Document LKjdBXdgYxonX9KXo64gXObRQ
TOXIKON FINAL REPORT: 01-7019-G1
_CHO/HGPRT FORWARD MUTATION ASSAY- ISO (T-6889.7)
Author DevaldSadhu,Ph.D.
Final Report Date: March 28, 2002
MANAGEMENT OF THE STUDY Performing Laboratory ToxikonCorporation 15 Wiggins Avenue Bedford,/VIA 01730 Sponsor: 3M 3M Center 220-2E-02 Saint Paul, MN 55144
Toxikon Corporation
CHO/HGPRT Forward Mutation Assay - ISO (T-6889.7) Project Nnmher: 01-7019-G1
Title Page Table of Contents
TABLE OF CONTENTS
StudySummary
Quality .Assurance Statement
Study Director Signature and Verification Dates
1.0 Purpose 2.0 References
3.0 Compliance 4.0 Identification of the Test and Control Articles
5.0 Identification of the Test System 6.0 Justification of Test System and Route of Administration 7.0 Experimental Design 8.0 Dosage 9.0 Evaluation Criteria 10.0 Results 11.0 Conclusion 12.0 Records
13.0 Confidentiality Agreement
Table I: Table II:
Table I/I:
Table IV: Table V: Table VI: Table VII:
Parallel Cytotoxicity Assay Parallel Cloning Efficiency (Non-Activated)
Parallel Cloning Efficiency (Activated)
HGPRT Mutagenesis Assay (Non-Activated) HGPRT Mutagenesis Assay (Activated) Summary of Mutant Colony Frequency in HGPRT Summary of Mutant Colony Frequency in HGPRT
Assay Assay
(Non-Activated) (Activated)
Page 2 of 18
Toxikon Corporation CHO/HGPRT Forward Mutation Assay - ISO (T-6889.7) Project Number: 01-7019-G1
STUDY SUMMARY The test article, AmmoniumPerfluorooctanoate (-FL-143),did not induce mutations at the HGPRT locus as evidenced by the absence of a statistically significant increase in the number of mutant colonies at the highest analyzable test concentration as compared to the negative controls in the presence or absence of an exogenous mammalian metabolic activation system. The statistically significant increase in the number of mutant colonies in positive controls in both the activated and non-activated assays verified the proper functioning of the test system. Therefore, based on the evaluation criteria of the protocol, the test article is considered non-mutagcnic, under the experimental conditions employed.
Page 3 of 18
Toxikon Corporation CHO/HGPRT Forward Mutation Assay - ISO (T-6889.7) Project Number: 01-7019-G1
QUALITY ASSURANCE STATEMENT
This study was conducted in compliance with U.S. Food and Drug Administration regulations set forth in 21 CFR, Part 58.
The sections of the regulations not performed by or under the direction of Toxikon Corporation, exempt from this Good Laboratory Practice Statement, included characterization and stability of the test article and its mixture with carriers, 21 CFR, Parts 58.105 and 58.113.
The Quality Assurance Unit conducted inspections on the following dates. The findings were reported to the Study Director and to Toxikon's Management.
INSPECTIONS
DATE OF INSPECTION
DATE REPORTED STUDY DIRECTOR
DATE REPORTED MANAGEMENT
DOSING CELL COUNTING RAW DATA
FINAL REPORT
01/02/02
02/07/02 03/28/02 03/28/02
01/02/02
02//07/02 03/28/02 03/28/02
01/02/02
02/07/02 03/28/02 03/28/02
. O: "
Mansi J_sLfi_ B.Sc. Qualit_ Assurance
0 519_ f ( ,!,__
I
t
Date
"
Page 4 of 18
ToxikonCorporation CHO/HGPRTForwardMutationAssay- ISO(]?-6889.7) ProjectNumber:01-7019-G1
STUDY DIRECTOR SIGNATURE AND VERIFICATION DATES
This study met with the technical requirements of the protocol. The study also met with the requirements of the Good Laboratory Practice Regulations, 21 CFR, Part 58, with the exemptions noted in the QA Statement.
Protocol Number:
3MC/VITRO/003-01/000
Study Director:
Devaki Sadhu, Ph.D.
Company:.
Toxikon Corporation
Signature:
__
_....__2 [(_
Oato:
Study Supervisor: VF,R 1FTCATTOND ATF,S:
Leigh Waugh-Cohert, B.A.
The study dates were as follows:
Protocol Effective Date:
Test Article Receipt: Project Log Date: Technical Initiation:
Technical Completion: Final Report Date:
12/12/01
12/12/01 12/12/01 02/05/02
02/20/02 03/28/02
Page 5 of 18
Toxikon Corporation CHO/HGPRT Forward Mutation Assay - ISO (T-6889.7) Project Number: 01-7019-G1
1.0 PURPOSE
The CHO/HGPRT Forward Mutation Assay evaluated the mutagenic potential of a test article via its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. The CHO assay system utilized toxic purine analogs to select for resistant cells that are presumed deficient in the purine salvage enzyme HGPRT.
2.0 REFERENCES
The test was conducted based upon the following references:
2.1 Biological Evaluation of Medical Devices - Part 3: Tests for Genotoxicity, Carcinogenicity and Reproductive Toxicity, ANSI/AAMI/ISO 10993-3: 1993.
2.2 OECD Guidelines for the testing of Chemicals, "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test," Test Guideline # 476, current version.
2.3 Hsie, A.W., Casciano, D.A., Couch, D.B., Krahn, D.F., O_leill, J.P., Whitefield, B.L. "EPA's Gene Tox Program," Mutation Research, 86:193-214 (1981).
