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FINAL REPORT
PURIFIED 8-2 ALCOHOL: SALMONELLA-ESCHERICHIA COX//MAMMALIANMICROSOME REVERSE MUTATION ASSAY WITH A CONFIRMATORY ASSAY
COVANCE STUDY 22900-0-4090ECD
STUDY SCHEDULE: Study Initiation Date: Initial Dose Date: Study Termination Date: Final Report Date:
23 October 2001 29 October 2001 10 December 2001 11 February 2002
STUDY DIRECTOR. SPONSOR. AND TESTING FACILITY
Study Director: Testing Facility:
Leon F. Stankowski, Jr., PhD Covance Laboratories, Inc. (Covance) 9200 Leesburg Pike Vienna, VA 22182
Sponsor:
Telomer Research Program Consortium administered by RAND Corp. 1700 Main Street Santa Monica, California 90407
This study was conducted in accordance with Covance Standard Operating Procedures; the Organisation for Economic Cooperation and Development (OECD) Principles of Good Laboratory Practice, ENV/MC/CHEM(98)17 adopted November 26, 1997; and the Environmental Protection Agency (EPA-TSCA), Title 40 of the US Code of Federal Regulations Part 792, issued November 29, 1983 (effective December 29, 1983 [revision effective September 18,1989]).
This document is the confidential property of the Telomer Research Program Consortium. No part of it may be transmitted, reproduced, published, or used by other persons without the permission of the Telomer Research Program Consortium.
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TABLE OF CONTENTS
2
PAGE
LIST OF TABLES.................................................................................................................... 5
LIST OF APPENDICES..........................................................................................................6
SUMMARY................................................................................................................................7
1. OBJECTIVE.....................................................................................................8
2. 2.1 2.1.1 2.1.2 2.1.2.1 2.1.2.2 2.1.3 2.1.3.1 2.1.3.2 2.1.4 2.1.4.1 2.1.4.2 2.1.4.3 2.1.5 2.1.5.1 2.1.5.2 2.1.5.3 2.2 2.3 2.3.1 2.3.2 2.3.2.1 2.3.3 2.3.3.1 2.3.3.2
MATERIALS....................................................................................................8 Tester Strains....................................................................................................8 Source of Tester Strains..................................................................................... 9 Storage of Tester Strain................................................................................... 10 Frozen Permanent Stocks................................................................................. 10 Master Plates.....................................................................................................10 Preparation of Overnight Cultures..................................................................10 Inoculation.........................................................................................................10 Harvest.............................................................................................................. 10 Confirmation of Tester Strain Phenotypes....................................................... 11 rfa Wall Mutation..............................................................................................11 pKMl 01 Plasmid R-factor............................................................................... 11 Characteristic Number of Spontaneous Revertants......................................... 11 Tester Strain M edia......................................................................................... 12 Culturing B roth.................................................................................................12 Minimal Bottom Agar Plates............................................................................ 12 Top Agar for Selection of Revertants.............................................................. 12 Test Article......................................................................................................13 Control Articles...............................................................................................13 Vehicle Controls...............................................................................................13 Positive Controls..............................................................................................13 Source and Grade of Positive Control Articles............................................... 14 Sterility Controls..............................................................................................14 lest Article........................................................................................................ 14 S9 M ix............................................................................................................... 14
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2.4 2.4.1 2.4.2
Liver Microsomal Enzyme Reaction Mixture (S9 Mix)............................ 15 S9 Homogenate................................................................................................15 S9 M ix.............................................................................................................. 15
3. 3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.2 3.2.1 3.2.2 3.3 3.4 3.4.1 3.4.2
EXPERIMENTAL DESIGN.................... .................................................. 15 Dose Rangefinding Study.............................................................................. 15 Design............................................................................................................... 16 Rationale........................................................................................................... 16 Evaluation of the Dose Rangefinding Study.................................................. 16 Selection of the Maximum Dose for the Mutagenicity Assay........................16 Mutagenicity Assay........................................................................................ 17 Design............................................................................................................... 17 Frequency and Route of Administration..........................................................17 Plating Procedures..........................................................................................17 Scoring the Plates.......................................................................................... 18 Bacterial Background Lawn Evaluation..........................................................18 Counting Revertant Colonies...........................................................................18
4. 4.1 4.2 4.2.1 4.2.1.1 4.2.1.2 4.2.1.3 4.2.2 4.2.3 4.2.3.1 4.2.3.2 4.2.4 4.3 4.3.1 4.3.2
DATA............................................................................................................... 18 Data Presentation...........................................................................................18 Assay Acceptance Criteria............................................................................ 19 Tester Strain Integrity...................................................................................... 19 rfa Wall Mutation............................................................................................. 19 pKMl 01 Plasmid............................................................................................. 19 Characteristic Number of Spontaneous Revertants.........................................19 Tester Strain Culture Density..........................................................................20 Positive Control Values................................................................................... 20 Positive Control Values in the Absence of S9 M ix........................................ 20 Positive Control Values in the Presence of S9 Mix (S9 Mix Integrity)........20 Cytotoxicity......................................................................................................21 Assay Evaluation Criteria.............................................................................21 Tester Strains TA98, TA100 and WP2wvrA................................................... 21 Tester Strains TA1535 and TA1537................................................................ 21
5. RECORDS TO BE MAINTAINED.............................................................22
6. STUDY RESPONSIBILITIES.....................................................................22
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7. RESULTS........................................................................................................22 7.1 Test Article Handling.................................................................................... 22 7.2 Dose Rangefinding Study (Appendix A, Tables 4 and 5 )......................... 23 7.3 Mutagenicity Assay (Appendix A, Tables 6, 8 - Individual Data,
Tables 7, 9- Summary Data).........................................................................23
8. CONCLUSIONS........................................................................................... 24
9. PROTOCOL DEVIATIONS........................................................................24
10. LIST OF REFERENCES.............................................................................. 25
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LIST OF TABLES
PAGE
Table 1. Table 2. Table 3. Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10
Tester Strain Genotypes...................................................................................... 8 Positive Controls.............................................................................................. 14 S9 Mix Components..........................................................................................15 Dose Rangefinding Study - TA100................................................................. 27 Dose Rangefinding Study - WP2wvrA............................................................ 28 Initial Mutagenicity Assay Results - Individual Plate Counts........................ 29 Initial Mutagenicity Assay Results - Summary............................................... 30 Confirmatory Mutagenicity Assay Results -Individual Plate Counts............31 Confirmatory Mutagenicity Assay Results - Summary.................................. 32 Historical Control D ata.................................................................................... 33
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LIST OF APPENDICES
PAGE Appendix A. Experimental Data Tables..............................................................................26 Appendix B. Definitions of Bacterial Background Lawn Evaluation Codes....................34 Appendix C. Quality Assurance and Compliance Statements............................................36
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SUMMARY
Objective: The objective of this study was to evaluate the ability of Purified 8-2 Alcohol to induce reverse mutations at the histidine locus of selected Salmonella typhimurium strains, or the tryptophan locus of Escherichia coli strain WP2vrA. in the presence and absence of an exogenous mammalian metabolic activation system (S9).
