Document Z82jLmdr9X7L08VOJkGQbw9Rp
AR226-3173
TRADESECRET
Study Title
H-24616: Bacterial Reverse M utation Test
Authors
Valentine O. Wagner, m , M.S. Michelle L. Klug, B.S.
Report Completion Date
25 January 2001
Performing Laboratory
BioReliance 9630 Medical Center Drive
Rockville, MD 20850 for
E. I du Pont de Nemours and Company Stine Haskell Research Center DuPont H askell Laboratory P.O. Box 50,1090 Elkton Road Newark, DE 19714-0050
Performing Laboratory Study Number AA37AZ.5011Q0.BTL
DuPont Project ID DuPont-5234
Work Request Number
[ B rr
r H il
DuPont-5234
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H -24616: B acterial R everse M utation Test
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C E R T IFIC A T IO N
We, the undersigned, declare dial this report provides an accurate evaluation o f data obtained from this study.
issued by Study Director:
Valentine O. W arier ID, M.S.
2 5 <3 e n 2 ^ 0 / Date
Approved by Study Monitor:
r t j M ---
Maria Donner, Fh.D. Senior Research Scientist
2% lo o i Date
B ioR eliance Study N o. AA37AZ.501100.BTL
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H-24616: Bacterial Reverse M utation T est
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TABLE OF CONTENTS
Page
C ertificatio n.............
Study Information...... Summary
i
Puroose
Characterization o f Test and Control Substances..........
M aterials and Methods
Results and Discussion.......*.....*....*.**9***#**i Conclusion.............
References
Data Tables
Table 1: M utagenicity Test in Salm onella typhim urium TA98 without S9.... Table 2: M utagenicity Test in Salm onella typhim urium TA98 with S9.... . Table 3: Mutagenicity Test in Salm onella typhim urium TA100 without S 9 l Table 4: M utagenicity Test in Salm onella typhim urium TA100 with S9.....1 Table 5: M utagenicity Test in Salm onella typhim urium TA1535 without S9, Table 6: M utagenicity Test in Salm onella typhim urium TA1535 w ith S9__ | Table 7: M utagenicity Test in Salm onella typhim urium TA1537 without S9' Table 8: Mutagenicity Test in Salm onella typhim urium TA1537 with S9.... '
....1Table 9: Mutagenicity Test in Escherichia coli WP2 uvrA without S9
Table 10: M utagenicity Test in Escherichia coli W P2 uvrA with S 9 ............... Table 11: Salm onella/E. coli Mutagenicity Test - Summary of Resuits..........
Appendix A: H istorical Control Data. Appendix B: Study Protocol
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H -24616: B acterial Reverse M utation T est
STUDY INFORMATION
DuPont-5234
Haskell N um ber 24616
P u n & tM H H l
Stability; The test substance appeared to be stable under the condition o f the study; do evidence o f instability w as observed.
Solubility: Aquatics: Dispersible in water All Others: Dimethylsulfoxide
Sponsor. E. I. du Pont de Nemours and Company W ilmington, Delaware 19898 U.S.A. November, 16,2000 / (see report cover page) November 28,2000 / December 06,2000
B io R elian ce Study N o. AA37A2LS01100.BTL
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H-24616: B acterial Reverse M utation T est
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SUMMARY
The lest substance,
was tested in the bacterial reverse mutation
test using S. typhimurium tester strains TA98, TA100, TA1535 and TA1537 and E coli tester
strain WP2 uvrA in the presence and absence of Aroclor-induced rat live- S9. The test was
performed using die plate incorporation method to evaluate the mutagenic potential o f the test
substance.
Water was selected as the solvent o f choice based on solubility o f the test ihsumr compatibility with the target cells, and stability of the test article in the solvent (per Sponsor supplied information). The test substance was soluble and but cloudy in water at approximately 10 mg/mL. Tire test substance was a workable suspension at 25 to SOm g/m i.
In the mutagenicity assay, the maximum dose tested was 5000 pg per plate; this dose was achieved using a concentration o f 50 mg/mL and a 100 pL plating aliquot. The test article diluted to a clear solution at 1.0 and 3.3 mg/mL. The dose levels tested were 100,333, 1000, 3333 and 5000 pg per plate. No positive responses were observed with any o f the tester strains in the presence and absence of rat S9 activation.
All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Test indicate that, under the conditions o f this study, H-24616 did not induce a positive responses with any of the tester strains in the presence and absence of Aroclor-induced rat liver S9.
B io R elian ce Study N o. AA37AZ.501100.BTL
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H-24616: B acterial Reverse M utation T est
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PURPOSE
The purpose o f this study was to evaluate the mutagenic potential of the test substance by measuring its ability to induce reverse mutations at selected loci o f several strains o f Salmonella typhimurium, and at die tryptophan locus of Escherichia coii WP2 uvrA in the presence and absence of S9 metabolic activation.
CHARACTERIZATION O F TEST AND CONTROL SUBSTANCES
The test substance, H-24616, was received by BioReliance on 06 November 2000 and was assignedths code number AA37AZ. The Sponsor characterized the test substance as a n | H f B ^ p t h a t s h o u l d be stored at ambient temperatore^A nexgration date was not provided Upon receipt, the test substance was described as a J H p p |||^ H H H |a n d stored at room temperature, protected from exposure to light.
The vehicle used to deliver H-24616 to the test system was water, (CAS# 7732-18-5), obtained from lif e Technologies.
Positive controls plated concurrently with the mutagenicity test are listed below:
Strain All Salmonella Strains
W P2vrA TA98
TA100.TA1535 T A 537
W P2kwA
S9 Metabolic Activation Rat
None
Positive Control
2-aminoanthracene (Sigma Chemical Co.)
2-nitrofluorene (Aldrich Chemical Co., Inc.)
sodium azide (Sigma Chemical Co.)
9-aminoacridine (Sigma Chemical Co.)
methyl methanesulfonate (Aldrich Chemical Co., Inc.)