3.0 COMPLIANCE
The present study conformed to all applicable laws and regulations. Specific regulatory requirements included the current FDA, 21 CFR, Part 58 - Good Laboratory Practice for Nonclinical Laboratory Studies.
, 4.0 IDENTIFICATION OF THE TEST AND CONTROL ARTICLES
The following information was supplied by the Sponsor wherever applicable. Confidential information does not apply. The Sponsor was responsible for all test article characterization data as specified in the GLP regulations.
4.1 Test Article: Ammonium Perfluorooctanoate (FL-143) CAS/Code #: 3825-26-1 / (T-6889.7) Lot/Batch#: Lot 332 Physical State: Solid Color: White / Crystal Density:. Not Supplied by Sponsor (N/S) pH: 4 - 7 Stability: Stable Solubility:. >1 g/mL Storage Conditions: Room Temperature, -20C or 4C Safety Precautions: Standard Toxikon Laboratory Safety Precautions
Page 6 of 18
ToxikonCorporation CHO/HGPRTForwardMutationAssay- ISO(T-6889.7) ProjectNumber:01-7019-G1
4.2 Control Articles - (Toxikon Supplied)
4.2.1 Negative Control Article Name: Ham's F-12 Complete Mediurri QC Inventory #: LPR-01-11-001-CC Physical State: Liquid Color: Colorless
Storage Conditions: Room Temperature Safety Precautions: Standard Laboratory Safety Precautions
4.2.2 Positive Control Article Name (Non-Activated Assay): Ethylmethanesulfoxide QC Inventory #: CSC-01-03-004-CC Physical State: Liquid Color: Clear
Storage Conditions: 4+2"C Safety Precautions: Standard Laboratory Safety Precautions
(EMS)
4.2.3 Positive Control Article Name (Activated Assay): Dimethylbenzanthracene QC Inventory #: CSC-97-09-013-CC Physical State: Liquid Color: Clear
Storage Conditions: 4+2"C Safety Precautions: Standard Laboratory Safety Precautions
(DMBA)
5.0 IDENTIFICATION OF THE TEST SYSTEM
Chinese Hamster Ovary (CHO) cells utilized in this assay were obtained from the American Type Culture Collection, Manassas, Virginia. The cells were derived from an ovarian biopsy of a chinese / hamster.
6.0 JUSTIFICATION OF TEST SYSTEM AND ROUTE OF ADMINISTRATION
6.1 This assay evaluated the mutagenic potential of atest article via its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (I-tGPRT) locus. This locus is responsible for production of HGPRT, an enzyme that is present in normal cells and allows cells to salvage hypoxanthine and guanine from the culture medium in order to synthesize DNA. In normal eeUs, when a toxic substance called 6-thioguanine is included in the growth medium, it is salvaged by the HGPRT enzyme along with hypoxanthine and guanine and incorporated into DNA, thereby causing cell death. Exponentially growing cells, sensitive to the toxic effects of 6-thioguanine, were exposed to a various concentrations of the test material. If the test material is potentially mutagenic, normal cells (which can utilize hypoxanthine, guanine and 6-thioguanine) mutate to become incapable of utilizing hypoxanthine, guanine, or 6-thioguanine from the culture medium. However, mutant cells retain their ability to grow as well as normal cells in culture medium because DNA synthesis is made possible by alternate synthetic pathways. Taken together, these findings indicate that the basis for selection of HGPRT mutants is the lack of any ability to utilize the toxic 6-thioguanine. Therefore, cells that grow to form colonies in the presence of 6-thioguanine are assumed to have undergone mutation either spontaneously or by exposure to the test material.
Page 7 of 18
Toxikon Corporation CHO/HGPRT Forward Mutation Assay - ISO (T-6889.7) Project Number: 01-7019-G1
The CHO/HGPRT Assay has been used extensively in the detection ofmutagenic activity of a wide range of chemical classes.
6.2 The test article was admi_stered in vitro, through a solvent compatible with the test system, per Sponsor's specification. This was the optimal route of administration available in this test system.
7.0 EXPERIMENTAL DESIGN
7.1 Cell Line
The CHO-K1 cell line was selected for its high cloning ability and rapid doubling time. It was not necessary to select the cell cultures to reduce the background frequency of HGPRT" mutants.
7.2 Maintenance of CHO Cells
The CHO cells were grown in complete Ham's F-12 medium, buffered with 10 mM HEPES. Complete medium consisted of Ham's F-12 medium containing 10% fetal bovine serum (FBS), 1-2 mM L-glutamine, 100-units/mL penicillin, and 100-_tg/mL streptomycin. Incomplete medium was serum-free complete medium. The cells were incubated at 371"C, 5+1% CO2, and saturated
humidity.
7.3 Forward Mutation Assay
7.3.1 Metabolic Activation System ($9): The $9 microsomal fraction was prepared from Sprague Dawley rat livers induced with Aroclor R 1254. The $9 rat liver homogenate was purchased from Molecular Toxicology Inc (157 Industrial Park drive, Boone, NC 28607) and stored at -80-a:5*Cuntil use. A combination of $9 fraction, isocitrie cofactors and incomplete medium was prepared just before exposure and used as the metabolic activation system. The cofactor mixture consisted of isocitrate (trisodium sal0 and NADP (disodium salt) at a final concentration of 4.5 mg/mL and 2.4 mg/mL, respectively. The $9 fraction was added to each flask at a concentration of 20 _tl_,/mL.
7.3.2 ParaUel Cytotoxicity Assay A parallel cytotoxicity assay was performed along with the Mutation assay. The cultures were treated exactly as the mutation plates, incubated for 9 days following the removal of the test article, fixed, stained, and colonies counted.
7.3.3 Preparation of Test Cultures: AP6Proximately 24 hours prior to exposure, duplicate 100 mm plates were seeded at a density of 1 x 10 cells/dish and triplicate parallel cytotoxicity plates were seeded at 200 cells/60 mm dish.
