Document mqB8n2KnMjk6v4R6e4RLdE1Vd
AR226-3173
TRADE SECRET
Study Title
H-24616: Bacterial Reverse Mutation Test
Authors
Valentine 0. Wagner. HI, M.S. MichelleL.Klug.B.S.
Report Completion Date
25 January 2001
Performing Laboratory
BioReliance 9630 Medical Center Drive
Rockville.MD 20850 for
E. L du Font de Nemours and Company Stine Hasken Research Center DuPont Haskell Laboratory P.O. Box 50,1090 Elkton Road Newark, DB 19714-0050
Performing Laboratory Study Number AA37AZ.501100.BTL
DuPont Project ID
DuPont-5234
Work Request Number
DuPont-5234
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H-24616: Bacterial Reverse Mutation Test
DuPont-5234
CERTIFICATION
We, the undersigned, declare that this report provides an accurate evaluation of data obtained
from this study.
Issued by Study Director:
M.S.Date ^<dw^j Q. M^^.3^_____ z5^L2fo/ Valentine 0. Warierni,
Approved by Study Monitor
rv-(3<^-^<?^- JL)CY^/^--
Maria Donner, Ph.D. SCTaor Research Scientist
^^ ^ftM 2.001
Date
BioReliance Study No. AA37AZ.501100.BTL
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H-24616: Bacterial Reverse Mutation Test
DuPont-5234
TABLE OF CONTENTS
Page
Certificadon...............................................................................................................................2 Study
Infonnation.........................,.....,...,........................,........,................................,...............4
Summary ......................................... ,,..,,....... ......................................................... ........5
Puipose......................................................................................................................................6
Characterization of Test and Control Substances..........................................,..........................^
Materials and Methods...............,..........,.............................................,..............,.......,..............?
Results and
1
Discussion,....,...................................,..,...............................................................!
Conclusion.......................,............,..........................................................................................ll 12
References......................................................................................................................
DataTables.......,...........................................,..........................................................................13
Table 1: Mutagenicity Test in Salmonella typhimurium TA98 without S9...................... 13
Table 2: Mutagenicity Test in Salmonella typhimurium TA98 with
14
Table 3:
Mutagenicity
Test
in
Salmonella
typhimurium
TA100
S9........................... without
15
Table 4: Mutagenicity Test in Salmonella typhimurium TA100 with S9....................
S9.........................16
Table 5: Mutagenicity Test in Salmonella typhimurium TA1535 without
17
Table 6: Mutagenicity Test in Salmonella typhimarium TA1535 with S9..................
S9..,......,..,..........18
Table 7: Mutagenicity Test in Salmonella typhimurium TA1537 without
S9..................19
Table 8: Mutagenicity Test in Salmonella typhimurium TA1537 with S9.......................20
Table 9: Mutagenicity Test in Escherichia coli WP2 uvrA without S9 ...........................21
Table 10: Mutagenicity Test in Escherichia coli WP2 uvrA with S9 ...............................22
Table 11: Salmonella/E. coli Mutagenicity Test - Summary of Results............................23
Appendix A: Historical Control Data..............................................,...............................,......,24
Appendix B: Study Protocol....................................................................................................26
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H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Substance Ti
STUDY INFORMATION
.2461
Haskell Number 24616
j| Punty,
Known Impurities: H
Physical Qiaracteaisdcs^J
Stability:
The test substance appeared to be stable under (fae conditions of the study; no evidence of instability was
observed.
Solubility: Aquatics: Dispersible in water All Others: Dimethyl sulfoxide
Sponsor.
E. I. du Font de Nemours and Company Wilnangton, Delaware 19898
U.S.A.
Study Initiated/Completed: November, 16,2000 / (see report cover page)
In-Life Initiated/Completed: November 28,2000 / December 06,2000
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H-24616: Bacterial Reverse Mutation Test
DuPont-5234
SUMMARY
The lest substance, H-24616^||mij|^|was tested in the bacteria! reverse mutation
test using S. typhinwriwn tester strains TA98, TAlOO, TA1535 and TA1537 and E. coU tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The test was perfbnned using die plate incorporation method to evaluate the mutagenic potential of the test
substance.
Water was selected as the solvent of choice based on solubility of the test substance, 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 lOmg/mL. The test substance was a workable suspension at 25 to 50 mg/mL.
In the mutagenidty assay, the maximum dose tested was 5000 fig per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 uL plating aliquot. The test article diluted to a clear solution at 1.0 and 3.3 mgteL. The dose levels tested were 100,333,1000, 3333 and 5000 pg per plate. No positive responses were observed with any of 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 of 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.
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H-24616: Bacterial Reverse Mutation Test
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PURPOSE
The purpose of this study was to evaluate the mutagenic potential of the test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhinwrwm, and at the tryptophan locus of Escherichia coli WP2 wrA in the presence and absence ofS9 metabolic activation.
CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES
The test substance, H-24616, was received by BioReliance on 06 November 2000 and was
agnedthe code number AA37AZ. The Sponsor characterized the test substance as an|----t
_JBrtpffiat should T)e storcd'at ambient temperature. AnTexpiration date was not provided.
Upon receipt, the test substance was described as aJIHHI^HHB^nd stored at room
temperature, protected from exposure to light.
The vehicle used to deliver H-24616 to me test system was water, (CAS^ 7732-18-5),
obtained Horn life Technologies.
