 5: CONCURRENT TOXICITY TEST USING H -24616 IN THE A BSENCE OF EXOGENOUS METABOLIC ACTIVATION; 20 HOUR CONTINUOUS TREATMENT........................................................ 19
TABLE 6: CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH H -24616 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION; 20 HOUR CONTINUOUS TREATM ENT. 20
TABLE 7: SUM M ARY............... ............... ....... . APPENDIX A H ISTO RICAL CONTROL D A T A ...
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A PPEND IX B ST U D Y PRO TO CO L......
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SUMMARY
H ie test substance, H-24616 m i m 0 [ ^ w a s tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence o f an Aroclor-induced S9 activation system in order to evaluate the clastogenic potential o f the test substance.
Water was determined to be the solvent o f choice based on information provided by the Sponsor and compatibility with the target cells. The test substance was soluble in water at a concentration o f 50 mg/mL, the maximum concentration tested.
In the chromosome aberration assay, the maximum dose tested was 5000 pg/mL. The cells were treated for 4 and 20 hours in die 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 < 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 pg/mL in die S9 activated 4 hour group and in the non-activated 20 hour exposure group. Dose levels < 500 pg/mL were soluble in treatment medium in die S9 activated 4 hour group and in the non-activated 20 hour exposure group.
Selection o f dose levels for the cytogenetics test was based upon post-treatment cell growth inhibition relative to the solvent control and solubility o f die test substance.
Recovery Time (hours)
Harvest S9 Cell growth
Time
inhibition at
(hours)
highest dose
scored
M itotic
Index
Reduction *
LED (lowest effective dose) for Structural
LED for Numerical Aberrations
(pg/mL)
Aberrations
4
16
20 - 15% at 5000 25%
None
None
20
20 - 36% at 5000 23%
None
None
4
16
20 *1" 40% at 2500 52%
None
None
* relative to solvent control at high dose evaluated for chromosome aberrations
The positive and rojventcontols fulfilled the requirements for a valid test. Based on the
findings o fthis s t u d y c o n c l u d e d to be negative for the induction o f structural and numerical chromosome aberrations in Chinese hamster ovary (CHO) cells in the pragRnra and absence o f Aroclor-induced rat liver S9.
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PURPOSE
The purpose o f this study was to evaluate toe clastogenic potential o f a trat snKgtnnTM based to n its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells.
CHARACTERIZATION OF TEST SUBSTANCE AND CONTROL ARTICLES
.; if0 ? SUj Stan^ ' H-24616>was received by BioRelianee on November 6,2000 and was
ass,iTMgn,ed the code j ber AA37AZ. The test substance was characterized by toe Sponsor as an _____ __ ; should be stored at ambient temperature. An expiration date was not y toe Sponsor. Upon receipt, toe test substance was described as ai was stored at room temperature, protected from exposure to light,
The solvent used to deliver H-24616 to toe test system was sterile water (CAS 7732-18-5) from toe Life Technologies Company. Mitomycin C (MMC; CAS No.: 50-07-7), was obtained rrom the Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations o f 1 and 2 pg/mL for use as toe positive control in toe non-activated test system. Cyclophosphamide (CP; CAS No.: 6055-19-2), was obtained fiom Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations o f 100 and 200 pg/mL for use as toe 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 tor toe test substance was used as toe solvent control at toe same concentration as that found in the test substance-treated groups.
Test System
M ATERIALS AND METHODS
Chinese ham ster ovary (CHO-K,) cells (repository number CCL 61) were obtained fiom Co" f f on>Manassas, VA, on May 29, 1997. In order to assure toe
karyotypic stability o f the cell line, working cell stocks were not used beyond passage 20. The freeze lot o f cells was tested using the Hoechst staining procedure and found to be free o f mycoplasma contamination. This cell line has an average cell cycle time o f 10-14 hours with a modal chromosome number o f 20. The use o f CHO cells has been demonstrated to be an eitective method o f detection o f chemical elastogens (Preston et al., 1981).
M etabolic A ctivation System
Aroclm l^ in d u c e d rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawiey rats induced with a single intraperitoneal injection o f Aroclor 1254,500 mg/kg, five days prior to sacrifice. The S9 was batch prepared and stored at
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^-70C until used. Each bulk preparation o f S9 was assayed for sterility and its ability to
metabolize 2-aminoanihracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to
Salmonella typhimurium TA100.
Immediately prior to use, die S9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 pL S9 per milliliter medium (McCoy's 5A serum-free medium supplemented with, 100 units penicillin and 100 pg streptomycin/mL, and 2 mM L-glutamine).
Chromosome A berration Assay
Selection o f dose levels for the cytogenetics test was based upon post-treatment cell growth inhibition relative to the solvent control and solubility o f 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 o f 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 ceIls/25 cm2 flask and were incubated at 371C in a humidified atmosphere o f 51% C 02 in air for 16-24 hours. Treatment was carried out by refeeding duplicate flasks with 4.5 mL complete medium (McCoy's 5A medium supplemented with 10% FBS, 100 units penicillin and 100 pg streptomycin/mL, and 2 mM L-glutamine) for the non activated study or 4.5 mL S9 reaction mixture for the S9 activated study, to which was added 500 pL o f dosing solution o f test or control article in solvent or solvent done. The osmolality o f the highest concentration o f dosing solution in the treatment medium was measured. The pH o f the highest concentration o f dosing solution in the treatment medium was measured using test tape.
In Ihe non-activated study, the cells were exposed to the test substance for 4 hours or continuously for 20 hours up to the cell harvest at 371C in a humidified atmosphere o f 51% C 0 2in 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 duplicate flasks for each treatment condition at a final concentration o f 0.1 pg/mL and the flasks returned to the incubator until cell collection.
In the S9 activated study, the cells were exposed for 4 hours at 371C in a humidified atmosphere o f 51% C 02 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 o f
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0.1 pg/mL and the flasks were returned to the incubator until cell collection.
A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems. After cell harvest an aliquot o f the cell suspension was removed from each culture and counted using a Coulter counter. The presence o f test substance precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The ceil counts and percent viability were used to determine cell growth inhibition relative to the solvent control.
Collection o f M etaphase Cells
Two hours after the addition o f Colcemid, metaphase cells were harvested for both the non-activated and S9 activated studies by trypsinization. Cells were collected approximately 20 hours after initiation o f treatment (Galloway et al., 1994). The cells were collected by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspended in 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 o f approximately 2 mL Camoy's fixative (methanokglacial acetic acid, 3:1, v/v). The cells were stored overnight or longer in fixative at approximately 2-8C.
