Document JJ7ebLRDNN5mJ7pZKgMbZbGrO
REPORT
EVALUATION OF THE MUTAGENIC ACTIVITY OF T-5869
IN AN IN VITRO MAMMALIAN CELL GENE MUTATION TEST WITH L5178Y MOUSE LYMPHOMA CELLS (WITH INDEPENDENT REPEAT)
NOTOX Project 115954 NOTOX Substance 38196
page I of 22 -
(,'VE,
NO',,' 9
T-5869 STATEMENT OF GLP COMPLIANCE
NOTOX Project 115954
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 was unknown.
Study Director
Ing. E.J. van de Waart
*7@
Date: (7k4&-"
page 2
T-5869 QUALITY ASSURANCE STATEMENT
NOTOX Project 115954
NOTOX B.V., 's-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
January 24, 1994 February 10, 1994 March 28, 1994
REPORTING DATES
January 24, 1994 February 14, 1994 March 28, 1994
Quality Assurance Manager
C.J. Mitchell B.Sc.
Date:20 L%-q
page 3
T-5869 REPORT APPROVAL STUDY DIRECTOR:
NOTOX Project 115954
Ing. E.J. van de Waart ... .................... DateC/o*&
MANAGEMENT:
Dr. I.C. Enninga Technical Director
...................... Date 20 0 L+ IC45 4
page 4
T-5869
NOTOX Project 115954
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 S.V. Hambakenwetering 3 5231 DO Is-Hertogenbosch The Netherlands
Ing. E.J. van do Waart
C.M. Verspeek
Start
: February 01, 1994
Completed : February 22, 1994
TEST SUBSTANCE
Identification Description Batch Purity Specific Gravity Instructionsfor 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
VEHICLE
The test substance was suspended or dissolved in dimethylsulphoxide of spectroscopic quality (Merck). Test substance concentrations were prepared directlyprior to use. The final concentrationof the solvent in the culture medium amounted to 0.8% (v/v).
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T-5869
NOTOX Project 115954
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. 476: NGenetic Toxicology: In Vitro Mammalian Cell Gene Mutation Tests', (adopted April 4, 1984).
- European Economic Community (EEC), Directive 87/302/EEC. Annex V of the EEC Directive 67/548/EEC, Part 8: Methods for the Determination of Toxicity; "Other Effects-Mutagenicity: In Vitro Mammalian Cell Gene Mutation Test'. EEC Publication no. L133 (adopt d May 30, 1988).
ARCHIVING
NOTOX B.V. will archive the following data for at least 10 years: protocol, report, test article reference sample, all specimens and raw data.
OBJECTIVE
Purpose of_the study
The objective of this study was to evaluate the test substance for its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse-lymphoma cells. The assay was conducted in the absence and presence of a metabolic system (S9-mix). The TK mutational system detected base pair mutations, frame shift mutations and small deletions.
Justificationfor selectionof the test system
L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes. The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions. Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistantto the cytotoxiceffectsof the pyrimidine analogue trifluorothymidine(TFT). TK deficient cells can not incorporate the analogue into its phosphorylatedderivative (nuclebtide);the nucleotides needed for cellular metabolism are obtained solely from de novo synthesis. In the presence of TK, TFT is converted into nucleot7des, which are lethal to the cells. Thus, cells which will survive in culture medium containing TFT are mutated, either spontaneously or by the action of the test substance, giving rise to a TK deficient phenotype. A test article which induces a positive response in this assay is presumed to be a potential mammalian cell mutagen.
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T-5869
NOTOX Project 115954
MATERIALS AND METHODS
TEST SYSTEM
Test System
L5178Y mouse lymphoma cells
Rationale
Recognized by the international guidelines as the recommended test system (e.g. EPA, OECD, EEC).
