Document MoGE6nEoexgJEVzr7g3kezZ4k
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MUTAGENICITY TEST ON
T-6342
MEASURING CHROMOSOME ABERRATIONS IN HUMAN WHOLE BLOOD LYMPHOCYTES
WITH A CONFIRMATORY ASSAY WITH MULTIPLE HARVESTS
FINAL REPORT
AUTHOR
Hemalatha Murli, Ph.D.
PERFORMING LABORATORY
Coming Hazleton Inc. (CHV) 9200 Leesburg Pike
Vienna, Virginia 22182
LABORATORY PROJECT IDENTIFICATION
CHV Study No.: 17073-0-449CO
SUBMITTED TO
3M Corporation Building 220-2E-02
3M Center St. Paul, Minnesota 55144-1000
STUDY COMPLETION DATE November 1, 1996
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C O R N IN G Hazleton QUALITY ASSURANCE STATEMENT
Project Title: Chromosome Aberrations in Human Whole Blood Lymphocytes With a Confirmatory Assay With Multiple Harvests
Project No.: 20990
Assay No.: 17073
Protocol No.: 449CO
Edition No.: 2, Modified for 3M Corporation
Quality Assurance inspections of the study and review of the final report o f the above referenced project were conducted according to the Standard Operating Procedures o f the Quality Assurance Unit and according to the general requirements o f the appropriate Good Laboratory Practice regulations. Findings from the inspections and final report review were reported to management and to the study director on the following dates:
Inspection/Date
Findings Reported
Auditor
Addition of Colcemid/08/15/1996
08/15/1996
C. Orantes
Draft Report Review/09/18-20/1996
09/20/1996
C. Orantes
Final Report Review/11/01/1996
11/01/1996
C. Smith
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STUDY COMPLIANCE AND CERTIFICATION
The described study was conducted in compliance with the Organization for Economic Cooperation and Development Principles o f Good Laboratory Practice C(81)30 (Final) Annex 2, issued 1979-1980 (effective 1981) with any applicable amendments. There were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrity o f the study or the interpretation o f the test results. The raw data have been reviewed by the Study Director, who certifies that the evaluation o f the test article as presented herein represents an appropriate conclusion within the context of the study design and evaluation criteria.
All test and control results in this report are supported by an experimental data record and this record has been reviewed by the Study Director. All raw data, documentation, records, protocol and a copy o f the final report generated as a result of this study will be archived in the storage facilities o f Coming Hazleton Inc. for at least one year following submission o f the final report to the Sponsor. After the one year period, the Sponsor may elect to have the aforementioned materials retained in the storage facilities o f Coming Hazleton Inc. for an additional period o f time, or sent to a storage facility designated by the Sponsor.
Submitted By:
Study Director:
Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Genetic and Cellular Toxicology
Date
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TABLE OF CONTENTS
Page No.
A B STR A C T............................................................................................................................................. 6
1.0 S P O N S O R ............................................................................................................................... 7
2.0 MATERIAL (TEST ARTICLE) ...........................................................................................7 2.1 Client's Identification/ 2.2 Date Received 2.3 Physical Description 2.4 Genetics Assay No.
3.0 TYPE OF A S S A Y .................................................................................................................. 7
4.0 PROTOCOL NO.......................................................................................................................7
5.0 STUDY DATES .................................................................................................................... 7 5.1 Initiation Date 5.2 Experimental Start Date 5.3 Experimental Termination Date
6.0 SUPERVISORY PERSONNEL ...........................................................................................7 6.1 Study Director 6.2 Laboratory Supervisor
7.0 OBJECTIVE ........................................................................................................................... 7
8.0 R A TIO N A LE........................................................................................................................... 8
9.0 EXPERIMENTAL D E S IG N ................................................................................................. 8
10.0 MATERIALS AND M ETHODS...........................................................................................9 10.1 Cells Used 10.2 Cell Culture Medium 10.3 Negative and Solvent Controls 10.4 Positive Control Agents 10.5 Dose Rangefinding Assay for Mitotic Suppression and Dose Determination 10.6 Aberrations Assay Without Metabolic Activation
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10.7 10.8 10.9 10.10
Aberrations Assay With Metabolic Activation Harvest Procedure Slide Preparation and Staining Aberrations Analysis and Assay Evaluation
11.0 RESULTS ............................................................................................................................13 11.1 Solubility and Dose Determination 11.2 Dose Rangefinding Assay Without Metabolic Activation 11.3 Dose Rangefinding Assay With Metabolic Activation 11.4 Chromosomal Aberrations Assay Without Metabolic Activation 11.5 Chromosomal Aberrations Assay With Metabolic Activation
12.0 CONCLUSION .....................................................................................................................16
13.0 REFERENCES .....................................................................................................................17
14.0 EXPERIMENTAL DATA TA B LES.................................................................................. 18
15.0 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED C E L L S ................................................................................................................................... 27
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ABSTRACT
The objective o f this in vitro assay was to evaluate the ability o f T-6342 to induce chromosomal aberrations in cultured whole blood human lymphocytes with and without metabolic activation.
The test article was dissolved in deionized water at a concentration of 500 mg/ml. The test article solutions and the vehicle control, deionized water, were dosed with a dosing volume o f 1% (10 pl/ml) for this assay.
For the dose rangefinding assay with and without metabolic activation, concentrations o f 0.167, 0.500, 1.67, 5.00, 16.7, 50.0, 167, 500, 1670, and 5000 pg/ml were tested. Reductions o f 35%, 6%, 38%, 15%, 18%, 57%, and 100% were observed in the mitotic indices o f the cultures dosed with 0.500, 1.67, 5.00, 16.7, 500, 1670, and 5000 pg/ml without metabolic activation, respectively, as compared with the solvent control culture. Reductions o f 8%, 5%, 4%, 1%, and 100% were observed in the mitotic indices o f the cultures dosed with 1.67, 50.0, 500, 1670, and 5000 pg/ml with metabolic activation, respectively, as compared with the solvent control culture.
Based on these data, the initial trial of the chromosomal aberrations assay was conducted testing concentrations o f 127, 253, 505, 1010, 1510,2010, and 2510 pg/ml without metabolic activation and o f 253, 505, 1010, 1510, 2010, 2510, 3010, and 4010 pg/ml with metabolic activation in 22.0 hour assays. Cultures dosed with 253, 505, 1010, and 1510 pg/ml without metabolic activation and with 505, 1010, 1510 and 2010 pg/ml with metabolic activation were evaluated for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed.
