p. 1

B051346

9. References

[1] Maron DM and Ames BN . Revised methods for the Salmonella mutagenicity test.

Mutat Res 1983 ; 113 : 173-215.

[2] Green MHL and Muriel WJ. Mutagen testing using Ti-p+ reversion in Escherichia

coli. Mutat Res 1976 ; 38 : 3-32.

[3] Chemical Substance Investigation Division of the Industrial Safety and Health

Department of the Ministry of Labor of Japan, Japan Industrial Safety and Health

Association, editors. Mutagenicity Tests in Industrial Safety and Health Law . 1991 .

10. Study Notes

10.1 Environmental conditions that many have had an effect on the re liability of

~ the study results

None

10.2 Deviations from the protocol

None

L~ 1A 1?1
p. 2

B051346

Table l Results of the Dose-finding Test

T s f he",. ce : MTDID366 3

Testing periods From April 17. 2006 to April 20, 2006

Metabolic Test material Number of revertants (mmnber of colonies/plate)

activation concentration Base-pair change ty e FrameshiR type

sy stem (p~ late) TA 100 TA 1535 WP2 uvrA - TA 98 TA 1537

110 10 21 16 14

Solvent control 107 109 13 12 17 19 15 16 12 13

101 11 21 11 15

0 .305 107 104 7 9 15 18 12 12 15 15

96 5 23 9 17

1 .22 99 98 10 8 13 18 12 11 16 17

101 9 16 15 11

S9mix 4 .88 95 98 7 8 19 18 8 12 19 15

85 10 22 11 13

(-) 19.5 103 94 10 10 23 23 15 13 9 11

110 11 20 16 17

78 .1 100 105 6 9 21 21 14 15 13 15

( 99 5 18 14 10

313 121 110 9 7 16 17 8 11 17 14

108 10 19 11 13

1250 99 104 6 8 14 17 12 12 14 14

112 11 23 14 19

5000 83 98 11 11 19 21 14 14 16 18

84 11 21 16 15

Solvent cont rol 92 88 8 10 22 22 21 19 12 14

117 8 23 10 17

0 .305 106 112 8 8 18 21 13 12 18 18

89 13 27 19 19

1 .22 86 88 10 12 23 25 19 19 14 17

91 5 19 16 15

s9mix 4.88 95 93 4 5 22 21 21 19 19 17

103 8 20 20 15

(+) 19.5 90 97 5 7 21 21 17 19 13 14

83 7 22 18 14

78 .1 90 87 6 7 20 21 18 18 16 15

96 7 27 17 12

313 86 91 9 8 21 24 18 18 11 1 2

102 7 19 18 14

1250 100 101 7 7 16 18 16 17 18 16

111 11 22 20 11

5000 115 113 6 9 23 23 13 17 16 14

Name and AF-2 NaN3 ENNG AF-2 9-AA

Positive concentration 0.01 0.5 2 0.1 80

contr ol -

not requiring Number of 859 315 605 484 530

S9 m ix reVertan late 788 824 302 309 590 598 537 511 569 550

Name and 2-AA 2-AA 2-AA 2-AA 2-AA

Positi~ concentrati on 1 2 10 0.5 2

control law

requiring Number of 716 278 720 455 115

;

S9 mix re.ertaatlplatn 678 6

97 267 273 698 709 425 440 101 108

AF-2 : 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, NaN3 : sodium azide, ENNG : N-ethyl-N-nitro-N-ntorosoguanid'me,

9-AA : 9-ammoacridine bydrochloride, 2-AA : 2-amaioanthracene

)" .

1 . The average number of colonies for each concentration is in the" (

2 . "Number of revertants" -

the observed value and average value in order beginning with the low concentr ations of the test subst ance .

Z`i.'

~ ~~
p. 3

B051346

Table 2 Results of the Main Test

Test substance : MTDID3663

Testing periods From April 25, 2006 to April 28, 2006

Metabolic Test material Number of revertants (number of colonies/plate)

activation concentration Base-pair changetype Frameshift type

system (μgJ late) TA 100 TA 1535 WP2rrvr,€' TA 98 TA 1537

107 11 22 24 8

Solvent control 91 ( 99 ) 5 8 18 ( 20 ) 20 ( 22 ) 9 9

105 5 26 21 13

313 78 ( 92 ) 5 5 21 ( 24 ) 26 ( 24 ) 7 10 )

92 9 18 24 3

s9mbx 625 89 ( 91 ) 6 8 17 ( 18 ) 20 ( 22 ) 7 5

87 10 26 24 12

(-) 1250 95 ( 91 ) 11 ( 11 ) 19 ( 23 ) 20 ( 22 ) 13 t 13 )

99 10 17 18 5

2500 79 ( 89 ) 7 9 18 ( 18 ) 19 ( 19 ) 6 6

99 5 16 20 8

5000 104 ( 102 ) 10 ( 8) 20 ( 18 ) 26 ( 23 ) 12 ( 10

80 9 26 26 12

Solvent control 81 ( 81 ) 12 ( 11 ) 22 ( 24 ) 17 ( 22 ) 12 ( 12

100 6 20 26 11

313 76 ( 88 ) 9 8 28 ( 24 ) 21 ( 24 ) 11 ( 11 )

80 7 27 20 15

S gm ix 625 90 ( 85 ) 9 8 22 ( 25 ) 22 ( 21 ) 8( 12 )

82 9 25 19 11

(+) 1250 92 ( 87 ) 8( 9 24 ( 25 ) 23 ( 21 ) 12 ( 1 2

82 5 25 14 7

2500 89 { 86 ) 8 7 26 ( 26 ) 17 ( 16 ) 7( 7

86 8 25 22 11

5000 89 ( 88 ) 12 ( 10 ) 23 ( . 24 ) 26 ( 24 ) 14 ( 13

Name and AF-2 NaN3 ENNG AF-2 9-AA

positive concentration 0.01 0.5 2 0.1 80

control (Awplate)

not requiring Number of 807 602 669 482 333

S9 mix revertant/plate 836 ( 822 ) 632 ( 617 ) 640 ( 655 ) 480 ( 481 ) 421 ( 377 )

Name and 2-AA 2-AA 2-AA 2-AA 2 -AA

~ Positive concentration

1 2 10 0.5 2

control (49/plate) I

requiring Numberof 787 238 756 437 132

S9 mix revertant/platlate 822 ( 805 ) 256 ( 247 ) 881 ( 819 ) 422 ( 430 ) 112 ( 122 e

AF-2 : 2-(2-fiuyl)-3-(5-nitro-2-fvrylJacry lamide, NaN3 : sodium azide, ENNG : N-ethyl-N-nitro-N-ntorosoguanidine ,

9-AA : 9-aminoac ridine hydrochloride, 2-AA : 2-aminoanthracene

1 . The average number of colonies for each concentra tion is in the

2. "Number ofrevertants" - the observed value and average value in order beginning with the low concentrations of the test substance.

GJ'

~ Sb
p. 4

B051346

(TA100~

200 200

.~- -S9mix ••-•©. . . .. +S9mi x

150 a)

150

~

CO

CO

~

y ~

'aW

0

° °

100 0° 100

-

~ `c

0 +~ -

4~

L

CD C

Q> °

D

50 50

D

~

0 0

0 313 625 1250 2500 5000 0 313 625 1250 2500 5000

Concentration ()1 g/plate)

Concentration ( 11g/Plate)

Fig.1 Dose-response curve of MTDID3663 (main test )

(TA1535~

40 40

-S9mix -•-- . .. . . +S9mix

----l---

30 30~

CD `

U

+ J Q.

R ~

CL

w

o

CO

O U

20 20

C i

o N

U ~

CO CD

CD 10 10

10- -Q • '(3 0

0 0

0 313 625 1250 2500 5000 0 313 625 1250 2500 5000

Concentration (u g/plate) Concentration (/1 g/plate )

Fig.2 Dose-response curve of MTDID3663 (main test)

^~-

IA~ 1
p. 5

B051346

~ WP2 uvrA- )

60 40

--~. .-- -S9mix -•©. •••• +S9mix

30 a) 45

4~

lC Q

cC

Q N

0)

y

U C

° 30

0 201 - ~

- 0 0 k<%I;v 4-1 -O- - -O - "

c _ ~ ©

c

cc 4.j

CD m

15

1 0

\

0

0 0 313 625 1250 2500 5000

0 0 313 625 1250 2500 5000

Concentration (/1 g/plate) Concentration (/1g/plate)

Fig.3 Dose-response curve of MTDID3663 (main test)

~ TA 98 1

40 40

-f--- -S9mix •-•-E3••••• +S9mi x

30 30

(D

CD

~ a

R

n C

O CO ~ a> c `©_ _ •

3 . ' . .

0 20 -°0 20

0 C

i*~. .+

~ U

w ~

G) d

CD> 10 10

0 0

0 313 625 1250 2500 5000 0 313 625 1250 2500 5000

Concentration Cu g/plate) Concentration (I1 g/plate)

Fig.4 Dose-response curve of MTD1D3663 (main test)

'21-

~ ~ ~
p. 6

( TA 1537 ~ B051346

40 40

--f-- -S9mix •••. ©. . . . . +S9mix

~ 30 30

41 CO c4

C L

m co w y

0 20 -o° 2

U U

0

4a 4-J

++

CD CD

~ ~

.{-~i

O

cc: 10 CIC 10

i i

\

er

0 0

0 313 625 1250 2500 5000 0 313 625 1250 2500 5000

Concentration (u g/plate) Concentration (g g/plate)

Fig.5 Dose-response curve of MTDID3663 (main test)

~ ~ ~
p. 7

~

B051346

(Appendix 1)

Acceptable ranges of negative (solvent) and positive control values

1 . Background data of the negative (solvent) control value

-S9mix +S9mix

Strain Average Range No. of Average Range No. of

assay assay

TA100 100 76-124 69 103 79-127 67

TA1535 11 5-127 68 11 5-16 67

W P2uvrA 26 14-38 66 25 14-37 66

~ TA98 17 8-26 67 21 11-31 67

TA1537 8 3-14 66 11 5-16 66

2. Background data of the positive control value

-S9mix +S9mix

Strain Average Range No. of Average Range No. of

assay assay

TA100 693 473-1386 37 802 592-1605 36

TA1535 358 266-715 36 271 179-542 35

WP2uvrA 763 491-1527 34 480 238-959 34

TA98 476 359-951 36 408 288-815 36

TA1537 509 120-1017 34 147 80-293 34

Range of the negative(solvent) control value was calculated by the average -L 2SD.

Range of the positive control value was calculated by the average ±3SD or multiplying

theby2.

Background data was calculated using the control values yielded April 5, 2005

to September 28, 2005 .

-z9-

LA S~ L-\
p. 8

B051346

Quality Assurance Statement

Title: Bacterial Reverse Mutation Test of MTDID3663

Study No.: B051346

By the inspections and audits listed below, I hereby certify that the study was carried

out in accord ance with its protocol and with our Standard Operating Procedures, and that

the report faithfully describes the methods and procedures applied, and that the reported

results accurately reflect the raw data of the study .

Standard Concerning Testing Facilities Implementing studies Related to New Chemical

Substances, (Pharmaceutical and Food Safety Bureau, Manufacturing Industries Bureau,

~ Environmental Policy Bureau, Ministry of the Environment, Pharmaceutic al and Food

affairs No . 1121003, November 17, 2003, No .3, November 21, 2003, Ministry of Health,

Labor, and Welfare, Ministry of Economy, Trade, and Industry, No.031121004)

Notification for Standard of Testing Facilities Based on Rule for Industrial Safety and

Health, Article 34-3- 2

(Notification No 76, Ministry of Labor, September 1, 1988 )

A Part Revision of Notification for Standard of Testing Facilities Based on Rule for

Industrial Safety and Health, Article 34(3)- 2

(Notification No 13, Ministry of Labor, March 29, 2000)

Date of reporting to

Inspection or audit Date of inspection the study director

or audit and to the management

Study Protocol April 5, 2006 April 5, 2006

Study Procedure

Dose-finding test, Pre-incubation April 18, 2006 April 19, 2006

Dose-finding test, Colony count April 20, 2006 April 24, 2006

Main test, Pre-incubation April 26, 2006 April 27, 2006

Main test, Colony count April 28, 2006 April 28, 2006

Draft Report June 2, 2006 June 2, 2006

Study Report June 8, 2006 June 8, 2006

Quality Assurance manager: Date: June 8, 2006 Hidetsugu Watabe sealed

General Testing Research Institute of

Japan Oilstuff Inspectors' Corporation

-3,9-

~SS
FINAL REPORT

M.S .I. Report No. A050397

Acute Toxicity Test of MTDID3663

~ with Medaka (Oryzias Wipes)

Submitted to:

Sumitomo 3M Limited

Prepared by:

Mitsubishi Chemical Safety Institute Ltd. (M.S.I.)

August 31, 2006

---~-,

~ 5 lp
AoS097

Acute Toxicity Test of MTDID3663

with Medaka (Oryzias latipes)

(English version)

Sponsor Sumitomo 3M Limited

Study Title Acute Toxicity Test of MTDID3663 with Medaka (Oryzias latipes)

Study No. A050397

This report is the English version of the original, which was wri tten in Japanese . I, the

undersigned hereby declare that this version faithfully reflects the original repo rt to the best of

my knowledge.

Translated by

SANITIZED

~-- -

~

~~~
AVJrJa l

Compliance with the GLP Standard s

Yokohama Laboratory

Mitsubishi Chemical Safety Institute Ltd.

Sponsor Sumitomo 3M Limited

Study Title Acute Toxicity Test of MTDID3663 with Medaka (Oryzias latipes)

Study No. A050397

I hereby certify that this study was carried out in accordance with its protocol and with

our standard operating procedures, and that the report accurately describes the test results .

( The study described in this report was conducted in compliance with the following Good

Laboratory Practice (GLP) standards.

The standards concerning the test facility in which the study of new chemical

substance is performed (Yakushokuhatsu No .1121003, Heisei 15.11 .17 Seikyoku

- No.3, Kanpokihatsu No .031121004, November 21, 2003 ; the latest revision, April

1, 2005)

Study Director

Midori MINO, B.Sc. Sealed date: August 31, 2006

~---

.~-3---

~-}
Quality Assurance Statement

Yokohama Laboratory

Mitsubishi Chemical Safety Institute Ltd.

Sponsor Sumitomo 3M Limited

Study Title Acute Toxicity Test of MTDID3663 with Medaka (Oryzias latipes)

Study No. A050397

We hereby certify that this study was carried out in accord ance with its protocol and

with our standard operating procedures. We certify that this report faithfully describes the

methods and procedures employed, and that the repo rted results reflect the raw data of the

study, accurately.

Date of Reporting to

Date of Inspection Management and

Study Director

Protocol: August 4, 2006 August 4, 2006

Amendment 01 August 9, 2006 August 9, 2006

In-progress study:

Preparation of test solutions : August 7, 2006 . August 7, 2006

Introduction of fish: August 7, 2006 August 7, 2006

Observation of fish: August 11 , 2006 August 11, 2006

Final report: August 31, 2006 August 31, 2006

Quality Assurance Personne l

Yumiko KASHIWAGI, B .Sc. Sealed date: August 31, 2006

Hiroko TOKIMI, B.Sc. Sealed date: August 31, 2006

Reiko TSUTSUI Sealed date: August 31, 2006

. 4

~~~
Aun"i

Introductio n

1. Study Titl e

Acute Toxicity Test of MTDID3663 with Medaka (Oryzias latipes)

(Study No . A050397)

2. Purpose

The acute toxicity test of the test substance with Medaka (Oryzias latipes) was conducted

in order to evaluate the toxicity of the test substance for notification under the Chemical

Substances Control Law of Japan.

3. Test Guidelines

The test method relating to new chemical substances "Alga growth inhibition test,

Daphnia sp. acute immobilisation test, and fish acute toxicity test" (Yakushokuhatsu

No.1121002, Heisei 15 .11 .13 Seikyoku No .2, Kanpokihatsu No .031121002, November

21, 2003 ; the latest revision, April 1, 2005 )

4. GLP Compliance s

The standards concerning the test facility in which the study of new chemical substance is

performed (Yakushokuhatsu No .1121003, Heisei 15 .11 .17 Seikyoku No .3, Kanpokihatsu

No.031121004, November 21, 2003 ; the latest revision, April 1, 2005)

5. Sponsor

Sumitomo 3M Limited

8-8 Minami-hashimoto 3-chome, Sagainihara, Kanagawa, Japan

~

6. Test Facility (Head Office)

Mitsubishi Chemical Safety Institute Ltd. (M.S .I.)

1-30 Shiba 2-chome, Minato-ku, Tokyo, Japan

7. Test Facility (Laboratory)

Yokohama Laboratory

Mitsubishi Chemical Safety Institute Ltd.

1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa, Japan

8. Study Director

Midori MINO, B.Sc.

Ecological Chemistry Division

.-3--

~(0d
A0MJ47

9. Personnel in Charge of the Study

Hiromitsu OOKUBO, Ph .D. Sealed date: August 31, 2006

(Experimental work and reporting)

Masamitsu NAKAGAWA, M .Sc. Sealed date: August 31, 2006

(Experimental work)

Kazuhisa OONISHI Sealed date: August 31, 2006

(Analytical work)

10. Date

Initiation of the study : August 4, 2006

Start of the exposure : August 7, 2006

End of the exposure : August 11 2006

Completion of the study: August 31, 2006

11. Storage

The following will be stored in the archives of the Yokohama Laboratory.

1) Protocol

2) Final report

3) Raw data

4) Test substance

5) Reference substance

6) Other requirements

~---- .

-6-=-

~~~
A05P13~

Contents

page

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

----------------------------------------••----•------------------------•- 8

1 . Test Substance 10 -------------------------------------------------------------------

1 .1 . Identification 10

1 .2 . Source 11

1 .3. Storage and Confirmation of Stability 1 1

2

. Test Species------------------------------------------------------------ -----------------•-- ------------- --------- ----------.12

3 . Test Methods________ 13

------------------------------------------------------------------------------------------------------- ~

3 .1 . Test Conditions 13

3 .2. Dilution Water 13

3 .3 . Apparatus 14

3 .4. Test Concentrations 14

3 .5. Preparation of Test Solutions 14

3 .6. Analysis of Test Solutions 15

3 .7. TestProcedure 15

3 .8. Calculation of Results 16

4. Results and Discussion 17 -------------------------------------•--------------------- ------------------------- ~

4 .1 . Circumstances That May Have Affected the Reliabilit y

of the Test Results 17

4.2 . Concentrations of Test Substance in Test Solutions 17

4.3 . Median Lethal Concentration (LC50) 17

4.4. Highest Concentration in 0% Mortality and Lowest Concentration

in 100% Mortality 17

4 .5 . Toxicological Symptoms 17

4.6 . Appearance, Temperature, Dissolved Oxygen Concentrations and

pH of Test Solutions 18

Tables 1-9 19-26

Figure 1 27

Appendix 1 . Infrared Absorption Spectra 28-29

Appendix 2 . Dilution Water Quality 30-31

Appendix 3 . Preparation of Test Solutions 32-33

Appendix 4. Analysis of Test Solutions 34-43

-~--

t--~ lp`~
AU -*3W

Summary

Sponsor Sumitomo 3M Limite d

Study Title Acute Toxicity Test of MTDID3663 with Medaka (Oryzias latipes)

Study No. A050397

Methods

The study was conducted in accordance with the test method relating to new chemical

substances "Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish

acute toxicity test" (Yakushokuhatsu No .1121002, Heisei 15 .11 .13 Seikyoku No .2,

Kanpokihatsu No .031121002, November 21, 2003 ; the latest revision, April 1, 2005).

~ 1) Exposure procedure: semi-static (batchwise renewal of the test solutions every

24-hour exposure)

2) Duration: 96 hours

3) Test concentrations: control, solvent control, 0 .540, 0.960, 1 .70, 3 .00, 5.40* mg/L

(nominal concentration) *: the maximum attainable concentration

Factor : 1 . 8

Solvent concentration: 95 μL/L (Tetrahydrofuran)

4) Volume of test solution : 5.0 L/vessel

5) Number of vessels: 1 vessel/concentration

6) Biomass loading : 10 fish/concentration

7) Temperature: 24± 1 °C

8) Light: fluorescent light

( 16 hours light (1000 lux or less)/8 hours dark

9) Analytical methods: gas chromatography / mass spectrometry (GC/MS)

LA ~3
AuDg:s~7

Results

1) Concentrations of test substance in test solutions :

The proportion of the measured concentrations to the nominal values were 86-109%

at the preparation and 53-76% after 24-hour exposure . It is considered that the main

cause of concentration reduction was volatilization of the test substance from test

soluitons.

2) Median lethal concentration (LC50) after 96-hour exposur e

>4.90 mg/L (95% confidence limits : impossible to calculate)

3) Highest concentration in 0% mortality after 96-hour exposur e

>4.90 mg/L

4) Lowest concentration in 100% mortality after 96-hour exposure

>4.90 mg/L

,-

9

~~o~
1 . Test Substance

1 .1 Identification

Name of the new chemical

substance

(by IUPAC nomenclature)

Alternate name

CAS RN

Structural formula o r

rational formula

(If both of them are

unknown, outline o f

preparation or

manufacturing

Molecular weigh t

Purity (%) of the new

chemical substance

subjected to the study

Lot number of the new SANITIZED

chemical substance

subjected to the study

Name and content of

impurities

Vapor pressure

Solubility in water

1-octanol/water partitio n

coefficient

Melting point

Boiling point

Appearance

at room temperature

Stability

Solubility in

solvents and the like

--i6--

LA
AURA-/

1 .2. Source

Supplier: Sumitomo 3M Limited

1 .3 . Storage and Confirmation of Stability

For the duration of the study, the test substance was stored in the refrigerator of M .S.I.

(refrigeration, dark place and sealing). At the end of the study, infrared absorption spectrum

of the test substance was measured . The spectrum was consistent with that measured at the

start of the study, indicating that the test substance was stable under the storage condition .

The infrared absorption spectrum is shown in Appendix 1 .

Apparatus: Infrared spectrophotometer, AVATAR 320, Nicolet Instrument Corporation

I

~~Q `P
tiu-%39

2 . Test Species

1) Common name : Medaka

2) Scientific name : Oryzias latipes

3) Source : Tsunashima Fishing Co.

(18-1 Tsunashima-nishi 5-chome, Kouhoku-ku, Yokohama,

Kanagawa)

4) Date of receipt: June 28, 2006

5) Lot No : : 06-H-0628

6) Total length* : 1.84 cm ( 1 .53-2.22 cm), n=10

7) Body weight: 0.060 g (0.043-0.097 g), n=10

8) Sensitivity to reference substance :

96-hour median lethal concentration (LC50) of reference

substance (CuSO4 • 5H20, guar anteed reagent) = 0.63 mg/L

(95% confidence limits : 0 .42-0.90 mg/L)

The average LC50 value in our laboratory (n=36, since

1995) is shown below. All of LC50 values are reduced

value to anhydrous of the reference subst ance.

average ± standard deviation=0.86±0.27 mg/L

minimum value-maximum value=0 .44-1 .5 mg/L

9) Age: Less than a year after hatching

10) Acclimation period : July 19, 2006 - August 7, 200 6

Mortality of fish during the week before exposure : <5 % .

The test fish used were in good health and free from any

apparent malformation. During the acclimation period, fish

were kept without medicine in the aquarium.

Acclimation conditions during 14 days before exposure

are shown below.

Medication before acclimation: sodium chloride (Jap an

Standard Salt Co., Ltd.); Elbarju for pet fish (sodium

nifurstyrenate, Ueno Fine Chemicals Industry, Ltd .)

11) Acclimation conditions :

Water: dilution water (see 3.2)

Procedure : flow-through

(biomass loading : 1.0 g fish/L/day)

Temperature : 24± 1 °C

Dissolved oxygen concentration : _> 80% of the saturation

pH: 6.5-8.5

Light: fluorescent light,

16 hours light (1000 lux or less) / 8 hours dark

Feed : TetraMin, TetraWerke

Feeding amount: Approx. 2% body weight of fish/day

(until 24 hours before exposure .)

--1 2 _--

~
A0583 3~

3. Test Methods

The study was conducted in accordance with the test method relating to new chemical

substances "Alga growth inhibition test, Daphnia sp . acute immobilisation test, and fish

acute toxicity test" (Yakushokuhatsu No .1121002, Heisei 15 .11 .13 Seikyoku No .2,

Kanpokihatsu No .031121002, November 21, 2003 ; the latest revision, April 1, 2005).

3.1 . Test Condition s

1) Exposure procedure: semi-static (Batchwise renewal of the test solutions

every 24-hour exposure .)

2) Duration: 96 hours

3) Volume of test solution : 5.0 L/vessel (Biomass loading : '_5 1 .0 g fish/L)

4) Number of vessels: 1 vessel/concentration

5) Biomass loading: 10 fish/concentration

6) Temperature : 24±1°C

7) Dissolved oxygen concentration: ? 60% of the saturation (not aerated)

8) pH: not adjusted

9) Light: fluorescent light,

16 hours light (1000 lux or less)/8 hours dark

10) Feeding: none

3.2 . Dilution Water

Dechlorinated tap water was used as dilution water in this test. Tap water supplied by

Yokohama City Water Service was filtrated with activated charcoal, and sodium

thiosulfate aqueous solution was added into the filtrated water for neutralization of

unreinoved trace amount of free-chlorine . This dechlorinated water was aerated, controlled

at 24±2°C, and used for the test . Water quality of the dechlorinated water has been

checked on the 35 parameters twice a year, and the results are shown in Appendix 2 .

Values of hardness and pH are usually 30-100 mg/L (CaCO3) and 6.5-8.5, respectively.

At the use of this water, residual chlorine was not detected. From these results, it was

considered that this water was suitable for the fish-breeding water.

---~-~

LA k-PIZO!,

~
A050'~

3.3. Apparatus

1) Vessel: 5.0 L glass vessel (Surface of test solution was covered with

Teflon sheet.)

2) Incubator : PVC-made tank (temperature control equipment : Coolnit CL80F, Taitec Co., Ltd.)

3) Thermometer: Check Temp, Hanna Instruments

4) pH/DO meter: D-55, Horiba Ltd.

5) Electrobalance : AG204, Mettler, K.K.

AE163, Mettler, K.K.

AB204-S, Mettler, K.K.

PB3002, Mettler, K.K.

AB204, Mettler, K.K.

3.4. Test Concentration s

The solubility of the test substance to the dilution water is 5 .4 mg/L (measured by

M.S.I.). Hence, the test concentration was set at 5 .4 mg/L or less.

A range-finding test was conducted before the definitive test, see the following table .

Consequently, the concentration range in the definitive test was set as follows :

Nominal concentrations of the definitive test : control, solvent control, 0 .540, 0.960, 1 .70,

3.00, 5 .40* mg/L

*: The maximum attainable concentration .

factor: 1 . 8

Result of range-finding test (1 vessel/concentration, 5 fish/concentration )

Concentration

(mg/L) Mortality for 96 hours (%) Toxicological symptoms

0.0540 0 none

(7- - 0.540 0 none

5.40 0 observed

3.5. Preparation of Test Solution s

The preparation methods of test solutions are shown in Appendix 3 . The control

solution contained dilution water only, and solvent control solution was dilution water

containing the solvent (95 μL/L).

The stock solution I was prepared at the start of exposure and stored under refrigeration

and dark (stable for 96 hours or more under this storage condition). Stock solution II and

stock solvent solution were prepared at each use and used for preparation of test solution .

-44-

Lk k.~:'C'\
Aos8R

3.6. Analysis of Test Solutions

During the exposure, the concentrations of the test substance were measured at two

renewal sets of the test solutions, at the beginning of exposure and before renewal at 24

hours, and after renewal at 24 hours and before renewal at 48 hours . The analysis samples

were collected form the test solutions. These samples were analyzed by gas

chromatography / mass spectrometry (GC/MS) . The test substance is diastereomeric

mixture, and 4 peaks were detected on a chromatogram . Hence, the concentration of the test

substance was determined by quantitative analysis using the total peak area of 4 peaks .

Details are shown in Appendix 4 .

3.7. Test Procedur e

Prior to the introduction of fish, the total length and body weight of ten fish out of the

same acclimation vessel were measured to confirm that total length of test fish was within

2 .3±1 .2 cm.

After observation or measurement of the appearance, temperature, dissolved oxygen

concentration (DO) and pH of each test solution, ten fish were introduced into each test

vessel. The appearance, temperature, DO and pH of each test solution were observed or

measured once a day (before and after renewal). Fish were not fed during exposure.

Number of dead fish, toxicological symptoms and abnormalities were recorded at 24,

48, 72 and 96 hours after the start of exposure . General toxicological symptoms are shown

below. If dead fish were found, they were removed immediately not to get water quality

worse.

General toxicological symptom s

Death: There is no reaction to stimulation.

Abnormal respiration : Gill movements are different from those of control fish.

Abnormal swimming: Swimming behaviors are apparently different from those of

control fish (slow or sensitive movement, convulsion,

overturn and so on).

Paralyzation: Although fish is moving on the bottom or near the surface of

water, it can not swim (including lying and apparent death).

~~ d
AU-1017

3.8. Calculation of Results

1) Determination of concentration of test substance for calculating median lethal

concentration (LC50)

Concentration of test substance to calculate LC50 was the average of measured

concentration (time-weighted mean).

Time-weighted me an:

ConcA„ - ConcB„

mc„ = ln(ConcA„) - In(ConcB„ )

me] + mc2 + + mc„ MC =

n

nic,, : Mean measured concentration during each exposure

ConcA,,: Measured concentration at the preparation of test solutio n

ConcB,, : Measured concentration at 24 hours after the preparation of test solution

_ (In the case of ConcA„=ConcB„, mc„=ConcA„=ConcB„)

MC: Mean measured concentration

2) Calculation of LC5 0

The cumulative mo rtality (%) for each concentration after 24, 48, 72 and 96-hour

exposure was calculated from the number of dead fish and biomass loading (10 fish),

and median lethal concentration (LC50) was determined according to the following table .

Mortality at maxi- > 50% <50% mum concentration

LC50 is selected among calculations

using the probit method, movin g

Detennination average method and binomial method . Estimated

{ method of LC50 95% confidence limits are calculated if concentration range.

possible .

Concentration- Included in the report. Included in the report. mortality curve

3) Highest concentration in 0% mortality and lowest concentration in 100% mo rtality

From the results at 24, 48, 72 and 96 hours a fter the start of exposure, the highest

concentration in 0% mortality and the lowest concentration in 100% mortality were determined if possible . The highest concentration in 0% mortality is the highest concentration in which no dead fish was observed, while the lowest concentration in 100% mortality is the lowest concentration in which all of fish were dead.

16 -

~~~
A0A3?~

4. Results and Discussion

4.1 . Circumstances That May Have Affected the Reliability of the Test Results

There was no significant matter that may have affected the reliability of the test results .

