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FINAL REPORT
ffp B tg g g g M g K g g ftsA L M O N E L L A -E S C H E R IC H IA CDL//MAMMA LIANV MICROSOME 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:
Sponsor:
ovance Laboratories, Inc. (Covance)
Ommr-)9200 Leesburg Pike Vienna, VA 22182
Santa Monica, California 90407
This study was conducted in accordance with Covance Standard Operating Procedures; the Organisation for Economic Cooperation and Development (OECD) Principles o f Good Laboratory Practice, ENV/MC/CHEM(98)17 adopted November 26, 1997; and the Environmental Protection Agency (EPA-TSCA), Title 40 o f the US Code o f Federal Regulations Part 792, issued November 29, 1983 (eifective December 29, 1983 [revision effective September 18, 1989]).
This document is the confidential property of thj No part of it ma^Ae transmitted, reproduced, published, or used by other persons without permission of t
not eoR i s t a TSCAC&i ggnftfted. s Compaq
Covance Study 22900-0-4090ECD 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 o f Overnight Cultures.............;.....................................................10
Inoculation.............................................................................................................10
H arvest.................................................................................................................. 10
Confirmation o f Tester Strain Phenotypes........................................................ 11
rfa Wall M utation.....................................................................'........................ 11
pKMIOl Plasmid R-factor.................................................................................. 11
Characteristic Number of Spontaneous Revertants...........................................11
Tester Strain M ed ia............................................................................................ 12
Culturing B ro th ....................................................................................................12
Minimal Bottom Agar Plates.............................................................................. 12
Top Agar for Selection of Revertants.................................................................12
Test A rticle.........................................................................................................13
Control Articles................................................................................................. 13
' Vehicle Controls............................................................ .'............................ .....13
Positive Controls................................................................................................ 13
Source and Grade o f Positive Control A rticles................................................ 14
Sterility Controls..............................
14
Test A rticle.......................................................................................................... 14
S9 M ix................................................................. :..............................................14
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Covance Study 22900-0-4090ECD
2.4 2.4.1 2.4.2
Liver Microsomal Enzyme Reaction Mixture (S9 M ix).............................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 o f the Dose Rangefinding Study.................................................... 16 Selection o f the Maximum Dose for the Mutagenicity Assay........................ 16 Mutagenicity A ssay............................................................................................ 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 M utation................................................................................................ 19 pK M l 01 Plasm id................................................................................................19 Characteristic Number of Spontaneous Revertants..........................................19 Tester Strain Culture Density............................................................................ 20 Positive Control Values......................................................................................20 Positive Control Values in the Absence o f 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 W P luvrA ..................................................... 21 Tester Strains TA1535 and TA1'537.............................................
5. RECORDS TO BE MAINTAINED.............................................................. 22
6. STUDY RESPONSIBILITIES....................................................................... 22
21
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Covance Study 2290-O-4O9OECD
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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 D ata)........................................................................... 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 Rangefmding Study - WP2vrA.............................................................. 28 Initial Mutagenicity Assay Results - Individual Plate Counts.........................29 Initial Mutagenicity Assay Results - Summ aiy................................................ 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 o f Bacterial Background Lawn Evaluation C odes.....................34 Appendix C. Quality Assurance and Compliance Statements..............................................36
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SUMIV^AJRY
Objective: The objective o f this study was to evaluate the ability induce reverse mutations at the histidine locus o f selected Salmonella typhimurium strains, or the tryptophan locus o f Escherichia coli strain WP2vrA, in the presence and absence o f an exogenous mammalian metabolic activation system (S9).
Study Design and Parameters: H ^ B H ^ I ^ H H U v a s tested in the plate incorporation
reverse mutation assay using S. typhimurium strains TA98, TA100, TA1535 and TA1537, and
E. coli strain WP2vrA, 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
with and without metabolic activation.
Doses of Purified 8-2 Alcohol evaluated, based on the results o f 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 o f 50 pL.
Results: No toxicity was observed in any tester strain in the presence or absence o f S9 up to the maximum dose tested o f 5000 pg/plate. The mean number o f 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 o f positive control values for TA1537 without S9 in Experiment 22900-C1, see Protocol Deviation), were fulfilled.
