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STATEMENT
FOR ENGLISH
TRANSLATION
Kashima Laboratory MitsubishiChemicalSafetyInstituteLtd.*
Sponsor Title
Sumitomo 3M Limited ChromosomalAberration Study of Sample D-1 in CulturedMammalian Cells
Study'No. 2LI62
This studywas conductedin Kashima Laboratoryof MitsubishiChemicalSafety Institute.The originalreportwas wriftenin Japanese. I hereby declare that this reportreflectsfaithfullythe originalreportas accuratelyas possible to my knowledge.
Translator
Tamotsu Nishitomi. M.S. Senior ResearchScientist
Date:
)Ifjlr
:As from October 1, 1994, the company name has been changed.
Submittedto: Sumitomo 3M Limited
c I:Z-IE 1=1 C) 1::;-"r D ChromosomalAberrationStudy of Sample D-1
in CulturedMammalian Cells
(StudyNo. 2 L 1 6 2
September 30, 1992 MITSUBISHI-KASEI INSTITUTE OF TOXICOLOGICAL
AND ENVIRONMENTAL SCIENCES
STATEMENT Kashima Laboratory Mitsubishi-KaseIinstituteof Toxicological and EnvironmentalSciences
Sponsor Title
Study No.
Sumitomo 3M Limited ChromosomalAberration Study of Sample D-1 in CulturedMammalian Cells 2LI62
This study has been conducted in accordancewith the GLP Standards applied to IndustrialChemicalsof Japan.
Management: Masanobu Katoh sealed Date: September30, 1992
QUALITY
ASSURANCE
STATEMENT
Kashima Laboratory Mitsubishi-KaseIinstituteof Toxicological and EnvironmentalSciences
Sponsor Title
Sumitomo 3M Limited ChromosomalAberration Study of Sample D-1 in CulturedMammalian Cells
Study No. 2LI62
Study procedureswere periodicallyinspectedand the report was audited by QualityAssuranceUnit. The standardsof inspectionadoptedwere in accordance with the GLP standardsapplied to IndustrialChemicalsof Japan.
Inspection or audit
Study procedure
Study report
Date of inspection or audit
April 13, 1992 April 20, 1992
July 3, 1992 September 30, 1992
Dateof reportingto the studydirector and to the management
April 13, 1992 April 20, 1992
July 3, 1992 September 30, 1992
Quality AssuranceUnit YoshihiroMiura
sealed Date: September30, 1992
Title
ChromosomalAberrationStudy of Sample D-1 in Cultured.Mammalian
Cells
(StudyNo. 2Ll62)
Purpose
To assess the clastogenicityof the test substance by the chromosomal aberrationtest in culturedmammaliancells
Guideline The Guidelinesfor ScreeningToxicityTestingof Chemicalsof Japan (KampogyoNo.700, YakuhatsuNo.1039, 61 Kikyoku No.1014, 1986)
GLP
The GLP Standardsapplied to IndustrialChemicalsof Japan
(KampogyoNo.39, YakuhatsuNo.229. 59 Kikyoku No.85, 1984)
Sponsor Sumitomo 3M Limited
Testing facility
Kashima Laboratory Mitsubishi-KaseIinstituteof Toxicologicaland Environmental
Sciences 14 Sunayama,Hasaki-machi,Kashima-gun,lbaraki
Study Director : Tamotsu Nishitomi
Other Contributors: AkihikoKido,Miyuki Tanaka Studydate : (Initiatioonf thestudy)
(Submissioonf the finalreport)
April 10, 1992 September 30, 1992
Unforeseeable circumstancethat may have affectedon the test resultsand deviationfrom the protocolhave not occurred.
Retentionof records All data,documents,the protocoland the finalreportwill be retainedin the
safekeepingfacilityof KashimaLaboratoryfor 10 years after the submissionof the final report.Furtherretentionwill be discussedwith the sponsor.