4.2. Concentrations of Test Substance in Test Solutions

The concentrations of test substance in test solutions are shown in Table 1, and

representative chromatograms are shown in Appendix 4.

Proportions of the measured concentrations to the nominal concentrations were 86-

109% at the preparation and 53-76% after 24-hour exposure . It is considered that the main

cause of concentration reduction is volatilization of the test substance from the test

solutions.

{ •

4.3. Median Lethal Concentration (LC50)

The cumulative mortalities for each exposure period are shown in Table 2, and the

concentration-mortality curve is shown in Figure 1 . The mortalities after 96-hour exposure

in the control and the solvent control were both 0%, and fulfilled the validity of the test.

The LC50s in each exposure period are shown in Table 3, and the value for 96-hour

exposure is shown below.

LC50 after 96-hour exposure : >4.90 mg/L

(95% confidence limits : impossible to calculate)

4.4. Highest Concentration in 0% Mortality and Lowest Concentration in 100%

Mortality

The highest concentration in 0% mortality and the lowest concentration in 100%

mortality of each exposure period are shown in Table 4, and the values for 96 hours are

shown below.

t--- Highest concentration in 0% mortality after 96-hour exposure : >4.90 mg/L;

Lowest concentration in 100% mortality after 96-hour exposure: >4.90 mg/L.

4.5. Toxicological Symptoms

The observed results of the test fish are shown in Table 5 . In control, solvent control

and concentration group 2, no abnormal symptom was observed during exposure . In

concentration group 1 and 3, surfacing was observed . In concentration group 4 and 5,

abnormal swimming (reduced activity) and surfacing were observed .

H

ILA
AoS~3;~

4 .6. Appearance, Temperature, Dissolved Oxygen Concentrations and pH of Test

Solutions

The appearance, temperature, dissolved oxygen concentrations and pH of test solutions

are shown in Table 6, 7, 8 and 9, respectively . Temperature of test solutions ranged within

24:L1°C, and concentrations of dissolved oxygen were over 60% of the saturation value

(8 .25 mg/L at 24.0°C). These conditions fulfilled the validity of the test . The pH values

were within proper range (6 .0-8.5) for Medaka.

.

~~~
A05~3P

Table 1 Measured Concentration of the Test Substance in Test Water

(Semi-Static Condition)

Nominal Measured Concentration (mg/L) Meana Measured

Concentration (Percent of Nominal) Concentratio n

(mg/L) 0 Hour (new) 24 Hours (old) 24 Hours (new) 48 Hours (old) (mg/L)

Control <0.0003 <0.0003 <0.0003 <0.0003 ----

Solvent Control <0.0003 <0.0003 <0.0003 <0.0003 ----

0.540 0.550 0.360 0.466 0.288 0.409

(102) (67) (86) (53) (76)

0.960 0.929 0.626 0.821 0.582 0.73 1

(97) (65) (86) (61) (76)

1 .70 1.69 1.26 1.46 1.03 1.35

(99) (74) (86) (61) (79)

3 .00 2.92 2.12 2.73 1.94 2.41

(97) (71) (91) (65) (80)

5 .40 5.89 3.97 5.85 4.12 4.90

(109) (74) (108) (76) (91)

a : Time weighted mean .

new : Freshly prepared test solutions.

old Test solutions on 24 hours after preparation of new solutions .

The test water for analysis was sampled at two renewal sets during 96-hour exposure .

-1 }

LA-~ x-\
A05~397

Table 2 Mortality of the Medaka (Ojyzias latipes) Exposed to the Test Substance

Nominal Meana Measured Cumulative Number of Dead (Percent Mortality) Concentration Concentratio n

(mg/L) (mg/L) 24 Hours 48 Hours 72 Hours 96 Hours

Control ---- 0 (0) 0 (0) 1 ( 10) 1 (10)

Solvent Control ---- 0 (0) 0 (0) 0 (0) 0 (0)

0 .540 0.409 0 (0) 0 (0) 0 ( 0) 0 (0)

0 .960 0.731 0 (0) 0 (0) 0 (0) 0 (0)

1 .70 1.35 0 (0) 0 (0) 0 (0) 0 (0)

( 3.00 2.41 0 (0).- 0 (0) 0 (0) 0 (0) . ........

5 .40 4.90 0 ( 0) 0 (0) 0 ( 0) 0 (0)

a : time weighted mean

---

--~9-~

~y~l S
aoM67

Table 3 Calculated LC50 Value s

Exposure LC50 95-Percent Statistical

Period Confidence Limits

(Hours) (mg/L) (mg/L) Method

. .. ... .. ... ... .. .. .. .... ... .. .. ..24 >4 ... .. ... .... ... .. ..... .. .. .. ... . ... .. .. .. ... ... .. ... .. .. .. .. .

.90 -- --

. .. .. ... .. ... ... .... .. .. .. ... .. .. ... .. .. ..... ... .. .. ... ....... ..... .. ... .. ... .. .. ... ... .. ... .. ... .. .. .. .... ... ... .. .. .. .. .. .... ... .. ... ... .. .. .. .. .. .. .. ... .. ... ...

.. .. ............ ... .. .. .. .. .. ... .48 >4 . ..... .. ... .. .. .. ... .... ... .. .. .. ....... .. ... ... .. .. .. .. .. ...

.90 - - - -

.... .. ..... .. ... .. .... .. ..... ... . ... .. .. .. ... .. .. ... ..... .. ... .. ... .... ... .. ... .. ... ... .. ..... .. .. ... .. .. ... .. .. .. ... . ... .. .. .. .. ... .. ... .. .... ... . .. ..... .. ... ...

... .. .. ... .. ... .. .. ... .. .. .. .. .. .72 >4 .. .. ... .. .. .. ... .. ..... .. .. .. ... .. .. .. ... .. .. ... .. ... .. .. ....

.90 - - - -

.. ... .. .. .. .. .... ... .. .. .. ... .. .. ... .. .. ..... ... .. ... .. . . .. .. ... .. ... .. ... .. ..... .. ........ .. ... .. .. ... .. .. .. ... .. .. .. .... .. .. ... ... .. ... .. .. .. .. .. .. .. ... .. ... ..

96 >4.90 - - - -

- - : Could not be determined .

~~ .

Table 4 Highest Concentration in 0% Mortality and the Lowest Concentration in 100% Mortality

Values

Exposure Highest Concentration Lowest Concentration

Period in 0% Mortality in 100% Mortality

(Hours) (mg/L) (mg/L)

. .. .. ... .. ... .. ... .. ...... .... ...24 >4 .. ... .. ... .. .. ... .... .. ... .... .. .... ... .. .. ... .. ... .. .... .... .. ... .. ... .. ......... .. ..

.90 >4 ..... .. ..... .. .. ... ..

.90 .. .. ... .. .. ... .. ... ....... ..... .. ... .... .. ..... .. ... ... .... .. .. ... . .. .. .. .. .. .. .. ... ... .. ... .. .. .. .. .. .. .. ... ... .

.... .. ... .. ... .... ... .. .. .. .. .. ...48 >4 .. ..... .. ... .. .. .. ... .. .. .. ... .. .. .... ... .. ... .. ..... .. .. .. .... ... .. ....... .. . ......

.90 >4 . .. ... . ... ..... .. .. .. ... ... ..... .. ..... ..... .. ... .. ..... ..... .. .. .. ... ... .. .. ..

.90 .. ... .. .. ..... .. .. .. .. .. ..... ... .. .. .. ... .. .... .. ..... ...

. ... .. .......... .. ... .... .. .. .. ..7... ..2... .. ....... .. ..... .. .. .. ... .. .. .. ...

. >4 ... .. .... .. .. .. .. .. ... .. ... .. .. .. .. ..90 ~ >4 .. .. .. .. ... .. .. .. ...... ..

.90

... .. ... ..

.. ... .. ... .. ..... .. ... .. ... .. ... .

.... .. .. ... .. .. .

.. .. .. .. .... ....... .. .. ... .. ... .. ...... .. ... .. .. .. ...

96 >4.90 >4.90

--24---

~ ~ ~p ,
AO

%

397

Table 5 Observed Toxicological Symptoms

Nominal Meana Measured Symptoms

Concentration Concentration (Symptom-number of fish)

(mg/L) (mg/L) 24 Hours 48 Hours 72 Hours 96 Hours

Control ---- N N N N

..

Solvent Control ---- N N N N

..... . .. ... ..... .. .. ... . ... .. .. ._...... ... .. ... .. ... ... .. .. .. ... .. .. ... .. ... .. ....... .. ..... .. .... ... .. .. .... .. ... .. ... .. ..... .. .. .. ... ... .. ...-... .. .. .. .. ... .. ..... .. .. .. .. .. ... ..... ... .... .. .. .. ... .. . . .. .. .. .. .. .. .. ... ... .. ... .... .. ... .. ... .. .. ... . ... .. .. .. ... ... .. .. .. ... .. ... .. .. ..--- .. ... .. .. ... ... .. ..

0.540 0.409 N N N SUR-1

0 .960 0.731 N N N N

1 .70 1.35 N N N SUR- 1

~ - - - - - _ -__ _ - - - ___ ._.......-.__.._._._...

ASR-2

3 .00 2.41 N N SUR-1 (SUR-2)

._....-- -

N SUR-10 ASR-10 ASR-10

5 .40 4.90 (SUR-10) (SUR-10)

a : Time weighted mean.

N No toxicological symptom was observed

. ASR Abnormal swimming (reduced activity) .

SUR : Surfacing.

~---

--23--

L_\__17
p. 31

A050397

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~~ `
A05PjA

Figure 1 Concentration-Mo rtality Curve

100

90

80

70

,-,

60

50

40

30

20

10 -t-- 24 hour

0 ~ 48 hour

0 .1 1 10 x 72 hour

Concentration(mg/L) --~- 96 hour

~---

~---

~~
AuPu;~ i

Appendix 1

Infrared Absorption Spectra

~j---,

~5--,

~~3
p. 37

~, ~

.1~Srcx..,-~~ 010~ Sa ✓`

0-\ pV-,,- e. C, vp~

SANITIZED

y<~ L\
AUSV3 $$

Appendix 2

Dilution Water Quality

~-36'--

LA lz;~
AO-M67

Table A-2 Dilution Water Qualit y

Parameter Concentration

BOD <2.0 mg/L

PH 8

COD <3.0 mg/L

.0 (20°C)

Coliform group bacteria N

Oil N

.D.

.D.

Cadmium <0.01 mg/L

Cyanide N.D.

Lead <0.1 mg/L

Chromium <0.05 mg/L

Arsenic <0.05 mg/L

~ Mercury <0.0005 mg/L

Free chlorine <0.02 mg/L

Bromide <1.0 mg/L

Fluoride <1.5 mg/L

Sulfide <0.3 mg/L

Total ammonium <1.0 mg/L

Copper <0.005 mg/L

Zinc <0.1 mg/L

Aluminum <0

Tin <1

.1 mg/L

.0 mg/L

Manganese <1.0 mg/L

Iron <1.0 mg/L

Nickel <0.1 mg/L

Total phosphorus <0.1 mg/L

Selenium <0.001 mg/L

Phenol <0.002 mg/L

Anionic surfactant <0.02 mg/L

Evaporation residue 88 mg/L

Electric conductivity 131 μS/cm

Total hardness (as CaCO3) 49 mg/L

Alkalinity 29 mg/L

Sodium 8.8 mg/L

Potassium 1.1 mg/L

Calcium 14 mg/L

Magnesium 3.6 mg/L

Sampling date : February 6, 2006

-3 i

~~~
Ao~A7

~. Appendix3

Preparation of Test Solutions

~--

r3~--

LA $-1
tiv~PAI

Preparation of Test Solutions

1 . Preparatio n

Preparation of stock solution I

Test substance __::> 540 mg -: _

------- - - -- - - - - - Solvent --- > tetrahydrofuran

- - - ------------------- --- -----•----- --•------------------------------------------------------

Final volume -- - > 10 mL

---- ----- - --- --- - -- ------ - - - - - - - -- ----

Vessel ----> 10 mL volumetric bottl e

------- --- - - - - ---- - ---- - ------------ - - - ----------------------------

Concentration - -- > 54000 mg/L -- - --- - -------------------------------- - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Mixing method - ---> inversio n

----- ----- - -------------------------•--------------------------------------------------------------

Preparation of stock solution I I

Stock solution I _____ ---------------------

:> 550 μL

----- -- -- -- -

Dissolving-agent ----> dilution water

- - ----- - - -----------=---------------------- - -----------------------------------------------

Final voluine ----> 5500 mL x 2

-- • in (e

Vessel ----> 5 L media bottle

------------•------------------------------------------------------------------•------------

> 5 . -----------------__ :40 mg/L

-------------------------------------------•-------- --

Solvent concentration -_ __.> __ 95 μg/L

Mixing method ----> add•stoclk-sol. I to dilutiori wafer witli sfu-rfiig-ancf --------------

-----------------------------------------_ (sealing) sonicate 10 min with stirring ----------------

Preparation of dissolving agent

Solvent -- _ -_ ____> tetrah~drofuran

----- -------------------------------------------

Solvent (volume) --

-

-

-

-> . . . . . . . . . .475

- -

0

----- tt.L__________________________

•------------------- ------=----- --- --- -- ---------------------

Dissolvin~=agent _-- :_:_> dilution water

----------------------------------------------------------------------

Final volume 50 mL

--- - -------------------------------------------------------------------------------------------------

Vessel ----> volumetric bo ttle

- - - ---------------------------- ----------------------------------------------------------------------

_Solvent -----------------------~9500

0

--------- μL/L ----- - -- --- ----- - - -

Mixin~ method __ :_ _> inversion

- -- - - -- -- -- --- -- - - -

2 . Preparation of test solution

Stock solutions II and dissolving agent are diluted according to following table, and used as the

test solutions. Control solution contains dilution water only .

Final volume ----> 5.0 L

------------------------------------------------------------------------------------------ ------

__ __ _____ __ ____> 5 L glass vesse - l

--------- - - --- -- -- - - - - - - - -- -

MixinQ method ----> stirring glass-rod _- __- -

-

-

-------------------- -------- ---------------------------------

Factor ----> 1.78

Stock sol. II Dissolving Solvent conc.

Nominal conc . Group No. agent

(mg/L) (abbreviation) (mL) (mL) (μL/L)

control C ----> 0 0 0

solvent control SC ----> 0 5.00 95

0.540 Conc.l ----> 500 4.50 95

0.960 Conc.2 ----> 889 4.11 95

1 .700 Conc.3 ----> 1574 3.43 95

3 .000 Conc.4 ----> 2778 2.22 95

5 .400 Conc.5 ----> 5000 0.00 95

-33--

~g~
AMh7

Appendix 4

Analysis of Test Solutions

~---

~ 3 4

~$ `
AuSk'r3Vi

1. Gas chromatography / mass spectronietry (GC/MS) Conditions

• System

Gas chromatograph/mass selective detector with headspace sampler,

Agilent Technologies, Inc ., No. 1

Gas chromatograph (GC): 6890, Agilent Technologies

Headspace sampler (HSS) 7694, Agilent Technologies

Mass selective detector (MSD) : 5973N, Agilent Technologies

Work station : ChemStation

• Conditions

GC

Column: Rtx-200 105 mx0.25 mm, 1 .0 μm df, RESTEK Co.

Carrier gas: helium, 1.0 mL/min (constant flow)

r Oven temperature: 60'C (1 min)-*5' C/min----> 110°C (10 .0 min)

Injector temperature : 230°C

MS interface temperature : 200°C

Injection method : split (split ratio=20.1 :1 )

Injection volume : 3.0 mL (HSS sampleloop volume)

HSS

Temperature: Oven: 60°C, Loop : 120°C, Transfer line : 200°C

Event time : GC cycle time: 35 min

Vial equilibration time : 20 min

Pressurization time: 0.2 min

Loop fill time : 0.03 min

Loop equilibration time : 0.2 min

Inject time : 0.2 min

Vial parameter: Shake 2 (HIGH)

MSD

f Temperature: Ion source: 230°C

Quadrupole mass filter: 150°C

SIM (Selected Ion Monitoring) :

Solvent delay : 5 min

Quant ion: TIC of m/z 151 .0, 195 . 0

The test substance is diastereomeric mixture, and 4 peaks were detected on a

chromatogram. Hence, the concentration of the test substance was determined by

quantitative analysis using the total peak area of 4 peaks .

~~d
Ao9b1~7

2. Calibration Curve

The test substance was dissolved in acetone to prepare standard solutions of 0, 0.1-500

mg/L. Analysis of standard solutions was performed as shown below. The peak area (count)

for each solution was plotted against its concentration (mg/L). The correlation coefficient

for the linear regression by the least square method of the calibration curve was 0 .9997. The

calibration curve is shown in Figure A-4-1 .

Collect 10 mL of purified water"

add 0 .1 mL of standard solution

mix

GC/MS analysis*2

1 : A4 grade in JIS K055 7

*2 : Measured value represents 1/100 of concentration of standard solution .

3. Detection limit

The minimum detectable total peak area of 4 peaks was set at 4000 count , and the

detection limit of concentration of the test substance was set at 0.0003 mg/L which

corresponds to the minimum detectable peak area .

4. Analysis of Test Solution

(1) Test solutions were analyzed as shown below . Representative chromatograms are

shown in Figure A-4-2 (2), (3), (4), (5), (7), (8), (9) and (10) .

Purified water

~- +

Acetone 0.1 mL

+

Analysis sample*' 10 mL (total volume of the test solution and purified water)

mix

GC/MS analysis

* 1 : By changing the mixing ratio of the test solution and purified water in each analysis vial, the

analysis samples are prepared to be within the range of the, calibration curve . The total volume

of the analysis sample is 10 mL each. In control and solvent control, test solutions are not

diluted, and 10 niL of them (0 mL of purified water) is collected .

LA `\1
(2) Standard solutions were analyzed by the same way in "2 . Calibration Curve".

Representative chromatograms are shown in Figure A-4-2 (1) and (6)

.

(3) The concentration of the test substance in the test solutions was determined by

comparing the peak area with that for the standard solution analyzed at each analysis.

5. Recovery test

Because the pre-treatment of the test samples for analysis was only to mix test solution

with acetone as shown in "4 . Analysis of Test Solutions", the recovery test was not required .

Therefore, measured concentration was not corrected for recovery rate .

(

!

LA C~,
tiupu46 i

Figure A-4-1 . Calibration Curve

No. Concentration Peak Area

1 0 0

(mg/L) (count)

2 0.001 35290

3 0.002 67064

4 0.005 159717

5 0.010 303044

6 0.020 590066

7 0.050 1457557

8 0.100 2930373

9 0.200 4917974

10 0.500 14673738

{ 12 2

11 1.00 25301800

.00 50711191

13 5.00 133789350

Y= 26,544,313X

r= 0.9997

1 .5E+08

!••

0

v

7 .5E+07

Cd

a

~

t-- ~.~. •

0 .0E+00

0 1 2 3 4 5 6

Concentration (mg/L)

.r-3-$-

~~
p. 47

~

SANITIZED

y~~ - 'A °\?~
p. 48

A050401

FINAL REPORT

M.S.I. Report No. A050401

Growth Inhibition Test of MTDID3663

with Pseudokirchneriella subcapitata

Submitted to:

Sumitomo 3M Limited

Prepared by:

Mitsubishi Chemical Safety Institute Ltd . (M.S.I.)

August 31, 2006

---~-~
A05046149

Growth Inhibition Test of MTDID366 3

with Pseudokirchneriella subcapitata

(English version)

Sponsor Sumitomo 3M, Ltd.

Study Title Growth Inhibition Test of MTDID3663 with Pseudokirchneriella subcapitata

Study No. A05040 1

{ This report is the English version of the original, which was written in Japanese . I,

the undersigned, hereby declare that this version faithfully reflects the original report to

the best of my knowledge.

Translated by

SANITIZED

~©b
A050481 50

Compliance with the GLP Standards

Yokohama Laboratory

Mitsubishi Chemical Safety Institute Ltd.

Sponsor Sumitomo 3M, Ltd .

Study Title Growth Inhibition Test of MTDID3663 with Pseudokirchneriella subcapitata

Study No. A050401

I hereby certify that this study was carried out in accordance with its protocol and

with our standard operating procedures, and that the report accurately describes the test

results.

The study described in this report was conducted in compliance with the following

Good Laboratory Practice (GLP) standards .

The standards concerning the test facility in which the study of new chemical

substance is performed (Yakushokuhatsu No.1121003, Heisei 15 .11 .17

Seikyoku No.3, Kanpokihatsu No .031121004, November 21, 2003 ; the latest

revision, April 1, 2005)

Study Director

~

Tatsuhiro NIINO, Ph.D. Sealed date: August 31, 2006

--~--.

So~
A050A51

Quality Assurance Statement

Yokohama Laboratory

Mitsubishi Chemical Safety Institute Ltd.

Sponsor Sumitomo 3M, Ltd.

Study Title Growth Inhibition Test of MTDID3663 with Pseudokirchneriella subcapitata

Study No. A05040 1

We hereby certify that this study was carried out in accordance with its protocol and

with our standard operating procedures. We certify that this report faithfully describes

the methods and procedures employed, and that the reported results reflect the raw data

of the study accurately.

Date of Inspection Date of Reporting

to Management and

Study Director

Protocol: August 4, 2006 August 4, 2006

Amendment (01): August 10, 2006 August 10, 2006

In-progress study:

Preparation of test solution : August 8, 2006 August 8, 2006

Introduction of Alga: August 8, 2006 August 8, 2006

Analysis of test solution : August 10, 2006 August 10, 2006

Measurement of cell concentration : August 10, 2006 August 10, 2006

Preparation of test solution : August 16, 2006 August 16, 2006

~ Introduction of Alga: August 16, 2006 August 16, 2006

Analysis of test solution : August 18, 2006 August 18, 2006

Measurement of cell concentration : August 18; 2006 August 18, 2006

Final report: August 31, 2006 August 31, 2006

Quality Assurance Personnel

Yumiko KASHIWAGI, B.Sc. Sealed date: August 31, 2006

Reiko TSUTSUI Sealed date: August 31, 2006

4---

~dID,
A0504(R' 52

Introductio n

1 . Study Title

Growth Inhibition Test of MTDID3663 with Pseudokirchneriella subcapitata

(Study No . A050401)

2. Purpose

The growth inhibition test of the test substance to algae was conducted in order to

evaluate the toxicity of the test substance for notification under the Chemical Substances Control Law of Japan.

3. Test Guideline

The test method relating to new chemical substances "Alg g growth inhibition test, a

Daphnia sp. acute immobilisation test, and fish acute toxicity test" (Yakushokuhatsu

No .1121002, Heisei 15.11 .13 Seikyoku No .2, Kanpokihatsu No .031121002, November 21, 2003 ; the latest revision, April 1, 2005)

4. GLP Complianc e

The standards concerning the test facility in which the study of new chemical

substance is performed (Yakushokuhatsu No .1121003, Heisei 15.11 .17 Seikyoku

No.3, Kanpokihatsu No.031121004, November 21, 2003 ; the latest revision, April 1,

2005).

~ 5. Sponsor

Sumitomo 3M, Ltd.

8-8 Minanii-hashimoto 3-chome, Sagamihara, Kanagawa, Japan

6. Test Facility (Head Office)

Mitsubishi Chemical Safety Institute Ltd. (M.S.I.)

1-30 Shiba 2-chome, Minato-ku, Toky o

So3
A05040?' 53

7. Test Facility (Laboratory)

Yokohama Laboratory

Mitsubishi Chemical Safety Institute Ltd .

1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa

8. Study Director

Tatsuhiro NIINO, Ph.D.

Ecological Chemistry Division of Yokohama Laboratory

9. Personnel in Charge of the Study

Kazuhisa OONISHI Sealed date: August 31, 2006

{ (Experimental and analytical work, and reporting)

Ruriko SAKURAZAWA, B .Sc. Sealed date: August 31, 2006

(Experimental work)

10. Date

Initiation of the study : August 4, 2006

Start of the exposure : August 16, 2006

End of the exposure: August 19, 2006

Completion of the study : August 31, 2006

It. Storage

The following will be stored in the archives of the Yokohama Laboratory.