Conclusion
as not mutagenic in this test system.
jritalu TSCA CEI
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Covance Study 22900-0-4090ECD
1. OBJECTIVE
The objective o f this study was to evaluate the test article for the ability to induce reverse mutations at the histidine locus in several strains o f Salmonella typhimurium (TA98, TA100, TA1535 and TA1537), and at the tryptophan locus in Escherichia coli tester strain WP2wvrA, 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 theiSalmonella typhijflurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 (Ames et al., 1975) and the Escherichia coli tryptophan auxotroph WP2vrA (Green and Muriel, 1976). The specific genotypes o f the strains are shown below (Table 1).
Table 1.
Tester Strain Genotypes
Tester Strain
TA98 TA 100 TA1535 TA1537 WP2vrA
his/trp Mutation toD3052 hisGA6 hisGA6
/SC3076 trp
Additional Mutations
Repair
LPS
uvrB rfa
uvrB rfa
uvrB
rfa
uvrB
rfa
uvrA
-
Plasmid
pKMIOl pKMIOl
-
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 o f 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 uvrQ 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 o f one o f the enzymes responsible for the synthesis o f part o f the lipopolysaccharide barrier that forms the surface o f the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes o f 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 W P luvrA are reverted from auxotrophy to prototrophy by base substitution mutagens.
2.1.1
Source o f Tester Strains
The Salmonella typhimurium tester strains were received from Dr. Bruce Ames, Department o f Biochemistry, University o f California, Berkeley. The Escherichia coli tester strain, WP2wvrA, was received from The National Collection o f Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).
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Tovauce Study 22900-0-4090ECD
2.1.2
Storage of Tester Strain
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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 o f 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 o f incubation was determined by spectrophotometric monitoring o f culture density. Cultures were harvested once a predetermined density was reached, which ensures that cultures have reached a density o f at least 0.5 x 109 cells/mL and that the cultures have not overgrown. Overgrown of
11
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 o f 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 o f the rfa wall mutation was confirmed by demonstration of the sensitivity o f the culture to crystal violet. An aliquot o f an overnight culture of each strain was overlaid onto plates containing selective media and an antibiotic sensitivity disk containing 10 pg o f crystal violet was added. Sensitivity was demonstrated by inhibition o f bacterial growth in a zone immediately surrounding the disk.
2.1.4.2
pKM l 01 Plasmid R-factor
The presence o f the pKMIOl plasmid was confirmed for cultures o f tester strains TA98 and TA100 by demonstration o f resistance to ampicillin. An aliquot o f an overnight culture of each strain was overlaid onto plates containing selective media and an antibiotic sensitivity disk containing 10 pg o f 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 o f spontaneous revertants per plate in the vehicle controls that is characteristic o f 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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ovance Study 22900-0-4090ECD
2.1.5
Tester Strain Media
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2.1.5.1
Culturing Broth
The broth used to grow overnight cultures o f 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 m L 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 o f 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 o f agar for selection o f 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 o f 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.
Avance Study 2290-0-409OECD 2.2 Test Article
13
Test Article:
Haskell Number: *24691
Physical Description: White solid
Date Received:
8 August 2001
The test a r t i c l e ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ v w a s 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 o f the analytical purity o f the test article. Any unused test article and/or the original test article container were returned to the Sponsor.
2.3 2.3.1
Control Articles Vehicle Controls
/as used as the vehicle. Vehicle controls were plated for all tester strains in the presence and absence o f S9. The vehicle control was plated on selective agar using a 50 pL aliquot (equal to the maximum aliquot o f test article plated), along with 100 pL o f 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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Covance Study 22! i0-0- 409OECD
Table 2.