Concentration (ftg/plate)
IQ 10 1.0
1.0
75
1,000
To determine the sterility o f tire test substance, the highest test substance dose level used in
the mutagenicity test was plated on selective agar with an aliquot volume equal to that used in
the te st
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H-24616: B acterial Reverse M utation Test
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T est System
M ATERIALS AND M ETHODS
The test- strains used were the Salmonella typhimurium histidine auxoteophs TA98,
TA100, TA1535 and TA1537 as described by Ames e t al. (1975), and Escherichia coli tester strain WP2 nvrA. Salmonella tester strains were received on 11 August 1998 and 10 November 1998 directly from Dr. Bruce Ames, University of California, Berkeley. E. coli was received on
01 July 1987 from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity o f die reversion mechanism in coli is sensitive to base-pair substitution mutations, rather than fawngghift mutations (Green and Muriel, 1976).
Overnight cultures were prepared by inoculating from tire appropriate master plate or from the appropriate frozen permanent stock into a vessel containing ~50 mL o f culture medium. To assure that cultures were harvested in late log phase, the length o f incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shakp-r/inraih^tm-at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 372C approximately 12 hours before the anticipated time of harvest Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent ransmittawi. yielding a titer o f greater than or equal to 0.3xl09 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates, and the data is on file but not presented in this report The study was conducted to comply with OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), adopted July 1997.
M etabolic A ctivation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 was purchased from MolTox, batch prepared on 18 October 2000 and stored at -7GC or colder until used. Each bulk preparation o f S9 was assayed for its ability to metabolize 2-aminoanthracene and 7,12dimethylbenz(a)anthracene to forms mutagenic to Salm onella typhimurium TA100.
The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM P-nicotinamide-adenine dinucleotide phosphate, 8 mM MgGh
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H-24616: Bacterial Reverse M utation T est
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and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix),
containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility o f the S9 and Sham mixes, a 0.5 mL aliquot o f each was plated on selective agar.
Solubility T est
A solubility test was conducted to select the vehicle using one or more of the following: water and dimethyl sulfoxide (DMSO). The test substance was tested to determine the vehicle, selected in order o f preference, that permitted preparation o f the highest soluble or workable stock concentration, up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents.
M utagenicity T est
The mutagenicity test was used to evaluate the mutagenic potential of the test substance. Five dose levels o f test substance (100, 333, 1000, 3333 and 5000 fig per plate) along with appropriate vehicle and positive controls were plated with tester strains TA98, TA100, TA1535, TA1537 and W P2 uvrA in the presence and absence o f rat liver S9 activation. All dose levels of test substance, vehicle controls and positive controls were plated in triplicate.
Plating and Scoring Procedures
In the mutagenicity test, the test system was exposed to the test substance via the plate incorporation methodology as described by Ames et al. (1975) and updated by Maron and Antes (1983).
On the day o f its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration o f 50 pM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL o f minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water produced by the Milli-Q Reagent W ater System. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).
Each plate was labeled with a code system that identified the test substance, test phase, dose level, tester strain, and activation, as described in detail in BioReliance's Standard Operating Procedures.
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Test substance dilutions were prepared immediately before use. O ne-half (0.5) m illiliter o f S9 or sham mix, 100 pL o f tester strain and 100 fiL o f vehicle or test substance w oe added to 2.0 mL o f molten selective top agar at 452C. After vortexing, die mixture was overlaid onto the surface o f 25 mL o f minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 pL aliquot o f appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 372C. Plates that were not counted immediately following the incubation period were stored at 2-8C until colony counting could be conducted (less than 10 days).
The condition o f the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree o f precipitation were scored relative to the vehicle control plates using the codes shown below.
Code 1 2
3
4 5 6 NP
IP
Description
Characteristics
Normal
Distinguished by a healthy microcolony lawn.
Distinguished by a noticeable thinning of the microcolony lawn and possibly a Slightly Reduced slight increase in the size o f the microcolonies compared to the vehicle control
plate.
Moderately Reduced
Distinguished by a marked thinning o f the microcolony lawn resulting in a pronounced increase in the size of die microcolonies compared to the vehicle control plate.
Distinguished by an extreme thinning of the microcolony lawn resulting in an Severely Reduced increase in the size of the microcolonies compared to the vehicle control plate
such that the microcolony lawn is visible to the unaided eye as isolated colonies.
Absent
Distinguished by a complete lack of any microcolony lawn over >90% o f the plate.
Obscured by Precipitate
Non-Interfering Precipitate
The background bacterial lawn cannot be accurately evaluated due to microscopic test substance precipitate.
Distinguished by precipitate on the plate that is visible to the naked eye but any precipitate particles detected by the automated colony counter total less than 10% of the revertant colony count (e.g., <3 particles on a plate with 30 revertants.)
Interfering Precipitate
Distinguished by precipitate on the plate that is visible to the naked eye and any precipitate particles detected by the automated colony counter exceed 10% o f the revertant colony count (e.g., >3 particles on a plate with 30 revertants.)
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Plates with sufficient test substance precipitate to interfere with automated colony counting were counted manually.
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Evaluation o f Results
For each triplicate plating, the mean and standard deviation of the number o f revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate o f at least one tester strain with a minimum o f two increasing concentrations o f test substance. Data sets for strains TA1535 and TA1537 w o e judged positive if the inmease in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA98, TA100 and WP2 uvrA were judged positive if die increase in mean revertants at the peak o f the dose response is equal to or greater than two times the mean vehicle control value.
C riteria fo r a V alid Test
The following criteria must be met for the mutagenicity test to be considered valid. All Salmonella tester strain cultures must demonstrate the presence o f die deep rough mutation (rfd) and the deletion in the w rB gene. Cultures o f tester strains TA98 and TA10Q must demonstrate the presence o f die pKMIOl plasmid R-factor. All WP2 wvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive}: TA98 (10 - 50); TA1Q0 (80 - 240); TA1535 (5 - 45); TA1537 (3 - 21); and W P2vrA (1 0 -6 0 ). To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to Q.3xl09 cells/mL. The mean o f each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value o f tire respective vehicle control. A minimum o f three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number o f revertants per plate as compared to the mean vehicle control value. This reduction m ust be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the background lawn.