Source
Dr. A.G.A.C. Knaap, Department of Radiation Genetics and Chemical Mutagenesis of the State Uni.versityof Leiden, The Netherlands (1981). This mouse lymphoma cell line was originally derived from the Fischer L5178Y line, isolatedby Clive (1975)*
Clive, D. and Spector, J.F.S., 1975, Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells, Mutation Res., 31, 17-29. -
Stock cultures of these cells were stored in liquid nitrogen (-1960C). The cultures were checked for mycoplasma contamination.
CELL CULTURE
Cell culture conditions
L517BY mouse lymphoma cells were cultured in F10 complete culture medium. Cultures were incubated in a humid atmosphere containing 5%
C02 in air at 370C. Cell dens*ty was preferably kept below 7 x 105 cellslml. Cells
were exposed to the test substance in F10 complete culture medium without serum, buffered with 20 mM HEPES.
F10 complete culture medium
F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 10% horse serum, Lglutamine (2 mM) and penicillin/streptomycin (50 U/ml and 50 ug/ml respectively).
Selective medium
Selective medium consisted of F10 complete culture medium which contained, in addition 0.33% Agar Noble (Difco) and 5 ug/ml TFT (Sigma),
Cloning medium
Cloning medium consisted of F10 complete culture medium, supplemented with 10% horse serum, which was transformed into a gel by addition of 0.33% Agar Noble.
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T-5869
NOTOX Project 115954
Environmental conditions
All incubationswere carried out in a humid
atmosphere (80-95%) containing 5% C02 in air in the dark at 37*C. The temperature and C02percentage were monitored during the experiment.
REFERENCE SUBSTANCES
Negative control: The vehicle of the test article.
Positive controls:
Without metabolic activation (-S9-mix)* Ethylmethanesulphonate (EMS; CAS no. 62-50-0; purity 98%; Janssen Chimica) (2 mM) was used. EMS causes direct alkylation of DNA.
With metabolic activation (+S9-mix): DimethyTn-itrosamine (DMN; CAS-no. 62-75-9,-purity 99%, Janssen Chimica) (0.5 mM) was used. DMN had to be activated by microsomal enzymes present in the S9-mix, resulting in a methyldiazonium ion which could react with cellular DNA.
Solvents for Reference Substances Hank's balanced salt solution (HBSS) without calcium and magnesium.
Solutions of reference substances were prepared immediately before use.
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 intraperitoneally with 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 12 hours 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).
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T-5869
NOTOX Project 115954
Ames, B.N., Mc Cann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonellt/mammalian microsome mutagenicity test. Mutation Res., 31, 347-364.
Preparation of S9-mix
S9-mix was prepared immediately before.use and kept on ice during the test. S9-mix contained per ml: 1.02 mg MgCl2.6H20; 2.46'mg KCI; 1.7 mg glucose-6phosphate; 3.4 mg NADP; 4 umol HEPES and 0.5 ml S9. The above solutions were mixed and filter (0.22 um)-sterilized(apart from the S9-fraction, which was added after filter-sterilizationof the S9-mix components). Metabolic activation was achieved by adding 0.2 ml liver S9-mix to each ml of cell suspension.
EXPERIMENTAL PROCEDURE
Selection of Dose Levels/CytotoxicityTest
Prior to the actual mutagenicitytest cytotoxicity data were obtained by treating 6 x 106 cells, suspended in 6 ml -ofF-10 medium buffered with 20 mM HEPES, in the absence of serum in a sterile 30 ml centrifuge tube with a range of test substance concentrations in approximately half log steps, both in the absence and presence of S9-mix. The centrifuge tubes were rotated for 3 h on a roller mixer at 370C. After the exposure, the cells were separated from the treatment solutions by 3 centrifugationsteps (115 g, 8 min), each followed by removal of the supernatant and resuspension of the cells, twice in Hank's balanced salt solution and finally in F-10 medium. The cells in the final suspension were counted in an 'Improved Neubauer' haemocytometer. Relative cytotoxicity, expressed as the reduction after approximately 24 h and 48 h growth compared to nontreated control cells, was used to determine a suitable concentration range (4 doses) of the test substance to be applied to cultures prepared for mutagenicity testing. Because the test substance was not toxic and difficultto dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium.