Positive Controls
Tester Strain
TA98 TA98 TA 100 TA 100 TA1535 TA 1535 TA1537 TA1537 WP2uvrA WP2uvrA
jf
/ S9 Mix 1
+
-
-
+
+ + -
jPositive Control
benzo [a]pyrene 2-nitrofluorene 2-aminoanthracene sodium azide 2-aminoanthracene sodium azide 2-aminoanthracene ICR-191 2-aminoanthracene 4-nitroquinoline-N-oxide
14
Dose (/xg/plate)
2.5 1.0 2.5 2.0 2.5 2.0 2.5 2.0 25.0 1.0
2.3.2.1
Source and Grade o f 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 Chemicf al 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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ovance Study 229CT0-0-409OECD 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/mLprotein) and 1296 (38.9 mg/mLprotein). 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/kg (Ames et a l, 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
Component
h 2o 1M NaH2P04/Na2HP04, pH 7.4 0.242M GIucose-6-phosphate 0.10M NADP 0.825M KC1/0.2M MgCl2 S9 Homogenate
Quantity
0.70 mL 0.10 mL 0.02 mL 0.04 mL 0.04 mL 0.10 mL
1.00 mL
3. EXPERIMENTAL DESIGN
3.1 Dose Rangefinding Study
The growth inhibitory effect (cytotoxicity) o f the test article to the test system was determined in order to allow the selection o f 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 WP2wwA in the presence and absence o f S9. Ten doses o f test article, up to 5000 pg/plate, were tested for cytotoxicity (one plate per dose).
3.1.2
Rationale
The cytotoxicity o f 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 WP2vrA does not possess the rfa wall mutation that the Salmonella typhimurium strains have and thus, a different range o f 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 o f 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 o f test article used in the subsequent mutagenicity assay was that used in the dose rangefmding 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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Avance Study 2290-0-4090ECD 3.2 M utagenicity Assay
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3.2.1
Design
The assay was performed using tester strains TA98, TA100, TA1535, TA1537 and W PluvrA in the presence and absence of S9. Doses o f the test article were selected based on the results o f the dose rangefinding study. The results o f 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 o f 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 o f test or control article were added to 2.5 mL o f molten selective top agar (maintained at 45 2C). When S9 was required, 500 pL o f S9 mix, 100 pL of tester strain and 50 pL of test or control article were
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'Covance Study 22900-0-4090ECD
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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 o f 25 mL o f 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 o f the bacterial background lawn was evaluated macroscopically and microscopically (using a dissecting microscope) for indications o f cytotoxicity and test article precipitate. Evidence o f 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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^^ovanc^tud^^TO 0-0-409O ECD 4.2 Assay Acceptance Criteria
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 o f 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 o f 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 TA1537 WP2vrA
Number of Revertants .
8 - 60 60- 240 4 - 45 2 - 25 5 - 40
.A.
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Covance Study 229t)0-0-4090ECD
4.2.2
Tester Strain Culture Density
20
The cell densities o f all tester strain cultures were greater than or equal to 0.5 x 109bacteria/mL (with the exception o f TA98 in Experiment 22900-B1, 0.4 x 109bacteria/mL), or the optical densities o f these cultures reached a target value demonstrated to produce cultures with at least 0.5 x 109 bacteria/mL, demonstrating that appropriate numbers o f bacteria were plated.
4.2.3
Positive Control Values
4.2.3.1
Positive Control Values in the Absence o f S9 Mix
The mean value o f the positive control for each tester strain exhibited at least a 3-fold increase over the mean value o f the vehicle control for that strain (with the exception o f 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 o f S9 Mix (S9 Mix Integrity)
The mean value o f the positive control for each tester strain exhibited at least a 3 -fold increase over the mean value o f the vehicle control for that strain, demonstrating that the S9 mix was capable o f metabolizing a promutagen to its mutagenic form(s).
An acceptable positive control in the presence o f S9 for a specific strain was evaluated as having demonstrated the integrity o f the S9 mix and the ability o f the tester strain to detect a mutagen.
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Covance Study 22900-TJ-4090ECD
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 o f 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 o f these tester strains over the mean revertants per plate o f 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 o f these tester strains over the mean revertants per plate o f 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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Covance Study 22900-0-4090ECD 5. RECORDS TO BE MAINTAINED
22
A ll raw data, documentation, records, the protocol, and the final report generated as a result o f this study will be archived in the storage facilities o f 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 o f time or sent to a storage facility designated by the Sponsor.
6. STUDY RESPONSIBILITIES
Function Study Director Laboratory Supervisor
7. RESULTS
7.1 Test Article Handling
formed a solution at 100 mg/mL in dimethylformamide aft er heating. In DMSO at 100 mg/mL, the test article formed a solution that foamed when h e ate d and vortexed. DMSO was selected as the vehicle. At 100 mg/mL, which was the m-ost concentrated stock prepared for the mutagenicity assay, the test article formed a transparent, colorless solution after heating to 45 C for 3 minutes; it remained freely soluble^ at all succeeding lower dilutions.
