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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 Nove mber 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

-

### SIGNATURE PAGE



### Compound: THE DOW CHEMICAL COMPANY STUDY ID: PAGE 4



### 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 LABORATO RY 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 Abe rration 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 Abe rration Assay in the Absence of S9 – Assay B1 ..............................................................................................32 Table 12. Historical Background Aberrati on Frequencies – Descriptive Statistics .......................................................................................................33 Table 13. Historical Background Aberration Fre quencies – Individual Values...34THE DOW CHEMICAL COMPANY STUDY ID: PAGE 8



### SUMMARY

was evaluated in an in vitro chromosomal aberration assay ut ilizing 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 trea ted 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 ch romosomal 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 trea ted with the positive control chemicals ( i.e.
, mitomycin C without S9 and cyclophospha mide 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 chro mosomal damage (clastogenicity) can be assayed in vitro using cultured mammalian cells. Am ong various cell systems that can be used for this purpose, cultu red rat lymphocytes have th e following advantages. The laboratory rat is widely used for toxicol ogical 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 envi ronmental factors on the endpoint being measured can be controlled. The karyotype of cultured rat lymphocytes (2N = 42) is stable as opposed to the relative kar yotypic 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 ly mphocytes have been developed in this laboratory ( Sinha et al. , 1985 ; Gollapudi et al. , 1986 ; Sinha et al. , 1988 ). The system
was validat ed in this laboratory by using va rious direct- and indire ct-acting clastogens and non-clastogens ( Sinha et al. , 1989 ). Analysis of the grow th characteristics suggested that rat lymphocytes in culture have an average generation time of approxima tely 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 exte rnal m etabolic activation system (S9) was
also used to facilitate the identif icatio n of agents requiring biotransformation for clastogenic activity.

Test Guidelines OECD Guidelines for testing of chemicals, #473: In vitro mammalian chrom osome aberration test, OECD publication service, 2, rue AndrePascal, 75775 Paris Cedex 16, France (1997).

USEPA Health effects testi ng 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 A ssurance Unit, Toxicology & E nvironmental 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 C onsulting archivist and stored at The Dow
Chemical Com pany, Midland, Michigan.
Safety
All personnel involved in the study were advise d of the safety precautions to follow when handling the test and positive control materials. Chemical safety inf ormation 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 (Metho d of Analysis and Reference)
The purity of the test ma terial was determined to be gas chrom atography with identification by Fourie r transfor m infrared spectroscopy and gas chromatography m ass 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 Cr l:CD(SD)) rats were selected because of their general acceptance and suitability for toxi city testing, availability of historical background data and the reliability of the commer cial 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 ve terinarian, or a trai ned animal/toxicology
technician u nder the direct supervision of a laboratory ve teri narian, to determine the general health status and acceptability for study purposes upon arrival at the laboratory (fully accredited by the Asso ciation for Assessment and Accreditation of Laboratory
Animal Care International - AAALAC Interna tional). The animals were housed two per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and accl imated 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 alphanum eric 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 performe d by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of sel ected contaminants. Drinking water obtained from the municipal water sour ce was periodically analyzed for chemical parameters and biological contaminants by th e 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 W elfare 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 act ivities were in full
accordance with these Final Rule s. 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 (GI BCO, 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 (HA 16, Murex Diagnostics Ltd., Dartford, England), and an additional 2 mM L-glutam ine (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 fl asks and incubated at
37°C.

Controls
The solvent selected for dissolving the test ma terial (dimethyl sulfoxide (DMSO)) was
used as the solvent control treatment. Mito mycin 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 monohydr ate (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 Ar oclor 1254 treated (500 mg/kgBW) male Crl:CD(SD) rats was purchased from a comm ercial 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 m ix consisted of 10 mM MgCl 2 

### ·6H 2 

### O (Sigma), 5 mM glucose-6-phosphate (Sigma), 4 mM nicotinamide adenine dinucleotide phosphate (Sigma), 10 mM CaCl 2 

### (Fisher, Fair Lawn, New Jersey), 30 mM KCl (Sigma), and 50 mM sodium phos phate (pH 8.0, Sigma and Fisher). The
reconstituted mix was added to the cultu re 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 fr esh 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 ob tain 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 mate rial in the stock dosing solutions used for treatment in Assay A1 were verified by th e Analytical Chemistry Laboratory, Toxicology
& Environmental Research and Consulti ng, The Dow Che mical 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-adhe sive labels containing a code system that identified the test material, experime nt 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 cen trifuge 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 e xposed 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 wa s terminated by washing the cells with culture medium. The cells were then placed in individual sterile disposable tissue culture flasks (T-25) along with approximate ly 4.5 ml of the orig inal 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 continuous ly with the test material for 1.5X normal cell cycle length. The solvent control, positive control, and test ma terial were added directly