Slide P reparation
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 centrifugation (at approximately 800 rpm for 5 minutes) the supernatant fluid was decanted and the cells resuspended to opalescence in fresh fixative. A sufficient amount o f cell suspension was dropped onto the center o f 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 o f M etaphase Cells
Slides were coded using random numbers by an individual not involved with the scoring process. To ensure that a sufficient number o f metaphase cells were present on the slides, the percentage o f cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 202 centromeres were examined under oil immersion without prior knowledge o f 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 the non-activated 4 hour exposure group, the non-activated 20 hour continuous exposure group was not evaluated for chromosome aberrations. Whenever possible, a minimum o f 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The number o f
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metaphase spreads that are examined and scored per duplicate flask may be reduced if the percentage o f 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 the absence o f 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 o f the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely
damaged cells (;>10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY coordinates for each cell with chromosomal aberrations were recorded using a calibrated microscope stage. Polyploid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored.
C o n tro ls
Mitomycin C was used as the positive control in the non-activated study at final concentrations o f 0.1 and 0.2 pg/mL. Cyclophosphamide was used as tire positive control in the S9 activated study at final concentrations o f 10 and 20 pg/mL. For both positive controls the dose level exhibiting a sufficient number o f 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 o f T est Results
Hie toxic effects o f treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the aberration studies. The number and types o f aberrations found, the percentage o f structurally and numerically damaged cells (perm it aberrant cells) in the total population o f 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 the total percentage o f cells with one or more aberrations or in tire frequency o f structural aberrations per cell.
Statistical analysis o f the percent aberrant cells was performed using the Fisher's exact te st Fisher's test was used to compare pairwise the percent aberrant cells o f each treatment group with that o f the solvent control. In the event o f a positive Fisher's test at any test substance dose level, the Cochran-Armitage test was used to measure dose-responsiveness.
All conclusions were based on sound scientific basis; however, as a guide to interpretation o f tiie data, the test substance was considered to induce a positive response when the percentage o f cells with aberrations is increased in a dose-responsive manner with one or more
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concentrations being statistically significant (p<0.05). If the test substance did not demonstrate a statistically significant increase in aberrations it will be concluded to be negative.
C riteria for a V alid Test
Ih e frequency o f cells with structural chromosome aberrations in toe solvent control must be within toe range o f toe historical solvent control. The percentage o f cells with chromosome aberrations in toe 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 nondneto f this study.
Archives
All raw data, the protocol, all reports, ancystainedand coded slides will be maintained according to Standard Operating Procedurep ^ l ^ M b y toe BioReliance RAQA unit headquartered at: BioReliance, 14920 Broschart Road, Rociville, MD 20850. Paper records will be retained for at least three years after which time the Sponsor will be eo n tirM for a decision as to the final disposition o f toe materials. All study materials returned to the Sponsor or destroyed w ill first be copied and the copy will be retained in toe BioReliance archives for a minimum o f 10 years.
Solubility
RESULTS AND DISCUSSION
Water was determined to be toe solvent o f choice based on information provided by toe Sponsor and compatibility with toe target cells. The test substance was soluble in water at a concentration o f 50 mg/mL, the maximum concentration tested.
Chromosome A berration Assay
Selection o f toe dose levels for toe cytogenetics test was based upon post-treatment cell growth inhibition relative to the solvent control and solubility o f tire test snhstanee Whenever possible, fee high dose to evaluate chromosome aberrations was selected to give 50% toxicity. Two additional dose levels, demonstrating little or no toxicity were also included. CHO cells were exposed to solvent alone ami to nine concentrations o f test substance ranging from 0.5 pg/mL to 5000 pg/mL in toe absence o f an S9 reaction mixture. CHO cells were exposed to
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solvent alone and to eight concentrations o f test substance ranging from 500 |ig/mT. to 4000 Hg/mL in the presence o f an S9 reaction mixture. The results o f the evaluation o f cell growth inhibition are presented in Tables 1, 3 and 5. Visible precipitate was observed in treatment medium at 5000 pg/mL 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 fig/mL in the S9 activated 4 hour group and in the non-activated 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 o f the toxicity study, the dose levels selected for evaluation in the chromosome aberration assay were as follows:
Treatment Condition
-S9
+S9
Time (hours) 4
20 4
Recovery Time (hours)
16
Dose levels (pg/mL)
500,1500,5000
0 500,1500,5000 16 1500,2000,2500
Chromosome A berration Assay
In the non-activated chromosome aberration assays the osmolality in treatment medium o f the highest concentration tested, 5000 pg/mL, was 259 mmol/kg. The osmolality in treatment medium o f the lowest precipitating dose, 1500 pg/mL, was 267 mmol/kg. The osmolality in treatment medium o f the highest soluble dose, 500 pg/mL, was 264 mmol/kg. The osmolality o f the solvent (water) in treatment medium was 273 mmol/kg. The pH o f tire concentration o f test substance in treatment medium was approximately 7. Changes o f higher than 50 mmol are not acceptable.
Cell growth inhibition relative to the solvent control o f H-24616 in CHO cells when treated for 4 hours in the absence o f S9 activation was 15% at 5000 pg/mL, the highest test concentration evaluated for chromosome aberrations (Table 1). The activity o f H-24616 in the induction o f 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 pg/mL, was 25% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 500, 1500 and 5000 pg/mL. The percentage o f cells with structural and numerical aberrations in tire test substance-treated groups was not significantly increased above that o f the solvent control (p>0.05, Fisher's exact test). The percentage o f structurally damaged cells in the MMC group was found to be statistically
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significant (11.5%).
There was no dose level with 50% toxicity (cell growth inhibition) in the S9 activated treatment group, so this portion o f the assay was repeated. Results from the initial S9 activated assay were recorded but are not reported. Toxicity o f H-24616 (cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in die presence o f S9 activation in the repeat assay was 40% at 2500 pg/mL, the highest test concentration evaluated for
chromosome aberrations (Table 3). The activity o f H-24616 in the induction o f chromosome aberrations is presented by treatment flask in Table 4 and summarized by group in Table 7. The mitotic index at the highest dose level evaluated for chromosome aberrations, 2500 jig/mt. vras 52% reduced relative to die solvent control. The dose levels selected for microscopic analysis who 1500,2000 and 2500 pg/mL. The percentage o f cells with numerical aberrations in the test substance-treated groups was not statistically increased above that o f the solvent control (p>0.05, Fisher's exact test). The percentage o f cells with structural aberrations in die test substance-treated groups was statistically increased above that o f the solvent control at dose level 2000 pg/mL (p<0.05, Fisher's exact test). However, the percentage o f cells with structural aberrations (4%) was within the historical solvent control range o f 0% to 6.5%. Therefore, it is not considered biologically significant The percentage o f structurally damagwt cells in the CP group was found to be statistically significant (14.5%).
hi the absence o f a positive response in the non-activated 4 hour exposure group, aliare from die non-activated 20 hour exposure group were evaluated for chromosome aberrations. Toxicity o f H-24616 (cell growth inhibition relative to the solvent control) was 36% at 5000 pg/mL, die highest test concentration evaluated for chromosome aberrations in the non activated 20 hour continuous exposure group (Table 5). The activity o f H-24616 in die induction o f chromosome aberrations is presented by treatment flask in Table 6 and summarized by group in Table 7. The mitotic index at the highest dose level evaluated for chromosome aberrations, 5000 pg/mL, was 23% reduced relative to die solvent control. The dose levels selected for microscopic analysis were 500,1500, and 5000 pg/mL. The percentage o f cells with structural or numerical aberrations in the test substance-treated groups was not significantly increased above that o f the solvent control (p>0.05). The percentage of structurally damaged cells in the MMC group was found to be statistically significant (13.5%).
