Document xjqrKve4747ZwzQ8d60zQJYvb
AR226-3094
TRADE SECRET
FINAL REPORT
Study Title
H-23005: IN VITRO MAMMALIAN CELL GENE MUTATION TEST WITH AN INDEPENDENT REPEAT ASSAY
LABORATORY PROJECT ID: HLO-1998-01211
AUTHORS:
Richard H.C. San, Ph.D. Jane J. Clarke, B.S.
STUDY COMPLETED ON: March 19, 1998
PERFORMING LABORATORY:
MA BioServices, Inc. 9630 Medical Center Drive, Rockville, MD 20850
for
E. I. du Font de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine
Elkton Road, P. 0. Box 50
Newark, Delaware 19714-0050
PERFORMING LABORATORY STUDY No.:
G97CK07.782001
I if* ^|
WORK REQUEST No.: H------ flX^^R^^B^H is
Page 1 of 31
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H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPontHLO-1998-01211
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
This study was conducted in compliance with EPA FIFRA (40 CFR 160) and EPA TSCA (40 CFR 792) Good Laboratory Practice Standards, and OECD Principles of Good Laboratory Practice (C(81)30(Final), Annex 2) with the following exception:
Treatment solutions or suspensions were not analyzed for identity, composition, uniformity, or stability of the test and control articles. The procedures used by trained personnel to prepare the treatment solutions intended to ensure:
A. The accuracy of concentration because the test and control articles were weighed on
an analytical balance accurate to 3 decimal places and the vehicle in which the test
and control substances were dissolved was accurately measured with graduated pipettes or flasks;
B. Uniformity because all treatment solutions or suspensions were mixed prior to administration to the test system; and
C. Stability because treatment solutions or suspensions were prepared just prior to administration to the test system.
This deviation did not affect the validity or the integrity of the study.
Submitter/Submitter: E. I. du Font de Nemours and Company
Study Director:
Study Monitor
^ ^ ^ ( / ^ Richard H.C. San, Ph.D.
Scientific Director
s/f'y/^s
Date
S/^/^g
Date
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H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
CERTIFICATION
We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study.
Reported Approved and Issued by Study Director:
/t^'^-^^)?
Richard H.C. San, Ph.D. Scientific Director
s/f7/ya
Date
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STUDY INFORMATION
Substance Tested: Synonyms/Codes:
* H-23005
DuPontHLO-1998-01211
Physical Characteristics: Tan liquid
Stability:
The test substance appeared to be stable under the
conditions of the study; no evidence of instability was
observed.
Sponsor:
E. I. du Font de Nemours and Company Wilmington, Delaware 19898
os^r"' " '
Study Initiated/Completed: December 11, 1997 / March 19, 1998
In-Life Initiated/Completed: December 16, 1997 / February 13, 1998
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H-2J005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
QUALITY ASSURANCE STATEMENT
Study Title:
Study Number:
Study Director:
IN VITRO MAMMALIAN CELL GENE MUTATION TEST WITH
AN INDEPENDENT REPEAT ASSAY
G97CK07.782001 Richard H. C. San, Ph.D.
This study has been divided into a series of in-process phases. Using a random sampling approach. Quality Assurance monitors each of these phases over a series of studies- Procedures, documentation, equipment records, etc., are examined in order to assure that the study is performed in accordance with the U.S. PDA Good Laboratory Practice Regulations (21 CFR 58), the U.S. EPA GLPs (40 CFR 792 and 40 CFR 160), the UK GLP Compliance Programme, the Japanese GLP Standard, and the OECD Principles of Good Laboratory Practice and to assure that the study is conducted according to the protocol and relevant Standard
Operating Procedures.
The following are the inspection dates, phases inspected, and report dates of QA inspections of this study.
INSPECT ON 11 DSC 97, TO STUDY DIR 11 DEC 97, TO MGMT 11 DEC 97 PHASE: Protocol Review
INSPECT ON 23 JAN 98-27 FEB 98, TO STUDY DIR 27 FEB 98, TO MGMT 27 FE3 98
PHASE: Scoring toxicity plates
INSPECT ON 23 FEB 98-24 FEB 98, TO STUDY DIR 24 FEB 98, TO MGMT 27 FEB 98
PHASE: Draft Report
INSPECT ON 19 MAR 98, TO STUDY DIR 19 MAR 98, TO MGMT 19 MAR 98
PHASE: Draft to Final Report
This report describes the methods and procedures used in the
study and the reported results accurately reflect the raw
data of the study.
/rW^ S^^c^L^^^_________ ^/Vg Becky(JD.
SchreckencosT^/ "
B.
S.
QUALITY ASSURANCE
DATE
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H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPR.T) with an Independent Repeat Assay
DuPont HLO-1998-01211
TABLE OF CONTENTS
Page
Good Laboratory Practice Compliance Statement
2
..........................
Certification .................................................... 3
Study Information ............................................... 4
Quality Assurance Statement ........................................ 5
Table of Contents ................................................ 6
Summary ..................................................... 7
Purpose ....................................................... 8
Characterization of Test and Control Articles ............................. 8
Materials and Methods ............................................ 8
Results and Discussion ........................................... 11
Conclusion ................................................... 12
Records and Sample Storage
13
........................................
References .................................................... 13
Data Tables ................................................... 14
Appendix I: Historical Control Data .................................. 21
Appendix II: Study Protocol ........................................ 23
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Sanitized. Dsee net contain TSCA C'
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
SUMMARY
The test article, H-23005, was tested in the CHO/HGPRT Mutation Assay in the absence and presence ofAroclor-induced rat liver S9. The preliminary toxicity assay was used to establish the dose range for the initial mutagenesis assay. The initial and independent repeat mutagenesis assays were used to evaluate the mutagenic potential of the test article. The dosing solutions were adjusted to compensate for the purity of the test article.
Sterile distilled water was determined to be the solvent of choice based on solubility information from the Sponsor and compatibility with the target cells. The test article was soluble in sterile distilled water at a concentration of 50 mg/mL, the maximum concentration
prepared in the preliminary toxicity assay.
In the preliminary toxicity assay, the maximum concentration of H-23005 tested was 5000 u.g/mL. There was visible precipitate in the treatment medium at concentrations ^1500 ug/mL at the end of treatment. Selection of dose levels for the mutagenesis assay was
based on the cloning efficiency relative to the solvent control and the solubility limitations.