1) Protocol

( 2) Final report

3) Raw data

4) Test substanc e

5) Reference substance

6) Other requirements

--6

LA
A05040 54

Content

page

Summary -------------- ------------------------------ - -•---------------------------------------------------------------------------- .8

I . Test Substance 10

---------------------------------------------------------

1 .1 . Identification 10

1 .2. Source 11

1 .3 . Storage and Confirmation . of Stability 11

2

. Test Species -------- -------------------- -----------.11 ----------------------------------------------------------------------

3 . Test Methods 12 --------•----------

3 .1 . Test Conditions 12

3 .2 . Medium 12

3.3. Apparatus 12

3.4. Test Concentrations 13

3 .5 . Preparation of Test Solutions 13

3 .6. Analysis of Test Solutions and Cultures -13

3.7. Test Procedure 14

3 .8 . Calculation of Results 14

4. Results and Discussion 18

--------•--------------------------------------•----------------------- 4 .1 . Circumstances That May Have Affected the Reliabilit y

of the Test Results 18

4.2 . Concentrations of Test Substance in Test Solutions and Cultures 18

4 .3. Growth Curves 18

4.4. 50% Growth Inhibition Concentration (EC50) and No Observe d

Effect Concentration (NOEC) 18

4.5 . Conditions in the Culturing Apparatus, pH of Test Solutions an d

Cultures, and Appearance of Test Solutions 19

4.6. Observation of Algae 19

Tables 1-8 20-25

Figure 1 26

Appendix 1 . Infrared Absorption Spectra 27-28

Appendix 2. Composition of Medium 29-30

Appendix 3 . Preparation of Test Solutions 31-32

Appendix 4. Analysis of Test Solutions and Cultures 33-41

~--=

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A05040q

. 55

Summary

Sponsor Sumitomo 3M, Ltd.

Study Title Growth Inhibition Test of MTDID3663 with Pseudokir-chneriella subcapitata

Study No. A05040 1

Methods:

The study was conducted in accordance with the test method relating to new

chemical subst ances "Alga growth inhibition test, Daphnia sp. acute immobilisation test,

and fish acute toxicity test" (Yakushokuhatsu No .1121002, Heisei 15 .11 .13 Seikyoku

No.2, Kanpokihatsu No.031121002, November 21,2003 ; the latest revision, April

1,2005).

1) Exposure procedure: static, closed system, and shake culture (100 rpm)

2) Duration: 72 hours

3) Test concentrations, nominal : control, solvent control, 6 .30* mg/L

* : limit test at the maximum attainable concentration

Solvent concentration: 94 μL/L (Tetrahydrofuran)

4) Volume of test solution: 100 mL/vessel

5) Number of vessels: 6 vessels/test groups

6) Biomass loading: 5x103 cells/mL

7) Temperature: 23 ±2°C

8) Light, continuous: 65 μE/m2/s (±8%, at the surface of test solutions,

in the center of the apparatus)

9) Analytical methods : gas chromatography / mass spectrometry (GC/MS)

Results

~ 1) Concentrations of test substance in test solutions and culture s

The proportion of the measured concentrations to the nominal values in test solution was 85% at the beginning of exposure . And this propo rtion in culture was 7% at the end of exposure . It is considered that the main cause of concentration reduction is

volatilization from the test solutions. Inhibition concentrations were estimated based

on the average of measured concentration (time-weighted average) .

2) Inhibition concentration by comparison of growth rate s

ErC50 (0-72h) : >1 .01 mg/L (95% confidence limits : impossible to calculate)*

NOECr (0-72h): >1 .01 mg/L

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A05040 p• 56

3) Inhibition concentration by comparison of areas under the growth curves

EbC50 (0-72h) : >1 .01 mg/L (95% confidence limits: impossible to calculate)*

NOECb (0-72h): >1 .01 mg/L

* The results are shown as "> maximum concentration" because the growth inhibition

at the maximum concentration level* was <50%.

~ nominal was 6.30 mg/L which was ceiling concentration, the average of

measured concentration was 1 .01 mg/L.

4) Observation of the shapes of algae

By microscopic observation at the end of exposure, in all concentration groups

neither unusual cell shape of algae (contraction, exp ansion, damaged cell etc.) nor

agglutination was examined, and the algae looked normal compared with the control .

-9 =

So~
A05040R• 57

1 . Test Substanc e

1.1. Identification

Name of the new chemical

substance

(by IUPAC nomenclature)

Alternate naine

CAS RN

Structural formula or

rational formula

(If both of them are

unknown, outline o f

preparation or

manufacturing )

Molecular weight ~+ N''~,~E~

Purity (%) of the new

chemical substance

subjected to the study

Lot number of the ne w

chemical subst ance

subjected to the study

Name and content of

impurities

Vapor pressure

Solubility in water

I-octanoUwater partition

coefficient

Melting point

Boiling point

Appearanc e

at room temperature

Stability

Solubility in

solvents and the like

~--~~-

J~©~
A0504(Pl• 58

1 .2. Source

Supplier: Sumitomo 3M Limited

1 .3. Storage and Confirmation of Stability

For the duration of the study, the test substance was stored in the refrigerator of M .S.I.

(refrigeration, dark place and sealing) . At the end of the study, the infrared absorption

spectrum of the test substance was measured. The spectrum was consistent with that measured at the start of the study, indicating that the test substance was stable under the storage

condition. The infrared absorption spectrum is shown in Appendix 1 .

Apparatus: Infrared spectrophotometer, AVATAR 320, Nicolet Instrument Corporation

2. Test Species

1) Organism : Unicellular green algae

2) Scientific name: Pseudokirchneriella subcapitata

3) Strain designation : ATCC22662

4) Source : American Type Culture Collection

5) Date of receipt : June 20, 1996

6) Laboratory culture : Subculture in Gorham cultures under sterile condition

Subculture has been confirmed to be sterile with bacteria test

every six month.

7) Sensitivity to reference substance :

Growth inhibition test of a reference substance (K2Cr2O7,

guaranteed reagent) has been conducted every six months

since November 1996. 72-hour 50% growth inhibition concentration by comparison of areas under the growth curves

~ (EbC50) was as follows:

average ± standard deviation : 0.426±0 .0671 mg/L, n=19

minimum value - maximum value : 0.285 - 0.543 mg/L

8) Pre-culture : Pre-culture period: August 12, 2006 - August 16, 2006

During the pre-culture, exponential growth of algae was observed. (The conditions were the same as the test.)

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A05040P• 59

3. Test Methods

The study was conducted in accordance with the test method relating to new chemical

substances "Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish

acute toxicity test" (Yakushokuhatsu No .1121002, Heisei 15 .11 .13 Seikyoku No.2,

Kanpokihatsu No.031121002, November 21, 2003 ; the latest revision, April 1, 2005).

Test vessels and other apparatuses were sterilized if necessary. Algae were introduced

into culture medium under an aseptic condition .

3.1. Test Conditions

1) Exposure procedure: static, closed system, and shake culture (100 rpm)

2) Duration: 72 hours

3) Volume of test solution: 100 mL/vessel

4) Number of vessels: 6 vessels/test groups

5) Biomass loading: 5xl03 cells/mL

6) Temperature: 23±2°C

7) Light: 65 μE/m2/s (±8%, at the surface of the test solutions,

in the center of the apparatus), continuous

8) pH: not adjusted

3.2. Medium

For the pre-culture and the test, the medium recommended by the test method relating

to new chemical substances for the Chemical Substances Control Law of Japan was

used after sterile filtration with 0 .22 μm of membrane filter. The composition of the

medium is given in Appendix 2 .

3.3. Apparatus

1) Test vessel: 500 mL Erlenmeyer flask with glass atopper, Iwaki

(head-space volume: 490 mL, measured by MSI)

2) Culturing apparatus: AGP-150RL, Itoh Seisakusho, Ltd.

3) Microscope: ECLIPSE TE300, Nikon Co., Ltd.

4) Electronic particle counter: CDA-500, Sysmex Co ., Ltd.

5) pH meter: HM-40V, Toa Electronics Corporation

6) Thermometer: TNA-120, Tasco Japan Co., Ltd.

7) Photometer: QMSS, Apogee Co., Ltd.

8) Electrobalance : AG204, Mettler K.K.

AE163, Mettler K.K.

AB204-S, Mettler K.K.

PB3002, Mettler K.K.

12

S\~
A0504M 60

3.4. Test Concentrations

The solubility of the test substance to the medium was 6 .3 mg/L (measured by M.S.I.).

Hence, the test concentration was set at 6 .3 mg/L or less . A range-finding test was conducted before the definitive test (see the following table) .

The concentration of the test substance was decreased in the range-finding test . The

main cause that reduced the concentration is considered to be a volatilization from the

test solution.

Nominal concentrations of the definitive test: control, solvent control, 6 .30* mg/L

* : limit test at the maximum attainable concentration

Results of range-finding test

The proportion of the The proportion of the measured

Concentration Inhibition (%) measured concentration concentration to the nominal

(mg/L) I y(0-72h)`'

to the nominal value value at 72 hours (% )

at 0 hour (%) with algae without algae

Solvent control

0.0630 -11 84 3 3

0 .630 4 -_

-- -

6.30 4 90 5 3

*I The percent inhibition in yield I y(0-72h) was calculated according to the following equation .

Iy= Y`Y~ x100

~

where:

Y,: yield at control (or solvent control)

Y,: yield at test substance concentratio n

Yield is value subtracted the nominal cell concentration (cells/mL) at the beginning of exposure

from the measured cell concentration (cells/mL) at the end of exposure .

3.5. Preparation of Test Solutions

The method of preparation of test solutions is shown in Appendix 3 . Control group

contained only medium, and solvent control solution was medium containing the solvent (94 μL/L). The stock solution was prepared at the beginning of exposure .

The appearance of each test solution was observed and recorded soon after preparation.

3.6. Analysis of Test Solutions and Culture s

The concentration of test substance in test solutions and cultures was measured at

the beginning of exposure, after 24-, 48- hour exposure and at the end of exposure by

--~- _-

S~\
A05040R• 61

gas chromatography / mass spectrometry (GC/MS) . The test substance is diastereomeric

mixture and 4 peaks were detected on a chromatogram . Hence, the concentration of the

test substance was determined by quantitative analysis using the total peak area of 4

peaks.

The analytical test solution was prepared independently at the beginning of exposure. After 24-, 48- hour exposure and at the end of exposure, equal amount of test culture which was taken from all vessels in each test group was mixed, and analyzed.

Details are shown in Appendix 4 .

3.7. Test Procedure

The cell concentration in the pre-culture was counted with electronic particle

counter, and hemacytometer on microscope . Then algal suspension was introduced into

each test solution so that the initial cell concentration in the test cultures would be

5x103 cells/mL.

. The culture flasks were placed according to the table of random sampling digits and

rearranged every 24 hour in the culturing apparatus controlled at 23+2°C to start the exposure. The cell concentration in each flask was determined at 24, 48, and 72 hours after the start of exposure . Aliquot of test culture (1 .0 mL) was suspended in an electrolyte (9 .0 mL) for electronic particle counter to count the algal cells by electronic particle

counter.

The color of the test solutions and cultures were observed with the naked eye at 0,

24 and 48 hours after the start of exposure. At the end (72 hours) of exposure, besides

visual observation of cell color, the cell shapes of algae were observed through the microscope.

At the beginning of exposure, the pH of the test solution were measured, while

those of the culture at the end of exposure . At the beginning of exposure, the pH of the

test solution were measured for the extra flask for exposure in each test group . At 72

hours after the start of exposure, pH of the culture were measured for one flask (No .1)

out of flasks for exposure in each test group. During 72-hour exposure period, the temperature and the light intensity in the culturing apparatus were measured at least once a

day.

3.8. Calculation of Results

1) Growth curves

The mean value of the cell concentration for each test concentration was plotted

against time to produce growth curves .

2) Calculation of the percentage inhibition of the cell growth

The percentage inhibition of the cell growth was calculated by comparison of growth

rates, or by comparison of areas under the growth curves .

H

S
A05040?' 62

i) Percentage inhibition of the cell growth by comparison of growth rates (L )

The average specific growth rate (μ) for exponentially growing cultures could be

calculated according to the following equation .

_ 1nN„ -1nN;

t" - ti

μ;_n : growth rat e

N; : measured cell concentration (cells/mL) at time t;

(cell concentration at the beginning of exposure is nominal cell concentration)

Nn: measured cell concentration (cells/mL) at time t„

t; : time of ith measurement

tn : time ofnth

measurement

The percentage reduction in average growth rate at each test subst ance concentration

(Iμ) was calculated according to the following equation .

X100

μc : growth rate at control (or solvent control)

lit : growth rate at each test substance concentratio n

ii) Percentage inhibition of the cell growth by comparison of areas under the growth

curves (IA)

The area below the growth curves were calculated according to the following equation.

N, -No

x

N, +Nz -2No N„_, +N,, -2No A 2 t, + 2 x(tz -t,)+•••+ x(t„-t„_,

2

A: area below the growth curve

No: nominal cell concentration (cells/mL) at time to

NI : measured cell concentration (cells/mL) at time tl

N,,: measured cell concentration (cells/mL) at time t„

tl : time of first measurement

t,,: time of nth measurement

The percentage inhibition of the cell growth at each test substance concentration (IA)

was calculated according to the following equation .

5~3
A05040 P• 63

IA= A`-`4' x100

A,

A,,: area under the growth curve at control (or solvent control)

At: area under the growth curve at each test substance concentratio n

3) Determination of concentration of test substance for calculating inhibition concentrations.

Concentration of test substance to calculate EC50 and NOEC was the average of

measured concentration (time-weighted average) .

(time-weighted average)

ConcA„ - ConcB„

mc„ = ln(ConcA„ ) - ln(ConcB„ )

MC mc, +mc2 +•••+mc „=

n

mc,,: Mean measured concentration during each exposur e

(In the case of ConcAn ConcB,,, Tn_c„=ConcA„)

ConcA,,: Measured concentration at the sta rt of the nth measurement

Measured concentration at the start, or at 24 or 48 hours exposur e

ConcB,,: Measured concentration at the end of the nth measuremen t

Measured concentration at 24 or 48 hours exposure, or at the end of exposure

MC: Mean measured concentration

~-~--

S ~~ .
A05040p• 64

4) Calculation of 50% Growth Inhibition Concentration (EC50 )

50% Growth inhibition concentration (EC50) was determined using the percentage

inhibition of the cell growth (Iμ or IA) by the following method .

Percentage inhibition in maxi- < 50%

mum concentration

Graph of concentration-growth

inhibition rates curves (plotted not included in the report

on the semilogarithmic graph)

Determination method of EC50 estimated concentration range

in case of calculating by Iμ : ErC50(0-72h) Denotation of EC50

in case of calculating by IA : EbC50(0-72h)

5) No Observed Effect Concentration (NOEC)

No Observed Effect Concentration (NOEC) was the highest concentration tested at

which the measured parameters showed no significant inhibition of growth relative to

the control (or the solvent control) values by the following table . The NOEC consequently is shown as "> test concentration" when measured parameters showed no significant inhibition of growth relative to the solvent control values, because the test was

conducted at one concentration in the definitive test . NOEC calculated from the growth

rate is expressed in NOECr (0-72h), while NOECb (0-72h) from the area under the

growth curves.

Comparison of two groups

[One group except control group]

~ F test for homogeneity of variances

When the variance is When the variance is

homogeneous not homogeneous

Student's Welch's

t-test t-test

Yukms Statlight #3 software (Yukms Corp ., Tokyo)

The NOEC values were determined by Student's t-test, subsequent of F test

(a=0.05).

~~~-- •

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A05040P• 65

4. Results and Discussio n

4.1 . Circumstances That May Have Affected the Reliability of the Test Results

There was no significant matter that may have affected the reliability of the test results.

4.2. Concentrations of Test Substance in Test Solutions and Culture s

At the beginning of exposure, the concentration of test substance in test solution

was analyzed. And the concentration in test culture was analyzed at 24, 48 and 72 hours

after the start of exposure. The results are shown in Table 1 and representative chromatograms are shown in Appendix 4 .

The proportion of the measured concentrations to the nominal values in test solution

was 85% at the beginning of exposure . And this proportion in culture was 7% at the end

of exposure .-It is considered that the main cause of concentration reduction is volatilization from the test solutions.

4.3. Growth Curve s

The cell concentrations of algae during exposure are shown in Table 2. The growth

rate of control and the growth curves are shown in Table 3 and Figure 1, respectively .

The cell concentration in control and solvent control increased by a factor of 152

and 148 (average values), respectively, during 72-hour culture. In control, the coefficient of variation of growth rates within days were less than 35%, while within replicates were less than 15% . These indicate that the cell in control had grown normally

under the test conditions.

4.4. 50% Growth Inhibition Concentration (EC50) and No Observed Effect Concentration (NOEC)

Percentage -inhibition of the cell growth or growth rate is shown in Table 4. EC50

and NOEC are shown in Table 5. The results concluded from percentage inhibition of

the cell growth and growth rate are shown as below .

1) Inhibition concentration by comparison of growth rate s

ErC50 (0-72h): >1.01 mg/L (95% confidence limits: impossible to calculate)*

NOECr (0-72h): >1 .01 mg/L

2) Inhibition concentration by comparison of areas under the growth curves

EbC50 (0-72h): >1.01 mg/L (95% confidence limits: impossible to calculate)*

NOECb (0-72h): >1.01 mg/L

* The results are shown as "> maximum concentration" because the growth inhibition

at the maximum concentration level* was <50% .

* nominal was 6.30 mg/L which was ceiling concentration, the average of measured

concentration was 1 .01 mg/L. .

6--

_ 5~40
A05040R• 66

4.5. Conditions in the Culturing Apparatus, pH of Test Solutions and Cultures, and

Appearance of Test Solutions

The conditions (temperature, light intensity and revolutions) in the culturing apparatus during 72-hour exposure period are shown in Table 6 . The pH values of test solutions at the beginning of exposure, and the values of test cultures at the end of exposure

are shown in Table 7.

The temperature in the culturing apparatus ranged within the nominal range of 23 ±

2°C. The pH of test solutions were 7.9 at the beginning of exposure, and 9 .9 - 10.1 at

the end of exposure. In this study, pH increased more than 1 .5 unit in all test concentration groups. When the growth of algae is very vigorous under closed condition, pH often should increases 1 .5 or more. Therefore, these increase were estimated to be acceptable.

The appearance of test solutions after preparation is shown in Table 8 . All test solutions were colorless and transparent, and showed no suspended solids, floating solids,

precipitations and oily substances.

4.6. Observation of Alga e

In all test concentration groups, color of test cultures observed with naked eye

showed a tendency to get more greenish overtime, as time advances .

At the end of exposure, the microscopic observation in all concentration groups

showed neither unusual cell shape of algae (contraction, expansion, damaged cell etc .)

nor agglutination was examined, and the algae looked normal compared with the control and solvent control .
A05040R• 67

Table 1 Measured Concentration of the Test Substance in Test Cultures

Nominal Measured Concentration (mg/L) Meana Measured

Concentration (Percent of Nominal) Concentration

(,n9/-)

(ing/L)

0 Hour 24 Hour 48 Hours 72 Hours (Percent of Nominal)

. .. .. ... .... .. .. .. .Control <0 .. .. ... ... ... .. ... .. .. ... .. ... . ... .. ... .. ... .. ... .. .. ... ..

.0002 <0 .. ....0002 <0.0002 <0.0002 .. ... .. .... ... ... .. ... .. .. .. _.. ..... .. .. ... .. .. .. ... .. .. ... . ... .. .... ... .. .. ... .. ... .. .. ... .. ..... ... .. .. ...... .. ... .... .. ... ... .. .. .. ... .. .. .. .. .. ..... .. ....... .. .. .. ... ... .. ... .. .. .. ... .. .. .... .. ... .. .. ..

. .. ...Solvent Control <0 .. .. .. .. ... .. ..... .. .. .. .. ... .. .. _. .. .. ... .. .. .. ... ... .. ... .. .. ..... .

.0002 <0 . ... .. ... .. .. ... .. ... .. ..... .. .. .. ..0002 <0.0002 <0.0002 .. .... .. .. ... .. .. .. ... .. .... ... .. .. .. . . .. .. .. ... ....... .. .......... .. ... .. ... ... .. .. .. ... .. ... .. .. ... .. ..... .. .. .. .. ... . ... .. .. ... .. .. .. ....... ... .. ... .. ... ...... ... ... .. ... ... .. .

6.30 5.34 0.553 0.433 0.424 1.01

(85) (9) (7) (7) (16)

a : time weighted mean

S ~~
A05040p

' 68

Table 2 Cell Densities ofPseudokirchneriella subcapitata during the 72-Hour Exposure

Nominal Concentration Vessel Cell Densities (cells/mL)

[Meana Measured Conc .]

(mg/L) No.

0 Hour* 24 Hours 48 Hours 72 Hours

1 5000 33400 155400 795700

2 5000 35700 150400 708700

3 5000 40500 142400 715700

Control 4 5000 39600 149400 786700

5 5000 32600 160400 776700

6 5000 39400 164400 77370 .. .. ... ... .. ... .. .... ... .. .. . 0 . .. . . .. .. ... .. ....... .. .. . ... .. ... .. .. ... .. .. .. .. .. .. ... Averag 5000 36900 153700 759500

SD 0 3400 8000 37500

1 5000 34700 161400 712700

Solvent 2 5000 37300 192400 711700

Control 3 5000 36800 159400 752700

4 5000 37500 152400 722700

5 5000 36100 178400 770700

.. .6 5000 38300 145400 77370 .. . . .. .. ... .. .. .. ..... .. ..... .. ..... ... .. . . ._. . ... .. ... .. ... ... .... ... .. .. .. ..... ... .. ... .... ..... .. .._. .. ... .... ..... .. .._. . .. .. 0 . ... .. . .. ..... .. .. ... .... ... .. ... .. .. .. .. . . . ... . . .. .. .. .... Averag 5000 36800 164900 740700

SD 0 1300 17400 28600

1 5000 37200 156400 834700

6.30 2 5000 35400 133400 761700

[1 .01] 3 5000 34200 158400 853700

4 5000 34500 141400 855700

5 5000 33500 155400 733700

. .. 6 5000 35100 141400 79470 . . .. . .. .. . .. ... .. .. .. .. ... ... .. ... .. ... .. .. ....... .. . . . .. .. . .. .. ..... ..... .. ... .. .. ..... .. . .. . . .. _ .. .. ... ... .. .. .. 0 . .. ... .. ....... .. ... .. . .. .. . .. . . .. ..... .. Averag 5000 35000 147700 805700

SD 0 1300 10300 50800

a : time weighted mean

SD Standard deviation

* : Nominal initial densities

S ~"1
A0504(9t 69

Table 3 Growth Rates of Control

Growth Rate s

Vessel No. ,u (0-2411) it (24-48h) a (48-72h)

1 0.0791 0.0641 0.0681

2 0.0819 0.0599 0.0646

3 0.0872 0.0524

11

0.0673

4 0.0862 0.0553 0.0692

5 0.0781 0.0664 0.0657

6 Average SD CV(%) 0.0860 0.0595 0.0645

Average 0.0831 0.0596 0.0666 0.0698 0.0121 17.3 ..... .. .. .. ..... .. .. .. ..... .. ... ..... _..... .. .. . .._ ... .. .... _. .. ... .... ... .. ..... .. .. .. . .. .. .. .. . ... .. .. .. .. ... .. .. .. ..... .. .. . . ... . ... ... .. ... .. ..... .. .. ...... .. .. ... .. .

SD 0.0039 0.0052 0.0019

CV(%) 4.7 8.7 2.9

SD : Standard deviatio n

CV: Coeffi cient of variation

~

~

S ~.C~ ,
A05040q' 70

Table 4 Growth Inhibition (%) of Pseudokirchneriella subcapitata

Nominal Concentration Growth Rates Areas under the growth curves

[Meana Measured Cone .] Rate Inhibition(%) Area Inhibition(%)

(mg/L) Vessel No. .c (0-72h) I (0-72h) A(0-72h) I A(0-72h)

1 0.0704 13780000

2 0.0688 12671000

3 0.0689 12678000

Control 4 0.0703 13676000

5 0.0701 13652000

6 0.0700 13876000

Average 0.0698 - 13389000 -

SD 0.0007 559000

1 0.0689 12959000

Solvent 2 0.0689 13753000

Control 3 0.0696 13441000

4 0.0691 12930000

5 0.0700 14096000

-___-_______-- --- -- -------------_-

6 0.0700 13393000 ._._..

.__------- --------------- .0694 - 13429000 - -Average 0

SD 0.0005 452000

1 0.0711 14363000

6.30* 2 0.0698 12892000

[1 .01] 3 0.0714 14567000

4 0.0714 14190000

5 0.0693 13038000

6 0.0704 13472000

Average 0 .0706 -1.7 ------ - 13754000 -----^ -2 .4 _------

SD 0.0009 715000

a time weighted mean ,

* 1 Values are the growth inhibition (%) relative to the solvent control .

SD Standard deviation

The maximum attainable concentration under the present test conditions and preparation

methods.

* Indicates a significant difference (a=0 .05) from the solvent control . (There was no sign in this

test.)

** Indicates a significant difference (a=0 .01) from the solvent control . (There was no sign in this

test.)

--z`~3---

s~l
A05040T' 71

Table 5 EC50 and NOEC

Based on I, (0-72h) value (Growth rates)

ErC50 (0-72h) 95-Percent NOECr (0-72h) (mg/L) Confidence Limits (mg/L)

(mg/L)

>1 .01 -- >1.01

Based on IA (0-72h) value (Areas under growth curves)

EbC50 (0-72h)

95-Percent

NOECb (0-72h)

(mg/L) Confidence Limits (mg/L)

(mg/L)

>1 .01 -- >1.01

The EC50 values and associated 95% confidence limits could not be determined by the least

squares linear regression analysis because the test was conducted at one concentration level .

-- not calculated

The NOEC values were determined by Student's t-test, subsequent to F test for homogeneity of

variances. Statistical analyses were performed using Yukms Statlight #3 software (Yukms Corp.,

Tokyo) and all tests of significance were at a=0 .05.

~ ~~
A0504('1• 72

Table 6 Temperature, Light Intensity and Revolutions in the Incubation Chambe r

Exposure Period Temperature Light Intensity Revolutions

(Hours) X) (a E/rn2/s) (rpm)

0 22.7 61-63 100

24 22.6 61-63 100

48 22.4 61-64 100

72 22.4 61-63 100

Table 7 pH Values of Test Culture s

Nominal Concentration pH

{ [Meana Measured Conc .]

(mg /L)

Hour 72 Hours (Vessel No.)

~-)

Control 7.9 10.1 (1)

.. .. ... .. .. ... .. ..... .. ..... .. .. .. .. ... .. ... .. .. ..... .. .. ... .. .... ... .. .. .. .... ..... ... . _ .. ..... .. .. ..... .. ... .. ... .. _... ..... .. .... ... .. ..... .. .. .. ... .. ....... .. ..... .. ....... ... .. .. ... .. .. ... .. .. ... .. .. ..... .. ....... .. .... ... .. .. .. ... ..

Solvent Control 7.9 10.0 (1)

.. .. ... .. ... .. .. ... .. ... .. .. .. .. .. ... .. .. ... .. .. .. .. ... .. .. .. ... .. .. .. ... . ... .. .. .. ... ... .. ... .. .... .. .. ... ... . .._ .. .. ....... .. ... .... ... .. .. .. ....... .. ... ....... .. .... ... .. ... .. .. ..... ... .. .. .. ... .. ..... .. ..... .. .. .. .. ... .. .. .. ....... ..

6 .30 [1 .01] 7.9 9.9 (1)

a : time weighted mean

Table 8 Appearance of Test Solutions after Preparation

Nominal Concentration

Suspended Floating Oily

[Meana Measured Conc.] Precipitation Color

solids solids Substances

(mg/L)

Control none none none none c-

_

~ _-

Solvent Control none none none none c-

._~.__.---~-~---~-----__- ____.-----._-....._ .

.---------.._..___.__---~--------------------........-

.___------

6.30 [1.01] none none none none ca : time weighted mean

c- : colorless

5 23
A05040R• 73

Figure 1 Algal Growth Curve ofPseudokirchneriella subcapitata

(Mean cell counts vs time during the 72-hour exposure)

1 .0E+07

Control

a Solvent control

~ 6.30 mg/L

~ 1.0E+06

0-11e,

4

:21 1 .0E+05

1 .0E+04

I .OE+03

0 24 48 72

Time (hr)

Value in legend is given in the nominal concentration .

-26

S
A050407• 74

Appendix 1

Infrared Absorption Spectra

{

- 27

S )-S;
p. 75

SANITIZED

~s 2k",
A05040p• 76

Appendix 2

Composition of Medium

(

-- z'u}--

~
A05040P

• 77

Table A-2 Medium

Nutrient salts Concentration (mg /L)

H3BO3 0.185

MnCI 2 • 4H2O 0.415

ZnCtz 0.003

FeC13 • 6H2O 0.08

Na2EDTA • 2H2O 0.1

CoCI 2 • 6H2O 0.0015

Na2MoO4 • 2H,O 0.007

CuC12 • 2H2O 0.00001

CaC12• 2H2O 18

NH4CI 15

K2HPO4 1.6

NaHCO3 50

MgCI2 • 6H2 0 12

MgSO4 7H2O 15

pH 8.3

--38~

~~~
A05040p• 78

Appendix 3

Preparation of Test Solutions

5 ~°~
A050404 79

Preparation of Test Solution s

1 . Preparation

Preparation of stock solution I

Test substance ----> 630 mg

- - - --- - ---- -- --- -- -- ---------•-•--•-- Solvent ----> tetrahydrofur an

- ----- ---- ---------------- -- ------ ------ - ----------------------------------------------- inal olume v ----> 10 mL

--- ------------------------ ------------------------------ I ---------------=-------------------------• Vessel ----> volumetric bottle

- - - ----- -- --- -- --------------- ---------- ----- - - - -- - ---- --- - - ---------------

. ._

Concentration ----> 63000 mg/L . .--•-------------------------------------------------------------------------------------------------•

ixing meth od -_ : _- - ----- stir by overtu rning with tight stopper -- -- -

Preparation of stock solution II

Stock solution I ----> __ _ ____ ____ _

---------------------•------•-- . 107------μl=-------------- Dissolving agent - - > medium, 23±2b-

-- --------------- --- . . .-- ----- ----------------------------------•-----------• Final volume -- -> 1070 mL

---- --------------------------- -------------•---------------------------------------------------------•

Vessel ----

-------------------y

er

-

flask

-----------------•------------ ----------------------------------------------

> Erlenme

Concentration ----> 6.30 mg/I

- -- -nt

------------------------------------------ μL/I --------------------------------------------• olve

----

__cflnceniratian --- - - ---- --- ------- -- -- -- -------- -- -------------

Mixing method ____> add stock so1 .I to media with stiring and sonicate for 30 min

with ti ht sto e r

-------------------•-------------- -_ - •---~------~p --------------------------------------------- . .

