Document 7OZr5GVLjzG9d0jRKoq131XDa
AR226-3093
TRADE SECRET
Study Title H-23005: Bacterial Reverse Mutation Test LABORATORY PROJECT ID: HL-1998-01316
AUTHOR: N. Lawrence Gladnick, B.A. STUDY COMPLETED ON: March 13,1998
PERFORMING LABORATORY:
E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050
WORK REQUEST No.: JiS--Z----wRJ
O.B^O'""""'80"081 Oon^.n'*'"--.0, 089"
Page 1 of 31
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
This study was conducted in compliance with TSCA (40 CFR 792) Good Laboratory Practice Standards and OECD Principles of Good Laboratory Practice (C(81)30(Final), Annex 2), with
the following exception:
1) Expired Oxoid nutrient plates were used in trial 1 for a portion of the phenotype confirmations. The plates were used a few days beyond their expiration, and all supported growth of viable bacteria, as was necessary for the phenotypic confirmations.
2) Treatment solutions or suspensions were not analyzed for identity, composition, uniformity, or stability-of the test and control articles. The procedures used by trained personnel to prepare the treatment solutions intended to ensure:
The accuracy of concentration because control articles were weighed on an analytical balance accurate to 3 decimal places and the test article and the vehicle in which the test and control substances were dissolved were accurately measured with graduated pipettes or flasks;
Uniformity because all treatment solutions or suspensions were mixed prior to administration to the test system; and
Stability because treatment solutions or suspensions were prepared just prior to administration to the test system.
This deviation did not affect the validity or the integrity of the study.
Submitter/Sponsor: E. I. du Pont de Nemours and Company
Study Director:
C^t^^^^^ ^'N Lawre^ce'Gladnick, B.A.
Toxicology Associate
3/^/^ Date
co^s^oo---------^"'
2-
QUALITY ASSURANCE DOCUMENTATION
Haskell Sample Number(s): 23005
Dates of Inspections: Conduct: January 21, 1998
Records, Reports: February 26-27, and March 2, 1998
Dates Findings Reported to: Study Director: March 2, 1998 Management: March 13, 1998
Reported by:
ChapmanDate \ ou^ MM^ <\ W
Ol^^a^-__________
<\P. Jeff
Quality Assurance Auditor
^fl^f^^
^3----------------------
.3-
CERTIFICATION
I, the undersigned, declare that this report provides an accurate evaluation of data obtained from
this study.
Reported Approved and Issued by Study Director:
^9 c^
/^
,,
/)' C^MA^ A^L^^-^^
. i
^//3/^y
N.
B . A . D a t e Lawrenceuladnick,
Toxicology Associate
Company Sanitized. Does not contain TSCA CB1
.4-
TABLE OF CONTENTS
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT................................................ 2 QUALITY ASSURANCE DOCUMENTATION.................................................................................... 3
4 CERTIFICATION......................................................................................................................................
STUDYINFORMATION..........................................................................................................................6
STUDY PERSONNEL....................................;.......................................................................................... 7
8
SUMMARY.................................................................................................................................................
9
INTRODUCTION......................................................................................................................................
MATERIALS AND METHODS............................................................................................................... 9
A. STUDY PROTOCOL..................................................................................................................... 9
B. TEST MATERIALS..................................................................................................................... 10
1. Test Substance.................................................................................................................. 10
2. Negative and Positive Controls......................................................................................... 10
C. TESTER STRAIN CHARACTERIZATION, STORAGE, AND CULTURE............................. 10
1. Tester Strain Characterization.......................................................................................... 10
2. Tester Strain Storage and Culture..................................................................................... 11
D. METABOLIC ACTIVATION SYSTEM..................................................................................... 12
E. CONCENTRATION SELECTION.............................................................................................. 12
F. STABILITY AND CONCENTRATION VERIFICATION......................................................... 12
G. BACTERIAL MUTAGENICITY ASSAYS................................................................................ 13
H. STATISTICAL ANALYSIS ........................................................................................................ 13
L ACCEPTABILITY CRITERIA..................................................................................................... 13
1. Tester Strain Phenotypes and Spontaneous Reversion..................................................... 13
2. Tester Strain Titer............................................................................................................. 14
3. Positive Control Values.................................................................................................... 14
4. Toxicity............................................................................................................................. 14
5. Rejection of Plates, Concentration Levels, or Assays...................................................... 14
J. CLASSIFICATION GUIDELINES............................................................................................... 15
RESULTS AND DISCUSSION............................................................................................................... 16
17
CONCLUSION.........................................................................................................................................
RECORDS AND SAMPLE STORAGE................................................................................................. 17
18
REFERENCES.........................................................................................................................................
19
TABLES....................................................................................................................................................
ABBREVIATIONS FOR TABLES
20
...................................................................................................
TABLE
1 -
Mutagenic Activity in Salmonella typhimurium TA97a in Trial
21 1..................................
TABLE 2 - Mutagenic Activity in Salmonella typhimurium TA97a in Trial 2.................................. 22
TABLE 3 - Mutagenic Activity in Salmonella typhimurium TA98 in Trial 1.................................... 23
TABLE 4 - Mutagenic Activity in Salmonella typhimurium TA98 in Trial 2.................................... 24
TABLE
5 -
Mutagenic Activity in Salmonella typhimurium TA100 in Trial
25 1..................................