–THE DOW CHEMICAL COMPANY STUDY ID: PAGE 15



### 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 Met abolic 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 centr ifuge tubes (5.5 ml/tube, two cu ltures per dose level). The cells were sedimented by centrifugation and th e 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 wash ing the cells with cu lture medium. The cells were then placed in indi vidual 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 concentra tions of the test mate rial and the selected concentration of the positive cont rol chemical s. The highest concentration of the test material assayed was based on a limit of solubi lity of the test material in the treatment
medium. The other concentrations tested we re 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 methano l: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 a nd expressed as percentages.
Initially, slides from the s hort treatment (with and wit hout S9) were evaluated for cytogenetic analysis. Slides from the con tinuous treatment without S9 were evaluated only when the results with short treatment yielded negative findings. Slides from the solvent controls, positiv e controls, and three concentratio ns of the test material were selected for cytogenetic analysis.
One hundred metaphases/replicat e 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 structur al 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 meta phases 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 accu rate 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 disinteg ration, chromosomal pulverization, etc.). Those cells having five or more aberrations/c ell 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 me taphases/replicate were ex amined 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 aberra tions (excluding gaps, miscellane ous and severely damaged) x 100 # metaphases evaluated



### Evaluation Criteria
For a test to be acceptable, the chromosoma l 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 reasona ble 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 incide nt 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 leve l, data from the replicates were pooled. A two-way contingency table wa s constructed to analyze th e frequencies of aberrant cells. An overall Chi-square statistic, base d on th e table, was partitioned into components of interest. Specifically, stat istics were generated to te st the global hypothesis of no difference in the average number of ce lls with aberrations among the dose groups ( Armitage, 1971 ). An ordinal metric (0, 1, 2, etc.) wa s 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 test s ( i.e. control vs. treatment) were performed at each dose level and evaluated at alph a = 0.05, again versus a one-sided alternative. If any of

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### the pairwise tests were signifi cant, a test for linear trend of increasing number of cells
with aberrations with incr easing dose was performed ( Armitage, 1971 ).
Polyploid cells were analyzed by th e Fisher Exact probability test ( Siegel, 1956 ). The number of polyploid cells were pooled across replicates for th e analysis and evaluated at alpha = 0.05. The data was analyzed separate ly based on the presence or absence of S9 and based on the exposure time.

RESULTS AND DISCUSSION
pH and Osmolality
The pH and osmolality o f treatment medium contain ing 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 th e pH or osmolality at this concentration as compared to the culture medium with solv ent alone (culture me dium with the test material, pH = 7.43, osmolality = 445 mOsm/kg H 2 

### 0; culture medium with 1% DMSO, pH = 7.42, osmolality = 444 mOsm/kg H 2 

### 0).
Assay A1
In the initial assay, cultures were treated with the test material in the ab sence and presence of S9 activation for 4 hours at conc entrations 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 cu ltures in Assay A1 had poor cell growth as indicated by less than no rm al total lymphocytes present. This observation is not reflected
in the mitotic indic es 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 A ssay 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 tr eatment) 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 displaye d excessive toxicity at the three highest concentrations (100, 150, and 200 μg/ml) while the remaining concentrations had
relative mitotic indi ces ranging from 13.7 to 81.7% comp ared to the solvent control values ( Table 5 ). In the presence of S9, the m itotic 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 concentra tions 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 dete rmination 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 se lected 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 comp ared 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 valu es at concentration levels of 12.5, 25, and 50 μg/ml were 1.5, 1.0, and 2.0%, resp ectively. In the presence of S9 ( Table
10 ), cultures treated with the test m aterial at concentrations of 25, 75, and 150 μg/ml had aberrant cell frequencie s of 0.0, 1.0, and 2.0%, respectively as

com pared to the solvent contro l value of 2.0%. Statistical analyses of these data did not identify significan t 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 ch emicals. 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 yielde d clearly nega tive results.

Continuous Treatme nt (24 hour treatment, Assay B1)
Based upon the negative findings in the 4 hour treatment in the absence of metabolic activation, slides from the continuou s 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 target ed concentrations of 0 (solvent control), 25, 50, and 100  g/ml were chosen for the determ ination of chromosomal aberration frequencies and incidence of pol yploidy. 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 comp ared to the solvent control values ( Table 8 ).
The frequency of aberrant cells in the solvent control was 2.0% and the corresponding values at concen tration levels of 25, 50, and 100  g/ml were 1.0, 0.5, and 1.0%, respectively ( Table 11 ). There were no statisti cally significant differences
between the test material treated cultures a nd 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 us ed, was non-genotoxic in this in vitro chromosomal aberration test.

