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### STUDY TITLE

EVALUATION OF IN THE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANINE -PHOSPHORIBOSYL TRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAY

Test Guidelines USEPA OPPTS 870.5300 (1998)

OECD, Guideline 476 (1997) EC, B.17 (2008)

Author(s)

Study Completion Date

Sponsor
The Dow Chem ical Company

Midland, Michigan 48674

Performing Laboratory Toxicology & Environmental Research and Consulting The Dow Chemical Company

Midland, Michigan 48674

Laboratory Project Study ID

-THE DOW CHEMICAL COMPANY STUDY ID: PAGE 2

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS

Compound:

Title: EVALUATION OF IN THE CHINESE HAMSTER OVARY CELUHYPOXANTHINE-GUANINEPHOSPHORIBOSYL TRANSFERASE (CHOIHGPRT) FORWARD MUTATION ASSAY

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 I. OECD

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

-'..:1

# ~=.-_S..L.- 9 0

# ~ 0 (Date)THE DOW CHEMICAL COMPANY STUDY ID: 101 DR PAGE 3

QUALITY ASSURANCE STATEMENT

Compound:

Title: EV ALUA nON OF IN THE CHINESE HAMSTER OVARY CELUHYPOXANTHINE-GUANINEPHOSPHORIBOSYL TRANSFERASE (CHOIHGPRT) FORWARD MUTATION ASSAY

This study was examined for conformance 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: 08 September 20 I 0

TYPE OF AUDIT:
Final protocol Study conduct
Protocol, data, and draft report DATE OF AUDIT:

14 September 20 I 0

27 September 20 I 0

02-07 December 2010 DATE FINDINGS REPORTED TO STUDY DIRECTORJMANAGEMENT:
14 September 20 I 0

27 September 20 I 0

08 December 2010

Final report 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: EVALUATION OF IN THE CHINESE HAMSTER OVARY CELUHYPOXANTHINE-GUANINEPHOSPHORIBOSYL TRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAY



# ld1Q/1O (Date)

Reviewed by:

USEPA TSCA 8(e)IFIFRA 6(a)(2) ASSESSMENT..**i~1** This test material has b<:en reviewed for possible TSCA 8(e)IFIFRA 6(a)(2) submission 

### q 110 InitialslDate**~**THE DOW CHEMICAL COMPANY STUDY ID: PAGE 5



### TABLE OF CONTENTS
Page


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

COMPLIANCE WITH GOOD LABORA TORY PRACTICE STANDARDS..................2
QUALITY ASSURANCE STATEMENT ..........................................................................3

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

SUMMARY.........................................................................................................................7

INTRODUCTION ...............................................................................................................8 Principle of the Test System ...........................................................................................8 Purpose............................................................................................................................8 Test Guidelines ...............................................................................................................8 Quality Assurance ...........................................................................................................9 Archiving ........................................................................................................................9 Safety ..............................................................................................................................9

TEST MATERIAL INFORMATION .................................................................................9 Test Material Name.........................................................................................................9 Chemical Name...............................................................................................................9 Synonyms........................................................................................................................9 Supplier, City, State (L ot, Reference Number)...............................................................9 Purity/Characterization (Method of Analysis and Reference)........................................9 Characteristics...............................................................................................................10

Molecular Formula .....................................................................................................10 Molecular Weight.......................................................................................................10 Chemical Structure .....................................................................................................10 CAS Number ..............................................................................................................10

STUDY DESIGN...............................................................................................................10 Indicator Cells ...............................................................................................................1 0 Media ............................................................................................................................11 Controls....................................................................................................................... ..11 Treatment Procedure .....................................................................................................11 Identification of the Test System ..................................................................................11 Preparation of the Treatment Solution ..........................................................................11 Analytical Verificati on of Dosing Solutions.................................................................12 In Vitro Metabolic Activation .......................................................................................12 Toxicity Assay ..............................................................................................................12 Selection of Dose Levels fo r the Gene Mutation Assay ...............................................13 Gene Mutation Assay....................................................................................................13 Evaluation Criteria ........................................................................................................14 Statistical Analysis........................................................................................................14

RESULTS AND DISCUSSION........................................................................................15

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### TABLE OF CONTENTS
Page

- 

### pH and Osmolality ........................................................................................................15 Assay A1 – Preliminary Toxicity Assay.......................................................................15 Assay B1 – Initial Mutagenicity Assay.........................................................................15 Assay C1 – Confirmatory Mutagenicity Assay ............................................................16

CONCLUSIONS................................................................................................................16

ACKNOWLEDGEMENTS...............................................................................................16

REFERENCES ..................................................................................................................17

TABLES ............................................................................................................................19 Table 1. Survival of CHO Cells Treated wi th – Assay A1 19 Table 2. Results of the Gene Mutation A ssay in CHO Cells Treated with in the Absence of S9 – Assay B1..................................20 Table 3. Results of the Gene Mutation A ssay in CHO Cells Treated with in the Presence of S9 – Assay B1 .................................21 Table 4. Results of the Gene Mutation A ssay in CHO Cells Treated with in the Absence of S9 – Assay C1..................................22 Table 5. Results of the Gene Mutation A ssay in CHO Cells Treated with in the Presence of S9 – Assay C1 .................................23 Table 6. Historical Laboratory Backgr ound Values of 6-Thioguanine Resistant (TG r 

### ) Mutant Frequency in CHO Cells – Individual Values ............24 Table 7. Historical Laboratory Backgr ound Values of 6-Thioguanine Resistant (TG r 

### ) Mutant Frequency in CHO Cells – Descriptive Statistics ............................................................................................................25 Table 8. Concentration Check Report – Assay B1 ....................................................26THE DOW CHEMICAL COMPANY STUDY ID: PAGE 7



### SUMMARY

was evaluated in the in vitro Chinese Hamster Ovary cell/hypoxanthine-guaninephosphoribosyl transferase (CHO/HGPRT) forwar d gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 10 to 200 μg/ml in the absence of S9 and from 2.5 to 200 μg/ml in the presence of S9. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The analytic ally determined concentrations of the test material in the dosing preparation used for the initial mutagenici ty assay ranged from 94.4 to 101.8% of the targeted values. The ad equacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the ab sence of S9 and 20-methylcholanthrene for assays in the presence of S9. Solvent contro l cultures were treated with the solvent used to dissolve the test material ( i.e. dimethyl sulfoxide). The results of the in vitro CHO/HGPRT forward gene mutation assay with indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activ ation (S9) system.