A rchives
Upon completion of the final report, all raw data and reports will be archived by BioReliance, Rockville, MD for a period of no less than 1 year from the study initiation date.
D eviations
No known deviations from tire protocol or assay-method SOPs occurred during the conduct o f this study.
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H -24616: B acterial R everse M utation T est
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Solubility Test
RESULTS AND DISCUSSION
W ater was selected as the solvent o f choice based on solubility o f die test substance, compatibility w ith the target cells and stability o f the test article in the solvent (per Sponsor supplied information). The test substance was soluble and but cloudy in water at approximately 10 mg/mL. The test substance was a workable suspension at 25 to 50 mg/mL.
M utagenicity T est
The results o f the mutagenicity test are presented in Tables 1 through 10 and summarized in Table 11. These data were generated in Experiment B l. In the mutagenicity assay, the maximum dose tested was 5000 pg per plate; this dose was achieved using %concentration of 50 mg/mL and a 100 pL plating aliquot. The test article diluted to a clear solution at 1.0 and 3.3 mg/mL. The dose levels tested were 100, 333, 1000, 3333 and 5000 pg per plate. Neither precipitate nor toxicity was observed.
hi Experiment B l, no positive responses were observed with any o f the tester strains in die presence and absence of rat S9 activation.
CONCLUSION
All criteria for a valid study were met as described in the protocol. The results o f the Bacterial Reverse Mutation Test indicate that, under the conditions of this study, H-24616 did not induce a positive response with any of the tester strains in the presence and absence of Aioclor-induced rat liver S9.
B io R elian ce Study N o. AA37AZ.501100.BTL
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H-24616: B acterial Reverse M utation T est
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REFERENCES
Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and Mutagens with the Salm onella/M am msiim Microsome Mutagenicity Test, Mutation Research, 31:347-364.
Green, M.H.L. and W J. Muriel (1976) Mutagen testing using trp+reversion in Escherichia coti, Mutation Research 38:3-32.
Maron, D.M. and B.N. Ames (1983) Revised Methods for tire Salm onella Mutagenicity Test, Mutation Research, 113:173-215.
OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), adopted July 1997.
Vogel, H.J. and D.M. Bonner (1956) Acetylomithinase o f E. coir. Partial Purification and Some Properties, J. Biol. Chem., 218:97-106.
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H -24616: B acterial Reverse M utation T est
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Bacterial Nutation Assay
_ Table 1
Test Article Id
Study Humber Strain
^ ^ S B B S R o O.BTL : TA98
Liver Microsomes ; None
Vehicle
: water
Plating Aliquot : 100 uL
Experiment No : B1 Cells Seeded : 0.6 X 10 Date Plated : 28 Nov 2000
Concentration Plate Revertants Background Average Standard
ug per plate Number per plate
Code
Revertants Deviation
Vehicle
01 02 03
10 1 21 1 iff 1
6 6
100 01 12 1 02 14 1 03 13 1
13 1
333 01 21 1 02 12 1 03 15 1
16 5
1000
01 14 1 02 10 1 03 22 1
15 6
3333
01 8 1 02 12 1 03 14 1
11 3
5000
01 11 1 02 13 1
03 10 1
11 2
Positive Control 2-nitrofluorene 1,.0 ug per plate
01 99 1 02 94 1 03 95 1
96
Background Lawn Code
l=Ncrmal; 2=Slightly reduced; 3=Severely reduced
4=Extremely reduced; 5=Absent; 6=0bscured by precipitate
NP=Non-Interfering precipitate; IP=Interfering precipitate
3
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H-24616: Bacterial Reverse Mutation Test
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Bacterial Mutation Assay
Table 2
Test Article Id Study Number
0
Strain
: T98
O.BTL
Liver Microsomes : Rat liver S9
Vehicle
; water
Plating Aliquot ; 100 uL______
Experiment No
Cells Seeded : S S X 108 Date Plated : 28 Nov. 2000
Concentration US per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
23 15 20
Background Code
1 1 1
Average Revertants
1-9
Standard Deviation
4
100 01 27 1 02 15 1 03 14 1
19 7
333 01 14 1 02 11 1 03 16 1
14 3
1000
01 18 1 02 21 1 03 19 1
19 2
3333
01 16 1 02 21 1 03 17 1
18 3
5000
01 16 1 02 24 1 03 18 1
19 4
Positive Control 2-aminoanthracene 1 .0 jig per plate
01 451 1
02 486 1
03 546 1
494
Background Lawn Code
---- ----
48
^Normai;^ 2=Slightly reduced; 3=Severely reduced
S0"000 5=Absent; 6=Obscured by precipitate NP-Non-Interfering precipitate; IP=Interfering precipitate
B io R eiian ce Study N o. AA 37AZ^01100.BTL
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H-24616: B acterial Reverse M utation Test
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Bacterial Mutation Assay