Preparation of solvent solution

--Solvent------------------- --

:>

- ------ -

tetrahydrofuran

- -----------------------•-----------

-

collected volume ----> 94 μL

-- - - - -- - - - - ------------------ • ----- b__________ . ------------------------------------- ---Dissolvin~ a~ent -- -- --___> medium, 23f2 C

----- - -- - - ----- - - ------- -- - --------- Final volume ----> 1000 mL

---- -----------• ---- V essel

--- - ----- -- -

----> ----- Erlenmeyer flask ----- - - -- - ------ . . . . - -- -- -

-- ----------------------------------------------------u --------------------------------•---------------• Dissolving agent ----> 94 L/

L

concentration ---------~ -------------------------- - ------ --- with stiring arid_sonicate for 30 MixinQ method add-dissolving agent to m-- e- ----

----------•----------------------------- mm with tight stopper-- - - - ---- -- -- - -------- - --

2. Preparation of test solutio n

Stock solution II and solution of dissolving agent are collected as shown in'the following table and

# diluted with medium .

Final volume ----> 0.10 L

---------------------------------------------------

Vessel ------------------------------•----------------- . .---

>

500 mL Erlenmeyer flask, Iwaki

--------------------------------- --- ----------- -------- --------- --- -- -- --- - -- - Mixing method --- ___>- -stir by overturning with tipht stopper ---- -- __

•---- -------

R pe licate > 6 vessels / test groups

------------------------------------------------ - -------------------------------------------------

Nominal Concentration Group No . Stock sol. II Solvent sol. Splvent conc .

mg/L (abbreviation) mL mL μL/L

Control C ----> 0 0 0

Solvent control SC ----> 0 100 94

6 .30 Conc.l ----> 100 0 94

•--32

530
A0504(pt 80

Appendix 4

Analysis of Test Solutions and Cultures

~

-- -

-33-

~3~
A0504181

1. Gas chromatography / mass spectrometry (GC/MS) Conditions

• System

Gas chromatograph/mass selective detector with headspace sampler ,

Agilent Technologies, Inc., No. 1

Gas chromatograph (GC) : 6890, Agilent Technologies

Headspace sampler (HSS) 7694, Agilent Technologies

Mass selective detector (MSD) : 5973N, Agilent Technologies

Work station: ChemStation

• Conditions

GC

Column: Rtx-200 105 mX0.25 mm, 1 .0 μm df, RESTEK

Co.

Carrier gas: helium, 1.0 mL/min (constant flow)

Oven temperature: 60°C (1 min)-6°C/min-->110°C (10.0 min)

Injector temperature : 230°C

MS interface temperature : 200°C

Injection method: split(split ratio=20.1 :1 )

Injection volume: 3.0 mL (HSS sampleloop volume)

HSS

Temperature : Oven: 60°C, Loop: 120°C, Transfer line : 200°C

Event time : GC cycle time: 35 min

Vial equilibration time : 20 min

Pressurization time: 0.2 min

Loop fill time : 0.03 min

Loop equilibration time : 0.2 min

Inject time : 0.2 min

Vial parameter : Shake 2 (HIGH)

MSD

Temperature : Ion source: 230°C

Quadrupole mass filter: 150°C

SIM (Selected Ion Monitoring):

Solvent delay: 5 min

Quant ion: TIC of m/z 151 .0, 195 . 0

The test substance is diastereomeric mixture and 4 peaks were detected on a .

chromatogram. Hence, the concentration of the test substance was determined by

quantitative analysis using the total peak area of 4 peaks

3 4

53~
A05048182

2. Calibration Curve

The test substance was dissolved in acetone to prepare standard solutions of 0, 0 .1-

500 mg/L. Analysis of standard solutions was performed as shown below. The peak area

(count) for each solution was plotted against its concentration (mg/L) . The correlation

coefficient for the linear regression by the least square method of the calibration curve

was 0.9997. The calibration curve is shown in Figure A-4-1 .

Collect 10 mL of purified water"

I <-- add 0.1 mL of standard solution

mix

1

GC/MS analysis*2

{

* 1 : A4 grade in JIS K055 7

*2 : Measured value represents 1/100 of concentration of standard solution.

3. Detection limit

The minimum detectable total peak area of 4 peaks was set at 4000 count , and the

detection limit of concentration of the test substance was set at 0 .0002 mg/L which

corresponds to the minimum detectable peak area .

4. Analysis of Test Solutions and Test culture s

(1) Test solutions and test cultures were analyzed as shown below . Representative

chromatograms are shown in Figure A-4-2 (2), (3), (4), (6), (7) and (8).

Purified water

+

Acetone 0.1 mL

+

Test solution and test culture

mix

GC/MS analysis

*1 : By changing the mixing ratio of the test solution and purified water in each analysis vial,

the analysis samples are prepared to be within the range of the calibration curve. The total

volume of the analysis sample is 10 mL each. In control and solvent control, test solutions

are not diluted, and 10 mL of them (0 mL of purified water) is collected .

33
A05048183

(2) Standard solutions were analyzed by the same way in "2 . Calibration Curve" .

Representative chromatograms are shown in Figure A-4-2 (1) and (5) .

(3) The concentration of the test substance in the test solutions was determined by

comparing the peak area with that for the st andard solution analyzed at each

analysis.

5. Recovery test

Because the pre-treatment of the test samples for analysis was only to mix test solution with acetone as shown in "4 . Analysis of Test Solutions", the recovery test was not

required. Therefore, measured concentration was not corrected for recovery rate .

~

--36

S 3~
A05040T' 84

Figure A-4-1 Calibration curv e

Input Dat a

No. Concentration Peak Area

1 0 0

(mg/L) (count)

2 0.001 35290

3 0.002 67064

4 0.005 159717

5 0.010 303044

6 0.020 590066

7 0.050 1457557

8 0.100 2930373

9 0.200 4917974

10 0.500 14673738

11 1.00 25301800

12 2.00 50711191

13 5.00 133789350

Y= 26,544,313X

r= 0.9997

1 .5E+08

0

U

7.5E+07

•'

O .0E+00 ~ .

1 0 1 2 3 4 5 6

Concentration (mg/L)

3~--

~'13> S
p. 85

1

C\...•, c> V.,~,-~-C~ .~G..N. s

SANITIZED

~ ~ ~ 3 le -' ~ 3 "1
A0583P

FINAL REPORT

M.S.I. Report No. A050399

Acute Immobilisation Test of MTDID3663

with Daphnia magna

Submitted to:

Sumitomo 3M Limited

~ --

Prepared by:

Mitsubishi Chemical Safety Institute Ltd. (M.S.I.)

August 31, 2006

.-~- .

S'-~ ~
A0?0399

Acute Immobilisation Test of MTDID3663

with Daphnia magna

(English version)

Sponsor Sumitomo 3M Limited

Study Title Acute Immobilisation Test of MTDID3663 with Daphnia magna

Study No. A050399

This report is the English version of the original, which was written in Japanese . I, the undersigned hereby declare that this version faithfully reflects the original report to the best o f

{ my knowledge.

Translated by

---- W1(Vt MED

-~--- . S; LA
p. 88

A050399

Compliance with the GLP Standard s

Yokohama Laboratory

Mitsubishi Chemical Safety Institute Ltd.

Sponsor Sumitomo 3M Limited

Study Title Acute Immobilisation Test of MTDID3663 with Daphnia magna

Study No. A050399

I hereby ce rtify that this study was carried out in accordance with its protocol and with

our standard operating procedures, and that the report accurately describes the test results .

The study described in this report was conducted in compli ance with the following

Good Laboratory Practice (GLP) st andards .

The standards concerning the test facility in which the study of new chemical

substance is performed (Yakushokuhatsu No .1121003, Heisei 1.5 .11 .17 Seikyoku

No.3, Kanpokihatsu No .031121004, November 21, 2003; the latest revision, April

1, 2005)

Study Directo r

Midori MINO, B.Sc. Sealed date: August 31, 2006

~-

---3---

S ~1c'~
A050399

Quality Assurance Statemen t

Yokohama Laboratory

Mitsubishi Chemical Safety Institute Ltd.

Sponsor Sumitomo 3M Limited

Study Title Acute Immobilisation Test of MTDID3663 with Daphnia magna

Study No. A050399

We hereby certify that this study was carried out in accordance with its protocol and

with our standard operating procedures. We certify that this report faithfully describes the

methods and procedures employed, and that the reported results reflect accurately the raw

data of the study.

Date of Inspection Date of Reporting

to Management and

Study Director

Protocol: August 8, 2006 August 8, 2006

Amendment 01 August 9, 2006 August 9, 2006

In-progress study:

Preparation of test solutions: August 9, 2006 August 9, 2006

Introduction of daphnid : August 9, 2006 August 9, 2006

Preparation of test solutions: August 10, 2006 August 10, 2006

Introduction of daphnid: August 10, 2006 August 10, 2006

( Observation of daphnid: August 11, 2006 August 11, 2006

Final report: August 31, 2006 August 31, 2006

Quality Assurance Personnel

Yumiko KASHIWAGI, B .Sc. Sealed date: August 31, 2006

Hiroko TOKIMI, B.Sc. Sealed date: August 31, 2006

Reiko TSUTSUI Sealed date: August 31, 2006

---4-

sy3
99

Introduction

1. Study Titl e

Acute Immobilisation Test of MTDID3663 with Daphnia magna

(Study No. A050399)

2. Purpos e

The acute toxicity test of the test substance with Daphnia magna was conducted in order

to evaluate the toxicity of the test substance for notification under the Chemical

Substances Control Law of Japan.

3. Test Guidelines

The test method relating to new chemical substances "Alga growth inhibition test,

Daphnia sp. acute immobilisation test, and fish acute toxicity test" (Yakushokuhatsu

No.1121002, Heisei 15 .11 .13 Seikyoku No.2, Kanpokihatsu No .031121002, November

21, 2003; the latest revision, April 1, 2005 )

4. GLP Compliance s

The standards concerning the test facility in which the study of new chemical substance

is performed (Yakushokuhatsu No .1121003, Heisei 15.11 .17 Seikyoku No.3,

Kanpokihatsu No.031121004, November 21, 2003 ; the latest revision, April 1, 2005)

5. Sponsor

Sumitomo 3M Limited

8-8 Minami-hashimoto 3-chome, Sagamihara, Kanagawa, Japan

6. Test Facility (Head Office)

Mitsubishi Chemical Safety Institute Ltd. (M.S.I.)

1-30 Shiba 2-chome,'Minato-ku, Tokyo, Japa n

7. Test Facility (Laboratory)

Yokohama Laboratory

Mitsubishi Chemical Safety Institute Ltd.

1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa, Japan

8. Study Director

Midori MINO, B.Sc.

Ecological Chemistry Division

--~-

syL~
P. 91

A050399

9. Personnel in Charge of the Study

Hiromitsu OOKUBO, Ph.D. Sealed date: August 31, 2006

(Experimental work and reporting )

Masamitsu NAKAGAWA, M.Sc. Sealed date: August 31, 2006

(Experimental work)

Kazuhisa OONISHI Sealed date: August 31, 2006

(Analytical work)

Tomie HANZAWA Sealed date: August 31, 2006

(Experimental work)

10. Date

Initiation of the study : August 8, 2006

Start of the exposure : August 10, 2006

End of the exposure: August 12, 2006

Completion of the study: August 31, 2006

11. Storage

The following will be stored in the archives of the Yokohama Laboratory .

1) Protocol

2) Final report

3) Raw data

4) Test substance

5) Reference substance

6) Other requirements

CSLlS
A050A

Content

page

Summary ----------------- ----------- ---------------------------------------------------------------------------------------------8

1 .Test Substance 10

1 .1 . Identification 10

1 .2 . Source 11

1 .3 . Storage and Confirmation of Stability 1 1

2. Test Species -------------- -------------------------------------- ----------------------- -------- --------------------- .12

3 . Test Methods 13 ------------------------------------------------------------------------------------------------------- -------- -

3 .1 . Test Conditions 13

3 :2. Dilution Water 13

3 .3 . Apparatus 14

3 .4. Test Concentrations 14

3 .5. Preparation of Test Solutions 15

3.6. Analysis of Test Solutions 15

3 .7. Test Procedure 15

3 .8 . Calculation of Results 16

4. Results and Discussion 17

~--------------------------------------------------------------------------------------------------~

4.1 . Circumstances That May Have Affected the Reliability

of the Test Results 17

4.2 . Concentrations of Test Substance in Test Solutions 17

4.3 . Median Effect Concentration (EC50) 17

4 .4. Highest Concentration in 0% Immobility an d

Lowest Concentration in 100% Immobility 17

4.5. Appearance, Temperature, Dissolved Oxygen Concentrations and pH

of Test Solutions 18

Tables 1-8 19-25

Figure 1 26

Appendix 1 . Infrared Absorption Spectra 27-28

Appendix 2. Composition of Dilution Water 29-30

Appendix 3 . Preparation of Test Solutions 31-32

Appendix 4. Analysis of Test Solutions 33-42

S L\ lA
A05 399

Summary

Sponsor Sumitomo 3M Limite d

Study Title Acute Immobilisation. Test of MTDID3663 with Daphnia magna

Study No . A050399

Methods:

The study was. conducted in accordance with the test method relating to new chemical

substances "Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish

acute toxicity test" (Yakushokuhatsu No.1121002, Heisei 15.11 .13 Seikyoku No .2,

Kanpokihatsu No .031121002, November 21, 2003 ; the latest revision, April 1, 2005 )

t 1) Exposure procedure: semi-static (batchwise renewal of the test solutions after 24-

hour exposure)

2) Duration: 48 hours

3) Test concentrations (nominal):

control, solvent control, 0.560, 1 .00, 1 .80, 3 .10, 5.60* mg/L

* : the maximum attainable concentration .

Factor: 1 . 8

Solvent concentration: Tetrahydrofuran, 94 μL/L

4) Volume of test solution : 130 mL /vessel

5) Number of vessels: 4 vessels /concentratio n

6) Biomass loading: 20 daphnids /concentration (5 daphnids /vessel)

7) Temperature: 20 ± 1 °C

8) Light: fluorescent light

{ 16 hours light (800 lux or less) /8 hours dark

9) Analytical methods: gas chromatography / mass spectrometry (GC/MS)

SL-~c-1
A050399

Results

1) Median Effect Concentration

The proportion of the measured concentrations to the nominal values were 81-

113% at the preparation, and 70-96% after 24-hour exposure . It is considered that

the main cause of concentration reduction was volatilization of the test substance

from test soluitons.

2) Results of after 24-hour exposure

EC50 (95% Confidence Limits) : >5.56 mg/L

(95% confidence limits: impossible to calculate)

Highest concentration in 0% immobility: 1.63 mg/L

Lowest concentration in 100% immobility : >5 .56 mg/L

3) Results of after 48-hour exposure

EC50 (95% Confidence Limits) : 3.30 mg/L

(95% confidence limits: 2.72-4.14 mg/L)

Highest concentration in 0% immobility: 0.857 mg/L

Lowest concentration in 100% immobility : >5 .56 mg/L

---

9~

S ~cSS
A05 399

1 . Test Substance

1.1. Identificatio n

Name of the new chemical --~

substance

(by IUPAC nomenclature)

Alternate name

CAS RN

Structural formula or

rational formul a

(If both of them are

unknown, outline of

preparation or

manufacturing

Molecular weight

Purity (%) of the new SMWED

chemical substance

subjected to the study

Lot number of the new

chemical substanc e

subjected to the study

Name and content of

impurities

Vapor pressure

Solubility in water

1-octanol/water partitio n

coefficient

~- -

Melting point

Boiling point

Appearanc e

at room temperature

Stability

Solubility i n

-solvents and the like

4 9

~~~
96

A050399

1 .2. Source

Supplier: Sumitomo 3M Limited

1 .3. Storage and Confirmation of Stability

For the duration of the study, the test substance was stored in the refrigerator of M.S.I.

(refrigeration, dark place and sealing). At the end of the study, infrared absorption spectrum

of the test substance was measured . The spectrum was consistent with that measured at the

start of the study, indicating that the test substance was stable under the storage condition .

The infrared absorption spectrum is shown in Appendix 1 .

Apparatus : Infrared spectrophotometer, AVATAR 320, Nicolet Instrument Corporation

{- -

-43--,

SSp
p. 97

A050399

2 . Test Species

1) Common name: Daphnia magna (Oomijinko in Japanese)

2) Scientific name : Daphnia magna

3) Source : National Institute for Environmental Studies, Japan

4) Date of receipt: July 18, 1995

5) Sensitivity to reference substance:

Acute immobilization test of a reference substance

(K2Cr2O7, guaranteed reagent) has been conducted every

six months since June 1998 . 48-hour median effect concentration (EC50) was as follows.

average ± standard deviation = 0 .75 ± 0 .14 mg/L, n=17

minimum value - maximum value = 0 .57 - 1 .02 mg/L

6) Growth stage : female juvenile (less than 24 hours old)

7) Culturing condition of parental daphnid for obtaining test juvenile s

Water: Elendt M4 (Reconstituted water recommended by OECD

Guidelines for Testing of Chemicals No .202 "Daphnia

magna Acute Immobilisation Test" (2004)) .

Biomass loading: <1 daphnid / 80mL (25 daphnids / 2 L)

Temperature : 20 ± 1°C

Dissolved oxygen concentration: 60% of the saturation

pH: 6.0-9.0

Light: fluorescent light,

16 hours light (800 lux or less) / 8 hours dark

Culturing period : July 19, 2006 - August 10, 2006

Mortality of parental daphnids during 2 weeks before test : <5 %

Presence of ephippia and males: none

Feed: Chlorella vulgaris (uniceller green algae)

Feeding amount: 0.2 mg C (organic carbon) / daphnid / day

~ Renewal of water in culturing vessel: periodically 3 times / week

Produced offsprings were removed daily.

S~ ~
p. 98

A050399

3. Test Methods

The study was conducted in accordance with the test method relating to new chemical

substances "Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish

acute toxicity test" (Yakushokuhatsu No .1121002, Heisei 15 .11 .13 Seikyoku No .2, Kanpokihatsu No.031121002, November 21, 2003 ; the latest revision, April 1, 2005 )

3.1. Test Conditions

1) Exposure procedure: semi-static (batchwise renewal of the test solutions after

24-hour exposure)

2) Duration: 48 hours

3) Volume of test solution : 130 mL /vessel

4) Number of vessels: 4 vessels /concentration

5) Number of test species: 20 daphnids /concentration (5 daphnids /vessel)

6) Temperature : 20 ± 1 °C

7) Dissolved oxygen concentration : ? 60% of the saturation; not aerated

8) pH: not adjusted

9) Light: fluorescent light,

16 hours light (800 lux or less) /8 hours dark

10) Feeding: none

3.2. Dilution Water

Elendt M4 (see "2, 7)") was used for dilution water. The composition of this water is

given in Appendix 2 . At the beginning of exposure test, hardness ( _,'S 250mg/L, CaCO3) and

pH (6.0-9 .0) were confirmed to be within proper range.

SS2
P. 99

A050399

3.3. Apparatus

1) Test Vessel: 100 mL Erlenmeyer flask with glass stopper (Because a great volatilization of the test substance from test solutions was considered,

test vessels were filled with the test solutions (130 mL) for minimize the void of them and were put the ground-in stopper .)

2) Incubator: PVC-made tank (Temperature control equipment: Coolnit CL-80F,

Taitec Co., Ltd.)

3) Thermometer: Check Temp, Hanna Instruments

4) DO meter: DOL-10, DKK Corporation

5) pH meter: HM-40V, Toa Electronics Corporation

6) Hardness test kit: HA-DT, Hach Company

~ 7) Electrobalance: AG204, Mettler K.K.

AE163, Mettler K.K.

AB204-S, Mettler K.K.

PB3002, Mettler K .K.

3.4. Test Concentrations

The solubility of the test substance to the dilution water is 5.6 mg/L (measured by

M.S .I.). Hence, the test concentration was set at 5 .6 mg/L or less.

A range-finding test was conducted before the definitive test. The result is as follows,

and enabled the selection of the concentration range in the definitive test .

Nominal concentrations of the definitive test : control, solvent control, 0 .560, 1 .00,

1 .80, 3 .10, 5.60*mg/L

*: The maximum attainable concentration .

Factor: 1 . 8

Result of range-finding test (2 vessels/concentration, 10 daphnids/concentration)

Concentration Immobility (%)

(mg/L) 48 hours

Solvent control 10*

0.0560 0

0.560 0

5 .60 90

* : This result was judged within normal limits .

--~4--

Ss3
p. 100

A050399

3.5. Preparation of Test Solutions

The method of preparation of test solutions is shown in Appendix 3 . The control solution was dilution water only, and solvent control solution was dilution water containing the

solverit (94 μL/L). An aliquot (130 mL) of each test solution was poured into four vessels .

A stock solution I (see Appendix 3) used for preparation of test solutions was prepared at

the start of exposure and stored under the refrigeration and dark (stable for 48 hours or

more under this storage condition). A stock solution II and a stock soluvent solution (see

Appendix 3) were prepared at each use.

3.6. Analysis of Test Solutions

At the start of the exposure, before and after renewal of test solutions, and at the end of

the exposure, test solutions were analyzed . Analysis samples were collected from one vessel out of four for each concentration group . Then, these samples were analyzed by gas

chromatography / mass spectrometry (GC/MS) . The test substance is diastereomeric mixture, and 4 peaks were detected on a chromatogram . Hence, the concentration of the test

substance was determined by quantitative analysis using the total peak area of 4 peaks . Details are shown in Appendix 4.

3.7. Test Procedur e

After measurement of the temperature, dissolved oxygen concentration (DO) and pH,

juvenile daphnids were introduced into each test vessel using a glass pipette to start exposure. When juvenile daphnids were introduced, the volume of total culture water in glass

pipette would not exceed 1% .or less of test solution in test vessels. Thereafter, daphnids

were kept untill the end of exposure .

The daphnids were observed at 24 and 48 hours after the start of exposure, and number

of immobile daphnids were recorded . Individuals which were not able to swim within 15

seconds after gentle agitation of the test vessel were considered to be immobilized .

The appearance, temperature, DO and pH of test solution in all concentration groups

( (only one vessel out of four for each concentration) were observed or measured at the start

of exposure, before and after renewal of test solutions, and at the end of the exposure .

-~-i-~--

`-{
p. 101

A050399

3.8. Calculation of Results

1) Determination of Concentration of Test Subst ance for Calculating Effect Concentration

Concentration of test substance to calculate the effect concentrations was the

average ofmeasured concentration (time-weighted me an).

Time-weighted mean:

ConcA,, - ConcB„

mc„ = ln(ConcA„) - ln(ConcB„ )

mcl +mc2 +•••+mc„ MC=

n

mc, : Mean measured concentration during each exposure .

ConcA,,: Measured concentration at the preparation of test solution .

ConcB,, : Measured concentration at the 24 hours after the preparation of test solution .

I (In the case of ConcA,=ConcB„, nzc„=ConcAn ConcB„ )

MC: Mean measured concentration.

2) Calculation of median effect concentration (EC50 )

The immobility (%) for each concentration was calculated using the number of immobile daphnids and biomass loading (20 daphnids), and median effect concentration (EC50)

was determined according to the following table.

Immobility at maximum concentration ~ 50% < 50%

EC50 is selected among calculations

Determination using the probit method, movin g

average method and binomial method. Estimated

method of EC50 95% confidence limits are calculated if concentration range.

~ possible.

Concentration- Included in the report. Included in the re ort. immobility curve p

3) Calculation of highest concentration in 0% immobility and lowest concentration in 100%

immobility

Highest concentration in 0% immobility is the highest concentration tested causing no

immobility. Lowest concentration in 100% immobility is the lowest concentration producing 100% immobility.

-1 G-~-

.S S 5
p. 102

A050399

4. Results and Discussion

4.1. Circumstances That May Have Affected the Reliability of the Test Results

There was no significant matter that may have affected the reliability of the test results .

4.2. Concentrations of Test Substance in Test Solution s

The concentrations of test substance in test solutions are shown in Table 1 and

representative chromatograms are shown in Appendix 4 .

The proportions of the measured concentrations to the nominal values were 81-113%

at the preparation and 70-96% after 24 hours. It is considered that the main cause of

concentration reduction was volatilization of the test substance from test solutions .

4.3. Median Effect Concentration (EC50)

The cumulative immobility for each exposure period is shown in Table 2, and the concentration- immobility curve is shown in Figure 1 . -

In the duration of the exposure, the immobilities in the control and solvent control

were both 0%, and also the rate of surface-trapped daphnid in these controls were both 0% .

Thus, these results fulfilled the validity of the test.

The EC50s are shown in Table 3 and as follows :

EC50 after 24-hour exposure: >5.56 ing/L (95% confidence limits: impossible to calculate); .

EC50 after 48-hour exposure : 3.30 mg/L (95% confidence limits: 2.72-4 .14 mg/L) .

4.4. Highest Concentration in 0% Immobility and Lowest Concentration in 100% Immobility

The highest concentration in 0% immobility and the lowest concentration in 100% immobility are shown in Table 4 and as follows :

Highest concentration in 0% immobility after 24-hour exposure : 1.63 mg/L;

Highest concentration in 0% immobility after 48-hour exposure : 0.857 mg/L;

Lowest concentration in 100% immobility after 24-hour exposure : >5 .56 mg/L;

Lowest concentration in 100% immobility after 48-hour exposure : >5.56 mg/L.

~-~---

S;7S k,,
p. 103

A050399

4.5. Appearance, Temperature, Dissolved Oxygen Concentrations and pH of Test Solutions

The appearance, temperature, dissolved oxygen concentrations and pH of test solutions

are shown in Table 5, 6, 7 and 8, respectively .

Temperature were ranged within 20±1°C during the exposure . Dissolved oxygen concentrations were over 60% of the saturation value (8 .8 mg/L at 20.0°C) . These values fulfilled the validity of the test. The pH were ranged within proper range (6 .0 - 9.0) for daphnids.

~

SS-7
p. 104

A050399

Table 1 Measured Concentration of the Test Substance in Test Wate r

(Semi-Static Condition))

Nominal Measured Concentration (mg/L) Meana Measured

Concentration

(Percent of Nominal, %) Concentration (mg/L)

(mg/L) 0 Hour 24 Hours 24 Hours 48 Hours (Percent of

New Old New Old Nominal, %)

Control <0.0004 <0.0004 <0.0004 <0.0004 --

Solvent <0

.0004 <0.0004 <0.0004 <0 .0004 -- Control

0.560 0.452 0.393 0.541 0.496 0.470

(81) (70) (97) (89) (84)

1 .00 0

.907 0.713 1 .04 0.787 0.857

(91) (71) (104) (79) (86)

1 .80 1

.62 1.53 1.85 1.52 1.63

(90) (85) (103) (84) (91)

3 .10 3.17 2.65 3.36 2.49 2.90

(102) (85) (108) (80) (94)

5 .60 6.33 5.39 5.83 4.77 5.56

(113) (96) (104) (85) (99)

a: time weighted mean

New: freshly prepared test solutions

~ Old: test solutions on 24 hours after preparation of new solutions

19

S S~
p. 105

A050399

Table 2 The Number of Immobilized Daphnia magna (Percent Immobility)

Nominal Meana Measured Cumulative Number of Immobilized Daphnia

Concentration Concentration (Percent Immobility)

(mg/L) (mg/L) 24 Hours 48 Hours

Control --. 0 0) 0 0)

Solvent Control -- 0 0) 0 0)

0.560 0

.470 0 0)-----------__ o....-(.--0)---------

--

._...---._........

1 .00 0.857 0 0) 0 0)

----

1 .80 1

.63 0 ( 0)------------ 1 (

5 )

-------------~---__

( 3.10 2.90 6( 30) 10 ( 50)

-------.--.---.__.__ .-. ... ... __....... -...-_........ -----.---..._...._ ..__._._.._ ..__..._._.-.._ .__.._ .....--.--- .._ .._._... ..........._._ ..------._

5 .60b 5.56 6 30) 16 ( 80)

a: time weighted mean

b: The maximum attainable concentration under the present test conditions and

preparation methods

rz9--

S~
p. 106

A050399

Table 3 Calculated EC50 Values

Exposure EC50 95-Percent

Period Confidence Limits Statistical Method

(Hours) (mg /L) (mg/L)

24 >5.56 --

. ..... .. .... .. .. .. .. ... .. ... .. .. .. ... .... .... ... .. .. ..... .. ... . .. ... .. .... ... .. ... .. .. ... .. .. ..... .. .. .. ... .. ... .. .. ... .. .. .. ... ... .. .. ..... .. .. .. ... .... ... .. ... .. ... ... .... .. ... . ... .. ... ... . .. ... .. .. ... .. .. ... .. .. .. ... ... .. ....... . ... .. .. .. ... .. .. .. ... .. ... .. .. ... .. .. .. ... ....... .. ... .

48 3.30 2.72 - 4 .14 Probit*1

* 1 : Using the concentrations of 0 .470 - 5.56 mg/L

--: Could not be determine d

Table 4 Highest Concentration in 0% Immobility and Lowest Concentration in 100%

Immobility

Exposure Highest Concentration Lowest Concentration

Period in 0% Immobility in 100% Immobility

(Hours) (mg/L) (mg/L)

24 1.63 >5.56

. .. ..... .. .... .. .. ... .. .. .. ... .. ... .. .. ....... .. ....... .. ... ... .. .. .. ..... .. .. ... ....... .. ....... .. .. .. ... .. ... .. .. ... .. .. ... .. ... .. ... .... .. .. ... .. ... . .. .. ... .. ... ....... .. ... .... .. ..... ... .. ... .. .. ... .. ..... .. ... .... ... .. ... .. .. .. .. .. ....... .. ..... .. ..... ... ...... ... .. .. ...

48 0.857 >5.56

--~-1--

