THE DOW CHEMICAL COMPANY 

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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 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 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 

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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: 

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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 

~ 
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TABLE OF CONTENTS 

Page 

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

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

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

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

SUMMARY.........................................................................................................................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 (Lot, 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 ...............................................................................................................10

Media ............................................................................................................................11

Controls.........................................................................................................................11

Treatment Procedure .....................................................................................................11

Identification of the Test System ..................................................................................11

Preparation of the Treatment Solution ..........................................................................11

Analytical Verification of Dosing Solutions.................................................................12

In Vitro Metabolic Activation .......................................................................................12

Toxicity Assay ..............................................................................................................12

Selection of Dose Levels for 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 with – Assay 

A1 19

Table 2. Results of the Gene Mutation Assay in CHO Cells Treated with 

 in the Absence of S9 – Assay B1..................................20

Table 3. Results of the Gene Mutation Assay in CHO Cells Treated with 

 in the Presence of S9 – Assay B1 .................................21

Table 4. Results of the Gene Mutation Assay in CHO Cells Treated with 

 in the Absence of S9 – Assay C1..................................22

Table 5. Results of the Gene Mutation Assay in CHO Cells Treated with 

 in the Presence of S9 – Assay C1 .................................23

Table 6. Historical Laboratory Background Values of 6-Thioguanine 

Resistant (TGr

) Mutant Frequency in CHO Cells – Individual Values ............24

Table 7. Historical Laboratory Background Values of 6-Thioguanine 

Resistant (TGr

) Mutant Frequency in CHO Cells – Descriptive 

Statistics ............................................................................................................25

Table 8. Concentration Check Report – Assay B1 ....................................................26
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SUMMARY 

was 

evaluated in the in vitro Chinese Hamster Ovary cell/hypoxanthine-guaninephosphoribosyl transferase (CHO/HGPRT) forward 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 analytically determined concentrations of the test 

material in the dosing preparation used for the initial mutagenicity assay ranged from 

94.4 to 101.8% of the targeted values. The adequacy of the experimental conditions for 

detection of induced mutation was confirmed by employing positive control chemicals, 

ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for 

assays in the presence of S9. Solvent control 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 activation (S9) system. 

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INTRODUCTION 

Principle of the Test System 

Hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) is a cellular enzyme that 

utilizes the substrates 5-phosphoribosyl-1-pyrophosphate and hypoxanthine or guanine to 

catalyze the synthesis of inosine- or guanosine-monophosphate. In mammalian cells, 

purine nucleotides are primarily synthesized de novo and the above pathway is a salvage 

pathway in the biosynthesis of these nucleotides. The gene for HGPRT is located on the 

mammalian X-chromosome. X-linked genes are 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 single mutational event. A mutation 

leading to the loss of HGPRT activity renders the cells incapable of utilizing 

hypoxanthine, guanine, or their analogs - thioguanine (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 activity would grow in the presence of these 

analogs because they are unable to form the lethal nucleotide analogs. Hence cells 

capable of performing clonal growth in the presence of the toxic purine analogs are 

assumed to have mutated from HGPRT+ to HGPRT- phenotype. The Chinese Hamster 

Ovary (CHO) cell line, CHO-K1-BH4 is suited for determining forward mutations at the 

HGPRT locus because it is a well characterized and validated system (Hsie et al., 1981; 

O'Neill et al., 1977a,b, 1979). These cells have a high cloning efficiency, rapid doubling 

time (12-14 hours), and a low spontaneous mutation frequency (1-20 mutants per 10

6

cells). 

These cells also show a dose-response with a variety of physical and chemical mutagens 

and a genetic basis for the presumptive mutational 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 mammalian gene 

mutation tests, OECD Publication Service, 2, rue Andre-Pascal, 75775 

Paris Cedex 16, France (1997). 

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USEPA United States Environmental Protection 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 Assurance Unit, Toxicology & Environmental Research and 

Consulting, The Dow Chemical Company, Midland, Michigan. 

Archiving 

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

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

Chemical Company, Midland, Michigan. 

Safety 

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

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

available. 