Table 3
L m m *Test Article Id
Study Number
: AA37AZ.501100.BTL
Strain
: TA100
Liver Hicrosomes : None
Vehicle
: water
Plating Aliquot : 100 pL
Experiment No : B1 Cells Seeded : 0.9 X 108 Date Plated : 28 Nov 2000
Concentration ug per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
88 83 84
Background Code
1 1 1
Average Standard Revertantsi Deviation
85 3
100 01 84 1 02 77 1 03 85 1
82 4
333 01 85 1 02 92 1 03 83 1
87 5
1000
01 79 1 02 71 1 03 86 1
79 8
3333
01 81 1 02 92 1 03 81 1
85 6
5000
01 116 1 02 90 1 03 100 1
102 13
Positive Control sodium azide 1.0 ug per plate
01 314 1 02 340 1
03 355 1
336
21
Background Lawn Code
l=Normal; 2=Slightly reduced; 3=Severely reduced
4=Extremely reduced; 5=Absent; 6=0bscured by precipitate
NP=Non-Interering precipitate; IP=Interfering precipitate
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Bacterial Mutation Assay
Table 4
Test Article Id
Study Number Strain
: TA100
LOO.BTL
Liver Microsomes Rat liver S9
Vehicle
: water
Plating Aliquot 100 uL
Experiment No : B1 Cells Seeded : 0.9 X 108 Date Plated : 28 Nov 2000
Concentration ug per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
98 82 89
Background Code
1' 1 1
Average Revertants
90
Standard Deviation
8
100 01 78 1 02 91 1 03 79 1
83 7
333 01 83 1 02 78 1 03 83 1
81 3
1000
01 89 1 02 79 1 03 81 1
83 5
3333
01 98 1 02 110 1 03 85 1
98 13
5000
01 85 1 02 95 1 03 91 1
90 5
Positive Control 2-aminoanthracene 1.0 ug per plate
01 621 1
02 668 1
03 520 1
603
76
l=Normal; 2=Slightly reduced; 3=Severely reduced
*e?ce<i> 5=Absent; 6=Obscured by precipitate NP-Non-Interfering precipitate; IP=Interfering precipitate
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Bacterial Mutation Assay
. Table 5
Test Article Id
Study Number
: AA^AZ.bOllO0.BTL
Strain
: TA1535
Liver Microsomes : None
Vehicle
: water
Plating Aliquot : 100 uL
Experiment No : B1 Cells Seeded : 2.7 X 10B Date Plated : 28 Nov 2000
Concentration Plate Revertants Background Average Standard
ug per plate Number per plate
Code
Revertants Deviation
Vehicle
01 02 03
14 1 18 1 19 1
17 3
100 01 16 1 02 21 1 03 14 1
17 4
333 01 21 1 02 24 1
03 13 1
19 6
1000
01 20 1
02 13 1 03 15 1
16 4
3333 5000
01 18 1 02 9 1 03 20 1
16
01 15 1
02 13 1
03
16 1
. 15
6 2
Positive Control sodium azide 1.0 ug per plate 01 188 1 02 187 1 03 172 1
182
Background Lawn Code l=Normal; 2=Slightly reduced; 3=Severely reduced 4=Extremely reduced; 5=Absent; 6=0bacured by precipitate
NP=Non-Interfering precipitate; IP=Interfering precipitate
9
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Bacterial Mutation Assay
Table 6
Test Article Id r g m m m '
Study Number
AA37ACTI 50.BTL
Strain
: TA1535
Liver Microsoznes Rat liver S9
Vehicle
water
Plating Aliquot 100 PL
Experiment No : B1
Cells Seeded : 2.7 X 10s Date Plated : 28 Nov 2000
Concentration pg per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
14 17 C
Background Code
1 1
Average Revertants
16
Standard Deviation
100 01 7 1 02 13 1 03 16 1
12
333 01 13 1 02 15 1 03 9 1
12
1000
01 17 1 02 21 1
03 16 1
18
3333
01 5 1 02 13 1
03 13 1
10
5000
01 17 1 02 18 1 03 21 1
19
tive Control 2-aminoanthraeene 1.0 jig 01 120 1
02 90 1
------ -------0-3---------1-0-5-- -----1------------ 105_________ 15
Background Lawn Code
~~ ~
l=Normal; 2=Slightly reduced; 3=Severely reduced 4Esrtrenjely reduced; 5=Absent; 6=Obscured by precipitate
C~ContaSnatedrin^ precxp^tate; IP=Interfering precipitate
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Bacterial Mutation Assay
Table 7
mTest Article Id
Stucty Number
; AA3'7AZ.501100.BTL
Strain
; TA1537
Liver Microsomes : None
Vehicle
: water
Plating Aliquot : 100 UL
Experiment No : B1 Cells Seeded : 1.6 X 10 Date Plated ; 28 Nov 2000
Concentration ug per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
7 4 8
Background Code
1 1 1
Average Revertants
6
Standard Deviation
2
100 01 10 1 02 9 1 03 6 1
82
333 01 8 1 02 8 1 03 10 1
91
1000
01 6 1
02 9 1
03 5 1
72
3333 01 3 1 02 6 1 03 7 1
52
5000 01 7 1 02 6 1 03 8 1
71
Positive Control 9-aminoacridine 75 ug per plate 01 689 i 02 708 i 03 699 i
699
10
Background Lawn Code l=Normal; 2=Slightly reduced; 3=Severely reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; 1P=Interfering precipitate
B io R elian ce Study No. A A 37A ZJ01100.BTL
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H-24616: Bacterial Reverse Mutation Test
D uPont-5234
Bacterial Mutation Assay
____
Table
Test Article Id s s s s s 3 ' ^
Study Humber
Strain
: TA1537
Liver Microsomes : Rat liver S9
Vehicle
; water
Plating Aliquot ; 100 uL
Experiment No Cells Seeded Date Plated
B1 1.6 X 10s 28 Nov 2000
Concentration tig per plate
Vehicle
Plate Number
01 02
Revertants per plate
Background Code
Average Revertants
Standard Deviation
03
