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STUDY TITLE 

EVALUATION OF IN AN IN VITRO

CHROMOSOMAL ABERRATION ASSAY UTILIZING RAT LYMPHOCYTES 

Test Guidelines 

USEPA OPPTS 870.5375 (1998) 

OECD, Guideline 473 (1997) 

EC, B.10 (2008) 

Author(s) 

Study Completion Date 

10 November 2010 

Sponsor 

The Dow Chemical Company 

Midland, Michigan 48674 

Performing Laboratory 

Toxicology & Environmental Research and Consulting 

The Dow Chemical Company 

Midland, Michigan 48674 

Laboratory Project Study ID 

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COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS 

Compound: 

Title: EVALUATION OF IN AN IN VITRO 

CHROMOSOMAL ABERRATION ASSAY UTILIZING RAT 

LYMPHOCYTES 

All phases of this study were conducted in compliance with the following Good 

Laboratory Practice Standards: 

US Department of Health and Human Services Food and Drug Administration - GLPs; 

Title 21 CFR, Part 58 - Good Laboratory Practice Regulations for Nonclinical Laboratory 

Studies, Final Rule 

Organisation for Economic Co-Operation and Development (OECD) -- OECD Series on 

Principles of Good Laboratory Practice and Compliance Monitoring, Number 1. OECD 

Principles on Good Laboratory Practice (as revised in 1997) ENV /MC/CHEM(98) 17 

Exception: the GLP characterization of the test material was conducted concurrently 

with the in-life of this study. 

(/-«-8010 

(Date) 

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QUALITY ASSURANCE STATEMENT 

Compound: 

Title: EVALUATION OF IN AN IN VITRO 

CHROMOSOMAL ABERRATION ASSAY UTILIZING RAT 

LYMPHOCYTES 

This study was examined for confonnance with Good Laboratory Practices as published 

by the USFDA and OECD. The final report was determined to be an accurate reflection 

of the data obtained. The dates of Quality Assurance activities on this study are listed 

below. 

Study Initiation Date: 24 May 2010 

TYPE OF AUDIT: 

Final protocol 

Study conduct 

Protocol, data, and 

draft report 

Final report 

DATE FINDINGS REPORTED TO 

DATE OF AUDIT: STUDY DIRECTORJMANAGEMENT: 

25-31 May 2010 02 June 2010 

25 May 2010 31 May 2010 

26-29 October 2010 05 November 2010 

The date of the signature below is the date of the final report audit. 

The final report accurately reflects the raw data of the study. 

(Date) 

Quality Assurance 

Toxicology & Environmental Research and Consulting 

The Dow Chemical Company 

1803 Building 

Midland, Michigan 48674 

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SIGNATURE PAGE 

Compound: 

THE DOW CHEMICAL COMPANY 

STUDY ID: 

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Title: EV ALUATION OF IN AN IN VITRO 

CHROMOSOMAL ABERRATION ASSAY UTILIZING RAT 

LYMPHOCYTES 

Reviewed by: 

(Date) 

(Date) 

USEPA TSCA 8(e)/FIFRA 6(a)(2) ASSESSMENT I 

This test material has been reviewed for possible TSCA 8(e)/FIFRA 6(a)(2) submission IDIIO 

InitialslDate 

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TABLE OF CONTENTS 

Page 

STUDY TITLE ...............................................................................................................1

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS.............2

QUALITY ASSURANCE STATEMENT .....................................................................3

SIGNATURE PAGE ......................................................................................................4

SUMMARY....................................................................................................................8

INTRODUCTION ..........................................................................................................9

Principle of the Test System ......................................................................................9

Purpose.......................................................................................................................9

Test Guidelines ..........................................................................................................9

Quality Assurance ....................................................................................................10

Archiving .................................................................................................................10

Safety .......................................................................................................................10

TEST MATERIAL INFORMATION ..........................................................................10

Test Material Name..................................................................................................10

Chemical Name........................................................................................................10

Synonyms.................................................................................................................10

Supplier, City, State (Lot, Reference Number)........................................................10

Purity/Characterization (Method of Analysis and Reference).................................10

Characteristics..........................................................................................................10

Molecular Formula ................................................................................................10

Molecular Weight..................................................................................................11

Chemical Structure ................................................................................................11

CAS Number .........................................................................................................11

TEST SPECIES AND HUSBANDRY.........................................................................11

Species and Sex........................................................................................................11

Strain and Justification.............................................................................................11

Supplier and Location ..............................................................................................11

Age at Study Start ....................................................................................................11

Physical and Acclimation.........................................................................................11

Housing ....................................................................................................................12

Identification ............................................................................................................12

Feed and Water ........................................................................................................12

Animal Welfare........................................................................................................12

STUDY DESIGN..........................................................................................................13

Lymphocyte Cultures...............................................................................................13

Controls....................................................................................................................13

In Vitro Metabolic Activation System .....................................................................13

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TABLE OF CONTENTS 

Page 

Preparation of the Treatment Solution .....................................................................14

Analytical Verification of Dosing Solutions............................................................14

Identification of the Test System .............................................................................14

Treatment Procedure without Metabolic Activation................................................14

Short Treatment.....................................................................................................14

Continuous Treatment ...........................................................................................14

Treatment Procedure using Metabolic Activation (S9) ...........................................15

Toxicity and Chromosomal Aberration Assay.........................................................15

Evaluation Criteria ...................................................................................................16

Statistical Analysis...................................................................................................16

RESULTS AND DISCUSSION...................................................................................17

pH and Osmolality ...................................................................................................17

Assay A1 ..................................................................................................................17

Assay B1 ..................................................................................................................18

Short Treatment (4 hour treatments, Assay B1)....................................................18

Continuous Treatment (24 hour treatment, Assay B1)..........................................19

CONCLUSIONS...........................................................................................................19

ACKNOWLEDGEMENTS..........................................................................................20

REFERENCES .............................................................................................................21

TABLES .......................................................................................................................22

Table 1. Concentration Check Report – Assay A1 .............................................22

Table 2. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with 

Various Concentrations of in the Absence of S9 

– Assay A1 ...................................................................................................23

