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25/JUN2.5.1999012:41PPMCFIIIEDI2C2A0L@E 0)<EN,WOLITER-q E?3--1?7:3
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REPORT
EVALUATION OF THE MUTAGENIC ACTIVITY OF' IN AN -IN VIIFTQ MAMMALIAN CELL GENE MUTATION TEST
WITH L5178Y MOUSE LYMPHOMA CELLS (WITH INDEPENDENT REPEAT)
NOTOX ProJect 223450 NOTOX Substance 79742
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4 67331?7:3
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T-6906
NQTOX Project 223458
STATEMENT OF OLP COMPLIANCE
NOTOX S.V., 's-Hertogenbosch. The Netherlands
The study described in this report has been correctly repe'rted and 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 AdministrRtion. Title 21 Code of Federal Regulations Part 58, The Umlted States Environmental ProtectlOn 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.
Study Director: C.M. Verspeek.Rlp
Date:
Management: Ing. E.J. van de Waart Head of Genetic Ecotoxicology
Da e
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25,,JUN1.1-1959.901@@:4[PlPiCFMEDICAL2L))O,EG@L <EN-WOUTERS 87-1-717?3
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T-6906 QUALITY ASSURANCE STATEMENT
NOTOX Project 223458
NDTOX S.V., 's-Hertogenbasch, The Netherlands
This rzpart was audited by the NOTOX Quality Assurance Unit to ensure that
the me had6 and results accuratelryeflectthe raw data,
The dates of QualityAssurance inspection&and audits are given below. During the on-site Inspectionsproceduresapplicabletalt@istype of study were Inspected.
DATES OF GAU INSPECTIONS/ AUDIT$
on-site inspection$
16-01-1998 27-01-1998 19-@02-1996 10-03-1098 19-03-1998
protocol inspection
31-12-1997
report audit
22-04-1998
REPORTING DATES
16-01-2998 27-01-1999 20-02-1998 12-03-IPOB 19-03-1998
31-12-1997
22-04-1998
Head of Quallty Assurance
C.i. MitchelI B.Sc.
. ......... Data:
page 3
25/,,1UN, 5. 1 9 9;J1l2 4 1Pyi PcFlvEDICAL^,20 0"',EN,WOUTERS E7:3317-,3
N't- i
E;04
T-6906
NOTOX Pro@ect 223458
SUMMARY
This report describes the offsets of T-6906 on the induction of forward mutation% at the thymidine-kinase locus (TK-locus) in.L5l78Y mouse lymphoms
cells in the presence and absence of SO-mix.
T-6906was tottedup to concentrationosf 42 and 45 @i'lmlin the absenceof S9-mix in the first and second experiment respectively.T-6906 was tested up to C*nCentratiOnt of 1000 and 750 pg/Ml in the presenct of 8% (vlv) S9. fraction. in the first and second experimentrespectively.
At these dose levels appropriate Cytot*XiCity wag Observed.
In the presence of 59-mix, in both mutation assays the!toxicityof T-6906 in the highest, precipitatingconcentrationis less severe than the toxicity in lower, non-precipitatingconcentrations.The toxicity observed in the cell count during the expression period is not observed in the cloming efficiency of the initiallysurviving cells. These observationswere confirmed in other experiments.Thereforethis rare finding In toxicity is probably a test substance related effect and not an accidentalfinding..
In the absence of $9 metabolic activation In the first.experiment7-6906 did not Induce a significantincreasein the mutant frequencyat the TK.Iocus, in the second experiment, T-6906 showed a 1.7-fold incresie In the mutant frequency at the TK-locus. However. this increase was observed only at one toxic concentration.The relative cloningefficiencyof the remainingcells was 22% compared to the solvent control, whereas 64% of the cells initially survived the treatment. This meant that the actual surv@:ivaolf the rells after treatment was only 10% (22% of 4A%). Since, no dose reg.ponserelationshipwas observed, the effect was observed only in one experimen'tand the increase was less than two-fold compared to the solvent control, thit increasewas considered not biologically relevant and 7-6906 Is conslideredto be riot mutagenic in the absence of $9-mix.
In the presence of 6% (Y/v) S9 metabolic activation,T-0906 showed 22- and 3.2-fold increases In the first and second experiment r@spectively in the mutant frequency at the TK-Iocus. In the first experiment, the Increase was observed only at one very toxic concentration.A severetdelayIn the cell growth at the concentrationof 1000 op/mi wet observed after 48 and 72 hours of subculture compared to the call growth of the solvent control and a redvction of 01% was observed in the cells plated for the determinationof tile cloning efficlancyof day 3.,In the second experiment,the Increase in the mutant frequency was observed at test substance concentrationsof 250 and 750 pe/ml. however no dose response relationship was observed. Since the increases were observed in two experiments and the increases in t@e second experiment were both in toxic and non toxic concentrations,these Increasesare considered to be biologicallyrelevant. Therefore,T-6906 is considered mutagenic in the presence of 69-mix.
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T-6906
NOTOX Project 223458
In the first experiment,both the number of small and.l,argemutant colonies were Increased compared to the solvent control values.'In the second experimert, mainly the large mutant colonies were increr'asecdompared to the solvent control values. The small colonies can be associlatedwith the induction of chromosomal mutations. The large coloniestppeared to result from mutants with single gone mutations (substitution&,deletions of base-pairs) affecting the TK gene,
mutant frequencies induced ty positive control cnamicais were Increased by 8.6 and 1S.7-fold for EMS in the first and second experiment respectively, and by 10.5- and 21.6-,Foldfor DMN, in the first and second experiment respectively. It was therefore concluded that the test conditions.both in the absence and presence of 69-mix, were appropriate for the detectionof a mutagenic response end that the metabolic activation system (89.mlx)functionedproperly.
It Is concluded that T-6906 is mutagenic in the mouse lymphome L517BY test system under the experimental conditions described in this report.
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(-)@..U)t U,)<F-N,iJOUTERS 4 87331773
NU.U'l'13 N F. J'/ rPE)s
T-6906
NOTOX Project 223458
PREFACE Sponsor Study Monltor
Testing Facility
Study Director Technical Coordinator Study Plan
3M USA
St. Paul, MINESOTA U.S.A.
55133-3220
Mr. R.H. Cox
3M Belgium Nv - Chemical EBC
Conadestraat 11 B-2070 ZWIJNDRECHT Belgium
NOTOX D.V. Hambakenwetering 3 5231 DO 's-Mortogemboach The Netherlands
C,M. Verspeek-Rip
G. van Oort
Start Completed
12 January 1998 14 April 1996
TEST SUBSTANCE
Identification Description Batch Purity Test substance storage
Stability under ctorage conditions Expiry date
Stabilityin vehicle
T-6906 Creme coloured solid 040016 97% At room temperature In the dark
Stable 31 December 1998 Dimethyloulphoxidea;t least96 h
The sponsor is responsiblefor the completenessand GLP@'ComPliancoef all test substance data.