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### INTRODUCTION
Principle of the Test System
Hypoxanthine-guanine-phosphoribosyl transfer ase (HGPRT) is a cellular enzyme that utilizes the substrates 5-phosphoribosyl-1-p yrophosphate and hypoxanthine or guanine to catalyze the synthesis of inosine- or gu anosine-monophosphate. In mammalian cells, purine nucleotides are prim arily synthesized de novo and the above pathway is a salvage pathway in the biosynthesis of th ese nucleotides. The gene for HGPRT is located on the mammalian X-chromosome. X-linked genes ar e hemizygous in females because of the fact that only one of the two X-chromosomes is genetically active. Hence, conversion of
HGPRT + 

### to HGPRT - 

### phenotype requires only a singl e mutational event. A mutation leading to the loss of HGPRT activity re nders the cells incapable of utilizing hypoxanthine, guanine, or their analogs - thi oguanine (TG) or azaguanine (AG). The wild type cells when grown in medium containing TG cannot survive because the incorporation into DNA of the nucleotides formed from TG is lethal to the cell. On the other hand, mutants deficient in HGPRT activ ity would grow in the presence of these analogs because they are unable to form th e lethal nucleotide analogs. Hence cells capable of performing clonal growth in th e presence of the toxic purine analogs are
assumed to have mutated from HGPRT + 

### to HGPRT - 

### phenotype. The Chinese Hamster Ovary (CHO) cell line, CHO-K 1 

### -BH 4 

### is suited for determining forward mutations at the
HGPRT locus because it is a well char acterized and validated system ( Hsie et al. , 1981 ; O'Neill et al. , 1977a,b

, 1979 ). These cells have a high cl oning efficiency, rapid doubling time (12-14 hours), and a low spontaneous m utation frequency (1-20 mu tants per 10 6


### cells).

These cells also show a dose-re sponse with a variety of phys ical and chemical mutagens and a genetic basis for the presumptive mu tational event has been well established.

Purpose
The purpose of this study was to determine the potential of to induce gene mutations at the HGPRT locus of Chinese Hamster Ovary (CHO) cells in
culture.

Test Guidelines OECD Guidelines for testing of chemicals, #476: In vitro ma mmalian gene mutation tests, OECD Publication Service, 2, rue Andre-Pascal, 75775 Paris Cedex 16, France (1997).

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### USEPA United States Environmental Prot ection Agency, Health Effects testing guidelines, OPPTS 870.5300 - ( In Vitro Mammalian Cell Gene Mutation Test), EPA712-C-98-221, August 1998.

EC B.17, Mutagenicity - In vitro mammalian cell gene mutation test. Official Journal of the European Communities, L142, 31/05/2008.
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 archiv ist and st ored at The Dow Chemical Company, 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. Ch emical safety information was made
available.

TEST MATERIAL INFORMATION

Test Material Name

Chemical Name

Synonyms

Supplier, City, State (Lot, Reference Number)

The Dow Chem ical Company, Midland, Michigan (Lot#
Purity/Characterization (Metho d of Analysis and Reference)
The purity of the test m aterial was determined to be
chrom atography with flam e ionization detection (GC-FID) and Karl Fi scher titration with

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### identification by Fourier Transform-In frared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS)

Characteristics

Molecular Formula

Molecular Weight

CAS Number

STUDY DESIGN

Indicator Cells

The cell line CHO-K 1 

### -BH 4 

### , originally obtained from Dr . Abraham Hsie, Oak Ridge National Laboratory, Oak Ridge, Tennessee, was used in this study. Stock cultures were
stored at about -80°C or below. The culture s were periodically checked for mycoplasma contamination (American Type Culture Collect ion, Manassas, Virginia). The cells were grown as monolayer cultures in plastic disposable tissue culture labware under standard conditions of approximately 5% CO 2 

### in air at 37  C in a humidified incubator.

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### Media
The cells were routinely maintained in Ha m's F-12 nutrient mix supplemented with 5% (v/v) heat-inactivated (56°C, 30 minutes), dialyzed fe tal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/ml; stre ptomycin sulfate, 0.1 mg/ml; fungizone, 25 μg/ml), and an additional 2 mM L-glutamine. The selection medium used for the
detection of HGPRT

- mutants was Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 μM 6-thioguanine, 5% serum, and the above-mentioned
antibiotics.

Controls

The solvent used to dissolve the test m aterial ( i.e. dimethyl sulfoxide (DMSO)) was used as the solvent control treatment. Ethyl methanesulfonate (EMS, CAS No. 62-50-0) was used as the positive control for the non-activat ion system (without S9 factor) at a final
concentration of 621  g/ml. The positive control for assays performed with S9 (activation system) was 20-methylcholan threne (20-MCA, CAS No. 56-49-5) at
concentrations of 4 and 12  g/ml. The dose levels of EMS and 20-MCA were based upon our unpublished findings.

