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005242
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STATEMENT FOR ENGLISH TRANSLATION
Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd.*
Sponsor : Sumitomo 3M Limited
Title
: Chromosomal Aberration Study of Sample D-l
in Cultured Mammalian Cells
Study' No. : 2L162
This study was conducted in Kashima Laboratory of Mitsubishi Chemical Safety Institute. The original report was written in Japanese. I hereby declare that this report reflects faithfully the original report as accurately as possible to my knowledge.
Translator
Tamotsu Nishitomi, M. S. Senior Research Scientist
* :As from October 1, 1994, the company name has been changed.
005243
-w*
Submitted to: Sumitomo 3M Limited
[ R E 3? O R T D Chromosomal Aberration Study of Sample D-l
in Cultured Mammalian Cells
(Study No. : 2 L 1 6 2)
September 30, 1992 M1TSUBI SHI-KSE I INSTITUTE OF TOXICOLOGICAL
AND ENVIRONMENTAL SCIENCES
005244
STATEMENT
Kashiraa Laboratory Mitsubishi-Kasei Institute of Toxicological and Environmental Sciences
Sponsor : Sumitomo 3M Limited
Title
: Chromosomal Aberration Study of Sample D-l
in Cultured Mammalian Cells
Study No. : 2L162
This study has been conducted in accordance with the GLP Standards applied to Industrial Chemicals of Japan.
Management:
Masanobu Katoh
sealed
Date: September 30. 1992
005245
QUALITY ASSURANCE STATEMENT
Kashima Laboratory Mitsubishi-Kasei Institute of Toxicological and Environmental Sciences
Sponsor : Sumitomo 3M Limited
Title
: Chromosomal Aberration Study of Sample D-l
in Cultured Mammalian Cells
Study No. : 2L162
Study procedures were periodically inspected and the report was audited by Quality Assurance Unit. The standards of inspection adopted were in accordance with the GLP standards applied to Industrial Chemicals of 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
Date of reporting to the study director and to the management
April 13. 1992 April 20. 1992
July 3. 1992 September 30. 1992
Quality Assurance Unit : Yoshihiro Miura
sealed Date: September 30. 1992
005246
Title
: Chromosomal Aberration Study of Sample D-l in Cultured Mammalian _
Cells
(Study No. 2L162)
Purpose
: To assess the clastogenicity of the test substance by the chromosomal aberration test in cultured mammalian cells
Guideline : The Guidelines for Screening Toxicity Testing of Chemicals of Japan (Kampogyo No. 700, Yakuhatsu No.1039. 61 Kikyoku No. 1014. 1986)
GLP : The GLP Standards applied to Industrial Chemicals of Japan (Kampogyo No. 39, Yakuhatsu No. 229. 59 Kikyoku No. 85, 1984)
Sponsor : Sumitomo 3M Limited
Testing : Kashima Laboratory facility Mitsubishi-Kasei Institute of Toxicological and Environmental
Sciences 14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki
Study Director
: Tamotsu Nishitomi
Other Contributors : Akihiko Kido, Miyuki Tanaka
Study date : (Initiation of the study) (Submission of the final report)
April 10, 1992 September 30, 1992
Unforeseeable circumstance that may have affected on the test results and deviation from the protocol have not occurred.
Retention of records : All data, documents, the protocol and the final report will be retained in the
safekeeping facility of Kashima Laboratory for 10 years after the submission of the final report. Further retention will be discussed with the sponsor.