Table 3. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with 

Various Concentrations of in the Presence of S9 

– Assay A1 ...................................................................................................24

Table 4. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with 

Various Concentrations of in the Absence of S9 

– Assay A1 ...................................................................................................25

Table 5. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with 

Various Concentrations of in the Absence of S9 

– Assay B1 ...................................................................................................26

Table 6. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with 

Various Concentrations of in the Presence of S9 

– Assay B1 ...................................................................................................27

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TABLE OF CONTENTS 

Page 

– 

Table 7. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with 

Various Concentrations of in the Absence of S9 

– Assay B1 ...................................................................................................28

Table 8. Incidence of Polyploidy (%PP) in Rat Lymphocyte Cultures 

Treated with Various Concentrations of in the 

Absence and Presence of S9 – Assay B1.....................................................29

Table 9. Results of the Chromosomal Aberration Assay in the Absence of 

S9 – Assay B1 ..............................................................................................30

Table 10. Results of the Chromosomal Aberration Assay in the Presence of 

S9 – Assay B1 ..............................................................................................31

Table 11. Results of the Chromosomal Aberration Assay in the Absence of 

S9 – Assay B1 ..............................................................................................32

Table 12. Historical Background Aberration Frequencies – Descriptive 

Statistics .......................................................................................................33

Table 13. Historical Background Aberration Frequencies – Individual Values...34
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SUMMARY 

was 

evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. 

Approximately 48 hours after the initiation of whole blood cultures, cells were treated 

either in the absence (4 and 24 hour treatments) or presence of S9 (4 hour treatment) with 

concentrations ranging from 0 (solvent control) to 200 g per ml of 

culture medium. The highest concentration was based on the limit of solubility of the test 

material in the treatment medium. Based upon the mitotic indices, cultures treated for 4 

hours with targeted concentrations of 0 (solvent control), 12.5, 25, and 50 g/ml in the 

absence and 0 (solvent control), 25, 75, and 150 g/ml in the presence of S9 were 

selected for determining the incidence of chromosomal aberrations. Cultures treated for 

24 hours in the absence of S9 with 0 (solvent control), 25, 50, and 100 g/ml were 

selected for determining the incidence of chromosomal aberrations. There were no 

significant increases in the frequencies of cells with aberrations in either the absence or 

presence of S9 activation. Cultures treated with the positive control chemicals (i.e., 

mitomycin C without S9 and cyclophosphamide with S9) had significantly higher 

incidences of abnormal cells in all assays. Based upon these results, 

 was considered to be non-genotoxic in this in vitro chromosomal aberration 

assay utilizing rat lymphocytes. 

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INTRODUCTION 

Principle of the Test System 

The ability of a compound to induce chromosomal damage (clastogenicity) can be 

assayed in vitro using cultured mammalian cells. Among various cell systems that can be 

used for this purpose, cultured rat lymphocytes have the following advantages. The 

laboratory rat is widely used for toxicological studies and hence the results of the in vitro

cytogenetic tests can be compared with other toxicological endpoints. Since rats can be 

maintained in well-controlled environments, the influence of environmental factors on 

the endpoint being measured can be controlled. The karyotype of cultured rat 

lymphocytes (2N = 42) is stable as opposed to the relative karyotypic instability of 

established cell lines. Furthermore, with the rat lymphocyte system, S9 preparations 

from the same species can be used for metabolic activation. 

Conditions for successfully culturing rat lymphocytes have been developed in this 

laboratory (Sinha et al., 1985; Gollapudi et al., 1986; Sinha et al., 1988). The system

was validated in this laboratory by using various direct- and indirect-acting clastogens 

and non-clastogens (Sinha et al., 1989). Analysis of the growth characteristics suggested 

that rat lymphocytes in culture have an average generation time of approximately 

16 hours (Sinha et al., 1985). The background aberration rate of cultured rat 

lymphocytes is low and no significant animal to animal variation in the aberration 

frequency was found (Sinha et al., 1989). 

Purpose 

The purpose of the study was to determine the clastogenic potential of 

 to cultured rat lymphocytes. An external metabolic activation system (S9) was 

also used to facilitate the identification of agents requiring biotransformation for 

clastogenic activity. 

Test Guidelines 

OECD Guidelines for testing of chemicals, #473: In vitro mammalian 

chromosome aberration test, OECD publication service, 2, rue AndrePascal, 75775 Paris Cedex 16, France (1997). 

USEPA Health effects testing guidelines, OPPTS 870.5375 - In vitro

mammalian Chromosome Aberration Test (1998). 

EC B.10 Mutagenicity – In Vitro Mammalian Chromosome Aberration 

Test. Official Journal of the European Communities, L142, 

31/05/2008. 

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Quality Assurance 

The study conduct, data, protocol, protocol changes/revisions, and final report were 

inspected by the Quality Assurance Unit, Toxicology & Environmental Research and 

Consulting, The Dow Chemical Company, Midland, Michigan. 

Archiving 

The data, protocol, protocol changes/revisions, and final report were archived by the 

Toxicology & Environmental Research and Consulting archivist and stored at The Dow 

Chemical Company, Midland, Michigan. 

Safety 

All personnel involved in the study were advised of the safety precautions to follow when 

handling the test and positive control materials. Chemical safety information was made 

available. 

TEST MATERIAL INFORMATION 

Test Material Name 

Chemical Name 

Synonyms 

Supplier, City, State (Lot, Reference Number) 

The Dow Chemical Company, Midland, Michigan (Lot# 

Purity/Characterization (Method of Analysis and Reference) 

The purity of the test material was determined to be gas 

chromatography with identification by Fourier transform infrared spectroscopy and gas 

chromatography mass spectrometry

Characteristics 

Molecular Formula

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Molecular Weight

Chemical Structure

CAS Number

TEST SPECIES AND HUSBANDRY 

Species and Sex 

Rats (Male) 

Strain and Justification 

Sprague-Dawley derived CD IGS (outbred Crl:CD(SD)) rats were selected because of 

their general acceptance and suitability for toxicity testing, availability of historical 

background data and the reliability of the commercial supplier. 