TEST MATERIAL INFORMATION 

Test Material Name 

Chemical Name 

Synonyms 

Supplier, City, State (Lot, Reference Number) 

The Dow Chemical Company, Midland, Michigan (Lot# 

Purity/Characterization (Method of Analysis and Reference) 

The purity of the test material was determined to be

chromatography with flame ionization detection (GC-FID) and Karl Fischer titration with 

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identification by Fourier Transform-Infrared (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-K1-BH4, 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 cultures were periodically checked for mycoplasma 

contamination (American Type Culture Collection, Manassas, Virginia). The cells were 

grown as monolayer cultures in plastic disposable tissue culture labware under standard 

conditions of approximately 5% CO2 in air at 37C in a humidified incubator. 

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Media 

The cells were routinely maintained in Ham's F-12 nutrient mix supplemented with 5% 

(v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum, antibiotics and 

antimycotics (penicillin G, 100 units/ml; streptomycin 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 material (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-activation system (without S9 factor) at a final 

concentration of 621 g/ml. The positive control for assays performed with S9 

(activation system) was 20-methylcholanthrene (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 106 cells/T-75 flask approximately 24 hours prior to 

treatment. At the time of treatment, the culture 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 treated 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 using self adhesive labels containing a code system that 

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

Preparation of the Treatment Solution 

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

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

diluted (1:100) with the treatment 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 dosing units were expressed in μg/ml. 

Analytical Verification of Dosing Solutions 

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

treatment in Assay B1 were verified by the Analytical Chemistry Laboratory, Toxicology 

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

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

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

In Vitro Metabolic Activation 

S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats 

were purchased from a commercial 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 MgCl2·6H2O, 5 mM glucose-6-phosphate, 4 mM 

nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 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 concentration of S9 in the culture, i.e., 2% v/v. Hence, the final 

concentration of the co-factors in the culture medium is 1/5th of the concentrations stated 

above.

Toxicity Assay 

The cytotoxicity of the test material was assessed by determining the ability of the treated 

cells to form colonies. This assay was conducted 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 

106 cells/flask) approximately 24 hours prior 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 factor. 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 days 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 for 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 top dose level was based on the solubility 

limitations of the test material in the treatment 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 toxicity, this spacing was adjusted to yield the desired levels of 

cytotoxicity. The cytotoxicity of the selected 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 completion of 

the assay. The cultures were trypsinized at the end of the treatment and replated at a 

density of 1 x 106 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 dishes/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 larger petri dishes were subcultured every 2-3 days and plated 

(at least two dishes/replicate) at a density of about 1 x 106 cells/100 mm petri dish. At each 

subculture, cells from various dishes within each 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 105

 cells/100 mm dish (a total of 10 dishes/treatment) in the selection media (Ham's F12 without hypoxanthine and with 6-thioguanine) for the determination of HGPRTmutants and 200 cells/60 mm dish (three dishes/treatment) in Ham's F-12 medium without 

hypoxanthine for determination of cloning efficiency. The dishes were incubated for about 

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7 days and the colonies were fixed/stained with methanol/crystal violet. The mutation 

frequency (expressed as mutants per 106 clonable cells) for each replicate were calculated 

by the following formula (Kirkland, 1989): 

MF = K x (m / c) 

Where: 

K = Pc x 106 / Pm

Pc = the number of cells plated for the survival plates 

Pm = 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 for mutation plates for each replicate 

Evaluation Criteria

For an assay to be acceptable, the mutant frequency in positive controls should have been 

significantly higher than the solvent controls. An additional criteria was that the mutant 

frequency in the solvent controls should have been within reasonable limits of the 

laboratory historical control values and literature values. The test chemical was 

considered positive if it induced a statistically significant, dose related, reproducible 

increase in mutant frequency. The final interpretation of the data took into consideration 

such factors as the mutant frequency and cloning efficiencies in the solvent controls. 

Statistical Analysis 

The frequency of mutants per 106 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 used as an estimate of variance (Kirkland, 1989). 

If the analysis of variance was significant 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). Linear dose-related trend tests were performed 

if any of the pairwise comparisons of test material with the solvent control yielded 

significant differences. 

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RESULTS AND DISCUSSION 

pH and Osmolality 

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

the test material and medium containing 1% 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). 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 this 

concentration as compared to the culture medium with solvent alone (culture medium 

with the test material, pH = 7.39, osmolality = 487 mOsm/kgH2O; culture medium with 

1% DMSO, pH = 7.34, osmolality = 477 mOsm/kgH2O). 