ARCHIVING NOTOX B.V. will archive the protocol,report, test artlc'lereference sample and raw date for at least 10 years.
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jjN. 9 5 p l@ KED[CrlL I'@10",E 0'-@
T-6906
NOTOX Project 223458
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: 'Genetic Toxicology: In Vitro Mammalian Cell Gene Mutation Tests', (adopted April 4, 1984). European Economic Community (EEC), Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; 'Mutagenicity: In Vitro Mammalian Cell Gene Mutation Test'. Directive 87/302/EEC, EEC Publication no. L133 (adopted May 30, 1988) and Directive 96/54/EEC, EEC P,Ublicationno. L248 (adopted September 30,
OBJECTIVE
Obiective
The objective of this study was to evaluate the mutagenic potency of T-6906 by testing its abllity.to induceforward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.
Background of 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+Ito TK'/-) are resistantto the cytotaxic effects of the pyrimidine analogue trifluorothymidine(TFT). TK deficient cells can not incorporatethe analogue Into Its phosphorylatedderivative (nuclectide);the nuclectldesneeded for r-ellularmetabolism are obtained solely from de novo synthesis, In the presence of TK, TFT is converted Into nucleotides,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. Furthermore, by applying the TFT-selection procedure it is possible to discriminate between the two different classes of TFT-resistant mutants (small and large colonies) which are believed to represent the different types of leslons induced in the DNA by the test substance. A test article which induces a positive response in this assay is presumed to be a potential mammalian cell mutagen.
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7-6906
NOTOX Project 223458
MATERIALS AND METHODS
TEST SYSTEM
Test System
L517BY mouse lymphoma cells
Rationale
Recommended test system in international guidelines (e.g. EPA, OECD, EEC).
Source
Dr. A.G.A.C. Knaap, Department of Radiation Genetics and Chemical Mutagenesis of the State Universityof Leiden,The Netherlands(1981). This mouse lymphomacell line was originally derived from the Fischer L5178Y line, isolated by Clive (1975).
Stock culturesof these cellswere stored in liquidnitrogen(-196'C). The cultures were checked for myceplasma contamination.
CELL CULTURE
F10 complete culture medium
FID complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco),supplementedwith 10% (vlv)horse serum, L-glutamine (2 mM) and penicillin/streptomyci(n50 U/ml and 50 Ag/ml respectively).
Cell cultureconditions
L517BY mouse lymphoma cells were cultured in FIO complete culture medium. Cell density was preferablykept below 7 x 105 cells/ml,
Exposition medium
Cells were exposed to T-6906 for 3 hours in F10 culture medium, buffered with 20 mM HEPES or for 24 hours in F10 completeculture medium.
Selective medium
Selective medium consisted of FIO complete culture medium, supplementedwith IOY,(v/v)
horse serum and I_Ulml TFT (Sigma).
Non-selective medium
Non-selective medium consisted of FIO complete culture medium, supplementedwith 10% horse serum.
Environmental conditions
All incubationswere carriedout in a humid atmosphere (80-9SYe)containing 5% C02 in air in the dark at 37*C. The temperature and C02percentage were monitored during the experiment.
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JJN2.5'.9.991":36PI,@ MEDICAL @"LO1,E 0'-"
T-6906
j
NOTOX Project 223458
TREATMENT OF THE TEST SUBSTANCE
The test substance was dissolved in dimethylsulphoxide of spectroscopic quality (Merck).Test substance concentrationswere prepared directly prior to use.
REFERENCE SUBSTANCES
NeQative control: The vehicle of the test article, being dimethylsulphoxide.
Positive controls:
Solvents for Reference Substances Hank's balanced salt solution without calcium and magnesium.
Solutions of reference substances were prepared immediately before use.
Without metabolic activation-f-S9-mix): @thylmethanesulphonate (EMS; CAS no. 62-50-0;-purity 98%; Janssen Chimica) (1 mM) was used. EMS causes direct alkylation of DNA@
With metabolic activation-L+S9-mix); Dimetbylnitrosamine (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.
METABOLIC ACTIVATION SYSTEM
Preparation of S9-fraction
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River Wiga, Sulzfeld, Germany-
The animals were housed at NOTOX In a special room under standard laboratory conditions, as described in the SOP'$. 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 12 hours preceding sacrifice).
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36 PI-@ MEDict@L
T-6906
NOTOX Project 223458
The livers of the rats were removed aseptically, and washed in cold (O'C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EOTA. 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.fraction)was transferred into sterile ampules, which were stored in liquid nitrogen (-196-C).
ELeparatlon of S9-mix
S9-mix was prepared immediately before use and kept on lee. 89-mix contained per ml: 1.63 mg MgC12-6H 0- 2.46 mg KC1; 1.7 mg glucose-6phosphate; 3,4 mg NADP; 4 pmol HEPFS' The above solution was filter(0.22 Am)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fractio' n (batch 97-8 or 98-1) was added (50% (viv) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 ml S9-mix to each ml cell suspension. The concentration of the S9-fraction in the exposition medium was 8% (V/V).
EXPERIMENTAL PROCEDURE
Cleansing
Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for.4 day in FIO complete culture medium containing 10*4 M hypoxanthine, 2 x 10 M aminapterin and 1.6 x 10-5 M thymidine (HATmedium) 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.
Dose range finding test
In order to select appropriate dose levels for mutggenicitytesting. cytotoxicitydata were obtained by treating 6 x 10 cells, suspended In 6 ml exposition medium, In a sterile 30 ml centrifuge tube, with a number of test substance concentrations increasing with approximately half log steps. T-6906 was tested in the absence and in the presence S9-fraction. The centrifuge tubes were rotated for 3 hours on a roller mixer at 370C.
The highest tested concentrationwas determined by the solubility of the test substance in the culture medium,
After 3 hours exposure, the cells were separated from the treatment solutions by 3 centrifugationsteps (215 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 complete culture medium. The cells in the final suspension were counted with a microscope with an 'Improved Neubauer' hasmacytometer.
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I,@36?1@ KEDICAILL2^0'ED@L
T-6906
0.J 3
NOTOX Project 223458
The cells were seeded for determination of the cloning efficiency (CE). For that, the cell suspensions were diluted and seeded in wells of a 96well dish. 1 cell was added per well (2 x 96-well microtiterplatesper concentration) in non selective medium. After 9 days the plates were scored for empty wells, i.e. wells in which no visible colonies were present. The microtiterplateswere scored with the naked eye or with a microscope at a magnification of 4x.
The calculation of the cloning efficiency was determined by deviding the number of empty wells by the total number of wells. This value.was called P(O), the zero term of the Poisson distribution. P(D) = number of empty wells/total number of wells.