TABLE 6 - Mutagenic Activity in Salmonella typhimurium TA100 in Trial 2.................................. 26
TABLE 7 - Mutagenic Activity in Salmonella typhimurium TA1535 in Trial 1................................ 27
TABLE 8 - Mutagenic Activity in Salmonella typhimurium TA1535 in Trial 2................................ 28
TABLE 9 -
Mutagenic Activity in Escherichia coli WP2 uvrA (pKMIOl) in Trial
29 1.......................
TABLE 10 - Mutagenic Activity in Escherichia coli WP2 uvrA (pKMIOl) in Trial 2..................... 30
TABLE 11-Historical Control Data.................................................................................................. 31
Company Sanitized. Does not contain TSCACBI
.5-
STUDY INFORMATION
^"*_
<
jMMBBi Substance Tested:
^^^ ------------------ &-
Synonyms/Codes: J^-23005,
Haskell Number: 23005 Composition:
Known Impurities:
Physical Characteristics: Tan liquid
Stability:
The test substance appeared to be stable under the
conditions of the study; no evidence of instability was
observed.
Sponsor:
E. I. du Pont de Nemours and Company Wilmington, Delaware 19898
U.S.A.
Study Initiated/Completed: January 12, 1998 / (see report cover page)
m-Life Initiated/Completed: January 16, 1998/February 6, 1998
Company Sanitized. Does no! contain TSCA CB!
-6-
STUDY PERSONNEL
Management:
Matthew S. Bogdanffy, Ph.D. Scott E. Loveless, Ph.D.
Study Director: N. Lawrence Gladnick, B.A. Primary Technician: Lisa M. Sulecki, A.S. Technical Publisher: Debra L. Dacey
Company Sanitized. Does not contain TSCA CBj
7-
SUMMARY The active ingredient of the test substance, H-23005, was evaluated in the bacterial reverse mutation assay using Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and Escherichia coli strain WP2 uvrA (pKMIOl) in the presence and absence of an exogenous metabolic activation system (Aroclor-inducedrat liver S9). The test was performed in two phases. The first phase consisted of a preliminary toxicity assay that was used to establish test substance solubility in the selected solvent and the concentration range for the mutagenicity assay. The second phase, consisting of the initial and independent repeat assays, evaluated the mutagenic potential of the test substance. Concentrations of 10, 50,100, 500,1000, 2500, and 5000 /ig/plate, adjusted for the active ingredient of the test substance, using the Salmonella strains and Escherichia coli strain, were evaluated in comparison to negative (solvent) controls. In a second independent repeat assay, these same concentrations, adjusted for the active ingredient of the test substance, were tested in
comparison to negative (solvent) controls.
Under the conditions of this study, no evidence of mutagenic activity was detected in either of
two independent trials. The test substance was negative.
Company Sanitized. Does not contain TSCA CB!
.8-
INTRODUCTION
This study evaluated the mutagenic potential of the test substance, H-23005, in Salmonella typhimurium strains TA97a, TA98, TA100, and TA1535 and in Escherichia coli strain WP2 uvrA (pKMIOl). The bacterial reverse mutation test uses ammo-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs.(l'2^ The Salmonella tester strains are unable to synthesize histidine because of specific point mutations in genes coding for histidine biosynthesis. Additional mutation of the defective gene specific to the tester strain can result in individual bacteria regaining the ability to synthesize histidine. Tester strains TA97a and TA98 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) primarily by frameshift mutagens. Tester strains TA100 and TA1535 are reverted by mutagens that primarily cause base pair substitutions. E. coli WP2 uvrA (pKMIOl) is unable to synthesize tryptophan due to an ochre mutation in the gene required for tryptophan biosynthesis. E; eoli WP2 uvrA ^pKMIOl) insensitivetomutagens'that act at AT base pairs within the trpE gene and may also revert to prototrophy from suppressor mutations at a locus in a tRNA gene.-'^ By comparing the number of chemically induced revertants to the number of spontaneous revertants, the mutagenicity of the test substance can be assessed.
MATERIALS AND METHODS
A. STUDY PROTOCOL
ie study was designed to comply with:
" "w
U.S. EFA", Office of Prevention, Pesticides and Toxic Substances (OPPTS) Guidelines (Subpart H, 40 CFR Part 799.9510 [1997]);
Guidelines of the Organisation for Economic Cooperation and Development (OECD
Guidelines for Testing of Chemicals, No. 471/472 "Bacterial Reverse Mutation Test" (Draft Updated Guideline, February, 1997));
The study design met or exceeded the above guidelines with the following exceptions:
S. typhimurium tester strain TA97a using ICR 191 as a positive control was used as a substitute for strain(s) TA97 or TA1537 (OECD).
E. coli WP2 uvrA (pKMIOl) was the only E. coli tester strain used (OECD).
These exceptions did not affect study validity for the following reasons: 1) Salmonella typhimurium tester strain TA97a has improved growth properties and has been shown to be more sensitive to frameshift mutagens; 2) In the absence of clear evidence to suspect that the test substance was a DNA cross-linking agent, E. coli WP2 uvrA (pKMIOl) was used for an assessment of test substance mutagenicity at AT base pairs. The strains used in this study comply with harmonized guidelines designed to minimize variations among testing procedures worldwide (OECD, OPPTS).
Company Sanitized. Does not contain TSCA CBI
-9-
B. TEST MATERIALS
1. Test Substance
The test substance is H-23005. Additional information regarding the test substance is found on
the study information page of this report. The test substance was assumed stable during this assay and no evidence of instability was observed.