In the confirmatory assay, replicate cultures were incubated with 125, 250, 500, 900, 1200, 1600, and 2000 pg/ml in a 22.1 hour assay and with 62.5, 125, 250, 500, 900, 1200, 1600, and 2000 pg/ml in a 46.0 hour assay under nonactivation conditions. Replicate cultures were incubated with 250, 500, 1000, 1500, 2000, 2500, and 3000 pg/ml in 22.1 and 46.0 hour assays with metabolic activation. Cultures dosed with 125, 250, 500, and 900 pg/ml from the 22.1 and 46.0 hour nonactivation assays, with 250, 500, 1000, and 1500 pg/ml from the 22.1 hour activation assay, and with 500, 1000, 1500, and 2000 pg/ml from the 46.0 hour activation assay were evaluated for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed, except for a weak increase in endoreduplication at 2000 pg/ml, an extremely toxic dose level, from the 45.9 hour activation assay.
The test article, T-6342, was considered negative for inducing chromosomal aberrations in cultured whole blood human lymphocytes with and without metabolic activation. These results were verified in independently conducted confirmatory trials.
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Chromosomal Aberrations in Human Whole Blood Lymphocytes With a Confirmatory Assay With Multiple Harvests With T-6342
1.0 SPONSOR: 3M Corporation
2.0 MATERIAL (TEST ARTICLE):
2.1 Client's Identification: T-6342
2.2 Date Received: July 21, 1995
2.3 Physical Description: Clear, colorless liquid
2.4 Genetics Assay No.: 17073
3.0 TYPE OF ASSAY: Chromosomal Aberrations in Human Whole Blood Lymphocytes With a Confirmatory Assay With Multiple Harvests
4.0 PROTOCOL NO.: 449CO, Edition 2, Modified for 3M Corporation
5.0 STUDY DATES:
5.1 Initiation Date: July 9, 1996
5.2 Experimental Start Date: July 31,1996
5.3 Experimental Termination Date: August 26, 1996
6.0 SUPERVISORY PERSONNEL:
6.1 Study Director: Hemalatha Murli, Ph.D.
6.2 Laboratory Supervisor: Carol S. Spicer, B.S. 7.0 OBJECTIVE:
The objective o f this in vitro assay was to evaluate the ability o f the test article, T-6342, to induce chromosomal aberrations in cultured human whole blood lymphocytes, with and without metabolic activation.
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8.0 RATIONALE:
The assay is designed to establish whether the test article or its metabolites can interact with cells to induce chromosome breaks. Chemically induced lesions may result in breaks in chromatin that are either repaired by the cell in such a way as to be undetectable or result in visible damage. Aberrations are a consequence o f failure or mistakes in repair processes such that breaks do not rejoin or rejoin in abnormal configurations (Evans, 1962).
In order to detect chromosomal aberrations, cells must be in metaphase so that chromosomes are visible. The lymphocytes in blood do not usually divide and are stimulated to divide in culture by phytohemagglutinin (PHA). The cells are later arrested at the appropriate stage (metaphase) by Colcemid.
9.0 EXPERIMENTAL DESIGN:
Results from the dose rangefinding assay were used to determine the dose range to be used in the chromosomal aberrations assay. In the dose rangefinding assay, the cultures were harvested 20.1 hours after initiation o f treatment. Mitotic indices o f the cultures were evaluated for evidence of toxicity. A summary of the treatment schedule for the preliminary test is given below.
Summary of Dose Rangefinding Assay Treatment Schedule in Hours
Test - S9 + S9
Test Article 0 0
Wash 19.5
3
Colcemid 20.1 20.1
Fixation 22.1 22.1
In the chromosomal aberrations assays, replicate cultures were used at each dose level, and negative, solvent controls, and for each dose of the positive control. The aberrations assays were conducted with a 22.0 hour harvest time in the initial trial and with 22.1 and 46.0 hour harvest times in the confirmatory trials. Chromosomal aberrations were analyzed from the cultures treated at four dose levels and from one o f the positive control doses. A summary o f the treatment schedule for the chromosomal aberrations assays is given below.
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Summary o f Chromosomal Aberrations Assay Treatment Schedule in Hours
Test Culture Initiation
Initial Trial
- S9 -48
+ S9
-48
Confirmatory Trial
- S9
-48
+ S9 -48
- S9
-48
+ S9
-48
Chemical
0 0
0 0 0 0
Wash Colcemid Fixation
19.2 20.0 3.0 20.0
19.3 20.1 3.0 20.1 43.3 44.0 3.0 44.0
22.0 22.0
22.1 22.1 46.0 46.0
10.0 MATERIALS AND METHODS:
10.1 Cells Used:
Human venous blood from a single, normal, healthy male donor was drawn into sterile, heparinized Vacutainers. Cultures were initiated with 0.3 ml of blood per 5 ml culture for the dose rangefinding assay and 0.6 ml o f blood per 10.0 ml culture for the chromosomal aberrations assays in 15 ml centrifuge tubes.
10.2 Cell Culture Medium:
The cells were incubated at about 37C on a slope, with loose caps, in an atmosphere of about 5 % C 0 2 in air. The culture medium used was RPMI 1640 (JRH Biosciences) supplemented with 15% fetal bovine serum (FBS; Biochemed, Lot#E5331, dose rangefinding assay; Lot No.:T06024, chromosomal aberrations assay), 1% phytohemagglutinin (PHA-M; Gibco), penicillin (100 units/ml; Quality Biologicals) and streptomycin (100 pg/ml; Quality Biologicals), and 2mM L-glutamine (Quality Biologicals).
10.3 Negative and Solvent Controls:
In the nonactivation assays, negative controls were cultures which contain only cells and culture medium. Solvent controls were cultures containing deionized water at the highest concentration used in test cultures (1% or 10.0 pl/ml). In the
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activation assays, the negative and solvent controls were the same as described in the nonactivation assays but with the S9 activation mix included.
10.4 Positive Control Agents:
The positive control agents which were used in the assays were mitomycin C (MMC) for the nonactivation series and cyclophosphamide (CP) in the metabolic activation series. Mitomycin C (CAS# 50-07-7, Sigma, Lot # 40H2508) is a clastogen that does not require metabolic activation. Cyclophosphamide (CAS # 6055-19-2, Sigma, Lot # 43H0269) does not act directly but must be converted to active intermediates by microsomal enzymes. In the chromosomal aberrations assays, three concentrations o f MMC (0.1, 0.2, and 0.3 pg/ml) and CP (20, 30, and 50 pg/ml) were used to induce chromosomal aberrations. One of the dose levels was analyzed in each of the aberration assays. Both MMC and CP were dissolved in water.