Supplier and Location 

Charles River Laboratories Inc. (Portage, Michigan) 

Age at Study Start 

Approximately 10 weeks 

Physical and Acclimation 

Each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology 

technician under the direct supervision of a laboratory veterinarian, to determine the 

general health status and acceptability for study purposes upon arrival at the laboratory 

(fully accredited by the Association for Assessment and Accreditation of Laboratory 

Animal Care International - AAALAC International). The animals were housed two per 

cage in stainless steel cages, in rooms designed to maintain adequate conditions 

(temperature, humidity, and photocycle), and acclimated to the laboratory for at least one 

week prior to the start of the study. 

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Housing 

After assignment, animals were housed two per cage in stainless steel cages. Cages had 

wire mesh floors and were suspended above absorbent paper. Non-woven gauze was 

placed in the cages to provide a cushion from the flooring for rodent feet. The gauze and 

pair housing provided environmental enrichment. Cages contained a hanging feeder and 

a pressure activated lixit valve-type watering system. The following environmental 

conditions were maintained in the animal room. 

Temperature: 22°C with a tolerance of ± 1°C (and a maximum 

permissible excursion of ± 3C) 

Humidity: 40-70% 

Air Changes: 12-15 times/hour 

Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.) 

Identification 

Animals were identified with each animal receiving a unique alphanumeric metal ear tag. 

Feed and Water 

Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, 

St. Louis, Missouri) in pelleted form. Feed and municipal water were provided ad libitum. 

Analyses of the feed were performed by PMI Nutrition International to confirm the diet 

provides adequate nutrition and to quantify the levels of selected contaminants. Drinking 

water obtained from the municipal water source was periodically analyzed for chemical 

parameters and biological contaminants by the municipal water department. In addition, 

specific analyses for chemical contaminants were conducted at periodic intervals by an 

independent testing facility. Copies of these analyses are maintained in the study file. 

Animal Welfare 

In accordance with the U.S. Department of Agriculture Animal Welfare Regulations, 9 

CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of 

this study were reviewed and approved by the Institutional Animal Care and Use 

Committee (IACUC). The IACUC has determined that the proposed activities were in full 

accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities 

to be used for this study were Genetic Tox 02, Humane Endpoints 01, and Animal ID 01. 

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STUDY DESIGN 

Lymphocyte Cultures 

Blood samples were collected by cardiac puncture, following euthanasia with carbon 

dioxide. In the assays, blood samples from individual rats were pooled and whole blood 

cultures were set up in RPMI 1640 (GIBCO, Grand Island, New York) medium

supplemented with 10% dialyzed fetal bovine serum (GIBCO), antibiotics and 

antimycotics (Fungizone 0.25 μg/ml; penicillin G, 100 u/ml; and streptomycin sulfate, 

0.1 mg/ml; GIBCO), 30 μg/ml PHA-P (HA16, Murex Diagnostics Ltd., Dartford, 

England), and an additional 2 mM L-glutamine (GIBCO). Cultures were initiated by 

inoculating approximately 0.5 ml of whole blood/5 ml of culture medium. Cultures were 

set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 

37°C. 

Controls 

The solvent selected for dissolving the test material (dimethyl sulfoxide (DMSO)) was 

used as the solvent control treatment. Mitomycin C (MMC, Sigma, St. Louis, Missouri, 

CAS No. 50-07-7) was used as the positive control chemical for the non-activation assay 

at a concentration of 0.5 μg/ml (4 hour treatment) or 0.05 and 0.075 μg/ml (24 hour 

treatment), while cyclophosphamide monohydrate (CP, Sigma, CAS No. 6055-19-2) was 

the positive control for the activation assay at final concentrations of 2 and 4 μg/ml. 

In Vitro Metabolic Activation System 

S9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kgBW) male 

Crl:CD(SD) rats was purchased from a commercial source (Molecular Toxicology, Inc., 

Boone, North Carolina) and stored at -100°C or below. Thawed S9 was reconstituted at a 

final concentration of 10% (v/v) in a "mix" (O'Neill et al., 1982). The mix consisted of 

10 mM MgCl2·6H2O (Sigma), 5 mM glucose-6-phosphate (Sigma), 4 mM nicotinamide 

adenine dinucleotide phosphate (Sigma), 10 mM CaCl2 (Fisher, Fair Lawn, New Jersey), 

30 mM KCl (Sigma), and 50 mM sodium phosphate (pH 8.0, Sigma and Fisher). The 

reconstituted mix was added to the culture medium to obtain the desired final 

concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the cofactors in the culture medium was 1/5 of the concentrations stated above. 

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Preparation of the Treatment Solution 

All test material solutions were prepared fresh on the day of treatment and used within 

two hours of preparation. The test material was first dissolved in DMSO and further 

diluted (1:100) with the treatment media to obtain the desired concentrations. All dosing 

units were expressed in μg/ml. MMC and CP were dissolved in RPMI 1640 with HEPES 

and antibiotics. 

Analytical Verification of Dosing Solutions 

The selected concentrations of the test material in the stock dosing solutions used for 

treatment in Assay A1 were verified by the Analytical Chemistry Laboratory, Toxicology 

& Environmental Research and Consulting, The Dow Chemical Company, Midland, 

Michigan. Samples were diluted in an appropriate solvent and analyzed by high 

performance liquid chromatography with ultraviolet detection (HPLC/UV). 

Identification of the Test System 

All test cultures were identified using self-adhesive labels containing a code system that 

identified the test material, experiment number, treatment, and replicate. 

Treatment Procedure without Metabolic Activation 

Short Treatment

Forty-eight hours ( 1 hour) after initiation of the cultures, the cell suspension was 

dispensed into 15 ml sterile disposable centrifuge tubes (5.5 ml/tube, two cultures per 

dose level). The cells were sedimented by centrifugation and the culture medium 

removed and saved. The cells were exposed to medium (RPMI 1640, HEPES, and 

antibiotics) containing the test or positive or solvent control treatments for 4 hours 

( 30 minutes) at 37°C and the exposure was terminated by washing the cells with 

culture medium. The cells were then placed in individual sterile disposable tissue 

culture flasks (T-25) along with approximately 4.5 ml of the original culture medium 

until the time of harvest. The cultures were harvested 24 hours ( 1 hour) after 

treatment initiation (i.e., 20 hours,  1 hour after treatment termination). 