Study Design and Parameters: Purified 8-2 Alcohol was tested in the plate incorporation reverse mutation assay using S. typhimurium strains TA98, TA100, TA1535 and TA1537, and E. coli strain WP2wvrA, with and without a metabolic activation system (S9) containing induced hepatic microsomal enzymes from AroclorTM 1254-treated rats. Initial and confirmatory mutagenicity assays were conducted. Each assay included vehicle and positive controls, and six dose levels of Purified 8-2 Alcohol, with and without metabolic activation. Doses of Purified 8-2 Alcohol evaluated, based on the results of a dose rangefinding assay and selected in conjunction with the Sponsor, were 33.3,100, 333, 1000, 3330 and 5000 pg/plate with and without S9. Each test and control article dose was evaluated in triplicate plates in each strain. The dimethylsulfoxide (DMSO) vehicle control, as well as the test and positive control articles were administered in a volume of 50 pL.
Results: No toxicity was observed in any tester strain in the presence or absence of S9 up to the maximum dose tested of 5000 pg/plate. The mean number of revertants per plate did not meet the criteria for a positive response in any tester strain with or without S9. All criteria for a valid study, including appropriate vehicle and positive control responses (with the exception of positive control values for TA1537 without S9 in Experiment 22900-C1, see Protocol Deviation), were fulfilled.
Conclusion: Purified 8-2 Alcohol was not mutagenic in this test system.
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1. OBJECTIVE
The objective of this study was to evaluate the test article for the ability to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537), and at the tryptophan locus in Escherichia coli tester strain WP2vrA, in the presence or absence of an exogenous metabolic activation system (S9). The assay design was based on OECD Guideline 471, updated and adopted July 21, 1997.
2. MATERIALS
2.1 Tester Strains
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 (Ames et ah, 1975) and the Escherichia coli tryptophan auxotroph WP2wvrA (Green and Muriel, 1976). The specific genotypes of the strains are shown below (Table 1).
Table 1.
Tester Strain Genotypes
T ester Strain
TA98 T A 100 T A 1535 T A 1537 W P lu vrA
h is/tr p M utation /zA D 3052
hisG 46 hisG 46 AC3076
trp
A dditional M utations
R epair
LPS
uvrB
rfa
uvrB
rfa
uvrB
rfa
uvrB
rfa
uvrA
-
Plasm id
pK M IO l pK M IO l
-
In addition to a mutation in the histidine or tryptophan operons, the tester strains contain two additional mutations which enhance their sensitivity to some mutagenic compounds. A mutation of the uvrA gene (Escherichia coli) or the uvrB gene {Salmonella typhimurium), results in a deficient DNA excision repair system which greatly enhances the sensitivity of
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these strains to some mutagens. Since the vrB deletion extends through the bio gene, the
Salmonella typhimurium tester strains containing this deletion also require the vitamin biotin
for growth.
The Salmonella typhimurium tester strains also contain the rfa wall mutation which results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e., benzo[a]pyrene) that would otherwise be excluded by a normal intact cell wall.
Strains TA98 and TA100 also contain the pKMIOl plasmid, which further increases the sensitivity of these strains to some mutagens. The mechanism by which this plasmid increases sensitivity to mutagens has been suggested to be by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strains TA100, TA1535 and WP2wvrA are reverted from auxotrophy to prototrophy by base substitution mutagens.
2.1.1
Source of Tester Strains
The Salmonella typhimurium tester strains were received from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley. The Escherichia coli tester strain, WP2mvtA, was received from The National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).
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2.1.2
Storage of Tester Strain
10
2.1.2.1
Frozen Permanent Stocks
Frozen permanent stocks were prepared by growing fresh overnight cultures, adding DMSO (0.09 mL/mL of culture) and freezing small aliquots at <-70C.
2.1.2.2
Master Plates
Master plates of the tester strains were prepared by streaking each tester strain from a frozen permanent stock onto minimal agar appropriately supplemented with either histidine and biotin (S. typhimurium) or tryptophan (E. coli), and for strains containing the pKMIOl plasmid, ampicillin. Tester strain master plates were stored at 5 3C.
2.1.3
Preparation of Overnight Cultures
2.1.3.1
Inoculation
Overnight cultures for use in all testing procedures were inoculated by transferring a colony from the appropriate master plate to a flask containing culture medium. Inoculated flasks were placed in a shaker/incubator which was programmed to begin operation (shaking, 125 25 rpm; incubation, 37 2C) so that the overnight cultures were in log phase or late log phase when turbidity monitoring began.
2.1.3.2
Harvest
To ensure that cultures were harvested in late log phase, the length of incubation was determined by spectrophotometric monitoring of culture density. Cultures were harvested once a predetermined density was reached, which ensures that cultures have reached a density of at least 0.5 x 109 cells/mL and that the cultures have not overgrown. Overgrown of
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stationary cultures may exhibit decreased sensitivity to some mutagens. Cultures were
removed from incubation when the target density was reached and were placed at 5 3C
until used in the assay.