CSostpsny <
Covance Study 2290fUt-0-4090ECD 7.2 Dose Rangeflnding Study (Appendix A, Tables 4 and 5)
23
A dose rangeflnding 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 o f test article, from 6.67 to 5000 pg/plate, were evaluated in the presence and absence o f 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 pg/plate with and without S9.
i
7.3 Mutagenicity Assay (Appendix A, Tables 6, 8 - Individual Data, Tables 7, 9- Summary Data)
Based upon the results o f the dose rangefinding s t u d y J P ^ ^ | | H H | | ^ w a s evaluated in the initial mutagenicity assay in all five tester strains at doses o f 33.3, 100, 333, 1000, 3330 and 5000 pg/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 0 ^ H | | B H ^ ^ valuated 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 ^ p P B | j j j j j j | B H ^ n 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 pg/plate with and without S9. Revertant frequencies for all doses o ^ g B B 9 8 i a!l tester
Compan* S!in"
TSCACBt
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 co/i/Mammalian- Microsome Reverse Mutation
Assay indicate that under the conditions o f this stud
as not
mutagenic in any o f the tester strains in the presence or absence o f an exogenous metabolic
activation system containing induced hepatic microsomal enzymes from
AroclorTM 1254-treated rats.
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.
jSsisuTSCS--GBt
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 5'<3/moe//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. M utation Research 1983; 113:173-215.
Vogel and Bonner, 1956. Vogel HJ, Bonner DM. Acetylomithinase o f E. coli: Partial purification and so m e properties. J B io l Chem 1956; 218:97-106.
B ovance Study 2290-O-4O9OECD Appendix A. Experimental Data Tables
26
i.Sr' 'rA-
G'
Covance Study 229D-0-4090ECD
Table 4
Dose Rangefinding Study - TA100
Test Article ED:
Experiment ID:
Date Plated:
:9-Oct-01
Date Counted:
Vehicle:____________DMSO________________________Plating Aliquot:
22900-A1 01-Nov-01 50 pL
TA100 Revenants Per Plate
(ig/Plate
0.00 (Vehicle) (50 pL)
Test Article 6.67 10.0 33.3 66.7
With S9
Revenants Per Plate
Background Lawn
Evaluation3
113 N
107 N 113 N 136 N 97 N
Without S9
Revenants Per
Plate
Background Lawn
Evaluation3
82 N
86 N 107 N 101 N 94 N
100 333 667 1000 3330
139 N 97 N 93 N 106 N 108 N
87 N 67 N 98 N 89 N 107 N
5000
105 NP
103 RP
" Background Lawn Evaluation Codes: N = normal R = reduced A = absent P = precipitate O = obscured by precipitate
27
& C}
-CV riP
ovance Study 229( 1-0-409OECD
Table 5
Dose Rangefmding Study - WP2wvrA
Test Article ID:
Experiment ID:
Date Plated:
29-Oct-Ol
Date Counted:
Vehicle:____________DMSO________________________ Plating Aliquot:
22900-A1 01-Nov-01 50 pL
WP2wA Revertants Per Plate
pg/Plate
0.00 (Vehicle) (50 pL)
Test Article 6.67 10.0 33.3 66.7
100 333 667 1000 3330 5000
With S9
Revotants Per Plate
Background Lawn
Evaluation3
23 N
21 N 15 N 13 N 17 N 12 N 14 N 18 N 23 N 12 N 17 NP
Without S9
Revertants Per Plate
Background Lawn
Evaluation3
12 N
12 N 11 N 15 N 11 N 18 N 12 N 11 N 12 N 14 N 13 NP
*Background Lawn Evaluation Codes: N = normal R = reduced A = absent P = precipitate O = obscured by precipitate
28
A* &
s
{P
& 4?