100 01 6 1 02 5 1 03 3 1
333
0021
10 1 81
03 8 1
1000 01 4 1 02 8 1 03 5 1
3333 01 9 1
1
02 9 1 03 6 1
5000 01 9 1 02 8 1 03 6 1
Positive Control 2-aminoanthracene 1.0 pg per plate 01 79 i
02 88 1
-------------
03 ______ 68
Background Lawn Code
1
78
l=Normal; 2=siightly reduced; 3=Severelv reduce''
10
N p S n e^ t L f ^ "Ced'' 5=*bfent'- 6=Obscured by precipitate NP Non-Interfering precipitate; Interfering precipitate
Company Sanitized. Does not contain TSCA CB1
H-24616: B acterial Reverse M utation Test
D uPont-5234
Bacterial Mutation Assay
Table 9
Test Article Id
Study Number
Strain
: WP2 uvrA
Liver Microsomes : None
Vehicle
: water
Plating Aliquot ; 100 uh
OO.BTL
Experiment No : B1 Cells Seeded : 3.2 X 10 Date Plated : 28 Nov 2000
Concentration pg per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
17 13
9
Background Code
1 1 1
Average Revertants
13
Standard Deviation
4
100 01 12 1 02 9 1 03 11 1
11 2
333 01 12 1 02 18 1 03 "17 1
16 3
1000
01 14 1 02 14 1 03 9 1
12 3
3333
01 12 1 02 17 1
03 9 1
13 4
5000
01 17 1 02 18 1 03 18 1
18 1
Positive Control methyl methanesulfonate 1000 ug per plate 01 82 1 02 72 1
_________________ 03_________ 61_______1______________ 72_______ ll
Background Lawn Code l=Normal; 2=Slightly reduced; 3=Severely reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate
B io R elian ce Study N o. AA37AZ.501100.BTL
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H-24616: B acterial Reverse M utation Test
D uPont-5234
Bacterial Nutation Assay
Table 10
i m p ATest Article Id
Study Number
: AAJ/A2i.buxi00.BTL
Strain
: WP2 uvrA
Liver Microsomes : Rat liver S9
Vehicle
: water
Plating Aliquot : 100 pL
Experiment No ; B1 Cells Seeded : 3.2 X 10s Date Plated : 28 Nov 2000
Concentration Plate Revertants Background Average Standard
ug per plate Number per plate
Code
Revertants Deviation
Vehicle
01 02 03
18 1 13 1
12 1
14 3
100 01 14 1 02 13 1 03 13 1
13 1
333 01 10 1 02 11 1
03 15 1
12 3
1000
01 14 1
02 12 1 03 13 1
13 1
1 3333
01 10 1 02 13 1 03 10 1
11 2
5000
01 11 1 02 14 1 03 11 1
12 2
Positive Control 2!-aminoanthracene 10 ug per plate 01 204 1 02 217 1
03 204 1
208
Background Lawn Code
l=Normal; 2=Slightly reduced; 3=Severely reduced
4=Extremely reduced; 5=Absent; 6=0bscured by precipitate
NP=Non-Interfering precipitate; IP=Interfering precipitate
8
B io R etian ce Study N o. AA37A2L5Q1100.BTL
Company Sanitized. Does noi contain TSCA CBI
22
H-24616: Bacterial Reverse Mutation Test
Bacterial Mutation Assay Summary of Results
Table 11
Test Article Id
Study Number
AA37AZ .Spil'd J.BTL
Experiment Mo ; b i
T. ,, Lxver
Average Revertants Mxcrosomes: Mone
Per
Plate
Standard
Deviation
Dose (ug/platel TA98
TA100
Vehicle 100 333 1000 3333 5000
Positive
16 6 85 3
13 1 82 4
16 5 87 5
15
11 11
6 79 3 85 2 102
8
6 13
96 3 336 21
Liver Microsomes: Rat liver S9
TA1535
17 3 17 4 19 6 16 4 16 6 15 2 182 9
TA1537
62 82 91 72 52 71 699 10
WP2 uvrA
13 4 11 2 16 3 12 3 13 4
18 1
72 11
Dose (gg/plate) TA98
Vehicle = Vehicle Control Positive = Positive Control Platxng aliquot: 100 pL
B io R eiian ce Study N o. AA37AZ.501100.BTL
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H-24616: B acterial Reverse M utation T est
APPENDIX A Historical Control Data
D uPont-5234
BioReliance Study No. AA37AZ.501100.BTL
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H-24616: Bacterial Reverse M utation Test
D uPont-5234
Historical Negative and Positive Control Values 1997-1999
revenants per plate
Activation
Strain
Control
None
Rat Liver
Mean SD Min Max Mean SD Min Max
TA98
Neg
16 6
4 59
21 7
6 65
Pos 397 216 21 1536 649 330 56 2454
TA100
Neg 128 29 53 288 144 30 62 258 Pos 592 159 129 1920 785 300 106 2871
TA1535
Neg
11 5
1 45
12 5
1 42
Pos
424 145
6 1024
98 79
8 1640
TA1537
Neg
6 3 0 30
7 3 1 29
Pos
819 468 13 2786 102 128
2 1835
WP2 uvrA
Neg Pos
16 5 190 127
3 48 35 961
17 6 338 282
2 115 16 2632
SD=standard deviation; Min=minimum value; Max=maximum value; Neg=negative control
(including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Postpositive control
B io R elian ce Study No. AA37AZ.501100.BTL
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25
H -24616: B acterial Reverse M utation T est
APPENDIX B Study Protocol
D uPont-5234
B ioR eliance Study No. AA37AZ.501100.BTL
Company Sanitized. Does not contain TSCA CB
26
H-24616: B acterial Reverse M utation Test
DuPont-5234
Sponsor Project Number DaPnnt-5234 BioReliance Study Numb; AA37AZ.501 iflfl.RTt.
1.0 PURPOSE
Reverse Mutation Test
The purpose of tins study is to evaluate the mutagenic potential of the
w
measuring its ability to induce reverse mutations at selected loci of several strains erf
Salmonella typhununum and at die tryptophan locus of Escherichia coli WP2 wrA in toe presence and absence ofS9 activation.