Continuous Treatment

Cultures were treated continuously with the test material for 1.5X normal cell cycle 

length. The solvent control, positive control, and test material were added directly 

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to the culture flasks 48 hours ( 1 hour) after initiation of the cultures and the 

cultures were harvested 24 hours ( 1 hour) later. 

Treatment Procedure using Metabolic Activation (S9) 

Only the short treatment (4 hours  30 minutes) procedure was used with S9. Forty-eight 

hours ( 1 hour) after initiation of the cultures, the cell suspension was dispensed into 

15 ml sterile disposable centrifuge tubes (5.5 ml/tube, two cultures per dose level). The 

cells were sedimented by centrifugation and the culture medium removed and saved. The 

cells were exposed to medium (RPMI 1640, HEPES, antibiotics, and the S9 mix) 

containing the test or positive or solvent control treatments for 4 hours ( 30 minutes) at 

37°C and the exposure was terminated by washing the cells with culture medium. The 

cells were then placed in individual sterile disposable tissue culture flasks (T-25) along 

with 4.5 ml of the original culture medium until the time of harvest. The cultures were 

harvested at 24 hours ( 1 hour) after treatment initiation (i.e., 20 hours,  1 hour after 

treatment termination). 

Toxicity and Chromosomal Aberration Assay 

The cultures were treated with various concentrations of the test material and the selected 

concentration of the positive control chemicals. The highest concentration of the test 

material assayed was based on a limit of solubility of the test material in the treatment 

medium. The other concentrations tested were separated by a factor of 2. Colcemid 

(1 μg/culture) was added 2-3 hours prior to harvest. The cells were swollen by hypotonic 

treatment (0.075 M KCl), fixed with methanol:acetic acid (3:1), dropped on microscope 

slides, and stained in Giemsa. Mitotic indices were determined as the number of cells in 

metaphase among 1000 cells/replicate and expressed as percentages. 

Initially, slides from the short treatment (with and without S9) were evaluated for 

cytogenetic analysis. Slides from the continuous treatment without S9 were evaluated 

only when the results with short treatment yielded negative findings. Slides from the 

solvent controls, positive controls, and three concentrations of the test material were 

selected for cytogenetic analysis. 

One hundred metaphases/replicate were examined from coded slides for structural 

abnormalities (Buckton and Evans, 1973; Sinha et al., 1984; Gollapudi et al., 1986). The 

number of cells examined for structural abnormalities was reduced to 50 metaphases per 

replicate when high numbers of aberrations were observed (approximately 20%). The 

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microscopic coordinates of metaphases containing aberrations were recorded. Only those 

metaphases that contained 42 + 2 centromeres were scored with the exception of cells 

with multiple aberrations, in which case accurate counts of the centromeres would not be 

possible. Structural chromosomal abnormalities counted include chromatid and 

chromosome gaps, chromatid breaks and exchanges, chromosome breaks and exchanges, 

and miscellaneous (chromosomal disintegration, chromosomal pulverization, etc.). 

Those cells having five or more aberrations/cell were classified as a cell with multiple 

aberrations. Chromatid gaps and chromosome gaps were not included in calculations of 

total cytogenetic aberrations. 

In addition, a total of 100 metaphases/replicate were examined for incidence of 

polyploidy. The data was used to calculate the following parameters: 

% Cells with aberrations: Aberrant cells (excluding cells with gaps only) x 100

 # metaphases evaluated 

Aberrations/100 cells: Total aberrations (excluding gaps, miscellaneous and severely damaged) x 100

 # metaphases evaluated 

Evaluation Criteria 

For a test to be acceptable, the chromosomal aberration frequency in the positive control 

cultures should be significantly higher than the solvent controls. The aberration frequency 

in the solvent control should be within reasonable limits of the laboratory historical values. 

A test chemical will be considered positive in this assay if it induces a significant, doserelated increase in the frequency of cells with aberrations and the incident of aberrant cells 

and cultures is outside the recent historical solvent control range. 

Statistical Analysis 

The proportions of cells with aberrations (excluding gaps) were compared by the 

following statistical methods. At each dose level, data from the replicates were pooled. 

A two-way contingency table was constructed to analyze the frequencies of aberrant 

cells. An overall Chi-square statistic, based on the table, was partitioned into components 

of interest. Specifically, statistics were generated to test the global hypothesis of no 

difference in the average number of cells with aberrations among the dose groups 

(Armitage, 1971). An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical 

evaluation. If this statistic was found to be significant at alpha = 0.05, versus a one-sided 

increasing alternative, pairwise tests (i.e. control vs. treatment) were performed at each 

dose level and evaluated at alpha = 0.05, again versus a one-sided alternative. If any of 

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the pairwise tests were significant, a test for linear trend of increasing number of cells 

with aberrations with increasing dose was performed (Armitage, 1971). 

Polyploid cells were analyzed by the Fisher Exact probability test (Siegel, 1956). The 

number of polyploid cells were pooled across replicates for the analysis and evaluated at 

alpha = 0.05. The data was analyzed separately based on the presence or absence of S9 

and based on the exposure time. 

RESULTS AND DISCUSSION 

pH and Osmolality 

The pH and osmolality of treatment medium containing approximately 176.8 μg/ml of 

the test material and medium containing 1% solvent (DMSO) were determined using a 

Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A 

freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts), respectively. 

There was no appreciable change in either the pH or osmolality at this concentration as 

compared to the culture medium with solvent alone (culture medium with the test 

material, pH = 7.43, osmolality = 445 mOsm/kg H20; culture medium with 1% DMSO, 

pH = 7.42, osmolality = 444 mOsm/kg H20). 