7.3.4 Exposure Periods: The cells were exposed to the test article for 17 hours in the non-activated assay and 5 hours in the activated assay. After exposure, cells were washed twice with PBS and supplemented with complete medium.
Page 8 of 18
Toxikon Corporation CHO/HGPRT Forward Mutation Assay - ISO (T-6889.7) Project Number: 01-7019-G1
7.3.5 Phenotype Expression: Approximately 48 hours following termination of non-activated exposure period and approximately , 64 hours following termination of activated exposure period, cells were trypsinized, counted, and plated at 1 x 106 cells per 100 mm dish. The cells were passed every 48-72 hours to maintain exponential growth during phenotypic expression for 6 days.
7.3.6 Selective Growth: Following the phenotypic expression period, cells were grown in selective medium to select for mutant cells. The selective medium included hypoxanthine-free Ham's F-12 with 10% fetal bovine serum, penicillin-streptomycin (P/S) and 10 _tM 6-thioguanine. Ten dishes (five from each duplicate) were used for each test condition. Cell density was 2 x 105/100 mm petri dish. The cultures were incubated for 9 days to allow colonies to develop.
7.3.7 Parallel Cloning Efficiency (PCE): Concurrently, the cloning efficiency was determined by plating cells in selective mediurn without 6-thioguanine. Six dishes were seeded for each concentration (three from each duplicate) at 200cells/60 mm dish. The cultures were then incubated for 9 days to allow colony formation.
7.3.8 Termination: At the end of the incubation period, plates were rinsed with PBS, fixed in methanol and stained with Giemsa. Only colonies with 50 or more ceils were counted.
7.4 Control Articles
7.4.1 Positive Control Article: The positive control for the non-activated system was Ethylmethanesulfoxide (EMS). For the . activated system, Dimethylbenzanthracene (DMBA) was used as the positive control.
7.4.2 Negative Control Article: Ham's F-12 Complete Medium served as the negative control article for the non-activated and activated assays.
8.0 DOSAGE
8.1 The test article was dissolved in the culture medium at a concentration of 5 mg/mL as per OECD guidelines. 1:128, 1:256 and 1:512 dilutions of 5 mg/mL concentration of the test article were used for dosing.
8.2 Dose Selection
8.2.1 A cytotoxicity preliminary dose-range finding was performed using several dilutions of the 5 mg/mL test article and 1:128, 1:256 and 1:512 dilutions were selected for testing. The positive control for the non-activated assay, EMS, was used at a concentration of 244 _tg/mL. The positive control for the non-activated assay, DMBA, was used at a final concentration of 9 _tg/mL.
Page 9 of 18
ToxikonCorporation CHO/HGPRTForwardMutationAssay- ISO(T-6889.7) ProjectNumber:01-7019-G1
9.0 EVALUATION CRITERIA
9.1 Evaluation of Test Results
The results of the CHO/I-IGPRT Locus Mutation Assay were evaluated based on the number of TG-resistant mutants per 1 x 106 surviving cells. The significance of the test results were determined by using the statistical program, Tallarida, R.S. and R.B. Murray's Pharmacological Calculations Procedure, A.NOVA (analysis of variance) and Newman- Keuls Test for confimaation. This statistical method determined if there was a significant (p < 0.05) increase in the mutation frequency of the test article compared to the negative control article.
The test article was considered to have caused a positive response in the assay if the test article had exhibited a reproducible and statistically significant increase in the number of mutants per 1 x 106 cells over its concurrent negative control article.
10.0 RESULTS
A preliminary Range Finding Assay was conducted to determine the dilutions of the test article to be used in the Forward Mutation Assay. Exposure to dilutions of the test article resulted in cytotoxicity ranging from 50% cell survival at 1:128 and 80% greater at 1:256 and 1:512. Based on these findings, three dilutions ranging from 1:128 to 1:512 were utilized in the Forward Mutation Assay.
The parallel cytotoxicity assay indicated that the positive control article exhibited a cell survival percentage of 10% and 45% in the non-activated and activated assays respectively (Table I). The dilutions of the test article extract, 1:128, 1:256 and 1:512 exhibited ceil survival values of 105, 99 and 102 respectively in the non-activated assay and 94, 104 and 118% respectively in the activated assay. The negative control article exhibited values of 79 and 110% in the non-activated and activated assays respectively.
The test article was tested at the three dilutions (1:128, 1:256, 1:512) established in the Range Finding Assay in non-activated assay. None of the three dilutions tested showed a statistically significant increase in the number of mutants per 1 x 10 6 surviving cells as compared to the corresponding negative controls in both the non-activated and activated assays (Tables II-VIO. The positive controls did show a statistically significant increase in the number of mutants per 1 x 106 surviving cells as compared to the corresponding negati've controls.
11.0 CONCLUSION
The test article, Ammonium Perfluorooctanoate (FL-143), did not induce mutations at the HGPRT locus as evidenced by the absence of a statistically significant increase in the number of mutant colonies at the highest analyzable test concentration as compared to the negative controls in the presence or absence of an exogenous mammalian metabolic activation system. The statistically significant increase in the number of mutant colonies in positive controls in both the activated and non-activated assays verified the proper functioning of the test system. Therefore, based on the
Page 10 of 18
ToxikonCorporation CHO/HGPRTForwardMutationAssay- ISO(T-6889.7) ProjectNumber:01-7019-G1
evaluation criteria of the protocol, the test article experimental conditions employed.
is considered
non-mutagenic,
12.0 RECORDS
under the
12.1 Original raw data is archived at Toxikon Corporation.
12.2 A copy of the final report and any amendments is archived at Toxikon Corporation.
12.3 The original final report and a copy of any protocol amendments or deviations is forwarded to the Sponsor.
12.4 All unused test articles shall be returned to the Sponsor by Toxikon, per sponsor's specification.
12.5 Final reports will not be reproduced except in full, without the written authorization/approval from Toxikon.
13.0 CONFIDENTIALITY AGREEMENT
Statements of cortfidentiality were as agreed upon prior to study initiation.