Substantial toxicity, i.e., cloning efficiency $50% of the solvent control, was observed at S50 ug/mL without activation and at 5000 .ug/mL with S9 activation. Based on these findings, the doses chosen for the initial mutagenesis assay ranged from 15 to 2500 u,g/mL
for both the non-activated and S9-activated cultures.
In the initial mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies >40 mutants per 106 clonable cells, were observed. Visible precipitate was
observed in treatment medium at concentrations S1500 jig/mL at the end of treatment. Concentrations of $500 u,g/mL were soluble in treatment medium. Toxicity, i.e., cloning efficiency <50% of the solvent control, was observed at doses of >1500 ug/mL without S9
activation and at 2500 pg/mL with S9 activation. Based on these findings, the doses chosen for the independent repeat assay ranged from 50 to 1500 u.g/mL for the non-activated cultures and from 50 to 2500 u.g/mL for the S9-activated cultures.
In the independent repeat assay, no positive responses were observed. Visible precipitate was
observed in treatment medium at concentrations S750 u,g/mL at the end of treatment. Concentrations of $500 pg/mL were soluble in treatment medium. No toxicity was observed
at any dose levels, with or without S9 activation.
Under the conditions of this study, test article H-23005 was concluded to be negative in the CHO/HGPRT Mutation Assay.
-7ompany Sanded- Does mi contain TSCA CB;
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
PURPOSE
The purpose of this study is to evaluate the mutagenic potential of the test article based on quantitation of forward mutations at the hypoxanthine-guanine phosphoribosyi transferase (HGPRT) locus of Chinese hamster ovary (CHO) cells.
CHARACTERIZATION OF TEST AND CONTROL ARTICLES
The test article, H-23005, was received by MA BioServices, Inc. (MA) on December 9, 1997
and was assigned the code number 97CK.07. The test article was characterized by the
Sponsorjjg a clear, tan liquid, which should be stored at room temperature. Its purity was
^IIIHIlBBHBIBlffl^P011 given
receipt, the test article was described as pale
yellow liquid with very tiny, dark particles and was stored at room temperature, protected
from exposure to light.
The vehicle used to deliver H-23005 to the test system was sterile distilled water (CAS 773218-5) obtained from Life Technologies. The dosing solutions were adjusted to compensate for the purity of the test article.
Ethyl methanesulfonate (EMS), CAS 62-50-0, lot 04625BR, expiration 3/98, was obtained fro.'n Aldrich Chemical Company and was used at a stock concentration of 20 u.l/mL as the positive control for the non-activated test system. Benzo(a)pyrene (B(a)P), CAS 50-32-8, lot 96H2600, expiration 9/98, was obtained from Sigma Chemical Company and was used at a stock concentration of 400 u.g/mL as the positive control for the S9-activated test system.
MATERIALS AND METHODS
Test System
CHO-K,-BH4 cells were obtained from Dr. Abraham Hsie, Oak Ridge National Laboratories, Oak Ridge, TN. CHO cells were cleansed in medium supplemented with hypoxanthine,
aminopterin and thymidine (HAT) then frozen. The freeze lot of cells was tested and found to be free of mycoplasma contamination. Cells used in the mutation assay were within four
subpassages from frozen stock in order to assure karyotypic stability.
Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was
prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor-1254, 500 mg/kg, five days prior to sacrifice. The S9 was batch prepared and stored at ^ -70C until used. Each bulk preparation of S9 was assayed for sterility and its ability
to metabolize 2-aminoanthracene and 7,12-dimethyl-benz(a)anthracene to forms mutagenic
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H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
to Salmonella typhimurhim TA100.
Immediately prior to use, the S9 reaction mixture was prepared by mixing S9 and 10 mM calcium chloride (CaCI;) with a filter-sterilized cofactor pool to contain 100 ul S9/mL cofactor pool, 4 mM nicotinamide adenine dinucleotide phosphate (NADP), 5 mM glucose-6-phosphate, 30 mM potassium chloride (KC1), 10 mM magnesium chloride (MgCL), and 50 mM sodium phosphate buffer, pH 8.0. The S9 reaction mixture was stored on ice until used. The S9 reaction mixture for the preliminary toxicity assay contained only 2.5 mM glucose-6-phosphate due to a calculation error. Since the data from the preliminary toxicity assay provided a toxicity profile that was consistent with that in the initial mutation assay,
this protocol deviation was not considered to have adversely impacted the integrity or
conclusions of this study.
Preliminary Toxicity Assay
The preliminary toxicity assay was used to establish the optimal dose levels for the mutagenesis assay and consisted of evaluation of test article effect on colony-forming efficiency. CHO cells were exposed for 5 hours at 371C to the vehicle alone and nine
concentrations of test article ranging from 0.5 to 5000 u.g/mL in both the absence and presence of S9-activation.
Mutagenesis Assays
The initial and independent repeat mutagenesis assays were used to evaluate the mutagenic
' potential of the test article. CHO cells were exposed for 5 hours at 371C to the vehicle alone, appropriate positive controls and at least five concentrations of test article in duplicate
in both the absence and presence of S9.
Treatment of the Target Cells
The mutagenesis assay was performed according to a protocol developed from published
methodologies (Hsie et al; 1981; and O'Neill et al., 1977). Exponentially growing CHO-K,-BH4 cells were seeded in F12FBS5-Hx at a density of5xl05 cells/25 cm2 flask and were incubated at 371C in a humidified atmosphere of5l% CO^ in air for 18-24 hours.
The cells for the preliminary toxicity assay were seeded at 5.6x105 cells/25 cm2 flask due to a calculation error. Since the data from the preliminary toxicity assay provided a toxicity profile that was consistent with that in the initial mutation assay, this protocol deviation was not considered to have adversely impacted the integrity or conclusions of this study. F12FBS5-Hx is Ham's F12 medium without hypoxanthine supplemented with 5% dialyzed FBS, 100 units penicillin/mL, 100 ug streptomycin/mL and 2mM L-glutamine/mL.