005247
Report of Results of Chromosomal Aberration Test in Cultured Mammalian cells
1. General Items
N a m e of the n e w 2-DV-e thy 1-N-perf ruoloalkyl(C=l~8) su lfonyl amino] ethy lacry late chemical substance (IUPAC nomenclature)
Other name Sample D-l
Molecular weight
625
Structural formula or rational formula (or outline of manufacturing method, in case both are unknown)
A p p e a r a n c e at ordinary temperature
Stability
liquid stable
CzHs 1
0 II
c ,,f 2,,*,s o 2n -c h 2c h 2o -c -c h =c h 2
n=l---8 n=8(main component) : ca. 78%
n=l~7 : ca. 21%(total) (lot No. 101)
Purity of the new chemical substance tested
99%
Name
and
concentration of
impurizies
phenothyadine 6020ppm
hydroquinonemonomethylether
17035ppm
Melting point
27~42C
Phys ico chemical properties of the new substance
Boiling point
Vapor pressure Partition coefficient
ca. 150 C (ImmHg)
Solubility
soluble in oil
W a t e r insoluble
D M S 0 insoluble Solubility
Acetone soluble(^50%)
O t h e r s soluble in freon 1,1, 3
2. Cell line and culture condition
Name of cell line C H L / I U
Obtained from
Dai-Nippon Pharmaceutical Co., Ltd
Species
Chinese hamster
Date obtained
6/20/1989
Medium
Eagle's MEM
Manufacturer
Nissui Pharmaceutical Co., Ltd.
Serum
Calf serum, 10 %
Manufacturer (Lot) Gibco Lab. (50K7711) Gibco Lab. (31P6615)
Doubling time
ca. 18 h
Freezing condition in liquid nitrogen
Passage No.
Number of chromosomes(mode)
21~23* 25
Culture condition
Container Temperature CO-2
Plastic dish 37C 5%
Remark
* Cells were frozen at passage 20
1/1 1
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3. S9 Mix
(1) Source of S9 (Encircle the applicable number, and fill in the relevant entries)
1. Made in-house
PteflflXfilUa----
Supplier
Kikkoman Corporation
^2?)Purchase
Prepared on Purchased on
3/19/1992 4/ 7/1992
Lot No.
RAA-272
(2)Storage Temperature, etc. of S9
Storage temperature '
Below -80C
Name and model of storage apparatus
(3)Preparation of S9 (If purchased, fill in spaces to extent possible)
Deep freezer CL-60
Animal used
Inducing substance
Species, Strain Sex Age (in weeks) Weight
Rats, Sprague-Dawley Name
Male 7 weeks 192 - 227 g
Administration method
Administration period and amount(g/kg-b.w.)
phnobarbital (PB) 5, 6-benzoflavone(BF)
Intraperitoneal
BP 4 days 0.03---0.06 BF 1 day 0.08
(4)Composition of S9 Mix
Constituents
Amount in 1 ml
Constituents
Amount in 1 ml
S9
0.3 ml NADP +
4 mol
MgCh
5
mol
NADPH
mol
KC1
33
mol
Buffer (HEPES)
4 mol
D-Glucose 6-phosphate
5
mol
Others(deionized water)
ml
(5)Treatment condition with S9 Mix (Encircle the applicable number, and fill in the relevant entries)
2/1 1
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v>
4. Cell Growth Inhibition Test
(l)Test condition and preparation of the test substance solution
Period of experiment
from 4/10/1992 to 4/15/1992
Without metabolic activation
With metabolic activation
Number of cells seeded
4 x 103 /ml
4 x 103 /ml
Cell
Days of culture *
3 days 3 days
Plate
Form Size Manufacturer
Number of plates for each concentration
Plastic dish 6 cm in diameter Becton Dickinson k Co.
2 plates
Plastic dish 6 cm in diameter Becton Dickinson & Co.
2 plates
Amount of medium
5 ml/plate
3 ml/plate
Solvent
DMSO
DMSO
C o n c e ntration of the original solution of the test substance
1000 mg/ml
1000 mg/ml
Preparation of the test
substance solution
Amount of the test substance
Volume of the solvent
State of the test substance (encircle the applicable one)
5000 mg
5000 mg
5 ml
5 ml
Dissolved. (Suspended^) Dissolved, (Suspended^)
Others(
) OthersC
)
Time after preparetion
within 50 min.
within 65 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 ml/p late 6h
Amount of S9 Mix
0.5 ml/p 1ate
Method of counting of cell number
counting : with a hemocytometer
fixing : with
10X formaline staining : with
0.IX crystal violet
counting : with a hemocytometer
fixing : with 10% formaline
staining : with 0.IX crystal violet
Remark: * The initiation day of culturing was defined as 0 day.