2. Negative and Positive Controls
Based on information supplied by the sponsor and solubility testing, sterile water was chosen as the test substance solvent, diluent, and negative control. The negative control was assumed
stable during this study, and no evidence of instability was observed. Any impurities were not
expected to have interfered with the study. Positive controls included the following:
Positive Control Substances
Chemical Abstracts
Registry Number
_2_-N_i_tr_of_lu_o_re_n_e_(2_N_F_) ______________Serv[iCcAeS # 607-57-8]
2-Aminoanthracene (2AA)
[CAS # 613-13-8]
Sodium azide (NAAZ)
[CAS # 26628-22-8]
ICR 191 Acridine mutagen (ICR 191)
[CAS # 17070-45-0]
9,10-Dimethyl-l,2-benzanthracene(DMBA) .
[CAS # 57-97-6]
JV-Ethyl-JV-nitro-JV-nitroguanidine (ENNG)_____[CAS # 4245-77-6]
Deionized water was the solvent for NAAZ and ICR 191. The solvent for 2AA, 2NF, DMBA, and ENNG was dimethyl sulfoxide (DMSO). The positive controls were assumed stable in this study and no evidence of instability was observed. Any impurities were not expected to have
interfered with the study.
C. TESTER STRAIN CHARACTERIZATION, STORAGE, AND CULTURE
1. Tester Strain Characterization
S. typhimurium tester strains were obtained from Dr. Bruce Ames, Berkeley, CA, USA. E. coli WP2 uvrA (pKMIOl) was obtained from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland, UK.
The characteristics of S. typhimurium tester strains TA97a, TA98, TA100, TA102, TA1535 and E. coli WP2 uvrA (pKMIOl) are as follows:
Company Sanitized. Does net contain TSCA CBI 10-
Str,am
Gene Locus
Excision Repair
LPS
S. typhimurium TA97a
hisD66lQ
AuvrB
rfa
S. typhimurium TA98
hisD3052 Ayvrff
rfa
S. typhimurium TA100
hisG46
AuvrB
rfa
S. typhimurium TA102
hisG42S
(+)
rfa
S. typhimurium TA1535
hisG46
AuvrB
rfa
E. coli WP2 uvi^ (pKMIOl)
trpE
^uvrA
NA
LPS=lipopolysaccharide; NA=Not applicable; (+) proficient
R-factor
(pKMIOl)
Plasmid
Present
Present Present Present Absent Present
pAQI
Plasmid
Absent Absent Absent Present Absent Absent
The deletion (A) in uvrB (a gene that codes for one protein involved in DNA excision repair)
increases bacterial sensitivity to several mutagens/' The uvrB trait is confirmed by demonstrating an increased bacterial sensitivity to ultraviolet light. Because this deletion also
extends through a gene needed for biotin biosynthesis, the bacteria require exogenous biotin to be added to culture media or plates for growth. The rfa mutation causes a partial loss in the
integrity of the lipopolysaccharide (LPS) cell wall so that permeability to large molecules is increased. The presence of the pKMIOl or R-factor plasmid, conferring ampicillin resistance, also enhances an error-prone DNA repair system that is endogenous to these bacteria/75 The pAQI plasmid, conferring tetracycline resistance, is tested at aliquot date for TA102.
Although some testing guidelines require 6'. typhimurium strain TA1537, Salmonella strains TA1537, TA97 or TA97a may be used interchangeably.05Salmonella strain TA97 has been
demonstrated to be more sensitive to frameshift mutagens and the reconstructed strain, TA97a,
was used in place of TA97 due to its improved growth properties/85Although some testing guidelines require E. coli WP2 uvrA, in the absence of data indicating a difference in sensitivity between the plasmid and non-plasmid containing strains, E. coli WP2 uvrA (pKMIOl) was used to assess bacterial mutagenicity/35 E. coli WP2 uvrA (pKMIOl) possess a deletion (A) in uvrA (involved in DNA excision repair), that is confirmed by demonstrating an increased sensitivity to
ultraviolet light.
2. Tester Strain Storage and Culture
All bacterial strains were stored frozen in approximately 8% (v/v) DMSO in Oxoid Nutrient Broth No. 2 at approximately -70C. Overnight cultures were prepared by inoculating 20 mL of Oxoid Nutrient Broth No. 2 with 0.1 mL of a thawed bacterial suspension and incubating at ~37C with shaking. Overnight cultures were then stored on ice until used for mutagenesis assays. Bacterial strain phenotypes were confirmed concurrently with each trial. Results for
reported trials demonstrated the appropriate responses.