2.0 SPONSOR
2.1 Name: 22 Address:
2.3 Representative: '
2.4 Sponsor ProjectNo.:
E.I. du Pontde Nemours and Company
Stine Haskell Research Center DuPont Haskell Labotatoty P.0 . Box 50,1090 Elkton Road Newark. DE 19714-0050
Maria Donner, Ph.D. Phone: 302-366-5251 Fas: 302-235-7156 Email: miHia.donner@usa.dupont.eom
DuPont-5234
25 WR#;
[ it
2.6 Haskell#:
24616
2.7 ServiceCode:
I )
3.0 {DENTIFICATION OF TESTAND CONTROL SUBSTANCES
3.1 Test Substance Name:
32 Test Substance I.D.:
H-24616(to be used in the Kurorttext)
33 Controls:
Negative: Test substance vehicle (Water)
rno- s-to.*v___e_ :
9-aminoacridine
7> ino
methyl 2-nitrofluorene sbdium aride
f f C all &>
fe Aia/*"
Protocol SPGTJOUOO
03-Nov-2000
B io R elian ce Study No. AA37AZ.501100.BTnL
PageloflO
27
m B io Reliance* f o m e ri M icro b io lo g ical Associates
Company Sanitized. Does not contain TSCA CB1
"" 1
1
H-24616: B acterial Reverse M utation Test
D uPont-5234
Sponsor Project Number: DuPont-5234 BioReliance Study Number AA37AZ.501100.BTL
3.4 Determination of Streng*, Purity, etc.
Unless alternate anangements arc made, die testing ferility at BioReliance will not perform analysis of die dosing solutions. The Sponsor will be directly responsible for determination and documentation of the analytical purity and composition o fdie substance, and the stability and strength ofdie substance in the solvent (or vehicle).
3.5 Test Substance Retention Sample
The retentionofa reserve sample ofthe substance will be the responsibility ofthe Sponsor.
4.0 TESTING FACILITY AND KEY PERSONNEL
4.1 Nuse;
Toxicology Testing Facility BioReliance
42 Address:
9630 Medical CenterDrive Rockville, MD 20850
43 Study Director; Valentine 0 . WagnerQl, MS. Phone:301-610-2152 Fax:301-738-2362 Email: swagner@bioreliance.com
5.0 PROPOSED STUDY DATES
5.1 Experimental StartDate:
28 Nov 2000
5.2 Experimental TerminationDate: 19 Dec 2000
5.3 Draft ReportDate:
04 Jan 2001
5.4 Final Report Date: 6.0 TEST SYSTEM
18 Jan 2001
The tester strains will include the S. typhimurium histidine auxotrophs TA98, TA1Q0,
TA1535 and TA1537 as described by Ames et ai. (1975) and die E. coli tester WP2 uvrA as described by Green and Muriel (1976).
Protocol SPGTS01100 03-Nov-2000
B ioR eliance Study No. A A 37AZ301 Q0.BTL
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9 Bio Reliance* fo rm erly M icro b io lo g ical Asso ciai*
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H-24616: B acterial Reverse M utation Test
D uPont-5234
Sponsor ProjectNumber DuPont - 5234 BioReliance Study Number AA37A2.SQ1100.BTL
Genotype o f She Strains UJed for Mutagen Tejtoeg
Histifine Mutation
Tryptophan Mutation
Additional Mutations
tmG46 TA1535 TA 100
hisC3076 TAI537
-
HUD3052
TA9
TrpE
-
LPS Repair R-factor
Rfa uvrB
-
Rfc uvrB
+R
*"
W P2uvrA
-
uvrA
-
Each S. typhimurium tester strain contains, in addition to a mutation in die histidine
operon, additional mutations that enhance sensitivity to some mutagen, The rfo
mutation results in a cell wail deficiency that increases the permeability of the cell to
certain classes of chemicals such as those containing large ring systems that would
otherwise be excluded. The deletion in die uvrB gene results in a
DNA
excision-repair system. Tester strains TA98 and TA100 also contain the pKMIOl
plasmid (carrying the R-factor). It has been suggested that the plasmid increases
sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase
complex involved withthe mismatch-repairprocess.
TA98 and TA1537 are reverted from histidine dependence (auxotropby) to ht<atin
independence (prototrophy) by fiameshift mutagens. TANK) is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions.
The E. coii tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox tt al,, 1990). Tester strain WP2 uvrA has a deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revenants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base-pair substitution mutations (Green and Muriel,
The S. typhimurium tester strains were received directly from Dr. Bruce Ames, University o f California, Berkeley. The E. coli tester strain was received fiom the National Collection of Industrial and Mani Bacteria, Aberdeen, RTMttimvt (United Kingdom).
7.0 EXPERIMENTAL DESIGN AND METHODOLOGY
The test substance will be tested at a minimum of five dose levels along with appropriate negative and positive controls with tester strains TA98, TA100, TA1535, TA1537 and WP2 uvrA with and without S9 activation. All dose levels o f test mt*.TM- oegrve controls and positive controls will be plated in triplicate
Protocol SPGT501I00
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B ioR eliance Study No. AA37AZ.501100.BTL
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H-24616: B acterial Reverse M utation Test
DuPont-5234
SponsorProject Number DuPont - 5234 BioReliance Study Numb: AA37AZ.501100.BTL
7.1 Solubility Determination
The Sponsorhas indicated that the test substance is soluble in water. & c iu h 'k f
7.2
r J i i '11 te CwOooMl p ( S e t
DoseLeVeIs
Peroni h ^P en S er
1 Veui
The Sponsorwill provide information for selection ofthe dose levels. This will be based on a previous study the Sponsorhas conducted.
7.3 Frequency and Route ofAdministration
The test system will be exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maton and Ames (1983). This test system has been shown to detect a wide range of classes ofchemical mutagens (McCann et at., 1975; McCannand Antes, 1976).
If the Sponsor is aware of specific metabolic requirements (e.g., azo compounds), this information will be utilized in designing the test Verification of a clear positive response is not required. Negative results will not be retested when justification can be provided. Equivocal results will be retested in conmlrarirm with die Sponsor using an appropriate modification of the experimental dwrign
(e.g., dose levels, activation system or treatment method). This guidance is based on the OECD Guideline 471 (adopted July 1997) and ICH Guidance on Specific Aspects ofRegulatory GenotoxicityTests for Pharmaceuticals (1997).
