Document GX6zb2Ok560wEEnmK0Q3R9dx
REPORT
EVALUATION OF THE MUTAGENIC ACTIVITY OF T-5869
IN THE AMES SALMONELLA/MICROSOME TEST (WITH INDEPENDENT REPEAT)
NOTOX Project 115965 NOTOX Substance 38196
page 1 of 23 -
T-5869 STATEMENT OF GLP COMPLIANCE
NOTOX Project 115965
NOTOX B.V., Is-Hertogenbosch, The Netherlands
The study described in this report was conducted in compliance with the most recent edition of:
The OECD Principles of Good Laboratory Practice
which are essentially in conformity with: The United States Food and Drug Administration. Title 21 Code of Federal Regulations Part 58.
The United States Environmental Protection Agency (FIFRA). Title 40 Code,of Federal Regulations Part 160. The United States Environmental Protection Agency (TSCA). Title 40 Code of Federal Regulations Part 792.
With the exception that the stability of the test substance in the vehicle is unknown.
Study Director
Ing. E.J. van de Waart Date:
page 2
T-5869 QUALITY ASSURANCE STATEMENT
NOTOX Project 115965
NOTOX B.V., Is-Hertogenbosch, The Netherlands.
Study procedures were subject to periodic inspections and general non study specific processes were also inspected at periodic intervals.
This report was audited by the NOTOX Quality Assurance Unit and the methods and results accurately reflect the raw data.
DATES OF QAU INSPECTIONS/ AUDITS
REPORTING DATES
24-01-1994 26-01-1994 15-03-1994
24-01-1994 26-01-1994 15-03-1994
Quality Assurance Manager
C.J. Mitchell B.Sc.
Date:20 page 3
T-5869 REPORT APPROVAL STUDY DIRECTOR:
MANAGEMENT:
NOTOX Project 115965
Ing. E.J. van de Waart
Date:
Dr. I.C. Enninga Technical Director .........................
Date:22001 cOL>+ L1@l+1qq34l1.34
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T-5869
NOTOX Project 115965
PREFACE Sponsor
Study Monitor Testing Facility
Study Director Technical Coordinator Study Plan
3M Belgium Chemical EBC Canadastraat 11 B-2070 ZWIJNDRECHT Belgium
Mr. R.H. Cox
NOTOX B.V. Hambakenwetering 3 5231 DD 's-Hertogenbosch The Netherlands
Ing. E.J. van de Waart
G. van Oort
Start
: January 25, 1994
Completed : February 04, 1994
TEST SUBSTANCE
Identification
Description
Batch
Purity
Specific Gravity
Instructions for test substance storage
Stability under storage conditions
Expiry date
Stable for at least 4 hours in vehicle
T-5869 Cream solid 2408 95% 1.5
At room temperature in the dark
Stable January 01, 1996
Dimethylsulphoxide: not indicated
VEHICL:E
The test substance was suspended in dimethylsulphoxide of spectroscopic quality (Merck). Test substance concentrations were-prepared directly prior to use.
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T-5869
NOTOX Project 115965
GUIDELINES
The study procedures described in this report were based on the following guidelines:
- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 471: *Genetic Toxicology: Salmonella typhimurium Reverse Mutation Assay,, (adopted May 26, 1983).
- European Economic Community (EEC), Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.14: 'Other Effects-Mutagenicity:Salmonella typhimurium Reverse Mutation Assaym. EEC Publication no. L383 (adopted December, 1992).
ARCHIVING
NOTOX B.V. will archive the following data for at least 10 years: protocol, report, test article reference sample and raw data.
OBJECTIVE
Purpose of the study
The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains to produce histidine-independent strains of these micro-organisms. The assay was conducted in the absence and in the presence of a metabolic system (S9-mix).
Justificationfor selectionof the test system
The Ames test has been shown to be a rapid and inexpensive indicator for the mutagenic activity of a wide range of chemical compounds. The bacterial strains employed were capable of detecting both frameshift and base-pair substitution mutagens.
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T-5869
NOTOX Project 115965
MATERIALS AND METHODS
TEST SYSTEM
Test System Rationale
Source
Salmonella typhimurium bacteria
Recognized by the international guidelines as the recommended test system (e.g. EPA, OECD, EEC).
Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. (TA1535, TA1537 and TA98; 1987 and TA100; 1993)
The characteristicsof the individual strains were as follows:
Strain
TA1537 TA98 TA1535 TA100
R-factor
Histidine mutation
Mutation type
hisC3076
Frameshift
FisD3052/R-factor*
Frameshift
hisG46
Base-pair substitutions
Fi:'-sG46/R-factor*
Base-pair substitutions
plasmid pKM101 (increases error-prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharidecellcoat) Ta-1 : mutation in the galactose metabolism chl : mutation in nitrate reductase bia : defective biotin synthesis G-v-rB: loss of the excision repair system (deletionof the
repair B gene)
ultraviolet-
Stock cultures of the four strains were stored in liquid nitrogen (-1960C). Strains were regularly checked to confirm their histidinerequirement,crystal violet sensitivity,ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
CELL CULTURE
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth
(Oxoid no. 2) and incubated in a shaking bath (37*C, 150 spm), until the cultures reached an O.D. of 0.4 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a
test.
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T-5869
NOTOX Project 115965
Agar plates
Top agar Environmental conditions
Agar plates (o 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purifiedagar (Oxoid, code L28) in Vogel-Bonner Medium E (*), 10 g glucose, 0.5 mg biotin and 0.6 mg histidine. (*) Vogel, H.J. and Bonner, D.M., 1956, Acetylornithinaseof Escherichia coli: partial purificationand some propertiefJ.-. Biol. Chem., 218, 97-106.
Vogel-Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 1200C.
All incubationswere carried out in the dark at 370C. The temperature was monitored during the experiment.
REFERENCE SUBSTANCES
Negative control: The vehicle of the test article.
Positive controls:
Without metabolic activation (-S9-mix):
Strain TA1535
Chemical sodium azide (SA) (Fluka)
TA,1537
9-aminoacridine (9AC)
(Janssen Chimica)
TA98 TA100
daunomycine (DM) (Sigma) methylmethanesulfonate (MMS)
(Merck)
Concentration/plate 1 ug 60 ug
4 ug 650 jig
Solvent Saline Saline
Saline DMSO
With metabolic activation (+S9-mix):
Strain
Chemical
TA1535,
TA1537 2-amincanthracene (2AA)
TA98,
TA100 2-amincanthracene (2AA)
(Sigma)
Concentration/plate Solvent
5 ug
DMSO
0.5 ug
DMSO
Solver@tsfor reference substances Saline = physiological saline (Medital Pharma Ned. B.V.) DMSO = dimethylsulphoxideof spectroscopic quality (Merck).
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T-5869
NOTOX Project 115965
METABOLIC ACTIVATION SYSTEM
Preparation of S9-homogenate:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar or Sprague Dawley rats, which were obtained from BRL, Switzerland.
The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP'S. The rats were injected intraperitoneallywith a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later,they were killed by decapitation; (they were denied access to food for at least JL2hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (DOC) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-1960C).
Preparation of S9-mix:
S9-mix was prepared immediately before use and kept on ice during the test. S9-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose -6-phosphate in 5.5 ml aqua bidest; 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgC12 solution; 1 ml 0.33 M KC1 solution,'and 0.5 ml S9. The above solutions were mixed and filter (0.22 um)-sterilized(apart from the S9fraction, which was added after filter-sterilizationof the S9-mix components).
EXPERIMENTAL PROCEDURE
Selection of dose levels
Selection of an adequate range of doses was based on a preliminary test with strain TA100, both with and without S9-mix. Eight concentrations were tested in triplicate (this preliminarytest was reported as a part of the first experiment of the mutagenesis assay). The highest-concentration of test article used in the subsequent mutagenesis assay was 5 mg/plate.
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T-5869
NOTOX Project 115965
Direct plate incorporation assay
Five different doses (with approximatelyhalf-log steps) of the test substance were tested in triplicate in each strain. The test substance was tested both with and without S9-mix in each strain, in two independent experiments. Top agar in top agar tubes was melted and heated to 450C. The following solutions were successively added to 3 ml top agar: 0.1 ml of a fresh bacterialculture (109 cells/ml)of one of the tester strains, 0.1 ml of a dilution of the test substance in DMSO, and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activationassays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidificationof the top agar, the plates were turned and incubated in the dark at 370C for 48 h. After this period revertant (histidineindependent) colonies were counted.
Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res., 31, 347-364. Maron, D.M. and Ames, B.N., 1983, R-evisedmethods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Erratum, 1983, Mutation Res., 113, 533.
Colony counting
The revertant colonies (histidine independent) were counted automatically with an Artek model 880 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficienttest article precipitate to interfere with automated colony counting were counted manually.
ACCEPTABILITY OF ASSAY
An Ames test is considered acceptable if it meets the following criteria: a) The negative control data (number of spontaneous revertants per plate)
should reasonably be within the laboratory background historical range for each tester strain. b) The positive control chemicals should produce responses in all tester strains which also reasonably are within the laboratory historical range documented for each positive control'substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean. c) The selected dose range should-include a clearly toxic concentration as demonstrated by the preliminary test with strain TA10O.or should extend to 5 mg/plate.
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T-5869
NOTOX Project 115965
DATA EVALUATION AND STATISTICAL PROCEDURES
No formal hypothesis testing was done. A test substance is considered negative (not mutagenic) in the Ames test if: a) The total number of revertants in any tester strain at any concentration
is not greater than two times the solvent control value, with or without metabolic activation. b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic)inthe Ames test if: a) It induces at least a 2-fold, dose related increase in the number of
revertantswith respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.If the test substanceshowed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the firsttest. b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
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T-5869 RESULTS
NOTOX Project 115965
TOXICITY OF THE TEST SUBSTANCE
The survival of the TAIOO culture was determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plates. 'Both in the absence and presence of S9-mix the survival of strain TAIOO was not reduced up to and including test substance concentrations of 5000 ug/plate (Table 1). Based on these data, the test substance was tested up to and including a concentration of 5000 ug/plate in the absence and presence of S9-mix.
THE AMES SALMONELLA/MICROSOME PLATE TEST
Tables 1 and 2 show the results of the Salmonella/microsome plate test With T-5869. All bacterial strains showed negative responses over the entire dose range of the test substance,-i.e.no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.
CONCLUSION
Based'on the results of this study it is concluded that the test substance is not mutagenic in the Ames Salmonella/microsome assay.
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T-5869
NOTOX Project 115965
TABLE I MUTAGENIC RESPONSE OF T-5869 IN THE AMES SALMONELLA/MICROSOME TEST.
Experiment 1
PLATE
Dose (ug/plate)
Mean number of revertant (His*) colonies/ 3 replicateplates ( S.D.) with different
strains of S.typhimurium
TA1535
TA1537
TA98
TA100
Without S9-mix
3 10 33 100 333 1000 33302
50002
6+ 4 7+ 3 5+ 3 84 51
5+ 2
5+ 2 8+ 3 6+ 5 7 4
16 + 3 13 + 1 14 + 3 19 + 6 18 4
136 + 14 140 + 2 122 + 5 140 + 24 142 + 6 139 + 21 143 + 10 133 + 5
Solvent control'
7+ 2
6+ 3
17 + 2
163 + 11
Positive control
265 + 12 359 + 29 621 + 30
1372 + 29
----------------------------------------------------------------------
With S9-mix
3 10 33 100 333 1000 33302
50002
5+ 1 81 8+ 2 92 51
5+ 2 6+ 4 7+ 2 6+ 3 5 1
18 + 5 21 + 2 16 + 5 16 + 5 16 2
130 + 9 133 + 12 136 + 2 118 5 135 + 8 148 + 5 150 + 14 134 + 10
Solvent control'
8+ 3
7+ 1
16 + 2
170 + 11
Positive control 240 8 507 +- 18 441 + 38
601 + 31
----------------------------------------------------------------------
1 0.1 ml OMSO 2 Test-substance precipitated slightly on the plates
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T-5869
NOTOX Project 115965
TABLE 2 MUTAGENIC RESPONSE OF T-5869 IN THE AMES SALMONELLA/MICROSOME TEST.