Company SanrfeeA Dsuss roloawteffil TSCA CBI
11-
D. METABOLIC ACTIVATION SYSTEM
Because bacterial strains lack many of the enzymes required to convert various promutagens to a reactive state, the assay was performed in the presence and absence of an exogenous metabolic activation system similar to that described by Maron and Ames (1983). The exogenous
metabolic activation system was a cofactor-supplemented post-mitochondrial fraction,
(i.e., 9000 x g; homogenate of 1 g wet liver weight in 3 mL of an approximately 0.15 M KC1 solution) prepared from the livers of young male Sprague Dawley rats treated with the enzymeinducing agent Aroclor 1254 (500 mg/kg i.p.) for five days prior to sacrifice. The Aroclor-induced rat liver S9 (purchased from MOLTOXTM) was characterized for protein content and metabolic activity by the vendor. To confirm the sterility of the exogenous metabolic activation systems, the prepared S9 mixture was streaked on nutrient agar capable of supporting the growth of viable bacteria. The amount ofAroclor-induced rat liver S9 in the
exogenous metabolic activation system was as follows:
4.0 mg Aroclor-induced rat liver S9 protein -10 % [v/v] / mL
exogenous metabolic activation system
The component concentrations in the exogenous metabolic activation system were as follows:
8 mM MgClz 33 mM KC1
5 mM Glucose-6-phosphate (as a sodium salt) 4 mM NADP^^ (as sodium salt) 100 mM Sodium phosphate buffer pH 7.4
E. CONCENTRATION SELECTION
In accordance with testing guidelines, the highest concentration evaluated in this study was approximately 167 mg/mL or 5000 /Ag/plate of the active ingredient of the test substance.
F. STABILITY AND CONCENTRATION VERIFICATION
Solutions of the test substance were prepared immediately prior to treatment and were presumed to be stable under the conditions of the study. Treatment and control solutions were not analyzed for concentration, uniformity, or stability. Top agar was not assayed for stability nor were the concentrations of the test substance or control articles since this assessment was not considered necessary to achieve the objectives of the study. Test substance sterility was confirmed by streaking the stock concentration in triplicate onto the surface of nutrient agar plates capable of supporting the growth of viable bacteria.
Company 8anS$S;tA Does not contain TSCA CBS
12-
G. BACTERIAL MUTAGENICITY ASSAYS
This study consisted of two independent trials. For each trial, three replicates were plated for each tester strain in the presence and absence of the exogenous metabolic activation system (Aroclor-induced rat liver S9) at each test substance concentration. Positive and negative
controls were included for each strain and condition. Treatments with the exogenous metabolic activation system were conducted by adding 0.1 mL of negative or positive control or test substance solution, 0.5 mL of metabolic activation system, and 0.1 mL of an overnight culture containing approximately 1 x 108 bacteria to approximately 2 mL of top agar [0.6-0.7% agar
(w/v) and 0.5-0.6% NaCI (w/v)] containing 0.05 mM L-histidine, D-biotin and L-tryptophan. These components were briefly mixed and poured onto a minimal glucose agar plate (25-30 mL of a 0.4% [w/v] glucose with Davis salts, purchased from MOLTOXTM). Treatments in the absence of the metabolic activation system were as those in the presence of the exogenous metabolic activation system with the exception that 0.5 mL of sterile buffer was used as a replacement for the volume of the exogenous metabolic activation system. After pouring onto the surface of minimal glucose agar plates, the top agar was allowed time to solidify, and the individually labeled plates were inverted and incubated at approximately 37C for about
48 hours. When necessary, plates were refrigerated at approximately 4C prior to evaluation and counting ofrevertant colonies.
Bacterial background lawns were evaluated for evidence of test substance toxicity and precipitation. Evidence of toxicity was scored relative to the concurrent negative control plates
and recorded with the mean revertant count for the strain, condition, and concentration. Revertant colonies for a given tester strain and condition were counted entirely by automated colony counter unless otherwise documented in the study records. Plates were also examined for test substance precipitation. Plates with test substance precipitation that interfered with the automated colony counting were counted manually when possible.
H. STATISTICAL ANALYSIS
Data for each tester strain were evaluated independently. For each tester strain, the average number ofrevertants and the standard deviation at each concentration with or without an exogenous metabolic activation system were calculated.
I. ACCEPTABILITY CRITERIA
An individual trial must have included a negative and positive control and at least 5 concentration levels of the test substance for each tester strain and condition. A data point, concentration level or trial was excluded from analysis when acceptability criteria were not met. The acceptability criteria for reported data was as follows:
1. Tester Strain Phenotypes and Spontaneous Reversion
To demonstrate the presence of the rfa mutation, all S. typhimurium tester strain cultures must have exhibited sensitivity to crystal violet. To demonstrate the presence of the uvrB mutation, all S. typhimurium tester strain cultures must have exhibited sensitivity to ultraviolet light in
^^""^P^ not contain TSCAC,
13-
comparison to strain TA102 (which is proficient in the repair of small amounts of ultraviolet light-induced DNA damage). All S. typhimurium tester strain cultures must have exhibited L-histidine dependent growth and E. coli WP2 uvrA (pKMIOl) must have exhibited L-tryptophan dependent growth. To demonstrate the presence of the uvrA mutation, the E. coli tester strain must have exhibited sensitivity to ultraviolet light. Tester strain cultures of 5'. typhimurium TA97a, TA98, TA100 and E. coli WP2 uvrA (pKMIOl) must have exhibited resistance to
ampicillin.
Based on published data and historical data collected at the testing facility, all tester strain
cultures must have exhibited a characteristic number of spontaneous revertants per plate in the absence of the test substance. The mean revertants per plate in the presence or absence of
exogenous metabolic activation systems was within the following ranges: S. typhimurium strains TA97a (65-163); TA98 (8-40); TA100 (58-192); TA1535 (2-28), E. coli strain WP2 uvrA
(pKMIOl) (101-227). Historical data collected for the testing facility is presented in Table 11.
2. Tester Strain Titer
To ensure that appropriate numbers of bacteria were plated, all tester strain culture titers must
have been approximately 1 x lO^ells/mL.
3. Positive Control Values
Mean positive control values must have exhibited at least a three-fold increase over the respective
mean of the concurrent negative (solvent) control value for each tester strain and condition.