2.1.4
Confirmation of Tester Strain Phenotypes
Tester strain cultures were checked for the following genetic markers on the day of their use in the mutagenicity assay:
2.1.4.1
rfa Wall Mutation
For the Salmonella tester strains, the presence of the rfa wall mutation was confirmed by demonstration of the sensitivity of the culture to crystal violet. An aliquot of an overnight culture of each strain was overlaid onto plates containing selective media and an antibiotic sensitivity disk containing 10 pg of crystal violet was added. Sensitivity was demonstrated by inhibition of bacterial growth in a zone immediately surrounding the disk.
2.1.4.2
pKMIOl Plasmid R-factor
The presence of the pKMIOl plasmid was confirmed for cultures of tester strains TA98 and TA100 by demonstration of resistance to ampicillin. An aliquot of an overnight culture of each strain was overlaid onto plates containing selective media and an antibiotic sensitivity disk containing 10 pg of ampicillin was added. Resistance was demonstrated by growth in the zone immediately surrounding the disk.
2.1.4.3
Characteristic Number of Spontaneous Revertants
The mean number of spontaneous revertants per plate in the vehicle controls that is characteristic of the respective strains was demonstrated by plating 100 pL aliquots of each culture along with the appropriate vehicle on selective media.
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2.1.5
Tester Strain Media
12
2.1.5.1
Culturing Broth
The broth used to grow overnight cultures of the tester strains was Vogel-Bonner salt solution (Vogel and Bonner, 1956) supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2 (dry powder).
2.1.5.2
Minimal Bottom Agar Plates
Bottom agar (25 mL per 15 x 100 mm petri dish) was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956), supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose.
2.1.5.3
Top Agar for Selection of Revertants
Top (overlay) agar was prepared with 0.7% agar (w/v) and 0.5% NaCl (w/v) and was supplemented with 10 mL of 1) 0.5 mM histidine/biotin solution per 100 mL agar for selection of histidine revertants, or 2) 0.5 mM tryptophan solution per 100 mL of agar for selection of tryptophan revertants.
When S9 mix was required, 2.0 mL of the supplemented top agar was used in the overlay. However, when S9 mix was not required, water was added to the supplemented top agar (0.5 mL of water per 2 mL of supplemented top agar) and the resulting 2.5 mL of diluted supplemented top agar was used for the overlay. This dilution ensured that the final top agar and amino acid supplement concentrations remained the same both in the presence and absence of S9 mix.
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2.2 Test Article
13
Test Article: Haskell Number: Physical Description: Date Received:
Purified 8-2 Alcohol 24691 White solid 8 August 2001
The test article, Purified 8-2 Alcohol, was supplied as a white solid. The test article was stored in a container at ambient temperature. The Sponsor was responsible for the determination and documentation of the analytical purity of the test article. Any unused test article and/or the original test article container were returned to the Sponsor.
2.3 Control Articles
2.3.1 Vehicle Controls
Dimethylsulfoxide (DMSO, CAS# 67-68-5, Acros Organics, Lot Nos. A014587401 and A012097501) was used as the vehicle. Vehicle controls were plated for all tester strains in the presence and absence of S9. The vehicle control was plated on selective agar using a 50 pL aliquot (equal to the maximum aliquot of test article plated), along with 100 pL of the appropriate tester strain and 500 pL of S9 mix (when necessary).
2.3.2
Positive Controls
The combinations of positive controls, activation condition, and tester strains plated concurrently with the assay are indicated below (Table 2, next page).
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Table 2.
Positive Controls
T ester Strain
S9 M ix
Positive C ontrol
TA98 TA98 TA 100 T A 100 TA 1535 T1535 T A 1537 T A I 537 W P 2i/v/'A W P2uwA
+ benzo[a]pyrene - 2-nitrofluorene + 2-am inoanthracene - sodium azide + 2-am inoanthracene - sodium azide + 2-am inoanthracene - ICR-191
+ 2-am inoanthracene
- 4-nitroquinoline-N -oxide
14
D ose (pg/plate)
2.5
1.0
2 .5 2 .0 2.5 2 .0 2.5 2 .0 2 5 .0
1.0
2.3.2.1
Source and Grade of Positive Control Articles
Benzo[a]pyrene (CAS# 50-32-8; purity >97%), 2-nitrofluorene (CAS# 607-57-8; purity >98%), sodium azide (CAS# 26628-22-8; purity >99%), ICR-191 (CAS# 1707-45-0; purity >90%), and 4-nitroquinoline-N-oxide (CAS# 56-57-5; purity >99%) were obtained from Sigma Chemical Co. All were prepared in DMSO, except for sodium azide, which was dissolved in deionized water.
2.3.3
Sterility Controls
2.3.3.1
Test Article
The most concentrated test article stock solution was checked for sterility by plating a 50 pL aliquot (the same volume used in the assay) on selective agar.
2.3.3.2
S9 Mix
The S9 mix was checked for sterility by plating 0.5 mL on selective agar.
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2.4 Liver Microsomal Enzyme Reaction Mixture (S9 Mix)
15
2.4.1
S9 Homogenate
S9 homogenate containing liver microsomal enzymes was purchased from Molecular Toxicology, Inc. [Lot Nos. 1302 (41.3 mg/mL protein) and 1296 (38.9 mg/mL protein). The homogenate was prepared from male Sprague-Dawley rats that had been injected (ip) with AroclorTM 1254 (200 mg/mL in com oil) at 500mg/lcg (Ames et al., 1975).
2.4.2
S9 Mix
The S9 mix was prepared immediately prior to use and contained the components indicated below (Table 3).
Table 3.