10.5 Dose Rangefinding Assay:
In these tests, cultures were initiated with 0.3 ml o f blood per 5 ml culture and were incubated for two days prior to treatment.
10.5.1 Test Without Metabolic Activation:
The lymphocytes were incubated with the test article for 19.5 hours at =37C. Then, the test article was washed from the cells with phosphate buffered saline and fresh complete medium containing Colcemid (final concentration 0.1 pg/ml) was added. The cultures were then harvested 2.0 hours later. (See Sections on Harvest and Slide Preparation and Staining).
10.5.2 Test With Metabolic Activation:
In this test, the lymphocytes were incubated to the test article for three hours at =37C in the presence o f a rat liver S9 reaction mixture (S9 15 pl/ml, NADP 1.5 mg/ml, and isocitric acid 2.7 mg/ml). The S9 fraction (Molecular Toxicology, Inc., Lot #0667) was derived from the liver o f male Sprague-Dawley rats which had been previously treated with Aroclor 1254 to induce the mixed function oxidase enzymes which are capable o f metabolizing chemicals to more active forms. The three hour incubation time was used because prolonged exposure to the S9 mixture might be toxic to the cells and the enzyme activity o f S9 is lost rapidly at
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about 37C. The medium did not have FBS during the exposure period to avoid possible inactivation o f short lived and highly reactive intermediates produced by the S9 enzymes by binding to serum proteins.
After the exposure period the cells were washed twice with buffered saline. Complete RPMI culture medium was added to the cultures which were then incubated for 18.5 hours with Colcemid (final concentration 0.1 pg/ml) added for the last 2.0 hours to collect metaphase cells. The cultures were then harvested, fixed, and slides were prepared and stained as was described for the nonactivation dose rangefinding assay .
10.5.3 Assay Evaluation:
Mitotic index was analyzed from the surviving dose levels by analyzing the number o f metaphases present in 1000 consecutive cells.
10.6 Aberrations Assay Without Metabolic Activation:
Cultures were initiated two days prior to treatment with 0.6 ml o f whole blood per 10.0 ml culture in 15 ml centrifuge tubes. Two days after culture initiation, the cells were treated with the test article at predetermined concentrations for about 19.3 and 43.3 hours. The cultures were then washed with buffered saline and complete RPMI 1640 medium containing 0.1 gg/ml Colcemid was placed back onto the cells. Two hours later, the cells were harvested and air dried slides were made. The slides were then stained in 5 % Giemsa solution for the analysis of chromosomal aberrations.
10.7 Aberrations Assay With Metabolic Activation:
Cultures were initiated two days prior to treatment with 0.6 ml o f whole blood per 10.0 ml culture in 15 ml centrifuge tubes. Two days after culture initiation, the cultures were incubated at =37C for three hours in the presence o f the test article and the S9 reaction mixture. After the three hour exposure period, the cells were washed twice with buffered saline and the cells were refed with complete RPMI 1640 medium. The cells were incubated for the rest o f the culture period up to the time o f harvest with 0.1 pg/ml Colcemid present during the last 2.0 hours o f incubation. The metaphase cells were then harvested and prepared for cytogenetic analysis.
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10.8 Harvest Procedure:
The cell suspension was centrifuged, the supernatant was discarded and cells were treated with hypotonic KC1 (0.075 M) for approximately 10 minutes. This treatment helps to swell the cells and thus disperse the chromosomes. After centrifugation and removal o f the KC1, the cells were washed three times with freshly prepared fixative (absolute methanolrglacial acetic acid, 3:1, v:v). Airdried slides were prepared from the harvested cells.
10.9 Slide Preparation and Staining:
Slides were prepared by dropping the harvested cultures on clean slides. The slides were stained with 5% Giemsa solution for the analysis of mitotic index and chromosomal aberrations. All slides were then air-dried and cover slipped.
10.10 Aberrations Analysis and Assay Evaluation:
Cells were selected for good morphology and only cells with the number o f centromeres equal to the modal number 46 were analyzed.
One hundred cells, if available, from each replicate culture at four dose levels of the test article, the negative, solvent, and positive control cultures were analyzed for the different types o f chromosomal aberrations (Evans, 1962; See Section 15.0). At least 25 cells were analyzed for chromosomal aberrations from those cultures with >25% cells with chromosomal aberrations. For control o f bias, all slides were coded prior to analysis. Cells with aberrations were recorded on the data sheets by the microscope stage location. Mitotic index was assessed by analyzing the number o f mitotic cells in 1000 cells and the ratio was expressed as a percentage o f mitotic cells.
The following factors were taken into account in the evaluation o f the chromosomal aberrations data:
1. The percentage of cells with any aberrations. 2. The percentage o f cells with more than one aberration. 3. Any evidence for increasing amounts o f damage with increasing
dose, i.e., a positive dose response.
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Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation o f the ability o f the test article to induce chromosomal aberrations since they may not represent true chromosomal breaks and may possibly be induced by toxicity. Percent polyploidy and endoreduplication were analyzed and results were tabulated. Historical control data are presented in Table 8.
A cell classified as "GT" is considered to contain 10 aberrations for statistical purposes but a ">" is also included in the tables for this classification to indicate that it is a minimum number.
Statistical analysis employed a Cochran-Armitage test for linear trend and Fisher's Exact Test (Thakur et al., 1985) to compare the percentage o f cells with aberrations (and, if applicable, the percentage of cells with more than one aberration), polyploidy, and endoreduplication in treated cells with results from vehicle controls. Test article significance was established where p<0.01. All factors as stated previously were taken into account and the final evaluation o f the test article was based upon scientific judgement.
11.0 RESULTS:
11.1 Solubility and Dose Determination
Deionized water (Prepared at CHV, Lot # 20) was the solvent of choice for this assay. T-6342 was dissolved in deionized water at a concentration o f 500 mg/ml for the dose rangefinding assay . The test article solutions and the vehicle control, deionized water, were dosed with a dosing volume o f 1% (10 pl/ml) for this assay. Concentrations o f 0.167, 0.500, 1.67, 5.00, 16.7, 50.0, 167, 500, 1670, and 5000 pg/ml were tested in the dose rangefmding assay with and without metabolic activation. The stability of the test article under the preparation and dosing conditions used in this assay is the responsibility o f the sponsor.