~~~
p. 107

A050399

Table 5 Appearance of Test Solutions

Nominal Meana Measured Appearance of Test Solutions

'Concentration Concentration 0 Hour 24 Hours 24 Hours 48 Hours

(mg/L) (mg/L) New Old New Old

Control -- C- C- C- C-

-

Solvent Control -- C- C- C- C0.560 0.470 C- C- C- C-

- ----- _

1 .00 0.857 C- C- C- C-

..._--- .__..--

1 .80 1.63 C- C- C-. C-

-- _-_------ - - -- - - - -

3.10 2.90 C- C- C- C5.60 5.56 C- C- C- Ca : time weighted mean

New: freshly prepared test solution s

Old: test solutions on 24 hours after preparation of new solutions

Color:

C-; colorless

--~~-~

Slo~
p. 108

A050399

Table 6 Temperature of Test Solution s

Nominal Meana Measured Temperature (°C)

Concentration Concentration 0 Hour 24 Hours 24 Hours 48 Hours

(mg/L) (mg/L) New Old New Old

Control -- 20.4 19.9 20.0 20. 1

-~-----. ___ .----_._....-_ ------__ __..

.^_----~-----

.4 19.9 20.0 20- Solvent Control 20 . 1

_____,_.__.--.---.-_ .-------------

0.560 0.470 20.2 19.9 19.9 20.1

1 .00 0.857 20.2 19.9 19.9 ---~-_ 20 . 1

--..__..------.--.--... ..

.80 1.63 20.4 19.9 19 . 1 .9 20. 1

3 .10 2.90 20.3 19.9 19.9 20.1 .

.-----~-- ._---_.______---~--._ .__.

._------_----~---------__.____._ .-__..

.----__-___-_-------------__-.____-----

5 .60 5.56 20.3 19.9 19.9 20.1

a: time weighted mean

New: freshly prepared test solutions

Old : test solutions on 24 hours after preparation of new solutions

~

--~3--•

StP~
p. 109

A050399

Table 7 Dissolved Oxygen Concentrations in Test Solution s

Nominal Meana Measured Dissolved Oxygen Concentration (mg/L)

Concentration Concentration 0 Hour 24 Hours 24 Hours 48 Hours

(mg/L) (mg/L) New Old New Old

Control -- 8.8 8.5 8.7 8. 6

-__-----._...__...

.-------------------~---

.8 8.5 8_ Solvent Control ' -- 8 .6 8. 7

0.560 0.470 8.8 . ---------8.5 -----~-- 8.7 -----~

.~__ 8.7

------------------------------------~---

1 .00 0.857 8.8 8.5 8.7 8.7

1 .80 1.63 8

.7 ~----- 8 .4---____----8

.6 8

.5 -----

- - ----- --- - ---------------- -

3 .10 2.90 8.6 8.3 8.6 8.5

------- .-.----

5 . 60 5.56 8.2 8.3 8.5 8.4

a: time weighted mean

New: freshly prepared test solutions

Old: test solutions on 24 hours after preparation of new solutions

24

Sto3

1
p.110

A050399

Table 8 pH Values of Test Solution s

Nominal Meana Measured PH

Concentration Concentration 0 Hour 24 Hours 24 Hours 48 Hours

(mg/L) (mg/L) New Old New Old

Control -- 8.1 8.0 8.1 8.0

Solvent Control -- 8.2 8.1 8.1 8.1

---_

.....

.-_------ ----- -- .--_.-

0.560 0.470 8.2 8.1 8.1 8. 1

-._.___--.----.__.__._...__ .._.

1 .00 0.857 8.2 8.0 8.1 8.1

1 .80 1.63 8.2 8.1 ---- 8.1 8.1

~ _

3.10 2.90 8.2 8 .0 8.1 8. 0

......-----__-----------~--------------_._ .---- ------------..

.----___---~--____..__ ._ ..

.----____-----

5 .60 5.56 8.2 8 .0 8.1 8.0

a: time weighted mean

New: freshly prepared test solutions

Old: test solutions on 24 hours a fter preparation of new solutions

-~s-~

''~~`~
AO?0399

Figure 1 Concentration-Immobility Curv e

100

90

o 24 Hours

80 -■-- 48 Hours

70

60

50

40

30

20

10

0

0.1 1 10

Concentration (mg/L)

26

S~S
p.112

A050399

Appendix 1

Infrared Absorption Spectra

~

S lplo
p. 113

.! _ V-& V` d-,Y CG ~ Io S O~/' ~ 1 C~ V\ S e-C:~ V-

\

SANITIZED

S lo`t
p. 114

A050399

( Appendix2

Composition of Dilution Water

(

29

S lo$
115

A050399

Table A-2 Elendt M4 medium recommended by OECD Guideline No .202 used as dilution

water

Macro nutrients Concentration (mg/L)

CaC12•2H2O 293.8

MgSO4•7H2O 123

KCl 5

.3

.8

NaHCO3 64.8

Na2SiO3•9H2O 10.0

NaNO3 0.274

KH2PO4 0.143

K2HPO4 0.184

Trace elements Concentration (μg/L)

H3B03 2859.5

MnC12•4H2O 360 .5

LiCI 306.0

RbCI 71.0

SrC12•6H2O 152.0

NaBr 16.0

Na2MoO4•2H2O 63.0

CuC12•2H2O 16.8

ZnC12 13.0

CoC12•6H2O 10

KI 3

.0

.25

NaZSeO3 2.19

NH4VO3 0.575

Na2EDTA•2H2O 2500

( FeSO4•7H2O 995.5

Vitamins Concentration (μg/L)

Thiamine hydrochloride 75 .0

Cyanocobalamine (B 12) 1 .00

Biotine 0.750

S ~~
p. 116

A050399

( Appendix3

Preparation of Test Solutions

~--

--~1--

S
A055399

Preparation of Test Solutions

1 . Preparatio n

Preparation of stock solution I

Test substance ----> 560 mg

- ---- - - - - - ----------

. . .

Solvent ----tetrahydrofura > n

- - -

------------------------------------------------•-----------------------------------------------------------

Final volume ----> 10 mL

--- --------•-----------•--•-------------------------------------------------------------------

Vessel ----> volumetric bottle

-------------------------------------------------•-----•------------------------------------------------- Concentration ----> 56000 mg/L

---------------------------------•-----•------------------------------------------------------------------

Mixing method ----> inversion

------------------------------------------------------------------------------------------------•--•--- . ._ . .

Preparation of stock solution I I

Stock solution I ----> 220 μL

- - --- - -- -------- ---------- -----•----- -- - ------------ ---•--------•-

Solvent --> dilution water (Elendt M4 with fully aerated and controled at 20±1'C)

- -- -- - - ------------ -------------------------------------------------------------------------- Final volume ----> 2200 mL

- - ----- ------------- - -- - - - ---------

Vessel ----> media bottl e

------------------------------------------------------------------------------------------------------------

Concentration ----> 5.60 mg/L

- -- ----------- - ------ -----------------------------------------------------------------------------

~ Solvent concentration _ -____>- __ _ 94 μg/L _ _ _ _ _

Mixing method ----> add 'stbck o. I to clilutiori wafer with sturirig an d

sonicate for 10 min with stirring . (sealing)

------------------------------------------- -------------------•-----------•------------------------•-------

Preparation of dissolving agen t

Solvent ----> tetrahydrofuran

-- - -------------------------- ----------- -- ------- ------•-----------------------------------------

-Solvent (volume) - --> 940 k~L- - - - -- - -- -------- - --- - --------------------- ------

Dissolving-agent ----> dilution water (Elendt M4 with fully aerated and controled at 20f1 °C)

- - - - --•---------------------------------------------------------------------------------------

Final volume 100 mL

---------------------------------------------------------------------------------------------------------

Vessel ----> volumetric bottle

----- -----------------------------•-- - -------------------------------------------------------------

Solvent concentration ----> 9400 μL/L

------------------------- --------------------------------------------------------------------------

MixinRmethod -_ :____ :> inversio n

- -----------------------------------------------------------------------

2 . Preparation of test solution

Stock solutions II and dissolving agent are diluted according to following table, and used as the

test solutions. Control solution contains dilution water only .

Final volume ----> 1 .10 L --- --- - --- - - - - ---- - ------ - - - - - - -- - - -

. . .

Vessel ----> media bottle

.--•------•-------•--------------------------------------------- -----------------------------------------

Mixing method ----> stirrer (3 min )

-------• --•-----•-•-----------------•-------- -----------------------•-----------------------•-------•-•-

_ _ -Factor ----> 1 .7 8

------------------------- - - --------------------------------------------------------

Stock sol . II Dissolving Solvent conc.

Nominal conc . Group No. agent

(mg/L) (abbreviation) (mL) (mL) (μL/L)

control C ----> 0 0 0

solvent control SC ----> 0 11.00 94

0.560 Conc.1 ----> 110.0 9.90 94

1 .000 Conc.2 ----> 196.4 9.04 94

1 .800 Conc.3 ----> 353.6 7.46 94

3 .100 Conc.4 ----> 608.9 4.91 94

5 .600 Conc.5 ----> 1100.0 0 94
p. 118

A050399

Appendix 4

Analysis of Test Solutions

(

---3 3-~

. SZ~
p. 119

A050399

1 . Gas chromatography / mass spectrometry (GC/MS) Condition s

• System

Gas chromatograph/mass selective detector with headspace sampler,

Agilent Technologies, Inc ., No . 1

Gas chromatograph (GC): 6890, Agilent Technologies

Headspace sampler (HSS) 7694, Agilent Technologies

Mass selective detector (MSD): 5973N, Agilent Technologies

Work station: ChemStation

• Conditions

GC

Column: Rtx-200 105 mX0.25 mm, 1 .0 μm df, RESTEK Co.

Carrier gas: helium, 1.0 mL/min (constant flow)

Oven temperature : 60°C (1 min)-->5°C/min--),110°C (10.0 min)

~ Injector temperature: 230°C

MS interface temperature : 200° C

Injection method : split (split ratio=20.1 :1 )

Injection volume : 3.0 mL (HSS sampleloop volume)

HSS

Temperature : Oven: 60°C, Loop : 120°C, Transfer line : 200°C

Event time : GC cycle time: 35 min

Vial equilibration time : 20 min

Pressurization time : 0.2 min

Loop fill time : 0 .03 min

Loop equilibration time : 0.2 min

Inject time : 0.2 min

Vial parameter: Shake 2 (HIGH)

MSD

Temperature: Ion source: 230°C

Quadrupole mass filter: 150°C

SIM (Selected Ion Monitoring) :

Solvent delay : 5 min

Quant ion: TIC of m/z 151 .0, 195.0

The test substance is diastereomeric mixture, and 4 peaks were detected on a

chromatogram. Hence, the concentration of the test substance was determined by

quantitative analysis using the total peak area of 4 peaks.

-1 3
p. 120

A050399

2. Calibration Curve

The test substance was dissolved in acetone to prepare standard solutions of 0, 0 .1-500

mg/L. Analysis of standard solutions was performed as shown below . The peak area

(count) for each solution was plotted against its concentration (mg/L) . The correlation

coefficient for the linear regression by the least square method of the calibration curve was

0 .9997. The calibration curve is shown in Figure A-4-1 .

Collect 10 mL of purified water* I

I E- add 0.1 mL of standard solution

mix

GC/MS analysis*2

*1 : A4 grade in JIS K055 7

*2 : Measured value represents 1/100 of concentration of standard solution.

3. Detection limit

The minimum detectable total peak area of 4 peaks was set at 4000 count , and the

detection limit of concentration of the test substance was set at 0 .0004 mg/L which

corresponds to the minimum detectable peak area.

4. Analysis of Test Solution

(1) Test solutions were analyzed as shown below. Representative chromatograms are

shown in Figure A-4-2 (2), (3), (4), (5), (7), (8), (9) and (10) .

Purified water

+

~ Acetone 0.1 mL

+

Analysis sample" 10 mL (total volume of the test solution and purified water)

mix

GC/MS analysis

*1 : The analysis samples are prepared to be within the range of the calibration curve by

changing the mixing ratio of the test solution and purified water in each analysis vial . The

total volume of the analysis sample is 10 mL each . In control and solvent control, test

solutions are not diluted, and 10 mL of them (0 mL of purified water) is collected .

---33-•

S'l'-~
121

A050399

(2) Standard solutions were analyzed by the same way in "2 . Calibration Curve" . Representative chromatograms are shown in Figure A-4-2 (1) and (6).

(3) The concentration of the test substance in the test solutions was determined by

comparing the peak area with that for the standard solution analyzed at each analysis.

5. Recovery test

Because the pre-treatment of the test samples for analysis was only to mix test solution

with acetone as shown in "4 . Analysis of Test Solutions", the recovery test was not required. Therefore, measured concentration was not corrected for recovery rate.

~

9

6 SZS
A05p0399

Figure A-4-1 Calibration curve

No. Concentration Peak Area

1 0 0

(mg/L) (count)

2 0.001 35290

3 0.002 67064

4 0.005 159717

5 0.010 303044

6 0.020 590066

7 0.050 1457557

8 0.100 2930373

9 0.200 4917974

10 0.500 14673738

11 1.00 25301800

12 2.00 50711191

13 5.00 133789350

Y= 26,544,313X

r= 0.9997

1 .5E+08

~, .

,-.

❑

~

0

7.5E+07 ~..

{ O.0E+00 gig ,

0 1 2 3 4 5 6

Concentration (mg/L)

. ~~

~~ (o
p. 123
p. 124

FINAL REPORT

M.S.I. REPORT No.A050393

Ready Biodegradability Test of MTDID3663

{

Submitted to :

SUMITOMO 3M, LTD.

Prepared by :

Mitsubishi Chemical Safety Institute Ltd .

February 7, 200 6

-4-

S~~
p. 125

Ready Biodegradability Test of MTl?ID3663

(English version)

Sponsor SUMITOMO 3M, LTD.

Study Title Ready Biodegradability Test of MTDID3663

Study No. : A050393

This report is the English version of the original, which was written in Japanese . We, the

undersigned hereby declare that this version faithfully reflects the original report to the best of

our knowledge .

Translated by

SANITIZED

- -

~- i

Date : Uol

J o Koyasu, B_Sc .

Study Directo r

Environmental Science Group A of Yokohama Laboratory,

Mitsubishi Chemical Safety Institute Ltd .

. .~-. ,

S~3
p. 126

COMPLIANCE WITH THE GLP STANDARD S

Sponsor : SUMITOMO 3M, LTD .

Study Title : Ready Biodegradability Test of MTDID3663

Study No. : A050393

I hereby certify that this study was carried out in accordance with its protocol and with our

standard operating procedures, and that the reported results reflect the raw data of the study

accurately.

The study described in this report was conducted in compliance with OECD Principles of

( Good Laboratory Practice (as revised in 1997).

Study Director

Junko Koyasu, B .Sc . Sealed date: February 7, 2006

S ~SLA
p. 127

QUALITY ASSURANCE STATEMENT

Sponsor : SUMITOMO 3M, LTD .

Study Title : Ready Biodegradability Test of MTDID3663

Study No. : A050393

By the inspections and audits listed below, we verify that this study was carried out in

accordance with its protocol and with our standard operating procedures. We certify that this

report faithfully describes the methods and procedures employed, and that the reported results

reflect the raw data of the study accurately.

Date of Date of Reporting to Test Facility

Inspection Management and Study Director

Protocol : November 16, 2005 November 16, 2005

Amendment 01 : November 30, 2005 November 30, 2005

Amendment 02 : January 31, 2006 January 31, 2006

In-progress study : November 17, 2005 November 17, 2005

Addition of test substance : November 17, 2005 November 17, 2005

DO measurement : December 1, 2005 December 1, 2005

Measurement of residual test substance amount :

December 15, 2005 December 15, 2005

Final report . February 7, 2006 February 7, 2006

Quality Assurance Personnel

Yumiko Kashiwagi, B.Sc. Sealed date : February 7, 2006

Hiroko Tokimi, B.Sc . Sealed date: February 7, 2006

-~--

~~'s5
p. 128

1 STUDY TITLE

Ready Biodegradability Test of MTDID3663 (Study No . A050393)

2 PURPOSE

This study was conducted to evaluate the ready biodegradability of the test substance

for notification under the Chemical Substances Control Law of Japan .

3 TEST GUIDELINES

The test was conducted according to the method described as the "Ready Biodegradability,

Closed Bottle Test" in the OECD Guidelines for Testing of Chemicals No .301D (1992).

4 GLP COMPLIANCE

The test was conducted according to OECD Principles of Good Laboratory Practice

(as revised in 1997).

5 SPONSOR

SUMITOMO 3M, LTD.

8-8 Minami-hashimoto 3-chome, Sagamihara-shi Kanagawa, Japan

6 TESTI NG FACILITY (Head Office)

Mitsubishi Chemical Safety Institute Ltd. (M.S.I.)

1-30 Shiba 2-chome, Minato-ku, Tokyo, Japan

7 TESTING FACILITY (Laboratory)

Yokohama Laboratory

Mitsubishi Chemical Safety Institute Ltd. (M.S.I.)

1000 Kamoshida-cho, Aoba-ku, Yokohama, Japan

8 PERSONNEL

Study Director

Junko Koyasu, B.Sc

Environmental Science Group A

Experimental Scientist

Kayoko Morita Sealed date : February 7, 2006

Sumiko Kawashima, B .Sc. Sealed date: February 7, 2006

Hirokazu Kobayashi, M.Sc. Sealed date : February 7, 2006

S~~
p. 129

9 DATE

Initiation of the study : November 16, 2005

Start of the BOD measurement: November 17, 2005

End of the BOD measurement: December 15, 2005

Measurement of residu al test substance amount: December 15, 2005

Completion of the study : February 7, 2006

10 STORAGE AND RETENTION OF THE TEST SUBSTANCE AND RECORDS

The followings will be stored in the archives of M .S.I.

1) Protocol

2) Final report

3) Raw data

4) Test substanc e

5) Other requirements

~--

~

-6--

S 1Z.-7
p. 130

CONTENTS

Page

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . .

.•-- . . . . . . . . . . . . . . . . . . . . . . ..----•--. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 TEST SUBSTANCE . . . . . . . . . . . . . . . . . . . . . . . . . . .•-•------ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

2 CONFIRMATION OF STABILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3 TEST METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 0

3 .1 INOCULUM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

3 .2 EXPOSURE CONDITIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

3 .3 PROGRAM OF MEASUREMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

3 .4 MEASUREMENT OF DISSOLVED OXYGEN CONCENTRATION . . . . . . . . . . . . . . . . 11

3.5 MEASUREMENT OF RESIDUAL TEST SUBSTANCE AMOUNT . . . . . . . . . . . . . . . . . . 11

3.6 pH MEASUREMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

3 .7 EQUATIONS FOR CALCULATION OF DEGRADABILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 4 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .1 4

4.1 CIRCUMSTANCES WHICH MIGHT HAVE AFFECTED

THE RELIABILITY OF THE TEST RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

.2 VALIDITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 4

4.3 DEGRADABILITY BASED ON BOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

4.4 DEGRADABILITY BASED ON RESIDUAL TEST SUBSTANCE AMOUNT .14

4.5 pH MEASUREMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 4

5 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

TABLES AND FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15-28

-~--

S$~
p. 131

SUMMARY

STUDY TITLE

Ready Biodegradability Test of MTDID3663 (Study No. A050393)

METHOD

The test was conducted according to the method described as "Ready Biodegradability, Closed

Bottle Test" in the OECD Guidelines for Testing of Chemicals No .301D (1992) due to volatility

of the test substance .

Test bottles :

Bottle No.l : inoculum + mineral medium

Bottle No.2 : test substance + inoculum + mineral medium

Bottle No.3 : test substance + purified wate r

Bottle No.4 : reference substance + inoculum + mineral medium

Measurement

BOD based on dissolved oxygen concentration

Residual test substance amount

RESULTS

Measured values (day 28)

Bottle No. Theoretical

2-1 2-2 3-1 3-2 value

BOD, mgO2/mg 0.00 0.01 0.03 0.03 0.555

Test substance, mg 2.86 2.93 2.90 2.95 2.93

Degradability(day 28)

Bottle No.

2-1 2-2 Average

BOD,% 0 2 1

Test substance,% 2 0 1

CONCLUSION

From the results of the degradability based on the BOD and on the residual test substance

amount, it is concluded that the test substance is not readily biodegradable and is not transformed

under the conditions of the Closed Bottle Test.

-SS ~°'~
p. 132

3 TEST METHODS

The test was performed according to the method described in OECD Guidelines for Testing

of Chemicals as "No .301D Ready biodegradability, Closed Bottle Test" (1992) due to volatility

of the test substance .

The test substance was exposed to the inoculum in BOD bottles. The concentration of

dissolved oxygen (DO) in the test bottles were measured at days 0, 7, 14, 21 and 28, and the

biochemical oxygen demand (BOD) was calculated . The residual test substance amount in the

test bottles were measured at days 0 and 28 .

3.1 INOCULUM

1) Inoculum : Secondary effluent from domestic sewage treatment plant

2) Source : Naruse Clean Center, Machida city

3) Date of collection : November 17, 2005

3 .2 EXPOSURE CONDITIONS

1)Apparatus

Incubator : M-210, TAITEC (dark condition)

2)Conditions

Temperature : 20± 1 °C

Exposure period : 28 days

Test volume : 300 mL

Concentration : test substance (bottles No .2 and 3) : 9.77mg/L

Sodium benzoate* 1(bottle No .4) : 4.00 mg/L

Inoculum (bottles No. 1, 2 and 4) : 50 μL/L

* 1 Reference substance : Guaranteed Reagent, Lot No.407C2060

KANTO CHEMICAL Co., INC.

3)Test bottle contents :

Bottle No.1 : Inoculum + mineral medium*2

Inoculum (200μL) was added to 4L of mineral medium and the medium

was filled immediately into 12 BOD bottles with glass stoppers.

Bottle No.2 : Test substance + Inoculum + mineral medium * 2

Inoculum (300μL) was added to 6L of mineral medium and the medium

~-- was filled immediately into 18 BOD bottles*3 with glass stoppers . Then,

1 .90μL (2.93 mg*4) of the test substance was added to each bottle with

microsyringe.

Bottle No-3 : Test substance + purified water

Purified water*5 was filled into 18 BOD bottles*3 with glass stoppers .

Then, 1 .90μL (2.93 mg*4) of the test substance was added to each bottle

with microsyringe .

Bottle No.4 : Reference substance + Inoculum + mineral medium * 2

Inoculum (200~tL) was added to 4L of mineral medium and sodium

benzoate solution (4000 mg/L) was added at final concentration of 4 .00

mg/L to the medium. Then prepared solution was filled into 10 BOD

bottles with glass stoppers.

*2 Mineral medium described in OECD guidelines for testing of chemicals

301D was use.d. Mineral medium and purified water were saturated with

oxygen.

*3 Four of the 18 bottles were for spare.

*4 Calculated using 1 .543 as the specific gravity.

*5 Grade A4, Japanese Industrial Standards K0557.
p. 133

3 .3 PROGRAM OF MEASUREMENT

Concentration of DO, pH* and residual test substance amount were measured according to

the following program.

Bottle No. Measurement day 0 day 7 day 14 day 21 day 28

1 DO 2 2 2 2 2

test substance I - - - 1

2 DO 2 2 2 2 2

test substance 2 - - - 2

3 DO 2 2 2 2 2

test substance 2 - - - 2

4 DO 2 2 2 2 2

test substance - - - - -

*At days 0 and 28, pH was measured after the measurement of DO .