The time of initiation of chemical treatment was designated as day 0. Treatment was carried
out by refeeding the treatment flasks with 4.5 mL F12FBS5-Hx/25 cm2 flask for the nonactivated study and 3.5 mL F12FBS5-Hx and 1 mL S9 reaction mixture/25 cm2 flask for the
9-
ffompany Sanitized. Does not contain TSCA CBi
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
89-activated study, to which was added 500 ,al dosing solution of test article in vehicle or vehicle alone. The positive control cultures were treated with 50 uL of EMS or B(a)P in a total of 5 mL per 25 cm2 flask- Duplicate flasks of cells were exposed to at least five concentrations of the test article for 5 hours at 371C. After the treatment period, all media were aspirated, the cells washed with Ca""""- and Mg^-free Hanks' balanced salt solution (CMF-HBSS) and cultured in F12FBS5-Hx for an additional 18-24 hours at 371C. In the preliminary toxicity assay, the cultures treated with 5000 and 1500 ug/mL were rinsed twice with CMF-HBSS instead of once. This is a deviation from the Standard Operating Procedures of the testing facility; however, the Study Director has determined that this deviation did not affect the integrity or conclusions of the study. At this time, the cells were
subcultured to assess cytotoxicity and to initiate the phenotypic expression period.
Evaluation of Cytotoxicity
For evaluation of cytotoxicity, the replicates from each treatment condition were detached using trypsin and subcultured independently in F12FBS5-Hx, in triplicate, at a density of 100 cells/60 mm dish. After 7-10 days incubation, the colonies were rinsed with HBSS, fixed
with methanol, stained with 10% aqueous Giemsa, counted and cloning efficiency determined.
Expression of the Mutant Phenotype
For expression of the mutant phenotype, the replicates from each treatment condition were trypsinized and subcultured independently in F12FBS5-Hx, in duplicate, at a density no
greater than 106 cells/I 00 mm dish. Subculturing by trypsinizing at 2-3 day intervals was employed for the 7-9 day expression period. At the end of the expression period, selection for the mutant phenotype was performed.
Selection of the Mutant Phenotype
For selection of the TG-resistant phenotype, the replicates from each treatment condition were trypsinized and replated, in quintuplicate, at a density of 2xl05 cells/100 mm dish in F 12FBS5-Hx containing 10 uM 6-thioguanine (TG, 2-amino-6-mercaptopurine). For cloning efficiency determinations at the time of selection, 100 cells/60 mm dish were plated in triplicate. After 7-10 days of incubation, the colonies were fixed, stained and counted for
both cloning efficiency and mutant selection.
Evaluation of Test Results
The cytotoxic effects of each treatment condition were expressed relative to the solventtreated control (relative cloning efficiency). The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (usually 2xl06 cells: 10 plates at 2xl05 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 106 clonable cells. For experimental conditions in which no mutant colonies were observed,
C&wnany Sanifesc' .';;-;, -sot confsir; TSCA CBs
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
mutant frequencies were expressed as less than the frequency obtained with one mutant colony. Mutant frequencies generated from doses giving <: 10% relative survival are presented in the data but were not considered as valid data points.
Because spontaneous mutant frequencies are very low for the CHO/HGPRT assay, calculation of mutagenic response in terms of fold increase in mutant frequency above the background rate does not provide a reliable indication of the significance of the observed response. The wide acceptable range in spontaneous mutant frequency also suggests the need to set a minimum mutant frequency for a response to be considered positive. Hsie et al. (1981) refer to a level of 50 mutants per 106 clonable cells. In this laboratory, a more conservative approach is used which sets the minimum level at >40 mutants per 106 clonable cells.
All conclusions were based on sound scientific judgement; however, the following criteria are presented as a guide to interpretation of the data:
The test article was considered to induce a positive response if there was a concentration-
related increase in mutant frequencies with at least two consecutive doses showing mutant
frequencies of>40 mutants per 106 clonable cells.
If a single point above 40 mutants per 106 clonable cells was observed at the highest
dose, the assay was considered suspect.
If no culture exhibited a mutant frequency of>40 mutants per 106 clonable cells, the test
article was considered negative.
Criteria for a Valid Test
The cloning efficiency of the solvent control must be greater than 50%. The spontaneous mutant frequency in the solvent control must fall within the range of 0-25 mutants per 106 clonable cells. The positive control must induce a mutant frequency at least three times that of the solvent control and must exceed 40 mutants per 106 clonable cells. There must be at
least four analyzable test article concentrations with mutant frequency data.
RESULTS AND DISCUSSION
Solubility
Sterile distilled water was determined to be the solvent of choice based on solubility information from the Sponsor and compatibility with the target cells. The test article was soluble in sterile distilled water at a concentration of 50 mg/mL, the maximum concentration prepared in the preliminary toxicity assay.
11
lifaed. Does not contain TSCA CB?
H-23005: In Vitro Mammalian Cell Gene iMutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
Preliminary Toxicity Assay
The results of the preliminary toxicity assay are presented in Table 1. CHO cells were exposed to solvent alone and nine concentrations of test article ranging from 0.5 to 500'0 (Ag/mL in the absence and presence of S9 reaction mixture. There was visible precipitate in the treatment medium at concentrations >1500 ug/mL at the end of treatment. The osmolality of the solvent control was 319 mmol/kg and the osmolality of the top dose, 5000 u,g/mL (which was non-precipitating at the beginning of treatment), was 307 mmol/kg. Cloning efficiency relative to the solvent controls (RCE) was 0% at 5000 Hg/mL without activation and 1% at 5000 u,g/mL with S9 activation. Based on the results of the toxicity test,
the doses chosen for the initial mutagenesis assay ranged from 15 to 2500 ug/mL for both the non-activated and S9-activated cultures.
Mutagenesis Assays
The cytotoxic effects of the test article (concurrent cytotoxicity) from the initial mutagenesis
assay are presented in Table 2. Mutagenicity data are presented in Tables 3 and 4. In both the non-activated and S9-activated systems, cultures treated with concentrations of 15, 50, 150, 500, 1500 and 2500 u.g/mL were cloned. Visible precipitate was observed in treatment medium at concentrations >1500 Hg/mL at the end of treatment. Concentrations of <500 Hg/mL were soluble in treatment medium. Relative cloning efficiency was 0% and 24% at the highest dose tested in the non-activated and S9-activated systems, respectively. The non-activated cultures treated with 2500 u.g/mL were not seeded for mutant selection due to
toxicity. None of the treated cultures that were cloned for mutant selection exhibited mutant frequencies of greater than 40 mutants per 106 clonable cells.
The cytotoxic effects of the test article from the independent repeat assay are presented in Table 5. Mutagenicity data are presented in Tables 6 and 7. In the non-activated system, cultures treated with concentrations of 50,150, 500, 750, 1000 and 1500 ug/mL were cloned. In the S9-activated system, cultures treated with concentrations of 50, 150, 500, 1000, 2000 and 2500 Hg/mL were cloned. Visible precipitate was observed in treatment medium at concentrations ^750 j^g/mL at the end of treatment. Concentrations of <5QQ u,g/mL were soluble in treatment medium. Relative cloning efficiency was 68% and 73% at the highest dose tested in the non-activated and S9-activated systems, respectively. None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 106 clonable cells.