Report of Results of ChromosomalAberration Test in Cultured Mammalian cells
1. General Items
Name of the new 2-EN-ethyl-N-perrfuoloalkyI(C=1-8)suIfonylaminolethylacrylate
chemical substance (IUPAC nomenclature)
Other name Sample D-1
Molecular weight
625
Structuralformulaor rationalformula (or outline of manufacturingmethod. in case both are unknown)
A p p e a r a n c e at ordinary temperature
Stability
liquid stable
CzHs
0
1
11
CnFz,.,SOzN-CHzCHzO-C-CH=CHz
n=1-8 n=B(main component) : ca. 78% n=1-7 : ca. 21%(total)
(lotNo. 101)
Purity of the new chemical substance tested
z: 99%
Nam e a n d concentration of
impurizies
phenothyadine 6020ppm
hydroquinonemonomethylether
17035ppm
Melting point
27-42*C
Physico-
ch em ica I properties of the new substance
Boilingpoint
Vapor pressure Partition coefficient
ca. 150 *C (lmmhg)
Solubility
solublein oil
W a t e r insoluble
DMS0 Solubility
Acetone
insoluble soluble(?=50%)
Others soluble in freon 1.1,3
2. Cell lineand culturecondition Name of cell line C H t / I U
Obtained from
Dai-Nippon Pharmaceutical Co., Ltd
Species Medium
Chinese hamster Eagle's MEM
Serum
Calf serum, 10 %
Doubling time
ca. 18 h
Passage NO.
21-23*
Number of chi-omosomes(mode) 25
Date obtained
6/20/1989
Manufacturer
Nissui Pharmaceutical Co., Ltd.
Manufacturer (Lot) Gibco Lab. (50K7711) Gibco Lab. (3lP6615)
Fre.ezingcondition in liquidnitrogen
Culture condition
Container Temperature
Plastic dish 370C
C02
5%
R emat-k
Cells were frozen at passage 20
1
3. S9 Mix (1) Sourceof S9 (Encirclethe applicablenumber,and fill in the relevantentries)
1. Made in-house
Supplier
Kikkoman Corporation
2OPurchase
Prepared on Purchased on
3/19/1992 4/ 7/1992
Lot No.
RAA-272
(2)StarageTemperature,etc. of S9
Storage temperature
Below -80*C Name and model of storage Deep freezer
apparatus
CL-60
(3)Preparatioonf S9 (Ifpurchased,fill in spacesto extentpossible)
Animal used
Inducingsubstance
Species,Strain Sex Age (inweeks) Weight
Rats, Sprague-Dawley Male 7 weeks 192 - 227 g
Name
Administrationmethod Administrationperiod and amount(g/kg-b.w.)
phenobarbital (PB) 5,6-benzoflavone(BF)
Intraperitoneal
BP 4 days 0.03-0.06 BF 1 day 0.08
(4)Compositioonf Sg Mix
Constituents S9 mgclg KCI
Amount in 1 ml
0.3
ml
5
gmol
33
g mo I
Constituents NADP + NADPH Buffer (HEPES)
Amount in I ml
4
u mo I
-
g moI
4
umol
D-Glucose 6-phosphate
5
u mo I Others(deionizewdater)
ml
(5)Treatmenctonditionwith S9 Mix (Encirclethe applicablenumber,and fill in the relevant entries)
Amount of S9 (Finalconcentration)
C,)P late method
5
%
2. Suspension method
Amount of S9 protein (Finalconcentration)
1.39 mg/ml
Treatment time
6
h
Culturetime after treatment of the test substance
18
h
Remark
2/ I I
4. CellGrowthInhibitioTnest (1)Tesctonditioanndpreparationf thetestsubstancseolution
Period of experiment
from4/10/1992 to 4/15/1992
Without metabolic activation
With metabolic activation
Number of cells seeded
4 x 10, /Ml
4 x 10' /ml
Cell
Days of culture
3
days
3
days
Plate
Form Size Manufacturer Number of plates for each concentration
Plasticdish 6 cm in diameter Becton Dickinson& Co.
2
plates
Plasticdish
6 cm in diameter Becton Dickinson & Co.
2
plates
Amount of medium
5
ml/plate
3
ml/plate
Solvent
DMSO
vmso
Concentration of the orig.inalsolution of the test substance
1000 mg/mi
1000 mg/ml
Preparation of the test substance solution
Amount of the test substance
Volume of the solvent
S ta te of th e te s t substance(encirclethe applicableone)
5000 mg
5 ml Dissolved, pendedd
c US Others(
5000 mg
5 ml Dissolved,(CuspendedD Others(
Time after preparetion
within50 min.
within 65 min.