Positive controls plated concurrently with me mutagenicity test are listed below:
Strain AH Salmonella Stmas
WP2wrA TA98
TA100.TA1535 TA1537
WP2<wA
S9 Metabolic Activation Rat
None
Positive Control
2-aininoanthracene (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 (Hg/plate)
1-0 10 1.0
1.0
75
1,000
To determine the sterility of me 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
fhetest
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H-24616: Bacterial Reverse Mutation Test
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MATERIALS AND METHODS
Test System
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975), and Escherichia coli tester strain WP2 nwA. Salmonella tester strains were received on 11 August 1998 and 10 November 1998 directly from Dr. Bruce Ames, University of California, Berkeley. E. coU 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 (auxotropby) 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 of die reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Overnight cultures were prepared by inoculating from the appropriate master plate or from
the appropriate frozen permanent stock into a vessel containing -50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and
monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shakec/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 teansmittance yielding a titer of greater than or equal to 0.3xl09 cells per milliliter. The actual titeis were determined by viable count assays on nutrient agar plates, and the data is on file but not presented in mis report. The study was conducted to comply with OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), adopted July 1997.
Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 was purchased from MbITox, batch prepared on 18 October 2000 and stored at -70C or colder until used. Each bulk
preparation of S9 was assayed for its ability to metabolize 2-aminoanthracene and 7,12-
dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM
glucose-6-phosphate, 4mM P-nicotinamide-adenine dinucleotide phosphate, 8mM MgQz
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Study No. AA37A2501100.BTL
7
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
and 33 mM KC1 in a 100 mM phosphatebuffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphatebuffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on
selective agar.
Solubility Test
A solubility test was conducted to select the vehicle using one or more of the following: water and dimethyl sulfoxide (DNSO). The test substance was tested to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration, up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic
solvents.
MutagenidtyTest
The mutagenicity test was used to evaluate the mutagenic potential of the test substance. Five dose levels of test substance (100, 333, 1000, 3333 and 5000 ug per plate) along with appropriate vehicle and positive controls were plated with tester strains TA98, TA100, TA1535, TA1537 and WP2 uvrA in the presence and absence of 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 Ames(1983).
On me day of its use, TniNmal top agar, containing 0.8 % agar (WV) and 0.5 % NaCI (W/V),
was melted and supplemented with L-histidine, D-biotin and L-ttyptophan solution to a final
concentration of 50 uM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water producedby the Milli-Q Reagent
Water 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 % (WV) agar and supplementedwith 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 (he test substance, test phase, dose level, tester strain, and activation, as described in detail in BioReliance's Standard Operating Procedures.
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H-24616; Bacterial Reverse Mutation Test
DuPont-5234
Test substance dilutions were prepared immediately before use. One-half (0.5) milliliter of S9 or sham mix, 100 ^iL of tester strain and 100 uL of vehicle or test substance were added to 2.0 mL of molten selective top agar at 452C. After vortexing, me mixture was overlaid onto the surface of 25 mL of minimal bottom agar. .When plating the positive controls, the test substance aliquot was replaced by a 50 pL aliquot of 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 of 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 of precipitation were scored relative to the vehicle control plates using the codes shown below.
Code 1 2 3 4 5 6
NP
n>
Description
Characteristics
Normal SlightlyReduced
Distinguishedby a healthy microcolony lawn.
Distinguished by a noticeable thinning of the mierocolony lawn and possibly a slight increase in the size of die microcolonies compared to the vehicle control plate.
Moderately Reduced
Distinguished by a marked thinning of the microcolony lawn resulting in a pronounced increase in the size of the microcolonies compared to the vehicle
control plate.
Severely Reduced Absent
Distinguished by an extreme thinning of the microcolony lawn resulting in an increase in die size of die microcolonies compared to the vehicle control plate such that die niicrocolony lawn is visible to (he unaided eye as isolated colonies.
Distinguished by a complete lack of any microcolony town over 90% of die
plate.
Obscured by Precipitate
Noil-Interfering Precipitate
Interfering Precipitate
The background bacterial lawn cannot be accurately evaluated due to microscopic test substance precipitate.
Distinguishedby precipitate on die plate that is visible to the naked eye but any precipitate particles detected by die automated colony counter total less than 10% of die revertant colony count (e.g., <3 particles on a plate with 30
rcvertants.)
Distinguishedby precipitate on die plate mat is visible to die naked eye and any precipitate particles detected by die automated colony counter exceed 10% of die revertant colony count (e.g., >3 particleson 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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Study No. AA37AZ501100.BTL
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H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Evaluation of Results
For each triplicate plating, the mean and standard deviation of the number of revertants per
plate were calculated and are reported.
For toe test substance to be evaluated positive, it must cause a dose-related increase in me mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test substance. Data sets for strains TA1535 and TA1537 were judged
positive if the increase 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 Ac increase in mean revertants at the peak of the dose
response is equal to or greater than two times the mean vehicle control value.
Criteria for a Valid Test
The following criteria must be met for the mutagenicity test to be considered valid. All
Salmonella tester strain cultures must demonstrate the presence of me deep rough mutation {rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of me pKMIOl plasmid R-factor. All WP2 uvrA 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); TA100
(80-240); TA1535 (5-45); TA1537 (3-21); and WP2wrA (10-60). To ensure that
appropriate numbers of bacteria are plated, tester strain culture tilers must be greater than or equal to 0.3xl09 cells/mL. The mean of each positive control must exhibit at least a three-fold
increase in the number of revertants over the mean value of the respective vehicle control. A minimum of 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 of revertants per plate as compared to the mean vehicle control value. This
reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A
reduction in me background lawn.
Archives
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.
Deviations
No known deviations from the protocol or assay-method SOPs occurred during the conduct of mis study.
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Study No. AA37A2.501100.BTL
10
H-24616; Bacterial Reverse Mutadon Test
DuPont-5234
Solubility Test
RESULTS AND DISCUSSION
Water was selected as the solvent of choice based on solubility of the test substance, compatibility with the target cells and stability of the test article in the solvent (per Sponsor suppliedinformation). The test substance was soluble and but cloudy in water at approximately lOmg/mL. The test substance was a workable suspension at 25 to 50 mg/mL.