Treatment Procedure
Cells in logarithmic growth phase were trypsinized and placed in medium containing 5%

serum at a standard density of 3.0 x 10 6 

### cells/T-75 flask approximately 24 hours prior to treatment. At the time of treatment, the cu lture medium was replaced with serum-free medium, S9 mix (when applicable ), and the test chemical, or the solvent control, or the positive control chemical. The cells were tr eated for approximately 4 hours at 37°C and the exposure was terminated by washing the cells with Dulbecco’s phosphate buffered saline (Ca ++ 

### and Mg ++ 

### free).
Identification of the Test System

All test cultures were identified usin g self a dhesive labels containing a code system that identified the test material, experime nt number, treatment, and replicate.
Preparation of the Treatment Solution

All test material solu tions 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 medium to obtain the desired concentrations. EMS

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### was dissolved in treatment medium. 20-MCA was dissolved first in DMSO and further
diluted in the culture medium. All dos ing units were expressed in μg/ml.
Analytical Verification of Dosing Solutions

The selected concentrations of the test mate rial in the stock dosing solutions used for treatment in Assay B1 were verified by th e Analytical Chemistry La boratory, Toxicology
& Environmental Research and Consulti ng, 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).

In Vitro Metabolic Activation
S9 liver homogenates prepared from Aroc lor 1254-induced male Sprague-Dawley rats were purchased from a comm ercial source (Molecular Toxicology, Inc., Boone, North Carolina) and stored at approximately -80°C or below. Thawed S9 was reconstituted at a
final concentration of 10% (v/v) in a “mix” ( O’Neill et al. , 1982 ). The S9 mix consisted of the following co-factors: 10 mM MgCl 2 

### ·6H 2 

### O, 5 mM glucose-6-phosphate, 4 mM
nicotinamide adenine dinucle otide phosphate, 10 mM CaCl 2 

### , 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the culture medium to
obtain the desired final concentr ation of S9 in the culture, i.e. , 2% v/v. Hence, the final
concentration of the co-factors in the culture medium is 1/5 th 

### of the concentrations stated
above.
Toxicity Assay
The cytotoxicity of the test material was a ssesse d by determining the ability of the treated cells to form colonies. This assay was conduc ted for selecting concentrations of the test material to be used in the gene mutation assay. Cells were seeded into T-25 flasks (1.0 x
10 6 

### cells/flask) approximately 24 hours prio r to treatment. Treatment was for approximately 4 hours with various concentrations of the test material with one replicate per dose in the presence and absence of S9 f actor. After termination of treatment, the cells were trypsinized and replated at a density of 200 cells/dish into 60 mm dishes (three/dose) and the dishes incubated for 6 da ys to allow colony formation. The colonies were then fixed/stained with methanol/crystal violet. The number of colonies/dish were
counted and the mean colonies/treatment were expressed relative to the solvent control value. The highest concentration tested in this assay was based on the solubility
limitations of the test material in the treatment medium.

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### Selection of Dose Levels fo r the Gene Mutation Assay
Based upon the toxicity assay, the dose level of the test material that was expected to
result in relative cell survivals of 10-20% in the activation system was selected as the top dose. In the non-activation assay, the t op dose level was based on the solubility
limitations of the test material in the tr eatment medium. At least four additional concentrations of the test material, the lowest of which was expected to give cell survival comparable to solvent control, were also tested in the gene mutation assay. The concentrations were usually spaced apart by a factor of 2. However, depending upon the dose response curve for toxicit y, this spacing was adjusted to yield the desired levels of cytotoxicity. The cytotoxicity of the select ed concentrations was determined concurrent with the gene mutation assay.
Gene Mutation Assay
Each dose level was set up in duplicate from the time of treatment until the comp letion of the assay. The cultures were trypsinized at the end of the treatment and replated at a density of 1 x 10 6 

### cells/100 mm dish (at least two dishes/replicate) for phenotypic expression ( O'Neill et al. , 1977a ; O'Neill and Hsie, 1979 ). In addition, 200 cells/60 mm dish (three di shes/replicate) were also plated to determine the toxicity and incubated for approximately 7 days to permit colony formation. During the phenotypic expression period (8 days), cells in the la rger petri dishes were subcultu red every 2-3 days and plated (at least two dishes/replicate) at a density of about 1 x 10 6 

### cells/100 mm petri dish. At each subculture, cells from various dishes within ea ch replicate were pooled prior to replating. At the end of the expression period, the cultures were trypsinized and plated at a density of
2 x 10 5 

### cells/100 mm dish (a total of 10 dishes/tre atment) in the selection media (Ham's F12 without hypoxanthine and with 6-thi oguanine) for the determination of HGPRT 

### mutants and 200 cells/60 mm dish (three dishes/treatment) in Ham's F-12 medium without hypoxanthine for determination of cloning efficien cy. The dishes were incubated for about

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### 7 days and the colonies were fixed/stained with methanol/c rystal violet. The mutation frequency (expressed as mutants per 10 6 

### clonable cells) for each replicate were calculated by the following formula ( Kirkland, 1989 ): MF = K x (m / c)

Where: K = P c 

### x 10 6 

### / P m



### P c 

### = the number of cells plated for the survival plates P m 

### = the number of cells plated for the mutation plates c = the mean colonies per plate for survival plates for each replicate m = the mean colonies per plate fo r mutation plates for each replicate

Evaluation Criteria
For an assay to be acceptable, the mutant fr equency in positive cont rols should have been significantly higher than the solv ent controls. An additional c riteria was that the mutant frequency in the solvent cont rols should have been within reasonable limits of the laboratory historical control values and literature values . The test chemical was considered positive if it indu ced a statistically significan t, dose related, reproducible increase in mutant frequency. The final interpretation of th e data took into consideration such factors as the mutant frequency and cl oning efficiencies in the solvent controls.
Statistical Analysis
The frequency of mutants per 10 6 

### clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutation frequency variance. The actual plate counts are assumed to follow a Poisson distribution therefore the mean plate count was us ed as an estimate of variance ( Kirkland, 1989 ).
If the analysis of variance was signifi cant at alpha = 0.05, a Dunnett's t-test was conducted ( Winer, 1971 ), comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Line ar dose-related trend tests were perform ed if any of the pairwise comparisons of test material with the solvent con trol yielded significant differences.