Method of preservation
room temperature
room temperature
Methodof sterilization
not done
not done
Treatmentof the cells
Amount of each test substancesolution
Period of treatment
0.025 ml/plate 24 and 48 h
0.015 ml/plate 6h
Amount of S9 Mix
0.5 ml/plate
Method of countingof cell number
counting: with a hemocytometer
fixing: with 10% formaline
staining: with 0.1% crystalviolet
-counting: with a hemocytometer
-fixing: with
10% formaline -staining: with
0.1% crystalviolet
Remark:* The initiationday of culturingwas definedas 0 day.
3/ I I
(2)Cellgrowth index (Relativevalue when the value of the solvent-treatedgroup is 100%)
Concentration( g g/ml)
Cell growth index
0 (solvent)
100
10
92
50
61
Without metabolic activation
100
-X-
22
(24 h treatment)
500
-X.
34
1000
-X-
33
2000
-X-
37
5000
X.
36
0 (solvent)
1 00
10
89
50
78
Without metabolic activation
100
-X-
29
(48 h treatment)
500
X.
14
1 000
X.
12
2000
X.
16
5 000
X.
0 (solvent)
23
1 00
With metabolicactivation
10
50
100
X.
500
X.
97
100 19
22
1 000
X-
12
2000
X.
72
5 0 0 0 x- 1 6 4 X-: The test substanceprecipitatedor floated in culture medium.
4/ 1 1
5. ChrowsomalAberrationTest (1)Testconditionand preparatioonf the testsubstancesolution
Period of experiment
from 4/17/1992 to 5/15/1992
Cell Plate
Number of cells seeded
.Days of culture
Form Size Manufacturer Number of plates for each concentration
Without metabolic activation
4 x 103
/Ml
3
days
Plastic dish 6 cm in diameter Becton Dickinson & Co.
2
plates
With metabolic activation
.4 x 10' /ml
3
days
Plastic dish 6 cm in diameter Becton Dickinson & Co.
2
plates
Amount of medium
5
ml/plate
3
mi/plate
Solvent
D-MSO
DMSO
Concentration of the original solution of the test substance
20 mglml
20 mg/ml
Preparationof of the test substance solution
Amount of the test substance
Volume of the solvent
State of the test substance (encircle the applicableone)
100 mg
5 ml DissolvedS,cuspendedD Others(
100 mg
5 ml Dissolved,( uCspendedD Others(
Time after preparetion
within 45 min.
within 35 min.
Method of preservation
room temperature
room temperature
Method of sterilization
not done
not done
Treatment of the cells
Amount of each test substance solution
Period of treatment
0.025 ml/plate 24 and 48 h
0.015 mi/plate 6h
Amount of S9 Mix
0.5 mi/plate
Mitotic inhibitor
Name Concentration
Coleemid Final conc. 0. 1 A g/ml
Colcemid Final conc. 0.1u g/mI
Period of treatment
2h
2h
Remark:
The initiationday of culturingwas definedas 0 day.
5/ I I
(2)ResuIt Table 1, 2 show the results.
(3)Judgementof the result
Judgement (Encircleone) I Reason for judgement:
Positive
(::@egativeD
The test substance did 'notincrease the cells with structural chromosomal
aberrationsand polyploidcellswith or withoutmetabolicactivation.
These resultsled to the conclusionthat the test substancedid not have clastogenic
potential.
(4)Referenti.amlatters
1. The test substance was insoluble in water and,DMSO and was soluble in acetone. But the test substance in DMSO was dispersed in culture medium better than that in acetone. ThereforeDMSO was selectedfor solventof the test substance.
2. In the cell growth inhibitiontest,50% inhibitiondose of cell growth (TCID5o) was 60gglmt in 24-hourtreatment.73g g/mt in 48-hourtreatmentwithoutmetabolic activationand 71 gg/mt with metabolicactivation. In all treatmentgroups, cell growth indexat lOOg g/mt or more did not increaseor slightly increasedand this concentrationcoincidewith that beginningto precipitate. So it was considered that the testsubstancewas saturatedat lOOg glmt or more (Fig.1, 2). From these results, the highest concentration in chromosomalaberration test was set at lOOg g/mt with or withoutmetabolicactivation.
3. In chromosomalaberration test, the test substanceprecipitatedor floated in culturemediumat 100 g glmt
4. Data were statisticallyanalysedby the x '-testagainst the negative control. Structualaberrantcells only with gaps were excludedfrom this analysis.