4. Toxicity
A minimum of three non-toxic concentration levels was required to classify the test substance.
A concentration level was considered toxic if it caused a >50 % reduction in the mean number of
revertants per plate relative to the mean of the concurrent negative control value.
5. Rejection of Plates, Concentration Levels, or Assays
A plate may have been rejected if contamination, test substance precipitation or conditions
resulted on a treatment plate that prevented an accurate colony counting.
A concentration level (or a negative control) was rejected if there were less than two data points, or if variability between replicate plates was judged excessive. Excessive variability was
. determined using scientific judgment.
An assay for an individual strain was rejected if the negative control was rejected, ifmutagenic activity was absent on all positive control plates, or if the tester strain failed to exhibit the
appropriate phenotypes.
Only those trials that met the criteria of acceptability were included in this report. All data will
be maintained in the study records. Company Sanitized. Does noi contain TSCA CBI
------
_^_
J. CLASSIFICATION GUIDELINES
A test substance was classified as POSITIVE (i.e., mutagenic) if the average number of
revertants in any strain at any test substance concentration was at least two times greater than the
average number of revertants in the concurrent vehicle control and there was a
concentration-related increase in the mean revertants per plate in that same strain.
A test substance was classified as NEGATIVE (i.e., not mutagenic) if there were no test
substance concentrations with an average number of revertants that were at least two times greater than the average number of revertants in the concurrent vehicle control; or there was no positive concentration-related increase in the mean revertants per plate in that same strain. In consultation with the sponsor, negative results may be confirmed. Results not meeting criteria for positive or negative classification were evaluated using scientific judgment and experience. A study conclusion may be reported as EQUIVOCAL.
Company Sanitized. Does not contain TSCA CB!
15-
RESULTS AND DISCUSSION
The active ingredient of the test substance, H-23005, was evaluated in the bacterial reverse mutation assay using Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and Escherichia coli strain WP2 uvrA (pKMIOl) in the presence and absence of an exogenous
metabolic activation system (Aroclor-induced rat liver S9). The test was performed in two phases. The first phase consisted of a preliminary toxicity assay that was used to establish test substance solubility in the selected solvent and the concentration range for the mutagenicity assay. The second phase, consisting of the initial and independent repeat assays, evaluated the mutagenic potential of the test substance, with the test concentrations adjusted so that the active ingredient was assessed at 5000 /zg/plate.
Solubility and Preliminary Toxicity Assay
Based on information supplied by the sponsor on the test substance evaluation form and preliminary solubility determinations, sterile water was chosen as the test substance solvent, diluent, and negative control. The test substance dissolved completely at 50 mg/mL for the preliminary toxicity assays, and at approximately 167 mg/mL for the two independent trials. The solution appeared off-white and slightly opaque.
The preliminary toxicity assay was used to establish a concentration range over which the test
substance was assayed. Concentrations of 10, 50,100,250, 500,1000,2500, and 5000 p,g/plate of the test substance were evaluated, one plate per dose, in comparison to negative (solvent) controls. Salmonella typhimurium strains TA97a and TA100 and the Escherichia coli strain WP2 uvrA (pKMIOl) in the presence and absence of an exogenous metabolic activation system (Aroclor-induced rat liver S9) were used in the preliminary toxicity assay.
Trial 1 and Trial 2
The results from the preliminary toxicity assay showed no significant decrease in the number of colonies at the highest concentration (5000 ^g/plate) assessed in comparison to negative solvent controls in the Salmonella strains or Escherichia coli strain. Toxicity was not observed at any concentration, and only microscopic precipitate was observed.
The active ingredient of the test substance was to be evaluated at the highest concentration set
forth by applicable test guidelines in comparison to negative controls. Data from both
independent trials were consistent with that of the preliminary toxicity test. No toxicity was observed at any concentration. In the second independent repeat assay, the same concentrations
were tested as in the first assay in comparison to negative controls for all bacteria. Precipitate was observed in all assays with all strains, in plated concentrations as low as 100 ^g/plate and up to 5000 ^ig/plate. Data are presented in Tables 1-10.
Company Sanitized. Does not contain TSCA CB1
-16-
CONCLUSION Under the conditions of this study, no evidence ofmutagenic activity was detected in either of
two independent trials. The test substance was negative.
RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory,
Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware
,..-.----"
17-
REFERENCES
1.
Ames, B.N., et al. (1975). Methods for Detecting Carcinogens and Mutagens with the
iS'o/OTone//a/Mammalian-Microsome Mutagenicity Test. Mutation Research, 31,
347-364.
2.
Maron, D. M. and B. N. Ames (1983). Revised methods for the Salmonella mutagenicity
test. Mutation Research 113,173-215.
3.
Gatehouse, D., et al. (1994). Recommendations for the Performance of Bacterial
Mutation Assays. Mutation Research, 312, 217-233.
4.
Wilcox, P., et al. (1990). Comparison of Salmonella typhimurium TA102 with
Escherichia coli WP2 Tester strains. Mutagenesis, 5, 285-291.
5.
Brusick, D. J., et al. (1980). An evaluation of the Escherichia coli WP2 and WP2uvrA
reverse mutation assay. Mutation Research 76,169-190.
6.
Ames, B. N., et al. (1973). An improved bacterial test system for the detection and
classification ofmutagens and carcinogens. Proc. Natl. Acad. Sci. USA 70, 782-786.