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### RESULTS AND DISCUSSION
pH and Osmolality
The pH and osmolality of treatment m edium containing approximately 176.8 μg/ml of the test material and medium containing 1% DMSO were determined using a Denver Basic pH meter (Denver Instrument C o., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in in vitro genotoxicity assays ( Thilagar et al. , 1984 ; Galloway et al., 1985 ; Cifone, 1985 ). There was no appreciable change in either the pH or osmolality at th is concentration as compared to the culture me dium with solvent alone (culture medium with the test material, pH = 7.39, osmolality = 487 mOsm/kgH 2 

### O; culture medium with 1% DMSO, pH = 7.34, osmolality = 477 mOsm/kgH 2 

### O).
Assay A1 – Preliminary Toxicity Assay
In a preliminary toxicity assay, the te st ma terial was assayed at concentrations of 0 (solvent control), 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 μg/m l ( Table 1 ) in the absence and presence of an externally supp lied metabolic activation system (S9). The highest concentration tested was based upon the limitations imposed by the solubility of
the test m aterial in the treatment medium. The treated cultures without S9 showed little to no toxicity with relative cell survival (RCS) values ranging from 79.6 to 104.3% ( Table 1 ). In the presence of S9 activation, excessive toxicity was seen at the highest concentration of 200 μg/ml while the remaini ng concentrations had RCS values ranging from 13.8 to 93.4% ( Table 1 ). Based upon the results of th is assay, concentrations of 0 (solvent control), 10, 25, 50, 100, and 200 μg/ml of the test material were selected for the initial gene mutation assay in the absence of S9 and 0 (solvent control), 2.5, 10, 25, 50, 100, 150, and 200 μg/ml in the presence of S9.
Assay B1 – Initial Mutagenicity Assay
In the initial mutagenicity assay (Assay B1), in the absence of S9, little to no toxicity was observed with RCS values ranging from 85.1 to 103.2% ( Table 2 ). In the presence S9,
cultures treated with the tw o highest concentrations (150 and 200 μg/ml) showed excessive toxicity during the expression peri od, thus were not available to select for mutants ( Table 3 ). The remaining cultures had RCS values ranging from 17.0 to 95.7%. The mutant frequencies observed in cultures trea ted with the test m ate rial in the absence

-THE DOW CHEMICAL COMPANY STUDY ID: PAGE 16

- 

### ( Table 2 ) and presence of S9 ( Table 3 ) were not significantly different from the concurrent solvent control values. All mutant frequencies were with in a reasonable range of historical background values ( Table 6 and Table 7 ).
Assay C1 – Confirmatory Mutagenicity Assay

In a confirm atory assay (Assay C1), the concen trations ranged from 0 (solvent control) to 200 μg/ml without S9 and 0 (solvent control) to 140 μg/m l with S9. There was little to no toxicity observed, as indicated by RCS values, in the absence of S9 ( Table 4 , 92.9 to 102.9%). In the presence of S9, RCS values sh owed moderate to no toxicity with values ranging from 12.0 to 95.7% ( Table 5 ). The mutant frequencies observed in cultures
treated with the te st material in th e abse nce and in the presence of S9 were not significantly different from th e concurrent solvent control values and were within the range of the laboratory historical background ( Table 6 and Table 7 ).
In both the initial and confirmatory mutagenicity assays, the positive control chemicals induced significant increases in mutant freque ncies and this data confirmed the adequacy of the experimental conditions for detecting induced mutations.
The analytically observed concen trations of the test material in the stock dosing solutions in Assay B1 ranged from 94.4 to 101.8% of target ( Douglass, 2010; Table 8 ) and these values also indicated stability of th e test material in the solvent.

CONCLUS IONS

The results of the in vitro Chinese Hamster Ovary cell/hypoxanthine-guaninephosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay with
indicate th at under the conditions of th is study, the test article was

non-m utagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

ACKNOWLEDGEMENTS
The authors gratefully acknowledge the contri butions of the following individuals to the conduct of the study and report preparation:
J. M. Smalley Genetic Toxicology Technologist

A. J. Wood Genetic Toxicology TechnicianTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 17



### REFERENCES
Cifone, M. A. (1985). Relationship between increases in the mutant frequency in L5178Y TK +/- 

### mouse lymphoma cells at low pH and metabolic activation. Environ. Mutagen. 7 (Suppl. 3), 27.

Galloway, S. M., Bean, C. L., Armstrong, M. A., Deasy, D., Kraynak, A., and Bradley, M. O. (1985). False positive in vitro chromosome aberration tests with non mutagens at high concentrations and osmolalities. Environ. Mutagen