Assay A1 – Preliminary Toxicity Assay 

In a preliminary toxicity assay, the test material was assayed at concentrations of 0 

(solvent control), 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 μg/ml (Table 1) in the 

absence and presence of an externally supplied metabolic activation system (S9). The 

highest concentration tested was based upon the limitations imposed by the solubility of 

the test material 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 remaining concentrations had RCS values ranging 

from 13.8 to 93.4% (Table 1). Based upon the results of this 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 two highest concentrations (150 and 200 μg/ml) showed 

excessive toxicity during the expression period, 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 treated with the test material in the absence 

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(Table 2) and presence of S9 (Table 3) were not significantly different from the 

concurrent solvent control values. All mutant frequencies were within a reasonable range 

of historical background values (Table 6 and Table 7). 

Assay C1 – Confirmatory Mutagenicity Assay 

In a confirmatory assay (Assay C1), the concentrations ranged from 0 (solvent control) to 

200 μg/ml without S9 and 0 (solvent control) to 140 μg/ml 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 showed moderate to no toxicity with values 

ranging from 12.0 to 95.7% (Table 5). The mutant frequencies observed in cultures 

treated with the test material in the absence and in the presence of S9 were not 

significantly different from the 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 frequencies and this data confirmed the adequacy 

of the experimental conditions for detecting induced mutations. 

The analytically observed concentrations 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 the test material in the solvent. 

CONCLUSIONS 

The results of the in vitro Chinese Hamster Ovary cell/hypoxanthine-guaninephosphoribosyl transferase (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 activation (S9) system. 

ACKNOWLEDGEMENTS 

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

conduct of the study and report preparation: 

 J. M. Smalley Genetic Toxicology Technologist 

 A. J. Wood Genetic Toxicology Technician 
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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., Krahn, D. F., O'Neill, J. P., and Whitfield, B. 

L. (1981). The use of chinese hamster ovary cells to quantify 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 induction at the hypoxanthine-guanine 

phosphoribosyl transferase locus in chinese 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 transferase 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). Phenotypic expression time of mutagen-induced 6-

thioguanine resistance in chinese hamster 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 dimethylnitrosamine 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 (2nd Edition). 

McGraw-Hill, New York. 
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EVALUATION OF CATECH NE-GUANIN

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Table 1.

OL IN THE CHINESE HAMSTER OVARY CELL/HYPOXANTHI

TRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAY 

 Survival of CHO Cells Treated with – Assay A1 

Cell Line: CHO-K1-BH4 Passage: 35

Without S-9 With S-9

Treatment No. of Colonies/Dish No. of Colonies/D

μg/ml 1 2 3 RCS (%)a 123 

Solvent Controlb 212 185 202 100.0 191 184 189 

0.78 201 199 192 98.8 180 166 181 1.56 195 173 203 95.3 153 163 173 3.13 202 196 181 96.7 151 151 143 6.25 200 195 187 97.2 140 125 118 12.5 197 203 179 96.7 123 117 118 

25 208 200 185 99.0 118 99 112 

50c 159 143 175 79.6 40 38 55 

100c 214 204 207 104.3 29 22 27 200c 209 200 209 103.2 6 8 2 

a Relative Cell Survival (%) = 

Mean number of colonies/dish in the solvent control (avg. of replicat

b 1% DMSOc Cloudy in treatment medium

Mean number of colonies/dish in the treated x 100
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EVALUATION OF 

- 

Table 2.

IN THE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANIN

TRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAY 

 Results of the Gene Mutation Assay in CHO Cells Treated with in the Absence of S9 – 

Cell Line: CHO-K1-BH4 Positive Control: 621 μg/ml EMS

Passage: 35 Solvent Control: 1% DMSO

Treatment 

μg/ml 12 3 RCS (%)a 1 2 3 4 5 6 7 8 9 10 Total 1 2 3 CE (%)c 

Solvent Control 184 172 168 101.1 0 0 0 1 0 0 1 0 -d -d 2 168 140 143 75.2 

Solvent Control 172 171 170 98.9 2 0 0 2 2 0 0 1 1 -d 8 153 136 175 77.3 

10 169 160 184 98.9 0 0 0 2 0 0 0 0 2 -d 4 129 165 151 74.2 10 171 156 176 97.0 0 0 0 1 0 0 1 1 1 1 5 122 132 120 62.3 25 193 161 176 102.2 0 1 1 0 0 0 0 0 0 0 2 154 147 135 72.7 25 178 167 181 101.4 -d -d -d -d -d -d -d -d -d -d -d 135 135 132 67.0 