CONCLUSION
All criteria for a valid study were met as described in the protocol. The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, H-23005 did
not cause a positive response in the non-activated and S9-activated systems and was concluded to be negative.
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
RECORDS AND SAMPLE STORAGE
Upon completion of the final report, all raw data and reports are maintained by the Quality Assurance Unit of MA BioServices, Inc., Rockville, Maryland, in accordance with the
relevant Good Laboratory Practice Regulations.
REFERENCES
Hsie, A.W., D.A. Casciano, D.B. Couch, B.F. Krahn, J.P. O'Neill, and B.L. Whitfield (1981) The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals. A report of the Gene-Tox Program, Mutation Research 86:193-214.
O'Neill, J.P., P.A. Brimer, R. Machanoff, G.P. Hirsch, and A.W. Hsie (1977) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyi transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system. Mutation Research 45:91-101.
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H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
TABLE 1 CHO/HGPRT HUTATIOM ASSAY
Preliiminary Toxiciity Assay Using H-23005
-S'9
Treatment1 Colonies per disti
1
2
3
Cloning Efficiency2
Relative Cloning Efficiency'
(%)
+S9
Treatment' Co tonies per dish
(^g/inl.)
1
2
3
Cloning Efficiency2
Relative Cloning Efficiency3
(%)
Uater (100 pL/mL}:
82
77
87
H-Z3005 (pg/mL):
0.5 80
89
60
1.5 57
62
77
5.0 74
67
64
15
58
76
56
50
32
28
52
150
33
45
38
500
18
23
25
1500'
27
22
11
5000'
0
0
0
0.82
0.76 0.65 0.68 0.63 0.37 0.39 0.22 0.20 0.00
100
83
82 73
0.79
100
93
0.5
60
59 70
0.63
79
80
1.5
59
72 64
0.65
82
83
5.0
78
79 52
0.70
88
77
15
78
52 52
0.61
76
46
50
61
85 76
0.74
93
47
150
49
77 59
0.62
78
27
500
48
56 35
0.46
58
24
1500'
46
57 44
0.49
62
0
5000'
0
1
1
0.01
1
1 Cells w ere exposed tio the test article for 5 hours at 371C.
2
Cloning efficiency =
total colonies counted
nuinber of dishes x 100 cells/clish ' Relative cloning eff iciency = cloning efficiency of treatment group X 100
cloning efficiency of: solvent group
visible precipitation, as observed at the end of the treatment period
14
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPontHLO-1998-01211
fABLE 2
CHI3/HGPRT MUTATIOM ASSAY
Concurrent iCytotox:icity Test Using H-230015 Ini tial Assay
-S'?
Treatmen t1 Co 1on i es per dishi
1
2
3
Cloning Efficiency2
Re lative
Cl on ing
Effiiciency3
(%)
+S9
Treatment1 Ccilonies; per clish
1
2
3
Cloning
Efficiency^
Relative Cloning
Efficiency3
(%)
Water (1 00 PL/mi.)
A
47
75
63
8
56
65
36
EMS (O.Z PL/mi.):
A
23
31
38
B
27
34
25
H-23005 dug/nil.):
15 A
65
88
45
B
50
50
49
50 A
78
62
54
B
46
43
42
150 A
57
62
46
B
36
39
40
500 A
52
61
46
B
46
42
28
1500' A
B
9
12
12
1
5
2
2500' A
0
0
0
B
0
0
0
0.57
0.30
0.58 0.54 0.47 0.46 0.07 0.00
A
80
86 81
100
B
81
92 82
0.84
100
B(a)P (4.0 pg/mL):
A
10
17 28
52
B
14
12 19
0.17
20
15 A
87
93 77
101
B
89
96 73
0.86
103
50 A
91
78 76
95
B
80
86 76
0.81
97
150 A
110 113 109
82
B
76
85 99
0.99
118
500 A
91
73 98
80
B
80
77 82
0.84
100
1500" A
60
70 58
12
B
66
77 65
0.66
79
2500' A
11
13 16
0
B
27
30 23
0.20
24
A and B .are duplicate cultures
i
Cells were exposed to the test article for 5 hours at 371C.
2
Cloning efficiency =
total colonies counted
number of dishes X 100 cells/dish
3
Relative cloning efficiency = cloning efficiency of treatment qrouo X 100
* cloning efficiency of solvent group
visible precipitation, as observed at the end of the treatment period
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H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPontHLO-1998-01211
1FABLE 3 CHO/HGPRT MUTATION / aSAY
Mon-activated (-Si9) Study Using H-;'3005
Treatmen t1
C:toninq Ef ficiency at Siilection
Coloni es per Dis;h
1
Z
3
1rota I Colonies
Cloning
Efficienc y2
Mutaint Co 101nies/Sel.ectioli Dish
1
2
3
4
5
Total Mutants/10 Mutant Clonable Colonies Cells3
Water (100 (A/mi.):
A
43
40
B
56
57
EMS (O.Z fJL/mL);
A
65
71
B
75
68
H-23005 (yg/niL):
15 A
B
64
72
57
63
50 A
B
150 A
B
47
61
47
49
43
33
58
68
500 A
B
70
72
66
79
1500' A
B
56
58
71
81
59
63
318
57
77
413
78
73
407
63
46
313
43
62
307
71
58
416
47
68
381
0.53
0.69
0.68 0.52 0.51 0.69 0.64
4
2
a
a
2
0
1
3
2
1
15
17.7
32
44
57
56 37
41
37
43
41
35
423
307.3
0
1
0
0
0
0
2
2
1
0
6
0
0
0
0
0
3
4
3
6
1
17
0
0
0
0
2
0
0
0
1
0
3
1
1
a
0
0
0
0
0
1
1
4
4
2
4
4
2
0
0
1
0
0
17
4.4 16.3 2.9 3.2 13.4
A and B .are duplicate culturel Celts were exposed t o 2 Cloning efficiency =
the test ar tide for 5 hours at 371C.
total colonies counted number of dishes x 100 cells/dish
3
Hutants/106 clonable cells =
total mutant colonies
number selection dishes X cloning efficiency X 2 x 10' cells
X 10"
plate lost to contamination visible precipitation, as observed at the end of the treatment period
16-
^pw
^'"tHS'Sl?