Covance Study 229' I-0-4090ECD
Table 6
Initial Mutagenicity Assay Results - Individual Plate Counts
Test Article ID: Date Plated: Vehicle: ____
Experiment ID: Date Counted: DMSO______ ________________ Plating Aliquot:
22900-B1 26-Nov-01 50 pL________
29
Dose/Plate
Microsomes: Rat Liver Vehicle Control
Revertants Per Plate
TA98_________ TA10Q________ TA1535_______ TA1S37_______WP2 uvrA
12 3
1 2 3 12 3
12 3 1z 3
Back ground
Lawn"
33 30 27 115 125 112 14 8 18
7 7 10 27 14- 5 N
Test Article 33.3 Mg 32 24 30 100 Mg 25 20 28
333 Mg 34 35 18 1000 Mg 34 33 27 3330 Mg 23 45 28 5000 Mg 28 45 30
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
5 10 14 10 9 5 10 10 9 6 12 9 10 14 9 11 9 12
10 16 19 19 11 IO 8 26 23 19 20 20
18 19 21 15 19 21
N N N NP NP NP
Positive Controlb
574 512 542 984 1048 1090 141 165 138 181 148 178 233 241 293 N
Microsomes: None Vehicle Control
12 25 9 116 86 90 10 12 17
6 5 3 16 14
Test Article 33.3 Mg 18 14 11
100 Mg 12 17 15 333 Mg 21 18 8 1000 Mg 20 17 16
3330 Mg 23 11 29 5000 Mg 18 10 19
87 89 70 17 14 6 82 86 70 9 7 17 88 97 86 14 11 11 97 94 90 11 7 10 79 112 84 17 16 10 94- 93 97 11 15 16
3 5 9 17 12 7 9 2 15 17 10 8 2 10 7 8 6 10 11 9 3 7 12 10 21 6 9 3 18 26
Positive Control'
208 192 216 1021 1028 948 664 656 579 1064 867 898 158 128
17
18 17 12 12 20 19
89
N
N N N NP NP NP
N
*Background Lawn Evaluation Codes: N = normal R= reduced 0 = obscured
b TA98 TA100 TA1535 TA1537 WP2vrA
benzo[a]pyrene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene
2.5 pg/plate 2.5 pg/plate 2.5 pg/plate 2.5 pg/plate 25.0 pg/plate
A= absent P = precipitate
c TA98 TA100 TA1535 TA1537 WP2vrA
2-nitrofluorene sodium azide sodium azide ICR-191 4-nitroquinolone-N-oxide
1.0 pg/plate 2.0 pg/plate 2.0 pg/plate 2.0 pg/plate 1.0 pg/plate
A<9 P? &
Cp'
^^1Covance Study 22901 0 409OECD
Table 7
Initial Mutagenicity Assay Results - Summary
Test Article ID: Date Plated: Vehicle: ____
13-Nov-Ol DMSO
Experiment ID: Date Counted: Plating Aliquot:
22900-B1 26-Nov-01 50 M-L
Dose/Plate
Microsomes: Rat Liver Vehicle Control
Mean Revenants Per Plate with Standard Deviation
TA98________ TA100_______TA1535_______ TA1537 Mean S.D. Mean S.D. Mean S.D. Mean S.D.
WP2uwA Mean S.D.