CE = -ln P(O)Inumber of cells plated per well. The P(O) and the CE are expressed as percentage,
Mutagenicity test
Treatment of the cells T-6906 was tested both in the absence and ip the presepce of S9-mix in two independent experiments.Per culture 9 x lou cells (1001ml)were usc ed. If t;sl substance concentrations were expected to be toxic, 16 x 106 ells
0 iml) were used per culture.
The concentrationof the S9-fraction in the exoositico medium was 0% (v/v)
Cell cultures in the absence of $9-mix weee'exposed for 24 hours to the test substance in exposition medium (Flo complete culture medium) and the cell cultures In the presence of S9-mix were exposed for 3 hours to the test substance in exposition medium (Flo culture medium buffered with 20 mM KEPES). The cell cultures were placed in 30 ml centrifuge tubes on a roller mixer at 37'C. Solvent and positive controls were included.
After exposure, cells were separated from the exposition medium by 3 centrifugationsteps (115 g. 8 min), each followed by removal of the supernatant and resusponsion of the cell&, twice In Hank's balanced salt solution and finally in F-10 complete culture medium.
The cells in the final suspension were counted with a microscope with an 'Improved Neubauer' hatmocytometer.
Cloning efficiency directl-y--aftetreatment Immediately after exposure to the test substance 2 x 96-well microtiter plates with 1 celliwellwere plated for each dose to determine the survival of day 0. The plates were scored after 9 days incubationfor determination of'the CE.
Expression Period and determination of the mutant fre-quencx For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the 24 hours exposure time for the cell cultures without S9-mix. The cells of the cell cultures with S9.mix were culturedfor 3 days af er the 3 hours exposure time.'During this culture period at least 4 x 10i cell& (if possible)were subcultured.Three days after the start of the treatment with the test substance the cells were plated for determination of the CE (CE3) and the mutant frequency (MF).
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RtJ iC IL, 1-41'U t U
T-6906
NOTOX Project 223458
At least four doses of the test substan*cewere selected for the mutation assay, both in the absence and presence of S9.mix.
For determination of the CE3 the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 x 96-well microtiter plates/concentration)in non selective medium.
For determination of the MF a total number of 5.76 x 10-5cells/ concentrationwere plated in three 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection).The micratiter plates for CE3 and MF were incubated for 12 days at 37'C in humified air with 5% C02-
After this incubationperiod the plates for the TFT-selectionwere stained for 2 h, by adding 0.5 mg/ml MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5. diphenyltetrazoliumbromide, Sigma) to each well, The plates for the CE3 and MF were scared with the naked eye or with the microscope.
The calculation of the mutation frequency was determined, as follows: MF a i-In P(0)12000)/CE3The mutant frequency was expressed as the number of mutants per los surviving cells.
DETERMINATION OF THE MUTANT COLONIES
The colonies were divided in:
Small colonies Large colonies
Morphological dense colonies with a sharp contour and with a diameter less than a quarter of a well. Morphological less dense colonies with a hazy contour and with a diameter larger than a quarter of a well.
A well containing more than one small colony was classified as one small colony.
A well containing more than one large colony was classified as one large colony.
A well containing one small and one large colony was classified as one large colony.
The large colonies appeared to result from mutants with single gene
mutations (substitutions,deletions of base-pairs)affecting the TK gene.
The small colonies can be associated with the induction of chromosomal mutations.
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MhUl@PL'f'LIU @t U
T-6906
NOTOX Project 223458
ACCEPTABILITY OF ASSAY
A mutation assay was consideredacceptabl'eif it met the following criteria: a) The absolute cloning efficiencyof the solvent controls was > 50%, b) At leastat threeof th four doses of the test substance,an acceptable
number of survivingcelels (106)could be analysedfor expressionof the TK mutation. c) The sponttngousmutant frequency in the untreated or solvent control was < 10 per 0 clonablecells. d) The positive controls (ethylmethanesulfonateand dimethylnitrosamine) inducedsignificant(at leastthree-fold)increasesIn the mutant frequencies. e) In the absence of $9-mix the selected dose range had to includea clearlytoxic concentration.
DATA EVALUATION AND STATISTICAL PROCEDURES
No formal hypothesis testing was done. A test substancewas consideredpositive (mutagenic)in the mutation assay if: a) It inducedat least a 3-fold increasein the mutant frequencycompared
to the solvent controlIn a dose-deptndent manner; and b) The resultswere reproduciblein an i' ndependentlyrepeatedtest.
A test substance was considerednegative (not mu-ta@e6ic)in the mutation assay if: a) None of the tested concentrationsshowed a mutant frequencyof at least
three-foldcomparedto the solvent control. b) The results were confirmed*inan independentlyrepeatedtest.
The precedingcriteriawere not absolute and other modifyingfactors might enter Into the final evaluationdecision.
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MEDICAOLL20",0E16
'4
T-6906
NOTOX Project 223458
RESULTS
DOSE RANGE FINDING TEST
T-6906 precipitated in the exposition medium at a concentration of 1000 pp/ml. Therefore, this concentration was used as the highest test substance concentration,
In the dose range finding test, L5178Y mouse lymphoma cells were treated wit;l a test substance concentration range of 3 to 1000 gg/ml in the absence and in the presence of S9-mix (Table 2).
In Table I the first column, contains the relative cell counts (expressed as the percentage of the control culture) of cultures directly after 3 hours exposure to the various concentrations of the test substance, whereas the second column expresses the relative cell survival directly after exposure as the percentage cloning efficiency,of the remaining cells (individual plate counts are listed In Appendix 1, Table 4).
In the absence of S9-mix, the number of cells counted after 3 hours treatment was reduced at a test substance concentration of 333 pglml by 67%, whereas the number of cells counted at the concentration of 1000 gg/ml was reduced by 52% compared to the solvent control. The initially surviving cells at all concentrations tested showed no clear reduction in the cloning efficiency compared to the cloning efficiency of the solvent control.
In the presence of 39-mix, no reduction was observed in the cell count of the remaining cells directly after treatment. The initially surviving cells showed no clear reduction in the cloning efficiency compared to the solvent control.
In the absence of S9.mix the selected dose range did not include a concentration with toxicity up to 90% compared to the solvent control. Therefore, cell cultures in the mutagenicity test, in the absence of S9-mix, were exposed for 24 hours to the test substance in exposition medium (FIO complete culture medium).
MUTAGENICITY TEST
Tables 2 and 3 show the percentages of cell survival and the mutant frequencies for various concentrations of 7.6906. Individual colony counts of cloning and selective plates, and cell counts during subculturing are listed in Tables 5-12 of Appendix 1.