Cleansing
Prior to mutagenicity(and cytotoxicitv)testing,the cells were grown for 1 day in culture medium containing 10-4 M hypoxanthineb,2 x 10-7 M aminopterin and 1.6 x 10--5M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a.recovery period of 2 days on medium containing hypoxanthine and thymidine only. After this period cells were returned to normal medium at least for 1 day before starting the experiment.
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T-5869
NOTOX Project 115954
Mutagenicity test*
The test substance was tested both with and without S9-mix in two independent experiments. 6 x 106 cells (106/ml),or 12 x 106 cells (106/ml) for test substance concentrationsexpected to be strongly toxic, for iach selected dose cells were exposed for 3 h to the test substance in HEPESbuffered Ham's F-10 medium without serum. For this purpose the cell suspensions were placed in 30 ml centrifuge tubes on a roller mixer at 37*C. Solvent and positive controls were included. After exposure, cells were washed twice with HBSS, counted and seeded in F-10 culture medium for expression of the mutant phenotype. The cultures were subcultured at least every other day in order to maintain log phase growth. The expression period for TFT-resistant mutants was 3 days. Immediately after exposure to the test substance 3 x 200 cells of each dose were plated into P90 petri dishes containing 15 ml cloning medium to determine cell survival (cloning efficiency).The cell survival was counted after'10-14 days with the Artek colony counter or the naked eye.
Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G. and Brown, M.M.M., 1979, Validation and characterization of the L517SY/TK Mouse lymphoma muta.genassay system, Mutation Res., 59, 61-108. Amacher, D.E., Paillet, S.C., Turner, G.N., Re@y-V,.A. and Salsburg, D.S., 1980, Point mutations at the thymidine kinase locus in L517BY mouse lymphoma cells. II. Test validation and interpretation,Mutation Res., 72, 447-474. Jotz, R.-and Mitchell, A.D., 1981, Effects of 20 coded chemicals on the forward mutation frequency at the thymidine kinase locus in L517BY mouse lymphoma cells. In: Evaluation of short-term tests of carcinogens. F.J. de Serres and J. Ashby (Eds.), Elsevier-North Holland. Van der Hoeven, J.C.M., Bruggeman, I.M. and Debets, F.M.H., 1984, Genotoxicity of quercetin in cultured mammalian cells, Mutation Res., 136, 9-21. C7ive, D., taspary, W., Kirby, P.E., Krehl, R., Moore, M., Mayo, J. and Oberly, T.J., 1987, Guide for performing the mouse lymphoma assay for mammalian cell mutagenicity, Mutation Res., 189, 143-156.
Mutant selection
After the expression period a total number-of 1.5 x 106 cells,were plated in ten P90 petri dishes, each containing 15 ml selective medium (TFTselection). TK-deficient mutants formed microcalonies in 10-14 days, which were counted with the naked eye. Cloning efficiencies at the time of mutant selection were determined as described'above. The mutant frequency was expressed as the number of mutants per 105 survivingcells.
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T-5869
NOTOX Project 115954
ACCEPTABILITY OF ASSAY
A mutation assay was considered acceptable if it met the following criteria: a) The absolute cloning efficiency of the solvent controls was > 50%. b) At least three of the four doses of the test substance had an acceptable
number of surviving cells (106) analysed for expression of the TK mutation. c) The spontaneous mutant frequency in the untreated or solvent control was < 5 per 105 clonable cells. d) The positive controls (ethylmethanesulfonate and dimethylnitrosamine) inducedsignificant (at least 2-fold) increasesin the mutant frequencies.
DATA EVALUATION AND STATISTICAL PROCEDURES
No formal hypothesis testing was done. A test substance was considered positive (mutagenic) in the mutation assay if: a) It induced at.least a 2-fold increase in the mutant frequency compared
to the solvent control in a dose-dependent manner; and b) The results were reproducible in an independently repeated test.