7.
McCann, J., et al. (1975). Detection of carcinogens as mutagens: bacterial tester strains
with R factor plasmids. Proc. Natl. Acad. Sci. USA 72, 979-983.
8.
Levin, D. E., et al. (1982). A new Salmonella tester strain, TA97a, for the detection of
frameshift mutagens. A run ofcytosines as a mutational hot spot. Mutation Research 94,
315-330.
Company Samfeed. Does not contain TSCACBS 18-
TABLES
-19-
Company Sanitized. Does not contain TSCA CB!
ABBREVIATIONS PORTABLES
Evidence for test substance toxicity to the bacteria was documented by recording the appearance of the plates and background lawn using the following key:
TO ' Normal, background microcolony lawn appeared normal.
T 1 Slightly reduced, background microcolony lawn was noticeably thinner.
T2 Moderately reduced, background lawn was markedly thinner resulting in an
increase in the size ofmicrocolonies compared to the vehicle control plate(s).
T3
Severely reduced, background lawn was distinguished by an extreme thinning
resulting in an increase in the size of the microcolonies compared to the vehicle
control plate(s). Microcolonies were seen readily by the unaided eye and were
greatly enlarged relative to controls.
T4 Absent, plate(s) were distinguished by a complete lack of any microcolony lawn
over a majority of the area of the plate(s).
Formation of a precipitate by the test substance was documented using the following key:
PO No precipitate, no precipitate observed.
PI
Microscopic precipitate, precipitate present that did not interfere with
background lawn evaluation or automated colony counting.
P2
Non-interfering precipitate, precipitate present that was visible to the naked eye
that did not interfere with automated colony counting.
P3
Interfering precipitate, precipitate present that required plate to be
counted by hand.
P4
Heavy interfering precipitate, precipitate present that prevented accurate colony
counting and obscured the background lawn requiring plate rejection (R).
Additional abbreviations include the following:
N
Absence of any noteworthy observation
..,..,.----"""
-20-
TABLE 1
MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUM TA97a IN TRIAL 1
Concentration H-23005
(active ingredient)
(Hg/plate)
Revertants
Plate Plate Plate
1
2
3
Average (S.D.)
Observations
A. WITHOUT METABC)LIC^U:TIVATTON
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
126
154
129
130
157
162
137
119
162
144
163
160
154
170
132
151
140
122
141
188
161
118
139
135
136 (15) 150 (17) 139 (22) 156 (10) 152 (19) 138 (15) 163 (24) 131 (11)
ICR 191
2 ^g/plate
2421 2478 2450
2450 (29)
B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9)
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
154
161
143
177
174
147
176
153
145
166
163
159
172
156
174
168
178
166
170
165
156
138
151
134
153 (9) 166 (17) 158 (16) 163 (4) 167 (10) 171 (6) 164 (7) 141 (9)
DMBA
20 p,g/plate
1720 1703 1742
1722 (20)
TO,PO TO,PO TO,PO TO,PO TO.P1 TO,P1 TO,P2 TO,P2
N
TO,PO TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P3
N
Company Sanitized. Does not contain TSCA CBI
21-
TABLE 2
MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUAnA97a IN TRIAL 2
Concentration H-23005
(active ingredient)
(^g/plate)
Revertants
Plate Plate Plate
1
2
3
Average (S.D.)
Observations
A. WITHOUT MET'ABOLIC / ^CTIVATION
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
111
134
149
177
130
121
137
145
140
139
155
146
151
152
141
164
165
168
124
109
124
130
127
105
131 (19) 143 (30) 141 (4) 147 (8) 148 (6) 166 (2) 119 (9) 121 (14)
ICR 191
2 p,g/plate
799 852 786
812 (35)
B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9)
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
135
154
159
150
147
143
147
165
147
187
178
157
157
153
177
162
138
163
140
141
150
136
125
150
149 (13) 147 (4) 153 (10) 174 (15) 162 (13) 154 (14) 144 (6) 137 (13)
DMBA
20 ^g/plate
853
928
849
877 (45)
TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P1 TO,P2 TO,P2
N
TO,PO TO,PO TO.PO TO.P1 TO,P1 TO,P1 TO,P2 TO,P3
N
Company Sanitized. Does not contain TSCA CBI 22-
TABLES
MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUMTA98 IN TRIAL 1
Concentration H-23005
(active ingredient) (pg/plate)
Revertants
Plate Plate Plate
1
2
3
Average (S.D.)
Observations
A. WITHOUT METABOLIC ACTIVATION
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
14
13
15
17
17
24
16
19
17
15
16
12
13
19
13
13
21
18
21
20
20
8
10
11
14 (1) 19 (4) 17 (2) 14 (2) 15 (3) 17 (4) 20 (1) 10 (2)
2NF 25^g/plate
1046 960 1054
1020 (52)
B. WITH METABOLIC ACTIVATION (Aroclor-inducedrat liver S9)
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
29
24
35
32
31
28
29
35
28
29
32
24
25
21
24
28
22
12
20
22
12
16
10
10
29 (6) 30 (2) 31 (4) 28 (4) 23 (2) 21 (8) 18 (5) 12 (3)
2AA
2 p,g/plate
972 1012 1050
1011 (39)
TO,PO TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 T1.P2
N
TO,PO TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P3
N
Company Sanifced. Does not contain TSCACBI
-23-
TABLE 4
MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUMTA9S IN TRIAL 2
Concentration H-23005
(active ingredient)
(^ig/plate)
Revertants
Plate Plate Plate
1
2
3
Average (S.D.)