Exetriment I Based on the resultsof the dose range flnding test and the solubilitytest. the following dose range was selected for mutagenicitytesting, in the absence of S9 metabolicactivation:10, 33, 100, 175, 333, 560 and 1000 jlglml expositionmedium. However,the dose levels loo pgiml and upwardswere too toxic for furthertesting;since not enough dose groupswere leftfor mutagenicity testing,the experiment was repeated.
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999123:7FI@ MEDICALL@4-o'E O'L
T-6906
NOTOX Project 223458
The following dose range was selected for mutagenicitytesting:
Without S9-mix: 10, 18, 33, 42, 56. 75 and 100 pg/ml exposition medium With S9-mix : 33, 100, 333 and 1000 pg/ml expositionmedium
Evaluationof the toxicity In the absence of 69 metabolic activation,the dose levels of 56 pg T-6906/ml and upwardswere too toxic for further testing.In the presence of S9 metabolicactivation,no severe toxicitywas observedand all dose levelswere evaluated.
The dose levels selectedto measure mutant frequenciesat the TK-locus were: Without S9-mix: 10, 18, 33 and 42 gg/ml expositionmedium With S9.mix 33, 100, 333 and 1000 jig/mlexposition medium
In the absenceof S9-mix (Table 2), the cell count of all concentrations tested showed no reductioncompared to the cell count of the solvent control after 24 hours treatment.The cloning efficiencyof the remainingcalls was comparableto the solvent control even at the highesttested dose. -
In the presenceof S9-mix, after 3 hours treatment,the cell count of the test. substance concentrationof 1000 gg/plate showed no reduction compared to the cell count of the solvent control,whereasthe cells countedat the concentrationof 333 pg/ml showed a reduction of 44% compared to the solvent control.The cell count after48 hours of subculture(Table6) at test substance concentrationsof 333 and 1000 pg/ml showed reductions of 61% comparedto the cell count of the solventcontrol.The cloningefficiencyof the remainingcells (Table2), at the test substanceconcentrationof 333 gg/plate was reduced by 44%, the other dose levels showed no reduction In the
cloningefficiency.
Further Investigationshowed that at a concentration 7, 50@
6906
already precipitatedin the expositionmedium. Therefjo@re ig'tmlse-was
selectedas highestdose in the absence and presenceof 89-mix, all other
concentrationsdid not precipitateIn the expositionmedium.
ExReriment 2 Based an the resultsof experiment1 and the solubilitytest, the following dose levelswere selectedfor mutagenicitytesting.
Without S9-mix: 10, 20, 30, 40, 45, 50 and 55 gg/ml expositionmedium With S9-mix: 10, 33, 100, 175, 250, 375, 500 and 750 pg/ml exposition medium
Evaluationof the toxicity
In the absence of S9 metabolicactivation,the dose level of 10 ;Ag/mlwas not selected for mutant frequency measurement, since this concentrationwas considerednon toxic and not relevant for mutant frequencymeasurement, The dose levels of So and 55 ;ig/mlwere too toxic. In the presence of S9 metabolic activation,the dose level of 10 pg T-6906/mlwas not selectedfor mutant frequency measurement,since this concentrationwas not relevantfor mutant frequency measurement.The dose levels of 375 and 500 iLg/mlwere not selected for mutant frequency measurement,since these concentrationswere too toxic,
page 15
38Pl@ MEDICAL @LZ)O
T-6906
NOTOX Project 223458
The dose levels selected to measure mutant frequencies at the TK-locus were: Without S9-mix: 20, 30. 40 and 45 gg/ml exposition medium With S9-mix : 33, 100, 175, 250 and 750 pg/ml exposition medium
In the absence of S9.mix (Table 3), after 24 hours treatment the cell count of the test substance concentration of 45 gg/ml was reduced by 56% compared to the cell count of the solvent control. The cloning efficiency of the remaining cells directly after treatment of 45 gg/ml showed a reduction of 78%.
In the presence of 59-mix, the cell count of the test substance concentration of 750 jug/plateshowed no reduction compared to the cell count of the solvent control, whereas the cells counted at concentrations of 100, 175 and 250 pg/ml showed reduction& of 62, 49 and 40% respectively. In the cloning efficiency of the remaining cells of the test substance concentration of 750 jig/plateno reduction was observed compared to the cloning efficiency of the solvent control, whereas the cloning efficiency of the concentration of 175 pg/ml showed a reduction of 66%.
In the presence of S9-mix, in both mutation assays the toxicity of T-6906 In the highest,precipitatingconcentrationIs less severe than the.toxicityIn lower, non-precipitatingconcentrations.The toxicity observed in the cell count during the expression period is not observed in the cloning efficiency of the initially surviving cells. These observationswere confirmed in other experiments (Tables 13-16). However, the results of the mutagenicitytests were not acceptable and.the experiments were not used to evaluate the mutagenic activity of T-6906, the individual colony count of day 0 and the cell count during the expression period are reported in Appendix 2, Therefore this rare finding in toxicity is probablya test substancerelated effect and not an accidental finding.
Evaluation of muta_qoniciu
The spontaneous mutant rf@ri)eaeqrqutruseen@ancy tnn he solvent-treatedcontrol cultures in
the absence of S9-mix (first a we
s ond experiment) and in the presence of
89-mix (first experiment)we iLLs outside the maximum value of the
historicalControldata r n er (0s..77-andoi.8 x 105 (mean 3.0 x 105) in the
absence of S9-mix) and 0 aenaec .6-l61v)x 1 (mean 2.9 x 105) in the presence
of S9-mix);for n;66 an
reans'py(eoctively). But these values were below the
10 mutants per 10 clonable cells. which was defined in the 'Acceptability
of assay' as an acceptable background for this assay. Therefore, this test
is adequate and considered to be acceptable.
The spontaneous mutant frequency in the solvent-treated control culture, in the second experiment in the presence of S9-mix, was between the minimum and maximum value of the historical control data range ((1.0- and 6.6 x 105 (mean 2.9 x 10-5)in the presence of S9-mix); for n=63).
Mutant frequencies induced by positive control chemicals were increased by 8.6 and 15.7-fold for EMS in the first and second experiment respectively, and by 10.5- and 11.6-fold for DMN, in the first and second experiment respectively(Table 2 and 3). It was therefore concludedthat the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (SP-mix) functioned properly.
page 16
T-6906
NOTOX Project 223458
In the absence of $9 metabolic activation in.the first experiment (Table 2) T-6906 did not induce a significant increase in the mutant frequency at the TK-locus. In the second experiment (Table 3), T-6906 showed a 1.7-fold increase in the mutant frequency at the TK-locus. However, this increase was observed only at one toxic concentration.The relative cloning efficiency of the remaining cells was 22% compared to the solvent control, whereas 44Vsof the cells initially survived the treatment. This means that the actual survival of the cells after treatment was only 10% (22% of 44%). Since, no dose response relationship was observed, the effect was observed only in one experiment and the increase was less than two-fold compared to the solvent control, this increasewas considered not biologically relevant and T-6906 Is considered to be not mutagenic in the absence of S9-mix.