11.2 Dose Rangefinding Assay Without Metabolic Activation
Hemolysis was observed prior to wash in the cultures dosed with 5000 pg/ml. Mitotic indices were analyzed from the cultures dosed with 0.167, 0.500, 1.67, 5.00, 16.7, 50.0, 167, 500, 1670, and 5000 pg/ml (Table 1). Reductions o f 35%, 6%, 38%, 15%, 18%, 57%, and 100% were observed in the mitotic indices o f the cultures dosed with 0.500, 1.67, 5.00, 16.7, 500, 1670, and 5000 pg/ml. Based on these results, the initial trial of the aberrations assay without metabolic activation
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was conducted with a 22.0 hour harvest testing concentrations o f 127, 253, 505, 1010, 1510, 2010, and 2510 pg/ml.
11.3 Dose Rangefinding Assay With Metabolic Activation
Hemolysis was observed prior to wash in the cultures dosed with 5000 fig/ml. Mitotic indices were analyzed from the cultures dosed with 0.167, 0.500, 1.67, 5.00, 16.7, 50.0, 167, 500, 1670, and 5000 (ig/ml (Table 1). Reductions of 8%, 5%, 4%, 1%, and 100% were observed in the mitotic indices o f the cultures dosed with 1.67, 50.0, 500, 1670, and 5000 pg/ml, as compared with the solvent control culture. Based on these results, the initial trial o f the aberrations assay with" metabolic activation was conducted with a 22.0 hour harvest testing concentra tions o f 253, 505, 1010, 1510, 2010, 2510, 3010, and 4010 pg/ml.
11.4 Chromosomal Aberrations Assay Without Metabolic Activation
INITIAL TRIAL
Hemolysis was observed prior to wash in the cultures dosed with 2510 pg/ml. Reductions of 38%, 18%, 18%, 50%, 55%, and 95% in the mitotic indices as compared with the solvent control cultures were observed in the cultures treated with 127, 253, 505, 1010, 1510, and 2010 pg/ml, respectively. Chromosomal aberrations were analyzed from the cultures treated with 253, 505, 1010, and 1510 (jg/ml (Table 2). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed.
Based on these results, the confirmatory trial under nonactivation conditions were conducted testing dose levels o f 125, 250, 500, 900, 1200, 1600, and 2000 pg/ml with a 22.1 harvest and 62.5, 125, 250, 500, 900, 1200, 1600, and 2000 pg/ml with a 46.0 harvest.
CONFIRMATORY TRIAL
In the 22.1 -hour confirmatory trial, hemolysis was observed prior to wash of the cultures dosed with 1600 and 2000 pg/ml, and a slight evidence o f hemolysis was evident at harvest o f the cultures dosed with 1600 pg/ml. Reductions o f 2%, 14%, 64%, 74%, 93%, and 93% in the mitotic indices as compared with the solvent control cultures were observed in the cultures dosed with 125, 500, 900, 1200, 1600, and 2000 pg/ml, respectively. Chromosomal aberrations were ana
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lyzed from the cultures treated with 125, 250, 500, and 900 pg/ml (Table 3). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed.
In the 46.0-hour confirmatory trial, hemolysis was observed prior to wash o f the cultures dosed with 1600 and 2000 pg/ml. Reductions o f 59%, 93%, 98%, and 98% in the mitotic indices as compared with the solvent control cultures were observed in the cultures dosed with 900, 1200, 1600, and 2000 pg/ml, respectively. Chromosomal aberrations were analyzed from the cultures treated with 125, 250, 500, and 900 pg/ml (Table 4). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed.
The sensitivity of the cell culture for induction o f chromosomal aberrations is shown by the increased frequency o f aberrations in the cells exposed to MMC, the positive control agent. The test article is considered negative for inducing chromosomal aberrations, polyploidy, and endoreduplication under nonactivation conditions.
11.5 Chromosomal Aberrations Assay With Metabolic Activation
INITIAL TRIAL
Hemolysis was observed prior to wash in the cultures dosed with 2010, 2510, 3010, and 4010 pg/ml. No cells were visible prior to the addition o f Colcemid to the cultures dosed with 4010 pg/ml, and hemolysis was observed prior to the addition o f Colcemid in the cultures dosed with 3010 pg/ml. Reductions o f 15%, 20%, 15%, 43%, 77%, and 95% in the mitotic indices as compared with the solvent control cultures were observed in the cultures treated with 253, 505, 1010, 1510, 2010, and 2510 pg/ml, respectively. Chromosomal aberrations were ana lyzed from the cultures treated with 505, 1010, 1510, and 2010 pg/ml (Table 5). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed.
Based on these results, the confirmatory trial under activation conditions were conducted testing dose levels o f 250, 500, 1000, 1500, 2000, 2500, and 3000 pg/ml with 22.1 and 46.0 hour harvests.
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CONFIRMATORY TRIAL
In the 22.1-hour confirmatory trial, hemolysis was observed prior to wash and prior to harvest of the cultures dosed with 2000, 2500, and 3000 pg/ml. Reductions o f 15%, 5%, 69%, 82%, 97%, and 100% in the mitotic indices as compared with the solvent control cultures were observed in the cultures treated with 500, 1000, 1500, 2000, 2500, and 3000 pg/ml, respectively. Chromosomal aberrations were analyzed from the cultures treated with 250, 500, 1000, and 1500 pg/ml (Table 6). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed.
In the 46.0-hour confirmatory trial, hemolysis was observed prior to wash and prior to harvest o f the cultures dosed with 2000,2500, and 3000 pg/ml. Reductions o f 15%, 80%, 100%, and 100% in the mitotic indices as compared with the solvent control cultures were observed in the cultures treated with 1500, 2000, 2500, and 3000 pg/ml, respectively. Chromosomal aberrations were ana lyzed from the cultures treated with 500, 1000, 1500 and 2000 pg/ml (Table 7). Due to toxicity, only 52 metaphases were available for analysis in one o f the cultures dosed with 2000 pg/ml. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed, except for a weak increase in endoreduplication at 2000 pg/ml, an extremely toxic dose level.
The successful activation o f the metabolic system is illustrated by the increased incidence o f cells with chromosomal aberrations in the cultures induced with cyclophosphamide, the positive control agent. The test article is considered negative for inducing chromosomal aberrations, polyploidy, and endoreduplication under conditions of metabolic activation, except for a weak increase in endoreduplication at an extremely toxic dose level in the 46.0 hour assay.