S9 Mix Components
Com ponent
H20 1M N aH 2PC >4/N a2H P04, p H 7.4 0.242M G lucose-6-phosphate 0.10M N A D P 0 .8 2 5 M K C 1/0.2M M g C L S9 H om ogenate
Q uantity
0.70 m L 0.10 m L 0.02 m L 0.04 m L 0.04 m L 0.10 m L
1.00 m L
3. EXPERIMENTAL DESIGN
3.1 Dose Rangefinding Study
The growth inhibitory effect (cytotoxicity) of the test article to the test system was determined in order to allow the selection of appropriate doses to be tested in the mutagenicity assay.
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3.1.1 Design
16
The dose rangefinding study was performed using tester strains TA100 and WP2wvrA in the presence and absence of S9. Ten doses of test article, up to 5000 pg/plate, were tested for cytotoxicity (one plate per dose).
3.1.2
Rationale
The cytotoxicity of the test article observed in tester strain TA100 is generally representative of that observed on the other Salmonella typhimurium tester strains and because of the comparatively high number of spontaneous revertants per plate observed with this strain, gradations of cytotoxicity can be readily discerned from routine experimental variation. The Escherichia coli tester strain WP2uvrA does not possess the rfa wall mutation that the Salmonella typhimurium strains have and thus, a different range of cytotoxicity may be observed. Also, the cytotoxicity induced by a test article in the presence of S9 may vary greatly from that observed in the absence of S9. Therefore, this would require that different test article dose ranges be tested in the mutagenicity assay based on the presence or absence of the microsomal enzymes.
3.1.3
Evaluation of the Dose Rangefinding Study
Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn.
3.1.4
Selection of the Maximum Dose for the Mutagenicity Assay
No cytotoxicity was observed in the dose rangefinding study and the highest dose level of test article used in the subsequent mutagenicity assay was that used in the dose rangefinding study (the reduction in background lawn observed in tester strain TA100 without S9 was not accompanied by a decrease in revertant frequency and likely was artifactual).
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3.2 Mutagenicity Assay
17
3.2.1 Design
The assay was performed using tester strains TA98, TA100, TA1535, TA1537 and WP2uvrA in the presence and absence of S9. Doses of the test article were selected based on the results of the dose rangefinding study. The results of the initial mutagenicity assay were confirmed in an independent experiment.
3.2.2
Frequency and Route of Administration
The tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, the test article, the tester strain, and the S9 mix (where appropriate) were combined in molten agar which was overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All doses of the test article, the vehicle controls and the positive controls were plated in triplicate.
3.3 Plating Procedures
These procedures were used in the dose rangefinding study and the mutagenicity assay. Each plate was labeled with a code which identified the test article, test phase, tester strain, activation condition and dose level. The S9 mix and dilutions of the test article were prepared immediately prior to their use.
When S9 was not required, 100 pL of tester strain and 50 pL of test or control article were added to 2.5 mL of molten selective top agar (maintained at 45 2C). When S9 was required, 500 pL of S9 mix, 100 pL of tester strain and 50 pL of test or control article were
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added to 2.0 mL of molten selective top agar. After the required components had been added,
the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar
contained in a 15 x 100 mm petri dish. After the overlay solidified, the plates were inverted
and incubated for 52 4 hours at 37 2C.
3.4 Scoring the Plates
Plates which were not evaluated immediately following the incubation period were held at 5 3C until such time that colony counting and bacterial background lawn evaluation could take place.
3.4.1
Bacterial Background Lawn Evaluation
The condition of the bacterial background lawn was evaluated macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose on the data tables using the code system described below (Appendix B).
3.4.2
Counting Revertant Colonies
Revertant colonies were counted by automated colony counter.
4. DATA
4.1 Data Presentation
For all replicate platings, the mean revertants per plate and the standard deviation were calculated (Appendix A).
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4.2 Assay Acceptance Criteria
19
Before assay data were evaluated, the criteria for a valid assay had to be met. The following criteria were used to determine a valid assay:
4.2.1
Tester Strain Integrity
4.2.1.1
rfa Wall Mutation
All Salmonella typhimurium tester strain cultures exhibited sensitivity to crystal violet, demonstrating the presence of the rfa wall mutation.
4.2.1.2
pKMIOl Plasmid
Tester strains TA98 and TA100 exhibited resistance to ampicillin, demonstrating the presence of the pKMIOl plasmid.
4.2.1.3
Characteristic Number of Spontaneous Revertants
All vehicle control cultures exhibited their characteristic number of spontaneous revertants per plate, demonstrating the requirement for histidine (Salmonella typhimurium) or tryptophan (.Escherichia coli). The acceptable ranges for the mean vehicle controls were as follows:
Strain
TA98 TA100 TA1535 T A 1537 W P2wvrA
Number of Revertants
8 - 60 60 - 240
4-45 2 - 25 5 - 40
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4.2.2
Tester Strain Culture Density
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The cell densities of all tester strain cultures were greater than or equal to 0.5 x 109bacteria/mL (with the exception of TA98 in Experiment 22900-B1, 0.4 x 109 bacteria/mL), or the optical densities of these cultures reached a target value demonstrated to produce cultures with at least 0.5 x 109 bacteria/mL, demonstrating that appropriate numbers of bacteria were plated.
4.2.3
Positive Control Values
4.2.3.1
Positive Control Values in the Absence of S9 Mix
The mean value of the positive control for each tester strain exhibited at least a 3-fold increase over the mean value of the vehicle control for that strain (with the exception of tester strain TA1537 in Experiment 22900-C1, see Protocol Deviation), demonstrating that the tester strains were capable of identifying a mutagen.
4.2.3.2
Positive Control Values in the Presence of S9 Mix (S9 Mix Integrity)
The mean value of the positive control for each tester strain exhibited at least a 3-fold increase over the mean value of the vehicle control for that strain, demonstrating that the S9 mix was capable of metabolizing a promutagen to its mutagenic form(s).
An acceptable positive control in the presence of S9 for a specific strain was evaluated as having demonstrated the integrity of the S9 mix and the ability of the tester strain to detect a mutagen.
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4.2.4
Cytotoxicity
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A minimum of three non-toxic doses was used to evaluate assay data. Cytotoxicity can be detected as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn compared to the appropriate vehicle control.