C O N C L U S IO N The positive and solvent controls fulfilled die requirements for a valid te st
Based on the findings o f this s t u d y p H H H j |w a s concluded to be negative for the induction o f structural and numerical chromosome aberrations in Chinese hamster ovary (CHO) cells in the presence and absence o f Aroclor-induced rat liver S9.
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REFERENCES
Evans, H.J. (1976) Cytological methods for detecting chemical mutagens, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, vol 4. Plenum Press, New York.
Galloway, S.M., M.J. Aardema, M. Ishidate Jr., J.L. Ivett, D J. Kirkland, T. Morita, P. Mosesso and T. Sofuni (1994) Report fiom working group on in vitro tests for chromosomal aberrations, Mutation Research 312(3):241-261.
International Conference on Harmonization (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Genotoxicity: Guidance on Specific Aspects o f Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 o f the ICH process on July 19, 1995. Federal Register 61:18198-18202, April 24,1996.
International Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing o f Pharmaceuticals. S2B document recommended for adoption at step 4 o f the ICH process on July 16, 1997. Federal Register 62:16026-16030, November 21,1997.
OECD Guideline for the Testing o f Chemicals, Guideline 473 (In Vitro Mammalian
Chromosome Aberration Test), July 1997.
Preston, R.J., W. Au, M A Bender, J.G. Brewen, A.Y. Carrano, J A Heddle, A.F. McFee,
S. W olff and J.S. Wassom (1981) Mammalian in vivo and in vitro cytogenetic assays: a
report o f the Gene-Tox Program, Mutation Research, 87:143-188.
Scott, D., N.D. Danford, B.J. Dean and D.J. Kirkland, 1990. Metaphase Chmmosome Aberration Assays In Vitro. In: Basic Mutagenicity Tests: UKEMS Recommended Procedures. D.J Kirkland (ed). Cambridge University 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 o f current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sisterchromatid exchange in Chinese hamster ovary, V79 Chinese lung and human lymphocyte cultures, Mutation Research 246:301-322.
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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
Treatm ent (MflAnL)
C e l Count
CricImISKI,
A \W IQ 6S OrtO*)
Cell Viability
(% )
Mean Cells per Flask
(x10)
C e l Growth Cell Growth
Index
fnnlDfOOfl
(%> (%)
W ater
A 2.62 B 2.59
H -2 4 6 1 6 0 .5
A 2.63 B 2.69
1.5 A 2.52 B 2.62
5 A 2.50 B 2.65
15 A 2.36 B 2.65
50 A 2.53 B 2.51
150 A 2.54 B 2.84
500 A 2.21 B 1.89
1500
A 2.04 B 2.02
5000
A 2.22 B 2.22
M M C .0.1 A 2.37 B 2.77
M MC, 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 2.18 2.50 2.1
100
101 -1 98 2 99 1 96 4 97 3 101 -1 79 21 78 22 85 15 99 1 81 19
Treatment: CHO cells were treated In the absence of an exogenous source of metabolic activation for 4 hours at 371C, Cell Viability determined by trypan blue dye exclusion. Mean Calls per Flask * cell count x % viable cells, reported as a mean o fflasks A and B. Cell Growth Index (cells per flask In treated groupfoells per flask In control group), expressed as a oercentaoe.
C ell Growth Inhibition 100% - cell growth index; not calculated for negative controls.
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TABLE 2
CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH H-24618 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION
4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD
Treatment (jg/mL)
Mitotic
% Aberrant Cells
Index Cells
Flask (%> Scored Numerical Structural
W ater
A 6.4 100 B 8.2 100
4 3
1 0
H-24816 500
A 6.0 100 B 5.6 100
7 8
1 1
1500
A 7.6 100 B 7.8 100
3 4
1 1
5000
A 6.0 100 B 6.4 100
5 7
1 1
M M C0.2
A 5.6 100 B 5.2 100
4 4
12 11
Total Number o f StructuralAberrations
Gaps Chromatid Br Ex
Chromosome Br Die Ring
Severely Damaged
CeRs
00 10
0 0
01 0 00 0
0 0
10
0
01 0
0 0 1 00 0
00 00
0 0
01 0 01 0
30 10
0 0
01 0 01 0
16 18
6 4
21 10
1 0
0 0
0 0
0 0
0 0
Average Aberrations
Per Cell
0.010 0.000
0.010 0.010
0.010 0.010
0.010 0.010
0.160 0.130
Treatment: CHO cells were treated for 4 hours at 371C In the absence of an exogenous source of metabolic activation. Additional dose levels of 0 .5 ,1 .5 , 5 ,1 5 , 50 and 150 pg/mL were tested as a safeguard against excessive toxicity at higher dose levels but were not required for microscopic examination. M itotic Index = number of mitotic figures x 100/500 cells counted.
% Aberrant Celle: numerical includes polyploid and endoredupllcated cells; structural excludes cells with only gaps. Chromatid break* Include chromatid and Isochromatid breaks and fragments (Br); chromatid exchange figures (Ex) include quadrlradlals, triradiats and complex rearrangements. Chromosome breaks Include breaks and acentric fragments (Br); dicentric chromosome (Die).
Severely damaged calls Includes ceRs with one or more pulverized chromosomes and cels with 10 or more aberrations. Average Aberrations Per Ceil: severely damaged cells and pulverizations were counted as 10 aberrations.
Com pany S anitized. Does not contain TSCA CBI
BioR eliance Study N o. A A 37A Z.331.BTL
16
TABLE 3
CONCURRENT TOXICITY TEST USING H-2461B IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION
4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD
Treatm ent (pg/imL)
A;TcTCell Count
Flask
Cell Viability
<%>
Mean Cells per Flask
(x10>
C e l Growth C e l Growth
Index
inrstsHuon
(%) <%)
W ater H -2 4 6 1 6 500 1000 1500 2000 2500 3000 3500 4000 CP, 10 CP. 20
A 1.60 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 1.15
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 metabolic activation for4houm at371oc. Cell Viability determined by trypan blue dye exclusion.
Mean Celle per Flask - cell count x % viable cells, reported as a mean for flasks A and B. Cell Growth Index -(c e lls per flask in treated group/cells per flask In control group), expressed as a percentage.
Cell Growth Inhibition *1 0 0 % - cell growth Index; not calculated for negative controls.