In the presence of 8% (vlv) S9 metabolic activation, T-6906 showed 22- and
3.2-fold increases in the first and second experiment respectively in the
mutant frequency at the_TK.:.-Iz"s-.-4*-tts-fJresxtperiment, the increase was
observed only-at-.tm-e-i-etroyxic conceintrati@n:@>sAevere delay in the cell
gr_owth,at-the conc
a '100 149m was observed after 48 and 72 hours
@of ubeulture compared to the cell growth of the solvent control and a
redsuction of 81% was observed in the cells plated@for the determination of
the cloning efficiency of day 3. In the seco-n'edxperiment, the increase in
the mutant frequency was observed at test substance concentrations 0-f-
and 750 pglml, however no dose response rela'tionship was observed/@t;,,nic ethh@e
increases were observed in two experiments and the increases in the second
experiment were both in toxic and non'toxic concentrations, these increases
are considered to be biologically relevant. Therefore, T-6906 is conside
mutagenic in the presence of S9-mix.
, r@')
In the first experiment,b= the number of small and large mutant colonies were increased compared to the solvent control values. In the second experiment, mainly the large-mutant colonies were'increased compared to the solvent control values.-The difference between the numbers of small.and large mutant colonies in the first and second experimentis a rare finding, usually the amount of small and large colonies the independentexperiments are comparable. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appeared to result from mutants with single gene mutations (substitutions,deletionsof base-pairs) affecting the TK gene.
CONCLUSION
In conclusion, T-6906 is mutagenic in the TK mutation test system under the .experimentalconditions described in this report.
page 17
J'JN.@15.,999 12 38?M KEDICT@L 9G410"E 041
T-6906
3
NOTOX Project 223458
REFERENCES
I Amacher, D.E., Paillet, S.C., Ray, V. 'Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. 1. Application to genetic toxicology testing,' Mutation Research, 64:391-406 (1979).
2 Amacher, D.E., Faillet, S.C., Turner, G.N., Ray, V.A. and Salsburg, O.S., 196D, Point mutations at the thymidine kinase locus ln L517aY mouse lymphoma tells. 11. Test validation and interpretation,Mutation Res., 72, 447-474.
3 Ames, B.N., Me Cann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonellatmammalian microsome mutagenicity test. Mutation Res., 31, 347-364.,
4 Bradley, M.G., Bhuyan B., Francis, M.C., Langenback, R., Peterson, A., Huberman, E. 'Mutagenesis by chemical agents in V-79 Chinese hamster cells: a review and analysis of the literature:a report of the Gene. Tox Program,' Mutation Research, 87:81-242 (1981).
5 Clive, D., Caspary, W., Kirby, P-t-, Krehl, R., Moore, M., Mayo, J. and Oberly, T.J., 1967, Guide for performing the mouse lymphoma assay for mammalian cell mutagenicity, Mutation Res., 189, 143-156.
6 Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G. and Brown, M.M.M.,
1979, Validation and characterization of the L527BY/TK Mouse lymphoma mutagen assay system, Mutation Res., 59, 61-108. 7 Clive, D., Spector J.F.S. slaboratory procedures for assessing specific locus mutations at the TK locus in cultured L517BY mouse lymphome cells,' Mutation Research, 32;17-29 (1975). 8 Cole, 3., Arlett, C.F., Green, M.H.L., Lowe, J. and Muriel, W., 1983, A comparison of the agar cloning and microtitration techniques for assaying cell survival and mutation frequency In L5178Y mouse lymphoma cells, Mutation res., 111, 371-366. 9 Cole, J. and Arlett, C,F., 1984, The detection of gene mutations in cultured mammalian cells. In: Mutagenicity testing, a practical approach Ed.. Venit, S. and Parry, J,M., IRL press limited. 10 Jotz, M. and Mitchell, A.D., 1981, Effects of 20 coded chemicals on the forward mutation frequency at the thymidine kinase locus in L617BY mouse lymphoma cells. Ini Evaluation of short-term tests of carcinogens.F.J. de Serres and J. Ashby (Eds.), Elsevier-North Holland.* 11 Van der Hoeven, J.C.M., Bruggeman, I.M. and Debets, F.M.H., 1984, Genctoxicity of quercetin in cultured mammalian cells, Mutation Res., 136, 9-21.
page 18
T-6906
NOTOX Project 223458
TABLE I DOSE RANGE FINDING TEST WITH L517BY MOUSE LYMPHOMA CELLS Relative survival,% of control
DOSE (Agiml)
CELL COUNT AFTER EXPOSURE
CLONING EFFICIENCY AFTER EXPOSURE
Pcr-
fLC E:
Without metabolic act_ivation
J(Z!-5=;P,c r -.4
Solvent control 100
100
3
101
10
120
33
es
84
70
CiIli
86
q-
100
97
63
333
33
71
10001
48
113
.........................................................................
Solvent control 3 10 33
100 333 10001
With metabolic activation
100 96 110 99 107 @,214 90
100 132, 141' 120 117
90 136
Solvent control dimethylsulphoxide T-6906 precipitated in the exposition medium
page 19
J'JN'.S. .999 1":39Pi@ KEDICAL
T-6906
NOTOX Project 223458
TABLE 2 CYTOTOXIC AND MUTAGENIC RESPONSE OF 7-6906 IN THE MOUSE LYMPHOMA L5178Y TEST SYSTEM
EXPERIMENT I
Vt@b
I
r,A@.@A @
DOSE (Agiml)
CELL COUNT AFTER TREATMENT % OF CONTROL
C.E. AFTER TREATMENT % OF CONTROL
ACTUAL SURVIVAL OF THE CELL$ % OF CONTROL'
C.E. AT DAY 3 ABSOLUTE
TOTAL NO. OF WELLS % WITH MUTANTS
MUTATION FREQUENCY
X 105
Without metabolic activation
Solvent
control 100
100
100
133
46 (19s 271)
6.5
10
102
se
90
125
39 (18r. 211)
5.8
is
93
147
237
135
36 (21s 151)
4.9
33
90
69
62
152
32 (13s 191)
3.9
42
so
97
78
111
26 (12s 141)
4.3
!y6
ikI c
F-MS
107
77
62
79
169 (47s 1221_)'..) 35.9
........................................................................................