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPontHLO-1998-01211
TABLE 4 CHO/HGPRT MUTATION ASSAY
Activated (+S9) Study Using H-23005
Treatment1
Cloni ng Effici ency at Selection
Colonies p er Dish
1
2
3
Total Colonies
Cloniing
Efficiiency2
Total Hutants/10"
Hutaint Col onies/Set ection Dish Mutant Clonable
1
2
3
4
5 Colonies Cells3
Water (100 (A/niL):
A
78
84
B
63
67
B(a)P (4.0 pg/mL):
A
51
71
8
54
78
H-23005 ((/g/mL):
15 A B
78
84
67
55
50 A
B
86
74
62
51
150 A
B
71
76
72
76
500 A
B
63
66
54
75
1500' A
B
60
63
98
88
2500' A
B
67
63
60
63
70
75
437
53
60
367
69
47
400
72
59
404
67
72
434
65
64
387
69
53
431
67
49
369
0.73
0.61
0.67 0.67 0.72 0.65 0.72 0.62
1
2
0
3
1
0
0
0
0
0
7
24
26
22
30 23
30
22
36
27 23
263
4.8
215.0
0
0
0
0
0
0
1
0
1
0
2
1
1
1
4
1
2
4
2
3
1
20
0
0
0
0
0
0
0
1
1
2
4
0
1
0
0
0
0
1
3
2
3
10
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1.5 14.9 2.8 7.8 0.7 <Q.S'
A and B are duplicate cultures
i
Cells were exposed tia the test article for 5 hours at 371 C.
2
Cloning efficiency =
total colonies counted
number of dishes x 100 cells/dish
3
Mutants/106 clonable cells =
total mutant colonies
x 106
n umber selection dishes X clon ing efficiency X 2 x 10 cells
4
Calculated ian the ba;iis of <1 inutant colony observed in the total number of dishes prepar ed.
visible p(ecipitation, as observred at the end of the treatment period
- 17Company San?(?zed. Does no; contain TSCA CB?
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
TABU: 5
CH0/HGPRT MUT AT ION ASSAY
Coricurrent Cytotoxicit y Test Using H-23005 independent Repeat Assay
-S9
Treatment1 Colonies oer dish
1
2
3
Cloning Efficiency2
Re lative
Cl on ing
Eff iciency3
(%)
+S9
Treatment' Colonies per dish
1
2
3
Cloning Efficiency2
Relative Cloning Efficiency3
m
Water (100 yL/mL):
A
64
54
60
B
52
72
56
EMS (0.2 yL/raL):
A
33
34
46
B
24
42
37
H-23005 (A'g/im-):
50 A
63
75
46
B
80
80
61
150 A
36
48
29
B
39
43
63
500 A
56
54
62
B
59
48
55
750' A
57
73
52
B
58
35
45
1000' A
39
44
53
B
50
42
64
1500' A
38
36
51
B
38
39
42
0.60
0.36
0.68 0.43 0.56 0.53 0.49 0.41
A
65
57 74
100
B
61
69 71
0.66
100
B(a)P (4.0 lUg/inL):
A
28
35 29
60
B
18
19 12
0.24
36
50 A
55
59 82
113
B
62
65 58
0.64
96
150 A
80
81 65
72
B
49
67 46
0.65
98
500 A
76
75 79
93
B
56
66 65
0.70
105
1000' A
52
68 60
89
B
46
60 51
0.56
85
2000' A
61
73 46
82
B
60
48 58
0.58
87
2500' A
55
65 59
68
B
28
42 39
0.48
73
A and B .ire duplicate cultures
l
Celts were exposed toi the tes1: article for 5 hour s at 371"C.
2
Cloning efficiency =
total colonies counted
number o f dishes x 100 cells/dish
3
Relative cloning effi ciency = cloning efficiency of treatment group X 100
cloning efficiency of solvent g roup
visible' precipitation, as obsierved at the end of the treatment period
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
TABLE 6
CHO/HGPRT MUTATION ASSAY
Non-activated (-S9) Study Using H-23005
Independent Repeat Assay
Treatmen t1
C toning Ef-ficiency at Select ion
Coloniiss per Ois,h
rota I
lSloni ng
1
2
3 Colonies Ef ficie'ncy"
Mut;ant Colorlies/Sel.ection [lish
1
2
3
4
5
Total Mutant Colonies
1lutants/105
Clonable Cells3
Uater (100 yL/mL):
A
63
60
B
36
66
EHS (0.2 PL/mL):
A
36
46
B
31
36
H-23005 (Pg/mL):
50 A
B
52
76
56
50
150 A
B
60
96
60
56
500 A
B
750' A
B
48
50
55
49
57
69
54
61
1000' A
B
63
44
84
61
1500' A
B
68
63
49
60
58
65
348
51
32
232
56
74
364
69
59
400
46
73
321
73
67
381
63
73
388
82
53
375
0.58
0.39
0.61 0.67 0.54 0.64 0.65 0.63
2
0
0
1
0
0
0
0
0
0
3
84
75
79
76 85
56
54
49
61
54
67
1
0
0
0
0
1
0
1
1
0
4
1
1
0
0
1
0
1
0
0
0
4
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
2
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
2.6
870.3
3.3 3.0
<0.94 3.1
<0.8"
0.8
A and B <are duplicate cultures
l
Cells were exposed tiD the test article for 5 hours at 3 71C.
2 Cloning efficiency =
total colonies counted
number of dishes x 100 cells/dish
3 Mutants/105 clenable cells =
total mutant colonies
X 10
number selection dishes X cloning efficiency X 2 x W cells
4
Calculated on the basis of <1 mutant colony observed in the total number of dishes prepared.
visible precipitation, as observed at the end of the treatment period
- 19eompany Sanitized. Does not contain TSCA CBI
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
TABLE 7
CHO/HGPRT MUTATION ASSAY
Activated (+S9) Study Using H-23005 Independent Repeat Assay
Treatment
Cloning Effici ency at Sielection
Colon ies per I)ish
Total
Cloning
1
2
3 Colonies Efficiency2
Mut,ant Coloniies/S<"lectio n
1
2
3
4
Total
Dist1 Mutant 5 Colonies
Mutants/10
Clonable Cells'
Water (100 pL/mL):
A
108
127
B
75
82
B(a)P (4.0 yg/ml.):
A
121
81
B
72
96
H-23005 (|Ug/mL):
50 A
B
84
115
76
92
150 A
B
97
110
75
99
500 A
B
1000' A
B
87
79
76
89
76
80
79
81
2000' A
B
86
91
86
71
2500' A
B
55
77
104
91
118
64
574
103
82
555
109
82
558
115
100
596
113
96
542
98
65
479
95
79
508
66
111
504
0.96
0.93
0.93 0.99 0.90 0.80 0.85 0.84
0
0
0
0
0
0
0
1
0
1
2
33
26
19
24 30
15
17
21
21
19
225
2
1
4
4
1
0
0
0
1
1
14
0
2
1
2
0
0
0
0
0
0
5
3
3
2
2
2
0
0
0
0
0
12
0
0
0
0
0
1
0
1
1
1
4
0
0
0
0
0
0
3
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
1.0
121.6
7.5 2.5 6.6 2.5 1.8 <0.64
A and B are duplica'Ce cultures
l
Cells WE"re exposed to the test article for 5 hou rs at 371C.