Expe riment 2
PLATE
Dose (ug/plate)
Mean number of revertant (His+) colonies/ 3 replicateplates ( S.D.) with different strains of S.typhimurium
TA1535
TA1537
TA98
TAIOO
100 333 1000 33302
50002
8+ 4
51 10 + 4 10 4
8+ 2
13 + 5 9+ 1
8+ 3
10 2 10 + 3
19 + 3 18 + 4 20 2 29 + 7 .29 3
171 + 20 159-+ 4 153 ; 14 168 + 17 129 - 9
Solvent control'
6+ 2
7+ a
21 + 2 160 + 8
Positive control
274 +- 28 577 + 89 643 + 54 1143 38
------------------------------------------------------------------------
With S9-mix
100 333 1000 33302
50002
7+ 1 12 + 5 9+ 4 8+ 3 7+ 1
9+ 2 5+ 4 7+ 2 5+ 3 7+ 3
22 + .4 25 + 3 26 + 2 26 + 5 23 + 1
136 + 13 128 + 14 134 + 7 136 + 14 137 ; 5
Solventcontrol'
7+ 2
8+ 4
25 + 3 139 + 8
Positive control
218 + 34 665 ; 86 253 -+ 7 490 +100
-----------------------------------------------------------------------
0.1 ml DMSO 2 Test substance precipitated slightly on the plates
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T-5869
NOTOX-Project 115965
APPENDIX 1
Evidence of test article precipitatean the plates is recorded by addition of the following precipitationcode.
PRECIPITATION CODE
Definition
Characteristics
Slight Precipitate
Moderate Precipitate Heavy Precipitate
Distinguishedby noticeable precipitate on the plate. However, the precipitate does not influence automated counting of the plate. Distinguished by a marked amount of precipitate on the plate, requiring the plate to be hand counted. Distinguished by a large amount of precipitate on the plate, making the required hand count difficult.
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NOTOX Project 115965
APPENDIX 2
Individualplate counts; (followingpages)
Strain Experiment
TA1535 1
----------------------------------------------------------
WITHOUT S9-MIX
plate
123
Dose(ug/plate)
MEAN SD
100 333 1000 33301 50001
11 4 3 10 5 5 8 43 4 7 12 64 5
6+4 7+3 5+3 8+4 5+I
Solvent control
857
7+ 2
Positive control
274 269 251
265 + 12
----------------------------------------------------------
WITH S9-MIX
plate
12 3
Dose(ug/plate)
MEAN SD
100 333 1000 333011 50002
556 887 689 8 11 8
545
5+1 8+ 1 8+ 2 9+ 2 5+1
Solvent control
5 11 9
8+ 3
Positive control
231 242 246
240
8
----------------------------------------------------------
Test substance precipitated slightly on the plates
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NOTOX Project 115965
Strain Experiment
TA1537 1
---------------------------------------------------------
WITHOUT S9-MIX
plate
123
Dose(ug/plate)
MEAN SD
100 333 1000 333011 50001
653 547 7 5 11 12 3 3 4 11 5
5+2
5
2
8+3
6+5
7+4
Solvent control
469
6+ 3
Positive control
326 370 380
359 29
---------------------------------------------------------
WITH S9-MIX
plate
123
Dose(ug/plate)
MEAN SD
100 333 1000 333011 50001
547 926 759
78 565
5+2 6+4
7 ;2 6+3 5;1
Solvent control
866
7+ 1
Positive control
486 513 521
507 -+ 18
---------------------------------------------------------
Test substance precipitated slightly on the plates
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NOTOX Project 115965
Strain Experiment
TA98 1
-----------------------------------------------------------
WITHOUT S9-MIX
plate
123
MEAN SO
Dose(ug/plate)
100 333 1000 3330L 50001
17 18 12 13 12 13 14 17 12 26 16 15 15 16 22
16 + 3 13 t 1 14 + 3 19 t 6 18 + 4
Solvent control
18 19 15
17 + 2
Positive control
637 586 639
@21 -+ 30
-----------------------------------------------------------
WITH S9-MIX
plate
1 -2 3
MEAN SD
Dose(ug/plate)
100 333 1000
33301 50001
14 24 17 20 24 20 21 11 17 20 17 10 18 16 15
18 + 5 21 t 2 16 + 5 16 t 5 16 + 2
Solvent control
17 13 17
16 + 2
Positive control
485 419 420
441 + 38
-----------------------------------------------------------
Test substance precipitated slightly on the plates
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NOTOX Project 115965
Strain Experiment
TA100 1