A test substance was considered negative (not mutagenic) in the mutation assay if:' a) None of the tested concentrations showed a mutant frequency at least
twice that of the solvent control. b) The results were confirmed in an independently repeated test.
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 115954
CYTOTOXICITY TEST/DOSAGE SELECTION
Preliminarysolubility tests indicated that T-5869 precipitatedin the exposition medium at concentrations of 100 pg/ml and upwards. Table 1 shows the results of the preliminary cytotoxicity test with T-5869 in the presence and absence of a metabolic activation system (S9-mix). In the absence of S9-mix T-5869 inhibited the growth of the lymphoma cells in suspension at a test substance concentration of 33 ug/ml by 22% and at 100 ug/ml by 72%. In the presence of S9-mix the test substance did not inhibite the growth of the lymphoma cells in suspension.
MUTAGENICITY TEST
Based on the resultsof the cytotoxicitytest (Table 1), the following
dose range was selected for mutagenicity testing:.
Without S9-m.ix:3 to 100 ug/ml culture medium
With S9-mix
3 to 100 ug/ml culture medium
EMS (2 mM) was used as a positive control in the test without metabolic activation, whereas DMN, (0.5 mM) served as a positive control for the assay with metabolic activation. Tables 2 and 3 show the percentages of cell survival and the mutant frequencies for various concentrations of T-5869. Individual colony counts of cloning and selective plates, and cell
counts during subculturing are listed in Tables 4-9 of the appendix.
Both in the presence and absence of S9-mix the test substance induced no significant increase in the mutant frequency in both independent experiments.
The spontaneous mutant frequencies in the solvent-treated control cultures were within our historical control data range (1.9 + 1.2 in the absence of S9-mix and 1.4 + 0.7 in the presence of S9-mix; incicated are means + S.D. for n=44 and 45-respectively).
Mutant frequencies induced by positive control chemicals were increased by 7- to 9-fold for EMS and by 21- to 23-fold for DMN. It was.therefore concluded that the test conditions were optimal and that the metabolic activation system (S9-mix) functioned properly.
CONCLUSION
In conclusion, T-5869 was found to be not mutagenic in the TK mutation test system under the experimental conditions described in this report.
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T-5869
NOTOX Project 115954
TABLE 1 PRELIMINARY CYTOTOXICITY DETERMINATION IN L517BY MOUSE LYMPHOMA CELLS
Dose (ug/ml)
Cells/ml (x 1C)5)
After After
After
0h
24 h
48 h
Suspension growth Total' % of control
.Withoutmetabolic activation (-S9-mix)
Solvent control 6.1
4.9
6.1
71.2
100
1
6.4
5.1
6.5
82.9
116
3
7.4
4.5
5.9
76.7
108
10
5.7
5.3
5.6
".l
93
33
4.9
4.7
6.2
55.8
78
1002
5.0
2.5
4.1
20.0
28
-----------------------------------------------------------------------
Solvent control 7.9