3/1 1
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V
(2)Ce11 growth index (Relative value when the value of the solvent-treated group is 100%)_
Concentration ( g/ml)
Cell growth index 00
0 (solvent)
10 0
10 92
50 61
Without metabolic activation (24 h treatment)
10 0 500
38 38
22 34
1000
3
33
2000
38
37
5000
38
36
0 (solvent)
100
10 89
50 78
Without metabolic activation (48 h treatment)
100 5 0 0'
38 38
29 14
1000
>8
12
2000
38
16
5000
38
23
0 (solvent)
100
10 97
50 100
With metabolic activation
10 0
38
19
500
38
22
10 0 0
38
12
2000
38
72
5000
38
64
38 : The test substance precipitated or floated in culture medium.
4/1 1
& 05251
5. Chromosomal Aberration Test
(l)Test condition and preparation of the test substance solution
Period of experiment
from 4/17/1992 to 5/15/1992
Without metabolic activation
With metabolic activation
Number of cells seeded
4 x 103 /ml
4 x 103 /ml
Cell
Days of culture *
3 days 3 days
Plate
Form
Size Manufacturer Number of plates for each concentration
Plastic dish 6 cm in diameter Becton Dickinson & Co.
2 plates
Plastic dish 6 cm in diameter Becton Dickinson & Co.
2 plates
Amount of medium
5 ml/plate
3 ml/plate
Solvent
DM50 DMSO
Concentration of the original solution of the test substance
20 mg/ml
20 mg/ml
Preparation of
of the test substance solution
Amount of the test substance
Volume of the solvent
S t a t e of the test substance (encircle the applicable one)
100 mg
100 mg
5 ml
5 ml
Dissolved. (Suspended^) Dissolved, (^Suspended^)
Others(
) OthersC
)
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 ml/plate 6h
Amount of S9 Mix
0.5 ml/plate
Mitotic inhibitor
Name Concentration
Colcemid Final cone. 0.1 izg/ml
Colcemid Final cone. 0.1/zg/ml
Period of treatment
2h
2h
Remark: * The initiation day of culturing was defined as 0 day.
5/1 1
005252
V'
(2) Result Table 1, 2 show the results.
(3) Judgement of the result
Judgement (Encircle one)
Positive
(^Negative^)
Reason for judgement:
The test substance did not increase the cells with structural chromosomal
aberrations and polyploid cells with or without metabolic activation.
These results led to the conclusion that the test substance did not have clastogenic
potential.
(4) Referential matters
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. Therefore DMSO was selected for solvent of the test substance.
2. In the cell growth inhibition test, 50% inhibition dose of cell growth (TCIDso) was Qtig/-mi in 24-hour treatment, T h u i/ t d in 48-hour treatment without metabolic activation and 71 p.g/mi with metabolic activation. In all treatment groups, cell growth index at 100//g/ml or more did not increase or slightly increased and this concentration coincide with that beginning to precipitate. So it was considered that the test substance was saturated at 100a g/ml or more (Fig. 1, 2). From these results, the highest concentration in chromosomal aberration test was set at 100M g h d with or without metabolic activation.
3. In chromosomal aberration test, the test substance precipitated or floated in culture medium at 100 lig lid
4. Data were statistically analysed by the x 2-test against the negative control. Structual aberrant cells only with gaps were excluded from this analysis.