----------------------------------------------------------
WITHOUT S9-MIX
plate
123
MEAN SD
Dose(ug/plate)
3
10 33 100 333 1000 33301 50001
123 134 151 140 138 142 121 127 117 121 131 167 146 145 136 162 122 132 133 153 144
138 133 128
136 + 14 140 + 2 122 + 5 140 + 24 142 + 6
139 + 21
143 + 10 133 + 5
Solvent control
150 168 170
163 + 11
Positive control
1383 1339 1395
1372 + 29
----------------------------------------------------------
plate
WITH S9-MIX 123
MEAN SD
'Dose(ug/plate)
3
10 33
100 333 1000 33301
50001
140 127 124 120 14 3 135 137 134 136 113 122 118 138 141 126
142 150 151 135 162 153 129 128 145
130 + 9 .133 + 12 136 + 2 118 + 5 135 + 8 148 t 5 150 + 14 134 + 10
Solvent control Positive control
162 182 166 637 586 581
170 + 11 601 + 31
7 --------------------------------------------------
Test substance precipitated slightly on the plates
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NOTOX Project 115965
Strain Experiment
TA1535 2
----------------------------------------------------------
WITHOUT S9-MIX
plate
12 3
MEAN SO
Dose(pg/plate)
100 333 1000 33301 50001
13 5 6 664 7 15 8 5 13 11 6 8 10
8+4 5+1 10 + 4 10 + 4 8+ 2
Solvent control
8 47
6+ 2
Positive control
296 283 242
274 + 28
----------------------------------------------------------
WITH S9-MIX
plate
1 2- 3
MEAN SD
Dose(ug/plate)
100 333 1000 33301 50001
886 9 10 18 4 11 12 10 5 10 8 67
7 +1
12 + 5
9
4
8+ 3
7+ 1
Solvent control
9 58
7+2
Positive control
185 217 253
218 + 34
---------------------------------------------------------
Test substance precipitated slightly on the plates
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Strain Experiment
TA1537 2
----------------------------------------------------------
WITHOUT S9-MIX
plate
123
MEAN SD
Dose(ug/plate)
100 333 1000 33301 50001
13 17 8 8 8 10 6 12 7 9 12 9 8 13 8
13 + 5 9+I 8+3 10 + 2 10 + 3
Solvent control
5 11 6
7+ 3
Positive control
679 516 535
577 + 89
----------------------------------------------------------
WITH S9-MIX
plate
1 2- 3
MEAN SD
Dose(pg/plate)
100 333 1000 33301 50001
11 8 7 10 2 3 8 95 338 7 49
9+2 5+4 7+2 5+3 7+3
Solvent control
3 11 10
8 +4
Positive control
645 760 591
665 + 86
----------------------------------------------------------
Test substance precipitated slightly an the plates
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Strain Experiment
TA98 2
----------------------------------------------------------
WITHOUT S9-MIX
plate
123
MEAN SD
Dose(ug/plate)
100 333 1000 33301 50001
16 20 22. 14 22 19
18 22 19 33 21 32 31 26 29
19 + 3 18 + 4 20 + 2 29 ; 7 29 + 3
Solvent control
22 19 21
21 + 2
Positive control
696 588 644
643 54
----------------------------------------------------------
WITH S9-MIX
plate
1 .2 3
MEAN SD
Dose(ug/plate)
100 333 1000 3330.1 50001
19 20 27 22 27 26 28 25 25 20 27 30 22 24 24
22 + 4 25 + 3 26 ; 2 26 + 5 23 + 1
Solvent control
28 23 23
25 + 3
Positive control
260 246 254
253 + 7
----------------------------------------------------------
Test substance precipitated slightly on the plates
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Strain Experiment
TA100 2
-----------------------------------------------------------
WITHOUT S9-MIX
plate
123
MEAN SD
Dose(ug/plate)
100 333 1000 33301 50001.
165 194 155 160 155 163 152 168 140 160 1.57 188 139 121 127
171 + 20
159
4
153 + 14
168 17
129 + 9
Solvent control
155 156 170
160 + 8
Positive control
1124 1118,1186
1143 + 38
-------------------------------------------:---------------
WITH S9-MIX
plate
1 -2 3
MEAN SD
Dose(ug/plate)
100
333 1000 3330150001
125 132 151 141 129 114 141 134 128 119 145 143
141 132 137
136 + 13
128 + 14
134
7
136 + 14
137
5
Solvent control
138 148 132
139 + 8
Positive control
392 591 488
490 + 100
-----------------------------------------------------------
Test substance precipitated slightly on the plates
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