12.0 CONCLUSION:
The test article, T-6342, was considered negative for inducing chromosomal aberrations in cultured whole blood human lymphocytes cells with and without metabolic activation. These results were verified in independently conducted confirmatory trials.
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13.0 REFERENCES:
Evans, H.J. (1962) Chromosomal aberrations produced by ionizing radiation, International Review o f Cytology, 13, 221-321.
Thakur, A.J., Berry, K.J., and Mielke, P.W. Jr (1985) A FORTRAN program for testing trend and homogeneity in proportions, Computer Programs in Biomedicine, 19, 229-233.
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TABLE 1
DOSE RANGEFINDING ASSAY
Assay No.: 17073 Trial No.: I Date: 07/31/96
Compound: T-6342
Lab No : CY7316
Metabolic Activation: -S9
Treatment
NEGATIVE CONTROL
RPMII640
SOLVENT CONTROL
Water
TEST ARTICLE
Metabolic Activation: +S9
% Mitotic Index
5.9
10.0 fxl/ml
6.S
0.167 ng/ml
7.4
0.500 ng/ml
4.4
1.67 ng/ml
6.4
5.00 ng/ml
4.2
16.7 ng/ml
5.8
50.0 ng/ml
7.2
167 ng/ml
7.1
500 (ig/ml
5.6
1670 ng/ml 5000 ng/ml*
2.9
0.0
Treatment
NEGATIVE CONTROL
RPMI 1640
SOLVENT CONTROL
Water
TEST ARTICLE
'
% Mitotic Index
7.4
10.0 nl/ml
8.3
0.167 ng/ml
9.4
0.500 ng/ml
8.6
1.67 ng/ml
7.6
5.00 ng/ml
8.3
16.7 ng/ml
8.4
50.0 ng/ml
7.9
167 ng/ml
8.6
500 ng/ml
8.0
1670 ng/ml 5000 ng/ml*
8.2
0.0
` Toxic dose level RPMI 1640 = Culture medium
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TABLE 2 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES
Cells Fixed 22.0 Hours After Treatment
Assay No.: 17073
Trial #: I
Date: 08/07/96
Lab#:CY8066
Metabolic Activation:-S9
Compound: T-6342
CONTROLS NEGATIVE:
RPMI 1640
SOLVENT:
Water
i0.0gl/ml
POSITIVE:
MMC
0.300 gg/ml
NUMBER AND TYPE OF ABERRATION
# OF %
%
%
NOT ABERRA CELLS CELLS % ENDO-
COMPUTED SIMPLE
COMPLEX
OTHER TIONS WITH WI TH >1 POLY REDUPLI- %
CELLS
PER ABERRA ABERRA PLOID CATED MITOTIC
SCORED TG j SG j UC TB j SB ID TR 1QR CR i D j R j Cl j DF GT CELL TIONS TIONS CELLS CELLS INDEX
A 100 B 100
A+B 200
A 100 B 100
A+B 200
A 25 B 25
A+B 50
21 1
31
3 3
6 42 71
11 3
1 1
14 3 i 524 5 16 3 5
0.01 1.0 0.00 0.0
0.01 0.5
0.00 0.0 0.00 0.0
0.00 0.0
0.36 32.0 0.44 44.0
0.40 38.0*
0.0 0.0 0.0 0.0
0.0 0.0
0.0 0.0 0.0 0.0
0.0 0.0
4 0 0.0 0.0 0.0
2.0 0.0
0.0 0.0
0.0
0.0 0.0
0.0
0.0 0.0
0.0
2.0 3.7
2.9
4.2 38
4.0
3.3 2.2
2.8
TEST ARTICLE
127 gg/ml** A+B 0
253 gg/ml 505 gg/ml 1010 gg/ml 1510 gg/ml
A 100 7 B 100 2
A+B 200 9
A 100 5 B 100 6
A+B 200 11
A 100 B 100 3 1
A+B 200 3 1
A 100 4 3 B 100 2
A+B 200 6 3
1 1 1
1 12 1 22 2 l 3
2010 gg/ml*** A+B 0
Kl'MI 1640 = Culture medium MMC = Mitomycin C * Significantly greater than the solvent controls, p<0.01. O ** Chromosome aberrations not analyzed due to higher doses available for analysis.
0.00 0.0 0.01 1.0
0.0 0.0 0.0 0.0
0.01 0.5
0.0 0.0
0.01 10 0.00 0.0
0.0 0.0 0.0 0.0
0.01 0.5
0.0 0.0
0.03 2.0 0.01 1.0
1.0 00 0.0 0 0
0.02 1.5
0.5 0.0
0.02 2.0
0.0 00
l
0.02 20
00 00
1
0.02 2.0
0.0 00
*** Chromosome aberrations not analyzed due to excessive toxicity.
00 0.0
0.0
00 0.0
0.0
00 0.0
00
00 0.0
00
2.5
3.5 3.0 3.3 34 32 33 22 1.7 20 2.1 15 18
0.2
C O R N IN G Hazleton
000285
CHV Studv No.: 17073-0-449( 0
TABLE 3 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES
Cells Fixed 22.1 Hours After Treatment
Assay No.: 17073
Trial #: 2
Date. 08/14/96
Lab#:CY8166
Metabolic Activation:-S9
Compound: T-6342
CONTROLS NEGATIVE:
RPMI 1640
SOLVENT:
Water
10.0 nl/ml
POSITIVE:
MMC
0.300 gg/ml
NUMBER AND TYPE OP ABERRATION
NOT COMPUTED
CELLS SCORED TG i SG i UC
SIMPLE TB I SB
tt OF
%
%
%
ABERRA CELLS CELLS % ENDO-
COMPLEX
OTHER TIONS WITH WITH >1 POLY REDUPLI- %
PER ABERRA ABERRA PLOID CATED MITOTIC
ID j TR !qr : CR ; D j R j CI j DF GT CELL TIONS TIONS CELLS CELLS INDEX
A 100 2 B 100 1
A+B 200 3
A 100 2 B 100
A+B 200 2
A 25 5 B 25 1 I
A+B 50 6 1
1 1
2
624 323
947
21 1
31
0.00 0.0 0.00 0.0
0.0 0.0 0.0 0.0
0.00 0.0
0.0 0.0
0.01 1.0 0.0 0.0 0.01 1.0 0.0 0.0
0.01 1.0 0.0 0.0
0.60 40.0 0.36 32.0
20.0 0.0 4.0 0.0
0.48 36.0* 12.0* 0.0
0.0 0.0
0,0
0.0 0.0
0.0
0.0 0.0
0.0
4.2 4.4
4.3
4.0 4.3
4.2
12 2.2
1.7
TEST ARTICLE
125 gg/ml
A 100 6 B 100 3 1
A+B 200 9 1
250 gg/ml
A 100 B 100
A+B 200
500 gg/ml
A 100 6 l B 100 3
A+B 200 9 1
900 gg/ml
A 100 3 1 B 100 4
A+B 200 7 1
1 1
1 t
1200 gg/ml** A+B 0 1600 gg/ml** A+B 0 2000 gg/ml** A+B 0
Rl'Ml 1640 = Culture medium MMC = Mitomycin C * Significantly greater than the solvent controls, p<0.01. ** Chromosome aberrations not analyzed due to excessive toxicity.