4.3 Assay Evaluation Criteria
Once the criteria for a valid assay had been met, responses observed in the assay were evaluated as follows:
4.3.1
Tester Strains TA98, TA100 and WP2wvrA
For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
4.3.2
Tester Strains TA1535 and TA1537
For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
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5. RECORDS TO BE MAINTAINED
22
All raw data, documentation, records, the protocol, and the final report generated as a result of this study will be archived in the storage facilities of Covance-Vienna for at least one year following submission of the final report to the Sponsor. After the one year period, the Sponsor may elect to have the aforementioned materials retained in the storage facilities of Covance-Vienna for an additional period of time or sent to a storage facility designated by the Sponsor.
6. STUDY RESPONSIBILITIES
Function
Study Director Laboratory Supervisor
Responsible Personfsi
Leon F. Stankowski, Jr., PhD Magnus A. Evertson, BS
7. RESULTS
7.1 Test Article Handling
Purified 8-2 Alcohol formed a solution at 100 mg/mL in dimethylformamide after heating. In DMSO at 100 mg/mL, the test article formed a solution that foamed when heated and vortexed. DMSO was selected as the vehicle. At 100 mg/mL, which was the most concentrated stock prepared for the mutagenicity assay, the test article formed a transparent, colorless solution after heating to 45C for 3 minutes; it remained freely soluble at all succeeding lower dilutions.
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7.2 Dose Rangefmding Study (Appendix A, Tables 4 and 5)
23
A dose rangefinding assay was conducted on the test article using tester strains TA100 and WP2wvrA (one plate per dose; Experiment 22900-A1; Tables 4 and 5). Ten doses of test article, from 6.67 to 5000 pg/plate, were evaluated in the presence and absence of S9. Apparently normal growth was observed in both tester strains at all doses evaluated with and without S9 (the reduction in background lawn observed in tester strain TA100 without S9 was not accompanied by a decrease in revertant frequency and likely was artifactual). However, the test article was found to be incompletely soluble in the aqueous top agar at a dose of 5000 ug/plate with and without S9.
7.3 Mutagenicity Assay (Appendix A, Tables 6, 8 - Individual Data, Tables 7, 9- Summary Data)
Based upon the results of the dose rangefinding study, Purified 8-2 Alcohol was evaluated in the initial mutagenicity assay in all five tester strains at doses of 33.3, 100, 333, 1000, 3330 and 5000 ug/plate with and without S9 (Experiment 22900-B1, Tables 6 and 7). All doses of the test article, as well as the concurrent positive and vehicle controls, were evaluated using three plates per dose. Apparently normal growth again was observed in all tester strains at all doses of Purified 8-2 Alcohol evaluated with and without S9. In addition, the test article precipitated from solution at doses >1000 pg/plate with and without S9. Revertant frequencies for all doses of Purified 8-2 Alcohol, in all tester strains with and without S9, approximated or were less than those observed in the concurrent vehicle control cultures.
The test article was re-evaluated in an independent confirmatory experiment under identical conditions, and similar results were observed (Experiment 22900-C1; Tables 8 and 9). Normal growth was again observed in all tester strains at all doses evaluated with and without S9, and the test article again precipitated from solution at doses >1000 ug/plate with and without S9. Revertant frequencies for all doses of Purified 8-2 Alcohol, in all tester
000052
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
24
strains with and without S9, again approximated or were less than control values. Except for
tester train TA1537 without S9 in Experiment 22900-C1 (see Protocol Deviation), all positive
and vehicle control values were within acceptable ranges in both assays. All criteria for a
valid study were met.
8. CONCLUSIONS
The results of the Salmonella - Escherichia col//Mammalian- Microsome Reverse Mutation Assay indicate that under the conditions of this study, Purified 8-2 Alcohol was not mutagenic in any of the tester strains in the presence or absence of an exogenous metabolic activation system containing induced hepatic microsomal enzymes from AroclorTM 1254-treatedrats.
9. PROTOCOL DEVIATIONS
In Experiment 22900-C1, the test article-treated cultures for tester strain TA1537 without S9 were scored even though the concurrent positive control values were below acceptable limits (all plates had no revertants). However, the vehicle control values for tester strain TA1537 without S9 were within acceptable ranges. In addition, the positive control values for tester strain TA1537 with S9 also were within acceptable limits, indicating that the tester strain and S9 were functioning properly. Thus, the observed low values were likely due to a technical error. This deviation is not considered to have had an adverse impact upon the integrity of the study or the conclusions derived from it.
000053
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
10. LIST OF REFERENCES
25
Ames et al., 1975. Ames BN, McCann J, Yamasaki E. Methods for detecting carcinogens and mutagens with the .Sh/mone//a/Mammalian-Microsome Mutagenicity Test. Mutation Research 1975; 31:347-64.
Green and Muriel, 1976. Green MHL, Muriel WJ. Mutagen testing using trp+reversion in Escherichia coli. Mutation Research 1976; 38:3-32.
Maron and Ames, 1983. Maron DM, Ames B. Revised methods for the Salmonella Mutagenicity Test. Mutation Research 1983; 113:173-215.
Yogel and Bonner, 1956. Vogel HJ, Bonner DM. Acetylomithinase of E. coli: Partial purification and some properties. JB iol Chem 1956; 218:97-106.