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– 

### ACKNOWLEDGEMENTS
The authors gratefully acknowledge the contri butions of the following individual to the conduct of the study and report preparation:
J. M. Smalley Genetic Toxicology TechnologistTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 21



### REFERENCES
Armitage, P. (1971). Statistical Methods in Medical Resear ch, 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 Depriv ation 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 Dimethylnitros amine 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 Cu ltured from the Spra gue-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 succes sive 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.

–THE DOW CHEMICAL COMPANYSTUDY ID:PAGE 22EVALUATION OFIN ANIN VITROCHROMOSOMAL ABERRATION ASSAY UTILIZING RAT LYMPHOCYTESConcentr–Table 1.ation Check Report – Assay A1*Dose LevelDate Mixed: 05/27/10Overall Overallmg/mLACL Report #: 2010-79Mean Std.Dev.Target Conc. (mg/mL):0.00Control Mean Measured Conc:<LLQ% o f Target Conc:(n): (2)Target Conc. (mg/mL):0.1600.16 Mean Measured Conc:0.145% o f Target Conc: 90.8%90.8% NA(n): (2)Target Conc. (mg/mL):0.3100.31 Mean Measured Conc:0.293% o f Target Conc: 94.5%94.5% NA(n): (2)Target Conc. (mg/mL):0.6300.63 Mean Measured Conc:0.609% o f Target Conc: 96.7%96.7% NA(n): (2)Target Conc. (mg/mL):1.251.25 Mean Measured Conc:1.21% o f Target Conc: 96.8%96.8% NA(n): (2)Target Conc. (mg/mL):2.502.50 Mean Measured Conc:2.44% o f Target Conc: 97.6%97.6% NA(n): (2)Target Conc. (mg/mL):5.005.00 Mean Measured Conc:4.96% o f Target Conc: 99.3%99.3% NA(n): (2)Target Conc. (mg/mL):10.010.00 Mean Measured Conc:9.83% o f Target Conc: 98.3%98.3% NA(n): (2)Target Conc. (mg/mL):20.020.00 Mean Measured Conc:21.8% o f Target Conc: 109.2%109.2% NA(n): (2)LLQ . (mg/mL):0.004040.00404NA<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.THE DOW CHEMICAL COMPANYSTUDY ID:PAGE 23EVALUATION OF–Table 2.IN ANIN VITROCHROMOSOMAL ABERRATION ASSAY UTILIZING RAT LYMPHOCYTESMitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations ofin the Absence ofS9 –Assay A1Concentration(μg/ml) 1 2 Average RMIa1% DMSO7.99.48.7100.03.17.05.56.372.46.36.29.07.687.412.58.89.29.0103.4256.68.17.485.150b4.65.55.158.6100b3.11.72.427.6200b0.02.71.416.1MMC 0.52.94.63.843.7aRelative M itotic Index:Average of Treated X 100Average of Solvent ControlbCloudy in treatment medium.Replicates% M.I.Treatment Duration: 4 hoursTHE DOW CHEMICAL COMPANYSTUDY ID:PAGE 24EVALUATION OF–Table 3.IN ANIN VITROCHROMOSOMAL ABERRATION ASSAY UTILIZING RAT LYMPHOCYTESMitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations of in the Presence of S9 –Assay A1Concentration(μg/ml) 1 2 Average RMIa1% DMSO5.69.67.6100.03.15.76.56.180.36.36.87.57.294.712.56.310.58.4110.5255.12.63.951.350b3.84.94.457.9100b2.63.63.140.8200b0.20.00.11.3CP 25.73.94.863.2CP 43.21.02.127.6aRelative Mitotic Index: Average of Treated X 100Average of Solvent ControlbCloudy in treatment medium.