. 7 (Suppl. 3), 48-49.
Hsie, A. W., Casciano, D. A., Couch, D. B., Kr ahn, D. F., O'Neill, J. P., and Whitfield, B. L. (1981). The use of chinese hamster ovary cells to quan tify specific locus mutation and to determine mutagenicity of chemicals: A report of the U.S. EPA's Gen-Tox Program. Mutat. Res. 86, 193-214.
Kirkland, D. J. (editor) (1989). Statistical Evaluation of Mutagenicity Test Data
, Cambridge University Press, New York, Pgs. 78-87.
O'Neill, J. P., Brimer, P. A., Machanoff, R., Hirsch, G. P., and Hsie, A. W. (1977a). A quantitative assay for mutation induc tion at the hypoxanthine-guanine phosphoribosyl transferase locus in chin ese hamster ovary cells (CHO/HGPRT): Development and definition of the system. Mutat. Res. 45, 91-101.
O'Neill, J. P., Couch, D. B., Machanoff, R ., San Sebastian, J. R., Brimer, P. A., and Hsie, A. W. (1977b). A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transf erase locus in chinese hamster ovary cells (CHO/HGPRT system): Utilization with a variety of mutagenic agents.
Mutat. Res. 45, 103-109.
O'Neill, J. P. and Hsie, A. W. (1979). Ph enotypic expression time of mutagen-induced 6- thioguanine resistance in chinese hams ter ovary cells (CHO/HGPRT system).
Mutat. Res. 59, 109-118.
O'Neill, J. P., Machanoff, R., and Hsie, A. W. (1979). The use of a rat liver S9 activation system in the CHO/HGPRT mutation induction assay. Environ. Mutagen. 1, 145.
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.

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- 

### Thilagar, A. K., Kumaroo, P. V., and Kott, S. (1984). Effects of low pH caused by glacial acetic acid and hydrochloric acid on chromosomal aberrations in CHO Cells. Toxicologist 4, 51.
Winer, B. J. (1971). Statistical Principles in Experimental Design (2 nd 