6/1 1
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6. Others
Testing facility
Name Address
Kashima Laboratory, Mitsubishi-Kasei Institute of Toxicological and Environmental Sciences
14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki Tel.0479(46)2871
Study director Name title Taraotu Nishitomi Senior Research Scientist
signed & sealed
Test dates from 4/10/1992 to 9/30/1992
7/1 1
00525.4
V*
Fig. 1 Cell growth index of Sample D-l (without metabolic activation)
Fig.2 Cell growth index of Sample D-l (with metabolic activation)
8/1 1
005255
TABLE 1 RESULTS OF THE CHROMOSOMAL ABERRATION STUDY OF Sample D-l IN THE DIRECT ASSAY
Test substance : Sample D-l
Treatment Exposure Dose No. of Polyploids
No. of cells with chromosomal structural aberrations (\)
time levels cells
Judge- Gaps
Chromatid type Chromosomal type Others
Total
Judge-
group
(h) (u g/ml)analysed ( * i ment 100 0
e
0
ctb 1
cte 1
csb 0
cse 0
~g *g ment * 022
Negative control
24
0 100
0/
0 0 0 1 0 0 1 1/
200 0(0.0)
0(0.0) 1(0.5) 1(0.5) 1(0.5) 0(0.0) 0(0.0) 3(1.5) 3(1.5)
100 0
000 000 00
(DMSO)
48
0 100
0/
0 0 0 0 0 0 0 0/
200 0(0.0)
0(0.0) 0(0.0) p(0.0) p(o.o) 0(0.0) 0(0.0) 0(0.0) 0(0.0)
100 1
010100 2 2
25 100
0-
0 1 0 0 0 0 1 1-
200 1(0.5)
0(0.0) 2(1.0) 0(0.0) 1(0.5) 0(0.0) 0(0.0) 3(1.5) 3(1.5)
100 0
000 100 1 1
24 50 100
0-
0 0 0 0 0 0 0 0-
200 0(0.0)
0(0.0) 0(0.0) 0(0.0) 1(0.5) 0(0.0) 0(0.0) 1(0.5) 1(0.5)
100 1
000100 11
# 100 100
0-
0 0 0 1 0 0 1 1-
Teat
200 1(0.5)
0(0.0) 0(0.0) 0(0.0) 2(1.0) 0(0.0) 0(0.0) 2(1.0) 2(1.0)
substance
100 1
00 1000 1 1
25 100
1-
0 0 0 0 0 0 0 0-
200 2(1.0)
0(0.0) 0(0.0) 1(0.5) . 0(0.0) 0(0.0) 0(0.0) 1(0.5) 1(0.5)
\ 100 1
100 100 12
46 50 100
0-
0 0 1 0 0 1 1-
200 1(0.5)
1(0.5) 0(0.0) 0(0.0) 2(1.0) 0(0.0) 0(0.0) 2(1.0) 3(1.5)
100 1
000 000 0 0
# 100 100
0-
0 0 0 1 0 0 1 1-
200 1(0.5)
0(0.0) 0(0.0) 0(0.0) 1(0.5) 0(0.0) 0(0.0) 1(0.5) 1(0.5)
100 0
0 12 12 1 0 0 23 23
Positive 24
0.03 100
0-
2 20 8 1 0 0 27 2B +++
control
200 0(0.0)
2(1.0) 32(16. 0) 20(10. 0) 2(1.0) 0(0.0) 0(0.0) 50(25.0) 51(25. 5)
9S2S00
(MMC)
48
0.03
100 100
0 0-
2 7 19 7 0 4 32 34 0 16 34 1 1 5 49 49 +
200 0(0.0)
2(1.0) 23(11. 5) 53(26. 5) 8(4.0) 1(0.5) 9(4.5) BK40.5) B3(41. 5)
ctb : chromatid break, cte : chromatid exchanee, csb : chromosome break, cse : chromosome exchange,
others : fragmentation etc. (except pulvalization)
Aberrant cells(-g) were statistically compared with the control by the * 2-test; +*+(p<0.001)
s The test substance precipitated or floated in culture medium when it was added.