BioR eliance Study N o . A A37A Z.331.BTL
17
Com pany Sanitized. Does not contain TSCA CBI
TABLE 4
CYTOGENETIC ANALYSIS O F CHO CELLS TREATED WITH H-24818 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION
4 HOUR TREATMENT, 18 HOUR RECOVERY PERIOD
Treatment (pgftnL)
Mitotic
% Aberrant Cells
Index Cells
Flask <%) Scored Numerical Structural
W ater
A 9.4 100 B 8.6 100
9 7
1 0
H-24616 1500
A 7.8 100 B 8.8 100
6 6
1 2
2000
A 7.2 100 B 8.8 100
11 10
4 4
2500
A 4.4 100 B 4.2 100
4 5
2 2
CP, 10
A 6.8 100 B 7.8 100
4 3
16 13
Total Number of Structural Aberration
Gaps Chromatid Br Ex
Chromosome Br Die Ring
Severely Damaged
Cells
00 0 10 0
01 0 00 0
0 0
210 110
24 04
1 0
12
0
211
5 12 9 28 8
00 00
0 1
00 00
0 0
00 0 00 0
21 0 11 0
0 0
0 0
0 0
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 371C to the presence of an exogenous source of metabolic activation. Additional dose levels o f500 and 1000 pg/mL were tested as a safeguard against excessive toxicity at higher dose levelsbut were not required for microscopic examination. Dose levels 3000,3500 and 4000 pg/mL were not analyzed due to excessive toxicity.
Mltoths Index = number of mitotic figures x 100/900 cells counted. % Aberrant Cells: numerical Indudes polyploid and endoreduplicated cells; structural excludes cells with only gaps. Chromatid breaks Include chromatid and Isochromatid breaks and fragments (Br); chromatid exchange figures (Ex) include quadrlradlats, trlredials and complex rearrangements. Chromosome breaks Include breaks and acentric fragments (Br); dicentric chromosome (Die). Severely damaged cells Includes calls 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.
BioR eliance Study N o . A A 37A Z.331.BTL
"15
Com pany Sanitized. D oes not contain TSCA CBI
tables
CONCURRENT TOXICITY TEST USING H-24616 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION
20 HOUR CONTINUOUS TREATMENT
Treatment (pg/mL)
Cell Count Averages Flask (x10)
Cell Viability
<%)
Mean Cells per Flask
(X101)
C e l Growth Cell Growth
Index
IitntknlilotUffjUonn
<%> (%>
w ater
A 2.08 B 2.19
H -2 4 6 1 6 0 .5
A 2.19 B Z25
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
M MC, 0.1
A
1.82
B 1.80
M MC, 0.2
A
1.56
B 1.48
100% 99%
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
Treatm ent: CHO calls were treated In the absence of an exogenous source of metabolic activation fb r4 h o u r8 a t3 7 1 0C. Cell Viability determined by trypan blue dye exclusion.
Mean Celia per Flask cell count x % viable cells, reported as a mean for tasks A and B. Cell Growth Index *(cells per flask in treated group/cells per flask In control group), expressed as a percentage. Call Growth Inhibition -1 0 0 % - call growth Index; not calculated for negative controls.
BioR eliance Study N o. A A37A Z.331.BTL
W
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TABLE 6
CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH B-248T6 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION
20 HOUR CONTINUOUS TREATMENT
Treatment (pgftnL)
Mitotic
% Aberrant Cells
Index Cells
Flask <%) Scored Numerical Structural
W ater
A 7.2 100 B 8.4 100
3 4
0 1
H-24618 500
A 5.8 100 B 8.8 100
4 4
0 2
1500
A 6.0 100 B 6.6 100
2 2
1 1
5000
A 6.2 100 B 5.8 100
4 5
1 2
MMC, 0.1
A 6.6 100 B 7.4 100
4 4
13 14
Total Number of Structural Aberrations
Gaps Chromatid Br Ex
Chromosome Br Die Ring
Severely Damaged
Cells
10 10
0 0
00 0 01 0
0 0
10
0
11 0
00 00
0 0
110 11 0
18
3
3 14 3
00 0 01 0
02 0 01 0
00 0 01 0
50 0 11 0
0 0
0 0
0 0
0 0
Average Aberrations
Per Cell
0.000 0.010
0.000 0.020
0.020 0.010
0.010 0.020
0.160 0.190
Treatment: CHO calls wars treated tor 20 hours at 371C in the absence of an exogenous source of metabolc activation. Additional dose levels of 0 .5 ,1 .5 , 5 ,1 5 , 50 and 150 ug/mL were tested as a safeguard against excessive toxicity at higher dose levels but were not required tor microscopic examination. M itotic Index " number of mitotic figures x 100/500 cells counted.
% Aberrant Cells: numerical includes polyploid and endoredupHcated cells; structural excludes cells with only gaps. Chromatid breaks Include chromatid and Isochromatid breaks and fragments (Br); chromatid (change figures (Ex) Include quadrlradlals, triredials and complex rearrangements.
Chromosome breaks Include breaks and acentric fragments (Br); dicentric chromosome (Die). Severely damaged cells Indude cells with one or mom pulverized chromosomes and cells with 10 or more aberrations. Average Aberrations P er Cell: severely damaged cells and pulverizations were counted as 10 aberrations.
BioReiiance Study N o . A A 37A Z.331.BTL
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Company Sanitized. Does not contain TSCA CBf
TABLE 7 SUMMARY
T reatm ent (Mg/mL)
W ater
H-24616 500 1500 5000
MMC 0.2
so Activation
-
w
1
I
Time
4
4 4 4 4
Mean
Aberrations C eils With Aberrations
Mitotic C ells
Per Ceil Numerical Structural
Index Scored (Mean +/- SD) (%)
(%>
8.3 200 0.005 0.071 3.5
0.5
5.8 200 0.010 0.100 7.7 200 0.010 0.100 6.2 200 0.015 0.122
5.4 200 0.145 0.442
7.5 3.5 6.0
4.0
1.0 1.0 1.0
11.5"
W ater
+
4 9.0 200 0.005 0.071 8.0
0.6
H-24616
1500
+
4 7.3 200 0.015 0.122 6.0
1.5
2000
+ 4 6.9 200 0.045 0.271 10.5 4.0*
2500
+
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
W ater
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 6.3 200 0.015 0.158 6.0 200 0.015 0.122
7.0 200 0.175 0.475
4.0 2.0 4.5
4.0
0.5
1.0 1.0 1.5
13.5**
Treatm ent: cells from an treatment conditions were hanrested at 20 hours after the Initiation of the treatments. Aberrations Per Cell: A severely damaged cell was counted as 10 aberrations.