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Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
Appendix A. Experimental Data Tables
26
000055
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
Table 4
Dose Rangefinding Study - TA100
T est A rticle ID : D ate P lated: V ehicle:
P u rified 8-2 A lcohol 2 9 -O ct-0 1 DM SO
E x p erim en t ID : D ate C ounted: P latin g A liquot:
22900-A 1 0 1 -N ov-01 5 0 juL
TA 100 R evenants Per Plate
H g /P Iate
0.00 (V ehicle) (50 pL)
T est A rticle 6.67 10.0 33.3 66.7
100 333 667 1000 3330 5000
W ith S9
R evenants Per P la te
B ackground Lawn
E valuation3
113 N
107 N 113 N 136 N 97 N 139 N 97 N 93 N 106 N 108 N 105 N P
W ithout S9
R evenants Per
P la te
B ackground L aw n
E valuation3
82 N
86 N 107 N 101 N 94 N 87 N 67 N 98 N 89 N 107 N 103 R P
3Background Lawn Evaluation Codes: N = normal R = reduced A = absent P = precipitate O = obscured by precipitate
27
000056
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
Table 5
Dose Rangefinding Study - W PluvrA
T est A rticle ID : D ate P lated: V ehicle:
P u rified 8-2 A lco h o l 2 9 -O ct-0 1 DM SO
E x p erim en t ID : D ate C ounted: P latin g A liquot:
22900-A 1 01 -N ov-01 50 uL
W P2vrA R evenants; P er Plate
(Xg/Plate
0.00 (V ehicle) (5 0 mL )
T est A rticle 6.67 10.0 33.3 6 6 .7
100 333 667 1000 3330 5000
W ith S9
R evenants Per P late
B ackground Lawn
E valuation3
23 N
21 N 15 N 13 N 17 N 12 N 14 N 18 N 23 N 12 N 17 N P
W ithout S9
R evenants Per
P la te
B ackground Lawn
E valuation3
12 N
12 N 11 N 15 N 11 N 18 N 12 N 11 N 12 N 14 N 13 N P
3 Background Lawn Evaluation Codes: N = normal R = reduced A = absent P = precipitate O = obscured by precipitate
28
000057
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
Table 6
Initial Mutagenicity Assay Results - Individual Plate Counts
Test Article ID: Date Plated: Vehicle:
Purified 8-2 Alcohol 13-Nov-Ol DMSO
Experiment ID: Date Counted: Plating Aliquot:
22900-B1 26-Nov-01 50 pL
29
Dose/Plate
M icrosom es: R at L iver V ehicle C ontrol
TA98 12 3
33 30 27
Revertants Per Plate
TA100 12
3
TA1535 12 3
115 125 112 14 8 18
TA1537 12 3
WP2wwA
12 3
Background Lawn"
7 7 10 27 14 5
N
T est A rticle
33.3 P g 32 24 30
100 Pg 25 20 28
333 P g 34 35 18
1000 Pg 34 33 27
3330 Pg 23 45 28
5000 Pg 28 45 30
P o s itiv e C o n tr o l13
574 512 542
112 91 103 108 102 117
88 114 108 119 115 116 115 99 104 111 120 116
16 17 16 11 9 10 12 9 14 18 9 17 16 14 19 14 16 8
984 1048 1090 141 165 138
5 10 14 10 9 5 10 10 9
6 12 9 10 14 9 11 9 12
10 16 18 19 19 19 11 10 21
8 26 15 23 19 19 20 20 21
181 148 178 233 241 293
N N N NP NP NP
N
M icrosom es: N one V ehicle C ontrol
12 25 9 116 86 90 10 12 17
6 5 3 16 14 17
N
T est A rticle
3 3 .3 P g 18 14 11
100 P g 12 17 15
333 P g 21 18 8
1000 P g 20 17 16
3 3 3 0 P g 23 11 2 9
5 0 0 0 P g 18 10 19
87 89 70 17 14 6
82 86 70
9 7 17
88 97 86 14 11 11
9 7 94 9 0 11 7 10
79 112 84 17 16 10
94 93 9 7 11 15 16
3 5 9 17 12 18 7 9 2 15 17 17 10 8 2 10 7 12 8 6 10 11 9 12 3 7 12 10 21 20 6 9 3 18 26 19
Positive C ontrol0
208 192 216 1021 1028 948 664 656 579 1064 867 898 158 128 89
N N N NP NP NP
N
" Background Lawn Evaluation Codes'. N = normal R = reduced 0 = obscured
b TA98 TA 100 TAI 535 TA 1537 WP2wvrA
benzo[a]pyrene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene
2.5 ng/plate 2.5 (ig/plate 2.5 (ig/plate 2.5 gg/plate 25.0 pg/plate
A = absent P = precipitate
TA98
2-nitrofluorene
TA100 sodium azide
TA1535 sodium azide
TA1537 ICR-191
WP2vi-A 4-nitroquinolone-N-oxide
1.0 (ig/plate 2.0 pg/plate 2.0 ,ug/plate 2.0 (Ig/plate 1.0 pg/plate
000058
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
Table 7
Initial Mutagenicity Assay Results - Summary
Test Article ID: Date Plated: Vehicle:
Purified 8-2 Alcohol 13-Nov-Ol DMSO
Experiment ID: Date Counted: Plating Aliquot:
22900-B1 26-Nov-Ol
50 uL
30
D ose/Plate
M icrosom es: R at Liver V ehicle C ontrol
M ean R evenants P er Plate w ith Standard D eviation
TA98 M ean S.D.
TA100 M ean S.D.
TA1535 M ean S.D .
T A 1537 M ean S.D .
W P2wvrA M ean S.D .