Back ground Lawn3
30 3 117 7
13 5
8L
15 11 N
Test Article
33.3 Mg 100 Mg 333 Mg 1000 Mg 3330 Mg 5000 Mg
29 4 24 4 29 10 31 4 32 12 34 9
102 11 109 8 103 14 117 2 106 8 116 5
16 1 10 1 12 3 15 5 16 3 13 4
10 5 83 10 1 93 11 3 11 2
15 4 N 19 0 N 14 6 N 16 9 NP 20 2 NP 20 1 NP
Positive Controlb
543 31 1041 53
148 15
169 18
256 33 N
Microsomes: None Vehicle Control
15 9
Test Article
33.3 Mg
14 4
100 Mg 15 3
333 Mg
16 7
1000 Mg 18 2
3330 Mg 21 9
5000 Mg 16 5
Positive Control3
205 12
97 16
82 10 79 8 90 6 94 4 92 18 95 2
999 44
13 4
12 6 11 5 12 2 92 14 4 14 3
633 47
52
63 64 74 82 75 63
943 106
16 2 N
16 3 N 16 1 N 10 3 N 11 2 NP 17 6 NP 21 4 NP
125 35 N
*Background Lawn Evaluation Codes: N = normal R = reduced 0 = obscured
b TA98 TA 100 TA1535 TA1537 WP2uvrA
benzo[a]pyrene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene
2.5 pg/plate 2.5 pg/plate 2.5 pg/plate 2.5 pg/plate 25.0 pg/plate
A = absent P = precipitate
c TA98 TA 100 TA1535 TAI 537 WP2vrA
2-nitrofIuorene sodium azide sodium azide
ICR-191 4-nitroquinolone-N-oxide
1.0 pg/plate 2.0 pg/plate 2.0 pg/plate 2.0 pg/plate 1.0 pg/plate
Table 8
Confirmatory Mutagenicity Assay Results - Individual Plate Counts
Test Article ID: Date Plated: Vehicle:
Experiment ID: Date Counted: DMSO________________________ Plating Aliquot:
22900-B1 07-Dec-01, 10-Dec-01 50 pL________________
Revenants Per Plate
Dose/Plate
Microsomes: Rat Liver Vehicle Control
TA98 123
29 27 26
TA100 12 3
97 98 122
TA1535 123
16 17 16
TA1537 12 3
11 8 11
Back ground WP2uvrA Lawn* 1 23
15 23 15 N
Test Article 33.3 100 333 1000 3330 5000
Mg 24 34 26 Mg 39 34 38 Mg 19 40 25 Mg 18 26 28 Mg 40 32 42
Mg 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 9 789 12 12 7 11 11 16
18 8 20 N 15 19 18 N 17 9 24 N 17 12 20 NP 26 25 29 NP 23 19 21 NP
Positive Control11
398 321 180 1131 1228 1189 138 192 125 156 180 141 468 389 461 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
Mg 20 21 26 Mg 24 14 18 Mg 14 24 18 Mg 18 13 18 Mg 21 18 20 Mg 23 30 24
110 116 85 72 97 89 96 93 95 105 91 103 92 81 84 90 84 111
7 8 10 10 12 10 9 16 8 8 13 14 10 10 8 13 3 13
678 872 375 643 423 6 84
12 12 21 N 16 17 21 N 11 29 20 N 17 17 16 NP 20 14 17 NP 16 18 20 NP
Positive Control*
287 263 220 1100 1089 1216 821 881 832 0 0 0 298 321 328 N
` Background Lawn Evaluation Codes: N = normal R = reduced 0 = obscured
bTA98 TA100 TA1S35 TA 1537 WP2uvrA
benzo[a]pyrene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene
2.5 pg/plate 2.5 pg/plate 2.5 (ig/plate 2.5 pg/plate 25.0 pg/plate
A = absent P = precipitate
c TA98 TA100 TA1535 TA1537 WP2uvrA
2-nitrofluorene sodium azide sodium azide ICR-191 4-nitroquinoIone-N-oxide
1.0 pg/plate 2.0 (ig/plate 2.0 (ig/plate 2.0 (ig/plate 1.0 |ig/plate
Covance Study 22900-U-4090ECD
32
Table 9
Confirmatory Mutagenicity Assay Results - Summary
Test Article ID: Date Plated: Vehicle:
29-Nov-Ol DMSO
Experiment ID: Date Counted: Plating Aliquot:
22900-B1 07-Dec-Ol, 10-Dec-01 50 uL_______________
Dose/Plate
Microsomes: Rat Liver Vehicle Control
Mean Revertants Per Plate with Standard Deviation
TA98 Mean S.D.
27 2
TA100 Mean S.D.
106 14
TA1535 Mean S.D.
16 1
TA1537 Mean S.D.
10 2
WP2vrA Mean S.D.