*, pO.05; " , psO.01; Fisher's exact test
BioR eliance Study N o. A A37A Z.331.BTL
C om pany S anitized. D oes not contain TSCA CB1 21
H-24616: In Vitro Chromosome Aberration Test in Chinese Ham ster Ovary (CHO) C ells________
D u P ont-5235
APPENDIX A H istorical C ontrol D ata
BioR eliance Study N o. A A 37A Z .33i.B T L
C om pany Sanitized. D oes not contain TSCA CBI 22
H -24616: In Vitro Chromosome Aberration Test in Chinese Ham ster Ovary (CHO) C ells________
D uPont-5235
IN VITRO MAMMALIAN CYTOGENETIC TEST USING CHINESE HAMSTER OVARY (CHO) CELLS
HISTORICAL CONTROL VALUES STRUCTURAL ABERRATIONS 1997-1999
H istorical V alues
M ean SD` Range
U n treated / M edium (% ) 1.5
1.4
0 .0 -6 .0
Water (%)
1.3 1.4 0 .0 -5 .5
NON-ACHVATED TEST SYSTEM
Saline (% )
DMSO (%)
A cetone (% )
Ethanol (%)
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
A ll Solvents
(% ) 1.5
1.4
0 .0 -6 .0
P o sitiv e control2
(%) 2 4 .0
17.7
6 .5 -1 0 0 .0
H istorical V alues
M ean SD` Range
U n treated / M edium (% ) 1.8
1.3
0 .0 -6 .0
W ater (%)
1.8 1.6 0 .0 -6 .5
S9-ACTIVATED TEST SYSTEM
Saline (% )
1.9 1.4 0 .0 -4 .5
DMSO (% )
1.8 1.5 0 .0 -5 .5
A cetone (%>
1.9 2 .2 0 0 -5 .0
Ethanol (% )
1.5 1.4 0 .0 -4 .0
Other* (% )
2.1 1.3 0 .0 -4 .5
A ll Solvents
(% ) 1.8
1.5
0 .0 -6 .5
P o sitiv e Control3
(%) 32.5
16.1
6.5-94.5
SD - standard deviation. P ositive control for non-activated studies, N -raethyl-N'-nitro-N-nitrosoguanidine (M NNG , 0 .75-2 pg/m l), and M itom ycin C (M M C, 0.08-0.15 pg/m l). Positive control for S9-activated studies, cyclophospham ide (C P, 10-50 pg/m l), and benzo(a)pyrene, (B [a]P, 3 0 pg/m l). Other solven ts include non-standard solvents and Sponsor supplied vehicles.
BioReliance Study N o.: AA37AZ.331.BTL
23
Company Sanitized. Does not contain TSCA CBI
H-24616: In Vitro Chromosome Aberration Test in Chinese Ham ster Ovary (CHO) Cells______
D u P on t-5235
IN VITRO MAMMALIAN CYTOGENETIC TEST USING CHINESE HAMSTER OVARY (CHO) CELLS
HISTORICAL CONTROL VALUES COM BINED NUMERICAL ABERRATIONS (POLYPLOID A N D ENDOREDUPLICATED CELLS)
1997-1999
H istorical V alu es
M ean SD` Range
U n treated / M edium (% ) 2 .5
1.6
0 .0 -9 .5
W ater (% )
2.3
12
0 .0 -7 .5
NON-ACTIVATED TEST SYSTEM
Saline (% )
2 .4 1.3 0 .0 -6 .0
DMSO (%)
2 .4 1.4 0 .0 -8 .0
A ceton e (%)
3 .0 1.7 1 .5 -6 .0
Ethanol (%)
2 .0 1.4 0 .0 -6 .0
Other4 (% )
2 .3 1.4 0 .0 -5 .5
AU S o lv e n ts
(% ) 2 .3
1.3
0 .0 -8 .0
P o sitiv e Control1
(% ) 3.2
1.8
0 .0 -9 .5
H istorical V alues
M ean S !? Range
U n treated / M edium (% ) 2 .9
1.8
0 .0 -9 .5
W ater (%)
4 .0 2 .4 0 .0 -1 1 .5
S9-ACTIVATED TEST SYSTEM
Saline (% )
3 .6 1.4 0 .5 -5 .5
DMSO (% )
2 .6 1.3 0 .0 -6 .0
A ratone (% )
3 .2 1.2 2 .5 -4 .5
Ethanol (% )
3.3 1.5 0 .5 -5 .5
Other4 (% )
2 .9 1.2 1.5-6.5
A ll S o lv e n ts
(% ) 3 .2
1.8
0 .0 -1 1 .5
P o sitiv e Control3
(%) 3.7
2 .0
0 .0 -1 0 .5
SD = standard deviation. Positive control for non-activated studies, N -m ethyl-N'-nitro-N-nitrosoguanidine (M NNG, 0 .7 5 -2 pg/m l), and M itom ycin C (M M C, 0.08-0.15 pg/m l). Positive control for S9-activated studies, cyclophospham ide (C P, 10-50 pg/m l), and benzo(a)pyrene, (B [o]P , 30 pg/m l).
Other solven ts include non-standard solvents and Sponsor supplied veh icles.
BioReliance Study N o.: AA37AZ.331.BTL
24
Com pany Sanitized. D oes not contain TSCA CBI
H -24616: In Vitro Chromosome Aberration Test in Chinese Ham ster Ovary (CHQ) C ells________
D u P ont-5235
APPENDIX B Study Protocol
BioReliance Study N o.: AA37AZ.331.BTL
25
Company Sanitized. Does not contain TSCA CBI
H-24616: In Vitro Chromosome Aberration Test in Chinese Ham ster Ovary (CHO) C ells________
D u P ont-5235
Sponsor Project Num ber D uPont-52 15 BioReliance Study Num ber AA37AZ.331 rtt
1.0 PURPOSE
Vitro M am m alian Chromosome A berration T est
The purpose o f this study is to evaluate the clastogenk potential o f a test substance based qpon its ability to induce chromosome aberrations in Chinese hamster ovary (CHO)
2.0 SPONSOR
2.1 INSXtte:
R l. du Pont & Nemouraand Company
2 2 Address:
Stine Haskell Research Center DuPont Haskell Laboratory P .O .B ox 50 1090 Elkton Road Newark, DE 19714-0050
23 Representative:
Maria Donner, Ph.D. Phone: 302-366-5251 Fax: 302-235-7156
maria.donner@usa.dupontcam
2.4 Sponsor Project #: DuPont-5235
2.5 W R#:
2.6 H askell#: 2.7 Service Code:
3.0
24616
IPO
3.1 Test Substance: Q H H H P
i"l l Test Substance I.D.: H-24616 (to be used in die report text)
3.3 Controls:
Solvent: Positive:
Test Substance Solvent (or V ehicle) Mitomycin C (MMC) Cyclophosphamide (CP)
3.4 Determination o f Strength, Purity, etc.
U n ^ a fte m a le arrangements are made, the testing facility at BioReliance w ill not perform analysis o f the dosing solutions. The Sponsor w ill be directly responsible ror determination and documentation o f die analytical purity am) com position o f die
Protocol No. SPGT33I 03-Nov-2000 BioReliance Study N o.: A A 37A Z.331.BTL
of 9 26"
SnBTioWRzeellibaonescneQ"
H-24616: In Vitro Chromosome Aberration T est in Chinese Ham ster Ovary (CHO) C ells
D u P on t-5235
, Sponsor Project Number: DuPont - 523S
BioReliance Study Num ber A A 37A 2.33i.B T L test substance, and the stability and strength o f the test substance in the solvent (or vehicle).