0.00 0.0 0.00 0.0
0.00 0.0
0.00 0.0 0.01 1.0
0.01 0.5
0.00 0.0 0.00 0.0
0.00 0.0
0.00 0.0 0.01 1.0
0.01 0.5
0.0 0.0 0.0 0.0
0.0 0.0
0.0 0.0 0.0 0.0
0.0 0.0
0.0 0.0 0.0 0.0
0.0 0.0
0.0 0.0 0.0 00
0.0 0.0
0.0 0.0
0.0
0.0 0.0
0.0
0.0 0.0
0.0
0.0 00
0.0
3.7 4.5 4.1
4.0 4.4 4.2
4.0 3.1
3.6 14 1.6
1.5
1.1 0.3 0.3
C O R N IN G Hazleton
GO0 2 3 6
CHV Study No.: 17073-0-449CO
TABLE 4 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES
Cells Fixed 46.0 Hours After Treatment
Assay No.: 17073
Trial #: 2
Date: 08/14/96
Lab#:CY8166
Metabolic Activation: -S9
Compound: T-6342
CONTROLS NEGATIVE:
RPMI 1640
SOLVENT:
Water
10.0gl/ml
NUMBER AND TYPE OF ABERRATION
NOT COMPUTED
CELLS SCORED TG ! SG i UC
SIMPLE TB j SB
# OF %
%
%
ABERRA CELLS CELLS % ENDO-
COMPLEX
OTHER TIONS WITH WITH >1 POLY REDUPLI- %
PER ABERRA ABERRA PLOID CATED MITOTIC
ID j TR j QR j CR j D 1 R j CI j DF GT CELL TIONS TIONS CELLS CELLS INDEX
A 100 B 100
A+B 200
A 100 B 100
A+B 200
1 2 3
2 2
3 11
41
2 l1
31
0.03 2.0 0.02 2.0
0.03 2.0
0.02 2.0 0.02 2.0
0.02 2.0
1.0 0.0 0.0 0.0
0.5 0.0
0.0 0.0 0.0 0.0
0.0 0.0
00 0.0
0.0
0.0 0.0
0.0
5.3 4.5
4.9
6.0 4.8
5.4
'TEST ARTICLE
62.5 ng/ml* A+B 0
125 ng/ml
A 100 5 B 100 6
A+B 200 11
250 ng/ml
A 100 5 B 100 4
A+B 200 9
500 ng/ml
A 100 4 B 100 5 1
A+B 200 9 1
900 ng/ml
A 100 4 1 B 100 4
A+B 200 8 1
1 1
11 11 1 1
1200 ng/ml** A+B 0 1600 ng/ml** A+B 0
us 2000 ng/ml** A+B 0
-a Rl'MI 1640 Culture medium
to * Chromosome aberrations not analyzed due to higher doses available for analysis. to
0.01 1.0 0.00 0.0
0.01 0.5
0.00 0.0 0.00 0.0
0.00 0.0
0.02 2.0 0.00 00
001 1.0
0.01 1.0 0.00 0.0
0.01 0.5
0.0 0.0 0.0 0.0
0.0 0.0
0.0 0.0 0.0 0.0
0.0 0.0
0.0 00 0.0 00
0.0 00
0.0 0.0 0.0 0.0
0.0 0.0
0.0 0.0
0.0
0.0 0.0
00
00 00
0.0
0.0 0.0
0.0
** Chromosome aberrations not analyzed due to excessive toxicity.
5.6
6.3 6.0 6.2 54 7.2 63 59 56 58 1.9 2.4 2.2
0.4 01 0.1
C O R N IN G Hazleton
0C 02
CUV Siudv No.: 17073-0-449CO
TABLE 5 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES
Cells Fixed 22.0 Hours After Treatment
Assay No.: 17073
Trial #: 1
Date: 08/07/96
Lab#:CY8066
Metabolic Activation:+S9
Compound: T-6342
CONTROLS NEGATIVE:
RPM1 1640
SOLVENT:
Water
POSITIVE:
CP
10.0gl/ml SO.Ong/ml1
NUMBER AND TYPE OF ABERRATION
NOT COMPUTED
CELLS SCORED TG j SG 1UC
SIMPLE TB i SB
ID j TR
It OF
%
%
%
ABERRA CELLS CELLS % ENDO-
COMPLEX
OTHER TIONS WITH WITII>I POLY RKDUPLI- %
PER ABERRA ABERRA PLOID CATED MITOTIC
QR CR j D j R j Cl j DF GT CELL TIONS TIONS CELLS CELLS INDEX
A 100 1 1 B 100
A+B 200 1 1
A 100 3 B 100 3 1
A+U 200 6 1
A 25 4 B 25 4 5
A+B 50 8 5
2
2 71 14 l 21 2
23 232
462
0.00 0.0 0.00 0.0
0.00 0.0
0.02 2.0 0.00 0.0
0.01 1.0
0.52 44.0 0.88 52.0
0.70 48.0*
0.0 0.0
0.0
0.0 0.0
0.0
8.0 28.0
18.0*
0.0 0.0
0.0
0.0 0.0
0.0
0.0 0.0
0.0
0.0 0.0
0.0
0.0 0.0
0.0
0.0 0.0
0.0
4.8 5.0
4.9
6.8 5.2
6.0
1.7 1.2
1.5
TEST ARTICLE
253 ug/mt** A+B 0
505 pg/ml I010ng/ml 15l0pg/ml 2010 gg/ml
A 100 B 100
A+B 200
A 100 B 100
A+B 200
A 100 B 100
A+B 200
A 100 B 100
A+B 200
2 2 4
6 6 41 7l 11 2 2 72 92
3 1
4
1 1
2
0.00 0.0 0.00 0.0
0.00 0.0
0.00 0.0 0.00 0.0
0.00 0.0
0.03 2.0 0.01 1.0
0.02 1.5
0.01 1.0 0.01 1.0
0.01 1.0
0.0 0.0 0.0 0.0
0.0 0.0
0.0 0.0 0.0 0.0
0.0 0.0
1.0 1.0 0.0 10
0.5 1.0
0.0 0.0 0.0 00
0.0 00
2510pg/ml*** A+B 0
Rl'MI 1640 = Culltire medium CP = Cyclophosphamide
! J * Significantly greater than the solvent controls, p<0.01.
vj ** Chromosome aberrations not analyzed due to higher doses available for analysis.