3 .4 MEASUREMENT OF DISSOLVED OXYGEN CONCENTRATIO N

Concentration of dissolved oxygen (DO) in the BOD bottles were measured at days 0, 7, 14,

21 and 28, and BOD was calculated.

1) Apparatus : DO meter, 5100, YSI/Nanoteeh Inc .

3 .5 MEASUREMENT OF RESIDUAL TEST SUBSTANCE AMOUNT

Amount of the residual test substance was measured with gas chromatograph (GC) as

follows:

1)Apparatus

(-- GC : 6890A , Hewlett-Packard

2)Conditions

Column : RESTEK Rtx-200, 105mX0 .25 mm i.d.Xl .O V n(film thickness)

Temperature : [Column Oven] 60°C (2min)-10°C /min --~ 200°C (5min)

[Injector] 250°C, [Detector] 250°C

Carrier gas : He

Detector flow : H2 40 mL/min, Air 450 mL/min

Makeup gas : He 45 mL/min

Column flow : 1 .0 mL/min (constant flow mode)

Injector : Split (Split 5 :1 )

Detector : FID

Injection volume : I μL

-~-t75 °12
p. 134

3)Calibration curv e

Standard solutions of the test substance with 0, 293, 586, and 1172 mg/L in chloroform

were prepared and analyzed with GC .

The total peak areas (pA • sec) of the test substance were plotted against the concentrations

(mg/L). The correlation coefficient was calculated to be 1 .00. Concentration of the test

substance for each bottle was calculated from ratio of peak area for the sample to that for the

standard solution with 1172 mg/L in chloroform .

[Figure 2 (p20) and Figure 3 (p21-22)]

4)Measurement of test substance amount in test bottle s

Each BOD bottle was cooled in an ice bath over 20 min and left in 25 °C for 20 min . After

removal of sealing water around the glass stopper, 5 mL of chloroform was poured onto the

collar of the BOD bottle .

The glass stopper was slightly slid up and chloroform was poured into the BOD bottle,

then 3 g of sodium chloride was added into the bottle .

The content in the BOD bottle whose opening was covered with wrap film was stirred for

5 min, then chloroform layer was analyzed with GC for determination of the test substance

amount.

The detection limit of the test substance was calculated to be 0 .01 mg based on the

minimum detectable peak area of I pA - sec for the peak in the GC chromatogram.

The recovery of the test substance in bottles No.2-1 and 2-2 at day 0 were both 104 % . The

recovery of the test substance in bottles No .3-1 and 3-2 at day 0 were both 105 %.

The amount of the residual test substance measured for bottles No .2 and 3 at day 28 were

corrected with the average recovery value of 104 and 105%, respectively .

[Table 3 (pl7) and Figure 4 (p23-24)]

3.6 pH MEASUREMENT

At days 0 and 28, pH was measured after measurement of DO .

1) Apparatus : pH meter, 720A,ORION Research

3 .7 EQUATIONS FOR CALCULATION OF DEGRADABILITY

Equations for calculation of degradability based on BOD and residual test substance amount

are as follows :

l)Degradability based on BOD

Degradability (%) = BOD / ThOD x 10 0

where BOD : Biochemical oxygen consumption (mg) in bottles No .2, 3 and 4

ThOD : Theoretical oxygen demand (mg) of the test substance

(or reference substance)

-3~-

s°~3
p. 135

Calculation of BOD

Bottles No.2 and 4

BOD (mgO2/g) = {(DOO - DOx) - (DObO - DObx)} / C

Bottle No.3

BOD (mgO2/g) = (DOO - DOx) / C

Where C : Concentration (mg/L) of the test substance (or reference substance)

DOO : Average of DO concentration (mgO2/L) at day 0

DOx DO concentration (mgOz/L) at day x

DObO : Average of DO concentration (mgO2/L) in bottle No . I at day 0

DObx : Average of DO concentration (mgO2/L) in bottle No .l at day x

ThOD

Sodium benzoate : C7H5O2Na + 15/202 - 7C02 + 5/2H20 + 1/2Na2O

ThOD : 1 .67 mgO2/mg

Test substance : C8H6OFi2+ 602 + 3H2O--+ 8C02 +12HF

ThOD : 0.555 mgO2/mg

2)Degradability based on residual test substance amount

(calculated on day 28 )

Degradability (%) _(1 - C281C) X 10 0

where C : Maximum theoretical value (mg)

C28 Amount (mg) of the test substance at day 28

--t-3-

s°\y
p. 136

4 RESULTS

4.1 CIRCUMSTANCES WHICH MIGHT HAVE AFFECTED THE RELIABILITY OF

THE TEST RESULTS

There was no specific circumstances which might have affected the reliability of the test

results.

4.2 VALIDITY

1) The degradability of reference substance (sodium benzoate) based on oxygen consumption

were 83 and 77% at day 14, and therefore satisfied the validity criterion (?60 % at day

14) given in the OECD Guidelines.

2) Oxygen consumption in the blank did not exceed 1 .5 mgOz/L after 28 days.

3) Oxygen concentration in all the bottles were not lower than 0 .5 mgO2/L.

Therefore, the biodegradability test was judged valid .

{ [Table I (p 15) and Table 2 (p 16)]

4.3 DEGRADABILITY BASED ON BO D

Oxygen consumption*' in bottles No.2-1 and 2-2 were 0 .00 and 0.01 mgOz/mg at day 28 .

(*' :Maximum theoretical value = 0.555 mgOz/mg)

Degradability based on BOD were 0 and 2 % in bottles No . 2-1 and 2-2.

[Table 1(p 15) and Table 2(p 16)]

4.4 DEGRADABILITY BASED ON RESIDUAL TEST SUBSTANCE AMOUNT

The residual test substance*2 was detected at amount of 2 .86 and 2.93 mg in bottles No .2-1

and 2-2, and 2.90 and 2 .95 mg in bottles No.3-1 and 3-2 .

(*2 : Maximum theoretical value = 2 .93 mg)

Degradability based on residual test substance amount were calculated to be 2 and 0 % in

bottles No.2-1 and 2-2. [Table 1(p15), Table 4(p18) and Figure 5 (p26-28)]

4.5 pH MEASUREMENT

At day 28, pH of the test solutions were 7 .0 in bottles No .2-I and 2-2, and 5.1 and 5.0 in

bottles No.3-1 and 3-2. [Table 1(p15)]

5 CONCLUSIONS

From the results of the degradability based on the BOD and on the residual test substance

amount, it is concluded that the test substance is not readily biodegradable and is not transformed

under the conditions of the Closed Bottle Test.

--~~-

s°~
p. 137

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Figure 1 Infrared absorption spectra of the test substance

'll

SANITIZED

a~. ~©o
p. 142

Figure 2 Calibration Curve of the Test Substanc e

Curve Fitting [Least Square Method]

Input Data

--- - -------------------------------------------------------------

Concentration Peak Area

No. X (mg/L) Y (pA • sec)

•------------------------------------------------------------------

1 - 0 0.000

2 293 267.729

3 586 524.443

4 ______1172____ 1067.980

------------------------------

Y = -1 .6158E+00 + 9.1010E-01 x X

r = 0.99994

Calibration Curve

1200.000

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p. 143

C ~.~

SANITIZED

~°°2--(,OC5
p. 144

Translation Statement

Sponsor: Sumitomo 3M Limited

Title : Chromosomal Aberration Study of MTDID3663 in Cultured

Mammalian Cells

Study No. : B051348

This study was conducted in the Kashima Laboratory of Mitsubishi Chemical Safety

Institute Ltd. The original report was written in Japanese . I hereby declare that this report

faithfully reflects the original report as accurately as possible .

Study director:

Date: ZOd

Kazushige Hori

Toxicology Division I); Kashima Laboratory

Mitsubishi Chemical Safety Institute Ltd .

~--

~~
p. 145

B051348

FINAL REPORT

Chromosomal Aberration Study of MTDID3663 in Cultured Mammalian Cells

(Study No . : B051348)

July 28, 2006

Mitsubishi Chemical Safety Institute Ltd.

~-~--

~\~
p. 146

B051348

1 . GLP Compliance Statement

Sponsor: Sumitomo 3M Limited

Title: Chromosomal Aberration Study of MTDID3663 in Cultured

Mammalian Cells

Study No. : B051348

This study was conducted in compliance with the following Good Laboratory Practice

Standards.

~ Standards Concerning Testing Facilities Implementing Studies Related to New Chemical

Compounds

(November 21, 2003, Pharmaceutical and Food affairs No. 1121003, Pharmaceutical and

Food Safety Bureau, Ministry of Health, Labor, and Welfare, November 17, 2003,

Manufacturing Industries Bureau, No. 3, Ministry of Economy, Trade, and Industry, No .

031121004, Environmental Policy Bureau, Ministry of the Environment )

Study director:

Signed & sealed Date: July 28, 2006

Kazushige Hori

Toxicology Division I1; Kashima Laboratory

Mitsubishi Chemical Safety Institute Ltd .

-2-

Co~~
p. 147

B051348

2. Table of Contents

1 . GLP Compliance Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. Table of Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 . Study Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3 .1 Title . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3 .2 Study number. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3 .3 Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3 .4 Guideline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3 .5 GLP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3 .6 Sponsor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3 .7 Organization under contract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3 .8 Testing facility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

( 3

. . . . . . . . . . . . 6

.9 Study director. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3 .10 Study contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3.11 Study schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3 .12 Retention. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3 .13 Retention materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4. Study Director Signature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 5 . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 6. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

6.1 Test substance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 6.1.1 Name . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6.1 .2 IUPAC name . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6 .1 .3 Structural formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6 .1 .4 Molecular weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6.1.5 Lot No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6.1 . 6 Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6 .1 .7 Impurity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6 .1 .8 Melting point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6.1 .9 Boiling point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6.1 .10 Vapor pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6 .1 .11 Partition coefficient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6.1 .12 Appearance at normal temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6.1 .13 Stability . . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 6.1 .14 Supplier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 6 .1 .15 Storage condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 6.1 .16 Storage area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

6.2 Control substances. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 6.2.1 Negative control substance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 6.2 .2 Positive control substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 2

6.3 Indicator cell line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 .1 Cell line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 6.3 .2 Supplier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 6 .3 .3 Date of receipt. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 6.3.4 Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 6.3.5 Specificity of cell line . . . . . . . . . . . . . . . . . . . . . . . . . . .

. • 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

--3-

~~3
p.148

B051348

6 .3 .6 Passage number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

6.3 .7 Culture condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

6.3.8 Reason for cell line selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

6.4 Culture medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

6 .4.1 Preparation of MEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

6.4 .2 Preparation of MEM culture medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 3

6.5 S9 mix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

6.5 .1 S9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

6.5 .2 Preparation of S9 mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

6.6 Preparation of the test substance solutions and positive control substance

solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

6.6.1 Preparation of the test substance solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 6 .6.2 Preparation of the positive control substance solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

6.7 Cell growth inhibition test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

6.7 .1 Treatment condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

6.7.2 Test substance concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

6 .7.3 Reason for concentration selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

6 .7.4 Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

6 .7.5 Measurement of cell growth index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

6.7 .6 Calculation of 50% cell growth inhibition concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6

6 .8 Chromosomal aberration test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

6 .8 .1 Treatment condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

6 .8 .2 Test substance concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 6.8-:3 - Positive control substance concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

6.8.4 Reason for positive control substance concentration selection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

6 .8.5 Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

6 .8 .6 Preparation of specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

6 .8 .7 Measurement of cell growth index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

6. 8 .8 Observation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

6.8.9 Judgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 9

6.9 Expression of the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

6 . 9 .1 Table. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

6.9.2 Figure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20

7. Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

7.1 Cell growth inhibition test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

7.2 Chromosomal Aberration test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1

8. Discussion and Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

9. Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 10. Study Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

10.1 Unforeseeable circumstances that may have affected the quality and integrity of

the data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 10 .2 Deviation from the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Table 1 Results of the cell growth inhibition test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Table 2 Results of the chromosomal aberration test (short-term treatment assay)

. . . 24

Table 3 Results of the chromosomal aberration test (continuous treatment assay)

. . 24

Figure 1 Cytotoxicity in the treatment with MTDID3 66 3

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[short-term treatment assay (-S9 mix)] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Figure 2 Cytotoxicity in the treatment with MTDID366 3

[short-term treatment assay (+S9 mix)] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Figure 3 Cytotoxicity in the treatment with MTDID3663

[continuous treatment assay] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Figure 4 Incidence of cells with structural aberrations

in the treatment with MTDID3663 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Figure 5 Incidence of cells with numerical aberration s

in the treatment with MTDID3663 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Quality Assurance Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

(Final page : 29)

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3. Study Outline

3.1 Title

Chromosomal Aberration Study of MTDID3663 in Cultured Mammalian Cells

3.2 Study number

B051348

3.3 Purpose

To assess the potential of MTDID3663 to induce chromosomal aberration in a

chromosomal aberration test using cultured mammalian cells .

3.4 Guideline

Testing Method Concerning Designated Chemical Substance s

(November 21, 2003, Pharmaceutical and Food affairs No. 1121002, Pharmaceutical and

Food Safety Bureau, Ministry of Health, Labor, and Welfare, November 13, 2003,

Manufacturing Industries Bureau, No . 2, Ministry of Economy, Trade, and Industry, No .

031121002, Environmental Policy Bureau, Ministry of the Environment)

3.5 GLP

Standards Concerning Testing Facilities Implementing Studies Related to New Chemical

Compounds

(November 21, 2003, Pharmaceutical and Food affairs No. 1121003, Pharmaceutical and

Food Safety Bureau, Ministry of Health, Labor, and Welfare, November 17, 2003,

Manufacturing Industries Bureau, No. 3, Ministry of Economy, Trade, and Industry, No .

031121004, Environmental Policy Bureau, Ministry of the Environment)

3.6 Sponsor

Sumitomo 3M Limite d

8-8, Minamihashimoto 3-chome, Sagamihara-shi, Kanagawa, Japan

r

r--

3.7 Organization under contract

Mitsubishi Chemical Safety Institute Ltd.

1-30, Shiba 2-chome, Minato-ku, Tokyo, Japan

3.8 Testing facility

Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd .

14 Sunayama, Kamisu-shi, Ibaraki, Japan

3.9 Study director

Kazushige Hori

Toxicology Division II, Kashima Laboratory

Mitsubishi Chemical Safety Institute Ltd.

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3.10 Study contributors

Seeding of cells: Kazushige Hori, Akihiko Kajiwara

Preparation of the test substance solution :

Kazushige Hori, Akihiko Kajiwara, Katsuaki Yasunaga,

Toru Fujimot o

Preparation of the positive control substance solution :

Emi Takashima, Katsuaki Yasunaga, Yumi Murakami

Preparation of S9 mix : Akihiko Kajiwara, Katsuaki Yasunaga, Toru Fujimoto

Treatment of cells: Kazushige Hori, Kazunori Narumi, Akihiko Kajiwara ,

Katsuaki Yasunaga, Toru Fujimoto, Yumi Murakami

Measurement of cell growth index :

Kazushige Hori, Akihiko Kajiwara, Kastuaki Yasunaga,

Toru Fujimoto, Yumi Murakami

Preparation of specimens: Kazushige Hori, Kazunori Narumi, Akihiko Kajiwara,

Katsuaki Yasunaga, Toru Fujimoto, Yumi Murakami

Preliminary observation: Katsuaki Yasunaga, Toru Fujimoto, Yumi Murakami

Observation of specimens: Kazushige Hori

3.11 Study schedule

Initiation of the study : May 10, 2006

Cell growth inhibition test: May 12, 2006 - May 16, 2006

Chromosomal aberration test May 19, 2006 - June 20, 2006

Completion of the study : July 28, 2006

3.12 Retention

The materials listed in the following section will be retained in the archives of the Kashima

Laboratory. The specimens will be retained for whichever is shorter of the two periods [(1)

or (2)] described below.

(1) Ten years after the notice coming under the law concerning examination and

regulation of manufacture of chemical substances (No .117, 1973), Article 4-1 or 2,

4(2)-2, 3, or 8, 5(4)-2, 24-2, or 25(3)- 2

(2) The time period that the materials remain stable

All the others will be retained for the period described in (1) . Further retention will be

determined in consultation with the sponsor .

3.13 Retention materials

(1) Protocol

(2) Protocol amendment

(3) Test substance and control substance records

(4) Cell record s

(5) Study result records

(6) Correspondence documents

(7) Specimens

(8) Final report

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B05134 8

4. Study Director Signature

Sponsor: Sumitomo 3M Limited

Title: Chromosomal Aberration Study of MTDID3663 in Cultured Mammalian

Cells

Study No. : B051348

~

Study Director:

Signed & sealed Date: July 28, 2006

Kazushige Hori

Toxicology Division I); Kashima Laboratory

Mitsubishi Chemical Safety Institute Ltd.

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B051348

5. Summary

An in vitro chromosomal aberration study of MTDID3663 was conducted using CHL/IU

cells derived from the lung of female Chinese hamsters .

Based on the result of a preliminary test, a cell growth inhibition test was conducted at

313, 625, 1250, 2500, and 5000 μg/mL in the short-term treatment assay with S9 mix

(+S9 mix assay) and without S9 mix (-S9 mix assay), and in the continuous treatment

assay for 24 hours (24-hour assay).

The results from these assays indicated that the concentration of 50% cell growth

inhibition was detected 325 μg/mL in the -S9 mix assay, but not in the +S9 mix assay and

the 24-hour assay.

Based on the result of the cell growth inhibition test, the chromosomal aberration test

was conducted at 78 .1, 156, 313, 625, 1250, 2500, and 5000 μg/niL in the -S9 mix assay,

{ at 1250, 2500, and 5000 μg/mL in the +S9 mix assay and the 24-hour assay .

The incidence of cells with chromosome aberration in structure and polyploidy induction

was less than 5% in each treatment condition .

In conclusion, MTDID3663 was not considered to have the ability of chromosome

aberration under the present study condition.

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6. Materials and Methods

6.1 Test substance

i

SANITIZED '

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p. 155

B051348

6.1 .13 Stability

Stable

6.1 .14 Supplier

Sumitomo 3M Limited

6.1.15 Storage condition

Stored in a refrigerator, shielded from light and airtight stopper.

Test substance used for the test was kept under the following conditions.

Test substance storage area 59 :

September 28, 2005 - October 3, 2005 actual temperature : 4.00 to 6.5°C

Test substance storage area 54 :

October 3, 2005 - December 28, 2005 actual temperature : 2.5° to 7.2°C

Test substance storage area 55 :

December 28, 2005 - May 22, 2006 actual temperature : 3 .5° to 6.2°C

6.1.16 Storage area

The test substance storage area 5 5

(The test substance was received on September 28, 2005, and was stored in test substance

storage area 59 from October 3, 2005, in test substance storage area 54 from October 3,

2005 to December 28, 2005 . After December 28, 2005, the test substance was stored in

test substance storage area 54.)

6.2 Control substances

6.2.1 Negative control substance

6.2.1.1 Name

Dimethyl sulfoxide (DMSO)

6.2.1.2 Manufacturer

Kanto Chemical Co., Inc.

6.2.1.3 Lot No.

510F1666

6.2.1.4 Purity

100.0%

6.2.1.5 Specification

For spectrometry

6.2.1.6 Reason for selection of the negative control substanc e

In a preliminary test for solubility, the test substance did not dissolve in saline at 50

mg/mL. However, it dissolved in DMSO at 500 mg/mL. Increasing in temperature,

discoloration or foaming was not observed in the DMSO solution . Therefore, DMSO was

selected as the vehicle for the test substance and was used as the negative control

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B051348

substance in this study.

6.2.2 Positive control substances

6.2.2.1 Name

Mitomycin C (MMC) [for the test without S9 mix]

Benzo [a] pyrene (BP) [for the test with S9 mix]

6.2.2.2 Supplier

MMC : Kyowa Hakko Kogyo Co ., Ltd.

BP : Tokyo Kasei Kogyo Co., Ltd.

6.2.2.3 Lot No.

MMC: 454ADK

BP : GG0 1

6.2.2.4 Contents

MMC : 103%

BP : 95 .6%

6.2.2.5 Reason for selection of the positive control substance s

These substances are widely used in chromosomal aberration tests using cultured

mammalian cells and are recommended in the applied guideline .

6.3 Indicator cell line

6.3.1 Cell line

CHL/IU (derived from lung of female Chinese hamster)

6.3.2 Supplier

Dainippon Pharmaceutical Co ., Ltd.

V7 6.3.3 Date of receipt

May 17, 2005

6.3.4 Storage

The cells were suspended in the culture medium with one-tenth volume of DMSO, and

then, the cell suspension was subdivided into tubes in 1-mL aliquots, frozen on May 24,

2005, and stored in liquid nitrogen .

The cells were thawed and cultured for use in the experiments before the study, and were

used in common with other studies.

6.3.5 Specificity of cell line

The specificity described below was confirmed for the frozen cells in Section 6 .3 .4 by

June 9, 2005 .

Modal chromosome number : 25

Doubling time : 14 .9 hours

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B051348

Mycoplasma infection : Negative

6.3.6 Passage number

At receipt: 1 4

At freezing for stock : 1 7

At treatment : 20-22 (11 to 18 days after thawing, thawing day equaled 0 day)

6.3.7 Culture condition

Culture dish : Plastic plate (diameter: 6 cm or 10 cm, Becton Dickinson and Company)

Temperature : 37°C

CO2 concentration : 5%

Humidity: Under moist atmosphere

Incubator: Programmable incubator (Model 6301C or 7300, Jouan S .A.)

~

6.3.8 Reason for cell line selectio n

This cell line is widely used for chromosomal aberration studies in cultured mammalian

cells, because it has advantages such as a small number of chromosomes (25) and

relatively large chromosome size . This cell line is recommended in the applied guideline .

6.4 Culture medium

MEM and MEM culture medium were prepared before or during the study, and were

used in common with other studies .

6.4.1 Preparation of MEM

(1) Eight-point-three grams of Eagle's minimum essential medium (Eagle's MEM

"Nissui"(1); Nissui Pharmaceutical Co., Ltd., Lot No. 540512) were dissolved in 880

mL of purified water.

(2) This solution was autoclaved at 121 °C for 20 min .

(3) This solution was supplemented with the following sterilized solutions, 8 .8 mL of

2 .92 w/v% L-glutamine solution and 11 .2 mL of 10 w/v% sodium bicarbonate

solution.

6.4.2 Preparation of MEM culture medium

One hundred milliliters of heat-inactivated (56°C, 30 min .) Bovine serum (Invitrogen

Corp., Lot No. 444175) was added to 900 niL of MEM .

6.5 S9 mix

6.5.1 S9

6.5.1.1 Supplier

Kikkoman Corporation

6.5.1.2 Lot No.

RAA-542

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B051348

6.5.1.3 Date of preparation

April 21, 2006

6.5 .1 .4 Date of receipt

May 12, 2006

6.5.1 .5 S9 preparation

The S9 was prepared from livers of 7-week-old male Sprague-Dawley rats (body weight :

210-245 g) treated with phenobarbital (PB, 4 times at 24-hour intervals i .p. at doses of 30,

60, 60, and 60 mg/kg, respectively) and 5, 6-benzoflavone (once i.p. at a single dose of

80 mg/kg on the same day of the third PB injection).

6.5.1.6 Protein content

25.48 mg/mL (final concentration at the cell treatment : 1 .27 mg/mL)

6.5.1.7 Storage condition

-80°C or below (actual temperature : -84° to -81°C)

6.5.1.8 Expiration date

October 20, 2006 (6 months after preparation at the manufacturer)

6.5.2 Preparation of S9 mix

The S9 mix was prepared just before use and kept in an ice bath until use. The

composition per milliliter of the S9 mix preparation was as follows

S9 0

.

.3mL

D-glucose 6-phosphate 5 Eunol

R-NADP+ 4 μmol

HEPES (pH 7 .2) 4 μmol

MgC12 5 μmol

~ KCl 33 μmol

Purified water remainder

6.6 Preparation of the test substance solutions and positive control substance

solutions

6.6.1 Preparation of the test substance solutions

(1) Test substance was weighed at 2500 mg and dissolved in DMSO by vortex mixing .

(2) The volume was made up to 5 mL with DMSO to give a 100-fold concentration of

the highest concentration in the treatment (500 mg/mL) .

(3) A portion of the highest concentration solution was serially diluted with DMSO to

give a series of 100-fold concentrations of the final application concentrations

selected (cell growth inhibition test : 250, 125, 62 .5, and 31 .3 mg/mL, chromosomal

aberration test: 250, 125, 62.5, 31 .3, 15 .6, and 7.81 mg/mL) .

(4) The above operation was performed under yellow light at room temperature . The

test substance solutions were prepared on the day of use . The storage period of the

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B051348

test substance preparation from preparation to use was as follows.

Cell growth inhibition test: 35 to 45 minutes

Chromosomal aberration test: 20 to 55 minutes

6.6.2 Preparation of the positive control substance solutions

6.6.2.1 Preparation of MMC solution

(1) MMC in a 2-mg vial was dissolved in 5 mL of water (water for injection, Otsuka

Pharmaceutical Factory Inc ., Lot No . K5E85) at use.

(2) This solution was serially diluted with saline (Otsuka Pharmaceutical Factory Inc .,

Lot No. K4L84) to prepare 10-fold concentrations of the prescribed concentrations

for each treatment condition (short-term treatment assay : 1 μg/ML, continuous

treatment assay: 0 .5 μg/mL) .

6.6.2.2 Preparation of BP solution

(1) BP was weighed at 20 mg and dissolved with 5 mL of DMSO (Kanto Chemical Co .,

Inc., Lot No. 510F1666) to prepare a 200-fold concentration of the prescribed

application concentration (4 mg/mL) .

(2) The solution was subdivided into tubes in 0 .2-mL aliquots, and stored in a freezer

(Test substance storage area 66 ; actual temperature : -28 .2° to -21 .6°C).

(3) The stocks frozen on January 6, 2006 were thawed and used in each test .

6.7 Cell growth inhibition test

6.7.1 Treatment condition

-S9 mix assay: Short-term treatment assay (6-hour treatment and 18-hour recovery)

without S9 mix

+S9 mix assay : Short-term treatment assay (6-hour treatment and 18-hour recovery) with

S9 mix

24-hour assay : Continuous treatment assay for 24 hours without S9 mix

6.7.2 Test substance concentratio n

-S9 mix assay: 313, 625, 1250, 2500, 5000 μg/mL

+S9 mix assay : 313, 625, 1250, 2500, 5000 μg/mL

24-hour assay: 313, 625, 1250, 2500, 5000 μg/mL

6.7.3 Reason for concentration selectio n

Before the cell growth inhibition test, a preliminary test was conducted at 50, 500, and

5000 μg/mL for each treatment condition . All assays were performed with one plate per

concentration. Cell treatment was conducted as described in Section 6 .7 .4. The plates

were observed under an inversed phase-contrast microscope to determine cell densities

after the treatment. The cell density was presented as the relative percentage (%)

compared to the negative control plate .

As a result, cytotoxicity was not finding in each test substance treatment group in each

treatment condition (density : 100%) . Precipitation of the test substance was observed in

the treatment medium at more than 500 μg/mL in any test substance treatment group .

Based on these results, the highest concentration in the cell growth inhibition test was set at

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B051348

5000 μg/mL, which is required in the applied guideline, and the concentrations described in

Section 6.7.2 were selected .

6.7.4 Treatment

( 1) Cells were seeded in 5 mL of culture medium (4x103 cells/mL) using plastic plates

(diameter: 6 cm) and incubated for 3 days .

(2) After the MEM culture medium was removed from the plate, the test substance

solution, S9 mix, and MEM culture medium were added to the plate as shown in the

table below. As the negative control group, DMSO was used in place of the test

substance solution. Two plates per concentration were used .