3.4 Test Substance Retention Sample
The retention o f a reserve sample o f the test substance w ill be tire responsibility o f 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 atucty inrector
Ramadevi Gudi, Ph.D.
Phone: (301)610-2169 Fax: (301)738-2362 E-mail: rgudl@bioreUance.com
1
5.0 TEST SCHEDULE
5.1 Proposed Experimental Initiation Date:
28 Nov2000
5.2 Proposed Experimental Completion Date: 19 Jan 2001
5.3 Proposed Report Date:
31 Jan 2001
6.0 TEST SYSTEM
The CHO-K, ceil line is a proline auxotroph with a modal chromosome number o f 20 and a population doubling tim e o f 10-14 hours. CHO-K, cells were obtained from Ore American Type Culture Collection (repository number CCL 61), Manassas, VA. The stability o f the modal chromosome number o f Orecell litre is routinely checked and the ceU line is routinely tested and determined to be free from mycoplasma contamination. This system has been demonstrated to be sensitive to die clastogenic activity o f a variety o f chem icals (Preston et al., 1981).
7.0 EXPERIMENTAL DESIGN AND METHODOLOGY
The chromosome aberration test w ill be conducted using standard procedures (Evans,
1976), by exposing cultures o f CHO cells to a minimum o f four concentrations o f the test
substance as w ell as to positive and solvent controls. In the non-activated test system,
treatment w ill be for 4 hours and for 20 hours; in the S9 activated test system , exposure w ill
be for 4 hours (Swierenga et al., 1991). To ensure evaluation o f first division metaphase
cells the dividing cells w ill be arrested in metaphase and harvested f~or microscopic
Protocol No. SPGT331 03-Nov-2M0
2 of9
m Bio Reliance"
BioReliance Study N o.: A A 37A Z331.BTL
27
Company Sanitized. Does not contain TSCA CBI
H-24616: In Vitro Chromosome Aberration Test in Chinese Ham ster Ovary (CHO) C eils
D u P on t-5235
Sponsor Proje Num ber DuPont-5 2 3 5
BioReliance Study Num ber AA37AZ.331.BTL evaluation o f chromosome aberrations at approximately 20 hours (1.5 normal cell cycles) after the initiation o f treatment (Galloway et al,, 1994). The clastogenic potential o f tee test substance w ill be measured by its ability to increase structural chromosome aberrations in a dose-responsive manner when compared to tee solvent control group. In tee event o f a positive response in tee 4 how non-activated study, the prolonged exposure non-acdvated study may not be scored. The test substance w ill also be assessed for its ability to induce numerical chromosome aberrations.
7.1 Solubility Determination
U nless tee Sponsor has indicated the test substance advent, a solubility determination w ill be conducted to determine the solvent and tee maximum soluble concentration up to a maximum o f 500 m g/ml. Solvents compatible with this test system , in order o f preference, include but are not lim ited to sterile water (CAS 7732-18-5), dimethylsulfoxide (CAS 67-68-5), ethanol (CAS 64-17-5), and acetone (CAS 67-64-1). The solvent w ill be the test substance advent, selected in order o f preference, that permits preparation o f tee highest soluble stock concentration, up to 500 mg/ml.
72 Preliminary Toxicity Test for Selection o f D ose Levels
Selection o f the dose levels for the cytogenetics test w ill be based upon post treatment toxicity (cell growth inhibition relative to the solvent control) and solubility o fthe test substance. CHO cells w ill be exposed to solvent alone and to at least nine concentrations o f test substance. The highest concentration tested w ill be 5 mg/ml or 10 mM whichever is lower for freely soluble test substances, or the maximum concentration resulting in a workable suspension for poorly soluble test substances not to exceed 5 mg/ml. The pH w ill be measured at the highest test substance treatment condition and w ill be adjusted, if necessary, in order to maintain a neutral pH in the treatment medium. The osm olality o f the highest dose level, low est precipitating dose level (where applicable) and the highest soluble dose level (where applicable) in treatment medium w ill also be measured. C ells seeded 16-24 hours earlier w ill be exposed for 4 hours in the absence and presence o f S9 and for 20 hours in tee absence o f S9. Just prior to trypsinization the cell cultures w ill be visually inspected for the extent o f monolayer confluency relative to the solvent control. Twenty hours after treatment initiation the cells w ill be harvested by bypsmizatfon and counted using an automatic cell counter and the cell viability w ill be assessed using trypan blue dye exclusion. The cell counts and percent viability w ill be used to determine cell growth inhibition relative to tee solvent control.
Whenever possible, tee high dose to evaluate chromosome aberrations w ill be selected to give at least 50s/ toxicity (cell grow * inhibition relative to the solvent control) irrespective o f solubility but not to exceed 5 mg/ml or 10 mM. A t least two additional dose levels, demonstrating minimal or no toxicity w ill be included. In the event the test substance cannot be dissolved at a high enough concentration in an appropriate solvent to be toxic, then the highest dose to be tested in tee chromosome
Protocol No. SPGT331 03-Nov-2Q00
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BioReliance Study N o.: AA37AZ.331.BTL
28
B Rio eliancf to u n tr ir M ie iA b io i i'C ii
Com pany ^n mlltiizzeecHf.lIDBooeessnnoot'ccoonnliain T SC A CBI
H -24616: In Vitro Chromosome Aberration Test in Chinese Hamster Ovary (CHO) C ells
D u P on t-5235
Sponsor Project Number: DuPont-5 2 3 5
BioReliance Study Number: AA37A2.331.BTL aberration test w ill be the concentration resulting in minimum precipitation in test medium. Precipitation w ili be determined by direct visual inspection. In the event the test substance demonstrates a dose-responsive increase in toxicity at concentrations that exceed solubility in treatment medium, then the highest dose to be tested w ill be the maximum concentration that results in at least 50% toxicity. In the event that neither cytotoxicity nor insolubility is observed in the preliminary test, the highest dose in die chromosome aberration test w ill be 5 mg/ml orlO m M whichever is lower. If excessive precipitation o f the test substance-solvent solution occurs upon addition to treatment medium, or if the osm olality o f the treatment medium is considered excessive, the Sponsor w ill be consulted.
7.3 Frequency and Route o f Administration
Target ceils w ill be treated for 4 hours in the absence and presence o f S9 and for 20 hours in die absence o f S9, by incorporation o f die test substance-solvent mixture into die treatment medium. This technique has been demonstrated to be an effective method o f detection o f chemical clastogens in this test system (Evans, 1976).