With metabolicactivation
Solvent
control 100
100
100
93
36 (23s 131)
7.2
33
92
95
87
95
21 (15s 61)
4.0
100
131
69
117
ill
20 (17s 31)
3.2
333
56
56
31
94
32 (16s 241)
63
1000,
121
lag
127
131 (72s 591) 160'0
DMN
102
65
66
31
108 @(53s 5i l@) 75.8
C.E. c Cloning Efficiency Solvent control= dimethylaulphoxide EMS = Ethylmethan*sulphonate DMN = Dimethylnitressmine
T-6906 precipitatedin the exposition.medium The actual survivalof cells (% of control)=thecell countafter treatment(% of control)x a the C.E. after treatment(% of control) = small colonies I = large colonies
page 20
JUN.'?@59.99 1 39PY MID[CAL t@4)0"'EO'L@
T-6906
@@Qi.li@ ,. @l NOTOX Project 223458
TABLE 3 CYTOTOXIC AND MUTAGENIC RESPONSE OF T-6906 IN THE MOUSE LYMPHOMA L5178Y TZST SYSTEM
EXPERIMENT 2
DOSE
(09/ml)
CELL COUNT AFTER TREATMENT
OF CONTROL
C.E. AFTER TREATMENT
% OF CONTROL
ACTUAL SURVIVAL OF THE CELLS % OF CONTROLI
C.E, AT DAY 3 ABSOLUTE
TOTAL NO. OF WELLS WITH MUTANT$
MUTATION FREQ?ENCY
x 10
Without metabolic activation
Solvent
control 100
100
loo
20
111
67
74
30
64
84
54
40
78
71
55
45
44
22
10
95
36 (10s 261) 7.0
74
31 7s 241) 7.7
as
21 65 151) 4.3
61
25 6s 191)
7.4
49
32 2s 301) 12.1
EMS
79
10
is
94 (12s
log a
...........................................................................................
Solvent
control 33 100 175 250 /7501
DMN
With metabolicactivation
100
100
100
97
113
96
38
85
32
51
34
1I
60
95
57
es
lie
100
so
41
33
147
37 2s 351)
4.7
192
77 6s 721)
8.5
162
38 4s 341)
4.4
165
39 4s 351)
4.4-
147
102 Ss 971) 14.9
1$7
85 (10s 751) 11.1
32
85 (12S 731)" 54.6
C.E. n Cloning Efficiency Solvent control m dimethylsulphoxide EMS Ethylmethanesulphonate DMN Dimethyinitrosamine
T-6906 precipitatedIn the expositlonmedium The actualsurvivalof cells (% of control)athecell count after treatment the C.E. aftertreatment(% of control)
small colonie& I @a large colonies
of control)x
page 21
39?It kiDICAL
T-6906
NOTOX Project 223458
APPENDIX 1
INDIVIDUAL COLONY COUNTS AND CELL COUNTS DURING EXPRESSION PERIOD
Cloning efficiencyof the dose range findingtest Call counts during expressionperiod
Cloning efficiencyimmediatelyafter exposure
Mutation experiments,Individual colony counts
Table 4 Tables 5-6 Tables 9-12
Dose range finding test Experiment I Experiment 2
Abbreviations used: DMN, dimethylnitrosamine EMS, ethylmethanesulphonate
Solvent control: dimethylaulphoxide
TABLE 4 DOSE RANAE RINDING TEST
Do&* 1) Emptywells/ (iiglm Cloningplate
12
Totalno.of emptywells
Cloning efficiency absolutt relative
(%of control)
Withoutmetabolicactivation
Solvent
control 28 41
69
102
loo
3
33 48
$1
66
84
10
51 43
94
71
70
33
41
39
so
as
86
loo
52 49
101
64
63
333
45 48
93
72
71
10001
25 36
61
115
113
..........................................................................
Solvent
INithmetabolic activation
control 49 47
96
69
100
3
39 38
10
37
36
77
91
132
33
39 4S
73
97
141
04
83
120
100
44 41
323
47 56
as 103
al
117
62
90
1000,
36 39
75
94
136
T-6906 precipitatedin the expositionmedium
page 22
j
T-6906
NOTOXProjeCt 223458
APPENDIX EXPERIMENT
1 continued 1
TABLE 5 CELL COUNTS AND SUBCULTURE DCA Withoutmetabolicactivation
I
DAY 0 1
DAY I
I
DAY 3
1
ltotalamountofftetalamount ofi I su:culluro I
I
Dos# leellsbefore lcellsafter I I log
lcellcouni1.
(pa/ml)Itreatmentx 1061treatmentx 2061%1)ITotalamount3)lc/mlx 10 1 %2)
Solvent I
control I a
10 1 a
Is - i a
33 1 a
42 1 1
56 1 16
75 1 16 100 5) 1 16
ENS
Ia
1 4.1
51
1 4.2
53
1 3.6
48
1 3.7
46
1 3.3
41
1
4)
1
4)
1
4)
1 4.4
55
(1) cellsaftertreatment x 100% tellsbefore treatment
(2) Coll count
1 100%
callcountof-z;;Ml
(3) celldensity 0.4 x 105 ciml (4) calldeath -(5) not used for mutation assay
3.7 1 1.9
100
3.8 1 3.3 174 3.4 1 4.7 247
3.3 1 3.0 ISO
3.0 1 4.2 221
4.0 1 3.0 ISO
TABLE 6 CELL COUNTS AND SUBCULTURE DATA With metabolicactivation
DAY a
DAY 2
1
DAY 3
IT*talamountofITatalamountofl I Subeul ure I
I I Subcultre I
I
Dose ]cellsbefore leellsafter I I x 201
[Cellcounil I x 109 Icellcounil
(pg/ml)ltreatnenxt1061treatmenxt 1061%1)iTotaalmount3)lc/mlx 10 1%2)lTotalamount4)IC'/Ixol10 1 %2)
Solventi
comtroll a
33 1
a
loo I
a
333 1
a
1000 S)i a
DMN I
a
1 5.2 1 4.0
1 6.a 1 2.9 1 6.3 1 5.3
65
4.0 1 4.6 100 410
60
4.0 1 A.9 107 4.0
as
4.0 1 4.3 93 4.0
36
2.8 1 1.9 39 4.0
79
4.0 .1 1.0 39 4.0
66
4.o 1 1.9 41 4.0
(1) cellsaftertreatmentx 100% cellsbefore treatment
(2) callcount
x 100%
Coll countof control
(3) calldensity0.4 105 c/ml (4) telldensity 1.6 Xx 105 cimi (5) T-6906precipitatedIn the expositionmedium
1 6.1 too
1 6.0
98
1 4.8
79
f 4.4 72
1 0.7
It
1 3.P
64
page 23
T-6906
L 4/U
NOTOX Project 223458
APPENDIX 1 continued
EXPERIMEN'T I
TABL@E7 CLONING EFFICIENCY DAY 0
Dose
Empty wellsl
(Agiml) Cloningplate
12
Totalno.of empty wells
Cloningefficiency absoluterelative
(sof central)
Withoutmetabolicattivation
Solvent control 45 35
10
44
45
is
20 33
33
55
49
42
46
36
00
is
100
89
77
aa
53
129
147
IOA
61
69
82
as
97
56
73
loo
C14S
54
43
97
Go
77
..........................................................................