Mutageoidty Test
The results of the mutagenicity test are presented in Tables 1 through 10 and summarized in Table 11. These data were generated in Experiment Bl. In the mutagenicity assay, the maximum dose tested was 5000 ng per plate; mis dose was achieved using a concentration of 50 mg/mL and a 100 uL 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 ug per plate. Neither
precipitate nor toxicity was observed.
In Experiment Bl, no positive responses were observed with any of the tester strains in me
presence and absence of rat S9 activation.
CONCLUSION
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 of this study, H-24616 did not induce a positive response with any of the tester strains in me presence and absence of Aroclor-induced rat liver S9.
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11
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
REFERENCES
Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and Mutagens with the Salmonetta/MazsaasSiaa Microsome Mutagenicity Test, Mutation Research, 31:347-364.
Green, M.HL. and WJ. Muriel (1976) Mutagen testing using up1'reversion in Escherichia coU,
Mutation Research 38:3-32.
Maron, D.M. and B.N. Ames (1983) Revised Methods for the Salmonella Mutagenicity Test, Mutation Research, 113:173-215.
OBCD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), adopted July
1997.
Vogel, H.J. and D.M. Bonner (1956) Acetylomithinase ofE. coli: Partial Purification and Some
Properties, J. Biol. Chem., 218:97-106.
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H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Bacterial Mutation Assay
I T'able I
Test Article Id
Study Number
: AA37AZ. 501100. BTL
Strain.
; TA98
Liver Microsomes : None
Vehicle
: water
Plating Aliquot : 100 UL
Experiment No ; B1 Cells Seeded : 0.6 X 108 Date Plated : 28 Nov 2000
Concentration
ug per plate
Plate Revertants Background Average Standard
Number per plate
Code
Revertanfci3 Deviation
Vehicle
01
02
03
10
1
21
1
16
1
i6
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 pig per plate
01
99
1
02
94
1
03
95
1
96
Background Lawn Code
l=Konnal; 2=Slightly reduced; 3=Severely reduced 4=Extreniely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interering precipitate
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H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Bacterial Mutation Assay
Table 2
Test Article Id
Study Number
Strain Liver Microsomes Vehicle Plating Aliquot
/a BMWJ
^AA3^7A2.501l1O.BTL
TA98
Rat
"
liver
S9
water
100 V"rL.
Experiment So ! B1
Cells Seeded 0.6 X 108 Date Plated ; 28 Nov. 2000
Concentration
ug per plate
Plate Revertants Background Average Standard
Number per plate
Code
Revertantfs Deviation
Vehicle
01
02
23
1
15
1
03
20
1
1-9
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-aniinoanthracene 1..0 ug per plate
01
451
1
02
486
1
_
_
03
546
1
494
48
Background Lawn Code
l=Mormal; 2=Sligbtly reduced; 3=Severely reduced 4=Extremely reduced? 5=Absent; 6=0bscured by precipitate MP=Non-Interfering precipitate; Ipslnterfering precipitate
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H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Bacterial nutation Assay
Table 3
Test Article Id
Study Number
Strain Liver Microsomes Vehicle Plating Aliquot
I----I : AA37AZ.5OUOO.BTI.
; TAlOO
: None : water : 100 uL
Experiment No : B1
Cells Seeded : 0.9 X 10" Date Plated : 28 Nov 2000
Concentration
ug per plate
Plate Revertants Background Average Standard
Number per plate
Code
Revertants Deviation
Vehicle
01
02
88
1
83
1
03
84
1
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=Nonnal; 2=Slighfcly reduced; 3=Severely reduced
4=Extremely reduced; 5=Absent; 6=0bscured by precipitate MP=Kon-Interering precipitate; IP=Interfering precipitate
BioReliance
Study No. AA37AZ501100 STL
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H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Bacterial Mutation Assay
Table 4
Test Article Id.