### Edition). McGraw-Hill, New York.THE DOW CHEMICAL COMPANYSTUDY ID:PAGE 19EVALUATION OFCATECHNE-GUANINE-PHOSPHORIBOSYL-Table 1.OL IN THE CHINESE HAMSTER OVARY CELL/HYPOXANTHITRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAYSurvivalof CHO Cells Treatedwith – Assay A1Cell Line:CHO-K1-BH4Passage: 35Without S-9 With S-9Treatment No. of Colonies/DishNo. of Colonies/Dishμg/ml 1 2 3RCS (%)a123RCS (%)aSolvent Controlb212 185 202 100.0191 184 189 100.00.78201 199 192 98.8180 166 181 93.41.56195 173 203 95.3153 163 173 86.73.13202 196 181 96.7151 151 143 78.96.25200 195 187 97.2140 125 118 67.912.5197 203 179 96.7123 117 118 63.525208 200 185 99.0118 99 112 58.350c159 143 175 79.640 38 55 23.6100c214 204 207 104.329 22 27 13.8200c209 200 209 103.26 8 2 2.8aRelative Cell Survival (%) =Mean number ofcolonies/dishin the solvent control(avg. ofreplicates)b1% DMSOcCloudy in treatment mediumMean number of colonies/dish in the treated x 100THE DOW CHEMICAL COMPANYSTUDY ID:PAGE 20EVALUATION OFORIBOSYL-Table 2.INTHE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANINE-PHOSPHTRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAYResults of the Gene Mutation Assay in CHO Cells Treated within the Absence of S9 – Assay B1Cell Line:CHO-K1-BH4Positive Control: 621 μg/ml EMSPassage: 35Solvent Control: 1% DMSOTGrMutantsTreatmentper 106μg/ml12 3RCS (%)a1 2 3 4 5 6 7 8 9 10 Total 1 2 3CE (%)cClonable CellsSolvent Control 184 172 168 101.1 0 0 0 1 0 0 1 0-d-d2 168 140 143 75.2 1.7Solvent Control 172 171 170 98.9 2 0 0 2 2 0 0 1 1 -d8 153 136 175 77.3 5.710 169 160 184 98.9 0 0 0 2 0 0 0 0 2-d4 129 165 151 74.2 3.010 171 156 176 97.0 0 0 0 1 0 0 1 1 1 1 5 122 132 120 62.3 4.025 193 161 176 102.2 0 1 1 0 0 0 0 0 0 0 2 154 147 135 72.7 1.425 178 167 181 101.4 -d-d-d-d-d-d-d-d-d-d-d135 135 132 67.0 -d50 155 135 151 85.1 0 1 0 0 1 1 1 1 0 1 6 157 143 156 76.0 3.950 170 149 166 93.5 0 1 0 1 0 1 0 1 0 0 4 165 156 143 77.3 2.6100e178 169 177 101.1 0 0 0 0 0 0 1 0 0 0 1 138 123 157 69.7 0.7100e172 188 173 102.8 1 0 0 0 0 0 0 0 1 0 2 144 141 150 72.5 1.4200e158 181 165 97.2 0 0 1 0 0 1 0 0 0 0 2 165 146 150 76.8 1.3200e173 175 187 103.2 0 1 0 0 0 0 0 0 0 0 1 143 158 156 76.2 0.7Pos.Control 119 96 121 64.8 22 20 21 14 19 23 16 15 18 28 196 82 98 107 47.8 204.9fPos.Control 100 107 116 62.3 16 23 19 19 20 24 17 18 22 17 195 82 82 82 41.0237.8faRelative cell survival (%) =Mean number of colonies/dish in the treated x 100Mean number of colonies/dish in the solvent control (avg. of replicates)bTGr= 6-Thioguanine resistantcCE (%) = Mean number of colonies/dish x 100No. of cells seeded/dishdLost due to contamination.eCloudy in treatment medium.fThe frequency of TGrmutants is significantly higher than the concurrent solvent control value (alpha=0.05).No. of Colonies/DishToxicity AssayNo. of Colonies/DishCloning Efficiency (CE)Mutation AssayTGrColonies/DishbTHE DOW CHEMICAL COMPANYSTUDY ID:PAGE 21EVALUATION OFORIBOSYL-Table 3.INTHE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANINE-PHOSPHTRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAYResults of the Gene Mutation Assay in CHO Cells Treated with in the Presence of S9 – Assay B1Cell Line:CHO-K1-BH4Positive Control: 4 and 12 μg/ml MCAPassage: 35Solvent Control: 1% DMSOTGrMutantsTreatmentper 106μg/ml123RCS (%)a1 2 3 4 5 6 7 8 9 10 Total 1 2 3CE (%)cClonable CellsSolvent Control 170 160 178 98.2 1 0 0 0 0 0 2 0 0 2 5 177 157 149 80.5 3.1Solvent Control 171 180 176 101.8 1 0 0 0 0 0 0 0 2 0 3 149 139 152 73.3 2.02.5167 154 174 95.7 0 0 0 0 0 0 1 1 1 0 3 161 123 155 73.2 2.02.5174 164 149 94.1 0 1 1 1 2 2 0 0 0 1 8 129 142 147 69.7 5.710146 151 148 86.0 0 0 0 0 1 0 1 0 0 0 2 156 155 157 78.0 1.310154 154 164 91.2 0 0 0 0 0 0 0 0 0 0 0 158 147 138 73.8 0.025128 115 129 71.9 2 1 0 1 0 0 0 0 0 0 4 151 148 143 73.7 2.725131 138 121 75.4 0 0 0 1 0 0 0 0 0 0 1 157 133 130 70.0 0.75067 73 67 40.0 0 0 0 0 0 0 0 0 0 0 0 117 107 137 60.2 0.05066 76 75 41.9 0 0 0 0 0 0 0 0 1 1 2 134 97 108 56.5 1.8100d29 34 31 18.2 1 0 1 0 0 0 0 0 1 0 3 94 111 109 52.3 2.9100d28 31 29 17.0 0 0 0 0 0 0 0 0 0 0 0 109 92 106 51.2 0.0150d8 12 7 5.2 -e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e150d7 5 6 3.5 -e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e200d3 2 3 1.5 -e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e200d2 2 5 1.7 -e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-ePos.Control (4 μg/ml) 179 189 160 102.0 12 13 10 9 18 14 10 18 8 7 119 136 129 106 61.8 96.2fPos.Control (4 μg/ml) 178 166 191 103.4 14 15 15 9 13 20 15 13 8 14 136 122 137 141 66.7102.0fPos.Control (12 μg/ml) 155 149 152 88.1 12 18 17 18 9 23 13 21 30 17 178 109 84 107 50.0 178.0fPos.Control (12 μg/ml) 174 149 156 92.6 15 10 12 23 18 16 22 17 24 14 171 135 106 136 62.8136.1faRelative cell survival (%) =Mean number of colonies/dish in the treated x 100Mean number of colonies/dish in the solvent control (avg. of replicates)bTGr= 