Back-
ground L aw n8
30 3 117 7
13 5
82
15 11
N
Test A rticle
33.3 Pg 100 Pg 333 Pg 1000 Pg 3330 Pg 5000 Pg
29 4 24 4 29 10 31 4 32 12 34 9
102 11 109 8 103 14 117 2 106 8 116 5
P o s itiv e C o n tr o l15
543 31 1041 53
16 1 10 1 12 3 15 5 16 3 13 4
148 15
10 5 83
10 1 93
11 3 11 2
169 18
15 4
N
19 0 N
14 6 N
16 9 N P
20 2 N P
20 1 NP
256 33
N
M icrosom es: N one V ehicle C ontrol
15 9
97 16
13 4
52
16 2 N
T est A rticle
33.3 P g
14 4
100 Pg
15 3
333 Pg
16 7
1000 Pg
18 2
3330 Pg 21 9
5000 Pg
16 5
P ositive C ontrol8
205 12
82 10 79 8 90 6 94 4 92 18 95 2
999 44
12 6 11 5 12 2
92 14 4 14 3
633 47
63 64 74 82 75 63
943 106
16 3 N 16 1 N 10 3 N 11 2 N P 17 6 N P 21 4 N P
125 35
N
n Background Lawn Evaluation Codes: N = normal R - reduced O = obscured
b TA98 TA 100 TAI535 TA1537 WP2iiv/-A
bcnzo[a]pyrene
2.5 gg/plate
2-aminoanthracene 2.5 pg/platc
2-aminoanthracene ' 2.5 |i.g/plate
2-aminoanthracene 2.5 ixg/plate
2-aminoanthracene 25.0 ug/plate
A = absent P = precipitate
c TA98 TA100 TA1535 TA1537 WP2UV/-A
2-nitrofluorene sodium azide sodium azide ICR-191 4-nitroquinoIone-N-oxide
1.0 |Xg/plate 2.0 pg/plate 2.0 |xg/plate 2.0 gg/plate 1.0 gg/plate
000059
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
31
Table 8
Confirmatory Mutagenicity Assay Results - Individual Plate Counts
Test Article ID: Purified 8-2 Alcohol
Experiment ID:
Date Plated:
29-Nov-01
Date Counted:
Vehicle:__________ DMSO_______________________Plating Aliquot:
22900-B1 07-Dec-01, 10-Dec-01 50 pL_______________
Revertants Per Plate
Dose/Plate
Microsomes: Rat Liver Vehicle Control
TA98 i 23
29 27 26
TA100 123
97 98 122
TA1535 123
16 17 16
TA1537 12 3
11 8 11
WP2vrA 1 23
Back ground Lawn8
15 23 15 N
Test Article 33.3 100 333 1000 3330 5000
pg 24 34 26 pg 39 34 38 pg 19 40 25 pg 18 26 28 pg 40 32 42 pg 49 32 39
114 98 132 126 115 122 111 111 112 106 113 99 85 97 103 93 97 97
9 21 11 14 15 15 14 17 12 8 5 15 11 11 15 15 13 10
8 14 7 10 5 14 10 10 9 7 89 12 12 7 11 11 16
Positive Control1*
398 321 180 1131 1228 1189 138 192 125 156 180 141
18 8 20 15 19 18 17 9 24 17 12 20 26 25 29 23 19 21
468 389 461
N N N NP NP NP
N
Microsomes: None Vehicle Control
24 17 12 95 96 85 6 8 5 11 3 5 21 20 16 N
Test Article 33.3 100 333 1000 3330 5000
pg 20 21 26 n o 116 85 pg 24 14 18 72 97 89 pg 14 24 18 96 93 95 pg 18 13 18 105 91 103 pg 21 18 20 92 81 84 pg 23 30 24 90 84 111
7 8 10 10 12 10 9 16 8 8 13 14 10 10 8 13 3 13
Positive Control
287 263 220 1100 1089 1216 821 881 832
678 8 72 375 64 3 423 684
12 12 21 N 16 17 21 N 11 29 20 N 17 17 16 NP 20 14 17 NP 16 18 20 NP
0 0 0 298 321 328 N
8Background Lawn Evaluation Codes: N = normal R = reduced O = obscured
b TA98 TA 100 TA1535 TA 1537 WP2wA
benzo [a]pyrene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene
2.5 pg/plate 2.5 pg/plate 2.5 pg/platc 2.5 pg/plate 25.0 pg/plate
A = absent P = precipitate
TA98 TA100 TA1535 TA1537 WP2uwA
2-nitrofluorcne sodium azide sodium azide ICR-191 4-nitroquinolone-N-oxidc
1.0 pg/plate 2.0 pg/plate 2.0 pg/plate 2.0 pg/plate 1.0 pg/plate
000060
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32
Table 9
Confirmatory Mutagenicity Assay Results - Summary
Test Article ID: Purified 8-2 Alcohol
Experiment ID:
Date Plated:
29-Nov-01
Date Counted:
Vehicle:__________ DMSO______________________ Plating Aliquot:
22900-B1 07-Dec-01, 10-Dec-01 50 uL______________________
Dose/Plate
Microsomes: Rat Liver Vehicle Control
Mean Revertants Per Plate with Standard Deviation
TA98
TA100
TA15 35
TA1537
Mean S.D. Mean S.D. Mean S.D. Mean S.D.
WP2uvrA Mean S.D.