2
Cloning efficiency =
total colonies counted
number of dishes x 100 cells/dish
3
Mutants/100 clonable cells = ____________total mutant colonies________1_0_3 ____ x 10
number selection dishes X cloning efficiency X 2 x
cells
4
Calculated on the basis of <1 mutant colony observed in the total number of dishes prepared.
visible precipitation, as observed at the end of the treatment period
-20-
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
APPENDIX I Historical Control Data
-21 Sompany Sanitized. Does not contain TSCA CB?
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
CHO/HGPRT Assay Historical Control Data
1995 - 1997
Mean MF SD
Maximum Minimum
Non-activated
Solvent Control
0.2uL/mL EMS
7.0 6.2 22.9 0.0
312.3 97.3
725.7 144.0
89-activated
Solvent Control
4.0u,g/mL B(a)P
6.1 6.0 23.1 0.5
180.3 63.0
349.0 38.1
Solvent control (culture medium, distilled water, saline, DMSO, ethanol, acetone or vehicle supplied by Sponsor)
EMS B(a)P MF SD
Ethyl methanesulfonate Benzo(a)pyrene Mutant frequency per 106 clonable cells Standard deviation
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
APPENDIX II
Study Protocol
-23 -
Sempany SanWzedL Does not contain TSCA C8S
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
MA Study Number: ^9^Ci<07. 7?AOO 1
1.0 PURPOSE
In Vitro Mammalian Cell Gene Mutation Test with an Independent Repeat Assay
The purpose of this study is to assess the mutagenic potential of a test article based on quantitation of forward mutations at the hypoxanthine-guanine phosphoribosyi transferase (HGPRT) locus of Chinese hamster ovary (CHO) cells.
2.0 SPONSOR
2.1 Name:
E. I- da Font de Nemours and Company
2.2 Address:
Haskell Laboratory for Toxicology and Industrial Medicine
P.O. Box 50, Elkton Road Newark, DE 19714
2.3 Representative:
Brian H.'Mathison, Ph.D.
|jlBHH| pas'
--%
2.4 Sponsor Project #:
--^^^^^^^Tfc^
3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES
3.1 Test Article: 3.2 Controls:
H-23005.
Negative: Positive:
Test article solvent (or vehicle) Ethyl methanesulfonate (EMS) Benzo(a)pyrene (BaP)
3.3 Determination of Strength, Purity, etc.
The Sponsor will be directly responsible for determination and documentation of the analytical purity and composition of the test article and the stability and strength of the dosing solutions.
3.4 Test Article Retention Sample
The retention of a reserve sample of the test article will be the responsibility of
the Sponsor.
4.0 TESTING FACILITY AND KEY PERSONNEL
4.1 Name:
Toxicology Testing Facility
MA BioServices, Inc.
Protocol No. SPGT7S2001
12/02/97
(MA I of 8
BIOSERVICES'
-^
-24-
.'S_SQlTr .uStWs Ssad..RrE.ui-..yzee..-,' . ^--.,.Q- ^ ^
esnigin T^ ^
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPontHLO-1998-01211
4.2 Address:
9630 Medical Center Drive Rockville, MD 20850
4.3 Study Director:
Richard H. C. San, Ph.D.
5.0 TEST SCHEDULE
5.1 Proposed Experimental Initiation Date:
/-2//6 /?7
5.2 Proposed Experimental Completion Date: 5.3 Proposed Report Date: 6.0 TEST SYSTEM
i//s /ys
^ /^ 7 /y 3
The CHO-K1-BH4 cell line is a proline auxotroph with a modal chromosome number of 20, a population doubling time of 12-14 hours, and a cloning efficiency of usually greater than 80% (Li et a!., 1987). This subclone (Dl) was derived by Dr. Abraham Hsie. Oak Ridge National Laboratories, Oak Ridge, TN. CHO cells were cleansed in medium supplemented with HAT (hypoxanthine, aminopterin and thymidine) then frozen. Cells
used in the mutation assay will not exceed four subpassages from frozen stock. Each
freeze lot of cells has been tested and found to be free of mycoplasma contamination.
The CHO/HGPRT assay was designed to select for mutant cells which have become resistant to such purine analogues as 6-thioguanine (TG) and 8-azaguanine as a result of mutation at the X-chromosome-linked HGPRT locus (O'Neill et a/.. 1977; Hsie et al., 1981; Machanoffe/ al., 1981; Li et al., 1987). This system has been demonstrated to be sensitive to the mutagenic action of a variety of chemicals (Hsie et al., 1981).
7.0 EXPERIMENTAL DESIGN AND METHODOLOGY
The assay will be performed by exposing CHO cells for 5 hours to five concentrations of test article as well as positive and the solvent controls in the presence and absence of an exogenous source of metabolic activation. After a seven to nine day expression period, the treated cells will be cultured in the presence of 10 U.M TG for selection of mutant colonies. The mutagenic potential of a test article will be determined by its ability to
induce a dose-related increase in the number of TG-resistant mutant colonies when
compared to the solvent control.
7.1 Selection of Solvent
Unless the Sponsor has indicated the test article solvent, a solubility determination will be conducted to measure the maximum soluble concentration in a variety of
solvents. Solvents compatible with this test system, in order of preference, include, but are not limited to, culture medium or distilled water (CAS 7732-18-5), dimethylsulfoxide (CAS 67-68-5), ethanol (CAS 64-17-5) and acetone (CAS 6764-1). The solvent of choice will be that solvent, selected in order of preference,
Protocol No. SPGT7S2001
12/02/97
2 of 8
^MA BlOSERVICES-
-25-
eompany Sawf-f^ ""^ no! c-.r'Sam TSCA CBi
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPontHLO-1998-01211
that permits preparation of the highest soluble stock concentration, up to a maximum of 500 mg/rnl.