With'metaboliacctlvatiom
Soly2nt
control
50
49
33
49 53
100
51 55
333
65
67
10001
40
47
99
66
100
102
63
95
206
sp
59
132
37
56
96
69
105
DMN
62
63
125
43
65
i T-6906pr*cipitst*d in the exposition medium
Cell death directly after treatment
page 24
J'J'N,.6';5.9.91 40PK KiD [CAL fl-t@"-,0E 0)6
T-6906
jI NOTOX Project 223458
APPENDIX 1 continued EXPERIMENT I
TABLE 8 SELECTION DATA AND CLONIOS EFFICIENCY
I
Mutant colonies
ICIoning efficiency (at dsy 3)
1
1
Dose
INumber
of wells with mutants
ITatal
INo. of empty welisl
Total
I
I
lagiml
I per selectic-n plate
lNo. of I cloning plate I No. off CE I w
I..............................I.m.u.t.a.ntsI................I .em.pty I
I
11
2
3
1
11
2 1 wells I
I
Solvent control
10 is 33 42 EMS
I
I 15 9 1a a 16 7 12 6 15 5 113 43
Without metabolic
actlvation
7 10
56
82 6s 45
11 43
70
S7
76 5a
34 23 36
I
1 46 1 29
1 39 1 31
1 36 1 24
1 32 1 24
1 26 1 30
1 169
1 42
I
I
I
22 1 51 iIS3 1 6.5
24 t 55 1125
1 5.6
26 1 50 1135
1 49
ia 1 42 1152 1 3 9
33 1 63 fill 1 4.3
46
1 87 1 79
1 55.9
With metabolic
activotion
Sciv control lint
33
100
333
1000, DMN
i 15 5 12 5 16 1 16 5 122 14 117 17
62 A1
01 77 21 23 24 15
12 6
1 36 1
41
90
1 21 1
39
11 1
1 20 1
32
52
1 32 1
41
29 22
1 131 1 75
12 23
1 108 1
74
MF 2 Mutant frequency per 105 survivors T-6906 precipitated in the exposition medium
35 1 76 1 93 35 1 74 1 95 31 1 63 1111 34 1 75 1 94
83 1158 1 ly 67 1141 1 31
1 7.2 1 A.0
1 3.2 1 6.3 liso.D 1 75.8
page 25
40PE@ 9EDICAL 9020 "'E OL@
ii0@ i"i 3
T-6906
NOTOX Project 223458
APPENDIX 1 continued
EXPERIMENT 2
TABLE 9 CELL COUNTS AND SUBCULTURE DATA Withoutmetabolicactivation
I DAY 0 1 1
DAY I I $Ubt
I DAY 3
ITotalamountofitatalamountofl
ullure I
I
Dose lcelisbefore Icellasfter I I x low
ipgiml)ltreatmenit iolsitreatmexnt1061%2)lTotaalmount3I)cImCl;lcxlo1u0n;11%2)
Solvent I
cont@rilI a
20 1 8
20 1 a
30 1 a
40 1 a
45 1 so 31 1
0 16
55 5 ( 16
EMS I a
1 4.5 1 3.9 1 5.0 1 2.9 1 3.5 1 2.0 1 1.7 1 1.7 1 3.5
56
4.0 1 4.2 100
49
3.7 1 2.8
67
63
4.0 1 3.0
71
36
2.7 1 3.1
74
44
3.3 1 2.9
69
25
1.9 1 1.7
40
11
1.7 1 4)
11
1.7 1 4)
3.3 1 1.1
26
(1)* cellsaftertroatmentx 100% cellsbeforetreatment
(2) cellcount
x 100%
call countof i7ant-rol
(3) celldensity0.4 x 105c/ml (4) celldeath (5) not usedfor mutationassay
P690 26
5.
2 :4 0Pit MEDICAL L20 -.'EO@D
T-6906
NOTOX Project 223458
APPENDIX 1 continued EXPERIMENT 2
TABLE 10 CELL COUNTS MO SUGCULTLIRE DATA With metabolicactivation
I
DAY 0
DAY 2
1
DAY 3
1
ITotal amount of]Tctml amount ofl I Subtul ure I
I I Subcult re I
I
Dose leolisbefore lcellsafter I I
ioi
lcellcounil
109
J, ceIll counil
(pi/ml)ltrestmontx 1061trestmentx 1061$Al)lTotlaamlount3)lclmlx 10 1%2)1'Tat.'almtun%4) c ml x 10 1%2)
Solventl
contirill 8
10 1
a
33 1
8
100 1
a
175 1
a
250 1
a
37S 6)1 16
Boo 671)1
is
750 1
16
DMN 1
6
1
5.5
1
4.5
1
4.8
1
2.1
1
2.8
1
3.4
1
4.9
1
4.9
1
9.3
1
4.4
do
4.0
1
4.8 100
4.0
56
4.0 1
3.8 79
4.0
to
4.0 1
3.0 79
4.0
26
1.9 1
2.0 sa
4.0
3S
2.S
1
1.6 33
5)
43
3.0
1
2.7 56
4.0
31
4.7
1
0.2
A
5)
31
4.7 1
0.2 4
5)
so
8.0 1 3.5 73 4.0
35
4,0
1
1.4 29
5)
(1) cellsaftertreatment x 100% cells before treatment
(2) cell count
x 100%
tellcountof control
(3) cell density 0.4 x 105 c/ml
(4) celldensity 1.6 x 10 ciml (5) no subculture
(6) not used in the mutation assay (7) T-6906 precipitatedIn the expositionmedium
1 4.4 100
1 4.6 105
1 3.7
64
1 4.4 100
1 3.8
66
1 2.5
57
1 0.2
5
1 0.2
5
1 4.1
93
1 1.9
43
page 27
i,@),j
T-6906
NOTOX Project 223458
APPENDIX 1 continued E)CPERIMENT 2
TABLE 11 CLONING EFRICIENCY DAY 0
Dos* Emptywellsi (pgiml) cloningplate
12
Totalno. of empty wells
Cloningefficiency absoluterelative
(%of control)
withoutmetabolic-activation
Solvent
control 36 41
77
91
100
10
56 41
97
68
75
20
54 so
104
61
67
30
S2 38
90
76
84
40
55 45
IGO
63
71
45
82 75
157
20
22
so
P3 96
189
2
2
55
96 95
191
1
1
&$
Be 67
175
9
la
..........................................................................