6-Thioguanine resistantcCE (%) =Mean number of colonies/dish x 100No. of cells seeded/dishdCloudy in treatment medium.eLost due to toxicity during the expression period.fThe frequency of TGrmutants is significantly higher than the concurrent solvent control value (alpha=0.05).No. of Colonies/DishTGrColonies/DishbNo. of Colonies/DishToxicity AssayMutation AssayCloning Efficiency (CE)THE DOW CHEMICAL COMPANYSTUDY ID:PAGE 22EVALUATION OFORIBOSYL-Table 4.INTHE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANINE-PHOSPHTRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAYResults of the Gene Mutation Assay in CHO Cells Treated within the Absence of S9 – Assay C1Cell Line:CHO-K1-BH4Positive Control: 621 μg/ml EMSPassage: 36Solvent Control: 1% DMSOTGrMutantsTreatmentper 106μg/ml12 3RCS (%)a1 2 3 4 5 6 7 8 9 10 Total 1 2 3CE (%)cClonable CellsSolvent Control 170 159 173 98.6 0 1 0 0 1 2 1 0 0-d5 109 86 101 49.3 5.6Solvent Control 154 189 173 101.4 0 0 0 0 0 1 0 0 0 1 2 119 99 102 53.3 1.910 183 172 155 100.2 3 2 1 2 3 1 0 1 0 2 15 104 74 105 47.2 15.910 175 168 168 100.4 0 0 1 0 1 1 2 2-d-d7 111 106 96 52.2 8.420 177 152 181 100.2 2 1 1 0 0 0 0 0 1 0 5 130 121 118 61.5 4.120 146 178 171 97.2 0 0 1 2 0 0 1 2 1 1 8 108 101 117 54.3 7.460 171 169 184 102.9 0 1 2 1 2 2 0 1 0 0 9 107 101 110 53.0 8.560 183 165 163 100.4 1 1 0 0 1 1 0 1 3 0 8 139 106 118 60.5 6.6100e172 177 162 100.4 0 0 0 1 1 1 1 0 0 0 4 115 122 96 55.5 3.6100e167 170 155 96.7 0 0 0 0 0 1 0 0 1 1 3 121 121 123 60.8 2.5200e172 147 162 94.5 1 0 1 0 0 1 1 0 0 1 5 116 127 116 59.8 4.2200e147 169 157 92.9 1 0 1 1 1 0 0 0 1 1 6 134 146 136 69.3 4.3Pos.Control 120 103 111 65.6 13 11 16 15 14 17 19 15 15 17 152 96 92 88 46.0165.2fPos.Control 109 104 122 65.8 16 21 19 21 15 19 17 15 22 -d165 87 92 86 44.2 207.5faRelative cell survival (%) = Mean number of colonies/dish in the treated x 100Mean number of colonies/dish in the solvent control (avg. of replicates)bTGr= 6-Thioguanine resistantcCE (%) = Mean number of colonies/dish x 100No. of cells seeded/dishdLost due to contamination.eCloudy in treatment medium.fThe frequency of TGrmutants is significantly higher than the concurrent solvent control value (alpha=0.05).No. of Colonies/DishTGrColonies/DishbNo. of Colonies/DishToxicity Assay Mutation AssayCloning Efficiency (CE)THE DOW CHEMICAL COMPANYSTUDY ID:PAGE 23EVALUATION OFORIBOSYL-Table 5.INTHE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANINE-PHOSPHTRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAYResults of the Gene Mutation Assay in CHO Cells Treated with in the Presence of S9 – Assay C1Cell Line:CHO-K1-BH4Positive Control: 4 and 12 μg/ml MCAPassage: 36Solvent Control: 1% DMSOTGrMutantsTreatmentper 106μg/ml123RCS (%)a1 2 3 4 5 6 7 8 9 10 Total 1 2 3CE (%)cClonable CellsSolvent Control 164 152 140 101.2 2 1 2 3 2 1 0 1 0 1 13 152 137 133 70.3 9.2Solvent Control 159 141 145 98.8 0 0 0 0 0 0 0 0 0 2 2 128 125 138 65.2 1.55 131 144 139 91.9 2 0 0 0 2 1 2 1 1 2 11 93 135 111 56.5 9.75 161 136 134 95.7 0 1 1 2 2 1 1 1 1 0 10 119 135 139 65.5 7.610 149 116 124 86.3 3 1 0 2 1 3 1 2 0 1 14 114 157 128 66.5 10.510 120 107 120 77.0 1 1 0 2 2 0 0 2 2 2 12 153 152 145 75.0 8.020 95 103 94 64.8 1 0 0 0 2 1 0 1 0 1 6 116 125 130 61.8 4.920 73 89 68 51.1 0 3 0 0 1 0 0 1 0 0 5 135 113 122 61.7 4.140 44 53 58 34.4 2 2 0 2 1 1 3 4 2 3 20 98 109 89 49.3 20.340 55 48 52 34.4 0 0 1 0 1 3 1 2 1 0 9 103 98 116 52.8 8.560 48 52 42 31.5 0 0 0 1 0 1 1 1 0 1 5 88 95 97 46.7 5.46052 46 40 30.6 1 0 0 0 3 0 1 1 1 0 7 118 120 101 56.5 6.280d47 35 20 22.6 1 1 0 0 1 0 2 2 1 2 10 84 79 86 41.5 12.080d38 27 38 22.9 0 1 1 0 0 1 0 0 1 0 4 93 96 94 47.2 4.2100d25 29 26 17.8 1 0 0 0 0 0 0 1 0 1 3 83 102 85 45.0 3.3100d28 30 29 19.3 1 1 0 0 0 2 0 0 1 0 5 90 92 88 45.0 5.6120d34 26 25 18.9 0 0 0 0 0 0 0 0 1 1 2 124 117 103 57.3 1.7120d19 22 34 16.6 0 0 0 0 0 0 0 1 0 0 1 100 101 102 50.5 1.0140d24 22 12 12.9 0 0 0 0 1 0 0 0 0 0 1 93 96 92 46.8 1.1140d20 13 21 12.0 0 0 1 0 0 0 0 0 2 0 3 80 100 90 45.0 3.3Pos.Control (4 μg/ml) 136 148 153 97.0 23 19 29 23 26 29 28 34 19 27 257 105 125 114 57.3224.1ePos.Control (4 μg/ml) 151 144 137 95.9 32 30 20 27 29 27 27 48 31 27 298 115 126 122 60.5 246.3ePos.Control (12 μg/ml) 123 131 135 86.3 10 11 12 18 10 11 10 8 9 16 115 125 120 125 61.793.2ePos.Control (12 μg/ml) 130 129 141 88.8 15 19 8 12 11 17 10 15 13 13 133 136 138 131 67.5 98.5eaRelative cell survival (%) =Mean number of colonies/dish in the treated x 100Mean number of colonies/dish in the solvent control (avg. of replicates)bTGr= 6-Thioguanine resistantcCE (%) =Mean number of colonies/dish x 100No. of cells seeded/dishdCloudy in treatment medium.eThe frequency of TGrmutants is significantly higher than the concurrent solvent control value (alpha=0.05).No. of Colonies/DishTGrColonies/DishbNo. of Colonies/DishToxicity AssayMutation AssayCloning Efficiency (CE)THE DOW CHEMICAL COMPANY STUDY ID: PAGE 24