Page 11 of 18
Toxikon Corporation CHO/HGPRT Forward Mutation Assay - ISO ('17-6889.7) Project Number: 01-7019-(]1
Table I: Parallel Cytotoxicity Assay (Mutagen Assay)
Dilution of Extract
Negative Positive (EMS) 1:128 1:256 1:512
Metabolic Activation Non-Activated Non-Activated Non-Activated Non-Activated Non-Activated
Total # of Foci
147 155 170 10 12 35 , 232 194 201 187 202 202 200 213 19.9
Average # Foci per plate 157 19 20.,9
. 19.7 204
Percentage Survival*
79 10 105 99 102
Negative
..
Positive (DMBA)
1:128
1:256
1:512
Activated Activated Activated Activated Activated
222 208 230 95 90 87 182 184 200 194 232 198 2.3,,.2....240 237
220 90 ..... 188 208 236
" 110 45 94 104 118
* Percentage Survival= Average colonies per plate / number ofceUs plated (200 cells) x 100 %
Page 12 of 18
Toxikon Corporation
CHO/HGPRT Forward Mutation Ashy - ISO (T-6889.7)
Project Number: 01-7019-G1
"-
TABLE II PARALLEL CLONING EFFICIENCY
NON-ACTIVATED
NEGATIVE CONTROL {MEDIUM)
# COLONIES/PLATE
SURVIVING
1
2
3
4
5
6 FRACTION*
70
80
106 103 117 110
0.488
POSITIVE CONTROL (EMS)
20 19
8
8
5
7
0.056
TEST ARTICLE EXTRACT 79 98
91
89 87 75
(1:128)
0.433
TEST ARTICLE EXTRACT 94 99
112
91 116 102
(1:256)
0.512
TEST ARTICLE EXTRACT 109 84
93
116 117 72
(1:512)
0.493
AVERAGE COLONIES
98
11
87
102
99
* SURVIVING FRACTION- AVERAGE COLONIES PER PLATE/NUMBER OF CELLS PLATED (200 CELLS/PLATE)
Page 13 of 18
Toxikon Corporation
CHO/HGPRT Forward Mutation Assay - ISO (T-6089.7)
Project Number: 01-7019-G1
"
TABLE III PARALLEL CLONING EFFICIENCY
ACTIVATED
NEGATIVE CONTROL
(MEDIUM)
POSITIVE CONTROL (EMS)
# COLONIES/PLATE
SURVIVING
1
2
3
4
5
6 FRACTION*
116 113 91
87 101 106
0.512
6
10
15
12
2
13
0.048
TEST ARTICLE EXTRACT 76 72
71
72 96 93
(1:128)
0.400
TEST ARTICLE EXTRACT 101 95
108
79
77 106
(1:256)
0.472
TEST ARTICLE EXTRACT 108 75
112
76 103 113
(1:512)
0.489
AVERAGE COLONIES
102
10
80
94
98
* SURVIVING FRACTIONffi AVERAGE COLONIES PER PLATE/NUMBER OF CELLS PLATED (200 CELLS/PLATE) Page 14 of 18
ToxikOn Corporation
CHO/HGPRT Forward Mutation Assay - ISO ('1"-6889.7)
Project Number: 01-7019-G1
".
TREATMENT
NEGATIVE (ACTUAL) NEGATIVE (NORMALIZED)+
POSI_'IVE (ACTUAL) POSITIVE (NORMALIZED)+
ITEST ARTICLE EXTRACT (1:128 ACTUAL) TEST ARTICLE EXTRACT (NORMALIZED)+
TEST ARTICLE EX'_RACT (1:256 ACTUAL) TEST ARTICLE EXTRACT. (NORMALIZED)+
TEST ARTICLE EXTRACT (1:512 ACTUAL) TEST ARTICLE EXTRACT {NORMALIZED.)+
TABLE IV HGPRT MUTAGENESIS
(NON-ACTIVATED)
ASSAY
# MUTANT COLONIES/PLATE
1 '-
2
3
4
5
6
7
8
9
10"
1
1
0'
0
2.05
2:05
0.00
0.00
0
0
0
0,00
0.00
0.00
1
1
0
2.05
2.05
0.00
40
39
33
716,42 698.51 591.04
54 967.16
'47
12
12
19
14
841.79 214.93 214,93 340.30 250,75
11 197,01
0
'0
0
0
0
2
1"
1
0
1
0.00
0:0Q 0.00
0.00
0.00
4.62
2.31
2.31
0.00.
2.31
1
0
0
0
1
1
0
2
0
0
1.95
0.00
0.00
0.00
1,95
1.95
0.00,
3.91
0.00
0,00
0
0
0
2
"0
I
I
0
0
0
0.00
0.00
0.00
4.06
0.00
2.03
2.03
0.00
0.00
0.00
+ NORMALIZED MUTATION FREQUENCY- NORMALIZED TO 1 X 106 CELLS PER PLATE BASED UPON CORRESPONDING SURVIVAL FRACTION (SEE TABLE II) Page ] 5 of 18
Toxikon Corporation
CHO/HGPRT Forward MtRation Amy - ISO ('1"-6889.7)
Project Number: 01-7019-G1
".