*** Chromosome aberrations not analyzed due to excessive toxicity.
' Due to toxicity, only 83 metaphases from the B culture were analyzed for polyploidy and endoreduplication.
0.0 0.0
0.0
00 0.0
0.0
0.0 0.0
0.0
00 0.0
0.0
5.1
4.7 4.9 4.8 4.2 5.9 5.1 3.1 3.7 3.4 1.1 1.7 1.4
0.3
C O R N IN G Hazleton
G 002S8
n
< vZ> -cG<oZ Assay No.: 17073
Compound: T-6342
-->ipGC.
on CONTROLS NEGATIVE:
RPMI 1640
SOLVENT: Water
POSITIVE: CP
Trial #: 2
TABLE 6 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES
Cells Fixed 22.1 Hours After Treatment
Date: 08/14/96
Lab #: CY8166
Metabolic Activation: +S9
NUMBER AND TYPE OF ABERRATION
# OF %
%
%
NOT ABERRA CELLS CELLS % ENDO-
COMPUTED SIMPLE
COMPLEX
OTHER TIONS WITH WITH >1 POLY REDUPLI- %
CELLS
PER ABERRA ABERRA PLOID CATED MITOTIC
SCORED TG ; SG i UC TB j SB ID TR j QR j CR j D j R j Cl j T GT CELL TIONS TIONS CELLS CELLS INDEX
A 100 8 B 100 6
A+B 200 14
lO.Ogl/ml
A 100 3 B 100 5 2
A+B 200 8 2
50.0gg/ml*** A 25 B 25
42 51
A+B 50 9 3
1 1 2
2
7 62
13 2
2 2
1 1
2
0.00 0.0 0.01 1.0
0.0 0.0 0.0 0.0
0.0 0.0
4.5 4.7
0.01 0.5
0.0 0.0
0.0
4.6
1
0.03 3.0
0.0 0.0
0.0
41
0.00 0.0
0.0 0.0
0.0
3.6
1
0.02 1.5
0.0 0.0
0.0
3.9
0.40 32.0 0.36 36.0
8.0 0.0 0.0 0.0
0.0 0.0
0.7 0.3
0.38 34.0*
4.0 0.0
00
0.5
TEST ARTICLE
Occ
C
250gg/ml 500 gg/ml 1000 gg/ml 1500 gg/ml
A 100 5 1 1 B 100 2 1
A+B 200 7 2 1
A 100 3 B 100 5 1
1 2
A+B 200 8 1
3
A 100 7 B 100 7
1 1
A+B 200 14
2
A 100 9 2 B 100 10
21 4
A+B 200 19 2
61
2000 gg/ml** AHI 0 2500 gg/ml** A+B 0 3000 gg/ml** A+B 0
0.00 0.0 0.00 0,0
0.00 0.0
0.01 1.0 0.02 2.0
0.02 1.5
0.01 1.0 0.01 1.0
0.01 1.0
0.03 2.0 0.04 4.0
0.04 3.0
RPM1 1640 = Culture medium CP = Cyclophosphamide * Significantly greater than the solvent controls, p<0.01. ** Chromosome aberrations not analyzed due to excessive toxicity. *** Due to toxicity, only 56 metaphases from the A culture and 36 metaphases from the B culture were analyzed for polyploidy and endoreduplication.
0.0 0.0 0.0 0.0
0.0 0.0
0.0 0.0 0.0 1.0
0.0 0.5
0.0 0.0 0.0 0.0
0.0 0.0
1.0 0.0 0.0 1.0
0.5 0.5
0.0 0.0
0.0
0.0 0.0
0.0
0.0 0.0
0.0
0.0 0.0
0.0
4.4 4.2
4.3 2.1 4.4
3.3 3.6 3.8
3.7
1.2 1.1
1.2
0.7 0.1 0.0
C O R N IN G Hazleton
CHV Study No.: 17073-0-449CO
TABLE 7 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES
Cells Fixed 46.0 Hours After Treatment
Assay No.: 17073
Trial #: 2
Date: 08/14/96
Lab#:CY8166
Metabolic Activation: +S9
Compound: T-6342
CONTROLS NEGATIVE:
RPMI 1640
SOLVENT:
Water
10.0pl/ml
NUMBER AND TYPE OF ABERRATION
NOT COMPUTED
CELLS SCORED TG i SG 1UC
SIMPLE TB I SB
U OF
%
%
%
ABERRA CELLS CELLS % ENDO-
COMPLEX
OTHER TIONS WITH WITH POLY REDUPL1- %
PER ABERRA ABERRA PLOID CATED MITOTIC
ID i TR 1QR j CR j D j R j Cl j DF GT CELL TIONS TIONS CELLS CELLS INDEX
A 100 3 B 100 2
A+B 200 5
A 100 l l B 100 3
A+B 200 4 1
1 1 1 1
2
0.00 0.0 0.01 1.0
0.01 0.5
0.01 1.0 0.01 1.0
0.01 1.0
0.0 1.0 0.0 0.0
0.0 0.5
0.0 0.0 0.0 0.0
0.0 0.0
0.0 8.0 0.0 8.1
00 8.1
0.0 6.3 0.0 . 6.9
0.0 6.6
TEST ARTICLE
250 pg/ml** A+B 0
7.7
500 pg/ml
A 100 B 100
A+B 200
1000pg/ml
A 100 B 100
A+B 200
1500 pg/ml
A 100 B 100
A+B 200
2000 pg/ml1
A 52 B 148
A+B 200
3 11
41
3 2
5
4 91
13 1
22 2
42
1 1
1 1 11 2 31
11 11
0.00 0.0 0.00 0.0
0.00 0.0
0.00 0.0 0.01 1.0
0.01 0.5
0.00 0.0 0.01 1.0
0.01 0.5
0.04 3.8 0.03 2.0
0.03 2.5
0.0 1.0 0.0 0.0
0.0 0.5
0.0 0.0 0.0 1.0
0.0 0.5
0.0 1.0 0.0 1.0
0.0 1.0
0.0 0.0 0.7 0.0
0.5 0.0
0.0 0.0
0.0
0.0 0.0
0.0
1.0 0.0
0.5
7.4 5.2
6.3*
7.8 8.1 8.0
6.4 7.7
7.1
5.6 5.5
5.6
0.8 1.8
1.3
2500 pg/ml*** A+B 0 3000 pg/ml*** AH) 0
0.0 0.0
RPMI 1640 = Culture medium
* Significantly greater than the solvent controls, p<0.01.