EVALUATION O F IN THE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANIN E-PHOSPHORIBOSYL TRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAY

Table 6. Historical La boratory Background Values of 6-Thioguanine Resistant (TG r ) Mutant Frequency in CHO Cells – Individual Values

S-9 Year (+ or -) No. TG r Mutants Per 10 6 Clonable Cells 2005 - 11.5, 7.2, 10.7, 8.7, 7.4, 6.2, 2.8, 6.1, 15.0, 3.9, 3.2, 26.3

+ 2.6, 3.7, 7.7, 2.9, 2.3, 8.7, 1.9, 5.6, 1.2, 5.4, 9.2, 6.6

2006 - 4.5, 7.3, 11.4, 10.2, 3.4, 6.5, 10.9, 12.5, 6.0, 7.0, 11.1, 7.1

+ 2.7, 6.4, 5.3, 6.4, 7.1, 6.0, 20.7, 13.2, 5.5, 5.5, 0.6, 6.5, 7.5, 7.5

2007 - 10.8, 14.3, 13.5, 12.6, 13.2, 7.7, 4.1, 9.8, 5.1, 4.4, 9.2, 14.5, 8.5, 10.7, 4.6, 13.7, 5.1, 10.5, 10.3, 3.7, 2.9, 6.9, 11.4, 9.5,10.0, 5.4, 9.2, 3.9, 3.9, 0.7, 13.4, 12.2

+ 20.5, 18.1, 14.9, 32.2, 9.3, 12.0, 6.7, 4.1, 7.4, 11.3, 3.3, 10.0, 7.6, 2.2, 4.5, 4.3, 21.9, 14.9, 17.9, 10.9, 13.6, 9.6, 3.2, 7.1, 4.4, 11.9, 9.0, 1.3, 5.5, 5.0, 4.9, 6.8

2008 - 4.1, 7.2, 7.6, 5.4, 26.0, 18.6, 4.0, 12.3, 8.8, 21.2, 2.4, 2.2, 4.3, 17.3, 6.6, 9.4

+ 6.0, 5.6, 10.0, 2.3, 12.0, 10.1, 24.2, 16.5, 6.5, 8.8, 10.0, 6.1, 7.4, 10.0, 2.6

2009 - 11.7, 2.9, 7.4, 8.7, 8.5, 8.4, 7.1, 13.9, 15.1, 7.3, 4.9, 4.7

+ 13.5, 13.1, 11.9, 3.4, 6.9, 9.4, 15.6, 3.7, 4.1, 3.6, 6.7, 4.6

2010 - 4.5, 3.3, 4.6, 7.6, 6.3, 7.5, 8.0, 5.5, 9.6, 6.1, 2.3, 5.5, 6.6, 3.5, 7.0, 7.1, 6.6, 4.6, 6.8, 10.1, 8.0, 8.4, 12.3, 14.5, 9.1, 15.2, 3.4, 8.6, 5.7, 3.4, 5.3, 3.8, 3.2, 1.6, 5.4, 4.1, 4.7, 9.8, 7.4, 1.5, 1.8, 7.9

+ 5.1, 6.4, 4.8, 8.7, 9.4, 11.1, 1.8, 6.4, 10.1, 7.0, 2.8, 9.1, 7.5, 7.1, 2.9, 1.6, 4.1, 4.6, 6.6, 9.6, 8.2, 12.5, 14.3, 4.1, 9.4, 5.1, 3.6, 5.0, 4.5, 5.6, 9.4, 11.5, 10.3, 10.0, 4.5, 1.7, 5.5, 2.8, 7.8, 4.1, 4.0, 5.5, 0.0, 5.5

-THE DOW CHEMICAL COMPANY STUDY ID: PAGE 25

EVALUATION O F IN THE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANIN E-PHOSPHORIBOSYL TRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAY

2 Table 7. Historical Laboratory Background Values of 6-Thioguanine Resistant (TG r ) Mutant Frequency in CHO Cells – Descriptive Statistics

S-9 Year (- or +) Mean Std. Dev. Minimum Maximum N b

2005 - 9.1 6.5 2.8 26.3 12 + 4.8 2.8 1.2 9.2 1

2006 - 8.2 3.0 3.4 12.5 12 + 7.2 4.8 0.6 20.7 14

2007 - 8.6 3.9 0.7 14.5 32
+ 9.9 6.8 1.3 32.2 32

2008 - 9.8 7.2 2.2 26.0 16
+ 9.2 5.5 2.3 24.2 15

2009 - 8.4 3.6 2.9 15.1 12
+ 8.0 4.5 3.4 15.6 12

2010 - 6.4 3.1 1.5 15.2 42
+ 6.4 3.2 0.0 14.3 44

a Frequencies TG r mutants per 10 6 cells

b Number of experiments FREQUENCIES a

-THE DOW CHEMICAL COMPANYSTUDY ID:PAGE 26EVALUATION OFIN THE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANINEPHOSPHORIBOSYL TRANUTATION ASSAY-Table 8.SFERASE(CHO/HGPRT) FORWARD MConcentration Check Report – Assay B1*Dose LevelDate Mixed: 09/28/10Overall Overallmg/mLACL Report #: 2010-149Mean Std.Dev.Target Conc. (mg/mL):0.00Control Mean Measured Conc:<LLQ% o f Target C onc:(n): (2)Target Conc. (mg/mL): 0.2500.25 Mean Measured Conc:0.236% o f Target C onc: 94.4%94.4% NA(n): (2)Target Conc. (mg/mL): 1.001.00 Mean Measured Conc: 0.962% o f Target C onc: 96.2%96.2% NA(n): (2)Target Conc. (mg/mL): 2.502.50 Mean Measured Conc:2.40% o f Target C onc: 96.1%96.1% NA(n): (2)Target Conc. (mg/mL): 5.005.00 Mean Measured Conc:4.84% o f Target C onc: 96.7%96.7% NA(n): (2)Target Conc. (mg/mL): 10.010.00 Mean Measured Conc:9.75% o f Target C onc: 97.5%97.5% NA(n): (2)Target Conc. (mg/mL): 15.015.00 Mean Measured Conc:15.3% of Target Con c: 101.8%101.8% NA(n): (2)Target Conc. (mg/mL): 20.020.00 Mean Measured Conc:19.9% o f Target C onc: 99.4%99.4% NA(n): (2)LLQ . (mg/mL):0.05050.0505 NA<LLQ = less than lower limit of quantit ationNA= Not Applicable*The dose levels indicated in this table refer to the stock treatment solutions. The actual treatment concentrations were 100-fold lower.