Study Number
Strain Liver Microsoaes Vehicle Plating Aliquot
HJ ('1----1
:A&37AZ.5iSTloo BTL ; TA100
; Rat liver S9
; water
: 100 ill,
Experiment No : B1
Cells Seeded : 0.9 X 10" Date Plated : 28 Mov 2000
Concentration ug per plate
Plate Revertants Background Average Standard
Number per plate
Code
Revertants Deviation
Vehicle
01
02
98
1
82
1
03
89
1
90
8
100
01
78
1
02
91
1
03
79
1
83
7
333
01
63
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-aminoantbracene 1.0 ug per plate
01
621
1
02
668
1
03
520
1
603
76
Background Lawn Code
l=Monnal; 2=Slightly reduced; 3=Severely reduced 4=Sxtremely reduced; 5=Absent; 6=0bscured by precipitate MP=Mon-Interfering precipitate; IP=Interering precipitate
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H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Bacterial Mutation Assay
W 9 l \ Test Article Id
Study Number
Strain Liver Microsome9 Vehicle Plating Aliquot
Table 5
; AA37AZ. 501100.BTL ; TA1535 : None : water : 100 pL
Experiment No : B1 Cells Seeded : 2.7 X 108 Date Plated : 28 Mov 2000
Concentration
ug per plate
Plate Revertants Background Average Standard
Number per plate
Code
Revertants Deviation
Vehicle
01
02
14
1
18
1
03
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
01
18
1
02
9
1
03
20
1
16
6
5000
01
15
1
02
13
1
03
16
1
15
2
Positive Control sodium azide 1.0 ug per plate
01
188
1
02
187
I
03
172
1
182
Background Lawn Code
l=Konnal; 2=Slightly reduced? 3=Severely reduced 4=Extremely reduced; 5Absent; 6=0bacured by precipitate MP=Mon-Interfering precipitate; IP=Interfering precipitate
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H-24616: Bacterial Reverse Mutation Test
DuPoat-5234
Bacterial Mutation Assay
Table 6
Test Article Id
Study Number
Strain Iiiver Microsomes Vehicle Plating Aliquot
i--------------9J
ASdTAZ.StHCTO .BTL TA1535
Rat liver S9
water
100 uL
Experiment No Bl
Cells Seeded 2.7 X 10s
Date Plated
28 Nov 2000
Concentration
pg per plate
Plate Revertants Background Average Standard
Number per plate
Code
Revertants Deviation
Vehicle
01
02
14
1
17
1
03
C
16
2
100
01
7
02
13
03
16
12
333
01
13
02
15
03
9
12
1000
01
17
02
21
03
16
18
3333
01
5
02
13
03
13
10
5000
01
17
02
18
03
21
19
Positive Control 2-aminoanthracene 1.0 ug per plate
01
120
1
02
90
1
___ 03
Background Lawn Code
__ _l05_
1
_
_ 105
15
IsNormal; 2=Slightly reduced; 3"Severely reduced 4=Exfcremely reduced; 5=Absent; 6=obscured by precipitate MP=Non-Interfering precipitate; IP=Interering precipitate
C=Contaminated
BioReliance StudyNo. AA37AZ501100.BTL
Company Sanitized. Does not contain TSCA CBl
18
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Bacberial Mutation Assay
I Table ;r
Test Article Id
Study Number
Strain Liver Microsomes Vehicle Plating Aliquot
fi^^^^^H "AA'^ffi 01100.BTL
: TA1537
: None
: water
: 100 uL
Experiment No : Bl Cells Seeded : 1.6 X 10" Date Plated ; 28 Nov 2000
Concentration
ug per plate
Vehicle
Plate
Number
01 02
Revertants Background Average
per plate____Code___Revertants
Standard Deviation
03
100
01
10
02
9
03
6
333
01
8
02
8
03
10
1000
01
02
03
3333
01
02
03
5000
01
02
03
Positive Control 9-aminoacridine 75 ug per plate
01
689
1
02
708
1
699
10
B_a_c_kg_r_o_un_d__La_w_n__Co_d0e3______699_____1_______
l=Nonnal; 2=Slightly reduced; 3=Severely reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Mon-Interfering precipitate; IP=Interfering precipitate
BioReliance
Study No. AA37AZ501100.BTL
company Sanitized. Does not contain TSCA CB1
19
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Bacterial I lutation Asse13r
1----3Table 8
Test: Article Id
Study Number
: AA37AZ.!501100.BTL
Strain
: TA1537
Liver Microsomes ; Rat liver S9
Vehicle
: water
Plating Aliquot ; 100 uL
Experiroenfc No : Bl Cells Seeded : 1. 6 X 10" Date Plated : 28 Nov 2000
Concentration
ug per plate
Vehicle
Plate
Number
01 02
Revertants
per plate
6
8
Background Code
1
1
Average Revertants
Standard Deviation
03
6
1
7
1
100
01
6
1
02
5
1
03
3
1
5
2
333
01
10
1
02
8
1
03
8
1
9
1
1000
01
4
1
02
8
1
03
5
1
6
2
3333
01
9
1
02
9
1
03
6
1
8
2
5000
01
9
1
02
8
1
03
6
1
8
2
Positive Control 2-aminoanthracene 1.0 ug per plate
01
79
1
02
88
1
03_______68_____1___________78
10
Background Lawn Code
Isllormal; 2=slightly reduced; 3=Severely reduced
4=s33xtremely reduced; 5=Absent; 6=0bscured by precipitate
NP=Kon-Interfering precipitate; XPInterfering precipitate
BioReliance Study No. AA37AZ.501100.BTL
Company Sanitized. Does not contain TSCA CB1
20
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Bacterial Mutation Assay
"1r w Test Article 1
Study Number
Table 9 ''AA?7SZ.501100.BTL
Strain Liver Microsomes Vehicle Plating Aliquot
WP2 uvrA None
water 100 uL
Experiment No ; B 1
Cells Seeded ^ 3 .2 X 10" Date Plated 28 Nov 2000
Concentration pg per plate
Pla^e Severfcants Background Average
Standard
Number per plate
Code
Revertants Deviation
Vehicle
01
02
17
1
13
1
03
9
I
13
4
100
01
12
1
02
9
I
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
I
02
72
1
03
61
1
72
11
Background Lawn Code
l=Norinal; 2=Slightly reduced; 3=Severely reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate? IP=Interfering precipitate
BioReliance
Study No. AA37AZ501100ATL
Company Sanitized. Does not contain TSCA CB1
21
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Bacterial Mutation Assay
Table 10
Test Article Id
Study Number
Strain Liver Microsomes Vehicle Plating Aliquot
IJ--------------i
"TASAM'J'/IMR.oiiOHlloOoO.BTL
WP2 uvrA
Rat liver S9
water
100 ul>
Experiment No Cells Seeded Date Plated
Bl 3.2 X 10" 28 Nov 2000
Concentration
ug per plate
Plate Rev<srtants Background Average Standard
Number per plate
Code
Revertants Deviation
Vehicle
01
02
18
1
13
1
03
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
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
208
Backg_r_ou_n_d__La_w_n__C_od0e3 ____204_____1_____ ___
l=Norrnal; 2=Slightly reduced; 3=Severely reduced 4=Exfcremely reduced; 5=Absent; 6=0bscured by precipitate KP=Kon-Interfering precipitate; IP=Interfering precipitate
BioReliance Study No. AA37AZJ01100.BTL
Company Sanitized. Does not contain T5CA CB1
22
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Bacterial Mutation Assay Summary of Results
Test Article Id
Study Number
19 Table 11
: AA3 7A?SfflLOO.BTI.