1
6.6
3
7.0
10
6.4
33
6.3
1002
6.6
With metabolic activation (+S9 mix)
5.7
6.8
119.6
100
6.3
5.7
92.6
77
6.4
6.0
105.0
88
7.7
5.8
111.7
93
6.9
6.2
105.3
88
6.8
6.4
112.2
94
Solvent control DMSO Subculture a 1.6 x 105 cells/ml
Total growth n Cell-countafter 0 h x (24 h cells/ml) x (48 h cells/ml
cells subcuttu-red cells subcultured
directlyafter
after 24 h
treatment
Test substance precipitated slightly in the exposition medium
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T-5869
NOTOX Project 115954
TABLE 2 CYTOTOXIC AND MUTAGENIC RESPONSE OF T-5869 IN THE MOUSE LYMPHOMA L5178Y TEST SYSTEM
EXPERIMENT I
DOSE (jig/ml)
C.E. AT DAY 0 (% OF CONTROL)
C.E. AT DAY 3 (ABSOLUTE %)
MEAN NO. OF MUTANTS PER PLATE
MUTATION
FREQUENCY x 105
Without metabolic activation(-S9-mix)
Solvent
control
100
84
3
98
77
10
101
80
33
82
89
.1001
89
90
2.0
1.6
1.0
0.9
1.2
1.0
1.8
1.3
1.5
1.1
2 mM EMS
104
79
14.1
11.9
--------------------------------------------------------------------------
With metabolic activation (+S9-mix)
Solvent
control
100
88
3
86
82
10
76
84
33
96
87
1001
80
88
0.5 mM OMN
44
49
1.0 1.0 0.8 1.1 1.9
13.6
0.8 0.8 0.6 0.8 1.4
18.5
C.E. = Cloning Efficiency Solvent control = OMSO EMS a Ethylmethanesulphonate DMN u Dimethylnitrosamine
Test substance precipitated slightly in the exposition medium
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T-5669
NOTOX Project 115954
TABLE 3 CYTOTOXIC AND MUTAGENIC RESPONSE OF T-5869 IN THE MOUSE LYMPHOMA L5178Y TEST SYSTEM
EXPERIMENT 2
DOSE
C.E. AT DAY 0
C.E. AT DAY 3
MEAN NO. OF MUTATION
(% OF CONTROL)
(ABSOLUTE %)
MUTANTS PER FREQUENCY
-PLATE
x 105
Without metabolic activation (-S9-mix)
Solvent
control
100
86
3
ill
88
10
102
79
33
58
88
1001
44
95
1.2
0.9
1.1
0.8
0.6
0.5
2.4
1.8
2.9
2.0
2 mM EMS
83
81
9.3
7.7
--------------------------------------------------------------------------
Solvent control
3 10 33 1001
0.5 mM DMN
With metabolic activation (+S9-mix)
100
87
95
81
75
88
91
81
82'
75
33
49
0.7 0.6 0.9 .0.6 0.7
7.9
0.5 0.5 0.7 0.5 0.6
10.7
C.E. c Cloning Efficiency Solvent control m DMSO EMS = Ethylmethanesulphonate DMN = Dimethylnitrosamine
Test substance precipitated slightly in the exposition medium
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T-5869
NOTOX Project 115954
APPENDIX
Individual Colony Counts and Cell Counts during Expression Period
FORMULAS AND CALCULATIONS FOR L517BY MOUSE LYMPHOMA TEST SYSTEM.
Mutant frequency per 105 survivors
Total number of mutant
100
X colonies on selective plates
cloning efficiency
number of seeded cells
Cloning efficiency Average No. of colonies on cloning plates X 100% 200
L5178Y MOUSE LYMPHOMA TEST SYSTEM
- Cell counts during expression period - Cloning efficiency immediately after exposure - Mutation experiments, individual colony counts
Tables 4-6 Tables 7-9
Experiment 1 Experiment 2
Abbreviations used: OMN, dimethylnitrosamine EMS, ethylmethanesulphonate