TREATMENT
NEGATIVE (ACTUAL) NEGATIVE (NORMALIZED)+
POSITIVE (ACTU_J_)
"'
POSITIVE (NORMALIZED)+
TEST ARTICLE EXTRACT 0:128 AcTUAL) TEST ARTICL E EXTRACT (NORMAL.IZEB)+
TEST ARTICf_E ExTRAcT (I :256 ACTUAL) ..... TEST ARTICLE EXTRAcT (NORMAL_IZED)+
TEST ARTICLE EXTRACT (1:512 ACTUAL) TEST ARTICLE EXTRACT (NORMALIZED)+
HGPRT
TABLE V MUTAGENESIS (ACTIVATED)
ASSAY
# MUTANT COLONIES/PLATE
1
2 .... 3 ....
4
5
6
7
8
'9
I0
0 0.00
45 931.03
0' 0.00
32 662.07
1
1
1,95 I t.95
26 537.93
22 455.17
i'" t.9S
20 413.79
1 1.95
18 372.41
0 0.00
15 310,34
0 ..... 0.00
36 744.83
0 0.00
31 641.38
0 0.00
29 600.00
2
0
0'
2
i'
0
0
3
0
0
5.00
0.00
0.00
5.00
2.50 , 0.00
0.00
7.50
0.00
0.00
0
0
1
0
2
0
0
0""
0
2"
0.00
0.00
2.12
0.00
4.24
0.00
0.00
0.00'
0.00
4.24
1
1
0
0
0
1
0
"0
1
0'
2.04
2.04
0.00
0.00
0.00
2.04
0.00 , 0.00,
2.04
0.00
+ NORMALIZED MUTATION FREQUENCY- NORMALIZED TO 1 X 106 CELLS PER PLATE BASED UPON CORRESPONDING SURVIVAL FRACTION (SEE TABLE In) Page 16 of 18
Toxikon Corporation
CHO/HGPRT Forward Mutation Assay-/SO (T-6889.7)
Project Number: 01-7019-GI
".
SUMMARY
TABLE VI OF MUTANT COLONY FREQUENCY
(NON-ACTIVATED ASSAY)
IN HGPRT ASSAY
TREATMENT
NEGATIVE CONTROL POSITIVE CONTROL TEST ARTICLE (1:8) rEST ARTICLE (1:16) YEST ARTICLE (1:32)
TOTAL # PLATES
TOTAL # FOC!
10
4
10
281
AVERAGE FOCI PER PLATE
0.4
28.1
AVERAGE FOCI* PER PLATE
(NORMALIZED) 0.82
503.28
10
5
0.5
1.16
10
5
0.5
0.98
10
4
0.4
0.81
* NORMALIZED TO I X I0' CELLS PER PLATE BASED UPON THE CORRESPONDING SURVIVAL FRACTION (SEE TABLE II)
Page 17 of 18
Toxikon Corporation CHO/HGPRT Forward Mutation Project Number. 01-7019-G1
Asuy - I$O (T-6889.7)
SUMMARY
TABLE VII OF MUTANT COLONY FREQUENCY
(ACTIVATED ASSAY)
IN HGPRT ASSAY
TREATMENT
NEGATIVE CONTROL POSITIVE CONTROL"
TOTAL # PLATES
TOTAL # FOC|
10
4
i'0 " 274
AVERAGE FOCI PER PLATE
. ,, 0,4 27.4
AVERAGE FocI* PER PLATE
(NORMALIZED)
p. 0.78
- 566.90
TEST ARTICLE (1:8)
10
8
_ i 0.8
2.00
TEST ARTICLE (1:16)'
10
5
0.2 "
1.06
TEST ARTICLE (1:32)i
I0
4
0,4
0.82
* NORMALIZED
TO 1 X 1Ol CELLS PER PLATE BASED UPON THE CORRESPONDING
SURVIVAL FRACTION (SEE TABLE HI)
Page 18 of 18
DE0-12-200N1ED12:32 PHT0[KON
F_XNO.7812711133
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TOXIKON TEST PROTOCOL FDA GLP GUIDELINES
CHO/HGPRT FORWARD MUTATION ASSAY - ISO (T-6889.7)
TO3_gON PROTOCOl N'tIMI_R 3MC/VITRO/003-01/000
PROTOCOr. nATF: 12/03101
F._'F_eTTV'I_13ATe.. 12/12/01
CNt_r.t_,NCF. 21 CFR, Part 58 Good Laboratory Practice for Nonclinical Laboratory Studies
MA_IAt_I_J]_fI_.N_T1_ _ _TTTI3Y
Performing Laboratory: Toxikon Corporation 15 Wiggins Avenue Bedford, MA 01730
Spo_or:
3M Company
3M Center
Corporate Toxicology
.... B_.ui!din0g220-02-E-02
St. Paul, MN 55144
"
........
DEC-12-200I ,-'_
- "--,," _--'
WED12:32 "..................
P_ TOXIKON
Toxikon Corporation CHO/HGPRT Fomard Mutation Assay - ISO Protocol Number: 3MCIVITROIO02-OIIO00 Protocol Date: 12/03/01 Effective Date: 12/12/01
(T_5889.7)
FAX NO, 7812711133
PROTOCOL ACCEPTANCE
P. 03/12
Devaki Sadhu, Ph.D.
Date
Study Director
Toxikon Corporation
15 Wiggins Avenue
Bedford, MA 01730
Toxikon Cozpoi_ion 15 Wiggins Avenue
Bedford, MA 01730
Sponsor's Representative
Date
3M Company 3M Center
Corporate Toxicology
Building 0220-02-E-02
St. Paul, MN 55144
Page 2 of 11.