Observations
A. WITHOUT MET.ABOLIC ACTIVA'riON
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
25
23
23
20
27
27
19
14
12
19
28
14
19
14
23
20
16
22
20
20
23
20
21
23
24 (1) 25 (4) 15 (4) 20 (7) 19 (5) 19 (3) 21 (2) 21 (2)
2NF
25 p,g/plate
1651 1574 1561
1595 (49)
B. WITH METABOLIC ACTIVATION (Aroclor-inducedrat liver S9)
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
18
13
28
19
14
18
18
14
15
26
25
20
19
13
21
27
17
22
24
23
24
15
17
21
20 (8) 17 (3) 16 (2) 24 (3) 18 (4) 22 (5) 24 (1) 18 (3)
2AA
2 ng/plate
748 710 794
751 (42)
TO,PO TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P2
N
TO.PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P2 TO,P3
N
Company Sanitized. Does not contain TSCA CBi -24-
TABLES
MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUMTAIQQ IN TRIAL 1
Concentration H-23005
(active ingredient)
(Hg/plate)
Revertants
Plate Plate Plate
1
2
3
Average (S.D.)
Observations
A. WITHOUT METABCme ALCTIVAT ION
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
115
106
100
93
91
95
103
92
86
126
104
104
137
99
91
90
96
106
103
89
87
95
68
76
107 (8) 93 (2) 94 (9) 111 (13) 109 (25) 97 (8) 93 (9) 80 (14)
NAAZ
2 p,g/plate
617 696 679
664 (42)
B. WITH METABOLIC ACTIVATION (Aroclor1"1-induced irat liver S9)
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
122
98
129
124
107
100
121
115
106
107
100
111
102
128
115
101
115
135
110
120
109
80
99
118
116 (16) 110 (12) 114 (8) 106 (6) 115 (13) 117 (17) 113 (6) 99 (19)
2AA
1 ^ig/plate
362 358 430
383 (40)
TO,PO TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P2
N
TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P2 TO,P3
N
Company Sanitized. Does not contain TSCA CBI
-25-
TABLE 6
MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMUKIUMTAIW IN TRIAL 2
Concentration H-23005
(active ingredient)
(^g/plate)
Revertants
Plate Plate Plate
1
2
3
Average (S.D.)
Observations
A. WITHOUT METABOLIC ACTIVATION
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
102
130
103
125
97
126
118
99
103
128
101
108
143
113
134
135
126
131
123
114
122
101
113
97
112 (16) 116 (16) 107 (10) 112 (14) 130 (15) 131 (5) 120 (5) 104 (8)
TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P1 TO,P2 TO.P2
NAAZ
2|ig/plate
843
854
832
843 (11)
B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9)
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
117
136
139
124
111
149
125
145
127
158
135
116
133
117
139
117
129
130
148
106
117
112
96
102
131 (12) 128 (19) 132 (11) 136 (21) 130 (11) 125 (7) 124 (22) 103 (8)
N
TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P1 TO,P2 TO.P3
2AA
2 p.g/plate
896 914 885
898 (15)
N
Company Sanitized. Does not contain TSCA CBI
-26-
TABLET
MUTAGEMC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUMTA1535 IN TRIAL 1
Concentration H-23005
(active ingredient)
(Hg/plate)
Revertants
Plate Plate Plate
1
2
3
Average (S.D.)
Observations
A. WITHOUT METABOLIC A CTIVA-:riON
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
12
14
14
20
14
6
9
11
15
10
12
14
10
13
9
16
9
15
11
11
10
9
10
12
13 (1) 13 (7) 12 (3) 12 (2) 11 (2) 13 (4) 11 (1) 10 (2)
NAAZ
2^ig/plate
479
561
585
542 (56)
B. WITH METABOLIC ACTIVATION (Aroclor-inducedrat liver S9)
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
6
7
5
4
11
12
12
6
8
13
9
9
8
8
15
10
7
7
12
10
10
9
8
8
6 (1) 9 (4) 9 (3) 10 (2) 10 (4) 8 (2) 11 (1) 8 (1)
2AA
2 (ig/plate
170
183
223
192 (28)
TO,PO TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P2
N
TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P2 TO.P3
N
Company Sanitized. Dees not contain TSCA CBj -27-
TABLES
MUTAGENIC ACTIVITY OF H-23005 IN SALMONELLA TYPHIMURIUMTA1535 IN TMAL 2
Concentration H-23005
(active ingredient)
(Hg/plate)
Revertants
Plate Plate Plate
1
2
3
Average (S.D.)
Observations
A. WITHOUT MET.ABOLIC ACTIVA'TON
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
9
6
10
9
13
6
10
8
9
9
15
9
13
12
8
9
8
8
12
12
10
7
15
13
8 (2) 9 (4) 9 (1) 11 (3) 11 (3) 8 (1) 11 (1) 12 (4)
NAAZ
2^g/plate
628
571
623
607 (32)
B. WITH METABOLIC ACTIVATION (Aroclor-induced rat liver S9)
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
4
9
10
12
16
12
7
11
17
7
9
7
14
13
11
11
9
12
10
9
14
8
8
12
8 (3) 13 (2) 12 (5)
8 (1) 13 (2) 11 (2) 11 (3) 9 (2)
2AA
2 |j,g/plate
79
96
123
99 (22)
TO,PO TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P2
N
TO,PO TO,PO TO.PO TO,P1 TO,P1 TO,P1 TO,P2 TO.P3
N
Company Sanitized. Does not contain TSCA CBJ
-28-
TABLE 9
MUTAGENIC ACTIVITY OF H-23005 IN ESCHERICHIA COLIWP2 uvrA (pKMIOl) IN TRIAL 1
Concentration H-23005
(active ingredient)
(l^g/plate)
Revertants
Plate Plate Plate
1
2
3
Average (S.D.)