6
6. Others
Name Testing facility Address
Kashima Laboratory,Mitsubishi-KaseiInstituteof Toxicological and Environmental Sciences
14 Sunayama, Hasaki-machi, Kashima-gun, lbatak.iTel.0479(46)2871
Study director Name title Tamotu Nishitomi Senior Research Scientist
signed & sealed
Test dates from 4/10/1992 to 9/30/1992
7
100 t
50 CL)
10
0
10
50
100
500
1000
2000
5000
Concentration (g glmt)
0 24-hourtreatment 0 48-hourtreatment
Fig.1 Cellgrowthindexof SampleD-1 (withoutmetabolicactivation)
100 50 -
10
L
0
10
50
100
500
1000
2000
5000
Concentratio(ng 9/rd)
0 With S9 mix (6-hourtreatmentand 18-hourrecovery)
Fig.2 Cell growthindexof SampleD-1 (withmetabolicactivation)
TABLE 1 RESULTS OF THE CHROMOSOMAL ABERRATION STUDY OF Sample,D-1 IN THE DIRECT ASSAY
Test substance : Simple D-1
rreatmentexposure Doze No. of Polypiolds
No. of cells with chromosomal structural aberrations -(%)
time levels cells
Judge- Caps
Chromatid type Chromosomal type Others
Total
group
(h) (.ug/ml)analysed
mentat
9
etb
cto
cob
cae
g
100
0
0
1
1
0
0
0
2
2
Negative
24
0 100
0
0
0
0
1
0
0
1
1
control
200 ID(O.0)
0(0.0) l(O.5) i(o.s) 1(0.5) 0(0.0) (o.o) 3(j.5) 3(1.5)
100 1
0
0
0
0
0
0
0
0
(DMSO)
48
0 100 1
o
0
0
0
0
0
0
0
0
200 o(O.0)
0(0.0) 0(0.0) 0(0.0) o(O.0) D(O.0) D(O.0) @(O.o) 0(0.0)
100
1
0
1
0
1
0
0
2
2
25 100
0
0
1
0
0
0
0
1
1
200 l(O.5)
o(O.0) 2(l.0) 0(0.0) 1(0.5) o(O.0) o(O.0) 3(l.5) 3(1.5)
ioo
0
0
0
0
1
0
0
1
1
24
so 100
0
0
0
0
0
0
0
0
200 0(0.0)
0(0.0) O(D.0) 1(0.5) 0(0.0) 0(0.0) 1(0.5) 1(0.5)
100
1
0
0
0
1
0
0
1
1
100 100
0
0-
0
0
1
0
0
1
1
rest
200 l(O.5)
D(o.o) 0(0.0) o(O.0) 2(1.0) o(o.o) 0(0.0) 2(1.0) 2(1.0)
substance
.100
IL
0
0
1
0
0
0
1
1
25 100
1
a
0
0
0
0
0
0
0
200 12(1.0)
.0to.0) 0(0.0) 1(0.5) 0(0.0) 0(0.0) D(O.0) .1(0.5) 140.5)
100
1
0
0
1
0
0
1
2
48
50 100
0
0
0
0
1
0
0
1
1
200 1(0.5)
1(0.5) .0(0.0) 0(0.0) 2(i.o) o(O.0) o(O.0) 2(i.o) 3(j.5)
100
1
0
0
0
0
0
0
0
0
100 100
0
0
0
0
1
0
0
1
1
200 1(0.5)
o(o.o) 0(0.0) p(O.0) 1(0.5) 0(0.0) 0(0.0) 1(0.5) 1(0.5)
100
0
0 --12
12
1
0
ol
23
23
Positive
24
0.03 100
0
2
20
a
1
0
0
27
28
control
200 0(0.0)
2(l.0) 32(l .0) 20(10.0) 2(l.0) 0(0.0) 0(0.0) 50(25.0) 51(25.5)
100
0
2
7
19
7
0
4
32
34
(MMC)
48
0.03 100
0
0
16
34
1
1
5
49
49
1
---1
1 200 p(O.0)
2(1.0) 23(11
(4.0) R(O.5) 9(4.5) 81(40.5) P3(4l.i)
ctb : chromatid break, cte chromatid exchange, cab : chromosome break. ese : chromosome exchange,
others : fragmentation etc. (except pulvalization)
Aberrant cells(-t) were statistically compared with the control by the X2-test; ***(p<0.001)
The test substance precipitated or floated In culture medium when It was added.