...... , DE0-12-2001 ..................... NED 12:32 PI"ITOXIKON
Toxikon Corl_rafion CHO/HGPRT Forward Mutation Assay - ][SO (T-6889.7) Protocol Number:. 3MCNITRO/002-01/000 Protocol Date: 12/03/01 Effective Date: 12/12101
.... FaX NO, 7812711133
TABLE OF CONTENTS
Title Page ProtocoAlcceptance TableofContents 1.0 Purpose 2.0 References 3.0 Compliance 4.0 IdentificaloifoTnestand ControAlrticles 5.0 IdentificatoifoTnestSystem 6.0 JustificatoifotnheTestSystemandRouteofAdministration 7.0 ExperimentaDlesign g.0 Dosage 9.0 Evaluation Criteria 10.0 Records 11.0 Confidentiality Agreement 12.0 Protocol Amendments/Deviations
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.DEC-12-2001N,..E. D12:.3..2. . PI"/TOXIKON
Toxlkon Corporation CHO/HGPRT Forward Mutation Assay- |SO Protocol Numbr: 3MCIVITROIO02-OI/O00 Protocol Date: 12/03/01 Effective Dine: 12/12/01
(T-fig89.7)
1,0 PURPOSE
FaX NO, 7812711133
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The CHO/HGPRT Forward Mutation Assay (with Confirmation) ex_aluatesthe mutageaie potential of a test article via its abilityto induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPKT) locus. The CLIOassay system utilizes toxic purine analogs to select for resistant ceils that are presumed deficient in the purine salvage enzyme HGPRT. The gone mutations are induced at the HGPRT locus in cultured Chinese hamster ovary (CHO) cells.
2.0 REFERENCES
The test will be conducted based upon the following references:
2. I Biological Evaluation of Medical Devices-Part 3: Tests for Genotoxieity, Carcinogenieity and Reproductive Toxicity, ANSI/AAMI//SO 10993-3: 1993.
2.2 OECD Guidelines for the testing of Chemicals, "Genetic Toxicology: Iv vitrn Mammalian Cell Gone Mutation Test," Test Guideline # 476, current version.
2.3 Hsie, KW., Casciano, D.A., Couch, D.B., Ks'aim, D.F., O%feill, J.P., Whitefield, B.L. "EPA's Gone Tox Program," Mutation Research, 86:193-214 (198 I).
2.4 Extraction procedures, if applicable, will be based upon the standard titled Biological Evaluation of Medical Devices-Part 12: Sample Preparation and Reference Materials, EN/ISO 10993-12 (1997).
3.0 COMPLIANCE
The study will conform to all applicable laws and regulations. Specific regulatory requirements
include the current Good Laboratory Practice for Nonclinical Laboratory Studies, FDA, 21 CFR, Part 58.
4.0 IDENTIFICATION OF THE TEST AND CONTROL ARTICLES
The following information will be supplied by the Sponsor on a test requisition form or other correspondence wherever applicable; it does not apply to confidential information. The Sponsor will be responsible for all test article characterization data as specified in the GLP regulations. Test and control articles (exclusive of extracts) that are mixed with carriers require verification of concentration, homogeneity and stability. Samples of test and control article mixtures will be returned to the Sponsor for characterization and verification, wherever applicable.
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Tosilm- Corpo_tlon CHOJHGPRTForward Mutation Assay - ISO (T-6_9.7) Protocol Number: 3MC/VITRO/002-01/000 ProtocolDate: 12/03/01 Effect/v+ Date: 12/12/01
FAXNO. 7812711133
4. l Test Article:To Be Determined (TBD) Lot/Batch #:
CAS/Code #:
PhysicalState: Color:
Density:
pH:
Stability:
Solubility:
Expiration Date: Storage Conditions: Safety Precautions:
4.2 ControlArticles (Toxikon Supplied):
4.2.1 Negative Control ArticleName: TBD Toxikon QC#"TBD Physical State: Liquid Color: Colorless Storage: Room Temperature Safety Precautions: Standard Laboratory Safety Precautions
4.2.2 Positive Control Articles:
4.2,2.1 Positive Control Article Name: TBD ToxikonQC #: TBD PhysicalState: Color:
Storage:
SafetyPrecautions: Known Mutagen.AppropriateLaboratory Safety Precautions
#..2.2,2Positive Control Article Name: TBD
Toxikon QC #: TBD
Physical State: Color:
Storage:
Safety Precautions: Known Mutagen. Appropriate Laboratory Safety Precautions
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Toxlkon Corporation CHO/HGPRT Forward Mu_tl_ _y - I$0 Protocol Number: 3MCIVITROIOO2-OI/O00 Protocol Date: 12/03101 Effe|ive Date: 12/12/01
(T-6_9,7)
_.0 IDENTIFICATION OF THE TEST SYSTEM
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Chinese Hamster Ovary (CHO) cells utilized in this assay are obtained from the American Type
Culture Collection, Rockville, Maryland. The cells are derived from an ovarian biopsy of a chinese hamster.
6.0 JUSTIFICATION OF TEST SYSTEM AND ROUTE OF ADMINISTRATION
6.1 This assay evaluates the mutagenic potential of a test article via its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. This locus is responsible for production of HGPRT, an enzyme that is present in normal ceils and allows cells to salvage hypoxanthine and guanine from the culture medium in order to synthesize DNA. In normal cells, when a toxic substance called 6-thioguanine is included in the growth medium, it is salvaged by the HGPRT enzyme along with hypoxanthine and guanine and incorporated into DNA, thereby causing cell death. Exponentially growing cells, sensitive to the toxic effects of 6-thioguanine, are exposed to several concentrations of the test material. If the test material is potentially mutagenic, normal cells (which can utilize hypoxanthine, guanine and 6-thioguanine) mutate to become incapable of utilizing hypoxanthine, guanine, or 6-thioguartine from the culture medium. However, mutant cells retain their ability to grow as well as normal cells in culture medium because DNA synthesis is made possible by alternate synthetic pathways. Taken together, these findings indicate that the basis for selection of HGPKT mutants is the lack of any ability to utilize the toxic 6thioguanine. Therefore, cells that grow to form colonies in the presence of 6-thioguanine are assumed to have undergone mutation either spontaneously or by exposure to the test material. The CHO/HGPRT Assay has been used extensively in the detection of mutagenic activity of a wide range of chemical classes.