7.4 Controls
.
7.4.1 Positive Controls
All combinations ofpositive controls and tester strains plated concurrently with the test are listed below:
a ra te
S9 Activation
ro iturv---e-u_nn sro i
Salmonella Strains W PZuvrA TASI
TA100.TA1S35 TA1537 W P2uvrA
Rat None
ira o tin re n tte n re n .
2-ntaofluofene Sodium azide 9-am tooacridine
t.0 10 1.0 1.0 7S 1,000
7.4.2 Negative Controls
Appropriate negative controls will be plated for each tester strain with and without S9 activation. The negative control will be the vehicle alone,
Protocol SPGT501100
03-Nov-2000
BioReliance Study No. AA37AZ.501 00.BTL
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H-24616: B acterial Reverse M utation T est
D uPont-5234
Sponsor ProjectNumber: DuPont 5234
BioReliance Study N un*: AA37AZ.50H00.BTL
unless there is no historical basis for use of die selected vehicle. In die latter case, both untreated and vehicle controls will be used.
7.4.3 Sterility Controls
The most concentrated test substance dilution and die Sham and S9 mixes will be checked for sterility.
7.5 Exogenous Metabolic Activation
Aroclor 1254-indueed sat liver S9 will be used as the metabolic activation system. The S9 homogenate will be prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection ofAroclor 1254,500 mg/kg, five days prior to sacrifice. Dm S9 will be batch prepared and stored frozen at approximately -70C until used. Each batch of S9 homogenate will be tested for its ability to metabolize 2-aminoamfaracene and 7,12-dimethylbenzanthracene to forms mutagenic to S. typftimurnan TA100.
Immediately prior to use, the S9 will be thawed and mixed with a cofector pool to contain 10% S9 homogenate, SmM glucose-6-phosphate, 4mM p-nicotinamide-adettiiM dinucleotide phosphate, 8 mM MgO, and 33 mM RC1 in a 100mM phosphatebufferatpH 7.4. This mixture is referred to as S9 mix. Sham mixwill be 100mMphosphate bufferatpH 7.4.
7.6 Preparation ofTester Strain
Overnight cultures will be inoculated from toe appropriate master plate or from toe appropriate frozen stock. To ensure that cultures are harvested in late log phase, the length ofincubation will be controlled and monitored. At the end ofthe working day, each inoculated flask will be placed in a resting shaketrtncubator at room temperature. The shaker/incubetor will be programmed to begin shaking at approximately 125 tpm at 372C approximately 12hours before the anticipated time ofharvest
All cultures will be harvested by spcctrophotometric monitoring o f culture turbidity rather than fry duration of incubation since overgrowth of cultures can cause loss of sensitivity to some mutagens. Cultures will be removed from incubation at a density ofapproximately 10*ceUs/mL.
7.7 Test System Identification
Each plate will be labeled with a code system that identifies the test substance, test phase, dose level, tester strain and activation type as described in BioReliance's Standard Operating Procedures. `
Protocol SPGT501100
03-Nov-2000
B ioR eliance Study No. AA37A2L501 00.BTL
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H-24616: Bacterial Reverse M utation T est
DuPont-5234
Sponsor Project Number DuPont-5234
BioReliance Study Number: AA37AZ.S01100.BTL
7.8 Test Substance Preparation
Unless specified otherwise, test substance dilutions will be prepared immediately prior to use. All test substance dosing will be at room temperature under yellow light.
7.9 TreatmentofTest Systran
One half milliliter (0.5 mL) of S9 mix or Sham mix, 100 |L of tester strain and 50 pL of vehicle, test substance dilution or positive control will be added to 2.0 mL of molten selective top agar at 452C. When necesswy to achieve the targetconcentrationor eliminate toxic vehicle effects, aliquotsofotherthan 50 ,,L of test substance/vehicle/posttive control will be plated. The mixture will be vortex mixed and overlaid onto die surfece of 25 mL of minimal bottom agar. After the overlay has solidified, die plates will be inverted and incubated for approximately 48 to 72 hours at 372C. Plates that are not counted immediately following the incubation period will be stored at 2-8<C.
7.10 Scoring
The condition of the bacterial background lawn will be evaluated for evidence of test substance toxicity and precipitate. Evidence of toxicity will be scored relative to the negative control plate and recorded along with the reverta count for that plate. Toxicity will be evaluated as a decrease in the numbero frevertant colonies per plate and/or a thinning or disappearance of the bacterial background lawn. Precipitation will be evaluated after the incubation period by visual examination wimora magmneanon.
7.11 Tester Strain Verification
On the day ofuse in t mutagenicity test, all tester strain cultures will be checked forthe appropriate genetic markets cited in 6.0.
8.0 CRITERIA FORDETERMINATION OF A VALID TEST
The following criteria must be met for foe mutagenicity test to be considered valid:
8.1 Tester Strain Integrity
To demonstrate foe presence of the rfa mutation, ail S. typhtomrtum tester strain cultures must exhibit sensitivity to ciystal violet To demonstrate foe presence of foe uvrB mutation, all S. typkimurium tester strain cultures mustexhibit sensitivity to ultraviolet light To demonstrate the presence of the uvrA mutation, all colt tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate
Protocol SPGT501100
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B io R elian ce Study N o. AA37AZ.501100.BTL
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H-24616: B acterial Reverse M utation T est
D uPont-5234
Sponsor Project Number: DuPont - 5234 BioReliance Study Number: AA37AZ.S01100.BTL
the presence of die pKMIOl plasmid R-factor, tester strain cultures of TA98 and TA100 must exhibitresistance to ampiciliin.
8.2 Spontaneous RevertantBackground Frequency
Based on historical control data, alltester strain cultures must exhibit characteristic number of spontaneous revertants per plate in the negative controls
(vehicle). The mean revertants per plate must be within die following ranges
(inclusive): TA98, 10-50; TA100, 80-240; TA1535, 5-45; TA1537, 3-21; WP2 uvrA, 10-60.