** Chromosome aberrations not analyzed due to higher doses available for analysis.
*** Chromosome aberrations not analyzed due to excessive toxicity.
1Hue to toxicity, only 54 metaphases from the A culture and 58 metaphases from the 13 culture were analyzed for polyploidy and endoreduplication.
IsJ 'J l
C O R N IN G Hazleton
000290
TABLE 8
CONTROL DATA OF CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES 4-95 THROUGH 12-95
Negative Control
Solvent Control
Positive Control Mitomycin C Negative Control
Solvent Control
Positive Control Cyclophosphamide
Activation
#O f Aberrations
Per Cell
Without
MIN MAX AVG
N
0.00 0.06 0.012 18
Without
MIN MAX AVG
N
0.00 0.06 0.011 29
Without
MIN MAX AVG
N
0.02 0.60 0.267 19
With MIN
0.00
MAX
0.03
AVG
0.010
N 18
With MIN
0.00
MAX
0.02
AVG
0.009
N 28
With MIN
0.28
MAX
2.52
AVG
0.647
N 17
% O f Cel Is With
Aberrations
0.0 2.5 0.72 18
0.0 2.5 0.78 29
1.5 39.5 22.22
19
0.0 2.0 0.75 18
0.0 2.0 0.66 28
21.7 40.0 31.45
17
% O f Cells With > I
Aberration
0.0 0.5 0.06 18
0.0 0.5 0.05 29
0.0 15.0 3.63 19
0.0 0.5 0.06 18
0.0 0.5 0.05 28
0.0 40.0 12.90
17
% Polyploidy
% Endoreduplicated
Cells
% Mitotic Index
0.0 0.0 1.0 1.0 0.18 0.03 17 35
0.4 7.1 3.57 18
0.0 0.0
0.5
1.0 2.0
10.9
0.14 0.10 4.52
18 35
29
0.0 0.0 0.0 0.0 0.00 0.00 11 31
0.1 6.6 2.46 19
0.0 0.0 1.0 0.5 0.28 0.02 17 27
0.6 9.6 4.68 18
0.0 0.0
0.5
2.0 0:0 10.7
0.25 0.00 6.13
17 27
28
0.0 0.0 1.0 0.0 0.22 0.00 9 25
0.1 5.9 1.35 17
CHV Study No.: 17073-0-449CO
26 000291
C O R N IN G Hazleton
15.0 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS
SIMPLE
TB Chromatid Break:
An achromatic region in one chromatid, larger than the width of a chromatid. The associated fragment may be partially or completely displaced.
SB Chromosome Break:
Chromosome has a clear break, forming an abnormal (deleted) chromosome with an acentric fragment that is dislocated. This classification now includes the acentric fragment (AF). An AF is different from a SB only in that it can not be related to any specific chromosome.
DM "Double Minute" Fragment: These are small double dots, which may represent terminal or interstitial deletions, or even small rings. These possible origins are not distinguishable.
COMPLEX
ID Interstitial Deletion:
Length of chromatid "cut out" from midregion o f a chromatid, resulting in a small fragment or ring lying beside a shortened chromatid or a gap in the chromatid.
TR Triradial:
An exchange between two chromosomes, or one chromosome and an acentric fragment, which results in a three-armed configuration.
QR Quadriradial:
An exchange like a triradial, but resulting in a four armed configuration.
CR Complex Rearrangement:
An exchange among more than two chromosomes or fragments, caused by the induction o f several breaks.
D Dicentric:
An exchange between two chromosomes which results in a chromosome with two centromeres. This is often associated with an acentric fragment in which case it is classified as DF.
DF Dicentric with fragment.
CHV Study No.: 17073-0-449CO
27 000292
C O R N IN G Hazleton
COMPLEX (Continued) TC Tricentric:
QC Quadricentric: PC Pentacentric: HC Hexacentric: R Ring:
RC Ring Chromatid: RF Cl Chromosome Intrachange:
T Translocation:
AB Abnormal:
An exchange involving three chromosomes and resulting in a chromosome with three centromeres. Often associated with two to three AF. Such exchanges can involve many chromosomes and are named as follows:
four centromeres, up to four AF five centromeres, up to five AF six centromeres, up to six AF
A chromosome which forms a circle containing a centromere. This is often associated with an acentric fragment, in which case it is classed as RF.
Single chromatid ring (acentric).
Ring with associated acentric fragment.
Exchange within a chromosome; e.g., a ring that does not include the entire chromosome.
Obvious transfer of material between two chromosomes resulting in two abnormal chromosomes. When identifiable, scored as "T" not "2AB".
Abnormal monocentric chromosome. This is a chromosome whose morphology is abnormal for the karyotype, and often the result of a translocation, pericentric inversion, etc. Classification used if abnormality cannot be ascribed to, e.g., a reciprocal translocation.
CHV Study No.: 7073-0-449CO
28 000293
OTHER
GT Greater than Ten:
PP Polyploid Cell: E Endoreduplication: NOT COMPUTED TG Chromatid Gap:
SG Chromosome Gap: UC Uncoiled Chromosome:
C O R N IN G Hazleton
Greater them 10 aberrations: A cell which contains more than 10 aberrations. Heavily damaged cells will be analyzed to identify the types o f aberrations because multiple fragments, such as those found associated with a tricentric, do not count as independent aberrations.
A cell containing multiple copies o f the haploid number (n) of chromosomes.
A failure o f chromosomes to separate, resulting in a 4n cell.
("tid gap"). An achromatic (unstained) region in one chromatid, the size o f which is equal to or smaller than the width of a chromatid. These are noted but not usually included in final totals o f aberrations as they may not all be true breaks.
("isochromatid gap, IG"). Same as chromatid gap but at the same locus in both sister chromatids.
Failure o f chromatin packing. Probably not a true aberration.
CHV Study No.: 17073-0-449CO
29 000294