6.2 The test article will be administered in vitro, directly or through a solvent compatible whh the test system. These are the only routes of administration available in this test system.
7.0 EXPERIMENTAL DESIGN
7.1 Cell Line The CHO-K1 cell line is selected for its high cloning ability and rapid doubling time. To reduce the negative control article frequency of HGPRT" mutants to as low as possible, cell cultures can be selected against this phenotype and returned to normal growth medium for three or more days before use.
7.2 Maintenance of CLIO Cells The CHO cells will be grown in complete Ham's F-12 medium, buffered with 10 mM L-/VP,ES. Complete medium will consist of 10% fetal bovine serum (FBS), 1-2 mM L-glutamine, 100 units/ml penicillin, and 100 ug/mi streptomycin. Incomplete medium is serum-free complete medium. The ceils will be incubated at 375:1C, 5+1% CO2, and saturated humidity.
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DEC-12-2001_/ED.I.2..:.3..3. PHTOXIKON
FAXNO. 7812711133
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To_rJk0n Cerporatiolm CHOfflGPRT Forward Mutation AssayProtocol Number:. 3MC/VITRO/002-01/000 Protocol Date: 12/03/01 Effective Date: 12/12/01
ISO (T-6889.7)
7.3 Cytotoxicity Assay The Cytotoxicity Assay (if necessary) is conducted without metabolic activation, prior to the Mutation Assay. The highest concentration is based on maximumsolubility, 10 mg/ml, or Sponsor specifications, Liquid test articles will be tested at 100 ul/ml-0.01 ul/ml. Each test substance concentration, negative control and solventcontrol (if applicable)is tested in triplicate.
Cultures are initiated in 60 nun dishes at 200 cells/plate approximately24 hours prior to dosing. The cultures are exposed to the test article and controls for 15=17hours. At the end ofthe exposure period, dishes ate rinsed with medium or Phosphate Buffered Saline(PBS) and incubated for an additional6-9 daysto allow colonies to develop. At the end of the incubation period, the dishes are fixed in methanol, stained with Giemsa and colonies counted. Relative survival is obtained by comparing the number of surviving colonies for each dose to that of the negative or solvent control.
7.4 Forward Mutation Assay
7.4.1 Metabolic Activation System (S9) The $9 microsomal fraction is prepared from SpragueDawley rat livers induced with Aroclor_ 1254. The $9 rat liver homogenate is stored at -805C until use. A combination of $9 fraction, isocitric cofactors and incomplete medium is preparedjust prior to exposure and used as the metabolic activation system. The cot'actormixture consists ofisocitrate (trisodium salt) and NADP (disodium salt) at a final concentration of 4.5 mg/ml and2.4 mg/ml, respectively. The $9 fraction is added to each flask or directly to the oct'actormixture at a concentration of 20 ul/ml.
7.4.2 ParallelCytotoxicity Assay
A parallelcytotoxicity assay is performed along with the Mutation assay. The cultures are treated
exactly as the mutation plates, incubated for 6-9 days following the removal of the test article, fixed, stained, and colonies counted.
7.4.3 Preparation of Test Cultures Approximately 24 hours prior to exposure, duplicate 100 mm mutation plates are seeded at a
density of 5 x 10s cell_/dish and triplicate parallel cytotoxicity plates are seeded at 200 cells/60 mm dish.
7.4.4 Exposure Periods The cells will be exposed to the test article for 15-17 hours (4-6 hours for highly reactive chemicals) in the non=activatedassay and 4=6hours in the activated assay. After exposure, cells are washed at least once with medium or PBS and supplemented with complete medium.
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ToxikonCorporation CHO/HGPRTForwardMumlionAssay- ISO(T-fSSg.'/) ProtocoNl umber.3MC/VITRO/007.-01/600 ProtocolDat_:12/03/01 EffectDiavtee:12112101
F,qNO, 7812711133
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7.4.5 Phenotype Expression Approximately 24--48 hours following termination of exposure period, cells are trypsinized, counted, and plated at 1 x 106 cells per 100 mm dish. The cells are passed every 48-72 hours to maintain exponential growth during phenotypic expression for approximately 7-9 days.
7.4.6 Selective Growth
Following the phenotypic expression period, cells are grown in selective medium to select for mutant cells. The selective medium includes hypoxanthine-free HAM'S F-I2 with 10% dialyzed fetal bovine serum, penicillin-streptomycin (P/S) and 10 _ 6-thioguanine. A total of ten dishes (five fi,om each duplicate) are used for each test condition. Cell density is 2 x 105 / 100 mm petri dish. The cultures are incubated for 6-9 days to allow colonies to develop.
7.4.7 Parallel Cloning Efficiency (PCE) Concurrently, the cloning et_eiency is determined by plating cells in selective medium without 6-thioguanine. Six dishes are seeded for each concentration (three from each duplicate) at 200 cells/60 mm dish. The cultures are then incubated for 6-9 days to allow colony formation.
7.4.8 Termination
At the end of the incubation period, plates are rinsed with PBS, fixed in methanol and stained with Giemsa. Only colonies with 50 or more cells are counted.