Dose Selection
The optimal dose levels for the mutation assay will be selected following a
preliminary toxicity test based upon colony-forming efficiency. Approximately 5 x 10s CHO cells will be seeded into 25 cm2 flasks and incubated at 371C in
a humidified atmosphere of5l% CO; in air. Eighteen to 24 hours later, cells will be exposed to solvent alone and to no less than nine concentrations of test
article, the highest concentration being the lowest insoluble dose in treatment
medium not to exceed 5000 u.g/ml. The pH of the treatment medium will be adjusted, if necessary, to maintain a neutral pH in the treatment medium. The osmolality of the highest soluble treatment condition will also be measured. Exposure will be for 5 hours at 371C in a humidified atmosphere of 51% CO; in air in the presence and absence ofS9 activation. After the treatment period, all
media will be aspirated, the cells washed, and cultures given fresh culture medium. Eighteen to 24 hours later, the treated cells will be trypsinized and reseeded at a density of 100 cells/60 mm dish. After 7-10 days incubation at 371C in a
humidified atmosphere of 51% CO; in air, colonies will be fixed with 95% methanol, stained with 10% aqueous Giemsa, and counted. The cell survival of
the test article-treated groups will be expressed relative to the solvent control (relative cloning efficiency).
Whenever possible, the high dose will be selected to give a cell survival of 1020%. Four lower doses will be selected, at least one of which will be non-toxic. For freely soluble, non-toxic test articles, the highest concentration will be 5000 u.g/ml. For relatively insoluble, non-toxic test articles, the highest concentration will be the lowest insoluble dose in treatment medium but not to exceed 5000 pg/ml. If dose-related cytotoxicity or mutagenicity is noted, irrespective of solubility, then the top concentration will be based on toxicity as described above. In all cases, precipitation will be evaluated at the beginning and at the end of the treatment period using the naked eye (ICH, 1995).
7.3 Frequency and Route of Administration
Cell cultures will be treated for 5 hours by way of a vehicle compatible with the system, both in the presence and absence of metabolic activation. This technique of administration has been demonstrated to be effective in the detection of
chemical mutagens in this system.
Exogenous Metabolic Activation
Aroclor 1254-induced rat liver S9 will be used as the metabolic activation system. The source of S9 will be adult male Sprague-Dawley rats induced by a single
injection of Aroclor 1254 at a dose level of 500 ing/kg body weight five days
prior to sacrifice. The S9 will be batch prepared and stored frozen approximately
-70C until used.
Protocol No. SPGT782001
12/02/97
3 of 8
^ MA BlOSERVICES-
26. EwaTpSft;,-Senitlzed. Dses sioi cc:::ai?,
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPontHLO-1998-01211
Immediately prior to use. the S9 will be thawed and mixed with a cofactor pool to contain 100 u.1 S9/ml reaction mixture of approximately 4 mM NADP, 5 mM glucose-6-phosphate, 10 mM MgCl;, 30 mM KC1. 10 mM CaCI;, and 50 mM sodium phosphate buffer, pH 8.0 (Machanoff et al., 19S1). The S9 reaction mixture will be stored on ice until used.
7.5 Controls
7.5.1 Negative Control
The solvent (or vehicle) for the test article will be used as the negative control.
7.5.2 Positive Control
Ethyl methanesulfonate (EMS) will be used at a concentration of 0.2 ul/ml as the positive control for the non-activated study. Benzo(a)pyrene (BaP) will be used at a concentration of 4 ug/ml as the positive control for the
S9 activated study.
7.6 Preparation of Target Cells
Exponentially growing CHO-K.1-BH4 cells will be seeded in F12 medium without hypoxanthine, supplemented with 5% dialyzed serum, 2mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 ug/ml) (F12FBS5-Hx), at a density of 5 x l05 cells/25 cm2 surface area and will be incubated at 371C in a humidified atmosphere of 51% CO; in air for 18-24 hours.
7.7 Identification of the Test System
Using a permanent marking pen, the treatment flasks will be identified by the study number and a code system to designate the treatment condition and test phase.
7.8 Treatment of Target Cells
The time of initiation of chemical treatment will be designated as day 0. Cells will be exposed, in duplicate cultures, to five concentrations of test article for 5 hours at 371C in a humidified atmosphere of 51% CO, in air. For each 25 cm2 of surface area treated, the treatment medium will consist of 5 ml F12FBS5-Hx and 50 pi of control or test article diluted to the appropriate concentration in solvent for the non-activated study, or 4 ml F12FBS5-Hx, 1 ml S9 reaction mixture, and 50 ul of control or test article diluted to the appropriate concentration in solvent for the activated study. After the treatment period, all
media will be aspirated, the cells washed with Hank's Balanced Salt Solution
(CMF-HBSS) and cultured in F12FBS5-Hx at 371C in a humidified atmosphere
of5l% CO; in air. After 18-24 hours incubation, the cells will be subcultured
to assess cytotoxicity and to continue the phenotypic expression period.
Protocol No. SPGT782001
12/02/97
4 of 8
^ MA BlOSERVICES"
-27-
Company Sanitized. -Does not con{a?n TSCA CB^
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPontHLO-1998-01211
7.9 Estimation of Toxicity
For evaluation ofcytotoxicity, the replicate cultures from each treatment condition
will be subcultured independently in F 12FBS5-Hx. in triplicate, at a density of 100
cells/60 nun dish. After 7-10 days incubation at 371C in 51% CO; in air, colonies will be fixed with 95% methanol, stained with 10% aqueous Giemsa, and counted. Cytotoxicity will be expressed relative to the solvent-treated control
cultures.
7.10 Expression of the Mutant Phenotype
For expression of the mutant phenotype, the replicates from each treatment condition will be subcultured independently in Fl 2FBS5-Hx, in duplicate, at a density of no greater than 106 cells/100 mm dish. Subculture as above at 2-3 day intervals will be performed for the 7-9 day expression period. At this time, selection for the mutant phenotype will be performed.