I f die Sponsor is aware o f specific metabolic requirements, dim this information w ill be utilized in the preparation o f the study design. Verification o f a clear positive response is not required. Negative results w ill not be confirmed when justification can be provided. Equivocal results may be confirm !, upon consultation w ith the Sponsor, and may employ a modification o f the study design. T his guidance is based on the OECD Guideline 473 (1997) and ICH Guidance on Specific Aspects o f Regulatory Genotoxicity Tests fi Pharmaceuticals (1996).
7.4 Activation System
Aroclor 1254-induced ret liver S9 w ill be used as the metabolic activation system. The S9 w ill be prepared from male Sprague-Dswley rats induced with a single intraperitoneal injection o f Aroclor 1 2 5 4 ,5 00 mg/kg, five d a p prior to sacrifice. The S9 w ill be batch prepared and stored frozen at approximately -7 0 C until used. Each batch preparation o f $9 w ill be tested for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dimethylbeia(a)antferacene to forms mutagenic to
Salmonella typhlmurlum TA 100.
Immediately priorto use, the S9 w ill be thawed and mixed with cofactnrs to contain 2 mM magnesium chloride (MgC!2,) 6 mM potassium chloride (KC1), ImM glucose-6-phosphate. 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 p i S9 per m l serum five medium.
Protocol No. SPGT331 (O-Nov-2000 BioReliance Study N o.: AA37AZ.331.BTL
29
iWeontain TSCA CBI
H -24616: In Vitro Chromosome Aberration Test in Chinese Hamster Ovary (CHO) C eils________
D u P on t-5235
Controls
Sponsor Project Number: D u P o n t-5235 BioReliance Study Number: A A37AZ.33i.BTL
7.5.1 Solvent (or V ehicle) Control
The solvent for dm test substance w ill be used as the solvent control. For solvents other than water, physiological buffer, or medium, the final concentration in treatment medium w ill not exceed 1%.
7.5.2 Positive Controls
M itomycin C w ill be used at a concentration within 0.05-0,3 pg/m l as tire positive control in the non-activated study. Cyclophosphamide w ill be used at a concentration within 10-50 pg/m l as the positive control in the S9activated study.
7.6 Preparation o f TargetC ells
Exponentially growing CHO-K, cells w ill be seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 2 mM L-ghitamine, 100 units penicillin/m l and 100 pg streptomydn/ml) for each treatment condition at
approximately 5 x 10* cells/25 cm* flask. The flasks w ill be incubated at 37 |oC in a humidified atmosphere o f 5 1%C 0 2 in air for 16-24 hours.
7.7 identification o f Test System
Using a permanent marking pen, foe treatment flasks w ill be identified by ti BioReliance study number and a code system to designate tin treatment condition and test phase.
7.8 Treatment o f Target Cells
Treatment w ill be carried out in duplicate by refeeding the flasks with 5 ml complete medium for the non-activated exposure or 5 m l S9 reaction mixture for the S9-activsted exposure, to which w ill be added 50 p i o f dosing solution o f test or control sufacttticc in solvent or solvent alone. Larger volum es o f dosing solution may be used i f water, physiological buffer, or medium is used as ti solvent
In ti non-activated study, ti cells w ill be treated fiar 4 hours and fix 20 hours; in the S9-8ctivated study the cells w ill be treated for 4 hours. Treatment w ill be carried
out at 3 7 !C in a humidified atmosphere o f 5 1% COj in air. After the .4 hour treatment period m toe non-activated and toe av-activated studies* tne treatment medium w ill he aspirated, ti cells washed with phosphite buffered saline, refed with complete medium and returned to the incubator.
concurrent toxicity test to determine cell growtn inniDition relative to tne solvent control w ill be conducted in both the non-activated and t i S9-activated studies.
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7.9 Collection o f Metaphase Cells
C ells w ill be collected approximately 20 hours after initiation o f treatment This post-treatment harvest time represents approximately 1.5 normal cell cycles and was selected to ensure that the cells are analyzed in the first division metaphase after initiation o f treatment Two hours prior to cell harvest Colcemid w ill be added to the cultures at a final concentration o f 0 .1 pg/m l.
C ells w ill be harvested by trypsinization, collected by centrifigation and an aliquot w ill be removed for counting using an automatic cell counter and trypan blue dye exclusion. The remainder o f die cells w ill be sw ollen w ith 0.075M KC1, washed with tw o consecutive changes o f fixative (methanohglacial acetic acid, 3:1 v/v), capped and stored overnight or longer at approximately 2-8C . The cell counts and percent viability w ill be used to determine ceil growth inhibition relative to die solvent control (% toxicity). To prepare slides, the cells w ill be collected by centrifugation and resuspended in fresh fixative. The suspension o f fixed cells w ill be applied to glass microscope slides and air-dried. The slides w ill be identified by the experiment number, treatment condition and date. The slides w ill be stained with Giemsa and permanently mounted.
7.10 Scoring for Metaphase Aberrations
To ensure that a sufficient number o f metaphase cells are present on the slides, the percentage o f cells in m itosis per 500 cells scored (m itotic index) w ill be determined and recorded for each coded treatment group selected for scoring chromosome aberrations. Slides w ill be coded using random numbers by an individual not involved with the scoring process. In the event o f a positive response in the 4 hour non-activated study, the prolonged exposure non-activated study may not be scored. Metaphase cells with 20 2 centromeres w ill be examined under o il immersion without prior knowledge o f treatment groups. Whenever possible, a minimum o f 200 metaphase spreads from each dose level (100 per duplicate flask) w ill be examined and scored for chromatid-type and chromosome-type aberrations (Scott ct aL, 1990). The number o f metaphase spreads that w ill be examined and scored per duplicate flask may be reduced if the percentage o f abenant 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 reanangemeuts. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence o f any exchange figure w ill be scored as a break (chromatid or chromosome). Fragments observed with an exchange figure w ill not be scored as an aberration but w ill be considered part o f the incomplete exchange. Pulverized
chromosome(sX pulverized cells and severely damaged cells fe 10 aberrations) w ill also be recorded. Chromatid mid isochromatid gaps w ill be recorded but not included in die analysis. The XY coordinates for each celt with a structural
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BioReiiance Study Num ber AA37AZ.331 RTT. aberration w ill be recorded using a calibrated microscope stage. The percent polyploid and endoreduplicated cells w ill be evaluated per 100 cells for each dose level analyzed for structural aberrations.
8.0 CRITERIA FOR DETERMINATION OF A VALID TEST
8.1 Solvent Control
The frequency o f cells with structural chromosome aberrations in the solvent control must be within the range o f die historical solvent control.
8.2 Positive Control
The percentage o f cells with aberrations must be statistically increased (p^O.05, Fisher's exact test) relative to the solvent control.