7.5 Control Articles
7.5.1 Positive Control Article
The positive control for the non- activated system is Ethylmethanesulfoxide (EMS) or 4Nitroquinoline- 1-oxide (4NQ). For the activated system, 9,10,- Dimethyl- 1,2-benzanthracene (DMBA) or Dimethylnitrosamine (])biN) can be used as the positive control.
7.5.2 Negative Control Article
The appropriate solvent, extractant or cell culture medium will serve as the negative control article.
7.6 Confirmatory Assay
The results of the Forward Mutation Assay will be confirmed through an independent Confirmatory Assay. The Confirmatory Assay will be performed as described in Sections 7.0 and 8.0.
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ToxikonCorporation CHO/HGPRTForwardMutationAssay- I$O(T-fi889,7) ProtocolNumbr:3MC/VITRO/002-0U000 ProtocoD| ate:12/03/01 EffectiveDage:12112/01
8.0 DOSAGE
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8, ! Preparation of Test Article
8.1.1 Medical Devices: The test article will be extracted at ratios specified by EN/ISO 10993-12. Extraction vehicles may be one of the following media: serum-fi'ce medium, complete medium, or 0.9% USP Sodium Chloride for Injection, USP (NaCI).
Extraction conditions will be as specified by Sponsor: (please aheck desired condition)
121+2C for one hour 70:i:2,Cfor 24 hours 50:_2,Cfor 72 hours 37+1C for 72 hours 371C for 24 hours
per Sponsor's directions (
C for __ hours).
Extracts prepared with medium will be _e_,'teda_ 100% (a_'_t)concentration. E._ract_ prepared with Sodium Chloride will be diluted with 2X medium and tested at 50% _ract concentration
(considered neat). Modifications to test article preparation will be as spccified by the Sponsor,
8.1,2 Solid/Liquid Test Articles Solid test articles will be dissolved or suspended in a vehicle appropriate for the test system or as specified by the Sponsor. Liquid test articles will be administered as received at predetermined concentrations or as specified by the Sponsor.
8.2 Dose Selection
8.2.1 The concentrations for the Mutation Assay are selected based on toxicity information, maximum solubility of the test article or Sponsor specifications. If necessary, a Cytotoxicity Assay will be performed to determine toxicity. If toxicity is evident, the doses should include the highest concentration which causes a low level of survival (approximately 10%) and four lower doses, including one dose with no apparent cytotoxicity, If no apparent cytotoxicity is observed, the maximum concentration will be based on the limit of solubility or 10 mg/ml, whichever is lower (or as specified by the Sponsor). Subsequent doses will decrease in approximate half-log increments. In the absence of toxicity, liquid test articles will be tested at 100 ul/ml-0.01 ul/ml. Dose selection modifications will be as specified by the Sponsor.
8.2.2 Medical device extracts will be analyzed at one concentration only (neat extract). Dilutions of medical device extracts arc not analyzed unless otherwise required, in which case the justification shall be indicated in the final report. An initial Cytotoxidty Assay is not performed for device extracts.
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To:Liken Corporation CHO/HGPRT Forward Mttlatioa Assay Protocol Number: 3MC/VITRO/002-Ol]000 Protocol Date: 12/03/01 Effective Date: 12/12/01
- ISO
(T_889,7)
FF_XNO, 7812711133
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9.0 EVALUATION CRITERIA
The results of the CHO/I-IGPRT Locus Mutation Assay will be evaluated on the basis of the number of TG-resistant mutants per 1 x 106 surviving cells. The significance of the test results will be determined by either one of the following methods:
9.1 The test results are analyzed using a statistical program such as Tallarida, R.S. and ]LB. Murray's Pharmacological Calculations Procedure, AN'OVA (analysis of variance) and NewmanKeuls Test for confirmation ofpairwise comparisons. This statistical method determines if there is a significant (p < 0.05) increase in the mutation frequency of the test article compared to the negative control article. The results obtained for the mutation frequency at the various dose levels will be analyzed by the method of Linear Regression using a program such as "Linear Regression I" by R.J. Tallarida and ]LB. Murray, (Manual of Pharmacologic Calculations with Computer Programs, Springer-Verlag, New York, 1986, pp 10-13). This will determine if there is a positive dose response.
9.2 The test article is considered to have caused a positive response if the test article shows a statistically significant positive dose response or at least one test article dose shows a reproducible statistically significant increase in the number of mutants per I x 104 surviving cells as compared to the corresponding negative control article.
9.3 Confirmation Assay The confirmation assay will validate the reproducibility of the mutagenesis assay.
/ 10.0 RECORDS
10.1
Original raw data will be archived at Toxikon Corporation.
10.2 A copy of the final report and any amendments will be archived at Toxikon Corporation.
10.3 The original final report and a copy of any protocol amendments or deviations will be forwarded to the Sponsor.
10.4 All unused test article will be handled as specified in the Test Requisition Form. Otherwise, all remaining test article will be discarded.
11,0 CONFIDENTIAIJTY AGREEMENT
Statements of confidentiality may be agreed upon prior to study initiation.
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Toxilo_nCQl'porn|ion CHO/HGPRT Forward Mutation Assay - ISO (T-_889.7) Protocol Number: 3MCNITRO/002-01/000 Protocol Date: 12/03101 Effective Date: 12/12/01
12.0 PROTOCOL AMENDMENTS/DEVIATIONS
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Allchangestotheapprovedprotocoalndthereasonforthechangeswillbe documentedin writings,ignedbytheStudyDirectord,ateda,ndmaintainewdiththeprotocolN.o protocol amendmentswillbemade withoutwritteanpprovailntheformofa SponsorCommunicatioLnog betweentheSponsorandtheStudyDirectowrhichwillbe generateadscloselayspossiblteothe timeofthechange.
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