TABLE 2 RESULTS OF THE CHROMOSOMAL ABERRATION STUDY OF Sample D-1 IN THE METABOLIC ACTIVATION ASSAY
Test substance Sample D-1
rreatment with or Done No. of PolyploiAn
without levels cells
Judge- caps
group S9 Mix (,ug/al)analysed
sent**
c
100
0
0
Negative
-
0 100
0
0
control
200 p(O.0)
o(o.o)
100
0
0
(DMSO)
0 100
0
0
200 0(0.0)
o(O.0)
No. of cells with
Chromatid type
ctb
eta
0
0
0
0
0(0.0) 1)(0.0)
0
0
0
0
D(O.0) o(O.0)
chromosomal structural aberrations *(%)
Chromosomal type others
Total
cob
Coe
*g
0
0
0
0
0
1
1
0
2
2
1(0.5) 1(0.5) o(O.0) 2(l.0) 2(l.0)
0
0
0
0
0
0
0
0
(0.0) 1)(0.0) D(O.0) 0(0.0) 0(0.0)
100
0
0
0
0
1
0
0
1
25 100
0-
0
0
0
0
0
0
0
200 0(0-0)
(o.o) p(o.0) 0(0.0) . (0.5) 0(0.0) o(o.o) 1(0.5) 1(0.5)
100
0
0
1
0
0
0,
0
1
50 100
0-
1
0
0
0
0
0
0
rest substance
200 100 0 100 100 200 100 25 100
o(O.0)
0 0(0.0)
0 0
l(O.5) l(C.5) (0.0) @(o.0) D(O.0) D(B.0) l(O.5) 2(l.0)
1
0
0
0
a
0
0
-
0
0
0
0
0
0
0
1(0.5) 0(0.0) (0.0) D(O.0) o(o.o) B(o.o) G(o.o) l(O.5)
0
0
0
0
0
0
0.
-
0
0
0
1
0
0
1
200
(0.0)
o(O.0) 0(0.0) (0.0) 1(0.5) 0(0.0) 0(0.0) 1(0.5) 1(0.5)
100
0
0
0
0
1
0
0
1
+
50 100
0-
0
0
0
0
0
0
0
200 o(O.0)
0(0.0) -0(0.0) o(O.0) 1(0.5) 0(0.0) 0(0.0) 1(0.5) 1(0.5)
100
0
1
0
1
1
0
0
2
100
0-
0
0
0
1
0
0
1
200
(0.0)
1(0.5) 0(0.0) 1(0.5) 2(1.0) 0(0.0) (0.0) 3(l.5) 4(2.0)
Positive control
100 20 100
200 100
1 0 (0.5) 0
0
0
0
a
0
0
0
-
0
0
1
a
0
0
1
0(0.0) lo(o.0). 1(0.5) p(O.0) o(O.0) (0.0) 1(0.5) l(O.5)
0
12
211
0
0
44
73
7
(BP)
+
20 100 :i(@0@.5) 200
-
6 @ 7(13.155) _
27 1
0
0
36
69
7
6(3.0)
48(24.0) p(O.0) p(O.0) 00(40.0) @42171.0)R44(72.
ctb Chromatid break, cte chromatid exchange. cab : chromosome break. Coe : chromosome others : fragmentation etc. (except pulvalization)
exchange,
Aberrant cells(-K) were statistically compared with the control by the X 2-tent; +**(p<0.001)
The test substance precipitated or floated In culture medium when It was added.
Cells were treated with S9 (5% of final conc.) or without S9 for 6 hours and recovered for is hours.
Appendix : List of ControlReagenCi
NegativeControl(Vehicle)
Reagent Name dimethylsulfoxide
Abbreviation Lot No.I
Supplier
DMSO I 206NIO841 Kanto Chemical Co. Inc.
PositiveControl Reagent Name
mitomycin C benzo[alpyrene
-AbbreviationLot No.
Supplier
wc
719AAB. Kyowa Hakko Kogyo Co., Ltd.
BP
AX01 Tokyo Kasei Kogyo Co., Ltd.