Assay Testsubstance solution S9 mix MEM culture

y or DMSO medium

-S9 mix assay 0.03 mL - 3.0 mL

+S9 mix assay 0.03 mL 0.5 mL 2.5 mL

24-hour assay 0.05 mL - 5.0 mL

(3) Cells were treated for 6 hours in the sho rt-term treatment assays and for 24 hours in

the continuous treatment assay.

(4) In the short-term treatment assays, the surface of the cells was washed three times

with MEM after 6-hour treatment and incubated in 5 mL of fresh MEM culture

medium for additional 18 hours .

(5) At the beginning and end of the treatment, all plates were examined macroscopically

for precipitation or recrystallization of the test substance.

6.7.5 Measurement of cell growth index

(1) The cells were washed with Ca2+-, Mg2+-free Dulbecco's phosphate buffer saline

(PBS(-), Nissui Pharmaceutical Co., Ltd.).

(2) The cells were fixed with 10 v/v% formalin solution for 5 min .

(3) After removed 10 v/v% formalin solution, the cells were stained with 3 v/v%

Giemsa's solution for 10 min .

(4) Washed with water and then dried .

(5) The cell density was measured with a cell density counter (Monocellator, Olympus

Co., Ltd.).

(6) The cell growth index was calculated as a relative cell number compared to the cell

number of the negative control plate (average) as 100%.

6.7.6 Calculation of 50% cell growth inhibition concentratio n

For the treatment condition (test substance inhibit cell growth more than 50%), the

concentration giving 50% inhibition of cell growth (IC5o) was calculated. IC50 was

calculated from the formula of the line through the two data points showing cell growth

indices higher and lower than, but both near 50%.

For the treatment conditions (test substance did not inhibit cell growth more than 50%),

cell growth was not inhibited more than 50%. Therefore, the 50% cell growth inhibition

concentration (IC5o) was not calculated .

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B051348

6.8 Chromosomal aberration test

6.8.1 Treatment condition

-S9 mix assay, +S9 mix assay and 24-hour assay

6.8.2 Test substance concentration

Based on the result of the cell growth inhibition test, the following concentrations were

set for the chromosomal aberration test by the highest concentrations .

-S9 mix assay: 78 .1, 156, 313, 625, 1250, 250, 5000 μg/mL

+S9 mix assay : 1250, 2500, 5000 μg/mL

24-hour assay: 1250, 2500, 5000 μg/mL

6.8.3 Positive control substance concentration

-S9 mix assay: MMC at 0 .1 μg/mL

+S9 mix assay : BP at 20 μg/mL

24-hour assay: MMC at 0.05 μg/mL

6.8.4 Reason for positive control substance concentration selectio n

These positive control substances are known to induce chromosomal aberrations at the

above concentrations .

6.8.5 Treatment

Cell treatment was conducted as described in Section 6 .7.4.

For the positive control gr , cells were treated with the mixture.

MMC BP MEM culture

Assay solution solution S9 mix medium

-S9 mix assay 0.3 mL - - 2.7 mL

+S9mixassay - 0.015mL 0.5mL 2.5mL

24-hour assay 0.5 mL - - 4.5 mL

The test was performed using four plates for each concentration. Two plates were used

for specimen preparation and the others were used for the cell growth rate measurement .

As for the positive control group, the test was performed using two plates, because the

cell growth rate measurement was not conducted .

6.8.6 Preparation of specimens

(1) At two hours before the end of culture, colcemid was added to the medium in the

plates at a final concentration of 0 .1 μg/mL to accumulate the metaphase cells.

(2) At the end of culture, the cells were washed with PBS(-).

(3) The cells were treated with 0 .25 w/v% trypsin and dissociated from the plate .

(4) The cell suspension was collected in a centrifuge tube and centrifuged (1000 rpm for

5 min; this condition was applied to the following centrifugation step) to collect the

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B051348

cells.

(5) After the resulting supernatant was removed, the cells were subjected to hypotonic

treatment with 4 mL of a 0 .075 mol/L potassium chloride solution at 37°C for

15 min.

(6) The cells and 0 .5 mL of the fixative mixture (ice-cold methanol-acetic acid mixture

[3 :1, v/v]) were mixed together. The mixture was centrifuged and the supernatant

was removed.

(7) The cells and 4 mL of the fixative mixture were mixed together . The mixture was

centrifuged and the supernatant was removed.

(8) The procedure in (7) was conducted again.

(9) The cells were suspended in a small amount of the fixative mixture .

(10) A drop of the cell suspension was placed at two sites on a slide glass put on wet

cloth and was dried . Two slides were prepared from each plate .

(11) The cells were stained for 20 min with 3 v/v% Giemsa's solution . The slides were

washed with water and dried .

(12) The slides were mounted with a cover glass and mounting medium .

6.8.7 Measurement of cell growth index

(1) Measurement of cell growth index in the chromosomal aberration test was

conducted in the same procedure as Section 6.7.5 using the plates for the cell growth

rate measurement .

(2) The cell growth index was calculated as a relative cell number compared to the cell

number of the negative control plate (average) as 100%.

(3) Measurement of cell growth was not conducted for the positive controls.

6.8.8 Observation

6.8.8.1 Preliminary observatio n

The specimens were preliminarily examined by microscopic observation to confirm

whether the test was performed appropriately . The items described below were

confirmed.

(1) In a total of two slides prepared from each treatment plate, 50 or more metaphase cells

per plate were observed.

(2) The chromosomal aberrations were properly induced at the expected levels in the

negative and positive control groups .

6.8.8.2 Observation

6.8.8.2.1 Method for coding of the specimens

The specimens were rearranged randomly. The identification part of the specimens was

covered with the labels and observed .

6.8.8.2.2 Criteria for selection of observable metaphase cells

(1) Chromosomes were well spread .

(2) Number of centromere was 25±2 or 35 or more (except for fragmentation) .

6.8.8.2.3 Number of cells analyzed

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B05134 8

100 cells per plate (200 cells per concentration)

6.8.8.2.4 Classification of structural aberration [1]

(1) Chromatid break

(2) Chromatid exchange

(3) Chromosome break

(4) Chromosome exchange (dicentric, ring etc .)

(5) Fragmentation

6.8.8.2.5 Gap

A "gap" was defined as an achromatic region in a single chromatid that was narrower

than the width of the chromatid .

The numbers of gaps were listed in a separate column from the other aberrations.

~ Gaps were not taken as structural aberrations .

6.8.8.2.6 Classification of numerical aberration

(1) Polyploid cells with 35 or more centromere s

(2) Endoreduplicated cells

6.8.9 Judgment

6.8.9.1 Chromosomal aberrant cel l

Structural aberrant cell: A cell with at least one structural chromosome aberration

Numerical aberrant cell: A cell with numerical chromosome aberratio n

6.8.9.2 Acceptance criteria for the data

The data of the plate in which 100 metaphase cells per plate could be observed was

accepted.

6.8.9.3 Acceptance criteria for the test

The test was accepted when the following criteria were satisfied for each treatment

~ condition.

(1) The acceptance criteria for the data were satisfied in the negative and positive

controls and at three or more concentrations of the test substance treatment group .

(2) Incidence of structural and numerical aberrant cells in the negative control group was

less than 5% .

(3) Incidence of structural aberrant cells in the positive control group was more than

10% .

6.8.9.4 Judgment [2]

Negative: Both structural and numerical aberrant cells were observed at lower than 5% in

the test substance treatment group.

Inconclusive: The structural or numerical aberrant cells were observed at 5% or higher,

but lower than 10% in the test substance treatment group.

Positive: The structural or numerical aberrant cells were observed at 10% or higher in the
p. 164

B051348

test substance treatment group and increased dose-dependently .

6.8.9.5 Statistical analysis

No statistical analysis was performed .

6.9 Expression of the results

6.9.1 Table

(1) Cell growth index

(2) Numbers of structural and numerical aberrant cells per plat e

(3) Sums and incidences of structural and numerical aberrant cells per concentration

6.9.2 Figure

(1) Cell growth index

(2) Incidence of chromosomal aberrant cells

-zf

- l0 3c~
p. 165

B051348

7. Results

7.1 Cell growth inhibition test (Table 1, Figure 1-3)

Precipitation of the test substance was observed in the treatment medium at the beginning and

end of the treatment, in each test substance treatment group in each treatment condition . IC50

value was 325 μg/mL in the -S9 mix assay . In the +S9 mix assay and the 24-hour assay,

cell growth was not inhibited more than 50% in any test substance treatment group .

7.2 Chromosomal Aberration test (Table 2, 3, Figure 1-5)

Precipitation of the test substance was observed in the treatment medium at the beginning of

the treatment, at 156 μg/mL in the -S9 mix assay . Also, precipitation of the test substance was

observed in the treatment medium at the beginning and the end of the treatment, at more than

313 μg/mL in the -S9 mix assay and at each concentration in the +S9 mix assay and the

~ 24-hour assay.

In the preliminary microscopic observation, 50 metaphase cells per plate could be obtained in

the specimens in each plate . Therefore, the specimens of all plates were used in the

microscopic observation.

The incidence of cells with chromosomal structural aberrations or numerical aberrations was

less than 5% in each test substance treatment group in each treatment condition .

In the negative control group in each treatment condition, the incidence of structural

aberrant cells and numerical aberrant cells was less than 5% . In the positive control group

in each treatment condition, the incidence of structural aberrant cells was more than 10%.

8. Discussion and Conclusion

A chromosomal aberration study using cultured mammalian cells was conducted to

assess the clastogenicity of MTDID3663 .

The incidence of cells with chromosome aberration in structure and polyploidy induction

was less than 5% in each treatment condition .

The incidences of cells with chromosomal aberration show the expected values in the

negative control groups and the positive control groups. This indicated that the present

study was conducted properly.

In conclusion, MTDID3663 was not considered to have the ability of chromosome

aberration under the present study condition.

-2+

c .e31
p. 166

B051348

9. Referenc e

[1] Working Group on Genotoxicity Studies, the Third Subcommittee of Basic Research

Taskforce, the Pharmaceutical Evaluation Committee of the Federation of

Pharmaceutical Manufactures' Association, (Ed .): "Q & A on Genotoxicity Testing

for Pharmaceuticals", Scientist Inc ., Tokyo, 2000 .

[2] Toshio Sofuni, editor. Data book of Chromosomal Aberration Test In Vitro ; Revised

Edition 1998. Tokyo, Life-science Information Center; 1999.