Assay A1 

In the initial assay, cultures were treated with the test material in the absence and 

presence of S9 activation for 4 hours at concentrations of 0 (solvent control), 3.1, 6.3, 

12.5, 25, 50, 100, and 200 μg/ml (Table 2 and Table 3). Cultures were also treated 

continuously for 24 hours in the absence of S9 with the above concentrations plus an 

additional lower concentration of 1.6 g/ml (Table 4). Analytically detected 

concentrations of the test material in the stock solutions (Assay A1) varied from 90.8 to 

109.2% of the target (Douglass, 2010; Table 1) and these values also indicated stability of 

the test material in the solvent. All cultures in Assay A1 had poor cell growth as 

indicated by less than normal total lymphocytes present. This observation is not reflected 

in the mitotic indices of the cultures (Table 2, Table 3, and Table 4). Therefore, the 

slides from this assay were deemed unsuitable for evaluation for chromosome 

aberrations. The assay was repeated as Assay B1 with a slight modification of the 

concentration levels. 

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Assay B1 

In the repeat assay, cultures were treated with the test material in the absence (4 and 24 

hour treatment) and presence of S9 (4 hour treatment) at concentrations of 0 (solvent 

control), 12.5, 25, 50, 75, 100, 150, and 200 μg/ml (Table 5, Table 6, and Table 7). 

Short Treatment (4 hour treatments, Assay B1)

In the absence of S9, the cultures displayed excessive toxicity at the three highest 

concentrations (100, 150, and 200 μg/ml) while the remaining concentrations had 

relative mitotic indices ranging from 13.7 to 81.7% compared to the solvent control 

values (Table 5). In the presence of S9, the mitotic indices of the treated cultures 

ranged from 23.1 to 100.0% as compared to the solvent control values (Table 6). 

Based upon these results, cultures treated with targeted concentrations of 0 (solvent 

control), 12.5, 25, and 50 μg/ml in the absence of S9 and 0 (solvent control), 25, 75, 

and 150 μg/ml were chosen for the determination of chromosomal aberration 

frequencies and incidence of polyploidy. 

Among the cultures treated with the positive control chemicals for 4 hour, 

0.5 μg/ml of MMC and 4 μg/ml of CP were selected for evaluation of aberrations in 

the absence and presence of S9, respectively. 

There were no significant increases in the incidence of polyploidy cells in any of the 

test material treated cultures as compared to the solvent control values (Table 8). 

In the absence of S9 (Table 9), the frequency of cells with aberrations in the solvent 

control was 0.5% and the corresponding values at concentration levels of 12.5, 25, 

and 50 μg/ml were 1.5, 1.0, and 2.0%, respectively. In the presence of S9 (Table 

10), cultures treated with the test material at concentrations of 25, 75, and 

150 μg/ml had aberrant cell frequencies of 0.0, 1.0, and 2.0%, respectively as 

compared to the solvent control value of 2.0%. Statistical analyses of these data did 

not identify significant differences between the solvent control and any of the 

treated cultures without or with S9. The frequencies of aberrant cells observed in 

the test material treated cultures were within the laboratory historical background 

range (Table 12 and Table 13). 

Significant increases in the frequency of cells with aberrations were observed in 

cultures treated with the positive control chemicals. Aberrant cell frequencies in 

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MMC (- S9, 4 hour treatment), and CP (+ S9, 4 hour treatment) cultures were 

40.0% (Table 9), and 16.7% (Table 10), respectively 

A second assay with treatment of cultures in the presence of S9 was not considered 

necessary since the results of the initial test yielded clearly negative results. 

Continuous Treatment (24 hour treatment, Assay B1)

Based upon the negative findings in the 4 hour treatment in the absence of metabolic 

activation, slides from the continuous 24 hour treatment were evaluated (Table 7). 

Cultures treated continuously for 24 hours in the absence of S9 activation had 

excessive toxicity at the two highest concentrations (150 and 200 μg/ml) while the 

remaining concentrations had moderate to no toxicity with relative mitotic indices 

ranging from 43.8 to 82.3% relative to the solvent control value (Table 7). Based 

upon these results, cultures treated with targeted concentrations of 0 (solvent control), 

25, 50, and 100 g/ml were chosen for the determination of chromosomal aberration 

frequencies and incidence of polyploidy. Cultures treated with 0.075 g/ml MMC 

were selected to serve as the positive control. 

There were no significant increases in the incidence of polyploid cells in any of the 

test material treated cultures as compared to the solvent control values (Table 8). 

The frequency of aberrant cells in the solvent control was 2.0% and the 

corresponding values at concentration levels of 25, 50, and 100 g/ml were 1.0, 0.5, 

and 1.0%, respectively (Table 11). There were no statistically significant differences 

between the test material treated cultures and the solvent control values and all values 

were within the laboratory historical background range (Table 12 and Table 13). 

A significant increase in the frequency of cells with aberrations was observed in 

cultures treated with the positive control chemical. Aberrant cell frequency in 

MMC treated cultures was 27.0% (Table 11). 

CONCLUSIONS 

It was concluded that under the experimental conditions used, was 

non-genotoxic in this in vitro chromosomal aberration test.

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STUDY ID: 

PAGE 20 

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ACKNOWLEDGEMENTS 

The authors gratefully acknowledge the contributions of the following individual to the 

conduct of the study and report preparation: 

 J. M. Smalley Genetic Toxicology Technologist 
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STUDY ID: 

PAGE 21 

REFERENCES 

Armitage, P. (1971). Statistical Methods in Medical Research, John Wiley & Sons, Inc., 

New York. 

Buckton, K. E. and Evans, H. J. eds. (1973). Methods for the Analysis of Human 

Chromosome Aberrations, World Health Organization, Geneva. 

Gollapudi, B. B., Sutcliffe, D. J., and Sinha, A. K. (1986). Assessment of Cytogenetic 

Response to Folic Acid Deprivation in Rat Lymphocytes. In Vitro 22, 681-684. 

O'Neill, J. P., Machanoff, R., San Sebastian, J. R., and Hsie, A. W. (1982). Cytotoxicity 

and Mutagenicity of Dimethylnitrosamine in Mammalian Cells (CHO/HGPRT 

system): Enhancement by Calcium Phosphate. Environ. Mutagen. 4, 7-18. 

Siegel, S. (1956). Non-Parametric Statistics for the Behavioral Sciences. McGraw-Hill, 

New York. 

Sinha, A. K., Linscombe, V. A., Gollapudi, B. B., McClintock, M. L., Flake, R. E., and 

Bodner, K. M. (1984). The Incidence of Spontaneous Cytogenetic Aberrations in 

Lymphocytes Cultured from Normal Humans for 48 and 72 hr. Can. J. Genet. 