9.0 EVALUATIONOF TEST RESULTS
The toxic effects o f treatment arc based upon cell growth inhibition relative to die solvent . coolrol and w ill be presented for the toxicity and aberration studies. The number and types
o f aberrations found, the percentage o f structurally and numerically damaged cells (percent aberrant cells) in the total population o f ceils examined, and the mean aberrations per cell w ill be calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in die data but are not included in the total percentage o f cells with one or more aberrations or in the frequency o f structural aberrations per cell. Statistical analysis o f the percentage o f aberrant cells w ill be performed using the Fisher's exact te st The Fisher's test w ill be used to compare pairwise the percent aberrant cells o f each treatment group with that o f the solvent control. In die event o f a positive Fisher's exact trot at any test substance dose level, the Cochran-Armitage test w ill be used to measure dose-responsiveness. A ll conclusions w ill be based on sound scientific basis; however, as a guide to interpretation o f the data, tire test substance w ill be considered to induce a positive response when the percentage o f cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (psO.05). However, values that are statistically significant but do not exceed die range o f historic negative or solvent controls m ay be judged as not biologically significant. Test substances not demonstrating a statistically significant increase in aberrations w ill be concluded to be negative.
10.0 REPORT
. A report o f the results o f this study w ill be prepared by BioReiiance and w ill accurately describe all methods used for generation and analysis o f tire data,
R esults presented w ill include, but not be limited to:
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H -24616: In Vitro Chrom osom e Aberration T est
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in Chinese Hamster O vary (CHO) C ells___________________________
D u P on t-5235
Sponsor Project Num ber DuPont - 5235
BioReliance Study Number: A A 37A ZJ31.BTL T est substance: identification and CAS no., if known; physical nature and purity, if known; physicochemical properties relevant to the conduct o f die study, i f known; stability o f test substance, if known.
Solvent/Vehicle: justification for choice o f vehicle; solubility and stability o f test substance in solvent/vehicle, i f known.
Source o f ceils, karyotype features (modal chromosome number) and suitability o f the cell type used, absence o f mycoplasma, cell cycle length, passage number.
T est conditions: composition o f medium; COj concentration; incubation time; cell seeding density; solvent and solvent selection rationale; concentration o f test substance and concentration selection rationale; composition and acceptability criteria for the metabolic activation (S 9) system; duration o f treatment; duration o f treatment with and concentration o f ColcemM*; type o f metabolic activation system used; positive and solvent controls; methods o f slide preparation; number o f ceil cultures; criteria for scoring aberrations and
orirarforoooaMeriffff pfttitiwr, mythm
R**ula: description o f precipitation; pH and osm olality o f the treatment medium; cell growth inhibition relative to the solvent control; m itotic index and number ofm ettphases analyzed; type and number o f aberration (structural and numerical) given separately for each treated and control culture; concentration-response relational#; statistical analysis; lu ston cd control dflis,
11.0 RECORDS AND ARCHIVES
U pon com pletion o f the final report, all raw data and reports w ill be archived by B ioR eliance, R ockville, M D fo ra period o f no less than 1 y e from the study initiation
12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE
This protocol has been written to comply with OECD Guideline 473 (In Vitro Mammalian
Chromosome Aberration Test), July 1997 and w ife fee International Conference on Harmonisation o f Technical Requirements fo r Registration o f Pharmaceuticals for Human U se (1996 and 1997).
U iis study w ill be performed using fee EPA (TSCA) Good Laboratory Practice R egulations for Nonclim cal Laboratory Studies as a guideline; however, fee study w ill not m eet GLP requirements.
U nless arrangements are made to the contrary, unused dosing solutions w ill be disposed o f follow ing administration to the test system and all residual test substance w ill be disposed o f follow ing finalization o f tire report
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? r n r p arty gan fflT arf D n a s rm t c o n ta in T S C A CB1
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13.0 REFERENCES
Sponsor Project Number: D uPont-5 2 3 5 BioR eiiance Study Num ber AA37AZ.33t.BTL
Evans, H J. (1976) C ytologies! methods for detecting chemical mutagens, in: A.
H ollander (Ed.), Chemical Mutagens, Principles and Methods for their Detection, voi. 4.
Plenum Press, N ew York, NY.
-
Galloway, SM,, M J. Aatdema, M . Ishidate Jr., J i . Ivett, D J. Kirkland, T. Monta, P.
M osem o and T. Solim i (1994) Report from working group mi in vitro tests for chromosomal aberrations, Mutation Research 312(3)241-261.
International Conference on Harmonisation (ICH) o f Technical Requirements for
Registretion o f Pharmaceuticals for Human U se. Guidance on Specific Aspects o f
Regulatory Genotoxicity T ests for Pharmaceuticals. S2A document
for
adoption at step 4 o f the ICH process on M y 19,1995. Federal Register 61:18198-18202,
April 2 4,1996.
International Conference on Harmonisation (ICH) o f Technical Requirements for
Registration o f Pharmaceuticals fat Human U se. Genotoxicity: A Standard Battery for
G eaotoxidty Testing o f Pharmaceuticals. S2B document recommended for adoption at
step 4 o f the ICH process on M y 16,1997. Federal Register 62:16026-16030, November 21 ,1 9 9 7 .
OECD Guideline for the Testing o f Chemicals, Guideline 473 {In Vitro Mammalian
Chromosome Aberration Test), M y 1997.
Preston, R J , W. Au, M A Bender, J.G. Bremen, A .V . C enano, J A Heddte, A E . McFee, S. W olffand J.S. Wassora (1981) Mammalian in vivo and in vitro cytogenetic assays: a report o f the Gene-Tox Program, Mutation Research, 87:143-188.
Scott, D ,, N .D . Danfotd, B J . Dean and D J. Kirkland. 1990. Metaphase Chromosome Aberration Assays ht Vitro. Ik Basic Mutagenicity Tests: UKEMS Recommended Procedures. D J Kirkland (cd). Cambridge University Press, N ew York, NY .
Swierenga S l l , J A Heddle, E.A. Sigai, J.P.W. Gilman, R i . Brillinger, O i l Douglas and EJL Nestraann (1991) Recommended protocols based on a survey o f current practice in getw taxfciiy testing laboratories, IV. Chromosome aberration end sister-chromatid exchange in Chinese hamster ovary, V 79 Chinese lung and human lym phocyte cultures, M utttion Research 246:301-322.
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in Chinese Ham ster Ovary (CHO) C ells_____________________________________________________ DuPont-5235
Sponsor Project Number; DuPont - 5235
14.0 APPROVAL
BioReliance Study Num ber A A37A2.331.BTL
SPONSOR REPRESENTATIVE
l i O ^ c \ ON 0 o o ^ < _ r
(Print or Type Name)
&c-a.'
BIORELIANCE STUDY DIRECTOR
0 1 N O v / Z-OOO
DATE
A&cJ X**>o
DATE
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* B R -io eliance ten a c ity Mtcrcfcicle#cal A sseciatti
Company Sanliltliized. Does not contain TSCA CHI