Withmetabolicactivation
Solvent control 42 34
10
40 40
33
37 30
100
41 46
175
66 73
250
40 40
375
95 93
76
93
loo
so
ae
95
67
105
113
87
79
as
139
32
34
80
as
95
Is$
2
2
Soo
S6 64
7SO'
35 29
DMN
68 63
120
47
st
64
lic
III
131
38
di
T-6906precipitateidn tho expositiomnedium
page 28
'999 1'6:40?11
KEDICAL
tiO.37-13 o@9
T-6906
NOTOX Project 223458
APPENDIX 1 continued
EXPERIMENT 2
TABLE 12 SELECTIONDATA AND CLONING EFFICIENCY
I Mutant colonies
Do$$
lnumber of wells with mutants
PAS/Ml
I per selection plate
...................................
1
2
3
IT*tal iNo. of lmutants 1
IClaningefficiency(at day 31
lNa.of emptywellstTotal I
I cloning plate I No. ofl CE
I ..................
I empty I
11
2 1 wells I
ilithout metabolic activation
s
Solvent
I
control
1 5 10
28
30
1 36 1 36
so 1 74 1 95
29
14 6
28
18
1 31 1 49
42 1 91 1 74
30
12 7
14
3 4 1 21 1 43 37 1 so I so
40
1 10
24
35
1 25 1 49
55 1104 1 61
45
1 10
11
0 13 1 32 1 60 so 1113 1 49
EMS
I s go
4 27
3 27
1 94 1 at 77 1161 1 is
1 -1 I I MF I I
1 7.0 1 7.7 1 4.3 1 7.4 1 12.1 tlo9.8
withmetabolicactivation
Solvent
control 33
100 175
250
750 DMN
I
I a to 1 0 26 1 2 10 11 8 1 1 40
1 5 20 1 3 2$
1 14
3 24
2 23 2 19
3 29 2 31
5 22
1 11
3 21
0 it 10 1 28 3 24 4 26
I 1 37 1 26
1 77 1 16 1 38 1 is 1 39 1 22 1 102 1 22 1 95 1 20 1 85 1 70
is 1 44 1147 is 1 31 1182 20 1 38 1162
15 1 37 1165 22 1 44 1147 20 1 40 1157 70 1140 1 32
1 4.7 1 8.5 1 4.4 1 4.4 1 14.9 1 11.1
1 $4.6
W . Mutant frequency
per 105 survivors
page 29
'.999l@i:40PY MEDICAL @-.)@OE 0(@
T-6906
tiO,17 1'3 ?. 30
NOTOX Project 223458
APPENDIX 2
INDIVIDUAL COLONY COUNTS 6ND CFLL COUNTS OF-NOT REPORTED EXPERIMENTS
EXPERIMENT 3
TABLE 13 CELL COUNTS AND SUBCULTURE DATA With metabolicactivatio"
I
DAY 0
DAY 2
1
DAY 3
1 IT*talamountaft7otalamountofl I Subcul ure I
-
I I Subcu-ltyro I
I
Dose icellsbefore ICSI12after I I x lei' ICGII coun
lcellcounil
(Ikg/ml)ltreatmexnt1061treatmentx 1061%2)lTotalamount3)lclmlx loi1'..12)'Tot'ama0ol0unt4)ICIMIX la I %2)
Solventl
contrall a
IGO 1
8
250 1
6
Soo I
a
750 S)i 1
DM 1
5
1 4.4 1 2.6 1 3.0 1 3.4 1 4.1
1 4.1
55
4.0 1 3.3 100 4.0
1 6.5 100
33
2.4 1 3.4 103 4.0
1 5.5
as
38
2.8 1 2 5 76 4
1 S.2
No
6?
1 3.5 Be
43
3.1 1 1:3 39
51
3-B 1 0.8 24
6)
1 I.S 23
51
3.B 1 2.1 64 4.0
1 3.1
46
(1)
t;eat=nt x 100%
Cc:;i:::Ifi;ereatment
(2) Colclount
x 100%
all count of C;n-tr-ai
(3) :911density 0.4 los C/mi (4) celldensity 1.6 Xx 105 ciml
(5) T-6PO6 precipitatedin the expositionmedium (6) no subculture
TABLE 14 CLONIND EFFICIENCY DAY 0
Dose
Emptywellsi
(pg/ml) Cloningplate
12
Totalno. of empty wells
Cloningefficiency absolute relative
(% of control)
With metab lic activation
Solvent
control 46 43
100
52 48
250
47 45
Soo
41 &1
750
57 45
DMN
50 52
89
77
loo
Ica
65
84
92
74
96
82
as
110
102
63
B2
102
63
92
(1)T-6906precipitateIdn the expositionmodium
page 30 -
J'JN.'@5.'.999 1z':41PIt MEDICAL @.)o -1,E 0)
"r-6906
tiO.37,3 ?. 31
NOTOX Project 223458
APPENDIX 2 continued
EXPERIMENT 4
TABLE 15 CELL COUNTS AND SUBCULTURE DATA
Withmetabolicactivation
DAY 0
DAY 2
1
DAY,3
IT*talamountofitatalamountofl II Subcullure I
I I Subcultre I
I
Dose leellebefore (jagiml)ltreatmexntI06
lcill&after I
x log
IC;llc
I
[treatmenxt 1061%2)fTotaalmount3)le ml X'ul@'I.2)I'T.tI.a"4m.u,t4jI"tIm"i
Cou0ni1 l%2,
Solvemtl a contrall
100 1
a
250 1
a
375 1
a
Sao 1 16
7SO 5)1 16
DW 1
8
1 S.6
1 5.2 1 3.3
1 3.1 1 9.3 1 11.1 1 4.7
70
4.0 1 3.5 100 4.0
1 6.4 loo
6S
2.4 1 3.1 99 A
1 6.6 103
41
3.0 1 1.6 46
4?
1 3.4 53
39
2.9 1 1.1 51 461
1 4.5 7S
so
3.1 1 1.4 40
1 4.0 sa
69
3.8 1 1.1 31
6)
1 0.6
9
S9
3.6 1 1.1 31
6)
1 1.4
22
(1) cellsaftertreatmentx 100% cellsbefore treatment
(2) Coll count
x 100%
cell countof control
13) cell den ity 0.4 x 10--cS/ml
(4) celldon:ity 1.6 x 105 elml
(5) T-6906 precipitatedin the expositionmedium (6) no subculture
TABLE 16 CLONING EFFICIENCY DAY 0
Dose
Empty wells/
(liolml)cloningplate
12
Totalno. of empty wells
Cloningefficiency absolute relative
of control)
solvent
With metabolicactivation
control 51 Se
109
57
100
lea
48 50
98
67
Ila
250
53 61
114
52
91
875
62 57
119
48
84
Soo
65 57
222
45
79
750
55 49
104
61
107
DMN
74 76
ISO
25
44
(1)T-6906precipitatedin the expositionmedium
page 31