Cytol. 26, 528-531. 

Sinha, A. K., Gollapudi, B. B., Linscombe, V. A., Bruce, R. J., and McClintock, M. L. 

(1985). Analyses for In Vitro Growth Conditions, Cytokinetics, and Sister 

Chromatid Exchanges in Lymphocytes Cultured from the Sprague-Dawley Rat. 

In Vitro 21, 583-587. 

Sinha, A. K., Bruce, R. J., and Gollapudi. B. B. (1988). Cytokinetics and SCE baselines 

in rat and human lymphocytes during successive divisions in the presence of 

different culture media. In Vitro 24, 927-930. 

Sinha, A. K., Gollapudi, B. B., Linscombe, V. A., and McClintock, M. L. (1989). 

Utilization of Rat Lymphocytes for the In Vitro Chromosomal Aberration Assay. 

Mutagenesis 4, 147-153. 

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EVALUATION OF IN AN IN VITRO CHROMOSOMAL ABERRATION ASSAY UTILIZING RA

Concentr

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Table 1. ation Check Report – Assay A1* 

Dose Level Date Mixed: 05/27/10 Overall Overall

mg/mL ACL Report #: 2010-79 Mean Std.Dev.

Target Conc. (mg/mL): 0.00

Control Mean Measured Conc: <LLQ

% o f Target Conc:

(n): (2)

Target Conc. (mg/mL): 0.160

0.16 Mean Measured Conc: 0.145

% o f Target Conc: 90.8% 90.8% NA

(n): (2)

Target Conc. (mg/mL): 0.310

0.31 Mean Measured Conc: 0.293

% o f Target Conc: 94.5% 94.5% NA

(n): (2)

Target Conc. (mg/mL): 0.630

0.63 Mean Measured Conc: 0.609

% o f Target Conc: 96.7% 96.7% NA

(n): (2)

Target Conc. (mg/mL): 1.25

1.25 Mean Measured Conc: 1.21

% o f Target Conc: 96.8% 96.8% NA

(n): (2)

Target Conc. (mg/mL): 2.50

2.50 Mean Measured Conc: 2.44

% o f Target Conc: 97.6% 97.6% NA

(n): (2)

Target Conc. (mg/mL): 5.00

5.00 Mean Measured Conc: 4.96

% o f Target Conc: 99.3% 99.3% NA

(n): (2)

Target Conc. (mg/mL): 10.0

10.00 Mean Measured Conc: 9.83

% o f Target Conc: 98.3% 98.3% NA

(n): (2)

Target Conc. (mg/mL): 20.0

20.00 Mean Measured Conc: 21.8

% o f Target Conc: 109.2% 109.2% NA

(n): (2)

LLQ . (mg/mL): 0.00404 0.00404 NA

<LLQ = less than lower limit of quantitationNA= Not Applicable

* The dose level indicated in this table refers to the stock treatment solutions. The actual treatment concentrations were 100-fold lower
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EVALUATION OF 

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Table 2.

IN AN IN VITRO CHROMOSOMAL ABERRATION ASSAY UTILIZING R

Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations of 

Assay A1 

Concentration

(μg/ml) 1 2 Average RMI a

1% DMSO 7.9 9.4 8.7 100.0

3.1 7.0 5.5 6.3 72.4

6.3 6.2 9.0 7.6 87.4

12.5 8.8 9.2 9.0 103.4

25 6.6 8.1 7.4 85.1

50b 4.6 5.5 5.1 58.6

100b 3.1 1.7 2.4 27.6200b 0.0 2.7 1.4 16.1

MMC 0.5 2.9 4.6 3.8 43.7

aRelative M itotic Index: Average of Treated X 100 

 Average of Solvent Control

b Cloudy in treatment medium.

Replicates

% M.I.

Treatment Duration: 4 hours
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EVALUATION OF 

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Table 3.

IN AN IN VITRO CHROMOSOMAL ABERRATION ASSAY UTILIZING R

Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations of 

Assay A1 

Concentration

(μg/ml) 1 2 Average RMI a

1% DMSO 5.6 9.6 7.6 100.0

3.1 5.7 6.5 6.1 80.36.3 6.8 7.5 7.2 94.7

12.5 6.3 10.5 8.4 110.5

25 5.1 2.6 3.9 51.3

50b 3.8 4.9 4.4 57.9

100b 2.6 3.6 3.1 40.8200b 0.2 0.0 0.1 1.3CP 2 5.7 3.9 4.8 63.2

CP 4 3.2 1.0 2.1 27.6

aRelative Mitotic Index: Average of Treated X 100 

 Average of Solvent Control

b Cloudy in treatment medium.

% M.I.

Replicates

Treatment Duration: 4 hours
THE DOW 

EVALUATION OF 

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Table 4.

IN AN IN VITRO CHROMOSOMAL ABERRATION ASSAY UTILIZING R

Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations of 

Assay A1 

Concentration

(μg/ml) 1 2 Average RMI a

1% DMSO 4.8 4.9 4.9 100.0

1.6 5.1 4.3 4.7 95.9

3.1 5.5 5.1 5.3 108.2

6.3 6.6 8.3 7.5 153.1

12.5 7.5 8.2 7.9 161.2

25 6.5 5.3 5.9 120.4

50b 4.5 4.4 4.5 91.8

100b 3.3 0.7 2.0 40.8200b 0.0 0.0 0.0 0.0

MMC 0.05 3.0 3.8 3.4 69.4

MMC 0.075 1.8 2.6 2.2 44.9

aRelative Mitotic Index: Average of Treated X 100 

 Average of Solvent Control

b Cloudy in treatment medium.

Replicates

% M.I.