Solvent control: Dimethylsulphoxide
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T-5869
NOTOX Project 115954
TABLE 4 L5178Y MOUSE LYMPHOMA TEST SYSTEM Experiment 1
CELL COUNTS AND SUBCULTURE DATA
DAY 0
DAY 2
DAY 3
itatal amount ofitatal amount atl I Subculture I
I Subculture
dose leells before
fter I I x 106
ICall countl I
x log
Icell count
(uglal)ltr*atnentx 1061itcletill:ln,t x 1061%1)lTotal aaount3)lclal x 1051%2) ITatal asount4)lclml x 103 1%2)
Without Metabolic Activation (-Sg-nix)
ONSO
6
3
6
la
6
33
6
100
12
EMS
6
5.2
87
3.0
6.6
100
4.0
3.9
65
3.0
6.3
96
4.0
5.5
92
3.0
3.9
ag
4.0
4.5
75
3.0
5.2
79
4.0
6.6
55
6.0
3.2
79
4.0
4.1
do
3.0
6.3
95
4.0
With Metabolic Activation (+S9-aix)
5.a
100
6.5
112
6.0
103
3.9
-102
6.0
103
6.2
207
omso
6
3
6
10
6
33
6
100
6
DMN
6
4.6
so
3.0
4.7
7a
3.0
4.7
78
3.0
3.6
60
3.0
4.3
73
3.0
5.4
90
3.0
(1) calls after treatment x 100% cells before treatment
(2) cell count
x 100%
cell count of control
(3) cell density 0.4 x 105 c/ma
(4) cell density 1.6 x 105 clal
6.6
100
4.0
7.1
108
4.0
5.7
86
4.0
6.9
105
4.0
6.1
92
4.0
2.4
36
4.0
5.9
loo
5.9
100
6.4
IDS
5.3
93
5.4
92
4.8
81
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T-5869
NOTOX Project 115954 I
TABLE 5 L5178Y MOUSE LYMPHOMA TEST SYSTEM - CLONING EFFICIENCY DAY 0
EXPERIMENT 1
Dose (ug/ml)
No. of coloniesl
cloning plate
1
2
3
Mean No. of colonies/plate
Cloning efficiency absolute relative
(% of control)
Without S9-mix
Solvent
control 154 151 169
158
79
100
3
161 149 INF
155
78
98
10
143 174 159
159
80
101
33
122 135 INF
129
65
82
100
167 M
126
141
71
89
EMS
163 165 INF
164
82
104
--------------------------------------------------------------------------
With S9-mix
Solvent
control 187 188 169
181
3
145 167 INF
156
10
125 140 145
137
33
163 167 188
173
100
148 147 138
144
DMN
80 76 84
so
91
100
78
86
69
76
87
96
72
80
40
44
INF Plate infected with fungi
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T-5869
NOTOX Project 115954
TABLE 6 L517BY MOUSE LYMPHOMA TEST SYSTEM EFFICIENCY
SELECTION DATA AND CLONING
EXPERIMENT I
Dose jig/ml
Solvent control
3 10 33 100 EMS
inumber of colonies/Selection platelTotaliNo.of colonies IMeanIMF I
I----------------------------------I No. I/cloning plate I I
I
1 1 2 3 4 5 6 7 8 9 101
11 2 3 1
1
1
1
I
Without Metabolic Activation (-S9-mix)
I
I
I 1 02 42 103 00
20 12 2 1- 0. 4 10 02 22 14 21 16
22 11 01 21 21 13 14
23 11 31 04 13 9 14
2 1 20 2 1 10 0 2 12 1 3 18 3 -2 15 8 10 141
161 173 171 162 153 148 161 159 156 177 177 181 174 188 177 156 161 157
I I 168 1.61 154 0.91 159 1.01 178 1.31 180 1.11 158 11.91
I
With Metabolic Activation (+S9-mix)
Solvent I
control 1 1 0 0 1 3 0 2 1 1 1 10
3
1 0 1 0 0 2 0 2 1 2 2 10
10
1 0 1 02 0 1 02 11 8
33
3 0 1 2 2 0 0 0 0 3 11
100
1 1 2 3 2 0 4 2 2 2 19
DMN
6 13 15 15 16 14 18 13 12 14 136
177 181 170 162 157 170 163 171 INF 182 167 169 171 178 179 94 94 105
176 0.81 163 0.81 167 0.61 173 0.81 176 1.41 98 18.51