Average Revertants Per Plate Liver Microsoroes; None
Experiment No B1 Standard DeviataLon
Dose (ug/plate)
Vehicle
100 333 1000 3333 5000
Positive
Liver Microsomes;
TA98
TA100
16 13 16 15 11 11 96
6
~85" "
3
1
82
4
5
87
5
6 79 *
8
3
85
6
2 102 t 13
3 336 * 21
Rat 1 iver S9
TA1535
17 A 3
17
4
19 *
6
16
4
16
6
15
2
182 9
TA1537
6 t
2
8 *
2
9
1
7
2
5
2
7
1
699 10
WP2
13 11 16 12 13 18 72
UvrA
4
2
*
3
t
3
*
4
1
11
Dose (ug/plate) TA98
Vehicle
100
19 *
4
19 t
7
333
14 *
3
1000
19 A 2
3333
18
3
5000
Positive
19
4
494 48
Vehicle = Vehicle Control Positive = Positive Control Plating aliquot; 100 ul<
TA100
"9"0
8
83
7
81
3
83
5
98 13
90
5
603 76
TA1535
16 *
2
12 t
5
12
3
18
3
10
5
19
2
105 * 15
TA1537
7
1
5
2
9 t
1
6
2
8
2
8
2
78 10
WP2
14 13 12 13 11 12 208
UV2-A
3
1
3
1
2
2
8
BioReIiance Study No. AA37AZ.501100.BTL
Company Sanitized. Does noi contain TSCA CBI
23
H-24616: Bacterial Reverse Mutation Test
APPENDIX A
Historical Control Data
DuPoat-5234
BioReliance Study No. AA37AZ.501100JBTL
Company Sanitized. Doss not contain TSCA CBI
24
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Historical Negative and Positive Control Values
1997-1999
tevertants per plate
Activation
Strain
Control Mean
None SD Min
Max
Mean
Rat Liver SD Min
Max
TA98
Neg Pos
16
6
397 216
4
59
21 1536
21
7
649 330
6
65
56 2454
TA100
Neg Pos
128
29
592 159
53
288
129 1920
144 30 785 300
62 258 106 2871
TA1535
Neg
Pos
11
5
424 145
1
45
6 1024
12
5
98 79
1
42
8 1640
TA1537
Neg
Pos
6
3
819 468
0
30
13 2786
7
3
102 128
1
29
2 1835
Neg
WP2wrA
Pos
16
5
190 127
3
48
35 961
17
6
338 282
2
115
16 2632
SD=standaid deviation; Min=ininimum value; Max=maxtmum value; Negsnegative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control
BioReliance Study No. AA37AZ.501100.BTL
Company Sanitized. Doss not contain TSCA CBS
25
H-24616: Bacterial Reverse Mutation Test
APPENDIX B
Study Protocol
DuPont-5234
BioRelianse Study No. AA37AZ.501100.BTL
Company Sanitized. Does not contain TSCA Cffl
26
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Sponsor Project Number: BioReliance Study Number:
DuPont-5234 AA37AZ.50nOO.BTL
Revert Mutation Tsst
1.0 PURPOSE
The purpose of this study is to evaluate the routagcnic potential of the test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhunurium and at the tryptophan locus of Escherichia coli WP2 uvrA m die presence and absence ofS9 activation.
2.0 SPONSOR
2.1
Name:
2.2
Address:
2.3
Representative:
E.I. du Font de Ncmours and Company
StineHaskeH Research Center DuPont HaskeU Labctatory P.O. Box SO. 1090 Elloon Rood Newark, DE 19714-0050
Maria Donner, PhJ). Phone: 302-366-5251 Fax: 302-235-7156 Email: maria.donnei@usa.dupoot.coni
2.4
Sponsor Project No.:
IS WSM-.
2.6 Haakell#;
DuPont-5234
2.7
Service Code:
3.0 IDENTIFICATION OF TESTANP CONTROL SUBSTANCES
3.1
Test Substance Name: J'J
32 Test Substance ID.:
H-24616 (to be used in the report tort)
33
Controls:
Negative: Positive:
Test substance vehicle (Water)
9-annnoaeridinc
^c,<- ^)m50
mMetlhroyl mme..trhtflnesuIlFfoMnate
2-nitrofluoreac sodium azide
P<r C(tl( fo
^tl^ Vo^ ifaAJtv-ieoo
Protocol SPGTiOl 100
03-Nov-2000
BioReliaace Study No. AA37AZ.501100.BTL
Page I of 10
&BIOREUANCF
^y roiffi* M(icrobiBlofirl Allocuiffi
Company Sanitized. Does not contain TSCA CB1
27
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Sponsor Project Number: BioReliance Study Number:
DuPont-5234 AA37AZ.50110Q.BTL
Determination of Strength, Purity, etc.
Unless alternate amngeaients are made, the testing facility at BioRciianee will
not perform analysis of die dosing solutions. The Sponsor will be directly responsible for determination and documentation of the analytical purity and composition of the substance, and the stabiUtyand strengthof the substance in me solvent (or vehicle).
3.5
Teat Substance Retention Sample
The retention of a reserve sample of the substance wiU be the responsibility of the
Sponsor.
4.0 TESTING FACILITY AND KEY PERSONNEL
4.1
Name:
Toxicology Testing Facility BioReliance
43.