8.3 Tester Strain Titers
'
To ensure that appropriate numbers of bacteria are plated, all tester strain culture titers mustbe equal to or greaterthan 0.3x10*cells permilliliter.
8.4 Positive Control Values
Each mean positive control value must exhibit at least a three fold increase over d respective mean negative control value (vehicle) foreach testa strain.
8.5 Toxicity
A minimum ofthree non-toxic dose levels will be required to evaluate test data. A dose level is consideredtoxic if it causes a >50% reduction in the meannumberof revertants per plate relative to the mean negative control value (this reduction must be accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in die background lawn. In the event that less than three non-toxic dose levels are achieved, die affected portion of the test will be repeated with an appropriate changein dose levels.
9.0 EVALUATION OF TEST RESULTS
For a test substance to be evaluated positive, it must cause a dose-related increase in the
mean revertants p a plate of at least one tester strain over a minimum of two increasing concentrations oftest substance as specified below:
9.1 SteamsTA1535 and TA1537
Data sets will be judged positive if die increase in mean revertants at die peak of
the dose response is equal to or greater than three times the mean negative control value (vehicle).
Protocol SPGT501100
03-Nov-2000
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H-24616: B acterial Reverse M utation T est
D uPont-5234
Sponsor Project Number DuPont - 5234
BioReiiance Study Number: AA37AZ.S0n00.BTL
92 Strains TA98,TA100 and WP2 iwA
Data sets will be judged positive if the increase in mean revertsnts at the peak of the dose response is equal to or greater than two times the mean negative control value (vehicle).
10.0 REPORT
A report of the results of this study will be prepared by the Testing Laboratory and will accurately describe all methods used for generation and analysis o f the data. The report will include:
Test Substance; identification and CAS no., if known; physical nature and purity, if known; physicochemical properties relevant to the conduct of die study, if known; stability oftest substance, ifknown.
Solveni/Vehicle: justification for choice of vehicle; solubility and stability of test substance in solvent/vehicle, ifknown.
Strains: strains used; numberofcells/mL per culture; straincharacteristics.
Test conditions: amountoftest substanceperplate withrationale fordose selection and number of plates per concentration; media used; type and composition o f metabolic activation system, including acceptability criteria; treatment procedures.
Results: signs of toxicity, signs of precipitation; individual plate counts; the mean number of revertant colonies per plate and standard deviation; dose-response relationship, where possible; statistical analysis, if any; concurrent negative and positive control data means and standard deviations; historical negative and positive control datawith ranges, means and standard deviation.
Discussionofresults.
fjonriimfrtn,
11.0 RECORDS AND ARCHIVES
Upon completion of the final report, all raw data and reports will be archived by BioReiiance, Rockville, MD for a period o fno less than 1 year from the study initiation date.
12.0 REGULATORY REQUIREMENTS/GOOD LABORATORYPRACTICE
This protocol has been written to comply with OECD Guideline 471 for Testing of Chemicals (Genetic Toxicology: Bacterial Reverse Mutation Assay), adopted July 1997.
This study will be performed using the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies as a guideline; however, the study will not meet GLP
Protocol SPGT50I100
0J-Nov-2000
B ioR eiiance Study No. A A 37A Z.5011003TL
10 Bio ReuanceFormally M icrobiological A t lt t li l e i
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H-24616: B acterial Reverse M utation T est
D uPont-5234
Sponsor Project Number DuPont-5234
BioReliance Study Number: AA37AZ.50I100.BTL
Unless arrangements are made to the contrary, unused dosing solutions will be disposed of following administration to the test system and all residual test substance will be disposed o ffollowing finalization ofthe report
13.0 REFERENCES
Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Sa/mOBeffatoammalian-microsome mutagenicity test Mutation Research 31:347-364.
Green, M.H.L., and Muriel, W.J. (1976). Mutagen testing using top* reversion in Escherichia coli. Mutation Research 38:3-32.
International Conference on Harmonisation (ICH) o f Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance cm Specific Aspects of Regulatory Genotoxicity Teste for Pharmaceuticals. S2A document recommended for adoption at step 4 of the ICH process on July 19, 1995. Federal Register 61:18198-18202, April 24,1996.
International Conference on Harmonisation (ICH) of Technical Requirements for Registration o f Pharmaceutical* for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. S2B document recommended for adoption at step 4 of the ICH process on July 16, 1997. Federal Register 62:16026-16030, November 21,1997.
McCann, J. and Ames, B.N. (1976). Detection of carcinogens as mutagens in the SalmoneUaimaosomt test: assay o f 300 chemicals: discussion. Free. Natl. Acad. Sci. USA 73:950-954.
McCann, J., Choi, E , Yamasaki, E. and Ames, B.N. (1975). Detection of carcinogens as mutagens in the Salmonellahmctosome test: assay o f 300 chemicals. Proc, Natl. Acad. Sci. USA 72:5135-5139.
Maron, D.M. and Ames, B.N. (1983). Revised Methods for the Salmonella Mutagenicity Test Mutation Research 113:173-215.
OECD Guideline 471 for Testing of Chemicals (Genetic Toxicology: Bacterial Reverse Mutation Assay), adopted July 1997.
Wilcox, P,, Naidoo, A., Wedd, D J. and Gatehouse, D.G. (1990). Comparison of Salmonella yphlmurhtm TA102 with Escherichia coll WP2 tester strains. Mutagen 5:285-291.
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H-24616: B acterial Reverse M utation Test
D uPont-5234
14.0 APPROVAL
Sponsor Project Number: DuPont - 5234 BioReliance Study Number: AA37AZ.SOnoo.BTL
k Q-.rC ov D o o r t t ;
(Print or Type Name)
0~\ N io y J t o a o Date
BioReliance Study Director
/to 300O
Date
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03-Nov-2000
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36