Treatment Duration: 24 hours
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EVALUATION OF IN AN IN VITRO CHROMOSOMAL ABERRATION ASSAY UTILIZING RA

Table 5. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations of 

– Assay B1 

Concentration

(μg/ml) 1 2 Average RMI a

1% DMSO 12.7 13.5 13.1 100.0

12.5 9.7 11.6 10.7 81.7

25 11.1 9.3 10.2 77.9

50b 5.2 6.5 5.9 45.0

75b 1.6 2.0 1.8 13.7

100b 0.5 1.3 0.9 6.9150b 0.4 0.7 0.6 4.6200b 0.0 0.0 0.0 0.0

MMC 0.5 8.1 6.7 7.4 56.5

aRelative Mitotic Index: Average of Treated X 100 

 Average of Solvent Control

b Cloudy in treatment medium.

Replicates

% M.I.

Treatment Duration: 4 hours

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EVALUATION OF IN AN IN VITRO CHROMOSOMAL ABERRATION ASSAY UTILIZING RA

Table 6. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations of i

– Assay B1 

Concentration

(μg/ml) 1 2 Average RMI a

1% DMSO 0b 10.4 10.4 100.0

12.5 11.3 9.5 10.4 100.0

25 7.2 8.5 7.9 76.0

50c 7.7 6.9 7.3 70.2

75c 6.4 7.1 6.8 65.4

100c 5.0 6.3 5.7 54.8150c 4.6 5.1 4.9 47.1200c 2.8 1.9 2.4 23.1

 CP 2 6.0 6.6 6.3 60.6

CP 4 4.8 5.4 5.1 49.0

a Relative Mitotic Index: Average of Treated X 100 

 Average of Solvent Control

b Inadvertantly sample lost during harvest due to a technical error. Therefore, this replicate was excluc Cloudy in treatment medium.

% M.I.

Replicates

Treatment Duration: 4 hours

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THE DOW CHE

EVALUATION OF IN AN IN VITRO CHROMOSOMAL ABERRATION ASSAY UTILIZING RA

Table 7. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations of 

– Assay B1 

Concentration

(μg/ml) 1 2 Average RMI a

1% DMSO 8.8 10.4 9.6 100.0

12.5 8.0 7.7 7.9 82.3

25 8.3 7.2 7.8 81.3

50b 6.7 7.1 6.9 71.9

75b 5.8 4.9 5.4 56.3

100b 4.0 4.3 4.2 43.8150b 0.9 0.0 0.5 5.2

200b 0.0 0.0 0.0 0.0

MMC 0.05 5.4 6.7 6.1 63.5

MMC 0.075 3.9 4.6 4.3 44.8

aRelative M itotic Index: Average of Treated X 100 

 Average of Solvent Control

b Cloudy in treatment medium.

Replicates

% M.I.

Treatment Duration: 24 hours

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STUDY ID: 

PAGE 29 

EVALUATION OF IN AN IN VITRO CHROMOSOMAL 

ABERRATION ASSAY UTILIZING RAT LYMPHOCYTES 

Table 8. Incidence of Polyploidy (%PP) in Rat Lymphocyte Cultures Treated with Various 

Concentrations of in the Absence and Presence of S9 – Assay B1 

Concentration Average

(μg/ml) 1 2

1% DMSO 2.0 1.0 1.5

12.5 0.0 1.0 0.5

25 1.0 1.0 1.0

50b 1.0 0.0 0.5

0.5 MMC 0.0 0.0 0.0

Concentration Average

(μg/ml) 1 2

1% DMSO 1.0 0.0 0.5

25 0.0 0.0 0.0

75b 0.0 0.0 0.0

150b 2.0 1.0 1.5

 CP 4 0.0 1.0 0.5

Concentration Average

(μg/ml) 1 2

1% DMSO 1.0 0.0 0.5

25 1.0 1.0 1.0

50b 0.0 0.0 0.0

100b 1.0 0.0 0.5

MMC 0.075 1.0 0.0 0.5

a

Based on 100 Metaphases

b

Cloudy in treatment medium.

4 Hour With S9

%PPa

Replicates

4 Hour Without S9

Replicates

24 Hour Without S9

Replicates

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EVALUATION OF ATE NG R

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Table 9.

C CHOL IN AN IN VITRO CHROMOSOMAL ABERRATION ASSAY UTILIZI

Results of the Chromosomal Aberration Assay in the Absence of S9 – Assay B1 

(4 Hour Treatment) 

Conc entration R MI # of Cells Total

Cells with

Multiple

C

M is

(μg/ml) (% )a Evaluated Chromatid Ch romosome Chro matid Chromosome Chromatid Chromosome Aberration sb A berration s Ab

`

1% D MSO 1001 0 0 0 0 0 0 0 

1% D MSO 1000 0 1 0 0 0 1 0 

Totals 100.0 2001 0 1 0 0 0 1 0 

12.5 1001 0 0 0 0 0 0 0 

12.5 1001 0 3 0 0 0 3 0 

Totals 81.7 2002 0 3 0 0 0 3 0 

25 1000 0 1 0 0 0 1 0 25 1000 0 1 0 0 0 1 0 

Totals 77.9 2000 0 2 0 0 0 2 0 

50d 1000 0 1 1 0 0 2 0 

50d 1000 0 2 0 0 0 2 0 

Totals 45.0 2000 0 3 1 0 0 4 0 

MMC 0.5 50 1 0 8 2 15 0 25 0 

MMC 0 .5 50 2 0 12 1 13 0 26 5 

Totals 56.5 100 3 0 20 3 28 0 51 5 

aRMI=Relative mitotic in dex

bE xcluding Gap scValues in p arentheses are p ercen tages.dCloud y in treatment medium.eSignificantly (alpha= 0.0 5) different from solvent control.

Gaps Breaks Exchanges
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EVALUATION OF ATE R

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Table 10.