Address:
9630 Medical Center Drive Roclkvillc,MD 20850
43
Study Director
Valentine 0. Wagner ffl,M.S.
Phone: 301-<S10-2152
Fax:301-738-2362
Email: swagner@bioreliance.com
5.0 PROPOSED STUDY DATES
5.1
Experimental Start Date:
28Nov2000
5.2
Experimental Termination Date: 19 Dec 2000
5.3
Draft Report Date:
04Jan2001
5.4
Final Report Date:
6.0 TEST SYSTEM
18 Jan 2001
The tester strains will include the S. typhimurium tustidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Aloes et al. (1975) and the E. coli tester strain WP2 uvrA as described by Green and Muriel (1976).
Protocol SPOT501100
OM-tov-2000
BioReliance
StudyNo. AA37AZ^01100BTL
Page 2 of 10
f t BlORELIANCF ^Br foriMrIyMiciahialoBic*(AIaecf
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28
H-24616: Bacterial Reverse Mutation Test
DuPoot-5234
Sponsor Project Number: BioReliance Study Number:
DuPont-5234 AA37A2.5QnOO.BTL
Genotype of the SirainsUttd for Mut*$CTTeitiag
Hidtidine Mutation
Tiyptophin Moeakm
Additional Mudti
bis046 TA1535 TA100
hisC307 TAIS37
HuD3052
TA98
npE
-
LPS
Rtptir
R-futor
Rft
A"wB
-
RA
AirwB
HI
-
-
WP2uvrA
-
AnwA
-
Each S. typhimurium tester strain contains, in addition to a mutation in die histidine opcron, additional mutations that enhance sensitivity to some mutagens. The ria
mutation results in a cell wall deficiency that increases the penneability of flu cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in me uvrB gene results in a deficient DNA excision-repair system. Tester strains TA98 and TA100 also contain (he pKMIOl plasmid (eanying (he R-factor). It has been suggested that the plasond increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymeftte
complex involved with the mismatch-repair process.
TA98 and TA1537 are reverted fiom histidine dependence (awotrophy) to histidine independence (protottopoy) by framesaift mutagens. TA100 is (everted by both fiameshift and base substitution mutagens and TA1535 is reverted only by mutagens
that cause base substitutions.
The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox ft al., 1990). Tester strain WP2uvrA has a deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants 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, 1976).
The S. typhinmrium tester strains were received directly fiom Dr. Bruce Aaies,
University of California, Berkeley. The B. coli tester strain was received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland (United
Kingdom).
7.0 EXPERIMENTAL DESIGN AND METHODOLOGY
The test substance will be tested at a niinunum of five dose levels along with appropriate negative and positive controls vim tester strains TA98, TA100, TA1535, TA1537 and WP2 uvrA with and without S9 activation. All dose levels of test substance, negative controls and positive controls will be plated in triplicate.
Protocol SPGT501100
03-Nov-2000
BioReliance
Study No. AA37AZJOI 100.BTL
Psgt3ofl0
fi| BIORELIANCE"
^y . FOfmrl)rM(crob)oia|cIAircm
Company Sanitized. Doss not contain TSCA CBS
29
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Sponsor Project Number BioReliance Study Number;
DuPont 5234 AA37AZ.501100.BTL
7.1
Solubility Determination
The Sponsor has indicated that the test substance is soluble in water. A- Sc'(uAi/'"r
^^^ fcr^^^^^^^ uitU to Ctl<6t(JS (SkC Scf>
7.2
^m6o
The Sponsor wiU provide information for selection of the dose levels. This will be
based on a previous study the Sponsorhas conducted.
7.3
Frequency and Route of Administration
The test system will be exposedto toe test substance via OK plate incorporation methodologyoriginally described by Amcs et al. (1975) and updated by Maron and Ames (1983). This test system has been shown to detect a wide range of
classes of chemical mutagcos (McCann et al., 1975; McCann and Ames, 1976).
If we Sponsor is aware of specific metabolic requirements (e.g., azo compounds),
mis information will be utilized in designing the test Verification of a clear positive reipooM is not required. Negative results win not be retested when
justification can be provided. Equivocal results will be retested in consultation with the Sponsor using an appropriatemodification of me experimental design (e.g., dose levels, activation system or treatment method). This guidanceis baaed on me OECD Guideline 471 (adoptedJuly 1997) and ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Phannaceuticals (1997).
7.4
Controls
7.4.1 Positive Controls
All combinations of positivecontrols and tester strains platedconcurrently with me test are listed below:
StniB
S9 Actividon
PoiitiveConiro!
Cooc<fltiroofl
(pg/pfate)
Salmonella Stffhu RM
WP2uwA TA91
TA100.TA1535 TAIS37
W2uvrA
2^^------
2'nhrofhiofNM Sodium ffiEide 9-aninotcridine methy! mtbjaesulfo<ute
1.0 10
1.0 1.0 7S 1,000
7.42 Negative Controls
Appropriate negative controls will be plated for each tester strain with and without S9 activation. The negative control will be me vehicle alone,
Protocol SPGT501100
03-Nov-2000
BioReliance
Study No. AA37AZ^01100.BTL
Pge4ofl0
6l BiORELIANCF
Company Sanitizsd. Doss not contain TSCA CBI
30
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Sponsor Project Number BioRcliance Study Number:
DuPont-5234 AA37AZ.50HOO.BTL
unless there is no historical basis for use of die selected vehicle. In <he
latter ease, both untreated and vehicle controls will be used.
7.4.3 SterilityControls
The most concentrated test substance dilution and (he Sham and S9 mixes will be checked for sterility.