% M.I.ReplicatesTreatment Duration: 4 hoursTHE DOW CHEMICAL COMPANYSTUDY ID:PAGE 25EVALUATION OF–Table 4.IN ANIN VITROCHROMOSOMAL ABERRATION ASSAY UTILIZING RAT LYMPHOCYTESMitoticIndices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations ofin the Absence ofS9 –Assay A1Concentration(μg/ml) 1 2 Average RMIa1% DMSO4.84.94.9100.01.65.14.34.795.93.15.55.15.3108.26.36.68.37.5153.112.57.58.27.9161.2256.55.35.9120.450b4.54.44.591.8100b3.30.72.040.8200b0.00.00.00.0MMC 0.053.03.83.469.4MMC 0.0751.82.62.244.9aRelative Mitotic Index: Average of Treated X 100Average of Solvent ControlbCloudy in treatment medium.Replicates% M.I.Treatment Duration: 24 hoursTHE DOW CHEMICAL COMPANYSTUDY ID:PAGE 26EVALUATION OFIN ANIN VITROCHROMOSOMAL ABERRATION ASSAY UTILIZING RAT LYMPHOCYTESTable 5.MitoticIndices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations ofin the Absence ofS9– Assay B1Concentration(μg/ml) 1 2 Average RMIa1% DMSO12.713.513.1100.012.59.711.610.781.72511.19.310.277.950b5.26.55.945.075b1.62.01.813.7100b0.51.30.96.9150b0.40.70.64.6200b0.00.00.00.0MMC 0.58.16.77.456.5aRelative Mitotic Index: Average of Treated X 100Average of Solvent ControlbCloudy in treatment medium.Replicates% M.I.Treatment Duration: 4 hours–THE DOW CHEMICAL COMPANYSTUDY ID:PAGE 27EVALUATION OFIN ANIN VITROCHROMOSOMAL ABERRATION ASSAY UTILIZING RAT LYMPHOCYTESTable 6.MitoticIndices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations of in the Presence of S9– Assay B1Concentration(μg/ml) 1 2 Average RMIa1% DMSO0b10.410.4100.012.511.39.510.4100.0257.28.57.976.050c7.76.97.370.275c6.47.16.865.4100c5.06.35.754.8150c4.65.14.947.1200c2.81.92.423.1CP 26.06.66.360.6CP 44.85.45.149.0aRelative Mitotic Index:Average of Treated X 100Average of Solvent ControlbInadvertantly sample lost during harvest due to a technical error. Therefore, this replicate was excluded from the average.cCloudy in treatment medium.% M.I.ReplicatesTreatment Duration: 4 hours–THE DOW CHEMICAL COMPANYSTUDY ID:PAGE 28EVALUATION OFIN ANIN VITROCHROMOSOMAL ABERRATION ASSAY UTILIZING RAT LYMPHOCYTESTable 7.MitoticIndices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations ofin the Absence ofS9– Assay B1Concentration(μg/ml) 1 2 Average RMIa1% DMSO8.810.49.6100.012.58.07.77.982.3258.37.27.881.350b6.77.16.971.975b5.84.95.456.3100b4.04.34.243.8150b0.90.00.55.2200b0.00.00.00.0MMC 0.055.46.76.163.5MMC 0.0753.94.64.344.8aRelative M itotic Index:Average of Treated X 100Average of Solvent ControlbCloudy in treatment medium.Replicates% M.I.Treatment Duration: 24 hours–THE DOW CHEMICAL COMPANY STUDY ID: PAGE 29