50 155 135 151 85.1 0 1 0 0 1 1 1 1 0 1 6 157 143 156 76.0 50 170 149 166 93.5 0 1 0 1 0 1 0 1 0 0 4 165 156 143 77.3 

100e 178 169 177 101.1 0 0 0 0 0 0 1 0 0 0 1 138 123 157 69.7 

100e 172 188 173 102.8 1 0 0 0 0 0 0 0 1 0 2 144 141 150 72.5 200e 158 181 165 97.2 0 0 1 0 0 1 0 0 0 0 2 165 146 150 76.8 200e 173 175 187 103.2 0 1 0 0 0 0 0 0 0 0 1 143 158 156 76.2 

Pos.Control 119 96 121 64.8 22 20 21 14 19 23 16 15 18 28 196 82 98 107 47.8 

Pos.Control 100 107 116 62.3 16 23 19 19 20 24 17 18 22 17 195 82 82 82 41.0 

a Relative cell survival (%) = Mean number of colonies/dish in the treated x 100

 Mean number of colonies/dish in the solvent control (avg. of replicates)

b TGr = 6-Thioguanine resistantc CE (%) = Mean number of colonies/dish x 100

 No. of cells seeded/dish

d Lost due to contamination.e Cloudy in treatment medium.

f The frequency of TGr mutants is significantly higher than the concurrent solvent control value (alpha=0.05).

No. of Colonies/Dish

Toxicity Assay

No. of Colonies/Dish

Mutation Assay Cloning Efficiency (CE)

TGr Colonies/Dishb
THE DOW 

EVALUATION OF 

- 

Table 3.

IN THE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANIN

TRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAY 

 Results of the Gene Mutation Assay in CHO Cells Treated with in the Presence of S9 – 

Cell Line: CHO-K1-BH4 Positive Control: 4 and 12 μg/ml MCA

Passage: 35 Solvent Control: 1% DMSO

TG

Treatment 

μg/ml 123 RCS (%)a 1 2 3 4 5 6 7 8 9 10 Total 1 2 3 CE (%)c Clo

Solvent Control 170 160 178 98.2 1 0 0 0 0 0 2 0 0 2 5 177 157 149 80.5 Solvent Control 171 180 176 101.8 1 0 0 0 0 0 0 0 2 0 3 149 139 152 73.3 

2.5 167 154 174 95.7 0 0 0 0 0 0 1 1 1 0 3 161 123 155 73.2 2.5 174 164 149 94.1 0 1 1 1 2 2 0 0 0 1 8 129 142 147 69.7 

10 146 151 148 86.0 0 0 0 0 1 0 1 0 0 0 2 156 155 157 78.0 10 154 154 164 91.2 0 0 0 0 0 0 0 0 0 0 0 158 147 138 73.8 

25 128 115 129 71.9 2 1 0 1 0 0 0 0 0 0 4 151 148 143 73.7 

25 131 138 121 75.4 0 0 0 1 0 0 0 0 0 0 1 157 133 130 70.0 

50 67 73 67 40.0 0 0 0 0 0 0 0 0 0 0 0 117 107 137 60.2 50 66 76 75 41.9 0 0 0 0 0 0 0 0 1 1 2 134 97 108 56.5 

100d 29 34 31 18.2 1 0 1 0 0 0 0 0 1 0 3 94 111 109 52.3 

100d 28 31 29 17.0 0 0 0 0 0 0 0 0 0 0 0 109 92 106 51.2 150d 8 12 7 5.2 -e -e -e -e -e -e -e -e -e -e -e -e -e -e -e 

150d 7 5 6 3.5 -e -e -e -e -e -e -e -e -e -e -e -e -e -e -e 200d 3 2 3 1.5 -e -e -e -e -e -e -e -e -e -e -e -e -e -e -e 200d 2 2 5 1.7 -e -e -e -e -e -e -e -e -e -e -e -e -e -e -e 

Pos.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 Pos.Control (4 μg/ml) 178 166 191 103.4 14 15 15 9 13 20 15 13 8 14 136 122 137 141 66.7 

Pos.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 Pos.Control (12 μg/ml) 174 149 156 92.6 15 10 12 23 18 16 22 17 24 14 171 135 106 136 62.8 

a Relative cell survival (%) = Mean number of colonies/dish in the treated x 100

 Mean number of colonies/dish in the solvent control (avg. of replicates)

b TGr = 6-Thioguanine resistant

c CE (%) = Mean number of colonies/dish x 100

 No. of cells seeded/dish

d Cloudy in treatment medium.

e Lost due to toxicity during the expression period.

f The frequency of TGr mutants is significantly higher than the concurrent solvent control value (alpha=0.05).