MF = Mutant frequency per 105 survivors INF = Plate infected with fungi
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T-5B69
NOTOX Project 115954
TABLE 7 L5178Y MOUSE LYMPHOMA TEST SYSTEM - CELL COUNTS AND SUBCULTURE DATA Experiment 2
DAY 0
DAY 2
DAY 3
itatal amount of[Total amount ofl I Subculture I
I I Subculture I
I
dose
leells b:fore icells after
I I x 106
ICall countl I
x 106
icell count I
(ug/ml)ltreatne t x 1061treatment x 1061%1)lTotal amount3)lclnl x 1051%2)lTotal amount4)lciml x 105 1%2)
omso
6
3
6
10
6
33
6
loo
22
EMS
6
DMSD
6
3
6
10
6
33
6
100
6
DMN
6
without Metabolic Activation (-Sg-mix)
4.3
72
3.0
6.3
100
4.0
3.9
65
3.0
6.4
102
4.0
3.9
65
3.o
5.2
83
4.0
3.1
52
2.9
3.4
54
4.0
3.6
30
3.5
2.0
32
4.0
3.3
63
3.0
5.9
94
4.0
With Metabolic Activation (+S9-mix)
3.8
63
3.0
5.4
loo
4.0
3.9
65
3.0
8.7
104
4.0
4.2
70
3.0
@.5
89
4.0
3.8
63
3.0
6.1
73
4.0
3.7
62
3.0
7.4
as
4.0
4.4
73
3.0
3.2
38
4.0
5.9
100
5.3
90
5.7
97
5.5
93
4.8
al
4.7
80
4.3
100
5.2
121
5.0
116
5.6
130
5.1
119
3.5
81
(1) calls after treatment x 100% cells before treatment
(2) cell count
x 100%
cell count of control
(3) cell density 0.4 x 103 ciml (4) cell density 1.6 x 105 clal
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T-5869
NOTOX Project 115954
TABLE 8 L5178Y MOUSE LYMPHOMA TEST SYSTEM CLONING EFFICIENCY DAY 0
EXPERIMENT 2
Dose (ug/ml)
No. of colonies/ cloning plate
1
2
3
Mean No. of colonies/plate
Cloning efficiency absolute relative
(% of control)
Without S9-mix
Solvent
control 130 140 129
133
67
100
3
135 154 154
148
74
ill
10
140 ;'.121 145
135
68
102
33
70 80 82
77
39
58
100
51 60 62
58
29
44
ENS
112 ;.101 116
110
55
83
-------------------------------------------------------------------------
With S9-mix
Solvent
control 204 180 198
194
3
192 170 191
184
10
152 138 149
146
33
177 179 174
177
100
159 162 155.
159
OMN
69 67 56
64
97
100
92
95
73
75
89
91
80
82
32
33
page 21
T-5869
NOTOX Project 115954
TABLE 9 L517BY MOUSE LYMPHOMA TEST SYSTEM EFFICIENCY
SELECTION DATA AND CLONING
EXPERIMENT 2
Dose
INumber of colonies/Selection platelTotallNo. of coloniesIMean[MF I
-----------------------------I-N-o-.--I/cloningplate I I
1 2 3 4 5 6 7 8 9 101
11 2 3 1
1
Without Metabolic Activation (-S9-mix)
Solvent I
control 1 0 0 1 4 1 1 0 1 2 2 12 161 179 173
3
0 0 0 2 0 2 . 4 2 1 0 11 183 170 172
10
0 0 0 0 0 0 2 0 2 2 6 159 149 165
33
0 4 4 4 3 1 1 4 2 1 24 170 185 171
100
1 4 2 4 6 2 3 3 2 .2 29 192 199 178
EMS
8 5 5 9 8 6 10 13 18 11 93 157 156 173
With Metabolic Activation (+S9-mix)
Solvent I
control 1 3 0 0 0 2 0 1 0 0 1 7 175 181 162
3
1 0 0 3 1 0 0 0 2 0 0 6 161 159 167
10
2 0 0 2 0 2 2 1 0 0 9 180 171 175
33
0 0 0 0 1 0 2 0 1 2 6 164 163 160
100
2 0 1 0 1 1 0 1 1 0 7 152 136 160
DMN
6 8 8 9 6 6 8 9 11 8 79 96 91 106
MF Mutant frequency per 10 survivors
171 0.91 175 0.81 158 0.51 175 1.81 190 2.01. -162 7.71
1 1 I I I I 173 0.51 162 0.51 175 0.71 162 0.51 149 0.61 98 10.71
1
page 22