C CHOL IN AN IN VITRO CHROMOSOMAL ABERRATION ASSAY UTILIZING 

Results of the Chromosomal Aberration Assay in the Presence of S9 – Assay B1 

(4 Hour Treatment) 

Concentration RMI # of Cells Total

Cells with

Multiple

C

Mi

(μg/ml) (%)a Evaluated Chromatid Chromosome Chromatid Chromosome Chromatid Chromosome Aberrationsb Aberrations Ab

`

1% DMSO 200 1 0 3 0 1 0 4 0 

Totals 100.0 200 1 0 3 0 1 0 4 0 

25 100 0 0 0 0 0 0 0 0 

25 100 0 0 0 0 0 0 0 0 

Totals 76.0 200 0 0 0 0 0 0 0 0 

75d 100 1 0 1 0 0 0 1 0 

75d 100 1 0 1 0 0 0 1 0 

Totals 65.4 200 2 0 2 0 0 0 2 0 

150d 100 0 0 2 0 0 0 2 0

150d 100 1 0 2 0 0 0 2 0 

Totals 47.1 200 1 0 4 0 0 0 4 0 

CP 4 100 0 0 5 0 14 0 19 1 

CP 4 50 1 0 6 3 7 0 16 0 

Totals 49.0 150 1 0 11 3 21 0 35 1 

aRMI=Relative mitotic index

bExcluding GapscValues in parentheses are percentages.dCloudy in treatment medium.eSignificantly (alpha= 0.05) different from solvent control.

Gaps Breaks Exchanges
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Table 11.

C CHOL IN AN IN VITRO CHROMOSOMAL ABERRATION ASSAY UTILIZI

Results of the Chromosomal Aberration Assay in the Absence of S9 – Assay B1 

(24 Hour Treatment) 

Concentration RM I # of Cells Total

Cells with

Multiple

(μg/ml) (%) a Evaluated Chro matid Ch romosome Chro matid Chromosome Chro matid Chromosome Aberrationsb Aberrations 

1% D MSO 1001 0 2 1 0 0 3 0 

1% D MSO 1002 0 1 0 0 0 1 0 

Totals 100.0 200 3 0 3 1 0 0 4 0 

25 1001 0 1 0 0 0 1 0 

25 1001 0 1 0 0 0 1 0 

Totals 81.3 2002 0 2 0 0 0 2 0 

50d 1002 0 0 1 0 0 1 0 

50d 1000 0 0 0 0 0 0 0 

Totals 71.9 2002 0 0 1 0 0 1 0 

100d 1001 0 2 0 0 0 2 0 

100d 1001 0 0 0 0 0 0 0 

Totals 43.8 2002 0 2 0 0 0 2 0 

MMC 0 .075 502 0 8 0 7 0 150 

MMC 0 .075 502 0 8 0 110 190 

Totals 44.8 100 4 0 16 0 18 0 34 0 

aRMI=Relative mitotic in dex

bExcluding Gap scValues i n parentheses are p ercen tages.dCloudy in treatment medium.eSignificantly (alpha= 0.0 5) different from solvent control.

Gaps Breaks Exchanges
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STUDY ID: 

PAGE 33 

EVALUATION OF IN AN IN VITRO CHROMOSOMAL 

ABERRATION ASSAY UTILIZING RAT LYMPHOCYTES 

8

1

4

0

1

9

Table 12. Historical Background Aberration Frequencies – Descriptive Statistics 

S-9

Year (- or +) Mean Std. Dev. Minimum Maximum N b

2005 - 1.5 0.9 0.0 3.0 18

+ 2.2 0.5 1.5 3.0

2006 - 1.4 1.0 0.5 4.0 19

+ 1.8 0.8 0.5 3.0 1

2007 - 1.2 0.7 0.0 3.0 27

+ 1.3 0.7 0.0 3.0 1

2008 - 1.3 1.0 0.0 3.0 18

+ 1.1 0.7 0.0 2.5 1

2009 - 1.2 0.8 0.0 2.5 17

+ 1.4 1.0 0.0 3.0 1

2010 - 1.3 1.0 0.0 3.0 14

+ 1.0 0.6 0.5 2.0

a

 Frequencies are % cells with aberrations excluding gaps

b

 Number of experiments

FREQUENCIES a

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EVALUATION OF IN AN IN VITRO CHROMOSOMAL 

ABERRATION ASSAY UTILIZING RAT LYMPHOCYTES 

Table 13. Historical Background Aberration Frequencies – Individual Values 

S-9

Year (+ or -) Frequencya

 of Cells with Aberrations

2005 - 1.5, 2.0, 1.5, 0.0, 1.5, 0.5, 2.5, 3.0, 1.5, 1.5, 0.5, 2.0, 2.0, 3.0, 0.0, 1.0, 1.5, 1.5

+ 1.5, 2.5, 2.0, 2.5, 2.5, 2.0, 1.5, 3.0

2006 - 1.0, 3.5, 1.5, 1.0, 1.0, 1.0, 1.0, 1.5, 1.0, 1.0, 0.5, 1.5, 0.5, 4.0, 1.5, 0.5, 2.0, 0.5, 2.5

+ 2.0, 0.5, 2.5, 2.0, 1.0, 1.0, 1.0, 3.0, 2.0, 2.5, 2.0

2007 - 3.0, 1.5, 1.0, 1.5, 1.0, 1.0, 2.5, 0.0, 1.5, 0.5, 1.0, 1.0, 2.5, 0.5, 0.0 1.5, 2.0, 1.0, 2.0,

1.5, 1.0, 0.5, 1.0, 0.5, 1.5, 1.0

+ 1.5, 1.0, 1.0, 1.0, 0.5, 1.0, 1.5, 1.0, 1.5, 1.5, 0.0, 1.5, 3.0, 1.5

2008 - 0.5, 1.0, 0.0, 1.5, 1.0, 2.5, 0.5, 1.5, 0.5, 2.0, 0.0, 3.0, 2.5, 2.0, 2.5, 0.5, 2.0, 0.5

+ 1.0, 0.5, 1.0, 0.5, 1.5, 1.0, 0.0, 1.5, 2.5, 1.0

2009 - 1.0, 1.5, 1.5, 0.5, 2.5,1.5, 1.0, 0.0, 0.0, 2.0, 0.5, 2.0, 2.0, 0.5, 0.5, 1.5, 2.5

+ 1.0, 0.5, 1.0, 2.5, 2.0, 3.0, 0.0, 1.5, 0.0, 2.0, 1.5

2010 - 2.5, 0.5, 3.0, 0.0, 2.5, 0.5, 1.5, 0.5, 0.5, 1.5, 1.5, 0.5, 0.5, 2.0

+ 1.0, 1.5, 0.5, 1.0, 0.5, 0.5, 1.5, 0.5, 2.0

a

 Frequency is % cells with aberrations excluding gaps

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