No. of Colonies/Dish TGr Colonies/Dishb No. of Colonies/Dish

Toxicity Assay Mutation Assay Cloning Efficiency (CE)
THE DOW 

EVALUATION OF 

- 

Table 4.

IN THE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANIN

TRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAY 

 Results of the Gene Mutation Assay in CHO Cells Treated with in the Absence of S9 – 

Cell Line: CHO-K1-BH4 Positive Control: 621 μg/ml EMS

Passage: 36 Solvent Control: 1% DMSO

Treatment 

μg/ml 12 3 RCS (%)a 1 2 3 4 5 6 7 8 9 10 Total 1 2 3 CE (%)c 

Solvent Control 170 159 173 98.6 0 1 0 0 1 2 1 0 0 -d 5 109 86 101 49.3 

Solvent Control 154 189 173 101.4 0 0 0 0 0 1 0 0 0 1 2 119 99 102 53.3 

10 183 172 155 100.2 3 2 1 2 3 1 0 1 0 2 15 104 74 105 47.2 10 175 168 168 100.4 0 0 1 0 1 1 2 2 -d -d 7 111 106 96 52.2 

20 177 152 181 100.2 2 1 1 0 0 0 0 0 1 0 5 130 121 118 61.5 20 146 178 171 97.2 0 0 1 2 0 0 1 2 1 1 8 108 101 117 54.3 60 171 169 184 102.9 0 1 2 1 2 2 0 1 0 0 9 107 101 110 53.0 60 183 165 163 100.4 1 1 0 0 1 1 0 1 3 0 8 139 106 118 60.5 

100e 172 177 162 100.4 0 0 0 1 1 1 1 0 0 0 4 115 122 96 55.5 

100e 167 170 155 96.7 0 0 0 0 0 1 0 0 1 1 3 121 121 123 60.8 200e 172 147 162 94.5 1 0 1 0 0 1 1 0 0 1 5 116 127 116 59.8 200e 147 169 157 92.9 1 0 1 1 1 0 0 0 1 1 6 134 146 136 69.3 

Pos.Control 120 103 111 65.6 13 11 16 15 14 17 19 15 15 17 152 96 92 88 46.0 

Pos.Control 109 104 122 65.8 16 21 19 21 15 19 17 15 22 -d 165 87 92 86 44.2 

a Relative cell survival (%) = Mean number of colonies/dish in the treated x 100

 Mean number of colonies/dish in the solvent control (avg. of replicates)

b TGr = 6-Thioguanine resistant

c CE (%) = Mean number of colonies/dish x 100

 No. of cells seeded/dish

d Lost due to contamination.

e Cloudy in treatment medium.

f The frequency of TGr mutants is significantly higher than the concurrent solvent control value (alpha=0.05).

No. of Colonies/Dish TGr Colonies/Dishb No. of Colonies/Dish

Toxicity Assay Mutation Assay Cloning Efficiency (CE)
THE DOW 

EVALUATION OF 

- 

Table 5.

IN THE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-GUANIN

TRANSFERASE (CHO/HGPRT) FORWARD MUTATION ASSAY 

 Results of the Gene Mutation Assay in CHO Cells Treated with in the Presence of S9 – 

Cell Line: CHO-K1-BH4 Positive Control: 4 and 12 μg/ml MCA

Passage: 36 Solvent Control: 1% DMSO

TGr Muta

Treatment per 10

μg/ml 123 RCS (%)a 1 2 3 4 5 6 7 8 9 10 Total 1 2 3 CE (%)c Clonable C

Solvent 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 1.5

5 131 144 139 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 7.6

10 149 116 124 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 5.460 52 46 40 30.6 1 0 0 0 3 0 1 1 1 0 7 118 120 101 56.5 6.2