TABLE 2 RESULTS OF THE CHROMOSOMAL ABERRATION STUDY OF Sample D-l IN THE METABOLIC ACTIVATION ASSAY
Test substance : Sample D-l
Treatment With or Dose No. of without levels cells
Polyploids Judge- Gaps
No. of cells with chromosomal structural aberrations ()
Chromatid type Chromosomal type Others
Total
Judge-
group
S9 Mix (fig/ml )analysed 100
<*) ment ** 0
e 0
ctb 0
cte 0
csb 0
cse 0
-e *z ment* 000
Negative control
-
0 100
0/
0 0 0 1 1 0 2 2/
200 0(0.0)
0(0.0) 0(0.0) 0(0.0) 1(0.5) 1(0.5) 0(0.0) 2(1.0) 2(1.0)
100 0
0000000 0
(DMSO)
+
0 100
0/
0 0 0 0 0 0 0 0/
200 0(0.0)
0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)
100 25 100
0 0-
0001001 1 0 0 0 0 0 0 0 -
200 0 (0 .0 )
0(0.0) 0(0.0) 0(0.0) 1(0.5) p(0.0) 0(0.0) 1(0.5) 1(0.5)
100 0
010 000 1 1
- 50 100
0-
1 0 0 0 0 0 0 1-
200 0(0.0) 100 0
1(0.5) 1(0.5) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(0.5) 2(1.0) 1000000 1
# 100 100
0-
0 0 0 0 0 0 0 0-
Test substance
200 0(0.0) 100 0
1(0.5) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(0.5) 00 0 0 000 0
25 100
0-
0 0 0 1 0 0 1 1-
200 0(0.0)
0(0.0) 0(0.0) 0(0.0) 1(0.5) 0(0.0) D(0.0) 1(0.5) 1(0.5)
100 0
0 0 0 1 0 0 1 1
\ + 50 100
0-
0 0 0 0 0 0 0 0-
200 0(0.0)
0(0.0) 0(0.0) 0(0.0) 1(0.5) 0(0.0) 0(0.0) 1(0.5) 1(0.5)
100 0
10 110023
# 100 100
0-
0 0 0 1 0 0 1 1-
200 0(0.0)
1(0.5) 0(0.0) 1(0.5) 2(1.0) 0(0.0) 0(0.0) 3(1.5) 4(2.0)
Positive
-
100 20 100
1 0-
00 000 00 0 0 0 1 0 0 0 1 1-
005257
control (BP)
+
200 1(0.5)
D(0.0) 0(0.0) . 1(0.5) 0(0.0) 0(0.0) 0(0.0) 1(0.5) 1(0.5)
100 0
0 12 21 0 0 44 73 73
20 100
1-
6 15 27 0 0 36 69 71
200 1(0.5)
6(3.0) 27(13. 5) 46(24. 0) 0(0.0) 0(0.0) 60(40.0) 142(71 .0) 144(72 .0)
ctb : chromatid break, cte : chromatid exchange, csb : chromosome break, cse : chromosome exchange.
others : fragmentation etc. (except pulvalization)
Aberrant cells(-g) were statistically compared with the control by the x 2-test; ++(p<0.001)
* The test substance precipitated or floated in culture medium when it was added.
Cells were treated with S9 (5% of final cone.) or without S9 for 6 hours and recovered for IB hours.
Appendix : List of Control Reagents
Negative Control(Vehicle)
Reagent Name
Abbreviation Lot No.
Supplier
dimethylsulfoxide
DMSO 206N1084 Kanto Chemical Co. Inc.
Positive Control Reagent Name
mitomycin C benzo[a]pyrene
Abbreviation Lot No.
Supplier
MMC 719AAB Kyowa Hakko Kogyo Co., Ltd.
BP AX01 Tokyo Kasei Kogyo Co., Ltd.
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