EVALUATION OF IN AN IN VIT R O CHROMOSOMAL ABERRATION ASSAY UTILIZING RAT LYMPHOCYTES

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

50 b 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

75 b 0.0 0.0 0.0

150 b 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

50 b 0.0 0.0 0.0

100 b 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 %PP a

Replicates 4 Hour Without S9

Replicates

24 Hour Without S9 Replicates

–THE DOW CHEMICAL COMPANYSTUDY ID:PAGE 30EVALUATION OFATENG RAT LYMPHOCYTES–Table 9.C CHOL IN ANIN VITROCHROMOSOMAL ABERRATIONASSAY UTILIZIResults of the Chromosomal Aberration Assay in the Absence of S9 – Assay B1(4 Hour Treatment)Conc entrationR MI # of CellsTotalCells withMultipleCells WithM iscellaneousTotalAberrant(μg/ml)(% )aEvaluated Chromatid Ch romosome Chro matid Chromosome Chromatid Chromosome Aberration sbA berration s Aberration sCellsc`1%DMSO 1001 0 0 0 0 0 0 0 0 01%DMSO 1000 0 1 0 0 0 1 0 0 1Totals 100.0 2001 0 1 0 0 0 1 0 0 1(0.5)12.5 1001 0 0 0 0 0 0 0 0 012.5 1001 0 3 0 0 0 3 0 0 3Totals 81.7 2002 0 3 0 0 0 3 0 0 3(1.5)25 1000 0 1 0 0 0 1 0 0 125 1000 0 1 0 0 0 1 0 0 1Totals 77.9 2000 0 2 0 0 0 2 0 0 2(1.0)50d1000 0 1 1 0 0 2 0 0 250d1000 0 2 0 0 0 2 0 0 2Totals 45.0 2000 0 3 1 0 0 4 0 0 4(2.0)MMC 0.550 1 082 15 025 0019MMC 0.5 50 2 0 12 1 13 0 26 5 0 21Totals 56.5 100 3 0 20 3 28 0 51 5 040 (40.0)eaRMI=Relative mitotic in dexbE xcluding Gap scValues in p arentheses are p ercen tages.dCloud y in treatment medium.eSignificantly (alpha= 0.0 5) different from solvent control.GapsExchangesBreaksTHE DOW CHEMICAL COMPANYSTUDY ID:PAGE 31EVALUATION OFATERAT LYMPHOCYTES–Table 10.C CHOL IN ANIN VITROCHROMOSOMAL ABERRATION ASSAY UTILIZINGResults of the Chromosomal Aberration Assay in the Presence of S9 – Assay B1(4 Hour Treatment)ConcentrationRMI # of CellsTotalCells withMultipleCells WithMiscellaneousTotalAberrant(μg/ml)(%)aEvaluated Chromatid Chromosome Chromatid Chromosome Chromatid Chromosome AberrationsbAberrations AberrationsCellsc`1% DMSO200 1 0 3 0 1 04 004Totals 100.0 200 1 0 3 0 1 04 00 4 (2.0)25100 0 0 0 00 00 00025100 0 0 0 00 00 000Totals 76.0 200 0 0 0 0 0 00 00 0 (0.0)75d100 1 0 1 0 0 01 00175d100 1 0 1 0 0 01 001Totals 65.4 200 2 0 2 0 0 02 00 2 (1.0)150d1000020002002150d100 1 0 2 0 0 02 002Totals 47.1 200 1 0 4 0 0 04 00 4 (2.0)CP 4100 0 0 5 0 14 0 19 10 12CP 4 50 1 0 6 3 7 0 16 0 0 13Totals 49.0 150 1 0 11 3 21 0 35 1025 (16.7)eaRMI=Relative mitotic indexbExcluding GapscValues in parentheses are percentages.dCloudy in treatment medium.eSignificantly (alpha= 0.05) different from solvent control.GapsBreaksExchangesTHE DOW CHEMICAL COMPANYSTUDY ID:PAGE 32EVALUATION OFATENG RAT LYMPHOCYTES–Table 11.C CHOL IN ANIN VITROCHROMOSOMAL ABERRATIONASSAY UTILIZIResults of the Chromosomal Aberration Assay in the Absence of S9 – Assay B1(24 Hour Treatment)ConcentrationRM I # of CellsTotalCells withMultipleCells WithMiscellaneousTotalAberrant(μg/ml)(%)aEvaluated Chro matid Ch romosome Chro matid Chromosome Chro matid Chromosome AberrationsbAberrations AberrationsCellsc1%DMSO 1001 0 2 1 0 0 3 0 0 31%DMSO 1002 0 1 0 0 0 1 0 0 1Totals 100.0 200 3 03100400 4 (2.0)25 1001 0 1 0 0 0 1 0 0 125 1001 0 1 0 0 0 1 0 0 1Totals 81.3 2002 0 2 0 0 0 2 0 02(1.0)50d1002 0 0 1 0 0 1 0 0 150d1000 0 0 0 0 0 0 0 0 0Totals 71.9 2002 0 0 1 0 0 1 0 01(0.5)100d1001 0 2 0 0 0 2 0 0 2100d1001 0 0 0 0 0 0 0 0 0Totals 43.8 2002 0 2 0 0 0 2 0 02(1.0)MMC0.075 502 0 8 0 7 0 150 0 12MMC0.075 502 0 8 0 110 190 0 15Totals 44.8 100 4 0 16 0 18 0 34 0 0 27 (27.0)eaRMI=Relative mitotic in dexbExcluding Gap scValues i n parentheses are p ercen tages.dCloudy in treatment medium.eSignificantly (alpha= 0.0 5) different from solvent control.GapsBreaksExchangesTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 33

EVALUATION OF IN AN IN VIT R O CHROMOSOMAL ABERRATION ASSAY UTILIZING RAT LYMPHOCYTES



### 8

1

4

0

1

9 Tabl e 12

. Hi st ori cal B a ckground 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

–THE DOW CHEMICAL COMPANY STUDY ID: PAGE 34

EVALUATION OF IN AN IN VIT R O CHROMOSOMAL ABERRATION ASSAY UTILIZING RAT LYMPHOCYTES

Tabl e 13

. Hi st ori cal B a c kground Aberration Frequencies – Individual Values

S-9 Year (+ or -) Frequency a 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

–