80d 47 35 20 22.6 1 1 0 0 1 0 2 2 1 2 10 84 79 86 41.5 12.0

80d 38 27 38 22.9 0 1 1 0 0 1 0 0 1 0 4 93 96 94 47.2 4.2

100d 25 29 26 17.8 1 0 0 0 0 0 0 1 0 1 3 83 102 85 45.0 3.3100d 28 30 29 19.3 1 1 0 0 0 2 0 0 1 0 5 90 92 88 45.0 5.6120d 34 26 25 18.9 0 0 0 0 0 0 0 0 1 1 2 124 117 103 57.3 1.7120d 19 22 34 16.6 0 0 0 0 0 0 0 1 0 0 1 100 101 102 50.5 1.0140d 24 22 12 12.9 0 0 0 0 1 0 0 0 0 0 1 93 96 92 46.8 1.1140d 20 13 21 12.0 0 0 1 0 0 0 0 0 2 0 3 80 100 90 45.0 3.3

Pos.Control (4 μg/ml) 136 148 153 97.0 23 19 29 23 26 29 28 34 19 27 257 105 125 114 57.3 224.1e

Pos.Control (4 μg/ml) 151 144 137 95.9 32 30 20 27 29 27 27 48 31 27 298 115 126 122 60.5 246.3e

Pos.Control (12 μg/ml) 123 131 135 86.3 10 11 12 18 10 11 10 8 9 16 115 125 120 125 61.7 93.2ePos.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 98.5e

a Relative cell survival (%) = Mean number of colonies/dish in the treated x 100

 Mean number of colonies/dish in the solvent control (avg. of replicates)

b TGr = 6-Thioguanine resistant

c CE (%) = Mean number of colonies/dish x 100

 No. of cells seeded/dish

d Cloudy in treatment medium.

e The frequency of TGr mutants is significantly higher than the concurrent solvent control value (alpha=0.05).

No. of Colonies/Dish TGr Colonies/Dishb No. of Colonies/Dish

Toxicity Assay Mutation Assay Cloning Efficiency (CE)
THE DOW CHEMICAL COMPANY 

STUDY ID: 

PAGE 24 

EVALUATION OF IN THE CHINESE HAMSTER OVARY 

CELL/HYPOXANTHINE-GUANINE-PHOSPHORIBOSYL TRANSFERASE (CHO/HGPRT) 

FORWARD MUTATION ASSAY 

Table 6. Historical Laboratory Background Values of 6-Thioguanine Resistant (TGr

) Mutant Frequency in 

CHO Cells – Individual Values 

S-9

Year (+ or -) No. TGr

 Mutants Per 106 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 OF IN THE CHINESE HAMSTER OVARY 

CELL/HYPOXANTHINE-GUANINE-PHOSPHORIBOSYL TRANSFERASE (CHO/HGPRT) 

FORWARD MUTATION ASSAY 

2

Table 7. Historical Laboratory Background Values of 6-Thioguanine Resistant (TGr) 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 TGr mutants per 106 cells

b

 Number of experiments

FREQUENCIES a

- 
THE DOW CHE

EVALUATION OF IN THE CHINESE HAMSTER OVARY CELL/HYPOXANTHINE-G

PHOSPHORIBOSYL TRAN UTATION ASSAY 

- 

Table 8.

SFERASE (CHO/HGPRT) FORWARD M

 Concentration Check Report – Assay B1*

Dose Level Date Mixed: 09/28/10 Overall Overall

mg/mL ACL Report #: 2010-149 Mean Std.Dev.

Target Conc. (mg/mL): 0.00

Control Mean Measured Conc: <LLQ

% o f Target C onc:

(n): (2)

Target Conc. (mg/mL): 0.250

0.25 Mean Measured Conc: 0.236

% o f Target C onc: 94.4% 94.4% NA

(n): (2)

Target Conc. (mg/mL): 1.00

1.00 Mean Measured Conc: 0.962

% o f Target C onc: 96.2% 96.2% NA

(n): (2)

Target Conc. (mg/mL): 2.50

2.50 Mean Measured Conc: 2.40

% o f Target C onc: 96.1% 96.1% NA

(n): (2)

Target Conc. (mg/mL): 5.00

5.00 Mean Measured Conc: 4.84

% o f Target C onc: 96.7% 96.7% NA

(n): (2)

Target Conc. (mg/mL): 10.0

10.00 Mean Measured Conc: 9.75

% o f Target C onc: 97.5% 97.5% NA

(n): (2)

Target Conc. (mg/mL): 15.0

15.00 Mean Measured Conc: 15.3

% of Target Con c: 101.8% 101.8% NA

(n): (2)

Target Conc. (mg/mL): 20.0

20.00 Mean Measured Conc: 19.9

% o f Target C onc: 99.4% 99.4% NA

(n): (2)

LLQ . (mg/mL): 0.0505 0.0505 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