7.5
Exogenous Metabolic Activation
Aroclorl254-induced rat liver S9wi!l be used as the metabolic activation iyttem. The S9 hgmogenate will be prepared from male Sprague-Dawley rats induced with a single uioaperitoacal injection ofAroclor 1254,500 mg/kg, five days prior to sacrifice. Hie S9 will be batch prepared and stored fiozea at approximately
-TOoC until used. Each batch of S9 homogenate will be tested for its ability to
metabolize 2-aminoaithrecene and 7,12<di!nemylbefi2anttBcene to forms
mutagemc to S. typhimurwm TA100.
Immediately pri(ff to uae, the S9 will be thawed and mixed with a co&ctor pool to
contain 10% S9 noniogcnatc, 5mM gtucoae-6-paospbate, 4mM p-mcotinamide-ademaedinucleotpihdoesphate,8 mMMgd; and 33 mMKCl in a 100 mM phosphatebuffer at pH 7.4. Thb mixture is referred to as S9 mix. Sham
mix will be 100 mM phosphatebuffer at pH 7.4.
7.6
Preparahoa of Tester Strain
Overnight cultures will be inoculated from the appropriate master plate or from the appropriatefrozen stock. To ensure that cultures are harvested in late tog
phase, the lengthof incubation wiU be coatroUed and monitored. At the end of the
woriar day, each inoculated flask wiU be placed in a resting shajkei^ncubator at
room temperature. The shakerAncubetor will be programmed to begin shakingat
approximately 125 tpm at 372oC approximately 12 hours before the anticipated
time ofharvest-
All cultures wiu be harvested by spcctrophotometric monitoring of culture tmbidity rather than by duration of incubation since overgrowth of cultures can
cause loss of sensitivity to some mutagens. Cultures will be removed from incubation at a densityof approximately 10* cefls/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 SPOT501100
03-Nov-2000
BioRfiliance Study No. AA37A2L501100.BTL
Page 5 of 10
fit BlOREUANCF
Company Sanitized. Does not contain TSCA CB3
31
H-24616: Bacterial Reverse Mutation Test
DPont-5234
Sponsor Project Number: BioRcliance Study Number;
DuPont-5234 AA37AZ.50nOO.BTL
7.8
Test Substance PrepBratxon
Unless specified otherwise, test substance dilutions will be prepared 'immu-^ii^y prior to use. All test substance dosing will be at room temperature under yellow light
7.9
Treatment of Test System
7.10
OiMhflSfmimiiter(0.5mL)ofS9nuxorShammix, 100 (iL 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 452<>C. When necessary to achieve the taigetconcentrarion or eliminate toxic vehicle effects, aliquotsof other ftan 50 uL of test substance/vehicle/positive control will be plated. The mixture will be vortex mixed and overlaid onto die surface of25mL of minimal bottom agar. After the overlay has solidified, die plates will be inverted aad incubated for anproxunateSy48 to 72 hours at 372C. Plates ftal are not counted immediately following the incubation period will be stored at 2-8C.
Scoring
The condition of die bacterial backgroundtown will be evaluated for evidence of test substance toxicity and precipitate. Evidence of toxfcitywiU be scored relative to the negative control plate and recoided along with the revertant count for that plate. Toxkitywm be evaluated as a decrease m the number of revertam colonies per plate and/or a thinning or disappearanceof the bacterial background lawn. Precipitationwill be evaluated after the incubation period by visual examination
without magnification.
7.11 Tester Strain Verification
Oa the day of use in the mutagcmcity test, all tester strain cultures will be checked for the appropriategenetic mattes cited in 6.0.
8.0 CRITERIA FOR DETERMINATION OF A VALID TEST
The foUowing criteria must be met for the mutagenirity test to be considered valid:
8.1
Tester Strain Integrity
To demonstrate Ac presence of the/?& mutation, all &/>pAftfrarii<m tester strain
cultures must exhibit sensitivityto crystal violet To demonstrate the presence of
the iwB mutation, all SOP^'""""" tester strain cultures must exhibit sensitivity to ultraviolet light. To demonstrate the presence of the uvrA. mutation, all E. coll
tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate
Protocol SPGT501100
03-Nov-ZOOO
BioReliancc Study No. AA37AZ.501100.BTL
PtgcSoflO
1j^
_
_
BlORELIANCF
Company Sanitized. Does rsot contain TSCA CBI
32
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Sponsor Project Number: BioRcliance Study Number:
DuPont - 5234 AA37AZ.501100.BTL
the presence of the pKMIOl plasmid R-factor, tester strain cultures ofTA98 and TA100 must exhibit resistance to ampiciliin.
8.2
Spontaneous Revertant Background Frequency
Based on historical control date, all tester 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; W2(wrA,10-60.
'
8.3
Tester Strain Titcrs
To ensue that appropriatenumbers of bacteria are plated, all tester strain culture tilers must be equal to or greater than 0.3x10'cells per milliliter.
8.4
Positive Control Values
Each mean positive control value must exhibit at least a three fold increase over we respectivemean negative control value (vehicle) for each tester strain.
8.5
Toxicity
A nunmum of three con-toxic dose levels wiH be required to evaluate test data. A
dose level is considered toxte if it causes a>50% reduction in the mean number of revertants per plate relative to we mean negative control vahie (this reduction muift be accompanied by an abrupt dose-dependentdrop in tfaerevertant count) or
a reduction in me backgroundlawn. In the event that less man three non-toxic
dose levels are achieved, me affected portion of the test mil be repeated with an appropriatechange in 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 rcvertants per plate of at least one tester strain over a minimum of two increasing
concentrations oftest substance as specified below:
9.1
Strains TA1535 and TA1537
Data sets will be judgedpositive if die increase in mean revcrtants at me peak of
the dose response is equal to or greater than three times the mean negative control vahie (vehicle).