7.11 Selection of the Mutant Phenotype
For selection of the TG-resistant phenotype, cells from each treatment condition
will be plated into a maximum of five dishes at a density of2x 105 cells/100 nun
dish in Fl 2FBS5-Hx containing 10 u,M TG. For cloning efficiency at the time of selection, 100 cells/60 mm dish will be plated in triplicate in medium free ofTG. After 7-10 days of incubation, the colonies will be fixed, stained and counted for
both cloning efficiency at selection and mutant selection.
7.12 Independent Repeat Assay
An independent repeat mutation assay, with and without a metabolic activation system, will be conducted.
8.0 CRITERIA FOR DETERMINATION OF A VALID TEST
8.1 Negative Controls
The cloning efficiency of the solvent (or vehicle) control must be greater than 50%. The spontaneous mutant frequency in the solvent (or vehicle) control must fall within the range of 0-25 mutants per 106 clonable cells.
8.2 Positive Controls
The positive control must induce a mutant frequency at least three times that of
the solvent control and must exceed 40 mutants per 106 clonable cells.
8.3 Test Article-Treated Cultures
A minimum of four analyzable concentrations with mutant frequency data will be
required.
Protocol No. SPGT782001
12/02/97
5 of 8
|l| MA BlOSERVICES"
28-
'&S UQi C.,.iffl^
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
9.0 EVALUATION OF TEST RESULTS
The cytoioxic effects of each treatment condition are expressed relative to the solventtreated control (relative cloning efficiency). The mutant frequency (MF) for each treatment condition is calculated by dividing the total number of mutant colonies by the number of cells selected, corrected for the cloning efficiency of cells at the time of mutant selection, and is expressed as TG-resistant mutants per 10' clonable cells. For experimental conditions in which no mutant colonies are observed, mutant frequencies will be expressed as less than the frequency obtained with one mutant colony. Mutant frequencies generated from doses giving Sl0% relative survival are not considered as valid data points and will not be included in the data analysis.
Spontaneous mutant frequencies in this assay range from 0 to 25 mutants per 10s clonable cells. As a result, calculation of mutagenic response in terms of fold increase in mutant frequency above the background rate does not provide a reliable indication of the
significance of the observed response. The wide acceptable range in spontaneous mutant
frequency also suggests the need to set a minimum mutant frequency for a response to be considered positive. Hsie et al. (1981) refer to a level of 50 mutants per 10' clonable cells. In this laboratory, a more conservative approach is used which sets the minimum significant level at >40 mutants per 10s clonable cells.
All conclusions will be based on sound scientific judgement; however, the following will be used as a guide to interpretation of the data. The test article will be considered to
induce a positive response if there is a concentration-related increase in mutant frequencies with at least two consecutive doses showing mutant frequencies of>40 mutants per 106 clonable cells. If a single point above 40 mutants per 106 clonable cells is observed at the highest dose, the assay will be considered suspect If no culture exhibits a mutant frequency of >40 mutants per 10' clonable cells, the test article will be considered
negative.
10.0 REPORT
A report of the results of this study will be prepared by the Testing Laboratory and will accurately describe all methods used for generation and analysis of data.
Results presented will include, but not be limited to:
test substance: identification and CAS no., if known; physical nature and purity, if
known; physicochemical properties relevant to the conduct of the study, if known; stability of test article, if known.
solvent/vehicle: justification for choice of vehicle; solubility and stability of test article
in solvent/vehicle, if known.
cell type used, number of cultures, methods for maintenance of cell cultures
rationale for selection of concentrations and number of cultures
Protocol No. SPGT78200I
12/02/97
6 of 8
^HMA BlOSERVICES"
-29 Company San^wd. Roes no? conia!rs TSCA CBF
H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
test conditions: composition of media. CO-, concentration, concentration of test substance, vehicle, incubation temperature, incubation lime, duration of treatment, cell density during treatment, type of metabolic activation system, positive and negative controls, length of expression period, selective agent
method used to enumerate numbers of viable and mutant cells dose-response relationship, if applicable
positive and solvent control historical data
11.0 RECORDS AND ARCHIVES
Upon completion of the final report, all raw data and reports will be maintained by the Quality Assurance Unit of MA BioServices, Inc., Rockville, MD in accordance with the
relevant Good Laboratory Practice Regulations.
12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE
This protocol has been written to comply with EPA Health Effects Testing Guidelines, Draft Document OPPTS 870.5300 (Detection of Gene Mutations in Somatic Cells in Culture), Fed. Register, vol. 61, June 1996, with OECD Guideline for the Testing of Chemicals, Guideline 476 {In Vitro Mammalian Cell Gene Mutation Test), Proposal for Updating Guideline 476, February 1997, and with the International Conference on Hannonisation of Technical Requirements for Registration ofPharmaceuticals for Human Use, Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, Step 4 Final Draft, July 18, 1995.
This study will be performed in compliance with the provisions of the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies.
Will this study be submitted to a regulatory agency? ^s
If so, to which agency or agencies? S'^itfv^^ij. " We if
Unless arrangements are made to the contrary, unused dosing solutions will be disposed of following administration to the test system and all residual test article will be disposed of following finalization of the report.
13.0 REFERENCES
EPA Health Effects Testing Guidelines, Draft Document OPPTS 870.5300 (Detection of Gene Mutations in Somatic Cells in Culture), Fed. Register, vol. 61, June 1996.
Hsie, A.W., Casciano, D.A-, Couch, D.B-, Krahn, B.F., O'NeilI, 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 Gen-Tox Program. Mutation
Research 86: 193-214.
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H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay
DuPont HLO-1998-01211
International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, Step 4 Final Draft. July 18, 1995.
Li, A. P., Carver. J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., Loveday. K..S-. O'Neill, J.P., Riddle, J.C., Stankowski. L.F. and Yang, L.L. (1987). A guide for the performance of Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyi transferase gene mutation assay. Mutation Research 189: 135-141.
Machanoff, R., O'Neill, J.P. and Hsie, A.W. (1981). Quantitative analysis ofcytotoxicity and mutagenicity of benzo(a)pyrene in mammalian cells (CHO/HGPRT). Chem. Biol. Interactions. 34: 1-10.
O'Neill, J.P., Brimer, P.A., Machanoff, R., Hirsch, J.P. and Hsie. A.W. (1977). A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyi transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system. Mutation Research 45: 91-101.
OECD Guideline for the Testing of Chemicals, Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test), Proposal for Updating Guideline 476, February 1997.
14.0 APPROVAL
^ / C^fe*^._____________/i/ff/^r -SPONSOR REPRESENTATIVEDATE
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(Print or Type Name) _
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MA STUDY DIRECTORDATE
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