Observations
A. WITHOUT METAB(3LIC^^CTIVAlTON
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
137
168
136
134
126
132
131
151
173
162
142
96
137
158
120
131
118
120
140
114
167
150
164
132
147 (18) 131 (4) 152 (21) 133 (34) 138 (19) 123 (7) 140 (27) 149 (16)
ENNG
2 (J,g/plate
1237 1384 1302
1308 (74)
B. WITH METABOLIC ACTIVATION (Aroclor^^induced rat liver S9)
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
182
154
185
154
171
214
151
140
146
142
170
156
126
134
146
175
148
150
156
165
153
133
129
130
174 (17) 180 (31) 146 (6) 156 (14) 135 (10) 158 (15) 158 (6) 131 (2)
2AA
25 p,g/plate
2222 2276 2172
2223 (52)
TO,PO TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P2
N
TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P2 TO,P2
N
29-
Company Sanded. Does not contam TSCA CBI
H-23005: Bacterial Reverse Mutation Test
DuPontHL-1998-01316
TABLE 10
MUTAGENIC ACTIVITY OF H-23005 IN ESCHERICHIA COLIWP2 uvrA (pKMIOl) IN TRIAL 2
Concentration H-23005
(active ingredient)
(^g/plate)
Revertants
Plate Plate Plate
1
2
3
Average (S.D.)
Observations
A. WITHOUT METAB(3LIC^\CTIVA1'ION
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
121
114
106
127
127
137
126
154
131
137
152
137
137
129
128
142
123
137
113
135
116
99
102
101
114 (8) 130 (6) 137 (15) 142 (9) 131 (5) 134 (10) 121 (12) 101 (2)
ENNG
2 i-ig/plate
1342 1296 - 1330
1323 (24)
B. WITH METABOLIC ACTIVATION (Aroclor^^-induced rat liver S9)
0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0
147
153
154
178
167
156
187
188
164
180
172
169
171
176 202
220
182
163
137
144
167
153
160
174
151 (4) 167 (11) 180 (14) 174 (6) 183 (17) 188 (29) 149 (16) 162 (11)
2AA
25 p,g/plate
1030 1161 1214
1135 (95)
TO,PO TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P2
N
TO,PO TO,PO TO,PO TO,P1 TO,P1 TO,P2 TO,P2 TO,P3
N
sinTSCACBI .30-
H-23005: Bacterial Reverse Mutation Test
DuPontHL-1998-01316
TABLE 11
HISTORICAL CONTROL DATA'
Tester Strain Control [Positive Control2]
Exogenous Metabolic Activation System
Mean(S.D.)
Range Minimum-Maximum
S. typhimurium TA100 Negative Negative
Positive [NAAZ-2] Positive [2AA-1] S. typhimurium TA 1535
Negative Negative
Positive [NAAZ-2] Positive [2AA-2] S. typhimurium TA97a
Negative Negative
Positive [ICR 191-2] Positive [DMBA-20] Positive [2AA-1] S. typhimurium TA98
Negative Negative
Positive [2NF-25] Positive [2AA-2] E. coli WP2 uvrA (pRMlOl^ Negative Negative
Positive [MMS-1000] Positive [ENNG-2] Positive [2AA-25] Positive [2AA-250]
Absent Absent
Present Present
Absent Absent
Present Present
Absent Absent
Present
Present Present
Absent Absent
Present Present
Absent
Absent Absent
Present
Present Present
121 128 853 1211
(31) (32) (252) (457)
16 (6) 15 (6) 576 (173) 387 (134)
101 117 1747
'1357
941
(18) (25) (671) (334) (316)
23 26 1391 1656
(7) (7) (364) (544)
156 172 1656 1662 1592 1682
(27) (28) (446) (155) (487) (372)
54- 218 65- 253 339- 2604 94- 2682
7- 46 5- 39
206- 1156 55- 1323
59- 151 67- 196 476- 3209 702- 1683 308- 2035
9- 46
11- 49 567-2373 437- 3114
90- 221 93-255 208- 2453
1353-1918
485--2484 929--2230
'Historical data for tester strains used in the reported study. Data are based on studies reported during the period 1996 up to and including November 26,1997. Data include all control solvents or diluents, metabolic activation
systems based on Aroclor-induced rat liver S9, and all forms of study modification (e.g., plate incorporation, preincubation/gas, waste water). abbreviations for positive controls: NAAZ (sodium azide); 2AA (2-aminoanthracene); 2NF (2-nitrofluorene); MMS (methyl methanesulfonate); ICR 191 (ICR 191 Aci-idine mutagen); DMBA (9,10-dimethyl-l,2benzanthracene), ENNG (//-ethyl-^v-nitro-Jv-nitrosoguanidine). The number following abbreviation is the
microgram (ug) amount per plate or vial used for the positive control.
CompanySanitized. ^s no? contain TSCACBI
-31-