10. Study Notes

10.1 Unforeseeable circumstances that may have affected the quality and integrity

of the data

None

10.2 Deviation from the protocol

None

-22-

~~~
p. 167

B051348

Table 1 Results of the cell growth inhibition test

Cell growth index (°/a)

Concentration Continuous treatment

(μg~~) Short-term treatment assay

assay

-S9 mix assay +S9 mix assay 24-hour assay

Negative control 100

.0 100.0 100.0 (DMSO)

313 P 50.3 81.2 71.4

~ 625 P 44.2 71.8 78.6

1250 P 38.7 83.4 65.6

2500 P 47.7 85.1 64.1

5000 P 81.9 89.0 106.8

DMSO: Dimethyl sulfoxide

P: Precipitation of the test substance was observed in the treatment medium at the beginning and the end of

the treatment.

~-

tp 33
p. 168

B051348

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p. 169

B051348

A v

m ^~

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p. 170

B051348

Figure 1 Cytotoxicity in the treatment with MTDID3663

[short-term treatment assay (-S9 mix)]

150

-O- cell growth inhibition test

-0-- chromosomal aberration test

100

...,

ao 50

...

..,

U

0

0 1250 2500 3750 5000

Concentration (μg/mL)

Figure 2 Cytotogicity in the treatment with MTDID3663

[short-term treatment assay (+S9 mix)]

100

,--.

~

~0

~

a

~. ..

~ 50

3

0

~.

bn

~ -4- cellgrowth inhibition test

U

--*-chromosomal aberration test

0

0 1250 2500 3750 5000

Concentration (μg/mL)

~03~
p. 171

B051348

Figure 3 Cytotoxicity in the treatment with MTDID3663

[continuous treatment assay]

150

--o-- cell growth inhibition test

.-~

-0- chromosomal aberration test

... .

~ 100

a

c

~~

o

b1D 50 ..

U

0

0 1250 2500 3750 5000

Concentration (μg/mL)

lP 3'~
p. 172

B051348

Figure 4 Incidence of cells with structural aberrations

in the treatment with MTDID3663

10

-0--S9 mix assay

-*-- +S9 mix assay

-6 24-hour assay

U

CW 5

'L 3 . .

.U

~

0

0 1250 2500 3750 5000

Concentration (μg/mL)

Figure 5 Incidence of cells with numerical aberrations

in the treatment with MTDID366 3

10

--o-- - S9 mix assay

---0-- + S9 mix assay

~- o

~-.

6-24-hour assay

. .~

WW 5 CD

10

tj

0

0 1250 2500 3750 5000

Concentration (μg/mL)

-2-8-

k,r-, 3$
p. 173

B051348

Quality Assurance Statemen t

Sponsor: Sumitomo 3M Limited. Title: Chromosomal Aberration Study of MTDID3663 in Cultured Mammalian Cells

Study No. : B051348

This study was carried out in accordance with the following standard . I hereby certify

that this final report faithfully describes the methods and results in this study . The inspection and reporting are as follows .

Standard Concerning Testing Facilities Implementing studies Related to New Chemical

Substances

(Pharmaceutical and Food Safety Bureau, Manufacturing Industries Bureau,

Environmental Policy Bureau, Ministry of the Environment, Pharmaceutical and Food

affairs No. 1121003, November 17, 2003, No.-3, November 21, 2003, Ministry of Health,

Labor, and Welfare, Ministry of Economy, Trade, and Industry, No . 031121004)

Inspection Reporting Date

Phase Inspector ~ate to the Study to the

Director Management Protocol

Protocol draft Tomoko Kanae Apr 26, 2006 Apr 26, 2006 Apr 26, 2006

Tomoko Kanae Apr 27, 2006 Apr 27, 2006 Apr 27, 2006

Protocol Tomoko Kanae May 10, 2006 May 10, 2006 May 10, 2006

Protocol Amendment draft (1) Tomoko Kanae Jul 14, 2006 Jul 14, 2006 Jul 14, 2006

Protocol Amendment (1) Tomoko Kanae Jul 27, 2006 Jul 27, 2006 Jul 27, 2006 Study procedure

Preparation of the test substance Tomoko Kanae May 15, 2006 May 15, 2006 May 15, 2006

solution

Cell treatment Tomoko Kanae May 22, 2006 May 22, 2006 May 22, 2006

Preparation of specimens and Michiko Sugaya May 23, 2006 May 23, 2006 May 23, 2006

measurement of cell growth index

Observation of specimens Tomoko Kanae Jun 1, 2006 Jun 1, 2006 Jun 1, 2006

Raw data, Final report

Raw data, Draft report Tomoko Kanae Jul 26, 2006 Jul 26, 2006 Ju126, 2006

Tomoko Kanae Jul 27, 2006 Ju127, 2006 Ju127, 2006

_Raw data, Final report Tomoko Kanae Ju128, 2006 Ju128, 2006 Ju128, 2006

Quality Assurance Manager: Signed and Sealed Date : July 28. 2006

Kazuo Morotome

Quality assurance unit of Kashima Laboratory

Mitsubishi Chemical Safety Institute Ltd.

-29-

k4:1 T-1
p. 174

FINAL REPORT

M.S.I. Report No. A050395

Bioconcentration Study of

MTDID3663

~ with Carp

Submitted to:

Sumitomo 3M Limited

~

Prepared by:

Mitsubishi Chemical Safety Institute Ltd. (M.S.I.)

August 29, 2006

~~ b
p. 175

Bioconcentration Study of MTDID3663

with Carp

(English version)

SMI'TUED

Sponsor Sumitomo 3M Limited

Study Title Bioconcentration Study of MTDID3663 with Carp

Study No. A050395

This report is the English version of the original, which was written in Japanese. We,

the undersigned, hereby declare that this version faithfully reflects the original report to

the best of our knowledge . ~_- ~ ~,.- • I

------------------

~

Approved by

~ Date:---"

; -------•-----•----------•------------------------ °p

Yasuo~ -----------------------------

Study Directo r

Ecological Chemistry Division of Yokohama Laboratory,

Mitsubishi Chemical Safety Institute Ltd.
p. 176

Compliance with the GLP Standards

Yokohama Laboratory,

Mitsubishi Chemical Safety Institute Ltd .

Sponsor Sumitomo 3M Limited

Study Title Bioconcentration Study of MTDID3663 with Carp

Study No. A050395

I hereby certify that this study was carried out in accordance with its protocol and

with our standard operating procedures, and that the reported results reflect accurately

the raw data of the study.

The study described in this report was conducted in compliance with the Good

Laboratory Practice (GLP) standards concerning the test facility in which the study of

new chemical substance is performed (Yakushokuhatsu No .1121003, Heisei 15.11 .17

Seikyoku No.3, Kanpokihatsu No .031121004, November 21,2003; the latest revision,

April 1,2005)

Study Directo r

Yasuo SATO Sealed date: August 29, 2006

~ Study Director

Ecological Chemistry Division of Yokohama Laboratory

--~-- (o ~ ~
p. 177

Quality Assurance Statement

Yokohama Laboratory,

Mitsubishi Chemical Safety Institute Ltd .

Sponsor Sumitomo 3M Limite d

Study Title Bioconcentration Study of MTDID3663 with Carp

Study No. A050395

By the inspections and audits listed below, we hereby certify that this study was

carried out in accordance with its protocol and with our standard operating procedures .

We certify that this report faithfully describes the methods and procedures employed,

and that the reported results reflect the raw data of the study accurately.

Date of Date of Reporting to

Inspection Management and

Study Director

Protocol: March 24, 2006 March 24, 2006

Amendment 01 March 30, 2006 March 30, 2006

Amendment 02 May 8, 2006 May 8, 2006

Amendment 03 May 9, 2006 May 9, 2006

Amendment 04 June 5, 2006 June 5, 2006

Amendment 05 July 5, 2006 July 5, 2006

Amendment 06 July 14, 2006 July 14, 2006

Amendment 07 July 24, 2006 July 24, 2006

( In-progress study:

Preparation of feed solutions: March 27, 2006 March 27, 2006

Preparation of feed solutions: May 8, 2006 May 8, 2006

Introduction of fish: May 11, 2006 May 11, 2006

Analysis of standard solutions: May 18, 2006 May 18, 2006

Analysis of test fish : May 18 and 22, 2006 May 22, 2006

Analysis of test water: June 1, 2006 June 1, 2006

Final report: August 29, 2006 August 29, 2006

Quality Assurance Personnel

Yumiko KASHIWAGI, B .Sc. Sealed date: August 29, 2006

Hiroko TOKIMI, B .Sc. Sealed date: August 29, 2006

Reiko TSUTSUI Sealed date: August 29, 2006

-~- ~ y 3
p. 178

Introduction

1 . Study Titl e

Bioconcentration Study of MTDID3663 with Carp

(Study No. A050395)

2. Purpos e

This study was conducted to evaluate the bioaccumulation potential of the test

substance for notification under the Chemical Substances Control Law of Japan.

3. Test Guideline

The study was conducted in accordance with the Test Method Relating to New

~ Chemical Substances <Bioconcentration test of chemical substances in fish and

shellfish> (Yakushokuhatsu No .1121002, Heisei 15 .11 .13 Seikyoku No .2, Kanpokihatsu

No.031121002, November 21,2003 ; the latest revision, April 1,2005), which prescribes

the procedure for testing new chemical substances as required by the Chemical

Substances Control Law of Japan .

4. GLP Compliance

The study described in this report was conducted in compliance with the Good

Laboratory Practice (GLP) standards concerning the test facility in which the study of

new chemical substance is performed (Yakushokuhatsu No .1121003, Heisei 15 .11 .17

Seikyoku No.3, Kanpokihatsu No .031121004, November 21,2003 ; the latest revision,

April 1,2005)

~ 5. Sponsor

Sumitomo 3M Limite d

3-8-8 Minami Hashimoto, Sagamihara, Kanagawa, Japan

6. Testing Facility (Head Office)

Mitsubishi Chemical Safety Institute Ltd. (M.S .I.)

1-30 Shiba 2-chome, Minato-ku, Tokyo, Japan

7. Testing Facility (Laboratory)

Yokohama Laboratory

Mitsubishi Chemical Safety Institute Ltd.

1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa, Japa n

~' tP~~
p. 179

8. Study Director

Yasuo SATO

Ecological Chemistry Division of Yokohama Laboratory

9. Experimental Scientist

Tomoyoshi AKIKUSA, M.Sc. Sealed date: August 29, 2006

(Experimental work)

Naoko SASANO, M.Sc. Sealed date: August 29, 2006

(Experimental work)

(_ = Shinichi TAKEUCHI, B .Sc. Leave of absence-from July 26, 2006

(Experimental work)

10. Date

Study initiation : March 24, 2006

Exposure starting: May 11, 2006

Exposure completion: July 10, 2006 -

Depuration initiation: July 10, 2006

Depuration completion : July 18, -2006

Study completion: August 29, 2006

11. Storage and Retention of Test Substance and Records

The following will be stored in the archives of the Yokohama Laboratory .

1) Protocol

2) Final report

3) Raw data

4) Test substance

5) Other requirements

-6-

lo LA S
p. 180

Contents

page

Summary ----------------------------------------------- 9

1

. Test Substance ------------------------- 13 ----------------------------------------

1 .1 . Identification 13

1 .2 . Source 14

1 .3 . Confirmation of Stability 14

2. Outline of Bioconcentration Test 14

~--------------------------------------------------------------------

3. Dilution Water 15 -------------------------------------------

4

. Acute Toxicity Test --------- --------- ----------------------------------------------------------------15 ----------------------

4.1 .Testfish 15

4.2. Test Conditions 16

4.3 . Preparation of Test Water 16

4.4. Results 16

5. Analytical Methods . ~ 17 . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . --------------

5 .1 . Apparatus and Reagents 17

5 .2. Gas Chromatography Mass Spectrometry System and Conditions 18

5.3 . Calibration Curve 19

5 .4. Analysis of Test Substance in Test Water 19

5 .5 . Analysis of Test Substance in Test Fish 20

5 .6. Blank and Recovery Tests 21

6. Bioconcentration Test Methods 22

------------------------------------------------------------------------------------

6.1 . Test Fish 22

6.2. Test Conditions 23

6 .3 . Test Apparatus 23

6 .4. Preparation of Test Water 24

6.5 . Introduction of Fish, Observations and Environmental Control 25

6.6. Measurement of Test Substance 25

6.7. Bioconcentration Factor 27

6.8. Calculation of biological half-life 27

-~-~y~
p. 181

7. Results and Discussion ------------------------------------------------------------------------------------------------- 28

7.1 . Circumstances That May Have Affected the Reliability of the Test Results 28

7.2 . Monitoring of Test Conditions 28

7.3. Concentration of Test Substance in Test Water 28

7.4. BCF of Test Substance in Test Fish 28

7.5. BCF on Fish tissue 29

7.6. Biological half-life 30

Tables & Figures 31-72

Appendix 1 Information on the test substanc e

Appendix 2 Dilution water quality

Appendix 3 Chromatograms (Analysis of the test substance in the test water)

Appendix 4 Chromatograms (Analysis of the test substance in the test fish during

exposure period)

Appendix 5 Chromatograms (Analysis of the test substance in the tissues)

Appendix 6 Chromatograms (Analysis of the test substance in the test fish during

depuration period)

~

~ (o ~-l
p. 182

Summary

Sponsor: Sumitomo 3M Limited

Study title: Bioconcentration Study of MTDID3663 with Carp

Study number : A050395

Study period: March 24, 2006 to August 29, 2006

Methods: The study was conducted in accordance with the Test Method Relating

to New Chemical Substances <Bioconcentration test of chemical

substances in fish and shellfish> (Yakushokuhatsu No .1121002, Heisei

15.11 .13 Seikyoku No .2, Kanpokihatsu No.031121002, November

21,2003 ; the latest revision, April 1,2005), which prescribes the

procedure for testing new chemical substances as required by the

Chemical Substances Control Law of Japan.

Supply of test water: Continuous flow-through dilution system (800 L/day)

~ Test substance in test water (nominal concentrations)

High concentration level: 0.05 mg/L

Low concentration level: 0.005 mg/L

Control: 0 mg/L

Dissolving agents in test water

High concentration level: 2-Methoxyethanol 25 ppm (v/v)

Low concentration level : 2-Methoxyethanol 25 ppm (v/v)

Control : 2-Methoxyethanol 25 ppm (v/v)

Exposure period : 60 days

Depuration period : 8 days

Analytical method: Gas Chromatography Mass Spectrometry (GC/MS)

As the test substance was mixture, many peaks were detected in GC/MS

chromatograms. Concentration of the test substance was derived from each peak

area. The approximate retention time of each peak (A-D) are shown below .

~ RT: 21 min. (Peak A)

22 min. (Peak B)

23 min, (Peak C)

24 min. (Peak D)

Concentration of each component was indicated as the weight concentration of

the test substance without consideration of their ratio .

96-hour LC50 to Medaka: >5 mg/L

Lipid content: 4.8% (n=3, 3 .7-5.9%) at the start of the exposure period

5.4% (n=3, 4.5-7.0%) at the end of the depuration period

lQy
p. 183

Results: The bioconcentration factors (BCF) obtained in this study were as follows :

Peak A

Exposure period (days) 7 14 28 35 60

High concentration level

Mean conc

BCF, No

. in test water (mg/L) 0.0479 0.0485 0.0492 0.0497 0.0493

. 1 1250 982 1230 1050 1660

BCFss 1210 No.2 1360 1230 1000 1130 1100

Low concentration level

BCF, No

Mean conc . in test water (mg/L) 0.00480 0.00486 0 .00488 0.00492 0.00498

. 1 1380 1300 1200 1220 1510

BCFss 1280 No.2 1190 1590 1190 1250 1530

Peak B

Exposure period (days) 7 14 28 35 60

High concentration leve l

BCF, No

Mean conc . in test water (mg/L) 0.0475 0.0482 0.0490 0 .0495 0 .0492

. 1 1310 1030 1290 1110 1760

BCFss 1270 No.2 1430 1280 1050 1200 1160

Low concentration level

Mean conc

BCF, No

. in test water (mg/L) 0.00479 0.00485 0.00489 0 .00492 0.00496

. 1 1430 1350 1250 1270 1610

BCFss 1360 No.2 1260 1670 1250 1310 1640

Peak C

Exposure period (days) 7 14 28 35 60

High concentration leve l

Mean conc . in test water (mg/L) 0 .0492 0.0495 0.0504 0.0506 0.0501

BCF, No. 1 924 719 878 760 1260

BCFss 891 No.2 1020 895 702 830 844

Low concentration level

BCF, No

Mean cone. in test water (mg/L) 0.00491 0.00494 0.00498 0.00500 0.00507

. 1 1040 919 866 862 1120

BCFss 932 No.2 901 1150 849 885 1160

Peak D

Exposure period (days) 7 14 28 35 60

High concentration level

Mean conc

BCF, No

. in test water (mg/L) 0.0498 0.0500 0.0507 0.0509 0.0500

. 1 874 667 843 731 1180

BCFss 863 No. 2 970 820 673 795 796

Low concentration level

Mean conc

BCF, No

. in test water (mg/L) 0.00495 0.00499 0.00499 0.00502 0.00504

. 1 987 861 820 824 1070

BCFss 898 No. 2 860 1070 811 849 1100
p. 184

Discussion

At the both concentration levels, the variation of mean BCF determined at the last

three consecutive measurements fell within ± 20% . It is confirmed that BCF values had

reached the steady-state . BCF at the steady-state (BCFss) of Peak A were 1210 at the high

concentration level and 1280 at the low concentration level, BCFss of Peak B were 1270

at the high concentration level, and 1360 at the low concentration level . BCFss of Peak C

were 891 at the high concentration level, and 932 at the low concentration level . BCFss of

Peak D were 863 at the high concentration level, and 898 at the low concentration level.

BCF on fish tissue

In order to locate highly-concentrated sites, the remaining two fish samples taken on

day 60 of exposure period each concentration level were dissected to head, viscera, edible

part (muscle and bone), and integument (fins and skin with scale), then analyzed with

GC/MS . As shown in the table below.

{. .__

Peak A

BCF

Part High concentration Low concentration

level (day 60) level (day 60)

Head 1600 2050

Viscera 2790 3870

Muscle and bone 676 786

Fin & skin (with scale) 1720 1570

Peak B

BCF

Part High concentration Low concentration

level (day 60) level (day 60)

Head 1690 2190

~ Viscera 2980 4150

Muscle and bone 718 835

Fin & skin (with scale) 1810 1670

Peak C

BCF

Part High concentration Low concentration

level (day 60) level (day 60)

Head 1170 1460

Viscera 2030 2700

Muscle and bone 496 559

Fin & skin (with scale) 1220 1080

.~r lo s C>
p. 185

Peak D

BCF

Part High concentration Low concentration

level (day 60) level (day 60)

Head 1100 1410

Viscera 1910 2620

Muscle and bone 467 534

Fin-i& skin (with scale) 1160 1060

The BCF of all peaks were larger on viscera, and smaller on muscle and bones than

that on the other parts in both high and low concentration levels .

Depuration test

The BCF on whole body exceeded 1000 and a depuration test was accordingly

conducted for 8 days. In the depuration test, the concentration of the test substance in fish

decreased with time. The biological half-life (BHL) of Peak A in fish was calculated to be

2.3 days at the high and 3 .2 days at the low concentration level, BHL of Peak B in fish

was calculated to be 2 .4 days at the high and 3 .3 days at the low concentration level, BHL

of Peak C in fish was calculated to be 2 .0 days at the high and 2 .6 days at the low

concentration level, BHL of Peak D in fish was calculated to be 1 .9 days at the high and

2 .5 days at the low concentration level.

~

~1Q51
p. 186

1 . Test Substance

1 .1. Identification

Name of the new

chemical substance

(by IUPAC

nomenclature)

Alternate name

CAS RN

Structural formula or

rational formula

(If both of them are

unknown, outline of

preparation or

manufacturing )

Molecular weight SANITIZED

Purity (%) of the new

chemical substance

subjected to the study

Lot number of the new

chemical substance

subjected to the study

Name and content of

impuritie s

Vapor pressure

Solubility in water

1-octanol/water partition

coefficient

Melting point

Boiling point

Appearance

at room temperature

Stability

Solubility in

solvents and the like

All data were provided by the sponsor

--~-

~S~
p. 187

E33l.lT'1MA2

.
p. 188

1 .2. Source

Supplier: Sumitomo 3M Limited

1.3. Confirmation of Stability

During the course of the study, the test substance had been sealed in a bottle and

stored in a refrigerator in the dark . At the end of the experiment, the infrared absorption

spectrum of the test substance was measured . The spectrum was consistent with that

measured before the exposure period, indicating that the test substance was stable under

the storage conditions.

(Appendix 1)

Apparatus: Infrared spectrophotometer, AVATAR 320, Nicolet Instrument Corporation

2. Outline of Bioconcentration Test

The study was conducted according to the Test Method Relating to New Chemical

Substances <Bioconcentration test of chemical substances in fish and shellfish>

(Yakushokuhatsu No.1121002, Heisei 15 .11 .13 Seikyoku No.2, Kanpokihatsu

No.031121002, November 21,2003 ; the latest revision, April 1,2005), which prescribes

the procedure for testing new chemical substances as required by the Chemical

Substances Control Law of Japan.

Prior to the bioconcentration test, an acute toxicity test with Medaka was conducted

for 96 hours to determine 50% lethal concentration (LC50) . It was confirmed that the

bioconcentration test was carried out at two concentration levels less than a hundredth and

a thousandth of the 96-hour LC50 .

The bioconcentration test system shown in Figure 1 (p . 62) was set up in triplicate . In

addition to the control, two groups of carp were exposed to the test substance at high and

low concentration levels. During the exposure period, the concentrations of the test

substance in water and fish were measured periodically. The bioconcentration factors

~ (BCF) calculated from the results were used to evaluate the bioaccumulation potential of

the test substance in fish tissues .

In order to locate highly-concentrated sites, fish samples taken at the end of exposure

period were dissected to four parts and then analyzed. Then followed the exposure period,

depuration test was conducted for 8 days to calculate the biological half-life by supplying

dilution water without test substance.

-~' ~oS 3
p. 189

3. Dilution Wate r

As dilution water, Yokohama municipal tap water was treated with activated charcoal

and sodium thiosulfate solution was added to neutralize trace free chlorine . This dilution

water was checked half-yearly and verified to satisfy the Water Quality Criteria for the

Protection of Aquatic Living Resources. (Appendix 2)

4. Acute Toxicity Test

The acute toxicity test with Medaka was conducted in accordance with the OECD

Guidelines for Testing of Chemicals No.203 "Fish, Acute Toxicity Test" (1992).

Concentration of the test substance in test water was not measured, and the 96-hour LC50

was calculated on the basis of nominal concentrations.

4.1. Test Fish

~_ . 1) Species : Medaka(Oryzias latipes)

Source: Tsunashima Fishing Co. ,

5-18-1 Tsunashimanishi, Kouhoku-ku, Yokohama, Kanagawa,

Japan

Date of purchase : August 26, 2005

Lot number: 05-H-0826

Pre-acclimation: At the start of acclimation, visual observation was made and

abnormal fish were removed. The remaining normal fish were

reared in an aquarium with flowing water. During the

acclimation period, fish were kept without receiving medicine

in the aquarium.

Total length: 2± 1 cm

Body weight: Approximately 0.2 g

2) Acclimation

Aquarium number: D-11

Water temperature: 24 ± 1 °C

Period: - >_ 2 weeks

Food: Tetramin® from Tetra Werke

Feeding : The food was given in an amount of 2% of the fish weight

everyday (Feeding was discontinued during the 24 hours

before testing).

Mortality during the 1 week before testing : <5%
p. 190

4.2. Test Condition s

To confirm that the nominal concentrations of the bioconcentration test were less than

a hundredth and a thousandth of the 96-hour LC50, the concentration of acute toxicity test

was decided based on the preliminary test. Since the concentration of the dissolving

agents exceeded 100 ppm, limit test was performed with the following conditions.

Concentration level: 0 (control), 5 mg/L

Number of fish: 7/ group _

Volume of test water: 5L

Water temperature : 24± 1 °C

Renewal of test water: Every 24 hours

Aeration: None (Covered with Teflon sheet)

Dissolved oxygen conc . : 60% of the saturation (? 5 mg/L at 24 °C)

Feeding: None

Duration: 96 hours

4.3. Preparation of Test Water

1) For exposure group

The test substance, 160 ,u L (250 mg, specific gravity : 1 .563 (g/mL)) added to 10 mL

of 50% (w/v) HCO-40/2-methoxyethanol and diluted with 2-methoxyethanol to 25 mL

(stock solution). The amount of 2 .5 mL of this stock solution was diluted to 5L with

dilution water.

(Concentration of the test substance : 5 mg/L

Concentration of HCO-40 : 100 mg/L

Concentration of 2-methoxyethanol: 395 ppm (v/v))

2) For control

The amount 10 mL of 50% (w/v) HCO-40/2-methoxyethanol was diluted with 2-

methoxyethanol to 25 mL . Amount of 2 .5 mL of this solution, was diluted to 5L with

dilution water.

(Concentration of HCO-40 : 100 mg/L

Concentration of 2-methoxyethanol: 400 ppm (v/v))

4.4. Results

The 96-hour LC50 of the test substance was >5 mg/L .

Concentration of Cumulative-mortality (%)

test substance (mg/1L) 24 hr 48 hr 72 hr 96 hr

0 0 0 0 0

5 0 0 0 0

The nominal concentration of bioconcentration test was confirmed to be less than a

hundredth and a thousandth of the 96-hour LC50 .

~'IeSS
p. 191

5. Analytical Methods

5.1 . Apparatus and Reagents

Apparatus

Electrobalance: PB3002, Mettler K.K.

PB3002-S, Mettler-Toledo K .K.

Homogenizer: AM, Nihon Seiki Kaisha Ltd.

Aspirator: VWP-15, Yamato Scientific Co . Ltd.

PP-11, Masuda Corporation

Reagents

Acetonitrile : Guaranteed reagent, Junsei Chemical Co., Ltd.

~

t

~ lQ S ~
p. 192

5.2. Gas Chromatography Mass Spectrometry System and Condition s

System

Gas Chromatography Mass Spectrometry (GC/MS) with Head Space Sampler No .1

Gas Chromatography (GC): Agilent 6890

Head Space Sampler (HSS): Agilent 7694

Mass Selective Detector (MSD) : Agilent 5973N

Work station : MSD Chemistation

Conditions

rGCl

Column : RESTEK Rtx-200 105m X 0.25mm, film thickness 1 .0 u m

Carrier Gas : Helium 1.0 mL/min(Constant flow)

Temperature Column :60°C (2min)-2°C/min->110°C (lmin)

Injector: 230°C

MS interface : 200°C

Injection method : Sprit (sprit ratio= 20.1 : 1)

Injection volume: 3 .0mL (HSS sampleloop volume)

FHSS1

Temperature: Oven 60°C, Loop 120°C, Transfer Line 200°C

Event time : GC Cycle Time 40min

Vial Equilibration Time 20min

Pressurization Time 0.2min

Loop Fill Time 0.03min

Loop Equilibration Time 0.2min

Inject Time 0 .2min

Vial parameter : Shake HIGH

[MSD

] ~ Temperature :Ion source 230°C, Quadrupole mass filter 150°C

SIM (Selected Ion Monitoring) method :

Solvent Delay 8.0min

Quant ion m/z 151 .0 [M-C5H5OF6]+

195 .0 [M-C3HF6] +

As the test substance was mixture, many peaks were detected in GC/MS

chromatograms. Concentration of the test substance was derived from four each peak area .

The approximate retention times of each peak (A-D) are shown below.

RT: 21 min. (Peak A)

22 min. (Peak B)

23 min. (Peak C)

24 min. (Peak D)

Concentration of each component was indicated as the weight concentration of the test

substance without consideration of their ratio .

-1-a" lp S -1
p. 193

5.3. Calibration Curve

The test substance was dissolved in acetonitrile at the concentration of 1000 mg/L .

Then it was diluted with acetonitrile to prepare stock solutions of 0, 0 .25, 0.5 and 1 mg/L.

Zero point one milliliter of these stock solutions were added to HS vials containing

l OmL purified water and capped immediately . These standard solutions were analyzed as

described in section 5.2. The peak area (count) for the test substance in each solution

was plotted against its concentration (concentrations of the test substance in standard

solution: 0 .0025-0.01 mg/L). The calibration curve drawn by the least square method

was a straight line almost passing through the origin and gave a satisfactory correlationcoefficient of 0.9995 (Peak A), 0 .9993 (Peak B), 0.9998 (Peak C) and 0.9997 (Peak D).

[Figure 2 (p. 63-66), Figure 3(p. 67)]

Before each set of analysis, chromatography of 0 .005 mg/L standard solution was

performed. The peak area was used to determine the test substance in test water and test

fish.

5.4. Analysis of Test Substance in Test Wate r

The test substance in test water was analyzed according to the following procedures .

Procedures varied according to concentration level .

High concentration level

HS vial containing purified water* 9mL and acetonitrile O lmL is capped

immediately after adding test water 1mL (Dilution ratio 10 )

Performed GC/MS analysis

Low concentration level

HS vial containing test water IOmL is capped immediately after adding

( acetonitrile 0.1mL.

I

Performed GC/MS analysis

* :Purified water: Grade A4 in Japanese Industrial Standards (JIS) K0557

~- 1~5b
p. 194

5.5. Analysis of Test Substance in Test Fish

The test substance in test fish was analyzed according to the following procedure .

Two fish weighed (gpprox . 16 g in total)

<-- Acetonitrile 40 mL

Cut into pieces with scissors

Homogenized for 5 minutes at approx. 8000 rpm

Filtered with suction through filter paper (S-60) on Buchner funnel

A cetonitrile layer Residue

E-- Acetonitrile 40 mL

Homogenized for 3 minutes at approx. 8000 rpm

I

Filtered with suction through filter paper (S-60) on

Buchner funne l

Acetonitrile layer Residue

IE-- Acetonitrile

Discarded

Diluted to 100 mL

I

After added 0 1 mL portion (Dilution ratio 1000), HS vial containing purified water* l OmL is

capped immediately

Performed GC/MS analysis

*:Purified water: Grade A4 in JIS K0557
p. 195

5.6. Blank and Recovery Tests

In the GC /MS analysis, detection limit was set at 0 .00025 mg/L, which corresponds

to 5% of the standard solution (0 .005 mg/L).

1) Test water

Blank test

Two samples of control water were analyzed according to the method of the low

concentration level described in Section 5 .4. Measured values were smaller than

detection limit, and the concentration of the test substance in the control water was

calculated to be <0.0003 mg/L (Peak A, B, C and D).

[Table 1(p. 31), Figure 4 (p. 68)]

Recovery test

The test water of high concentration level was diluted with purified water and

analyzed with GC/MS . The test water of low concentration level was directly analyzed

~ with GC/MS. Therefore the recovery test was not performed.

2) Test fish

Blank test

At the start of the exposure period, four control fish were analyzed two by two after

treatment by the method described in Section 5 .5 . At the end of the exposure period,

control fish were also analyzed in the same manner. Measured values were smaller than

detection limit, and the concentration in fish was calculated to be <0 .16 μg/g (Peak A, B,

C and D).

[Table 2 (p. 32), Figure 5 (p . 69-70)]

Recovery test

Zero point zero five milliliter of the 1000 mg/L acetonitrile solution of the test

substance was added to a pair of control fish . Then these fish were analyzed after treatment

by the method described in Section 5 .5 to evaluate recovery. Added to a fish weight of 16 g,

the test substance existed at 3 μg/g, which is 62-fold of the high concentration level (0 .05

~ mg/L). The four measurements of recovery gave the mean value of 84 .6% (Peak A), 84 .6%

(Peak B), 86.3% (Peak C) and 86 .3% (Peak D), the standard deviation of 3 .1% (Peak A),

3.2% (Peak B), 2.3% (Peak C) and 3 .9% (Peak D), and the range of 6.9% (Peak A), 7.0%

(Peak B), 5 .6% (Peak C) and 9.2% (Peak D), satisfactory for quantitative analysis. The

average recovery was used to correct the measured values.

[Table 3 (p. 33), Figure 6 (p . 71-72)]

ke C5
p. 196

6. Bioconcentration Test Methods

6.1. Test Fish

1) Species: Carp(Cyprinus carpio)

Source : Niikura Fish Farm

1217, Shimoya, Isehara-city, Kanagawa, Japan

Date of purchase : July 4, 2005

Lot number: 05-K-0704

Pre-acclimation : At the start of acclimation, visual observation was made and

abnormal fish were removed. The remaining normal fish were

then reared in an aquarium with flowing water. During the

acclimation period, fish were kept without receiving medicine

in the aquarium.

Total length: 8± 4 cm

Body weight: Approximately 5 g

Age: Less than a year after hatching

2) Acclimation

Aquarium number: A-1

Water temperature: 24 ± 2 °C

Period: > 2 weeks

Food: Babygold® from Kyorin

Feeding: The food was given in an amount of 2% of the fish weight .

Mortality during the 1 week before testing : <5%

i I
p. 197

6.2. Test Conditions

1) Concentration level (nominal concentration)

High concentration level : 0.05 mg/L

(2-methoxyethanol 25 ppm (v/v))

Low concentration level: 0.005 mg/L

(2-methoxyethanol 25 ppm (v/v))

Control: 0 mg/L

(2-methoxyethanol 25 ppm (v/v))

2) Supply of dilution water: 800 L/day (Turnover rate : 16 times/day)

3) Supply of feed solution : 20 mL/day

4) Number of fish (at the start of the exposure period) :

High concentration level : 44 fish in 50 L test water (<1 .0 g/L/day)

Low concentration level : 44 fish in 50 L test water (<1 .0 g/L/day)

Control: 28 fish in 50 L test water (<1 .0 g/L/day)

5) Water temperature : 24 ± 2°C

6) Dissolved oxygen: > 60% of the saturation (> 5 mg/L at 24 °C)

7) Aeration: None

8) pH: 6.0-8. 5

9) Lighting: Approximately 16 hr/day

(Hf fluorescent lamp with wavelengths of 400-700 nm)

10) Exposure period : 60 days

11) Depuration period : 8 days (By supplying dilution water without test substance)

6.3. Test Apparatus

The outline is shown in Figure 1 (p . 62) .

z~-~ ~ ~
p. 198

6.4. Preparation of Test Wate r

1) Preparation of feed solutions

High concentration level

Test substance, 1 g (640 ,u L)*

E-2-methoxyethanol

Dissolved and diluted to 500 mL (Feed solution 1 )

Concentration of test substance : 2000 mg/L

Low concentration level

Feed solution 1, 50 mL

E--2-methoxyethanol

Diluted to 500 mL (Feed solution 2)

Concentration of test substance : 200 mg/L

Control

2-methoxyethanol

Feed solutions were renewed approximately at intervals of 18 days .

*Specific gravity : 1 .563 (g/mL)

2) Preparation of test wate r

Each feed solution was supplied to mixing glass tube with metering pump for feed

solution, and diluted to each nominal concentration by mixing with dilution water

( delivered with metering pump for dilution water, then poured into each test chamber .

[Figure 1 (p. 62)]

~- lo ~e 3
p. 199

6.5. Introduction of Fish, Observations and Environmental Control

The bioconcentration test system was operated with no fish until GC/MS analysis

proved that the test substance concentrations in both test chambers were kept at respective

nominal concentrations. Fish were introduced into the test chamber and the

bioconcentration test was then started . During the exposure and depuration period, the

following observations and measurements were made .

1) Conditions of fish

Observations were made on the appearance, swimming and feeding behavior of fish .

2) Feeding

Babygold® from Kyorin was given in an amount of approximately 2% of the fish

weight everyday.

3) Dissolved oxygen and temperature

During the exposure period, the dissolved oxygen (DO) and temperature were

measured when test water was sampled. During the depuration period, they were

measured at the start of depuration period, and when test fish was sampled.

Apparatus: DO meter, DOL-10, DKK Corporation

Digital thermometer, TX1001, Yokogawa Meters & Instruments Corp.

4) Lipid content

At the start of exposure period and end of depuration period, five control fish were

taken and lipid content of three of them were measured (n=3) .

5) pH

At the start and end of the exposure and depuration period, the pH of test water was

measured.

t" Apparatus: pH meter, HM-40V, Toa Electronics

6.6. Measurement of Test Substance

1) Test water

On days 0, 7, 14, 21, 28, 35, 42, 49 and 60 of the exposure period, each test water

was analyzed as described in Section 5 .4.

2) Fish

On days 7, 14, 28 and 35 of the exposure period, four fish were taken, and on day 60

of the exposure period, six fish were taken from each concentration level. Four of them

were analyzed two by two after treatment by the method described in Section 5 .5 .

As the BCF exceeded a prescribed limit of 1000, a depuration test was conducted

using the remaining fish. On days 1, 2, 4 of the depuration period, four fish were taken,

and on day 8 of the depuration period, ten fish were taken from each concentration levels

and analyzed in the same manner.

' 2-5- la~ `~
p. 200

In order to locate highly-concentrated sites, the remaining two fish samples of eac h

concentration level taken on day 60 of exposure period were dissected to head, viscera,

edible part (muscle and bones), and integument (fins and skin with scales), then analyzed

in the same manner.

6.7. Bioconcentration Factor

1) Calculation of bioconcentration factor

The bioconcentration factor (BCF) is calculated by the following equation :

BCFn = Cfn / Cwn

The steady-state bioconcentration factor (BCFss) is calculated by the following

equation:

BCFss = Cfs / Cw,s

Where BCFn : The bioconcentration factor after n days

BCFss: The steady-state bioconcentration factor

Cf,,: Concentration of the test substance in test fish after n days (μg/g )

Cw,,,: Mean concentration of the test substance in test water over n days

(mg/L)

Cfs: Mean concentration in test fish at steady-state (μg/g)

Cw,s: Mean concentration in test water at steady-state (mg/L )

Steady-state : Steady-state is decided to be reached, when three successive BCF at

intervals of 48 hours or longer are within 20% of each other . Cfs and

Cw,s are calculated from last three successive data. When the obtained

BCF are all less than 100, it is considered that steady-state is reached .

If BCF values are smaller than 100 in all points and three successive

mean BCF at intervals of 48 hours or longer are not within 20% each

other, the BCFss is not calculated by above equation and BCFss is just

declared "< (maximum BCF value) "or <(maximum BCF value).

2) Determination limit of bioconcentration factor

As described in Section 5 .6.2), the lowest detectable amount of test substance in fish

was <0 .16 μg/g (Peak A,B,C and D). This indicated that BCF were obtained when the

test substance in test water of 0.05 and 0.005 mg/l, were concentrated by factors of 4

and 32, respectively. These factors depend on the fish weight used for analysis.

6.8. Calculation of biological half-life

The concentration of the test substance in depuration test is given by the following

equation:

Ct=Coxe~

Where Ct: Concentration in fish on day t of depuration (μg/g)

Co: Mean concentration in fish at the end of the exposure period (μg/g)

k: Depuration rate constant (day-1 )

1,6' ~c~e5
p. 201

t: Time in depuration period (day)

On each of the two concentration levels, the depuration rate constant, k, is determined

by the least square method, and the biological half-life (BHL) is calculated by the

following equation :

BHL =1n2/k

Where BHL: Biological half-life (day)

The persistence of the test substance in fish is calculated by the following equation :

R = Ct/Cox10 0

Where R: Persistence of the test subst ance in fish (%)

(

~ ~o lo ~P
p. 202

7. Results and Discussio n

7.1. Circumstances That May Have Affected the Reliability of the Test Results -

On May 20, 2006, a day in the exposure period, regular maintenance of facilities

was performed. The supplies of electricity, water and air were stopped from 7 :30 to 22:00.

During all that time, at high and low concentration level, the test conditions containing

supplies of dilution water and feed solution were kept constant by using auxiliary power

unit and another water source .

However at control level, the supplies of dilution water and feed solution were

stopped. During the period, test water was aerated to avoid the decrease of DO. DO and

temperature of the test water were checked in the morning and afternoon for confirmation

of the conditions of control chamber. The results satisfied the Test Condition in section

6.2 . It showed events derived from the regular maintenance had no influence on the result

of the bioconcentration test.

~ 7.2. Monitoring of Test Conditions

During the test period, the concentration of dissolved oxygen and the temperatur e

variation fulfilled the test conditions (DO : ? 60% of the saturation, temperature : 24 ~ I

2°C). The pH values were in the range from 6 .0 to 8 .5. The mortalities in both control and

treated fish were less than 10% at the end of the test. There was no abnormality in shape

of the body or in swimming and feeding behavior during the test period.

Lipid content: 4.8% (n=3, 3 .7-5.9%) at the start of the exposure period

5.4% (n=3, 4.5-7.0%) at the end of the depuration period

(The average at the end of the depuration period fell within ± 25%

range of the average at the start of the exposure period .)

7.3. Concentration of Test Substance in Test Wate r

The concentration during the exposure period averaged 0 .0493 mg/L (Peak A), 0 .0492

mg/L (Peak B), 0.0501 mg/L (Peak C) and 0 .0500 mg/L (Peak D) at the high

concentration level and 0:00498 mg/L (Peak A), 0 .00496 mg/L (Peak B), 0.00507 mg/L

(Peak C) and 0.00504 mg/L (Peak D) at the low concentration level . These were

sufficiently close to the nominal values. The coefficients of variation were 6.5% (Peak A),

5.1% (Peak B), 4.2% (Peak C) and 6.2% (Peak D) at the high concentration level, and

4

.0% (Peak A), 4.0% (Peak B), 3 .4% (Peak C) and 3 .2% (Peak D) at the low concentration

level. These concentrations of the test substance in the chamber at both concentration

levels were maintained within ± 20% of the mean of the measured values .

[Table 4-5 (p. 34-41), Figure 7-8 (p. 34-41), Appendix 3]

7.4. BCF of Test Substance in Test Fish

The bioconcentration factors (BCF) of Peak A were 982-1660 at the high

concentration level, and 1190-1590 at the low concentration level . BCF of Peak B were

1030-1760 at the high concentration level, and 1250-1670 at the low concentration level .

BCF of Peak C were 702-1260 at the high concentration level, and 849-1150 at the low

concentration level . BCF of Peak D were 667-1180 at the high concentration level, and

811-1100 at the low concentration level.

[Table 6-7 (p. 42-49), Figure 9-10 (p. 42-49), Appendix 4]

At the both concentration levels, the variation of mean BCF determined at the last
p. 203

three consecutive measurements fell within ± 20% . It is confirmed that BCF values had

reached the steady-state. Each BCF at the steady-state (BCFss) of Peak A was 1210 at the

high concentration level, and 1280 at the low concentration level . BCFss of Peak B were

1270 at the high concentration level, and 1360 at the low concentration level . BCFss of

Peak C were 891 at the high concentration level, and 932 at the low concentration level.

BCFss of Peak D were 863 at the high concentration level, and 898 at the low

concentration level.

7.5. BCF on Fish tissue

Shown in the table below.

BCF on fish tissue

Peak A

BCF

~_ . Part High concentration Low concentration

level (day 60) level (day 60)

Head 1600 2050

Viscera 2790 3 870

Muscle and bone 676 786

Fin & skin (with scale) 1720 1570

Peak B

BCF

Part High concentration Low concentration

level (day 60) level (day 60)

Head 1690 2190

Viscera 2980 4150

Muscle and bone 718 835

Fin & skin (with scale) 1810 1670

Peak C

BCF

Part High concentration Low concentration

level (day 60) level (day 60)

Head 1170 1460

Viscera 2030 2700

Muscle and bone 496 559

Fin & skin (with scale) 1220 1080

-~- ko le
p. 204

Pe ak D

BCF

Part High concentration Low concentration

level (day 60) level (day 60)

Head 1100 1410

Viscera 1910 2620

Muscle and bone 467 534

Fin & skin (with scale) 1160 1060

The BCF of all peaks were larger on viscera, and smaller on muscle and bones than

that on the other parts in both high and low concentration levels.

[Table 8-9 (p. 50-53), Appendix 5]

7.6. Biological half-life

The depuration test showed that the concentration of the test substance in fish

decreased with time . BHL of Peak A in fish was calculated to be 2 .3 days at the high and

3 .2 days at the low concentration level, BHL of Peak B in fish was calculated to be 2 .4

days at the high and 3 .3 days at the low concentration level, BHL of Peak C in fish was

calculated to be 2.0 days at the high and 2.6 days at the low concentration level, BHL of

Peak D in fish was calculated to be 1 .9 days at the high and 2.5 days at the low

concentration level.

[Tables 10-11 (p . 54-61), Figure 11-12 (p . 54-61), Appendix 6]

Cl
p. 205

Table 1 Blank test of the test substance in water

A B C

peak sample peak area conc.

No. No. count in water

sample std. mg/L

A 1 <948 18969 <0.0003

2 <948 18969 <0.000 3

B 1 <956 19115 <0.0003

2 <956 19115 <0.0003

C 1 <1146 22912 <0.000 3

2 <1146 22912 <0.0003

D 1 <1275 25498 <0.0003

2 <1275 25498 <0.0003

~

Concentration of std. solution (Cstd) : 0.005mg/L

Equation : C=A=BxCstd