Back ground Lawn'
18 5 N
Test Article 33.3 pg 100 Pg
28 37
5 115 17
3 121
6
14 15
6 1
10 10
4 5
15 17
6N 2N
333 f l g
28 11 111
1
14 3
10
1
17
8N
1000 pg 24 5 106 7
95
8 1 16 4 NP
3330 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 Control11
300 111 1183 49 152 36 159 20 439 44 N
Microsomes: None Vehicle Control
18 6 92 6
62
Test Article 33.3 Pg 22 3 104 16
82
100 Pg 19 5 86 13 11 l
333 Pg
19 5 95 2
11 4
1000 Pg 16 3 100 8 12 3
3330 Pg 20 2 86 6
91
5000 Pg 26 4 95 14 10 6
Positive Control'
257 34 1135 70 845 32
6 4 19 3 N
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
1 Background Lawn Evaluation Codes: N = normal R = reduced 0 = obscured
bTA98 TA 100 TA 1535 TA1537 WP2kwA
benzo[a]pyrene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene
2.5 pg/plate 2.5 pg/plate 2.5 pg/plate 2.5 pg/plate 25.0 pg/plate
A = absent P = precipitate
c TA98 TA100 TA1535 TA1537 WP2uvrA
2-nitrofluorene sodium azide sodium azide ICR-191 4-nitroquinolone-N-oxide
1.0 pg/plate 2.0 pg/plate 2.0 pg/plate 2.0 pg/plate 1.0 pg/plate
0 & 3^
vOl-
G$V
iCovance Study 2290-0-4090ECD
Table 10
Historical Control Data Plate IncorporationJMethod - Report Period 01E
33
Strain
Mean Revertants per Plate Standard Deviation Maximum Minimum Count
Vehicle Controls with S9 M ix
TA98
TA 100
TA1535
28.2 105.7 7.2 14.3 61 143 14 61 290 294
13.3 4.4 30
5 209
TA1537 WP2wvrA
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
WP2vrA 19.0 5.8 38 5 194
Strain Mean Revertants per Plate Standard Deviation Maximum Minimum Count
Positive Controls with S9 Mix***
TA98
TA 100
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
WP2vrA 568.4 173.3 1055 192 198
Strain Mean Revertants per Plate Standard Deviation Maximum Minimum Count
Positive Controls without S9 Mix**
TA98
TA 100
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
** 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 pg/plate 2.5 pg/p!ate 25.0 pg/plate
*** 7 A9g
TA100 TA 1535 TA1537 WP2uvtA
2-nitrofluorene
sodium azide sodium azide ICR-191 4-nitroquinoline-N-oxide
WP2wvrA
227.0 77.3 481
10
190
1.0 pg/plate 2.0 pg/plate 2.0 pg/plate 2.0 pg/plate 1.0 pg/plate
.-R6SS
Bits' * 6 0
Covance Study 229uU-0-4090ECD Appendix B. Definitions of Bacterial Background Lawn Evaluation Codes
34
Com Psn^ S s
Does not contai YSCA CB!
Covance Study 2290TP0-409OECD
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 o f the microcolony lawn and an increase in the size o f the microcolonies compared to the vehicle control plate.
A Absent
A complete lack o f 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 o f the microcolony lawn and an increase in the size o f the microcolonies compared to the vehicle control plate.
Evidence of macroscopic test article precipitate on the plates is recorded by addition o f 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.
Covance Study 22900-0-4090ECD Appendix C. Quality Assurance and Compliance Statements
36
XSOA. CBV
not con tai
I-409OECD
37
QUALITY ASSURANCE STATEMENT
Salmonella-Escherichia Co/z'/Mammalian-Microsome Reverse lutation Assay with a Confirmatory Assay
The report has been reviewed by the Quality Assurance Unit o f Covance Laboratories Inc., in accordance with the Good Laboratory Practice regulations as set forth in the Environmental Protection Agency (EPA - TSCA), Title 40 o f 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
Auditor
Director Management___________
24-Oct-2001
P. Cceres
14-NOV-2001
J. Howard
07- Jan-2002 C. Smith
08- Feb-2002 C. Smith
Representative, Quality Assurance Unit
// Date
2^
STUDY COMPLIANCE AND CERTIFICATION
Except that the test and control article dosing solutions were not analyzed for stability, homogeneity or accuracy o f 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 o f the U.S. Code o f Federal Regulations Part 792; and the Organization for Economic Cooperation and Development (OECD) Principles o f 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 o f the study or the interpretation of the test results. The raw data have been reviewed by the Study Director, who certifies that the evaluation o f the test article as presented herein represents an appropriate conclusion within the context o f the study design and evaluation criteria.
Bacterial Mutagenesis Genetic and Molecular Toxicology Covanee-Vienna
Testing Facility Management:
Study Completion Date
Bacterial Mutagenesis Genetic and Molecular Toxicology
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