Back ground Lawn5
27 2 106 14 16 1 10 2 18 5 N
Test Article 33.3 Pg 28 5 115 17 14 6 10 4 15 6 N
100 Pg 37 3 121 6 15 1 10 5 17 2 N
333 Pg 28 11 111 1 14 3 10 1 17 8 N
1000 Pg 24 5 106 7
95
8 1 16 4 NP
3330 Pg 38 5 95 9 12 2 10 3 27 2 NP
5000 Pg 40 9 96 2 13 3 13 3 21 2 NP
Positive Control1*
300 111 1183 49 152 36 159 20 439 44 N
Microsomes: None Vehicle Control
18 6 92 6
62
6 4 19 3 N
Test Article 33.3 Pg 22 3 104 16
82
100 Pg 19 5 86 13 11 1
333 Pg 19 5 95 2 11 4
1000 Pg 16 3 100 8 12 3
3330
Pg
20
-z>. 86
6
91
5000
Pg
26
4
95 14
10
6
Positive Control0
257 34 1135 70 845 32
7 1 15 5 N 6 3 18 3 N 5 2 20 9 N 4 2 17 1 NP 3 1 17 3 NP 6 2 18 2 NP
0 0 316 16 N
a Background Lawn Evaluation Codes: N = normal R = reduced O = obscured
b TA98 TA 100 TAI 535 TA 1537 WP2iwA
benzo[a]pyrene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene
2.5 pg/plate 2.5 gg/plate 2.5 gg/plate 2.5 gg/plate 25.0 gg/plate
A = absent P = precipitate
c TA98 TAI 00 TAI 535 TAI 537 WP2UV/-A
2-nitrofluorene sodium azide sodium azide ICR-191 4-nitroquinolone-N-oxide
1.0 pg/plate 2 .0 gg/plate 2.0 gg/plate 2.0 gg/plate 1.0 gg/plate
000061
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
Table 10
Historical Control Data
Plate Incorporation Method - Report Period 01E
33
Strain
Mean Revertants per Plate Standard Deviation Maximum Minimum Count
Vehicle Controls with S9 Mix
TA98
TA100
TA1535
28.2 105.7 7.2 14.3 61 143 14 61 290 294
13.3 4.4 30
5 209
TA1537 WP2mvt-A
10.4 20.0 4.6 6.5 26 41
29 185 203
Strain
Mean Revertants per Plate Standard Deviation Maximum Minimum Count
Vehicle Controls without S9 Mix
TA98
TA100
TA1535
16.4 86.8 12.6
5.4 13.8
4.5
36 128
28
3 46
3
289 282 207
TA1537 8.6 4.3 26 1 183
WP2wwA 19.0 5.8 38 5 194
Strain
Mean Revertants per Plate Standard Deviation Maximum Minimum Count
Positive Controls with S9 Mix***
TA98
TA100
TA1535
325.8
837.3
132.1
72.3 285.9
26.1
614 1985
203
146 317
77
278 281 204
TA1537 128.1 28.2 221 46 181
WP2wvrA 568.4 173.3 1055 192 198
Strain Mean Revertants per Plate Standard Deviation Maximum Minimum Count
Positive Controls without S9 Mix**
TA98
TA100
TA1535
255.6
990.5
708.4
59.8 166.9 119.1
464 1572 1037
130 386 333
276 273 202
TA1537 980.9 269.5 2135 522 178
WP2vrA 227.0 77.3 481 10 190
** TA98 TA100 TA1535 TA1537 WP2uvrA
benzo[a]pyrene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene
2.5 pg/plate 2.5 pg/plate 2.5 fig/plate 2.5 pg/plate 25.0 ig/plate
*** TA98 TA 100 TA1535 TAI 537 WP2uvt'A
2-nitrofluorene sodium azide sodium azide ICR-191 4-nitroquinoline-N-oxide
1.0 ng/plate 2.0 H-e/pIate 2.0 pg/plate 2.0 ue/plate 1.0 pg/plate
000062
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
Appendix B. Definitions of Bacterial Background Lawn Evaluation Codes
34
000063
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
35
Bacterial Background Lawn Evaluation Code
The condition of the background bacterial lawn is evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate as follows:
CODE DEFINITION CHARACTERISTICS OF BACKGROUND LAWN
N Normal
A healthy microcolony lawn.
R Reduced
A distinct thinning of the microcolony lawn and an increase in the size of the microcolonies compared to the vehicle control plate.
A Absent
A complete lack of any microcolony lawn.
0 Obscured by The background bacterial lawn cannot be accurately evaluated due to
Precipitate
microscopic test article precipitate, macroscopic test article
precipitate, or plate coloration.
E Enhanced
A distinct thickening of the microcolony lawn and an increase in the size of the microcolonies compared to the vehicle control plate.
Evidence of macroscopic test article precipitate on the plates is recorded by addition of the following precipitate code to the code number used to evaluate the condition of the background bacterial lawn.
P Precipitate
Macroscopic precipitate observed on the plate.
000064
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
Appendix C. Quality Assurance and Compliance Statements
36
000065
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
37
QUALITY ASSURANCE STATEMENT
Purified 8-2 Alcohol: Salmonella-Escherichia Co/z/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay
The report has been reviewed by the Quality Assurance Unit of Covance Laboratories Inc., in accordance with the Good Laboratory Practice regulations as set forth in the Environmental Protection Agency (EPA - TSCA), Title 40 of the U.S. Code of Federal Regulations Part 792; and the Organization for Economic Cooperation and Development (OECD) Principles of Good Laboratory Practice ENV/MC/CHEM (98)17; and any applicable amendments. The following inspections were conducted and the findings reported to the Study Director and study director management. Written status reports of inspections and findings are issued to Covance management according to standard operating procedures.
Inspection Dates
24-Oct-2001 13-Nov-2001 07-Jan-2002 08-Feb-2002
Phase
Protocol Review S9 Mix Preparation Draft Report Review Final Report Review
Dates Reported to Study Director and Study
Director Management 24-Oct-2001
14-Nov-2001
07-Jan-2002
08-Feb-2002
Auditor
P. Cceres J. Howard C. Smith C. Smith
:^ - W h - ~ r 7 .
........ ........................
Representative, Quality Assurance Unit
.. M
Date
000061
Purified 8-2 Alcohol Covance Study 22900-0-4090ECD
38
STUDY COMPLIANCE AND CERTIFICATION
Except that the test and control article dosing solutions were not analyzed for stability, homogeneity or accuracy of preparation, this study was conducted in compliance with the Good Laboratory Practice regulations as set forth by the Environmental Protection Agency (EPA - TSCA), Title 40 of the U.S. Code of Federal Regulations Part 792; and the Organization for Economic Cooperation and Development (OECD) Principles of Good Laboratory Practice ENV/MC/CHEM (98)17; and any applicable amendments. There were no other deviations from the aforementioned regulations or the signed protocol that would affect the integrity of the study or the interpretation of the test results. The raw data have been reviewed by the Study Director, who certifies that the evaluation of the test article as presented herein represents an appropriate conclusion within the context of the study design and evaluation criteria.
Leon F. Stankowski, Jr., PhD/ Bacterial Mutagenesis Genetic and Molecular Toxicology Covance-Vienna
Testing Facility Management:
Brian C. Myhr, PhD Jj Bacterial Mutagenesis Genetic and Molecular Toxicology
2 -/tt/ 2Study Completion Date
SM) O TLDate '
Report Reviewed and Accepted for E.I. du Pont de Nemours and Co. by:
Gerald Kennedy, PhD Sponsor Study Monitor
Date
Maria Donner, PhD Sponsor Technical Project Monitor
Date
000067