Protocol SPGT501100
03-Nov-2000
BioReliance Study No. AA37AZJ1100.BTL
Pgc7ofl0
11 BlOREUANCE- .fe. ^y Fafmfly MicrobialoifCil Airctjii
Company Sanitized. Does not contain TSCA CBI
33
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Sponsor Project Number BioReliance Study Number
DuPoat - 5234 AA37AZ.50UOO.BTL
92 Strains TA98,TA100 and WP2iwA
Data sets will be judgedpositive if the increase in mean revertanb at the peak of
the dose response is equal to or greater than two times &e mean negative conlrol value (vehicle).
10.0 REPORT
Areportoftheresultsofthisstudy will be prepared by the Testing Laboratory and will accurately describe all methods used for generation and analysis of the date. The report will include:
Test Substance: identification and CAS K)., if Imown; physical nature and purity, if known; physioocheroicalpropertiesrelevant to the conduct of OK study, if known; stabilityof test substance, if blown.
Solvent/Vehicle: justification for choice of vehicle; solubility and stability of test substance in solvent/vehicle, ifknown.
Strains: strahuus^ number ofcellar per culture; stram characteristics.
Te^ conditions: amouat of test substance per plate widiratioiiale for dose selection and number of plates per concentration; media used; type and compositkm of metabolic activation system, includingacceptabmtymterie; treatment procedures.
Results: signsof toxkity; signs of precipitation; individual plate counts; the mean number of revcrtant colooies per plate and standan) deviation; doae-response relationship, where possible;,statistical analysis, if say; coacaneot negative and positive control date means and standard deviations; historical negative and positive control data with ranges, means and standard deviation.
Discussion of results.
Conclusion.
11.0 RECORDS AND ARCHIVES
Upon completion of die final report, all raw data and reports will be archived by BioReliance, Rockvflle.MD for a periodof no less than 1 year froin the study initiation
date.
12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE
This protocol has been written to comply with OECD Guideline 471 for Testing of Chemicals (Genetic Toxicology: Bacterial Reverse Mutation Assay), adoptedJuly 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 requirements.
Protocol SFOTSOf 100
03-NOV-2000
BioReliance
Study No. AA37AZJOH(IO.BTL
PiBeloflO
19 BIOR-,ELIANCE- .afe^ _ ^y Ftinei)rMiecbiale|i(IAtfciji
Company SanSHzed. Does not contain T8CA CB^
34
H-24616: Bacterial Reverse Mutation Test
DuPont-5234
Sponsor Project Number BioRcliance Study Number:
DuPont-5234 AA37AZ.501100.BTL
13.0
Unless airangements are made to die contrary, unused dosing solutions will be disposed
of following administration to the test system and all residual test substance will be disposedof following finalhation of the report.
REFERENCES
Ames. BX, MeCann, 1. a& Yuoasaid, E. (1975). Methods for detecting carcinogens
and mutegens with the SalmoneKa/mammaSaa-micsosome mutageaicity test Mutation Research 31:347-364.
Green, M.H.L., and Muriel, W.J. (1976). Mutagen testing using tip* reveraion in EschtricUa coll. Mutation Research 38:3*32.
International Conference on Hmnomsation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance OB Specific Aspects of
Regulatory Genotoxicity Tests for Poazmaceimcals. 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 Hannonisation (ICH) of Technical Requirements for Registration of Pharmaceutical* for Human Use. Genotoxidty: A Standard Battery for Genotoxicity Testing ofPhannaceuticais. S2B document recommended for adoption at step 4 of the ICH process on July 16, 1997. Federal Register 62:16026-16030,
November 21,1997.
MeCann, J, and Ames, BX (1976). Detection of carcinogens as mutageos in the
SalmonelSafmicsmome test: assay of 300 chemicals: discussion. Proc. Nad. Acad. Sci.
USA 73:950-954.
MeCann, J., Choi, E., Yamasaid, E. aad Ames, B.N. (1975). Detection of carcinogens as mutagens in the Sa/wow/to/imcrosome test: assay of 300 chemicals. Proc. Nad. Acad.
Sci.USA72:5135-5l39,
Maron, DM and Amea, BX (1983). Revised Methods for the Salmonella Mutagenicity
Test. Mutetion Research 113:173-215.
OECD Guideline 471 for Testing of Chemicals (Genetic Toxicology: Bacterial Reverse Mutation Assay), adoptedJuly 1997.
WHcox, P., Naidoo, A., Wedd, DJ. and Gatehouse, D.G. (1990). Comparison of Salmonella Syphlmwhmf TA102 with Escherichia coll WP2 tester strains. Mutagenesis
5:285-291.
Protocol SPGT501100
03.Nov.2000
BioReliance
Study No. AA37A2.501 IOO.BTL
Page 9 of 10
1ig^ 'y
B_ lOR_,ELIANCE-
PBcrriT Mtcfftlflogic*) Auacfin
Company Sanitized. Does notconfainTSCACBl
35
H-24616: Bacteria! Reverse Mutation Test
DuPont-5234
Sponsor Project Number: BioReIiance StudyNumber:
DuPont-5234 AA37AZ.SOI100.BTL
14.0 APPROVAL
k<^-^ ^gy\Aa^- Sponsor Represcatatrve
0"\ ^^ovAoo o
"l55e------
M.o-c o>. Uoo/\<t.^ O'rirt or Type Name)
f^^SWSQ ~BS^
Protocol SPOTTOI 100
03.Nov.2000
BioReIiance
Study No. AA37AZ501100.BTL
PigelOoflO
fi| BlORELIANCE-
^y rofRrilir M*ciabrlcKl Aelcii
Company Sanitized. Does npl contain TSCA CB1
36